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Antioxidant and Antimicrobial Properties of Cashew (Anacardium occidentale L.) Leaf Extracts

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Cashew leaf extracts contain high phenolic compounds, flavonoids, tannins, and cinnamic acid. From the previous study, the antioxidant of the leaf extract obtained by 70 % methanol showed the highest values of TPC, FRAP and DPPH assays. For antimicrobial properties, it was reported that the 70% ethanolic and aqueous leaf extracts could inhibit the growths of Staphylococcus aureus, Escherichia coli, and Aspergillus niger. The aims of this research were to study the extraction of cashew leaves and to determine its antioxidant and antimicrobial properties. The cashew leaves were dried, ground, extracted with distilled water, 70% ethanol, 70% methanol, and 70% acetone (1:15 w/v), and then lyophilized. The percent yield of aqueous, 70% ethanolic, 70% methanolic and 70% acetone extracts were 3.33, 14.64, 15.72, and 12.39% (d.b.), respectively. The antioxidant activities of leaf extracts were analyzed by the FRAP and DPPH assays. The results showed that methanolic and ethanolic extracts had higher antioxidant activity and total phenolic content than aqueous and acetone extracts. The antimicrobial activities were determined by disk diffusion method, minimal inhibitory concentration (MIC), and minimum fungicidal concentration (MFC). One percent of methanolic, ethanolic, and acetone extracts could inhibit the growth of gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria. At 10% (w/v) of ethanolic and methanolic extracts could inhibit growths of Aspergillus niger and Colletotrichum gloeosporioides. However, only at 60% (w/v) of ethanolic and methanolic extracts showed inhibitory activities against Penicillium digitatum. The lowest MIC (6.25 and 12.5 µg/100µL) and MFC (25 and 50 µg/100µL) were obtained by an ethanolic extract.
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Antioxidant and Antimicrobial Properties of
Cashew (Anacardium occidentale L.) Leaf Extracts
Mooksupang Liangpanth and Wirongrong Tongdeesoontorn*
Program in Technology Management of Agricultural Produces School of Agro-Industry,
Mae Fah Luang University, Chiang Rai 57100, Thailand
*Corresponding author(s), e-mail: wirongrong.ton@mfu.ac.th
Abstract:
Cashew leaf extracts contain high phenolic compounds, flavonoids, tannins, and
cinnamic acid. From the previous study, the antioxidant of the leaf extract obtained by 70 %
methanol showed the highest values of TPC, FRAP and DPPH assays. For antimicrobial
properties, it was reported that the 70% ethanolic and aqueous leaf extracts could inhibit the
growths of Staphylococcus aureus, Escherichia coli, and Aspergillus niger. The aims of this
research were to study the extraction of cashew leaves and to determine its antioxidant and
antimicrobial properties. The cashew leaves were dried, ground, extracted with distilled
water, 70% ethanol, 70% methanol, and 70% acetone (1:15 w/v), and then lyophilized.
The percent yield of aqueous, 70% ethanolic, 70% methanolic and 70% acetone extracts
were 3.33, 14.64, 15.72, and 12.39% (d.b.), respectively. The antioxidant activities of leaf
extracts were analyzed by the FRAP and DPPH assays. The results showed that methanolic
and ethanolic extracts had higher antioxidant activity and total phenolic content than aqueous
and acetone extracts. The antimicrobial activities were determined by disk diffusion method,
minimal inhibitory concentration (MIC), and minimum fungicidal concentration (MFC). One
percent of methanolic, ethanolic, and acetone extracts could inhibit the growth of gram-
positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria.
At 10% (w/v) of ethanolic and methanolic extracts could inhibit growths of Aspergillus niger
and Colletotrichum gloeosporioides. However, only at 60% (w/v) of ethanolic and
methanolic extracts showed inhibitory activities against Penicillium digitatum. The lowest
MIC (6.25 and 12.5 µg/100µL) and MFC (25 and 50 µg/100µL) were obtained by an
ethanolic extract.
Keywords: Antioxidant, Antimicrobial, Cashew leaf extract.
