Abstract: Nymphaea odorata (Nymphaeaceae) is an old herbal recipe used in the treatment and or management of ocular, skin, gastrointestinal and urino-genital ailments amongst many others. However, its use in malaria control at the larval stage is yet to be investigated. Hence the larvicidal and anti-microbial studies were undertaken. The larvicidal assay determined in terms of percentage mortality showed that the crude leaf extract gave weak larvicidal activity (LA %) of 10 and 20% (at 5% w/v) and 20 and 30% (at 10% w/v) both at 12 and 24 h incubation, respectively. Surprisingly, the crude extract and fractions were inactive against the bacterial and fungal isolates tested. These results in particular render untenable claims in ethno-medicine of the uses of the plant in treating infections especially those of microbial origin.
INTRODUCTION
The genus; Nymphaea takes its name from the Greek word; numphe which means virgin or water nymphe and is reputed for the anti-aphrodisiac activity of its members. Nymphaea odorata (water-lily) is known by common names such as fragrant water-lily, American white-lily and Alligator Bonnet amongst many others (Josh and Emily, 2002). It belongs to the family; Nymphaeaceae which consists of six genera and seventy species (Trease and Evans, 1996) and grows in ponds, marshes and sluggish streams (Odey and Kingdersley, 1993).
Water-lily is an old fashioned herbal remedy which when made into a douche is used to treat eye troubles, leucorrhoea, diarrhea, scrofula, inflamed tissues, bronchial, kidney, bladder and prostrate-gland troubles. The leaves and roots are made into poultice to treat wounds, cuts, bruises, ulcers, boils and painful swellings (Harvey and John, 1898). Tea made with the roots makes a good gargle for irritation and or inflammation of mouth and throat. It can also be used to treat coughs, tuberculosis, remove freckles and pimples from the face and skin (George, 2001).
Compounds such as tannins [tannic acids and gallic acids (anti-microbial], alkaloids[nymphaerine and nupharine] and glycosides [cardenolide and myrictrin] which are antiseptic, astringent and demulcent have been reportedly isolated from this plant (Trease and Evans, 1996). In spite of these, the present study was designed with the aim of investigating into the larvicidal activity and also to confirm or otherwise the sensitivity of the plant extract and fractions to selected microbes.
MATERIALS AND METHODS
Collection of Plant
The fresh leaves of Nymphaea odorata were collected in the
month of November, 2006 from Idim Itak (Stream of Itak) in Itak Atap Ikono
Local Government Area of Akwa Ibom State, identified and authenticated
by R. Nia of the Department of Pharmacognosy and Natural Medicine, University
of Uyo, Uyo, Nigeria where voucher specimen NoH53 was deposited.
Chemicals, Micro-Organisms and Media
The chemicals reagents: Butanol, Ethanol, Chloroform and Hexane (all
of AnaLaR grade; Aldrich Chemicals Inc, USA) were purchased in Uyo. Silica
gel (254GF), Ampiclox and Ketoconazole were obtained from Unique Pharmaceuticals
Limited, Lagos, Nigeria. The micro-organisms (Bacillus subtilis,
Staphylococcus aureus, Escherichia coli, Salmonella typhi,
Klebsiella pneumoniae and Candida albicans) clinically isolated
from human specimens; urine, faeces, wounds and vaginal swabs were obtained
from the Medical Laboratory, University of Uyo Health Center. They were
collected in sterile bottles, identified and authenticated by convectional
biochemical tests (Gibson and Khoury, 1986; Murray et al., 1995)
and then refrigerated at 0-5 °C at the Pharmaceutical Microbiology
and Parasitology Unit, Faculty of Pharmacy, University of Uyo, Uyo, Akwa
Ibom State prior to use. Also, Mueller Hinton II Agar (Biotec Laboratory,
Ipswich, England), Sabouraud Dextrose Agar (International Diagnostic Group
PLC, Lancaster, England) and Nutrient Broth (Oxiod Limited, Basingstroke,
England) were used.
