Pharm. Méd. Trad. Afr. 2004, Vol.13, pp. 149-159
ANTI-CANDIDAL PROPERTY AND ACUTE TOXICITY OF GLADIOLUS
GREGASIUS BAKER (IRIDACEAE)
1. C. Assob Nguediaa, F.-X. Etoa_8 , V. Penlap Benga, D. Lontsi", Y. Kuete"; and R. Somo
Moyou C
a Laboratory ofApplied Microbiology and Molecular Pharmacology,Faculty ofScience,
University of Yaoundé I. P.o. Box 812, Yaoundé. Cameroon.
bLaboratory ofOrganic Chemistry, Faculty ofScience, University of Yaoundé I. P.o.
Box
812, Yaoundé, Cameroon.
c Laboratory of Parasitology, Faculty of Medicine, University of Yaoundé L
P.o. Box 8445. Yaoundé, Cameroon.
Corresponding author:
J. C. ASSOB NGUEDIA, Department of Biochemistry, Faculty of Science, University of
Yaoundé 1, P.O. Box 812, Yaoundé, Cameroon. Fax: (237) 223 1407 Ite!. : 9629452;
E-mail: juleclement@yahoo.fr.
AB8TRACT
Hydroethanolic 80% extract of the bulbs of Gladiolus gregasius Baker showed high
degree of anti-candidal activities against Candida albicans and Candida krusei; the minimal
inhibitory concentration on these strains were at equal concentration of 12.5 ug/ml compared
to nystatine (3.2 ug/ml) the reference drug. However this extract has no inhibitiory activity
against gram - and gram + bacterial strains: Escherichia coli, Pseudomonas aeruginosa,
Proteus vulgaris, Staphylococcus aureus and Streptococcusfaecalis. Phytochemical screening
revealed an important saponins content which may explain this very high level anticandidal
property of the extract. The evaluation of the acute toxicty on male (LD so=1O.5 glkg) and
female (LDso=14.5 g/kg) rats revealed that the crude extract was not toxic. Sorne serum
biochemical parameters were also evaluated to assess the liver (ALP, ASA T, ALAT,
proteins) and kidney (urea and creatinin) functions; Significant differences (p<O.OS) were
noted between the control and test groups in male (4 g/kg) and female (8 g/kg body weight).
These results suggest that Gladiolus gregasius can be used in the treatment of candidal
infections.
Key words: Anticandidal activity; Gladiolus gregasius Baker; Iridaceae; Acute toxicity
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Pharm. Méd. Trad. Afr. 2004, Vol. 13, pp. 149-159.
1 INTRODUCTION
The growing importance of fungi in the medical field has attracted a lot of interest and
has heightened awareness of the risks they pose as pathogens to humans. Yeast of the genus
Ca~dida is by far the most frequently isolated yeast from humans. It is found in the mouth,
gastrointestinal tract and women genital tract as commensal of these anatomie sites. However,
C. albicans is the most pathogenic of the opportunistic fungi and its transformation from a
commensal to pathogen is elicited by local and systemic factors which render the hum an host
susceptible to infections (Farell et al., 1983; Ryley, 1986; Odds, 1988).
Medical records from the CPC (2001) has highlighted candidiasis as the most
recurring causes of vaginal infection in women. Because CUITent comercialised antibiotics are
costly and have indeed considerable drawbacks in terms of serious side effects; Herbal drugs
are playing important role in healthcare in Cameroon. The demand by most people in
Cameroon for medicinal plants, as for the rest of Africa, have increased this last decade
(Herbal gram. 2003). Sorne naturally occuring substances found in higher plants: terpenoid,
flavonoids, alkaloids and saponins are an important group of molecules with antifungal
properties (Cotoras et al., 2001).
Gladio/us gregasius Baker belongs to the familly of Iridaceae. It is a bulbous slender
herb with very narrow leaves, flowers are purple or white with purple throat. The plant is
found in the West part of Cameroon and extend to the West African region (Hutchinson et
Dalziel, 1968). It is used by traditional healers in the West Province of Cameroon to cure
urogenital tract, skin and respiratory infections (Megne, 1997). But, up to now, nothing has
been reported conceming the chemistry and biological properties of the plant. The aim of this
paper is to present the phytochemical analysis of this plant and its antimicrobial properties as
weIl as its acute toxicity on male and female albinos rats.
