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Malaysian Journal of Analytical Sciences, Vol 23 No 4 (2019): 637 - 647
DOI: https://doi.org/10.17576/mjas-2019-2304-09
637
MALAYSIAN JOURNAL OF ANALYTICAL SCIENCES
Published by The Malaysian Analytical Sciences Society
ANTIBACTERIAL AND ANTIOXIDANT ACTIVITIES OF EXTRACTS
FROM Calophyllum ferrugineum AND Calophyllum incrassatum
(Aktiviti Antibakteria dan Antioksidan Terhadap Ekstrak daripada Calophyllum ferrugineum dan
Calophyllum incrassatum)
Nurul Iman Aminudin1*, Farediah Ahmad2, Muhammad Taher3
1Department of Chemistry, Kulliyyah of Science,
International Islamic University Malaysia, 25200 Kuantan, Pahang, Malaysia
2Department of Chemistry, Faculty of Science,
Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia
3Department of Pharmaceutical Technology, Kulliyyah of Pharmacy,
International Islamic University Malaysia, 25200 Kuantan, Pahang, Malaysia
*Corresponding author: nuruliman@iium.edu.my
Received: 19 August 2018; Accepted: 23 June 2019
Abstract
Calophyllum is a pan-tropical genus belongs to the Guttiferae family and locally known in Malaysia as ‘bintangor’. There has
been a continual interest to further investigate the phytochemistry of Calophyllum sp because this genus is a rich source of active
secondary metabolites which shows anti-HIV, cytotoxicity and antimicrobial properties. In this study, antibacterial and
antioxidant activities of barks and leaves of C. ferrugineum and C. incrassatum were investigated. Cold extraction method
employing dichloromethane, ethyl acetate and methanol as solvent was performed. All extracts were tested for their total
phenolic content and antioxidant activities by DPPH radical scavenging and Ferric Reducing Antioxidant Power (FRAP) assays.
The methanol extract from the leaves of C. ferrugineum showed the highest TPC value at 122.08 mg GAE/g and the lowest
DPPH SC50 value at 11.80 µg/mL. The methanol extract from the barks of C. ferrugineum was found to have the highest FRAP
value among all extracts. The antibacterial activity of all extracts was tested using minimum inhibition concentration (MIC) test
against Bacillus subtilis, Staphylococcus aureus, Escheria coli and Pseudomonas aeruginosa. Only the dichloromethane extract
from bark of C. ferrugineum showed moderate MIC value against Gram positive bacteria, B. subtilis and S. aureus at 125 µg/mL.
Keywords: Calophyllum, C. ferrugineum, C. incrassatum, antioxidant, antibacterial
Abstrak
Calophyllum adalah genus pan-tropika di dalam keluarga Guttiferae dan dikenali secara tempatan di Malaysia sebagai
‘bintangor’. Terdapat minat berterusan untuk mengkaji lebih mendalam tentang fitokimia Calophyllum sp kerana genus ini
adalah sumber yang kaya dengan metabolit sekunder aktif yang menunjukkan ciri-ciri anti-HIV, sitotoksiti dan antimikrob. Di
dalam kajian ini, aktiviti antibakteria dan antioksidan kulit batang dan daun C. ferrugineum dan C. incrassatum telah dikaji.
Kaedah pengekstrakan sejuk menggunakan diklorometana, etil asetat dan metanol sebagai pelarut telah dilakukan. Semua ekstrak
telah diuji untuk kandungan total fenolik dan aktiviti antioksidan melalui cerakin pemerangkapan radikal DPPH dan kuasa
antioksidan penurunan Ferik (FRAP). Ekstrak metanol daripada daun C. ferrugineum telah menunjukkan nilai TPC tertinggi
pada 122.08 mg GAE/g dan nilai SC50 DPPH terendah pada 11.80 µg/mL. Ekstrak metanol daripada kulit batang C. ferrugineum
telah menunjukkan nilai FRAP tertinggi antara semua ekstrak. Aktiviti antibakteria semua ekstrak telah diuji untuk kepekatan
rencatan minimum (MIC) terhadap Bacillus subtilis, Staphylococcus aureus, Escheria coli dan Pseudomonas aeruginosa. Hanya
ekstrak diklorometana kulit batang C. ferrugineum menunjukkan nilai MIC yang sederhana terhadap bakteria Gram positif, B.
subtilis dan S. aureus pada 125 µg/mL.
