Ethnobotanical
Leaflets 10: 24-40. 2006. Antifungal Activities of Ethanol and
Aqueous Crude Extracts of Four Nigerian Chewing Sticks *A. A ADEKUNLE
|
|
Zone of inhibition (mean +
S.D, mm |
|||
Sample (100mg/ml) extracts |
Aspergillus flavus |
Candida albicans |
Microsporum gyseumm |
Trichophyton mentagrophyte |
Control |
0.00 + 0.00a* |
0.00 + 0.00a |
0.00 + 0.00a |
0.00 + 0.00a |
Anogeissus schimperi |
9.40 + 0.21c |
0.00 + 0.00a |
2.50 + 0.20a |
4.60 + 0.22d |
Distemonathus benthmianus |
13.40 + 0.14e |
13.70 + 0.15e |
11.40 + 0.18f |
13.40 + 0.17e |
Vernonia amagdalina |
12.30 + 0.12f |
12.16 + 0.13f |
11.60 + 0.12f |
11.50 + 0.12f |
Xanthoxyllum zanthoxyloides |
10.90 + 0.10g |
11.20+0.11f,g |
12.40 + 0.14f |
11.50 + 0.12f |
Mixed extracts |
11.50 + 0.13f |
13.40 + 0.18e |
13.00 + 0.17e |
14.80 + 0.14k |
* Zone of inhibition with similar alphabets shows no
significant difference p = 0.05
* Zone of inhibition with different alphabets shows
significant difference p = 0.05
Table 2. Antifungal
Activity of the Aqueous Crude Extracts of Four Nigerian Chewing Sticks.
|
Zone of inhibition (mean +
S.D, mm |
|||
Sample (100mg/ml) extracts |
Aspergillus flavus |
Candida albicans |
Microsporum gyseum |
Trichophyton mentagrophyte |
Control |
0.00 + 0.00a* |
0.00 + 0.00a |
0.00 + 0.00a |
0.00 + 0.00a |
Anogeissus schimperi |
8.30 + 0.45c |
0.00 + 0.00a |
2.20 + 0.39a |
3.50 + 0.57d |
Distemonathus benthmianus |
12.00 + 0.19f |
12.62 + 0.74f |
10.60 + 0.30g |
12.60 + 0.49f |
Vernonia amagdalina |
11.90 + 0.92f |
11.84 + 0.16f |
11.40 + 0.20f |
10.80 + 0.40g |
Xanthoxyllum zanthoxyloides |
10.50 + 0.05g |
11.00 +0.10f,g |
11.90 + 0.36f |
11.30 + 0.12f,g |
Mixed extracts |
11.30+0.18f,g |
13.10 + 0.90e |
12.60 + 0.17f |
13.70 + 0.47e |
* Zone of inhibition with similar
alphabets shows no significant difference p = 0.05
* Zone of inhibition with different
alphabets shows significant difference p = 0.05
Table 3. Phytochemical Compounds in
the Crude Extracts (Aqueous and 70% Aqueous Ethanol) of Four Nigerian Chewing
Sticks.
|
Phytochemical Compound |
|||||||
Sample (100mg /ml) extracts |
Alkaloid |
Antraquinone |
Anthocy-anin |
Flavo-noids |
Phloba Tannin |
Sapo-nin |
Steroid |
Tannin |
Anogeissus schimperi Aqueous Ethanol |
– – |
– + |
– + |
+ + |
+ + |
– + |
+ + |
– – |
Distemonanthus benthmianus Aqueous Ethanol |
– – |
– + |
+ + |
+ + |
– – |
+ + |
– + |
+ + |
Vernonia amagdalina Aqueous Ethanol |
– – |
– + |
– + |
+ + |
+ + |
– + |
– + |
+ + |
Xanthoxylum zanthoxyloides Aqueous Ethanol |
– – |
– – |
– + |
+ + |
+ + |
+ + |
– + |
+ + |
Mixed extracts Aqueous Ethanol |
– – |
– + |
+ + |
+ + |
+ + |
+ + |
+ + |
+ + |
(+) Presence
of the phytochemical compound
(–) Absence
of the phytochemical compound
Ethnobotanical
Leaflets 10: 24-
Antifungal Activities of Ethanol and Aqueous Crude
Extracts of Four Nigerian Chewing Sticks
Department of Botany and
Microbiology
Email: aaded@yahoo.com
*Corresponding author
Issued
ABSTRACT
The antifungal activities of the ethanol and aqueous crude extracts of four Nigerian chewing sticks were investigated. Also a preliminary phytochemical analysis of the plants was done. The chewing sticks include Anogeissus schimperi, Distemonanthus benthmianus, Vernonia amagdalina and Xanthoxylum zanthoxyloides. All the plants tested, except Anogeissus Schimperi, displayed antifungal activities, zone of inhibition above the 10mm standard mark. The ethanol crude extracts of the chewing sticks had a greater zone of inhibition in comparison with the aqueous extract. Among the individual plant extracts, D. benthmianus had the highest antifungal activity against Candida albicans, Aspergillus flavus and Microsporium gypseum and Trichophyton metagrophytes. The chewing sticks contain antifungal agents, though the concentration and composition of the bioactive substances may differ amongst the plants. Distemonath benthmianus exhibited a better antifungal activity and thus made it more suitable for better dental care.