2. MATERIAL AND METHODS
Plant material: Bulbs of G. gregasius Baker (Iridaceae) were collected at Bafou (West
Province of Cameroon) in 2002. The plant was authenticated at the Department of Vegetal
Biology of the Yaoundé 1 University and at the National Herbarium of Yaoundé (Cameroon)
where voucher specimens are deposited under the number 52405/HNC.
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Preparation of extracts: Air-dried and ground powder (500 g) of G. gregasius bulbs was
macerated at room temperature with 1 liter of 80% hydroethanolic solvent and the filtrate was
evaporated ta dryness using a ventilated aven at 40°C ta produce 60 g of a brown extract.
Before testing, the dry residue was dissolved in distilled and sterilized water ta give a
concentration to be used in the tests.
Phytochemical analysis: The phytochemical analysis were done on the extract to caracterise
their biological properties using the common previously methods described by Harbone
(1976).
Screening of antimicrobial activity: The microorganisms used in this work, provided by the
Medical Bacteriology Laboratory of the "Centre Pasteur of Cameroon (Yaoundé)", included
yeasts strains: Candida albicans (C albicans (LMP0204U)), Candida krusei (C krusei
(LMP0304U)) and Gram + and Gram- bacteria strains: Staphylococcus aureus (s. aureus
(LMP 0106U)), Streptococcuspneumoniae ((s. pneumoniae) (LMP 021 OU)). Escherichia coli
(E. coli (LMP 0201 U)), Proteus vulgaris (P.
vulgaris (LMP 0210U)). Pseudomonas
aeruginosa ((P. aeruginosa) (LMP 0102U)), which were kept under the collection "LMP" in
our Laboratory at 4°C. The tested microorganisms were activated on Nutrient agar (bacteria
strains), or Sabouraud agar (yeasts strains) at 37°C for 24 hours. Iwo or three isolated
colonies were diluted aseptically in sterile physiological water to yield density matching with
the 0.5 Mac Far/and turbidity standard (1.5 108 Colony Forming Units (CFU)/ml), and 100
fold dilution done to obtain a suspension with a final concentration of approximately 106
CFU/ml for the tests. Different methods were applied for the screening.
For the hole-plate diffusion method (Berghe et Vlietnick, 1991), petridishes were filled
with the Agar Mueller Hinton (bacteria) medium or sabouraud dextrose agar (yeasts), and
inoculated with the test microorganism. Wells (6 mm diameter) were then made and filled
with 150 III of the test sample: 80% hydroethanolic extract of G. gregasius extract, nystatine
(yeasts) or gentamicine (bacteria) were included in every tests.
The Minimum inhibitory concentration (MIC), considered as the lowest concentration
of the sample whieh inhibits the visible growth of microbe, was determined by the macrobroth
dilution method (Carbonelle et al.,1987) in broth Mueller Hinton or Sabouraud medium
supplemented with glucose 10% and phenol red. In this method, 0.4 ml of a freshly prepared
cell suspension (lO 6 CFU/ml) was added to 3.6 ml of susceptibility test broth containing serial
twofold dilutions of each tested sample in glass tests tubes (13 by 100 mm) fitted with cotton
151
Pharm. Méd. Trad. Afr. 2004, Vol.13, pp. 149-159.
wood. These tubes were incubated in air at 37°C for 24 hours before being read. The MIe was
considered as the lowest concentration of the sample that prevented visible growth.
Minimum Fungicidal Concentration (MFC) (the lowest concentration yielding
negative sub cultures or only one colony) was also deterrnined by subculturing 100 III of each
negative growth tube and positive growth control for 24 hours on fresh free from antibiotic
agarised Mueller Hinton or sabouraud.
Animais: Adults Wistar albinos rats (120 - 140 g); were bred at the Departement of
Biochemistry, University of Yaoundé 1. They were ail clinically healthy and maintained in
standard environmental conditions oftemperature (27.0±0.5 "C). They were fed a standard
diet and tap water ad libitum. The bioassay was conducted in accordance with the
internationally accepted principles (WHO, 1992) for laboratory use and care. Rats were
deprived of food but not of water 12 h prior to administration of the test substance.
Acute toxicity: The animais were separated in six groups of four males and four females
including one control (groupl) and five treated. The remaining groups (2-6) received 4,8, 12.