Extraction and Processing
The leaves were air-dried and powdered in an electric mill. The resultant
ground powder was then extracted with cold 50% aqueous ethanol at room
temperature (27 ± 2 °C) for 72 h. The filtrate was evaporated
to dryness using a rotary evaporator (Buchi CH-920, Laboratorium Technic,
Flawk/SG, Switzerland). The dried crude ethanolic extract was subsequently
investigated for plant metabolites (alkaloids, tannins, cardiac glycosides,
terpenes, anthraquinones and flavonoids) according to laid down phytochemical
methods (Harborne, 1984; Sofowora, 1993; Trease and Evans, 1996). Also,
the dried crude ethanolic was chromatographed on silica gel (254GF) column
and gradient elution using hexane: chloroform: butanol (1:1:1) mixture.
Eluates which showed similar TLC profiles under UV (λ 366) were pooled
and bulked separately to obtain the hexane, chloroform and butanol fractions
were evaporated to dryness and stored in a refrigerator at -4 °C prior
to the biological tests.
Larvicidal Assay
The Breeding of Larvae of Anopheles gambiae
The larvae were bred by keeping outdoor basins of water under
growing shrubs near houses for about two weeks. After this period, at
least three groups of mosquitoes larvae were identified accurately in
a container using classical methods (Sievers et al., 1949). Anopheles
gambiae, Aedes agypti and Culex piper-fatigans responsible
for the transmission of malaria, yellow fever and filariasis, respectively
were so identified. The fourth instar larvae of Anopheles gambiae
were later selected, separated and the species authenticated at the Department
of Entomology, Michael Okpara University of Agriculture, Umidike, Abia
State, Nigeria before further work.
The method employed for the determination of larvicidal activity was adopted from that described by several researchers (Ojewole et al., 2000) and WHO directives on such assay with modifications (WHO, 1970). Thirty Anopheles gambiae larvae in their fourth stage were put in recovery cups (250 mL plastic jars) containing 10 mL de-ionized water (pH 7.0) at room temperature (27 ± 2 °C). Three (3 mL) volume each of the graded concentrations of the extracts (5 and 10% w/v) were added to 90 mL de-ionized water, mixed thoroughly and then poured into exposure cups (250 mL plastic jars containing larvae food). Each aqueous solution of the extract was set up in triplicates. Negative control (containing 90 mL de-ionized water and larvae food) as well as positive control (containing 3 mL absolute alcohol, 90 mL de-ionized water and larvae were also set up in triplicates. Both the test controls were set up and maintained at room temperature (27 ± 2 °C). The Anopheles larvae in each recovery cup were scooped and transferred by means of small nets into test exposure cups containing the sample solutions and or control, larvae food and de-ionized water. The larvae in the test and controls set up were incubated for a period of 12 and 24 h at room temperature (27 ± 2 °C). Therefore, the larvae were gently scooped into small nets, washed with de-ionized water, transferred into recovery cups containing 100 mL of de-ionized water, maintained at pH 7.0 and allowed to settle. Prior to mortality determinations, the larvae in recovery cups were gently disturbed and made to go below the water surface by agitating the water with a sterile pipette. The dead and dying larvae which started to float on the surface, were pushed down the recovery cups. The living larvae which were able to swim to the surface were allowed to do so within 5 min following addition. The larvae remaining and or staying at the bottom of the recovery cups unable to swim to the surface were regarded as dead.
Anti-Microbial Sensitivity Test
Determination of Zone of Inhibition
The media were prepared according to Manufacturers` instructions,
poured into sterile petri-dishes (diameter, 13.5 cm) and then allowed
to set. The bore-hole diffusion method was used for the anti-microbial
screening test. The bacteria were cultured in nutrient agar while the
fungus was cultured in the sabouraud dextrose agar. The inoculum of each
organism was introduced into each petri-dish. Cylindrical plugs were removed
from the agar plates by means of a cork borer to produce wells of approximately
6.0 mm. The wells were equidistant from each other and the edge of the
plate (Washington, 1995; National Committee for Clinical Laboratory Standards,
2003). Concentrations of 10 and 20 mg mL-1 of the crude ethanolic
extract and the fractions at 5 mg mL-1 dissolved in methanol:
de-ionized water (1:1 v/v) were separately introduced into wells. Also,
concentrations of 10 μg mL-1 of ampiclox (a standard antibiotic),
1 mg mL-1 of ketoconazole (a standard anti-fungal drug) and
methanol: de-ionized water (1:1 v/v) were introduced into other wells
as positive and negative controls, respectively. The experiments were
carried out in triplicates. The plates were left at room temperature (27
± 2 °C) for 2 h to allow for diffusion. The plates were then
incubated at 34 ± 2 °C for 24 h.
