IOSR Journal of Pharmacy and Biological Sciences (IOSR-JPBS)
e-ISSN:2278-3008, p-ISSN:2319-7676. Volume 12, Issue 3 Ver. VI (May – June 2017), PP 72-79
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Antimicrobial Activity of Seed Extracts from Albizia bernieri E.
Fourn. (Fabaceae)
Lovarintsoa Judicael Randriamampianina1,
Vahinalahaja Eliane Razafintsalama2, Danielle Aurore Doll Rakoto1,
Hanitra Ranjana Randrianarivo1,
Victor Louis Jeannoda1
1
Laboratory of Applied Biochemistry to Medical Sciences, Fundamental and Applied Biochemistry
Department, Faculty of Sciences, University of Antananarivo, P.O. Box 906, Antananarivo 101, Madagascar
2
National Center for Application of Pharmaceutical Research (CNARP), P.O. Box 702, Antananarivo 101,
Madagascar
Abstract: This work aimed at assessing the antimicrobial activity of Albizia bernieri seed extracts. Methanol
extract (SME), alkaloids extracted under basic (Alk1) and acidic (Alk2) conditions, polar saponosides (Sap1)
and less polar saponosides (Sap2) obtained by n-butanol fractionation were used. Their activities were tested
against 9 pathogenic germs including 4 Gram (+) bacteria, 5 Gram (-) bacteria and 1 yeast using disc diffusion
and microdilution methods. Regardless the method used to assess the antimicrobial activity, all extracts were
efficient but their effects depended upon the germs tested. They all displayed a broad spectrum activity.
Alkaloids were by far the most efficient with an excellent effect (Minimum Inhibitory Concentration or
MIC<100µg/ml) against all germs tested. Streptococcus pneumoniae, Streptococcus pyogenes, Clostridium
perfringens and Candida albicans were the most sensitive with MICs less than 10 µg/ml. SME and Sap 2 had
moderate or low effects (MIC between 100 and 1000 µg/ml) and Sap1 was the less efficient (MIC ≥1000 µg/ml).
All the extracts exerted bactericidal or fungicidal action on all sensitive germs. These preliminary results
revealed antimicrobial activity of Albizia bernieri seeds that could be used to treat different infectious diseases
and might lead to the development of pharmaceutical agents.
Keywords: Albizia bernieri, antimicrobial activity, disc diffusion method, microdilution method, minimum
bactericidal concentration, minimum inhibitory concentration.
I.
Introduction
Microbial strains developing stronger resistance to various antibiotics are growing in numbers which
constitutes a significant public health problem. There is an urgent need to find new disposable and affordable
remedies to face this problem [1]. A systematic screening of plant extracts as a source of anti-bacterial
compounds has been undertaken in different laboratories [2, 3].
Several Albizia species organ extracts from different countries had been reported displaying
antimicrobial properties. They include the extracts of A. zygia leaf [4], A. julibrissin leaf, stem and flower [5], A.
amara leaf, flower, pod and bark [6], A. lebbeck seed [7], A. anthelmintica root bark [8], A. amara and A.
saman leaves [9], wood [10], A. andianthifolia bark and root [11]. Seed extracts from some endemic Albizia of
Madagascar also exhibited antimicrobial effects [12]. The purpose of this study was to assess the antimicrobial
properties of A. bernieri seeds, one of the 24 Albizia species endemic to Madagascar [13]. The effects of seed
extracts were tested on pathogen germs responsible for serious infections in humans and many of which are
resistant to antibiotics.
II.
Materials And Methods
2.1 Plant Materials
A. bernieri is a large shrub or tree up to 25 m tall (Fig.1) growing throughout the western Part of
Madagascar, from the North to the South. The plant was identified by comparison of voucher specimens,
registered under 29133-SF, with herbarium samples of Department of Forest Research and Fish Farms of
FOFIFA Antananarivo. Dry fruits were collected on July 2010 from Mampikony in the North West of
Madagascar.
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Antimicrobial Activity of Seed Extracts from Albizia bernieri E. Fourn. (Fabaceae)
b
a
d
c
Figure 1: Albizia bernieri: a) the whole plant; b) fruits; c) leaves; d) seeds
Source: the authors
2.2 Microorganism Strains
The 10 microorganisms used in this study consisted of 4 Gram (-) bacteria, 5 Gram (+) bacteria and1
yeast (TABLE 1). They were maintained on agar slant at 4°C and cultured on a fresh appropriate agar plate 24 h
prior to any antimicrobial test.