16, 20 g/kg of body weight each respectively of oral single doses of the 80% hydroethanolic
extract. The control group received tap water at an equivalent volume. Observations were
made and recorded systematically 1 and 2h after extract administration. The visual
observations included changes in respiratory, motility and skin sensitivity, diarrhoeae,
behavioral pattern, weight gain, food and water consumption. The number of survivors was
noted after 48 hours and these were then maintained for a further 5 days, after which they
were sacrificed by decapitation by making an incision on the jugular vein to collect blood.
The medium letal dose (LDso) was deterrnined using the Behrens and Karber method (1983).
Evaluation of the toxicity degree was based on previously described method (Delongeas et al.,
1983 ; Shorderet, 1989; Lu, 1992).
Bioehemical estimation: Sera were assayed for Alkaline phosphatase (ALP) (Morgernstem
et al., 1965), Aspartate aminotransferase (ASA T) (Reitman and Frankel, 1957), Alanine
aminotransferase (ALAT) (Reitman and Frankel, 1957), creatinin (Cheesbrough, 1985), urea
(Cheesbrough, 1985).
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Pharm. Méd. Trad. Afr. 2004, Vol.i3, pp.i49-i59
Statistical analysis: One way analysis of variance (ANOVA) was applied for determining the
statatistical significance in various markers level between the control and the tested group.
The level of significance was set at 0.05 and 0.01 (Wonnacott and Wonnacott, 1995).
3 RESULTS AND DISCUSSIONS
The phytochemical studies revealed the presence of an important content of saponins
and glycosides and moderate level of polyphenol, phenols, triterpenes and steroids. Tannins,
alkaloids and anthranoids were not noticed. The results of the soIid diffusion method tests
showed an important antifungaI activity on C. albicans and C. krusei with inhibition zone of
21±O1 and 22±02 mm respectively (table 1); however no inhibition zone was noticed on ail the
bacteria strains (s. aureus, S. faecalis, E. coli, P. vulgaris and P. aeruginosa). The inhibition
pararameter ofthis extract on the two Candida strains presented in table 1 revealed an
important inhibition parameter. In fact the minimal inhibitory concentration on these strains
were at equal concentration of 12.5 ug/rnl compared to nystatine (3.2 ug/ml) the reference
drug. The minimal fungicidal concentration values also showed an important activity of the
total extract (25 ug/ml) as regarded to that obtained with nystatine (6.4 ug/ml), This specifie
anticandidal activity of the hydroethanolic extract could be attributed to saponosides which
have shown better inhibitory activity against fungi strains rather than against bacteria
pathogens (Bruneton, 1999).
The same extract was tested for its acute toxicity on male and female albinos rats
(Wistar).
Observations were made and recorded systematically 1 and 2h after extract
administration. The visual observations which included changes in respiratory, motility and
skin sensitivity, diarrhoeae, behavioral pattern, weight gain, food and water consumption
significantly changed in group 3 (8 glkg) in male and female rat as compared to the control.
This implies that at high dose (~8 glkg) ail the animais were physically affected and resulted
to sorne death recorded. We registered total mortality at 12 and at 20 g/kg body weight
respectively in male and female. The medium letal dose (L050) values were 10.5 g/kg and
14.5 g/kg ofbody weight on male and female rats respectively. It can be concluded that G.
gregasius is not toxic as regard to the threshold of toxic substances (5 g/kg) stipulated by
Delongeas et al. (1983) ; Shorderet, (1989) and Lu, (1992). On the other hand, these value of
the L050 showed that male rats were more susceptible to this extract than female. This
difference in rcsponse to substance may be due to sex hormones which act on enzymes
153
Pharm. Méd. Trad. Afr. 2004, Vo1.13,pp.149-159.
involved in the metabolism ofxenobiotics in mammalians; as this difference is always noticed
after puberty (Hodgson et Guthrie, 1980).
Blood chemistry of male and female rats are shown in table 2 and 3 repectively. In
male, it is observed that there is significant increased in ALP, ASAT, ALAT, urea (p<0.05)
",
creatinin (p<O.OI) values in the test groups (4 & 8 glkg) when compared with the control.
Significant increase (p<O.OI) in ALP, ASAT and urea value appeared in group 3 (8 g/kg).
