International Research Journal of Plant Science (ISSN: 2141-5447) Vol. 2(10) pp. 311-316, October, 2011
Available online http://www.interesjournals.org/IRJPS
Copyright © 2011 International Research Journals
Ogu G. I.
1*
,
Madagwu E. C
2
, Eboh, O. J.
1 and Ezeadila, J. O
3
1
Department of Biological Sciences, Novena University, P.M.B002 Ogume, Delta State.
2
Department of Public and Community Health, Novena University, P.M.B002 Ogume, Delta State.
3
Department of Applied Microbiology and Brewing, Nnamdi Azikiwe University, Awka, Anambra State.
Accepted 28 September, 2011
Diodia scandens leaf is applied topically for the treatment of various superficial skin infections among the Ukwuani aborigines of Delta state, South-South Nigeria. To obtain a scientific base for this practice, its phytochemistry and antidermatophyte potentials against three dermatophytes, Microsporium gypseum, Trichophyton mentagrophytes and Trichophyton rubrum isolated from infected hairs, nails and skins (mostly children) in Obiaruku, Ukwuani Local Government Area of Delta State, Nigeria were evaluated. Methanol and aqueous extracts of the leaves were tested using the agar diffusion method at extract concentrations 100mg/ml, while griseofulvin was used as the standard drug. The phytochemical investigation revealed the presence of saponin, flavonoid and tannins and cardiac glycosides. The antifungal activity showed that all the isolates were susceptible to both the organic and aqueous extracts, though the methanolic extracts had wider diameter of inhibitions than the aqueous extract. The highest susceptibility was observed against T. mentagrophytes (22.2mm) followed by T. rubrum (21.0mm), while
M. gypseum (18.5mm) was the least. Their minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) value was 3.13/6.25mg/ml for T. mentagrophytes and T. rubrum , and
6.25/12.5mg/ml for M. gypseum. The anti-fungal activity of the extract was not significantly affected by variations in temperature (30-100 o
C) and pH (2.0-10) for 1hour. This work, therefore may probably justify the traditional application of this plant in the management of skin diseases.
Keywords: Diodia scandens, superficial skin infections, antimicrobial activity, and agar diffusion method
INTRODUCTION
Dermatophytes are a group of closely related fungi known to cause human superficial mycoses in many tropical countries where their prevalence still remain a public health problem. They are classified into 40 species of three genera; Microsporium, Trichophyton, and
Epidermophyton based on the types of microconidia they produce (David et al ., 1997). Microsporium spp mostly affect hairs and skins, but not nails; Trichophyton spp infect hairs, skins and nails; while Epidermophyton spp infect skin and nails, but not hairs (David et al., 1997).
The mode of spread is either by direct or indirect contact with an infected particle which is usually a fragment of keratin containing viable fungus. Indirect transfer may occur via the floor of swimming pools, bath rooms or on brushes, combs, towels and animal grooming implements
*Corresponding E-mail: gideonikechuku@yahoo.com;
Phone: +2348065009432
(Shinkafi and Manga, 2011). The infections resulting from dermatophytes are hardly fatal but mostly debilitating and

312 Int. Res. J. Plant Sci. disfiguring diseases that can give rise to permanent deformations if untreated (Elewski, 1996; Yuanwu et al .
,
2009).
Several antifungal agents including various azoles, tolnafate cream and allylamine derivatives were introduced in the treatment. However, these antifungal agents are expensive and have varying degrees of toxicity (Dwivedi and Singh, 1998; Irobi and Daramola,
1993). Hence, there is the need to give greater attention to developing more anti fungal (anti-dermatophilic) drugs so as to effectively check the increasing prevalence of these infections, especially in the tropics and subtropics, where the climate make people more susceptible to the infections. Coincidentally, natural materials especially of plant products have been found to possess one or more medicinal properties (Farnsworth and Morris, 1976; Alves et al., 2000). Currently, several plants have been screened and discovered to possess significant antimycotic activities (Irobi and Daramola, 1993; Mann et al.
, 2008; Sule et al., 2010)
Diodia scandens SW (Rubaceae) is a straggling perennial herb with slender angular stem up to 3m high, with opposite to alternate ovate lanceolate leaves and white clustered flowers (Akobundu and Agyakwa, 1987).