RESULTS AND DISCUSSION
The plant material used in this present study was identified, authenticated
and collected observing basic guidelines of plant collection. The solvents
and reagents used were of analytical grade. The phytochemical screening
revealed the presence of alkaloids, saponins, tannins cardiac glycosides
and flavonoids while anthraquinones and terpenes were absent (Table
1). This confirms previous studies as contained in Trease and Evans
(1996). Secondary metabolites such as alkaloids, tannins, flavonoids and
cardiac glycosides present in the plant are the basis for the curative
and or management of many ailments such as wounds, digestive disorders,
coughs, ulcers, skin troubles and different kinds of inflammations claimed
in its ethno-medicine.
Larvicidal Activity
Preliminary larvicidal assay was carried out on the crude extract
at 5 and 10% w/v and at 12 and 24 h incubation. The larvicidal activity
(LA%) was calculated in terms of percentage mortality. The lethality furnished
after 12 and 24 h incubation was concentration and time-dependent (Table
2, 3). At 5% w/v (12 and 24 h) and 10% w/v (12 and
24 h), the plant demonstrated very weak larvicidal activity of 10 and
20%, 20 and 30%, respectively. These results have revealed that the potential
for larvicidal activity is very small in the crude extract of the plant.
However, research is presently ongoing in our laboratory to determine
if the fractions obtained from the crude extract will
| Table 1: | Phytochemical screening of leaf extract of Nymphaea odorata |
| L = Leaf extract of Nymphaea odorata, + = Trace (insignificant amounts), + + + = Abundant, - = Absent | |
| Table 2: | Larvicidal activity (LA%) of leaf extract of Nymphaea odorata at 5% w/v after 12 and 24 h incubation |
| L = Leaf extract of Nymphaea odorata, PC = Positive Control, NC = Negative Control | |
| Table 3: | Larvicidal activity (LA%) of leaf extract of Nymphaea odorata at 10%w/v after 12 and 24 h |
| L = Leaf extract of Nymphaea odorata, PC = Positive Control, NC = Negative Control | |
| Table 4: | Anti-microbial sensitivity of leaf extract and fractions of Nymphaea odorata at different concentrations in methanol: De-ionized water (1:1 v/v) *Zone of inhibition ± 0.5 mm |
| L = Leaf extract of Nymphaea odorata, Lh, Lc, Lb (hexane, chloroform and butanol fractions of leaf extract, respectively), A = Ampiclox (standard antibiotic or anti-bacterial drug), K = Ketoconazole (standard anti-fungal drug), C = Methanol: De-ionized water (1: 1v/v), NT = Not Tested, *Zone of inhibition recorded is diameter of zone and bore-hole cup size [zone diameter (mm) + 6 mm] | |
demonstrate any improvements in the larvicidal activity. Interestingly, the crude leaf extract tested positive to both alkaloids and saponins which have been shown in separate studies (Bentley et al., 1984; Ojewole et al., 2000; Oladimeji et al., 2006a, b, 2007; Nia et al., 2006) to be lethal to the fourth instar larvae of Anopheles gambiae which prevent the emergence of adult mosquitoes responsible for the transmission of malaria still scourging huge populations of people around the world.
Anti-Microbial Sensitivity Test
The extract and the fractions (obtained from the chromatographic purification
of the extract) were screened for antibacterial and anti-fungal activities
using B. subtilis, S. aureus, E. coli, S. typhi,
K. pneumoniae and C. albicans to represent a desirable spectrum
of microbes. The extract was tested at 10 and 20 mg mL-1 while
the fractions were screened at 5 mg mL-1 because of the possible
higher level of purity associated with fractions. The results presented
in Table 4 show that the activities elicited were slightly
concentration-dependent. Generally, the extract and the fractions were
inactive against all the microbes tested. Alternatively, it could be inferred
that the plant did not elicit any remarkable antibacterial and anti-fungal
activities.
These results are surprising because the phytochemical screening carried out on the extract revealed the presence of tannins and flavonoids which have been reported in previous studies (Lamikanra et al., 1990; Burapadaja and Bunchoo, 1995; Adesina et al., 2000) to be anti-microbial. The present study can not support the claimed uses of the plant in ethno-medicine especially for ailments of microbial origin.
ACKNOWLEDGMENT
The kind assistance of E.J. Akpan in the biological studies is gratefully appreciated.