ISSN
1394 - 2506
Nurul Iman et al: ANTIBACTERIAL AND ANTIOXIDANT ACTIVITIES OF EXTRACTS FROM
Calophyllum ferrugineum AND Calophyllum incrassatum
638
Keywords: Calophyllum, C. ferrugineum, C. incrassatum, antioksidan, antibakteria
Introduction
Calophyllum is a pan-tropical genus that comprises approximately 180 – 200 species. It is the largest genus in the
sub-family Calophylloideae and categorised under the Guttiferae family [1]. In Malaysia, Calophyllum is locally
known as ‘bintangor’. This plant is used traditionally to treat malaria, bronchitis, gastric and hepatic disturbances
pain, wound infections, inflammation, rheumatism, varicose, haemorrhoids, chronic ulcer and acts as diuretic [2].
Apart from medicinal values, Calophyllum is often used for decorative purposes such as furniture, parquet flooring,
solid door and plywood due to their distinctive colours. The poisonous latex from the bark can also be used to numb
fish and kill rats [3]. Calophyllum genus has been reported as a plant-rich source of phenolic compounds including
xanthones, flavonoids and coumarins. Numerous researches had been conducted on common Calophyllum species
such as C. inophyllum and C. lanigerum [4]. The discovery of a series of pyranocoumarins known as ‘the
calanolides’ with strong anti-HIV properties from C. lanigerum var. austrocoriaceum [5] has elevated the
phytochemical research of this species. This genus also produces chromanone carboxylic acids, terpenoids and
phloroglucinol derivatives.
In continuation of phytochemicals and bioactivities studies on several Calophyllum species from Malaysia [6–9],
the antioxidant and antibacterial activities of C. ferrugineum Ridley and C. incrassatum M. R. Henderson & Wyatt-
Smith were investigated. C. ferrugineum is an evergreen tree distributed around Southeast Asia especially in
Malaysia. It grows from 5 – 30 m and can be found in lowland or colline mixed dipterocarp forest. The bark is used
to make walls and masts for house-building. It is also used as decoction by mothers three days after the childbirth.
C. incrassatum is distributed in East Malaysia, Borneo and Sulawesi. This tree can grow up from 15 – 36 metres
and is usually found in well-drained, mixed dipterocarp lowland forest or sometimes in swamp forest [1]. To date,
there is no scientific report on the antioxidant and antibacterial activities for both species. Therefore, herein the
authors report the evaluation of antioxidant and antibacterial activities of crude extracts from the barks and leaves of
both species.
Materials and Methods
Chemicals and reagents
Folin-Ciocalteu’s phenol, gallic acid, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were
purchased from Sigma Aldrich. Ascorbic acid (AA) was purchased from Goodrich Chemical Enterprice (GCE)
while 2,2-diphenyl-1-picrylhydrazyl (DPPH) was purchased from Fluka. 2,4,6-Tri(2-pyridyl)-S-triazine (TPTZ) was
purchased from Merck while anhydrous sodium carbonate (Na2CO3), sodium acetate trihydrate
(CH3COONa.3H2O), iron (III) chloride hexahydrate (FeCl3.6H2O) and hydrochloric acid (HCl) were obtained from
Qrec. Nutrient agar (NA) and nutrient broth (NB) were purchased from Merck while streptomycin sulphate (SS) and
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium (INT) were obtained from Sigma Aldrich. Sodium
chloride (NaCl), tryptone, glycerol and H2SO4 (98%) were supplied by Qrec, yeast extract was obtained from
Scharlau and Tween 80 was purchased from Fischerbrand.