Traditionally, different parts of the plants- sap, leaf, stem and root, are used for various medicinal purposes. In
Western Africa, it is used as antidotes (venomous stings, bites and pain killers, venereal disease), the leaves are used as uricant (Burkill, 1985). In Nigeria, the leaf extracts or sap are used for curing eczema, stop bleeding, treat bruises and minor cuts and ear problems, and also as antiabortifacient (Etukudo, 2003). According to Essien et al 2010, tannins, saponins, and cardiac glycosides were identified as the phytochemical constituents of D. scandens leaf extract.
Recent personal interaction with the Ukwuani aborigines of Delta state, Nigeria, reveled the leaf extract of D. scandens is used by both the young and old to treat superficial skin disorders with good results. The procedure involves squeezing out the sap from freshly collected leaves, and applying the expressed fluid by scrubbing daily on the infected skin sites for about 3-
5days. In view of the traditional claim for the usefulness of the leaf of D.
scanden, it is pertinent to provide a scientific base for this practice. Currently, there is dearth of such information in the available arsenal of literature.
This work was therefore conducted to investigate the effect of D scandens leaf extracts against three dermatophytes: Microsporium gypseum, Trichophyton mentagrophytes and Trichophyton rubrum isolated from infected hairs, nails and skins (mostly children) in some towns in Ukwuani Local Government Area of Delta State,
Nigeria.
MATERIALS AND METHODS
Collection, Identification and Preparation of plant material
Fresh Diodia scandens plant was collected from within the premises of Novena University Campus, Delta State,
Nigeria, in September 2010. The plant was identified at the Department of Biological Sciences, Novena University
Ogume (Amai Campus) via the morphological appearance as described by Akobundu and Agyakwa,
(1987). The leaves were confirmed taxonomically at the
Botany unit of the Department courtesy of Prof. J. M. O.
Eze. The fresh leaves were manually separated from the plant and washed properly with tap water before rinsing with sterile distilled water. The cleaned leaf samples were air-dried at ambient temperature (30±2 o
C) to constant weight for about 7 days. The dried materials were reduced to coarse form using a pestle and mortar and further pulverized into very fine particles with an electric blender (Super Search Model 2815). The powdered sample obtained was stored in a polyethylene bag until needed for analysis.
Preparation of Extracts
Exactly two hundred grams (200g) of the powdered plant sample was exhaustively Soxhlet extracted using 500ml each of sterile distilled water and 95% ethanol for about
8hours. The extracts were evaporated to dryness (on water bath) at 45 o
C. The resulting dark brownish mass with respectively yields of 24.2g (12.1 %w/w) and 24.8g
(12.4%w/w) for the aqueous and ethanol extract were obtained. 1.0g of each extract was reconstituted in 10ml
20% Dimethyl sulfoxide (DMSO) to obtain working concentration of 100mg/ml. The stock solution was then sterilized using a 0.45
u m pore size membrane filtered by suction pump. The sterilized extract was stored inside sterile McCartney bottles and kept in refrigerator at 5 o
C.
Phytochemical screening of extracts
The extracts were qualitatively screened for alkaloids, tannins, soponins, flavonoids steroids, terpenoids and cardiac glycosides using standard procedure (Trease, and Evans, 1989; Edeoga, et al ., 2005)
Isolation of test Micro-organisms.
The test fungi ( Microsporium gypseum, Trichophyton

rubrum and Trichophyton mentagrophytes ) used in this study were isolated from children with dermatophyte infections( infected skin, hairs and nails) in primary schools in Obiaruku, Ukwuani Local Government area of
Delta state, Nigeria.
The sites of the infection were first cleaned with surgical spirit, before using scalpel to scrap the scales, hairs and nails into clean white paper and all samples labelled appropriately for analysis. The collected samples were examined microscopically (with 10-20%
KOH) for the characteristic macroconidia and microconidia, presence of hyphae and arthroconodia, and also plated on Sabouraud Dextrose agar (Lab M,
India)(SDA) supplemented with 0.05% chloramphenicol and incubated at ambient temperature (28-30 o
C) for 7-14 days. The fungal isolates were identified based on colonial appearance, pigment production on the underside and microscopic characteristics (Rebel and
Taplin, 1970; Campbell, 1980; Hartman and Rohde,
1980). The pure culture was further confirmed by comparing them with stock cultures kept at the National
Institute for Veterinary Research VOM, Nigeria.