1. Introduction
The cashew tree (Anacardium occidentale L.) has a native from Brazil, Mexico and
United States of America (USA), it is one of the exotic crops in Thailand. The characteristics
of cashew fruit are a bell shape, greenish-gray color, fleshy and swollen. Actually, Cashew
plants can separate into 2 parts as are edible part and medical part. The edible part consists of
seed, fruit, and leaf which contains many nutrients, fat, protein carbohydrate. It can be
consumed as fresh and pass processed products.1 The cashew plant can be used as one of the
traditions of the medical plant with many parts of the plant (seed, leaf, flower, and bark). Its
leaf contains the antimicrobial compound, such as phenolic compounds, tannin, vitamin C,
carotenoids, and organic acids.2 The cashew leaf extract has a higher antioxidant activity
than coconut, sweet orange, lemon, and papaya leaf extract.3 Many types of research
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reported the inhibitory ability of a cashew leaf to against foodborne pathogenic bacteria. The
leaves can inhibit growth of Staphylococcus aureus, Staphylococcus mutans, Esherichia coli,
Enterococcus faecalis, and Candida albicans3. From the study of antibacterial activity of
many edible plants, the result showed that cashew leaf extract had higher potential to against
Escherichia coli and Staphylococcus aureus than madachi (Khaya senegalensis), rumfu
(Cassia goratensis), and hararrabi (Boswellia dalzielli) leaf extract4. In vitro test research
showed cashew leaves had the highest inhibition of microbial growth than teak and mango
leaf extracts. It had antimicrobial properties because the leaf contained a lot of tannins,
flavonoids and, phenols5.
The cashew leaf extract was reported that it can also inhibit the growth of Aspergillus
niger, which can cause disease in citrus fruit.6 However, a few of researchers studied
inhibitory effect of cashew leaf extract against the other pathogenic fungi growths.
Additionally, the types of solvent can affect yield, antioxidant activities and antimicrobial
properties of the plant extract. The types of extracted antioxidant depend on the polarity of
the solvent extracts.7 So, the objectives of this research were to study the extraction of
cashew leaves and to determine its antioxidant and antimicrobial properties.
2. Materials and Methods
2.1 Extraction of cashew leaves
Cashew leaves (Anacardium occidentale L.) were harvested from the plantation „Ban
Bang Klang‟, Ranong, Thailand. After budding 1214 days. The leaves were washed with
tap water and dried at 50°C for 1 days. These dried leaves were ground into powder and
stored in the closed container. Dried cashew leaves were extracted with water, or 70%
ethanol, 70% methanol, or 70% acetone at ratio 1:15 (w/v), and then shaken at 150 rpm for
48 h. The solution was filtered and then evaporated by rotary evaporator at 45°C for
1520 min. The crude extract was dried using freeze dryer. The extract powder will be
dissolved in 20% dimethyl sulfoxide and storage at 4°C.8
2.2 Antioxidant activities of extracts
2.2.1 Total Phenolic Content (TPC)
The total phenolic content of the extracts was determined according to Folin-
Ciocalteu assay,9 gallic acid was used as a standard. The extract solution (500 µL) was
mixed with 2.5 mL of 10% (w/v) Folin-Ciocalteu reagent and 2 mL of 7.5% (w/v) sodium
carbonate. The mixture was stirred and incubated in darkness for 1 hour at room temperature
(25°C). The absorbance at 765 nm was measured using microplate spectrophotometer.
2.2.2 DPPH Free Radical Scavenging
The Free Radical Scavenging was analyzed following the method of Molyneux.10
DPPH solution (60 mM) was prepared by dissolving 0.00236 g in 95% ethanol (v/v). The
DPPH solution (1950 µL) was mixed with plant extracts (50 µL). Trolox (10,000 µM) was
used as a standard solution, and methanol was used as a blank. The mixtures were left at
room temperature for 30 min. The absorbance at 517 nm was measured using microplate
spectrophotometer.
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2.2.3 Ferric reducing antioxidant power (FRAP)
The ferric reducing antioxidant power was evaluated by the method of Benzie and
Strain.11 The extract (400 µL) was mixed with 400 µL of the freshly prepared FRAP
solution. Distilled water was used as a blank. The mixture was incubated at 37°C for 30 min
and then measured the absorbance at 595 nm using microplate spectrophotometer.
2.3 Antimicrobial properties Test
Five microbes were used to test the antimicrobial properties of cashew leaf extracts.
Bacteria strain of gram-positive is Staphylococcus aureus TISTR746, and gram-negative is
Escherichia coli TISTR 527. Three fungi were used i.e. Aspergillus niger TISTR3281,
Colletotrichum gloeosporioides, and Penicillium digitatum for investigation the antifungal
activity. All of microbial were obtained from Microbiological Laboratory, Mae Fah Luang
University.
2.3.1 Disk diffusion
Disk diffusion assay of cashew leaf extract (CLE) was determined using the method
of Tayel et al.12 with some modifications. The spores of fungal from P. digitatum,
C. gloeosporioides and A.niger were suspended in sterile distilled water. In part of bacterial
strains, they were activated in nutrient broth for 48 h before testing. The solution of
106 spores (200 μL) of fungi and 108 CFU/mL of bacteria were mixed with 150 mL of potato
dextrose agar (PDA) and nutrient agar (NA), respectively, and then 25 mL of the mixture
were poured into a petri dish. The plant extract (50 μL) was loaded into the sterilized paper
disk. The disks were put on a petri dish and incubated at 25ºC for 48 h.