Microorganism
The bacterial strains acquired were from the American Type Culture Collection (ATCC). The activities of the
samples were screened against four strains, two Gram-positive bacteria, Bacillus subtilis (ATCC 6633),
Staphylococcus aureus (ATCC 29737) and two Gram-negative bacteria, Escherichia coli (ATCC 10536) and
Pseudomonas aeruginosa (ATCC 9027).
Plant material
Sample of C. ferrugineum Ridley (SK2587/14) was collected at Hutan Lenggong, Perak on 28 August 2014 while
C. incrassatum M. R. Henderson & Wyatt-Smith (SK2612/14) was collected from Hutan Gunung Belumut, Kluang,
Johor on 1 October 2014. Plant specimens of C. ferrugineum and C. incrassatum were deposited at Herbarium
Universiti Putra Malaysia. All plant samples were identified by Dr Shamsul Khamis, a botanist from Universiti
Kebangsaan Malaysia.
Malaysian Journal of Analytical Sciences, Vol 23 No 4 (2019): 637 - 647
DOI: https://doi.org/10.17576/mjas-2019-2304-09
639
Extraction method
The dried and ground barks and leaves of C. incrassatum and C. ferrugineum were macerated sequentially with
dichloromethane, ethyl acetate and methanol for 3 days at room temperature. The extracts were then filtered and
similar extraction process was repeated twice. The extracts were then concentrated under reduced pressure by using
rotary evaporator. All extracts were stored at 4°C for further use.
Antibacterial activity
The antibacterial activity of all extracts was tested quantitatively by evaluating their minimum inhibition
concentration (MIC). Two Gram positive bacterial strains of Bacillus subtilis (ATCC 6633), Staphylococcus aureus
(ATCC 29737) and two Gram negative bacterial strains of Escheria coli (ATCC 10536) and Pseudomonas
aeruginosa (ATCC 9027) were chosen. The MIC was carried out by serial broth microdilution according to our
previous reported method [7]. The sample stock solution (1000 μg/mL) was prepared in 5% DMSO in nutrient broth
(NB) supplemented with 0.02% (v/v) Tween 80. Further twofold dilution with NB was performed to afford
concentration of samples from 1000-7.81 μg/mL. 50 μL of bacteria inocula was dispensed in the 96-well microplate
followed by 50 μL of sample solution. Streptomycin sulphate was employed as positive control in this assay. The
microplates were pre-incubated for 24 hours at 37 ºC for S. aureus, E. coli and P. aeruginosa and 30 ºC for B.
subtilis. 25 μL of p-iodonitrotetrazolium (INT) (0.2 mg/mL in sterile distilled water) solution was added to all wells
and were pre-incubated for at least 30 minutes. Bacterial growth in the wells was indicated by the formation of
reddish-pink color while clear well indicates inhibition of bacterial growth by the sample.
Antioxidant activity: Total phenolic content
The total phenolic content (TPC) of the extracts were determined by Folin-Ciocalteau’s assay [10]. In brief, sample
solution in MeOH (40 μL) with concentrations from 1000-7.81 μg/mL obtained from twofold dilution were mixed
with Folin-Ciocalteau’s reagent (20 μL) in the 96-well microplate and were incubated for 5 minutes at room
temperature in a dark condition. Sodium carbonate (80 μL) was added to all wells followed by distilled water (60
μL). The mixture was kept in the dark for 90 minutes and the absorbances were recorded at 760 nm. A calibration
graph of standard gallic acid at concentration 125-7.81 μg/mL versus absorbance was constructed. The total
phenolic contents were expressed as mg gallic acid equivalent (GAE)/g of extract.
DPPH radical scavenging assay
2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay of the extracts was carried out following the
method by Kassim et al. [10]. In brief, DPPH reagent (30 µL) with final concentration of 300 µM was added to the
samples (170 µL) with concentration ranging from 1000-7.81 µg/mL in MeOH obtained from twofold dilution. The
reaction mixture was allowed to incubate in dark condition at room temperature for 30 minutes. The DPPHblank wells
consisting of DPPH reagent and MeOH were reserved for each microplate. The absorbance for DPPH radical
inhibition was measured at 517 nm. The radical scavenging effect was examined and compared with ascorbic acid
(AA) and butylated hydroxytoluene (BHT) as the references. The percentage of DPPH radical scavenging inhibition
was calculated by using the following formula;
Scavenging Concentration (%SC) = [(ADPPH blank ˗ (ASample - Ablank sample)] / ADPPH blank x 100% (1)
where ADPPH blank is the absorbance of DPPH reagent with MeOH, Asample is the absorbance of sample solution with
DPPH reagent and Ablank sample is the absorbance of sample solution.