TABLE 1: List of germs used
Strains
Staphylococcus aureus
Bacillus cereus
Streptococcus pneumoniae
Streptococcus pyogenes
Enterobacter aerogenes
Enterobacter cloacae
Yersinia enterocolitica
Pseudomonas aeruginosa
Clostridium perfringens
Candida albicans
Reference
ATCC 25923
ATCC 14579
ATCC 6305
ATCC 19615
ATCC 13048
ATCC 13047
ATCC 23715
ATCC 10145
ATCC 13124
ATCC 10231
Gram
+
+
+
+
-
2.3 Chemicals for Antimicrobial Assay
Antibiotic and antifungal used as references in this study were Neomycin 30 µg/disc and Miconazole
500 µg/disc.
2.4 Preparation of Seed Extracts
2.4.1 Seed Methanolic Extract
Ground seed powder (250 g) was delipidated with hexan (3 X 500 ml), then extracted with methanol (3 X
500 ml). After filtration using a Whatman filter paper, extract was evaporated to dryness under reduced
pressure. The dry residue dissolved in sterile distilled water constituted seed methanol extract (SME).
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Antimicrobial Activity of Seed Extracts from Albizia bernieri E. Fourn. (Fabaceae)
2.4.2 Saponosides Extraction
SME was mixed with an equal volume of n-butanol. Aqueous phase was yet two times treated with an
equal volume of n-butanol. The three organic phases gathered and the aqueous phase were evaporated to dryness
under reduced pressure. Seven grams (7g) of the aqueous phase residue were dissolved in 100 ml of methanol.
The soluble fraction obtained was gradually added to 300 ml of acetone-diethyl ether mixture (v/v) cooled in an
ice bath. After a few minutes of maceration the precipitates formed were recovered by centrifugation (1000 rpm
during 5 minutes at +4°C). The supernatant was collected and treated under the same previous conditions. This
operation was repeated three times. All the precipitates were gathered and solvent was removed by evaporation.
The resulting residue (3.99 g) constituted the polar total saponosides named Sap1. Three grams (3g) of the
organic phase residue were dissolved in 100 ml of methanol. The resulting solution was also submitted to the
same treatment by acetone-diethyl ether mixture (v/v). After evaporation, 2.1 g of less polar total saponosides
(Sap2) were obtained.
2.4.3 Alkaloids Extraction
Two extraction methods were used.
Alkaloid Extraction under Basic Conditions
Four grams (4g) of SME powder were moistened with 20 ml of ammoniac 20%, then suspended in 300
ml of dichloromethane. The mixture was stirred at room temperature during 24 h. After filtration, the solution
was evaporated to dryness with a rotary evaporator at low pressure at 40°C. The residue obtained was dissolved
in 20 ml of water and the solution was acidified with sulfuric acid (10%) until pH 2-3 and then extracted with
ether to remove acidic and neutral lipophilic compounds. After adjusting its pH to 9-10 with NH4OH (20 %), the
aqueous solution was extracted with dichloromethane (3 x 20 ml). The lower organic phase was washed three
times with distilled water, then dehydrated with anhydrous sodium sulphate (Na 2SO4) and evaporated to dryness
under reduced pressure. The residue obtained was the crude total alkaloid Alk1.
Alkaloid Extraction under Acidic Conditions
SME powder (4g), moistened with HCL 1M (30 ml ),was suspended in 100 ml of methanol and stirred
for 24 h at room temperature. After filtration, the solution was evaporated to dryness under reduced pressure.
The aqueous acidic solution was alkalinized to pH 9-10 with sodium hydroxide (NaOH 20 %), then extracted
with3 x 20 ml of dichloromethane. The organic phase was dehydrated with anhydrous sodium sulphate
(Na2SO4) and evaporated to dryness under reduced pressure to yield the crude total alkaloid Alk2.
The five seed extracts tested are summarized in TABLE 2.
TABLE 2: List of seed extracts tested
Abbreviations
SME
Sap1
Sap2
Alk1
Alk2
Extracts
Seed methanolic extract
Total saponosides from aqueous butanol phase of SME
Total saponosides from organic butanol phase of SME
Total alkaloids under alkaline extraction
Total alkaloids under acidic extraction
2.5 Phytochemical Screening
The reactions of chemical group detection were those developed by [14, 15].
2.6 Antimicrobial Assays
All the materials and methods used for antimicrobial assay were detailed in our previous paper [16].