Standardization of Inoculum
Fungal spores were harvested after 7 days old SDA slant culture was washed with 10ml normal saline in 2% Tween
80 with the aid of glass beads to help in dispersing the spores. The spore suspensions were standardized to
10
5 spores /ml.
Antifungal Susceptibility Studies
Sabouraud Dextrose Agar (SDA) (Lab M, India) was prepared according to specifications, autoclaved (121 o
C for 15minutes) supplemented with 0.05% chloramphenicol and dispensed into 11cm diameter Petri dishes. 1ml of each standardized spore suspension
(10
5 spores/ml) was evenly spread on the surface of the gelled SDA plates. Then, sterile cork a borer (6mm in diameter) was used to make well at the centre of each seeded plates. Thereafter, 0.2ml of the reconstituted aqueous and methanol extracts (100mg/ml) was applied into each labeled well. 0.2ml each of 20% DLMSO and standard drug griseofulvin, (100mg/ml) (Clarion Medicals
Ltd. Lagos, Nigeria), served as negative and positive control respectively. The plates were incubated at ambient temperature for 1-7days and observed for growth. Anti-fungal activities of the extract as well as the controls were measured and recorded as means
Ogu et al. 313 diameter of zones of inhibition around the three wells.
Determination of Minimum Inhibitory Concentration
(MIC) of extracts
The MIC of the extracts was also carried out using broth dilution method as described in Ibekwe et al, 2001.
Sabouraud dextrose broth prepared according to the manufacturer’s instruction in separate test-tube sterilized at 121 o
C for 15minutes and then allowed to cool. Twofold serial dilutions of the aqueous and methanol extracts in the broth were made from the stock concentration of the extract to obtain 50, 25, 12.5, 6.25, 3.13 and 1.56mg.
0.1ml of the standardized inoculums (10
5 spores/ml) was then inoculated into the different concentrations of the extracts in the broth. Controls were also set up along the test experiment. The test tubes of the broth were incubated at 30 o
C for 1-7days and observed for turbidity of growth. The lowest concentration which showed no turbidity in the test tube was recorded as the MIC.
Determination of the Minimum Fungicidal
Concentration (MFC) of extracts
Fresh Sabouraud Dextrose agar media were prepared, sterilized at 121 o
C for 15mins and was poured into sterile
Petri-dishes and left to cool and solidify. The contents of the MIC in the serial dilution were then sub-cultured onto the media and incubated at 30 o
C for 1-7days and observed for colony growth. The MFC was the plate with the lowest concentration of extract and without colony growth.
Effect of temperature and pH on stability of extracts
The method of Doughari and Sunday, (2008) was followed in this analysis. 50mg/ml concentration of extracts was reconstituted in 20%DMSO, and 5ml of the suspension dispensed into five test tubes. The tubes were then treated at 4 temperature, 60 o
C, 70 o o
C in the refrigerator, 30 o
C and 100 o
C at room
C using water bath for
1hours. They were tested for antifungal activities as described earlier. The effect of pH was determined by treating the extract at pH ranges of 2.0-10.0 using 1N
HCL and 1N NaOH solutions respectively in a series of test tubes for 1hour. After 1hour treatment, each of the extract was neutralized (pH 7.0) once again using 1N
HCL and 1N NaOH as the case may be. They were also