Ferric reducing antioxidant potential (FRAP) assay
Experiment was carried out following the method by Arriffin et al. [11]. FRAP reagent was freshly prepared, consist
of stock solution with ratio 10:1:1 of acetate buffer (300 mM), TPTZ (10 mM) in HCl (40 mM) and FeCl3.6H2O (20
mM) solutions. Sample (5 µL) with concentration of 1000-100 μg/mL, methanol (15 µL) and FRAP reagent (150
µL) were added to the 96-well microtiter plate. The absorbance was recorded after 10 minutes of incubation at 37
°C at 573 nm. A calibration graph of standard FeSO4.7H2O solution with concentration of 1.0-0.1 mM vs
absorbance was constructed. The FRAP activity of extracts was expressed as mM FRAP equivalent to FeSO4.7H2O
Nurul Iman et al: ANTIBACTERIAL AND ANTIOXIDANT ACTIVITIES OF EXTRACTS FROM
Calophyllum ferrugineum AND Calophyllum incrassatum
640
Statistical analysis
Three replicates of each sample were used for statistical analysis with values reported as mean ± SD. Standard
curves were generated and calculation of the 50% inhibitory concentration (IC50) values was done using GraphPad
Prism for Windows (version 5.02) software. The Student’s t-test was carried out using SPSS (version 22) software
for comparison between treatment of samples and positive controls. Pearson’s correlation coefficient was used to
determine the correlation between two independent variables. A value of p < 0.05 was considered significantly
different.
Results and Discussion
Antibacterial activity
The antibacterial activity of crude extracts from two Calophyllum species was tested against two Gram-positive
bacteria, B. subtilis and S. aureus and two Gram-negative bacteria, P. aeruginosa and E. coli through MIC
determination (Table 1). Streptomycin sulphate was employed as a positive control. According to Sousa et al., the
antibacterial activity of MIC values of crude extracts over 1000 µg/mL is inactive, from 500 to 1000 µg/mL is
weak, from 100 to 500 µg/mL is moderate and less than 100 µg/mL is considered good [12]. Rios and Recio also
proposed that MIC values below 100 µg/mL for crude extracts are promising [13].
Table 1. Minimum Inhibition Concentration (MIC) of Extracts of C. ferrugineum and C. İncrassatum
Calophyllum Species
Part
Crude
(Abbreviation)
Minimum Inhibition Concentration (MIC)
(µg/mL)a
Gram-positive
Gram-negative
B. s
S. a
P. a
E. c
Crude Extracts
C. ferrugineum
B
CH2Cl2 (CFBD)
125
125
>1000
>1000
EtOAc (CFBE)
500
1000
>1000
>1000
MeOH (CFBM)
>1000
>1000
>1000
>1000
L
CH2Cl2 (CFLD)
1000
>1000
1000
1000
EtOAc (CFLE)
>1000
1000
500
>1000
MeOH (CFLM)
>1000
>1000
>1000
>1000
C. incrassatum
B
CH2Cl2 (CIBD)
>1000
>1000
>1000
>1000
EtOAc (CIBE)
>1000
>1000
>1000
>1000
MeOH (CIBM)
>1000
>1000
>1000
1000
L
CH2Cl2 (CILD)
1000
1000
>1000
>1000
EtOAc (CILE)
1000
>1000
>1000
>1000
MeOH (CILM)
>1000
>1000
>1000
>1000
SSb
1.56
50
6.25
0.78
a Data represent mean ± standard deviation of three replicate experiments; b Positive control; B: Barks; L: Leaves; B. s:
Bacillus subtilis; S. a: Staphylococcus aureus; P. a: Pseudomonas aeruginosa; E. c: Escherichia coli; SS: Streptomycin
Sulphate.