2.3.2 Minimal inhibitory concentration (MIC)
The MIC assays were investigated by using the method of Tayel12 with some
modifications. The plant extract was mixed with potato dextrose broth (1:1 v/v) that
containing in 96-well plate. The two-fold series dilution will be started from the highest
concentration (50 μg) to the lowest concentration (0.05 μg). The spores of fungi
(106 spores/mL) were loaded into each concentration (1:10 v/v). The 96-well plate was
incubated at 25°C for 48 h. The lowest concentration defended visible growth of fungi was
defined as MIC.
2.3.3 Minimal fungicidal concentration (MFC) determination
MFC was analyzed by using the same concentration of extract from MIC assay in
96-well plate which cannot see the growth of fungi. The selected concentration from MIC
was sub-cultured on a PDA and then incubated at 25°C for 48 h. The lowest concentration
against the growth of fungi was recorded as MFC.
3. Results and Discussion
3.1 Percent yield of plant extracts from different solvents
Table 1 showed effect of the ratio between cashew leaf extract and four solvents on
the percent yield of extract. The highest percent yield was obtained by using methanol
(15.72%) as a solvent, followed by ethanol (14.64%), acetone (12.39%) and, aqueous
extraction (3.33%) as shown in Table 1. The ability of extraction or dissolution related to
the polarity and chemical structure of the solvents.13 Normally, water, ethanol, methanol
belong in a group of polar protic solvent. Only acetone is dipolar aprotic solvent.14
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All solvents are most effective in the extraction of polar substances. However, the greatest of
solvent which can dissolve non-polar molecules was methanol. Actually, the structure of
methanol consists of a single carbon atom can link with OH group and three hydrogen atom.
Methanol contained both of a polar group (OH) and a non-polar group (CH).15 Cashew leaf
extract was contained polar and non-polar compounds which can be dissolved well in
methanol.
Table 1 Percent yield of the extracts
Solvents
Percentage yield (w/w)
Water
3.88±0.24
70% Ethanol
14.64±0.16
70% Methanol
15.72±0.28
70% Acetone
12.39±0.30
3.2 Antioxidant properties
The antioxidant activities were determined by using TPC, DPPH and FRAP assay
(Figure 1 and Figure 2). The result showed that methanolic and ethanolic extracts showed
the highest TPC. The methanolic extract also showed the highest FRAP. The lowest TPC -
was found in aqueous extract and acetone extract. Meanwhile, the highest DPPH was
obtained in aqueous extract. The results from this study were similar to other research which
studied the effect of water, ethanol, methanol and acetone extraction of selected plants.7
The polarities of solvents affected by rising of phenolic compounds which accumulated in
the leaf extract. In the other word, phenolics and triterpenoids were dissolved well in the
ethanol. Acetone was a great solvent for flavonoids and xanthoprotein. Water is polar,
consisting of OH-ions which can react with polar groups of tannins. However, different
compounds in cashew leaf extract showed the different antioxidant activities. Tannin had the
ability of alkylperoxyl radical scavenging, to decrease carcinogenesis.17 On the opposite
side, other phenolic compounds were reported to give the reducing power.18
Figure 1 Total phenolic content of cashew leaf extract (n=5) with different superscripts indicating significant
differences (p<0.05)
0.00
10.00
20.00
30.00
40.00
50.00
Aqueous Ethanolic Methanolic Acetone
b
a
b
a
Solvents
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Figure 2 Antioxidant activity by FRAP and DPPH assay of cashew leaf extracts (n=5)with different
superscripts indicating significantly differences (p<0.05)
3.3 Antimicrobial activity
The result of antibacterial effect showed in Table 2. The 1% of ethanolic, methanolic,
and acetone extract could inhibit the growth of E. coli and S. aureus. The highest inhibition
zone of the bacteria exhibited in the methanolic extract. Normally, S. aureus (gram-positive)
is more sensitive to the antimicrobial agent than E.coli (gram-negative) because of
differences in cell membrane structure between gram-positive and gram-negative bacteria.