Flavonoid was present in all the plant extracts. Tannin was present in all the plant extract, except that of Anogeissus schimperi. Alkaloids were absent in all the plant extract. The ethanolic extracts had more phytochemical compounds than the aqueous extracts.
KEYWORDS: Chewing Sticks; antifungal activity; Ethanol and aqueous extract; flavonoid; Tannin
In many African houses, teeth are cleaned
in the morning by chewing the root or skin stem of certain plants, until they
acquire brush like ends (EL-Said et al., 1971). The fibrous end is then cleaned and it
brushes the teeth thoroughly. In certain
parts of
Investigations carried out on some of these chewing sticks, shows that they all posses antimicrobial activity against oral microbial flora, but to varying degrees when tested by cup-plate agar method (Enwonwu, 1997). This could indicate therefore that the chewing sticks, in addition to providing mechanical stimulation of the gums, also destroy microbes, presenting the mouth a feature, which is absent in the common toothpaste and brush method (Enwonwu, 1997). The advantage of the chewing sticks over the conventional toothpaste and brush could explain why many Africans have strong teeth (Ugoji et al., 2000).
Some of the plants employed as chewing
sticks for dental care include Anogeissus schiemperi (Hochst); Distemonathus benthmianus
(Baill); vernonian amygdalina (Del.) and Xanthoxyllum
zanthoxyloides (Lam.) “formerly Fagara zanthoxyloides”
( Burkill,1997). A. schimperi is in the family combretaceae
found in southern
The aim of this paper is to report the antifungal activities of ethanol and aqueous extracts of Anogeissus schimperi, Distemonanthus benthmianus, Xanthoxyllum Zanthoxyloides and Vernonia amagdalina on oral pathogens and some dermatophytes using the paper disc method. Also the preliminary phytochemistry of the extracts will be examined.
Source of plant materials:
The stem or root of the plant used as
chewing stick were purchased from Ketu and Tejuosho markets of Lagos State, Nigeria. The plant species parts were authenticated by
a botanist at the Department of Botany and
Source of microorganisms:
Aspergillus flavus, Candida albicans,
Microsporum gypseum, Trichophyton metagrophytes were colleted from patients at the Department medical microbiology,
college of medicine, Idi-Araba,
Extract Preparation:
The ethanol and aqueous extracts were
carried out using modified methods of Saxena and Mathela (1996). The
stem or roots of the different plants (chewing sticks) were chopped into tiny
bits. They were then pounded with local
pistil and mortar before blending with an electric blender. Twenty grammes of
the ground plant part was soaked separately in 100ml, 70% aqueous ethanol, and
another twenty grammes in sterilised distilled water
(100ml) for 24 hours. The fluids were
then filtered using Whatman No. 1 filter paper. The ethanol and water extract were
concentrated using a Rotatory evaporator at 400C. The concentrated extracts were the ethanol
and aqueous extract of the plants. They
were kept in the fridge prior to use.
Antifungal tests:
The paper disc diffusion method if Irobi and Daramola (1994) was modified here.
Spore of conidia suspension of 105 – 107 cells,
counted with haemocytometre were made. About 10ml Sabouraud
dextrose agar, (SDA) water poured into Petri dishes and allowed to
solidify. A micropipette was used to
introduce 0.1ml of the spore or conidia suspensions on to the agar plate, and
spread with glass spreading rod under sterile conditions. Sterilised disc (6mm, Whatman
No AA2017006) were soaked in each of the extracts (100mg/ml) being assayed for 6
hours. Four of these soaked discs were
spread on a fungal spore or conidia seeded plate with the help of sterile
forceps. There was a control which
contains the SDA and fungal inoculums but the discs were soaked in sterilised
distilled water only without extracts.
Three replicates were produced for each fungus. All the plates containing the discs were then
incubated at 28 – 310C. Zone
of inhibition was measured after 48 – 72 hours.
Results were statistically analysed using standard deviation (S.D),
analysis of variance (ANOVA, F – Test) and Duncan multiple range test (Parker,
1979).