ALAT and creatinine level sgnificantly (p <0.01) increased in group 4 (12g/kg) and 5 (16
g/kg) as compared to the control. Estimating the activities of serum marker enzymes like
ASAT, ALAT and ALP can make assessment ofliver function. When liver cell plasma
membrane is damaged a variety of enzymes normally located in the cytosol are released in to
the blood stream. Theil' estimations in the serum are useful quantitative marker of the extent
and type of hepatocellular damage (Venukumar and Latha, 2002). The enhanced activities of
these enzymes in all the treated male groups and in groups 3,4 and 5 for female correspond to
the liver damage induced by the extract. Creatinine is a nitrogenous waste product from the
metabolism of creatine in the skeletal muscle. Creatinine diffuses freely throughout the body
water. It is filtered from the blood, by the kidney and excreted in the urine. Urea is the main
waste product of the protein breakdown. As the rate of production is generally fairly constant,
raised plasma urea levels indicate decreased renal function. The estimation of creatinine and
urea levels are therefore important tools for the investigation of the kidney damage
(Cheesbrough, 1985). These biochemical parameters similarly increased in the all our tested
group (male) and in groups 3, 4, 5 (female) indicated that the extract affected kidney function.
The high level anti-candidal and the non toxic properties of the 80 % hydroethanolic
extract of G. gregasius ensure the safety of the consumers. Further chromagraphic studies of
the fractions are needed in the search of new active molecules. The discovery of a potent
herbaI remedy that is safe is a big advantage in fungal infection therapy. It is vital in the
treatment of systemic fungal infection that are usually frequent in immuno-compromised
patients as toxicities induced by commercially antifungal drugs are often observed in these
patients due to high dosage and prolonged therapy (Somchit et al., 2003).
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Pharm. Méd. Trad. Afr. 2004, Vo!.l3, pp.l49-l59
Acknowledgments
We wish to express our profound gratitude to Ml. Guimapi Charles Moodem, the
traditional healer who provided the plant material, M. Koufani Analectus (National
Herbarium of Yaoundé), the "Centre Pasteur du Cameroun (Yaoundé)" and to the Chinese
Cooperation.
References
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Table 1: Antimicrobial parameters of the 80% hydroethanolic extract of G. gregasius and
nystatine against the tested Candida species
souches
BOO 80%
Nystatine
i
<1> (250 ug/ml)
MIC
MFC
<1> (2011g/ml)
MIC
MFC
(ug/ml)
(ug/ml)
(ug/rnl)
(ug/ml)
C. albicans
22±Ol
12.5
25
25±01
3.2
6.4
C. krusei
21±O2
12.5
25
25±Ol
3.2
6.4
<1>: inhibition diameter
157
Pharm. Méd. Trad. Afr. 2004, Vol. 13, pp.149-159
Table 2: Effect of the hydroethanolic extract of Gladiolus gregasius Baker on different
biochemical parameters in the serum of male rats.
Serum biochemical
Dose
pararneters
N=4
N=4
N=l
°g!kg
4 g/kg
8 g!kg
ALP (UI/l)
62,8±O,4
63,9±O,3*
67,4*
ASA T (UI/l)
40,8±O,6
43,1±O,5*
44,3*
ALAT (UI/I)
19,O±O,4
2l,9±O,5*
22,8*
Urea (mg/l)
166,3±O,8
167,8±O,06*
173,2*
Creatinine (mg/l)
7,21 ±O,06
7,33±O,15**
7,61 **
* Significantly different from control (p<O,05).
** Significantly different from control (p<O,Ol)
N : number of survivors
158
Pharm. Méd. Trad. Afr. 2004, Vol.13, pp. 149-159
Table 3: Effect of the hydroethanolic extract of Gladiolus gregasius Baker on different
biochemical parameters in the serum of female rats.
Serum biochemical
Dose
parameters
og/kg
4 glkg
8 g/kg
12g/kg
16g/kg
N=4
N=4
N=2
N=2
N=2
ALP (UIII)
63,4±O,2
62,6±O,3
6l,3±O,5"''''
66,4±O,6"""
67,2±O,5"""
ASAT (UIII)
40,7±O,3
4l,9±1.4
44,5±O,3"""
48,6±O4"""
50,l±O,2"""
ALAT (UI/l)
l8,9±O,7
l8,6±O,6
19,4±O,4
2l,O±O,3'"
23,3±O,4'"
Urea (mg/l)
l67,2±O,8
l68,4±1,l
l68,9±O,6"""
172,3±O,4 "'*
175,6±O,8"'*
Creatinin (mgll)
7,33±O,13
7,O9±O,45
7,45±O,21
7,63±O,18*
8,21±O,22'"
'" Significantlydifferent from control (p<O,05).
*'" Significantlydifferent from control (p<O,OI)
N : Number of survivors
159