Only gram-negative had an outer membrane which can prevent antimicrobial gent entering
the cell and showed as the less inhibition zone.19 Table 3 showed antifungal properties of the
extract as the minimum concentration of the CLE which can inhibit the growth of A. niger,
P. digitatum, and C. gloeosporioides. The result showed that 10% of aqueous, ethanolic,
methanolic and acetone extract could be used only against A. niger. The result was similar to
the previous research which reported the ability to kill A. niger of cashew leaf extract.6
Actually, antimicrobial substances in CLE can enter into the cytoplasm and destroyed DNA
and RNA of microbial.20 Moreover, the studied mechanism of antimicrobial showed three
major influence on the destroyed cell microbial. First, the antimicrobial agent can retard
replication of genetic information, then it transfers the degradation genetic to stop protein
synthesis. The antimicrobial can change function and structure of cell wall21. A lot of
antimicrobial agents in cashew leaf extracts such as phenol, phenolic compound, tannins,
and flavonoids have reported the ability to inhibit the growth of pathogenic bacteria and
fungi.22 The lowest MIC (6.25 and 12.5µg/100µL) and MFC (25 and 50 µg/100µL) was
obtained from ethanolic and methanolic extract, respectively (Table 4). This result indicated
that A. niger was more sensitive to cashew leaf extract than P. digitatum and
C. gloeosporioides.
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
DPPH FRAP
Water
Ethanol
Methanol
Acetone
mmol TAE/g dw
mmolFeSO4/g dw
basis
a
a
d
c
c
b
b
c
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Table 2 Growth inhibition of various pathogenic bacteria by cashew leaf extracts.
No.
Concentration of
extract
Zone of inhibition (mm)
Escherichia coli
Staphylococcus aureus
1
H2O (control)
0
0
2
0.5% Aqueous
0
0
3
0.5% Ethanolic
0
0
4
0.5% Methanolic
0
9.5±0.7
5
0.5% Acetone
0
0
6
1% Aqueous
0
8.4±0.6
7
1% Ethanolic
9.63±0.5
9.82±0.6
8
1% Methanolic
10.23±0.2
12.1±0.8
9
1% Acetone
8.33±0.6
9.36±0.5
Table 3 Growth inhibition of various fungi by cashew nut leaf extract
No.
Concentration of
extract
Zone of inhibition (mm)
A.niger
P. digitatum
C. gloeosporioides
1
Control
0
0
0
2
1% Aqueous
0
0
0
3
10% Aqueous
8.33±0.3
0
0
3
25% Aqueous
9.33±0.6
0
0
5
50% Aqueous
10.83±0.8
0
0
6
60% Aqueous
11.50±1.3
0
0
7
1% Ethanolic
0
0
0
8
10% Ethanolic `
11.17±3.4
0
7.67±0.29
9
25% Ethanolic
10.00±1.5
0
8.00±0.50
10
50% Ethanolic
11.50±1.5
0
12.67±0.76
11
60% Ethanolic
12.17±0.6
7.33±0.76
13.00±0.50
12
1% Methanolic
0
0
0
13
10% Methanolic
9.83 ±1.15
0
5.33±2.02
14
25% Methanolic `
9.67±1.61
0
7.83±0.58
15
50% Methanolic `
10.33±0.76
0
12.67±0.76
16
60% Methanolic
11.83±0.29
7.83±0.83
12.83±1.15
17
1% Acetone
0
0
0
18
10% Acetone
9.67±1.32
0
7.17±0.58
19
25% Acetone
9.50±0.71
0
8.00±0.50
20
50% Acetone
11.17±0.69
0
8.17±0.58
21
60% Acetone
11.83±0.58
0
10.17±0.58
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Table 4 Minimal inhibitory concentration (MIC) and minimum fungicidal concentration
(MFC) of cashew leaf extracts
Extracts
A.niger
C. gloeosporioides
P. digitatum
MIC
(µg/100 µL)
MFC
(µg/100 µL)
MIC
(µg/100 µL)
MFC
(µg/100 µL)
MIC
(µg/100 µL)
MFC
(µg/100 µL)
Aqueous
12.5
25
25
25
-
-
Ethanolic
6.25
25
12.5
25
6.25
50
Methanolic
6.25
25
12.5
25
12.5
50
Acetone
6.25
50
25
50
-
-
Note: *MIC and MFC: 10% (w/v) of the solvents extract were a test with A.niger and C. gloeosporioides while
P. digitatum was used 60 % (w/v) of the extract. (-) Mean cannot against growth of fungi
4. Conclusion
The highest percent yield of CLE obtained by 70% methanolic extraction which also
gave the highest TPC and FRAP activity. Whereas, the aqueous extract exhibited the highest
in DPPH scavenging activity. The growth of E. coli and S. aureus could be inhibited by
using 1% of ethanolic, methanolic and acetone cashew leaf extracts. All the extract at 10%
concentration showed the inhibitory activity against A. niger. Sixty percent of ethanolic and
methanolic extracts showed antifungal property against all the studied fungi (P. digitatum,
C. gloeosporioides and A. niger). The results indicated that the cashew leaf extract has a
potential to be used as an antibacterial and antifungal agent for the further application.
Acknowledgements
This research would not have been a success without the chemicals and equipment
support from Mae Fah Luang University. For cashew leaves, I would like to thank for the
plantation „Ban Bang Klang‟, Ranong, Thailand for the cashew leaves and give some useful
information for this study.
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