The dichloromethane extract from bark of C. ferrugineum showed moderate MIC value against Gram positive
bacteria, B. subtilis and S. aureus at 125 µg/mL. It was suggested that high content of bark resins in the
dichloromethane extracts of C. ferrugineum is responsible to the antibacterial activity. This justification is supported
by the previous finding on the strong antimicrobial properties of the bark resins from C. inophyllum and C.
antillanum [14]. Chromanone carboxylic acids compounds are presented as major compounds in the bark resin of
Calophyllum species and these compounds are reported to have antibacterial activities. Previous report on the
isolation of six chromanone acids from C. brasiliense also demonstrated a moderate-to-strong antibacterial activity
especially to Gram-positive bacteria [15], thus supporting these findings. In contrast, the dichloromethane extract
from bark of C. incrassatum showed inactive antibacterial activity against all strains. This result suggested that there
Malaysian Journal of Analytical Sciences, Vol 23 No 4 (2019): 637 - 647
DOI: https://doi.org/10.17576/mjas-2019-2304-09
641
were no chromanone carboxylic acids types of compound present in the bark extract. The bark of C. incrassatum
also did not contain resin, thus suggesting that no chromanone carboxylic acid can be isolated from this species.
Meanwhile, the other crude extracts showed weak or inactive inhibition towards all bacterial strains tested.
Antioxidant activity
Spectrophotometric assay that involves the use of specific chromophore such as Folin-Ciocalteu (FC) reagent is
commonly chosen, since it is simple and less time consuming as well as suitable for screening purposes. In general,
yellow coloured FC reagent containing molybdenum, Mo6+ will reduce to Mo5+ in the presence of reducing agent in
basic medium to form dark blue complex [16]. The standard calibration curve of gallic acid (Figure 1) was
constructed to calculate the total phenolic content expressed as mg gallic acid equivalent (GAE) per gram of extract
(y = 0.0107x + 2.6402, R2 = 0.9978). In general, the MeOH extracts showed the highest TPC value followed by
EtOAc extracts. It can be deduced from the trend that the TPC increased as the solvent polarity increased since the
phenolic compounds normally constitutes in more polar extract. Meanwhile, all dichloromethane extracts tested
were devoid to this assay and no phenolic content was detected.
Figure 1. Gallic acid calibration curve
The methanol extract of leaves and barks of C. ferrugineum showed the highest TPC value at 122.08 mg GAE/g and
118.48 mg GAE/g, respectively. These TPC values were higher compared to the barks and leaves of C. incrassatum
at 91.61 mg GAE/g and 45.68 mg GAE/g, respectively. A similar trend was also observed where ethyl acetate
extracts from C. ferrugineum showed higher TPC values compared to C. incrassatum. These results suggested that
C. ferrugineum is richer with phenolic constituents compared to C. incrassatum.
The concentration of sample needed to scavenge 50% of DPPH radical activity (SC50) of the extracts is summarised
in Table 2. All tested samples that showed (P < 0.05) were considered as statistically significant and different
compared to ascorbic acid as the positive control. Blois had classified the antioxidant activity of the tested samples
with the SC50 value as very strong (SC50 < 50 µg/mL), strong (50 µg/mL < SC50 < 100 µg/mL), moderate (100
µg/mL < SC50 < 150 µg/mL), weak (150 µg/mL < SC50 < 200 µg/mL), very weak (200 µg/mL < SC50 < 250 µg/mL)
and inactive (SC50 > 250 µg/mL) [17].
