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Phytochemical characterization of bioactive compounds extracted with
different solvents from Calophyllum inophyllum flower and activity
against pathogenic bacteria
Luksamee Vittaya
a,
*, Chakhriya Chalad
a
, Waraporn Ratsameepakai
b
, Nararak Leesakul
c
a
Faculty of Science and Fisheries Technology, Rajamangala University of Technology Srivijaya, Trang 92150, Thailand
b
Office of Scientific Instrument and Testing, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
c
Division of Physical Science and Center of Excellence for Innovation in Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90110,
Thailand
ARTICLE INFO
Article History:
Received 14 September 2022
Revised 16 January 2023
Accepted 30 January 2023
Available online 7 February 2023
Edited by: Dr P. Bhattacharyya
ABSTRACT
Calophyllum inophyllum is used in traditional medicine to treat several diseases and conditions. Several stud-
ies have attempted to isolate useful compounds from various parts of this plant. However, the phytochemical
constituents of C. inophyllum flower have not been extensively studied. This pioneering study focused on the
chemical composition of C. inophyllum flower analyzed by gas chromatography-electron ionization/mass
spectrometry (GC-EI/MS) and the antibacterial effects of C. inophyllum flower extracted with organic solvents.
Phytochemical compounds were obtained from C. inophyllum flower via maceration with sequential extrac-
tion using hexane, ethyl acetate, and methanol, respectively. Phytochemical components of total phenolic
(TP), total flavonoid (TF), and total saponin (TSC) contents were determined by colorimetric methods and
each extract was found to be rich in phenolic, flavonoid, and saponin constituents. The antibacterial activities
of the extracts were studied by disk diffusion method. The minimum inhibitory concentrations (MIC) and
minimal bactericidal concentrations (MBC) assays were used to address the potentials of extracts. All extracts
were active against pathogenic bacteria at different concentrations, and were especially active against Salmo-
nella tyhpi. In addition, the hexane extract exhibited the lowest MIC and MBC of 0.098 and 3.12 mg/mL,
respectively, against B. cereus based on the antibacterial dilution method. The correlation analysis indicated a
negative relationship between the flavonoid content and the inhibition zone of Salmonella typhi, with a sig-
nificant value of p<0.05.On the contrary, a positive relationship between the saponin content and the inhi-
bition zone Klebsiella pneumoniae. These results showed that C. inophyllum flower extracts are rich of
bioactive compounds such as phytol, eugenol, caryophyllene oxide, a-copaene, a-muurolene,
b-caryophyllene, b-amysin, farnesol, palmitic acid, and cadinene derivatives.
© 2023 SAAB. Published by Elsevier B.V. All rights reserved.
Keywords:
Calophyllum inophyllum
Antibacterial activity
FTIR
GC-MS
Phytochemical
Secondary metabolites
1. Introduction
Folk wisdom regarding the use of herbal medicine has been
passed down through generations. Some herbs exhibit specific heal-
ing qualities, which can be used to treat many symptoms. Research-
ers have extensively studied active biomolecules present in herbs. In
medicinal plants, these biomolecules can be primary or secondary
metabolites (Ghorbanpour et al., 2016a,2106b;Roessner and Beckles,
2009). The rich biodiversity of Thailand provides a vast resource of
medicinal plants, especially in mangrove zones. Plants found in man-
groves can contain several primary and secondary metabolites, such
as amino acids, vitamins, fatty acids, flavonoids, and phenolic acid,
which are associated with important medicinal profiles. In Thai tradi-
tional medicine, the mangrove plant, Calophyllum inophyllum, known
in English as mastwood among other names, is used to treat various
ailments such as wound healing, arthritis and skin diseases.
C. inophyllum is a medicinal plant in the Calophyllaceae family. It
dominantly grows in landsea transitional ecosystems of tropical
forest. It is a medium-sized tree up to 20 m in height with rough bark
and white flowers. The flower, with the average size of 25 mm in
width, occurs in racemose or paniculate inflorescences consisting of
415 flowers. The blooming is all-year-round. the fruit (the ballnut)
appears as rounded and green drupe with 24 cm in diameter and a
single large seed is located at the center. Previous research reported
that the plant exhibits anticancer activities (Shanmugapriya et al.,
2017) and antibacterial activities (Malarvizhi and Ramakrishnan,
2014). Several parts of the plant contain bioactive compounds such
as flavonoids, alkaloids, steroids, terpenoids, and saponins (Hapsari
* Corresponding author.