314 Int. Res. J. Plant Sci.
Table 1: Phytochemical analysis of D. scandens extracts
Phytochemical composition Aqueous extrract Methanol Extract
Alkaoids
Flavonoids
Saponins
Tannins
Cardiac glycosides
Steroids
Terpeniods
Anthroquinones
-
+
+
+
+
+
-
-
_
+
+
+
+
+
-
-
Key: + = detected, = Not detected
Table 2: Anti-fungal activities of aqueous and methanol leaf extract of D. scandens(100mg/ml)
Zones of Inhibition (mm)
Test Isolate Aqueous Extract Methanol extract Griseofulvin 20% DMSO
T. mentagrophytes
T. rubrum
19.5a**
19.2a**
22.2a*
22.1a*
25*
25*
0
0
M. gypseum 18.4a** 20.3a* 24 0
Results are means of three replicate values. Mean values followed by same letter are not significantly different by
Duncan multiple range test (a P>0.05; *P>0.05, **P<0.01), tested for antifungal activities as described above
Statistical analysis
The data was analyzed with ANOVA and the means were separated using Duncan Multiple Range Test (DMRT) at a probability level of 5%.
RESULTS AND DISCUSSION
The phytochemical analysis revealed that flavonoids, saponins, tannins, and cardiac glycosides were present in both aqueous and methanolic extracts of leaf of scandens. However, alkaloids, terpenoids
D. and anthroquinones were not detected in both extracts (Table
1). The antimicrobial potency of any plant is linked to the levels of secondary metabolites inherent in its extract.
Saponins, a special class of glycosides with soapy characteristic, were reported as active antifungal agents
(konkwara, 1976; Sodipo et al., 1991). Tannins have been reported to hinder the development of micro-organisms by their ability to precipitate and inactivate microbial adhesions enzymes and cell envelope proteins
(Konkwara, 1976; Cowan, 1999). The antimicrobial activity of flavonoids is due to their ability to complex with extracellular and soluble protein and to complex with bacterial cell wall, thereby disrupting their membrane integrity.(Tsuchiya et al., 1996) The significant antifungal activities observed in this study could thus be attributed to the interaction of one or more of the identified metabolites against the test organisms.
The result of antifungal activities showed that both aqueous and methanol leaf extract of D. scandens appreciably inhibited the growth of the three test dermatophytes at concentration of 100mg/ml (Table 2).
This result is in line with the work of Shinkafi and Manga,
(2011), who reported that the aqueous and organic leaf extracts of Mitracarpus scaber and Pergularia tomentosa exhibited significant ant-fungal activities against T. mentagrophytes, T. rubrum and M. gyseum at extracts concentrations of between 80 and 160mg/ml. The activities of the methanol extract were higher, though not significant (P>0.05) when compared with the aqueous extract. The reason for this slight difference may be attributed to the solubility level of the phytoconstituents in the extracting solvents. It means that the methanol dissolved more of more of the active ingredients than aqueous solvent. This reason is supported by Cowan
(1999), who reported that organic solvent were better extraction solvent over water. The Trichophyton spp

Ogu et al. 315
Table 3.
Minimum inhibitory concentration and Minimum fungicidal concentration of Extracts
Test Isolate
T. mentagrophytes
T. rubrum
M. gypseum
Aq Extract (mg/ml)
MIC /MFC
6.25/12.5
6.25/12.5
6.25/12.5
Meth Extract (mg/ml)
MIC / MFC
3.13/6.2
3.13/6.2
6.25/12.5
Griseofuvin (mg/ml)
MIC/ MFC
0.78/1.56
0.78/1.56
0.78/1.56
Table 4.
Effect of temperature on the stability of the extracts
Test Isolate Zone diameter of Inhibition (mm)**
Temperature ( o
C) pH
30* 4 60 80 100 5.6* 2 6 8 10
T. mentagrophytes 22.1 22.5 23.1 23.2 23.9 23.4 23.5 23.1 22.2 21.4
T. rubrum 21.3 21.0 22.3 22.4 22.7 22.5 22.9 22.1 21.7 20.1
M. gypseum 20.1 20.0 20.4 20.8 21.0 21.9 21.8 21.3 20.9 19.9
*Untreated sample, **Results are means of three replicate values
(19.5mm-22.2mm diameters) were more sensitive to the extracts than M. gyseum (18.4mm-20.3mm diameters). while only T. mentagrophytes and T. rubrum had similar
This result is similar to the findings of (Natarajan et al.,
2003; Ahmed et al., 2009) who separately reported that
Trichophton spp were the most susceptible test dermatopyhtes to the extracts of Azadirachta indica and
MIC/MFC values of 3.13/6.25mg/ml. Pant extracts that exhibited similar MIC/MFC against Trichophyton rubrum,
Trichophyton mentagrophytes, Microsporum nanum and
Epidermophyton flocosum had been reported in the past
Vitellaria paradoxa respectively. The slight variations in their susceptibility of the dermatophytes could be traced to the similar structural and chemical composition of
(Natarajan et al., 2003; Natarajan and Natarajan (2009).