Preliminary Phytochemical
Studies:
Supporting phytochemical
studies were carried out using methods described by Fadeyi
et al., (1987) and Harbone, (1998). Basic phytochemical
screening were performed on the chewing stick
extracts. They were screened for the
presence of anthocyanin, anthraquinone,
alkaloids, flavonoids, phlobatarnnin,
saponin, steroid and tannin.
RESULTS
The ethanolic, aquesous extract of each of the chewing sticks as well as
the missed extract inhibited the growth of all the fungi tested, except the
extract of Anogeissus schimperi
which did not inhibit the growth of C. albicans
(Tables 1 – 2). Only the extracts of A.
schimperi had zone of the inhibition less than
10mm (Tables 1 and 2). The ethanol crude extracts of the chewing sticks had a
greater zone of inhibition in comparison with the aqueous extract. Amongst the plant extracts D. benthmianus had the highest antifungal activity against
C. albicans and A. flavus. The mixed extracts had the highest zone of
inhibition on M. gypseum and T. mentagrophytes. There were significant differences
between the zones of inhibition among the extracts of some of the plant part
tested. The control, which was with
sterilised distilled water, yielded no zone of inhibition.
Table 3 shows the phychemical
compounds present in the aqueous and ethanol crude extracts of the four
plants. Flavonoids was present in
all four plants extracts. Tannin was
present in all the extract except in the aqueous and ethanol extract of Anogeissus schimperi. Alkaloid was not present in any of the plant
extract. The ethanolic
extracts had more phytochemical compounds than the
aqueous extracts. The mixed extracts (ethanolic) and ethanolic extracts
of Vernonia amagdalina
had the highest range of phytochemical compounds
(Table 3).
DISCUSSION
The result presented here shows the
presence of antifungal substances in all the ethanol chewing stick
extracts. The aqueous extracts had lower
fungal inhibition which might be because the bioactive components were more
soluble in the ethanol. This is in
agreement with findings of Barnabas and Nagarajan
(1988) on other plants. Of all the plant
extract tested, D. benthmianus was the most
active against the fungi tested, since it possessed the highest antifungal inhibition. A. schimperi
extracts do not contain much phytochemical compound,
especially tannin, which probably might be attributed to its low activity. Burapadaja and Bunchoo (1995) reported the presence of tannins in Terminalia citrina
extracts and explained that the tannins inhibited cell wall formation in fungi
leading to the death of the micro-organism.
A. schimperi extracts might not contain
potent antifungal substances even though Ugoji et
al., (2000) reported that the aqueous extract contain antibacterial
substances. This observation supported Valenciennes et al., (1999), who reported that
antibacterial agents might not necessarily be antifungal. It is of significance to note the high
sensitivity of some of the fungi to the D. benthmianus
extracts, because it is claimed locally that the use D. benthmianus might reduce or completely stop mouth
odour. The mixed extracts (ethanol or
aqueous) of all the chewing sticks proved to inhibit the fungi better than the
extracts of most be due to the presence of more phytochemical
compounds or bioactive components in the mixed extracts as observed in the
result of the phytochemical study.
Conclusively, this study has shown the
presence of antifungal substances in the chewing sticks. The concentration and composition of the
bioactive substances may differ from one plant to another as indicated in the
degree of potency and presence of phytochemical
compounds. The regular use of the
Nigerian chewing sticks may decrease the incidence of dental disease caused by microbes. The chewing stick of Distemonathus
benthmianus because of its better antifungal
properties might be recommended for good oral health.
REFERENCES
Barnabas,
C. G. and Nagarajan, S. (1988). Antimicrobial
activity of flavonoids of some medicinal plants. Fitoterapia 3;
508-510.
Buada, C. V. and Boakye – Yiadom, K. (1973). The
antibacterial activity of some Ghanian chewing
sticks.
Burapadja, S. and Bunchoo, A. (1995). Antimicrobial activity of Tannins from Terminalia citrina Planta medica 61(4):
365 – 366.
Burkhill, H. M. (1997). Usefull plants of Tropical
EL-Said,
F; Fadulu, S. O.; Kuye. J. O.; and Sofowora, E. A. (1971). Native cures in Nigeria Part II:
The antimicrobial properties of the buffer extract of chewing sticks. Lioydia 34: 172 – 174
Enwonwu, C. O. (1974). Socio –
economic factors in the dental caries prevalence and frequency. Nigerian carries res. 8; 155 –
177.
Fadeyi, M. O.; Adeoye, A. O. and Olowokudejo,
Harbone, J. B. (1998). Phytochemical
methods: A guide to modern techniques of plant analysis. 3rd Edition. Chapman and Hill,
Irobi,
O. N. and Daramola, S. O. (1994). Antifungal
activities of crude extracts of Mitracarpus villosus (Rubiaceae). Journal of Ethnopharmacology
38: 604 – 610.