y = 0.0107x + 2.6402
R² = 0.9978
0
1
2
3
4
5
050 100 150
Absorbance
Concentration (µg/mL)
Nurul Iman et al: ANTIBACTERIAL AND ANTIOXIDANT ACTIVITIES OF EXTRACTS FROM
Calophyllum ferrugineum AND Calophyllum incrassatum
642
Table 2. TPC, DPPH SC50 and Pearson correlation coefficient values
Calophyllum
Species
Part
Crude
(Abbreviation)
Antioxidant Activity
r coefficient
TPC
(mg GAE/g)a
DPPH
SC50 (µg/mL)a
C. ferrugineum
B
CH2Cl2 (CFBD)
ND
> 1000
0.607
EtOAc (CFBE)
84.09 ± 0.30*
26.72 ± 0.15***
0.810*
MeOH (CFBM)
118.48 ± 0.01*
18.82 ± 0.38***
0.473
L
CH2Cl2 (CFLD)
ND
> 1000
0.623
EtOAc (CFLE)
75.68 ± 0.16*
18.70 ± 1.50**
0.893**
MeOH (CFLM)
122.08 ± 0.04*
11.80 ± 1.03*
0.867**
C. incrassatum
B
CH2Cl2 (CIBD)
ND
> 1000
0.550
EtOAc (CIBE)
18.29 ± 0.20*
49.90 ± 1.97***
0.800*
MeOH (CIBM)
91.61 ± 0.01*
17.23 ± 1.21**
0.709*
L
CH2Cl2 (CILD)
ND
> 1000
0.509
EtOAc (CILE)
35.86 ± 0.01*
52.62 ± 1.38***
0.652
MeOH (CILM)
45.68 ± 0.10*
53.33 ± 1.98**
0.612
AAb
NA
8.90 ± 0.27
NA
BHTb
NA
15.30 ± 0.63
NA
a Data represent mean ± standard deviation of three replicate experiments; b Positive control; B: Barks; L: Leaves; * P < 0.05, **
P < 0.01,*** P < 0.001; AA = Ascorbic acid; BHT: Butylated Hydroxytoluene; NA = Not Available.
All dichloromethane crude extracts displayed inactive DPPH radical scavenging activity with SC50 values of more
than 1000 µg/mL. In contrast, all methanol and ethyl acetate extracts possessed significant DPPH radical
scavenging activity in dose-dependent manner as depicted in Figure 2 and Figure 3, respectively. The methanol
extract from the leaves of C. ferrugineum displayed the lowest SC50 value (11.80 µg/mL) compared to the positive
control, butylated hydroxytoluene (BHT) (SC50 15.30 µg/mL). In addition, the DPPH radical scavenging activities
of methanol extracts from barks of C. incrassatum and barks of C. ferrugineum, as well as the ethyl acetate extract
from the leaves of C. ferrugineum were comparable to BHT since their SC50 values lies in the range of 17.23 –
20.51 µg/mL. This shows that high TPC values of the methanol extracts are strongly associated with the present
findings. Phenolic compounds that are rich in hydroxyl groups serve as a hydrogen donor to the radical thus act as
good antioxidants.
Figure 2. Percentage ınhibition of DPPH radical scavenging activity of EtOAc extracts
0
20
40
60
80
100
0 500 1000
Percentage Inhibition (%)
Concentration (µg/mL)
CFBE
CFLE
CIBE
CILE
AA
BHT
Malaysian Journal of Analytical Sciences, Vol 23 No 4 (2019): 637 - 647
DOI: https://doi.org/10.17576/mjas-2019-2304-09
643
Figure 3. Percentage ınhibition of DPPH radical scavenging activity of MeOH extracts
The correlations between TPC and DPPH radical scavenging activity for each crude extract were evaluated using
Pearson’s correlation coefficient as tabulated in Table 1. In this study, the two variables were positively correlated
with the r coefficient value in the range of 0.473 – 0.893. This indicates a positive correlation whereby high total
phenolic content is proportional to strong DPPH radical scavenging activity. Previous work on the bioassay-guided
fractionation of the methanol and ethanol extracts from several Calophyllum species that gave high DPPH radical
scavenging activity led to the isolation phenolic compounds; flavonoids and xanthones [18,19].