E-mail addresses: luksamee.v@rmutsv.ac.th,nokluksamee@hotmail.com
(L. Vittaya).
https://doi.org/10.1016/j.sajb.2023.01.052
0254-6299/© 2023 SAAB. Published by Elsevier B.V. All rights reserved.
South African Journal of Botany 154 (2023) 346355
Contents lists available at ScienceDirect
South African Journal of Botany
journal homepage: www.elsevier.com/locate/sajb
et al., 2022;Mah et al., 2015;Susanto et al., 2019). In addition, antiox-
idant and antibacterial activities, due to the presence of phenolic sub-
stances, have been reported (Cassien et al., 2021;Saechan et al.,
2021). The phenolic compounds were confirmed by the NH and OH
components in two mangrove plants, Rhizophora apiculata and Rhizo-
phora annamalayana (Arulkumar et al., 2020). Their antioxidant activ-
ities involved functional groups that can eliminate radicals, reduce
stress, and prevent diseases. There have been reports on bioactive
components in extracts of C. inophyllum leaf, stem, bark, fruit, seed,
root, and flower. Petroleum ether, methanol, ethanol, acetone, ethyl
acetate, and water were among the solvents used for extraction (Hap-
sari et al., 2022;Kadir et al., 2015;Ojah et al., 2020;Sakthivel et al.,
2019;Saravanan et al., 2011). Compounds such as phytol, linoleic
acid, methylisostearate, and diphenylmethane extracted by metha-
nol, may prevent incurable diseases such as common cold, cancer,
asthma, and allergic diseases (Saravanan et al., 2015). Linoleic acid,
methyl ester and hexadecanoic acid (palmitic acid) obtained from the
slow pyrolysis of wood bark have found a wide range of applications
in treating ulcers, wounds, and burns. As enzyme inhibitors, they
have been used in the treatment of schizophrenia (Sakthivel et al.,
2019). The hydrodistillation of C. inophyllum leaf, flower, seed, pod,
and root yielded g-terpinene which was used for its antioxidant and
anti-inflammatory effects (Ojah et al., 2020;Wen et al., 2018). Differ-
ent bioactive compounds can be obtained by extraction using differ-
ent solvents. Gas chromatography-mass spectrometry (GCMS) has
been used for the evaluation of metabolites and volatile compounds
in plant extracts, such as Broussonetia luzonica (Franelyne et al.,
2016), Avicennia marina (Rozirwan et al., 2022), Calophyllum inophyl-
lum (Saravanan et al., 2015) and Dregea volubilis (Singamoorthy et al.,
2021).
Up to now, metabolite profiling by GCMS has not been used to
identify bioactive compounds obtained by solvent extraction of C.
inophyllum flower using hexane, ethyl acetate, and methanol. To the
best of our knowledge, this is the first report on the identification of
bioactive compounds from C. inophyllum flower extracted with sol-
vents of different polarities including hexane, ethyl acetate, and
methanol. GCMS analysis was used to identify the chemical compo-
sition of volatile bioactive compounds in the extracts and in vitro
assays were used to determine their antibacterial activity.
2. Materials and methods
2.1. Plant collection
Flowers of C. inophyllum were collected during September 2016
from coastal region around Rajamangala University of Technology
Srivijaya, Trang Province, Thailand. The plant material was identified
and authenticated by the Department of National Parks, Wildlife and
Plant Conservation, Thailand. A herbarium voucher was submitted as
BKF. 194,811.
2.2. Plant extraction
The collected flowers were dried in the shade for a week. Five
hundred grams of powdered flowers were extracted with 500 mL of
hexane by maceration for five days. The extract was then filtered and
concentrated at reduced pressure using a rotary evaporator at 45 °C.
The residue was macerated further with ethyl acetate and methanol
using the same procedure as for hexane. All extracts were stored in
vials at 4 °C until analyzed.
2.3. Total phenolic compounds
The Folin-Ciocalteu colorimetric method, adopted from Vittaya et
al. (2019), was used to determine the total phenolic content (TPC) in
the extracts of C. inophyllum flower. The calibration curve was
prepared using Gallic acid as the standard and the values were
expressed in mg of gallic acid equivalent (GAE) per gram of crude
extract. The calibration curve was linear, represented by a regression
equation of y= 0.0040x + 0.0086 with R
2
of 0.9977.
