African Journal of Agricultural Research Vol. 2(3), pp. 076-079, March 2007
Available online at http://www.academicjournals.org/AJAR
ISSN 1991- 637X © 2007 Academic Journals
Full Length Research Paper
Phytochemical characterization and the antimicrobial
property of Aframomum danielli extract
Fasoyiro, S.B1* and Adegoke, G.O2
1
Institute of Agricultural Research and Training, P.M.B. 5029, Moor – Plantation, Ibadan, Nigeria.
2
Department of Food technology, University of Ibadan, Ibadan, Nigeria.
Accepted 17 January 2007
Characterization of preliminary phytochemical components of Aframomum danielli seeds was
determined. Fractions of the seeds obtained by vacuum liquid chromatographic process were tested for
antimicrobial activities. Phytochemical screening revealed the presence of alkaloids, cardenolides,
carotenoids and polyphenols. All fractions obtained from the petroleum ether extract exhibited
antimicrobial activity on food-borne pathogens with minimum inhibitory concentrations in the range of
100 – 800 microgram per millilitres.
Key words: Phytochemicals, A. danielli fractions, antimicrobial properties.
INTRODUCTION
Food preservations in form of synthetic chemicals have
been known to be effective as antimicrobial agents and
antioxidants (Sherwin, 1990). However, the possible toxicity of synthetic chemicals has been a subject of study for
many years (Chang, 1977; Mishra and Dubey, 1994).
Commercial antioxidants such as butylated hydroxyanisole (BHA), tetra–butylated hydroquinone (TBHQ) are
effective antioxidants but their safety for human consumption is not assured (Ito et al., 1985). Questions concerning the safety of these chemicals in food products have
led to increased scrutiny and reappraisal (Inatani et al.,
1983). The use of natural occurring materials as presservatives is a promising alternative to the use of chemicals
(Howell, 1986).
The potential sources of natural preservative are spices, herbs, fruits, seed, leaves, barks and roots (Pratt
and Hudson, 1996). The strong association between increased consumption of these natural products and human
diseases prevention has been explained by the content of
the phytonutrients (Halliwell and Gutteridge, 1984).
Aframonum danielli is a spice belonging to the genus
Aframomum of the family Zingiberaceae (Dalziel, 1948).
The seeds are smooth, shining olive – brown with a turpentine – like taste and they are used medicinally. The
nutritional profile of A. danielli had been reported by
Adegoke and Skura (1994). The essential oils of the seed
were also reported by Adegoke et al. (2003). Antimicrobial activities of the crude extracts of A. danielli against a
number of microorganisms have been that the extracts of
the spice inhibited the growth of bacteria: Salmonella
enteriditis, Pseudomonas fragi, Pseudomonas fluorescens, Proteus vulgaris, Streptococcus pyogenes, staphyloccus aureus and molds Aspergillus flavus, Aspergillus
niger reported (Adegoke and Skura, 1994; Fasoyiro et al.,
2001). The antimicrobial properties of many other spices;
sage, oregano, allispice, onions, garlic, ginger on food –
borne pathogens and molds have been reported (Wu et
al., 1982; Zaika et al., 1983; Shelef, 1980). Some components reported in these spices include alcohols, esters,
terpenes, phenols and organic acids (Weiser, 1971). This
paper reports the preliminary phytochemical compounds
in A. danielli seeds and the antimicrobial properties of
fractionated components of the petroleum ether extract.
MATERIALS AND METHODS
A. danielli pods were obtained from Ogbagi, Ondo state, Nigeria.
The seeds were removed from the pods and cleaned of the extraneous material. The seeds were pulverized in a warring blender
and sieved (200 µm aperture) and packaged in a polythene bag.
Phytochemical screening
*Corresponding author. E-mail:
[email protected]
The ground spice was tested for alkaloids, saponins, polyphenols,
tannins, cardenolides, anthraquinones, and carotenoids as descrybed by Trease and Evans (2002).
Fasoyiro and Adegoke
077
Table 1. Phytochemical analysis of A. danielli ground spice.
Tests
Observations
Indication
Meyer
Dragendroff
+ ve (cream colour)
+ ve (red – brown colour)
present
Cardenolides
Keller-killani
Kedde
+ ve (green colour)
+ve (brown-purple colour)
present
+ ve (no pink colour)
absent
Frothing test
Emulsion test
+ ve (no frothing)
+ ve (no emulsion)
absent
FeCl3 test
Vanillin – HCL test
+ ve (green colour)
+ ve (no red colour)
absent
+ ve (dusky green colour)
present
+ ve (green colour)
present
Alkaloids
Anthraquinones
Saponins
Tannins
Flavonoids / polyhenols
FeCl3
Carotenoids
Conc. H2So4 test
Spice extraction and fractionation.
The ground spice was extracted with petroleum ether (40 - 60oC)
using the method described by Chang et al. (1977). Extract was
identified by thin-layer chromatography (TLC) and retention factor
(Rf) was determined using method of Hostettman et al. (1985). The
extract was fractionated using vacuum- liquid chromatographic
process as described by Odukoya et al. (1999).