Ferric Reducing Antioxidant Power (FRAP) involves the redox reaction between ferric (III) tripyridyltriazine
complex and a reducing agent to form ferrous (II) tripyridyltriazine complex. The reduction process increases the
absorbance value measured at 593 nm due to the colour changes from pale brown to intense blue [20]. The FRAP
values were determined using standard calibration curve of standard FeSO4.7H2O (y = 0.0005x + 0.0708, R2 =
0.9992) as shown in Figure 4 and expressed as mM FRAP equivalent to FeSO4.7H2O.
Figure 4. FeSO4.7H2O standard calibration curve
The FRAP values of the crude extracts for the concentrations of 1.0, 0.8, 0.6, 0.4, 0.2 and 0.1 mM were summarised
in Table 3. The trend for ferric ion reducing activities of the dichloromethane, ethyl acetate and methanol extracts
from the barks and leaves of C. incrassatum and C. ferrugineum are illustrated in Figures 5-7, respectively. The
0
20
40
60
80
100
0 500 1000
Percentage Inhibition (%)
Concentration (µg/mL)
CF
BM
CF
LM
CIB
M
y = 0.4695x + 0.0708
R² = 0.9992
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0 0.2 0.4 0.6 0.8 1
Absorbance
Concentration (mM)
Nurul Iman et al: ANTIBACTERIAL AND ANTIOXIDANT ACTIVITIES OF EXTRACTS FROM
Calophyllum ferrugineum AND Calophyllum incrassatum
644
methanol extracts showed the highest reducing activities followed by ethyl acetate and dichloromethane extracts for
all Calophyllum species. Similar trend was observed for the total phenolic content determination as discussed
earlier. Thus, it can be deduced that high concentration of phenolic compounds in more polar extracts acts as
reducing agent in this FRAP assay.
Table 3. FRAP equivalent of the extracts from Calophyllum species
Calophyllum Species
FRAP Equivalent to FeSO4.7H2O (mM)a
1.0
0.8
0.6
0.4
0.2
0.1
C. ferrugineum
CFBD
0.24 ± 0.01
*
0.18 ± 0.02
*
0.16 ± 0.01
*
0.16 ± 0.02
*
0.12 ± 0.01
**
0.11 ± 0.01
**
CFBE
2.98 ± 0.65
**
1.98 ± 0.17
**
1.88 ± 0.79
1.13 ± 0.06
**
0.87 ± 0.21
0.37 ± 0.02
*
CFBM
3.72 ± 0.46
***
2.74 ± 1.11
2.03± 0.63
1.41 ± 0.25
*
0.74 ± 0.19
0.40 ± 0.08
CFLD
0.37 ± 0.10
0.26 ± 0.02
*
0.20 ± 0.01
*
0.15 ± 0.01
*
0.13 ± 0.01
**
0.10 ± 0.01
**
CFLE
3.24 ± 1.04
1.24 ± 0.14
*
1.17 ± 0.42
0.68 ± 0.10
0.55 ± 0.05
0.22 ± 0.6
*
CFLM
2.57 ± 0.44
**
1.94 ± 0.72
1.37 ± 0.31
0.91 ± 0.12
0.72 ± 0.07
0.32 ± 0.03
*
C. incrassatum
CIBD
0.23 ± 0.07
*
0.23 ± 0.02
*
0.16 ± 0.02
*
0.12 ± 0.01
*
0.07 ± 0.01
*
0.05 ± 0.01
**
CIBE
1.35 ± 0.04
**
1.15 ± 0.25
0.65 ± 0.09
0.40 ± 0.02
0.18 ± 0.02
*
0.09 ± 0.01
**
CIBM
3.23 ± 0.22
***
2.44 ± 0.18
***
2.00 ± 0.22
**
1.40 ± 0.03
***
0.54 ± 0.04
0.20 ± 0.01
*
CILD
0.17 ± 0.05
*
0.11 ± 0.02
*
0.10 ± 0.02
*
0.10 ± 0.01
*
0.06 ± 0.01
**
0.05 ± 0.01
**
CILE
1.10 ± 0.27
0.62 ± 0.09
0.57 ± 0.11
0.39 ± 0.03
0.22 ± 0.02
*
0.14 ± 0.01
**
CILM
1.41 ± 0.29
*
0.98 ± 0.01
0.84 ± 0.09
0.57 ± 0.06
0.34 ± 0.04
0.22 ± 0.11
*
AAb
0.94 ± 0.24
0.29 ± 0.07
0.37 ± 0.09
0.17 ± 0.05
0.08 ± 0.01
0.07 ± 0.01
BHAb
1.30 ± 0.33
1.07 ± 0.14
0.83 ± 0.13
0.62 ± 0.11
0.33 ± 0.09
0.22 ± 0.06
BHTb
0.59 ± 0.21
0.36 ± 0.06
0.28 ± 0.05
0.21 ± 0.02
0.19 ± 0.04
0.09 ± 0.04