Parker,
R. E. (1979).
Introductory Statistics for biology. 2nd Edition. Edwards Arnold,
Saxena, J. and Methela, C. S. (1996). Antifungal activity of New compound from Nepeta
leucophylla and Nepeta
clarkeii. Applied
and Environmental microbiology 62(2): 702 – 704.
Sofowora, E. A. (1982). Medicinal plants and traditional medicine in
Ugoji, E; Egwari, L. O.; and Obisesan, B.
(2000). Antibacterial activities of aqueous extracts of ten African chewing
sticks on oral pathogens. Nig. Journal of Internal medicine 3(1) : 7-11.
Table 1. Antifungal
Activity of the Ethanol Crude Extracts of Four Nigerian Chewing Sticks.
|
Zone of inhibition (mean +
S.D, mm |
|||
Sample (100mg/ml) extracts |
Aspergillus flavus |
Candida albicans |
Microsporum gyseumm |
Trichophyton mentagrophyte |
Control |
0.00 + 0.00a* |
0.00 + 0.00a |
0.00 + 0.00a |
0.00 + 0.00a |
Anogeissus schimperi |
9.40 + 0.21c |
0.00 + 0.00a |
2.50 + 0.20a |
4.60 + 0.22d |
Distemonathus benthmianus |
13.40 + 0.14e |
13.70 + 0.15e |
11.40 + 0.18f |
13.40 + 0.17e |
Vernonia amagdalina |
12.30 + 0.12f |
12.16 + 0.13f |
11.60 + 0.12f |
11.50 + 0.12f |
Xanthoxyllum zanthoxyloides |
10.90 + 0.10g |
11.20+0.11f,g |
12.40 + 0.14f |
11.50 + 0.12f |
Mixed extracts |
11.50 + 0.13f |
13.40 + 0.18e |
13.00 + 0.17e |
14.80 + 0.14k |
* Zone of inhibition with similar alphabets shows no
significant difference p = 0.05
* Zone of inhibition with different alphabets shows
significant difference p = 0.05
Table 2. Antifungal
Activity of the Aqueous Crude Extracts of Four Nigerian Chewing Sticks.
|
Zone of inhibition (mean +
S.D, mm |
|||
Sample (100mg/ml) extracts |
Aspergillus flavus |
Candida albicans |
Microsporum gyseum |
Trichophyton mentagrophyte |
Control |
0.00 + 0.00a* |
0.00 + 0.00a |
0.00 + 0.00a |
0.00 + 0.00a |
Anogeissus schimperi |
8.30 + 0.45c |
0.00 + 0.00a |
2.20 + 0.39a |
3.50 + 0.57d |
Distemonathus benthmianus |
12.00 + 0.19f |
12.62 + 0.74f |
10.60 + 0.30g |
12.60 + 0.49f |
Vernonia amagdalina |
11.90 + 0.92f |
11.84 + 0.16f |
11.40 + 0.20f |
10.80 + 0.40g |
Xanthoxyllum zanthoxyloides |
10.50 + 0.05g |
11.00 +0.10f,g |
11.90 + 0.36f |
11.30 + 0.12f,g |
Mixed extracts |
11.30+0.18f,g |
13.10 + 0.90e |
12.60 + 0.17f |
13.70 + 0.47e |
* Zone of inhibition with similar
alphabets shows no significant difference p = 0.05
* Zone of inhibition with different
alphabets shows significant difference p = 0.05
Table 3. Phytochemical
Compounds in the Crude Extracts (Aqueous and 70% Aqueous Ethanol) of Four
Nigerian Chewing Sticks.
|
Phytochemical Compound |
|||||||
Sample (100mg /ml) extracts |
Alkaloid |
Antraquinone |
Anthocy-anin |
Flavo-noids |
Phloba Tannin |
Sapo-nin |
Steroid |
Tannin |
Anogeissus schimperi Aqueous Ethanol |
– – |
– + |
– + |
+ + |
+ + |
– + |
+ + |
– – |
Distemonanthus benthmianus Aqueous Ethanol |
– – |
– + |
+ + |
+ + |
– – |
+ + |
– + |
+ + |
Vernonia amagdalina Aqueous Ethanol |
– – |
– + |
– + |
+ + |
+ + |
– + |
– + |
+ + |
Xanthoxylum zanthoxyloides Aqueous Ethanol |
– – |
– – |
– + |
+ + |
+ + |
+ + |
– + |
+ + |
Mixed extracts Aqueous Ethanol |
– – |
– + |
+ + |
+ + |
+ + |
+ + |
+ + |
+ + |
(+) Presence
of the phytochemical compound
(–) Absence
of the phytochemical compound