Determination of antibacterial activity of A. danielli extract and
fractions was by the agar diffusion method as described by Hugo
and Russell (1983). Overnight both culture of test organism (2 ml)
was added to molten and cooled nutrient agar (45oC). This was
mixed and poured in a sterile petri-dish. The agar was allowed to
set and holes (8 mm cup size) were bored at the periphery and
centre of the agar. Extract or fraction (dissolved in l ml 50% ethanol)
in the range of 100 to 1000µg/ml were introduced into the holes.
Ampicillin (10µg/ml) was used as positive control. Standard deviation of the means was by SAS (1995).
RESULTS AND DISCUSSION
Table 1 shows the results of phytochemical tests for A.
danielli ground spice. A. danielli tested positive to both
the Meyer’s test and confirmatory Dragendroff’s test indicating the presence of alkaloids in the seeds. A. danielli
also tested positive to the two tests for cardenolides:
Keller-killani’s test and Kedde’s test indicating the presence of sugar as glycosides. These two tests revealed
the presence of glycosides in A. danielli.
Polyphenols in the forms of tannin, anthraquinones and
flavonoids were also tested in A. danielli. For both the
tannin and flavonoid tests with FeCl3, the spice tested
positive, but with further testing with vanillin-HCl, A.
danielli was confirmed to have no tannin but only flavornoids. Carotenoids were also confirmed present in the
seeds. Harbone (1984) reported that occasional phenolic
units are elucidated in alkaloids and that the existence of
sugar as glycosides usually occurs in the water soluble
fractions of phenolic compounds. The glycosides that
exist in A. danielli are likely to be in form of phenolic glycosides.
Table 2 shows the nature and retention factors of
fractions of petroleum ether extract obtained from A.
danielli. Fraction F1 existed as a yellow - orange oil with
Rf in the range of 0.75 - 0.88. Fraction F2 existed as a
dark brown viscous oil with a lower Rf value than fraction
F1. Fractions F3 and F4 are viscous brown oily solids
with lower Rf values than fraction F2. It was observed that
polarity of the fractions depends on the nature of the
eluting solvents. Rf value indicates the polarity of the
fractions. Lower Rf values shows higher polarity. Fraction
F4 had the highest polarity and F1, the lowest.
Table 3 shows the zones of inhibition of A. danielli crude petroleum ether extract on some food –borne pathogens in comparison with ampicillin. The crude extract had
higher antimicrobial activity on Bacillus subtilis followed
by Staphylococcus aureus. The extract had the least activity on Pseudomonas aeruginosa. This shows that A.
danielli extract had higher activity towards gram -positive
bacteria.
Table 4 shows the minimum inhibitory concentration
(MIC) of A. danielli petroleum ether fractions on some
078
Afr. J. Agric. Res.
Table 2. Fractions of A. danielli petroleum ether extracts obtained by vacuum liquid chromatography
(VLC).
Fractions
F1
Nature at room temperature
Yellow – orange oil
F2
Dark brown Viscous oil
F3
Dark brown viscous oily solid
F4
Dark brown solid
Eluting solvents
100% hexane
Hexane/EtoAC (90:10)
Hexane/EtoAC (80:20)
Hexane/EtoAC (70:30)
Hexane/EtoAC (60:40)
Hexane/EtoAC (50:50)
Hexane/EtoAC (40:60)
Hexane/EtoAC (30:70)
Hexane/EtoAC (80:20)
Hexane/EtoAC (10:90)
100% EtoAC
Retention factors (Rf)
0.75
0.88
0.65
0.63
0.62
Table 3. Zones of inhibition (mm) of A. danielli crude petroleum ether extract on some food-borne
pathogens in comparison with ampicillin.
Zones of inhibition (mm)
Food –borne pathogens
Bacillus subtilis
Bacillus cereus
Staphylococcus aureus
Escherichia coli
Pseudomonas aeruginosa
crude petroleum ether extract (100 µg/ml)
22.80± 2.20
8.52± 0.40
20.5± 1.40
11.7 ± 1.60
3.21 ± 0.44
Ampicillin (10 µg/ml)
39.2+ 0.40
10.7 ± 1.30
36.2 ± 1.70
20.3 ± 1.00
12.3± 0.06
Mean of three readings ± standard deviation
Table 4. Minimum Inhibitory Concentration (µg/ml) of A. danielli fractions on
food-borne pathogens.
Fractions
F1
F2
F3
F4
B.subtilis
400
400
400
400
B. cereus
800
800
800
800
S. aureus
200
200
100
200
E. coli
>800
800
400
400
Ps. aeruginosa
>800
>800
800
>800
cereus (800 µg/ml). Lower MIC range of 100 - 200 µg/ml
of the fractions was needed to inhibit S. aureus. Zones of
inhibition of different concentrations of fraction F3 is
shown in Figure 1. Higher MIC greater than 800 µg/ml
was needed for inhibition of P. aeruginosa,
Conclusion
Figure 1. Plates showing zones of inhibition of different
concentrations of fraction F3 on S. aureus.
food–borne pathogens. All the fractions had similar MIC
of 400 µg/ml on B. subtilis (400 µg/ml) and Bacillus
This study has been able to highlight some of the
phytochemicals present in A. danielli spice as alkaloids,
carotenoids and polyphenols which could possibly exist
as glycosides. Also, the antimicrobial properties of the
extracts and fractions show higher activities towards
gram -positive bacteria. This shows the possibility of the
use of A. danielli spice in reducing the incidence of food
spoilage and food toxins.
Fasoyiro and Adegoke
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