2.6.1 Antimicrobial Activity Test
The in vitro antimicrobial activity of the extracts was determined using disc diffusion method of Pyun
et al. [17] and Ngameni et al. [18]. The results were interpreted using the scale of Ponce et al. [19] and Celikel
et al. [20]. Bacteria are not sensitive for an inhibition zone diameter (IZD) less than 8 mm, sensitive for IZD of
9 -14 mm, very sensitive for IZD of 15 -19 mm and extremely sensitive for IZD larger than 20 mm.
Antifungal activity was evaluated by a method described by Favel et al. [21].
Negative controls were prepared by using the same solvents employed to dissolve the plant extract
samples while the standard antibiotics were used as positive controls. All the experiments were performed in
triplicate.
2.6.2 MIC, MBC and MFC Determination
Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Minimum
Fungicidal Concentration (MFC) were determined by microdilution method [22].
The standards used to interpret MIC results were those of Dalmarco et al. [2]. For crude extracts and
fractions, a MIC lower than 100 µg/mL was considered as an excellent effect, from 100 to 500 µg/ml as
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Antimicrobial Activity of Seed Extracts from Albizia bernieri E. Fourn. (Fabaceae)
moderate, from 500 to 1000 µg/mL as weak, and over 1000 µg/ml as inactive. The extract action is bactericidal
or fungicidal whether its ratio MBC/MIC or MFC/MIC is ≤4) and bacteriostatic and fungistatic when the ratio
is>4 [23, 24, 25].
III.
Results
3.1 Extraction yields
Extraction yields are shown in TABLE 3. They were calculated by reference to the starting material,
i.e. seed powder for SME and SME for Sap1, Sap2, Alk1 and Alk2. Extraction yields ranged from 1.16% (Alk1)
to SME (16.02%).
TABLE 3: Extraction yields of the different extracts
Extract
SME
Sap1
Sap2
Alk1
Alk2
Yield
16.02%
9.97%
5.5%
1.16%
2.78%
3.2 Phytochemical analysis
The major secondary metabolites identified in SME extracts can be seen in TABLE 4. Alkaloids,
saponosides, tannins, polyphenols, steroids, triterpenes and cardenolids were present, while flavonoids,
leucoanthocyanins, coumarins, iridoïds, steroids, unsaturated sterols and quinones were not detected. Sap1 and
Sap2 were contaminated with alkaloid traces, whereas Alk1 and Alk2 were free from any contamination.
TABLE 4: Phytochemical screening of A. bernieri seed extracts
Chemical groups
Alkaloids
Saponins
Flavonoids
Leucoanthocyanins
Cardenolids
Cyanogenetic glycosides
Unsaturated lactones
Coumarins
Tannins and Polyphenols
Tests
Mayer
Wagner
Dragendorff
Foam test
Hemolytic test
(hemolytic test)
Willstätter
Bate-Smith
Grignard
Kedde
Quinones
Steroids
Iridoïds
Triterpenes
Gelatin 1%
Gelatin-salt 10%
FeCl3
Borntrager
Liebermann-Burchard
Hot HCl
Liebermann-Burchard
Unsaturated sterols
Salkowsky
SME
+
+
+
+
+
+
+
+
+
Sap1
±
±
±
Sap2
±
±
±
Alk1
+++
+++
+++
Alk2
+++
+++
+++
+++
-
+++
-
-
-
-
-
-
-
-
+: positive test; -: negative test
3.3 Antimicrobial activity
At the concentration of 1mg/disc, a concentration often used in antimicrobial activity assessment in
plant extracts [26, 27, 28, 16], all the 5 extracts displayed antibacterial activity with IZD ranging from 8 to 27
mm (TABLE 5). However, activity depended on the extract and the microorganism used.
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Antimicrobial Activity of Seed Extracts from Albizia bernieri E. Fourn. (Fabaceae)
TABLE 5: Effects of A. bernieri seed extracts by disc diffusion method
Gram (+)
Gram (-)
Strains
Yeast
Enterobacter cloacae
ATCC 13047
Enterobacter aerogenes
ATCC 13048
Pseudomonas aeruginosa
Yersinia enterocolitica
Streptococcus
pneumoniae
Streptococcus pyogenes
Staphylococcus aureus
Clostridium perfringens
Bacillus cereus
Candida albicans
Inhibition zone diameter (mm)
Extracts (1000 µg/disc)
Neomycin
30 µg/disc
SME Sap1 Sap2 Alk1 Alk2
Miconazole
500 µg/disc
20
18
22
26
25
27
-
7
7
9
11.5
11
19
-
8
7
8
6
10
10
11
13
12
11
18
20
-
9
9
12
14
14
23
-
9
9
9
9
6
8
9
9
9
6
10
10
10
10
6
9
10
13
10
12
20
14
11
14
10
24
18
22
21
-
25
Alk1 and Alk2 were active against all the microorganisms tested and were the most efficient. Sap2 was
active against bacteria but not against Candida albicans. SME and Sap1 were the less active (IZD ≤ 9 mm)
against all the strains tested except Enterobacter cloacae. Enterobacter aerogenes, Yersinia enterocolitica and
Candida albicans were not sensitive to these 2 extracts.