2.4. Total flavonoid contents
The total flavonoid content (TFC) in the extracts of C. inophyllum
flower was determined by colorimetric assay as described by Vittaya
et al. (2020). Rutin was used as the standard for the calibration curve,
expressed in mg of rutin equivalents (RU) per gram of crude extract.
The linear calibration curve was represented by a regression equation
of y= 0.0016x + 0.0002 with R
2
of 0.9992.
2.5. Total saponin contents
The total saponin content (TSC) of the extracts of C. inophyllum
flower was determined according to the method reported by Sengut-
tuvan et al. (2014). Briefly, about 0.2 mL of extract (1 mg/mL) was
mixed with 0.5 mL of 0.8% (w/v) vanillin solution. Then 5 mL of 72%
sulfuric acid was added. The mixture was kept for a minute before
the incubation at room temperature for 10 min. The incubated mix-
ture was rapidly cooled in ice water to room temperature. The absor-
bance of the mixture was measured at 560 nm using UVvis
spectroscopy (U-1800 spectrophotometer, Hitachi High-Tech Science
Corp., Tokyo, Japan). Escin was used as the standard for preparing the
calibration curve, expressed in mg of escin equivalents (EC) per gram
of crude extract. The curve can be described by a regression equation
of y= 0.0007x + 0.0254 with R
2
of 0.9918. The experiment was per-
formed in triplicate and the obtained data were expressed as the
means §standard deviation.
2.6. Fourier transform infrared spectroscopy (FTIR) analysis
Using a mortar and pestle, about 1 mg of each extract of C. ino-
phyllum flower was mixed with 3 mg of dry potassium bromide (KBr)
to form a disk. All the IR spectra were recorded at 25 °C in the mid-
infrared range (4000 400 cm
1
) using the BX PerkinElmer FTIR
spectrophotometer (PerkinElmer, Massachusetts, USA.). Each spec-
trum was displayed in terms of absorbance of%T.
2.7. Gas chromatography-electron ionization/mass spectrometry (GC-
EI/MS)
Each C. inophyllum flower extract was analyzed using the 7890 B
GC gas chromatograph (Agilent Technologies, California, USA.)
equipped with HP-5 ms capillary column (30 mm length £0.25 mm
I. d. £0.25 mmfilm thickness). The analysis was carried out as fol-
lows. Briefly, 20 mg of crude extract were dissolved in 1 mL of solvent
(either hexane, ethyl acetate, or methanol) and then shaken by vor-
tex. The solution was sonicated for 2 min followed by filtration
through a nylon membrane (pore size: 0.2 mm) to remove debris.
One microliter of each sample was introduced via a split injector at a
temperature of 250 °C with a split ratio of 10:1. The oven tempera-
ture program was 40 °C for 2 min, raised at 5 °C/min to 320 °C, and
held for 10 min. The carrier gas was helium at a constant flow rate of
1 mL/min. The temperature of MSD transfer line was at 320 °C. The
solvent delay was 5 min. Tandem mass spectra (700D MS, Agilent
Technologies, California, USA.) were produced by electron ionization
(EI) at 70 eV. The ion source temperature was at 230 °C. The mass
spectrometer was operated in full scan mode from m/z 35550.
MassHunter software was used to control the GCMS and data acqui-
sition. The chemical constituents were identified after comparing the
mass spectral configurations obtained with the available mass spec-
tral database (WILEY10 and NIST14 libraries).
L. Vittaya, C. Chalad, W. Ratsameepakai et al. South African Journal of Botany 154 (2023) 346355
347
2.8. Determination of antibacterial activity
2.8.1. Bacterial strains and disk diffusion method
The test involved seven bacterial species. Three species were
Gram positive bacteria; namely Bacillus cereus TISTR 036, Staphylo-
coccus aureus TISTR 746, and Staphylococcus epidermidis TISTR 518.
Four species were Gram negative bacteria; namely Escherichia coli
TISTR 527, Salmonella typhi TISTR 2517, Klebsiella pneumoniae subsp.
pneumoniae TISTR 1383, and Pseudomonas aeruginosa TISTR 2370. All
bacteria were obtained from the National Center for Genetic Engi-
neering and Biotechnology, Thailand. All extracts were screened
three times for antibacterial activity using the paper disk diffusion
method (NCCLS, 1993). Bacteria were incubated in Mueller Hinton
broth at 35 °C for 2 6 h and the turbidity was measured at 0.5
McFarland standard (1.5 £10
8
colony forming units/mL) at 625 nm,
with the optical density (OD) between 0.08 and 0.10. The bacteria
were swabbed over the surface of the media (Mueller Hinton agar)
with a sterile cotton swab and allowed to solidify. Crude extracts
were dissolved in dimethyl sulfoxide (negative control) to obtain
stock solutions of 100 mg/mL and 10 mL of each sample was tested
on a sterile Whatman No. 1 filter paper disk (6 mm in diameter). After
incubation at 35 °C for 24 h, the zones of inhibition were measured in
millimeters. Gentamycin was used as a positive control.