However, the data in this study are not in agreement with that from the work of Ali-Shtayeh and Abu-Ghdeib (1999), who reported that aqueous extracts of 22 plants recorded fungal cells and cell wall structure, unlike the bacteria.
There was no inhibition recorded from the negative control (20% DMSO), while the standard drug, griseofulvin significantly inhibited the growth of all the test dermatophytes. The action of the aqueous, and methanol extracts of D. scandens against the dermatophytes tested may be due to inhibition of fungal cell wall, pore formation in the cell and leakage of cytoplasmic constituents by the active components such as alkaloids, saponins, protein, amino acid and sphingolipid biosynthesis and electron transport chain
(Hassan et al .
, 2007). The activities of the extracts were not significantly different from the standard drug at
P>0.05, though in their crude form. Similar result was reported by (Shinkafi and Manga, 2011). This is an indication of the antifungal efficacy of this plant extract as remedy for mycoses caused by the test dermatophytes.
Table 3, displays the MIC and MFC of the extracts and standard drug. The lowest MIC/MFC values of 3.13/6.25mg/ml were obtained from the methanol extract against T. mentagrophytes and T. rubrum , while
6.2512.5mg/ml was observed against M. gypseum . With the aqueous extract, similar MIC/ MFC value of
6.25/12.5mg/ml was recorded against all the test isolates, wide variations in their MIC/MFC values against
Microsporum canis, Trichophyton mentagrophytes and
Trichophyton violaceum. This could be attributed to the variations in the phytochemical properties of the plants and differences among the fungal species. The MIC and
MFC of the standard drug against the test organisms were 1.56mg/ml and 0.78mg/ml respectively. The comparable MIC/MFC variation between the plant extract and the standard drug shows that D. scandens extract actually possesses potent antifungal (anti-dermatophyte) properties that can be harnessed in the management of mycotic infections.
The result of effect of temperature and pH on the activity of the extract shows that temperature range of 4-
100 o
C as well as pH range of 2.0-10.0 did not significantly affect the activities and stability of the extract.
As the temperature increased from 4 to 60 o
C, the mean zone diameter of inhibition against T. mentagrophytes increased from 22.5mm to 23.1mm and then remained reasonably stable all through to 80 o
C (23.2mm), but again increased slightly to 23.9mm at 100 o
C. The same trend was observed aganinst the other test isolates. For the pH effect, relatively better activity were generally observed at acid pH (2-6), but decreased slightly as

316 Int. Res. J. Plant Sci. alkaline condition increased to 10.This is an indication that the bioactive ingredient in this plant extract are thermostable and to some extent stable against various ranges of pH, though acid pH seem to favour a slightly higher antimicrobial activity. The relatively higher activity of the extract at acid pH also indicates that the plant extract may possibly resist the acidic pH of the stomach, thus permitting the development of oral antibiotics from them. This finding is in consonance with the work of
Molan (1992) and Doughari and Obidah (2008).
In conclusion, this study has shown that the leaf extract of D. scandens possesses phytochemical principles that can inhibit the growth of some dermatophytes. This, therefore, may probably provide a good scientific backing for its traditional application as cure for superficial skin mycoses among some tribes in southern Nigeria. Further research is however recommended to isolate, purify and study the actual bioactive principle on a wide array of dermatophytes and other skin diseases with a view to synthesizing a novel antifungal agent for the management of various skin diseases in Nigeria and other African countries.
ACKNOWLEDGEMENTS.
We are grateful to Prof. E. O. Igumbor, and Prof. E. U.
Igumbor for their technical support throughout the project.
National Institute for Veterinary Research VOM, Nigeria, is also highly appreciated for their help in identification and confirmation of the test dermatophytes.
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