Neomycin 30 µg and miconazole 500 µg were more effective than all the A. bernieri seed extracts.
The antimicrobial activity of the 5 extracts assessed by microdilution method is shown in TABLE 6.
Regarding MIC values, 48% were <100 µg/ml, 20% between 100 and 500 µg/ml, 26% from 500 to
1000 µg/ml and 6% >1000 µg/ml. The values ranged from 31.25 to 500 µg/ml, 62.5 to >1000 µg/ml, 15.6 to
500 µg/ml, 3.9 to 62.5 µg/ml and 1.95 to 15.6 µg/ml for SME, Sap1, Sap2, Alk1 and Alk2 respectively.
About the MBC or MFC values, 44% were <100 µg/ml, 18% between 100 and 500 µg/ml, 28% from
500 to 1000 µg/ml and 10% >1000 µg/ml. These values were 125 to >1000 µg/ml, 125 to >1000 µg/ml, 31.25 to
1000 µg/ml, 7.8 to 62.5 µg/ml, 3.9 to 62.5 µg/ml for SME, Sap1, Sap2, Alk1 and Alk2 respectively. The ratios
MBC/MCI or MFC/MCI of all extracts varied from 1 to 4 which meant they had a bactericidal action against all
the microorganisms tested.
IV.
Discussion
A. bernieri seeds, like those of all Albizia species from Madagascar so far studied, mainly contained
saponosides and alkaloids. The n-butanol fractionation separated the A. bernieri saponosides into 2 chemical
groups: polar saponosides which have affinity to aqueous phase (Sap1) and less polar saponosides found in
organic phase (Sap2). Alkaloids were better extracted under acidic extraction as reported by George et al. [29].
Regardless the method used to assess the antimicrobial activity, all the A. bernieri seed extracts were
efficient but their effects depended upon the germs tested. They generally displayed a broad spectrum activity.
Activity levels were generally higher in liquid than in solid medium. In fact, activity considered as nonexistent (IZD <8 mm) or moderate (IZD between 9 and 14 mm) in solid medium were excellent
(MIC<100µg/ml) in liquid medium. As examples, against Candida albicans Alk1 had IZD = 7 mm and MIC =
62.5 µg/ml and against Enterobacter aerogenes, Clostridium perfringens and Candida albicans Alk2 had IZD =
10-11 mm and MIC = 3.9 -15.6 µg/ml. Bioactive compounds probably diffused little or not at all in solid
medium. SME contained different secondary metabolites such alkaloids, polyphenols, saponins and triterpenes
known for their antimicrobial properties. SME was substantially less active than saponosides and alkaloids it
contained. At least, that might be partially due to negative interactions between contaminants and the active
compounds. With the two methods used the alkaloids were more active than saponosides against all the strains
tested. Their MIC between 1.95 and 62.5 µg/ml and their MBC between 3.9 and 62.5 µg/ml allowed to rank
them among extracts with excellent effects [2]. Their activity on Enterobacter cloacae and Streptococcus
pyogenes, the most sensitive bacteria tested, was comparable with that of neomycine at 30 µg/ml. It is realistic
to expect that pure alkaloids could be at least as efficient as this standard antibiotic. Concerning the saponosides
group, the less polar saponosides (Sap2) were more efficient that the polar saponosides (Sap1). As noted above,
the standards used to interpret MIC results were those of Dalmarco et al. [2]. However, it should be noted that
there is no consensus on the inhibition level for natural products [30]. Compared to antimicrobial effects of
other Albizia species extracts from Madagascar, the A. bernieri extracts with MIC values between 1.5 and 1000
µg/ml, are by far more effective than the extracts of A. arenicola leaves on Candida albicans (MIC = 15620
µg/ml), the A. aurisparsa seeds on Bacillus cereus (MIC = 1980 µg/ml), the A. divaricata seeds on
Pseudomonas aeruginosa (MIC = 3120 µg/ml), the A. mahalao seeds (MIC = 3750 µg/ml) and the A. polyphylla
seeds (MIC = 2420 µg/ml) on Staphylococcus aureus[31]. In comparison with several plant extracts considered
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Antimicrobial Activity of Seed Extracts from Albizia bernieri E. Fourn. (Fabaceae)
as active by the authors, the A. bernieri seed extracts were more efficient. As illustrations, SME was by far more
efficient than other plant crude extracts (TABLE 7). All the A. bernieri extracts were bactericidal and fungicidal
against sensitive microorganisms at the concentrations tested. This property might be due to direct action of
bioactive compounds on membrane resulting in its lysis and cell death. Further chemical study will allow
knowing the number of active molecules and their originality compared with molecules from other Albizia
species or other plants.