a Data represent mean ± standard deviation of three replicate experiments; b Positive control; AA = Ascorbic acid;
BHT: Butylated Hydroxytoluene; BHA: Butylated Hydroxyanisole; * P < 0.05, ** P < 0.01,*** P < 0.001
The dichloromethane extract from the leaves of C. ferrugineum (CFLD) displayed the highest FRAP equivalent to
FeSO4.7H2O among all dichloromethane extracts followed by the bark extract of C. ferrugineum and C. incrassatum
as shown in Figure 5. Meanwhile, the ethyl acetate extract from the bark of C. ferrugineum followed by ethyl
acetate extract from the leaves of C. ferrugineum, ethyl acetate extract from the bark of C. incrassatum and ethyl
acetate extract from leaves of C. incrassatum showed the FRAP equivalent to FeSO4.7H2O in decreasing order
(Figure 6). Moreover, the methanol extracts from bark of C. ferrugineum, bark of C. incrassatum and leaves of C.
ferrugineum also depicted high FRAP equivalent to FeSO4.7H2O among all methanol extracts displayed in Figure 7.
Malaysian Journal of Analytical Sciences, Vol 23 No 4 (2019): 637 - 647
DOI: https://doi.org/10.17576/mjas-2019-2304-09
645
Figure 5. FRAP equivalent to FeSO4.7H2O of dichloromethane extracts
Figure 6. FRAP equivalent to FeSO4.7H2O of ethyl acetate extracts
Figure 7. FRAP equivalent to FeSO4.7H2O of methanol extracts
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 0.5 1 1.5
FRAP Equivalent to
FeSO4.7H2O (mM)
Concentration (µM)
CF
BD
CF
LD
0
0.5
1
1.5
2
2.5
3
3.5
0 0.5 1 1.5
FRAP Equivalent to
FeSO4.7H2O (mM)
Concentration (µM)
CF
BE
CF
LE
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.5 1 1.5
FRAP Equivalent to
FeSO4.7H2O (mM)
Concentration (µM)
CF
BM
CF
LM
Nurul Iman et al: ANTIBACTERIAL AND ANTIOXIDANT ACTIVITIES OF EXTRACTS FROM
Calophyllum ferrugineum AND Calophyllum incrassatum
646
The antioxidant powers of CFBM, CFLM, CIBM, CFBE and CFLE extracts that showed highest FRAP equivalent
to FeSO4.7H2O with respect to their respective type of extracts were compared with antioxidant power of ascorbic
acid (AA), butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) as the positive controls. From the
graph (Figure 8), all extracts display higher FRAP equivalent to FeSO4.7H2O compared to all positive controls. It
justified their stronger antioxidant power compared to the positive controls. The CFBM extract was found to have
the highest antioxidant power among all extracts followed by CIBM and CFBE.
Figure 8. FRAP equivalent to FeSO4.7H2O of selected extracts and positive controls
Conclusion
Based on the findings, both Calophyllum species showed a strong antioxidant activity especially from the methanol
extracts, suggesting potent antioxidant agents may be isolated. Meanwhile, the dichloromethane extract from the
bark of C. ferrugineum showed potent antibacterial activities selectively against the Gram-positive bacteria,
suggesting it may serve as possible source for new antibacterial agents. Mode of antibacterial action of the extracts
and isolated phytochemicals specifically against Gram-positive bacteria can be further studied.
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0
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