TABLE 6: MIC, MBC, MFC (µg/ml) and MBC/MIC or MFC/MIC A. bernieri seed extracts
Strains
Enterobacter cloacae
Gram (-)Bacteria
Enterobacter aerogenes
Pseudomonas aeruginosa
Yersinia enterocolitica
Streptococcus pneumoniae
Gram (+)Bacteria
Streptococcus pyogenes
Staphylococcus aureus
Clostridium perfringens
Bacillus cereus
Yeast
Candida albicans
Extract
MIC
MBC or MFC
SME
Sap1
Sap2
Alk1
Alk2
SME
Sap1
Sap2
Alk1
Alk2
SME
Sap1
Sap2
Alk1
Alk2
SME
Sap1
Sap2
Alk1
Alk2
SME
Sap1
Sap2
Alk1
Alk2
SME
Sap1
Sap2
Alk1
Alk2
SME
Sap1
Sap2
Alk1
Alk2
SME
Sap1
Sap2
Alk1
Alk2
SME
Sap1
Sap2
Alk1
Alk2
SME
Sap1
Sap2
Alk1
Alk2
500
1000
250
31.25
15.6
500
1000
250
31.25
15.6
1000
>1000
500
62.5
15.6
500
1000
250
31.25
15.6
31.25
62.5
15.6
7.8
1.95
125
125
31.25
3.9
1.95
250
1000
250
31.25
15.6
125
>1000
125
15.6
3.9
500
>1000
500
31.25
15.6
500
1000
125
31.25
7.8
>1000
1000
250
31.25
15.6
1000
>1000
500
62.5
62.5
1000
nd
1000
62.5
15.6
500
1000
250
31.25
15.6
125
125
31.25
31.25
7.8
125
125
62.5
7.8
3.9
500
1000
250
31.25
15.6
250
1000
125
31.25
7.8
500
>1000
500
31.25
15.6
1000
>1000
500
31.25
7.8
MBC/MIC or
MFC/MIC
nd
1
1
1
1
2
nd
2
2
4
1
nd
2
1
1
1
1
1
1
1
4
2
2
4
4
1
1
2
2
2
2
1
1
1
1
2
nd
1
2
2
1
nd
1
1
1
2
nd
4
1
1
nd: not determined
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Antimicrobial Activity of Seed Extracts from Albizia bernieri E. Fourn. (Fabaceae)
TABLE 7: Comparison of the MIC values of SME with those of other plant crude extracts on some
germs
Extracts
Germ
aqueous
trunk
extract
of
Harungana
madagascariensis [32]
hexanic leaf extract of Crotalaria retusa [33]
aqueous crude extract of Allium sativum [34]
methanolic leaf extract of Myrtusnivellei [35]
Pseudomonas aeruginosa
Bacillus cereus
Streptococcus pneumoniae
Candida albicans
V.
MIC
(µg /ml)
MIC of SME
(µg /ml)
6250
1000
1250
75 000
4500
500
31.25
500
Conclusion
The A. bernieri seeds contain interesting antimicrobial agents from various chemical groups but
alkaloids displayed the best activity. Alkaloids are a group of major therapeutic interest in terms of number,
structural diversity and range of pharmacological properties. A. bernieri seed extracts could be used to treat
gastrointestinal infections and skin diseases caused by the germs tested. For example, they could be
recommended to treat boils, wounds and injuries caused by or infected with Pseudomonas aeruginosa [36].
Further phytochemical research is needed to identify the active principles and to investigate other properties.
Acknowledgment
The authors are grateful to the Centre National d’Application deRecherchesPharmaceutiques (CNARP) for its
helpful support to this work.
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