2.8.2. Estimation of minimum inhibitory concentration (MIC) and
minimal bactericidal concentration (MBC)
MICs of all extracts were evaluated using the standard method
(CLSI, 2009) with a slight modification. Broth microdilution suscepti-
bility procedure was performed using 96-well microplates. Briefly,
200 mL of the different extract concentrations (100, 50, 25, 12.5, 6.25,
3.125, 1.563, 0.781, 0.391, 0.195, and 0.098 mg/mL) in 96-well culture
plates with 0.85% sodium chloride were prepared by two-fold serial
dilution with sterile Mueller Hinton Broth (MHB) in each well. The
last well was the positive control which contained only the culture
medium and bacterial suspension. Then, the inoculated microplates
were incubated at 35 °C for 24 h. All tests were performed in tripli-
cate. The MIC was considered as the lowest concentration of sample
that inhibited the growth of the bacteria. The MBC was the lowest
concentration of a crude plant extract that killed 99.9% of each bacte-
ria used (0.08 0.1 OD or 1.5 £10
8
colony forming units/mL).
2.9. Statistical analysis
The data were recorded from three replicates (n= 3) in each
experiment and all the results were presented as means with stan-
dard deviation. One-Way Analysis of Variance (ANOVA) was carried
out to compare the data and to further determine the statistically sig-
nificant differences (p<0.05). Correlations between the values for
phytochemical composition (TPC, TFC, and TSC) in each extract with
seven pathogenic bacteria were performed using Pearson’s correla-
tion coefficient.
3. Results and discussion
3.1. Extraction yield
C. inophyllum flower was extracted using hexane, ethyl acetate,
and methanol. Solvent polarity plays an important role on the solu-
bility of phytochemical constituents (Ramalingam and Rajaram,
2018;Nalimanana et al., 2022), as do the structures of the phyto-
chemical components. The polarity index of the solvents used in the
present study can be arranged as follows: hexane (0.1) <ethyl ace-
tate (4.4) <methanol (5.1) (Sadek, 2002). The highest yield was
obtained with methanol extraction and the yield decreased with
decreasing polarity of the extracting solvent (Table 1). The variation
in the yields can be explained by the greater solubility of proteins
and carbohydrates in methanol compared to hexane and ethyl ace-
tate. These results confirmed the richness of polar substances in this
plant. Our findings are in agreement with previous studies (Kadir et
al., 2015;Mahmoudi et al., 2016), which reported that the maximum
yield from Temnocalyx obovatus was obtained with absolute metha-
nol, as well as our previous work (Vittaya et al., 2022).
3.2. Phenolic compounds, flavonoids and saponin contents
The TPC, TFC, and TSC of the three extracts of C. inophyllum flower
ranged from 0.19 to 0.84 mg gallic acid equivalent per gram crude
extract (mg GAE/g CE), 0.91 to 1.48 mg rutin equivalent per gram
crude extract (mg Ru/g CE), and 2.99 to 5.05 mg escin equivalent per
gram crude extract (mg ES/g CE), respectively (Table 2). The extrac-
tion of C. inophyllum flower using ethyl acetate provided the highest
TPC and TFC, p<0.05. Extraction using hexane resulted in the highest
TSC. Phenolic and flavonoid compounds can be readily dissolved in a
medium polarity solvent since they have similar polar properties. It is
possible that the solubility of phytochemical compounds depends on
the extraction solvent used and the degree of polymerization pro-
duced by the interaction of these compounds with other phytochem-
icals or vitamins (Naczk and Shahidi, 2004). TPC and TFC in each
extract showed a positive trend with a correlation coefficient of
0.876, p= 0.002. On the other hand, TPC and TSC, and TFC and TSC
showed negative trends with correlation coefficients of 0.794,
p= 0.011 and 0.441, p= 0.235, respectively. The negative correla-
tions between phenolic contents and flavonoid and saponin contents
were attributed to the presence of other primary and secondary
metabolites in each extract of this plant (Sajid et al., 2012).
Table 1
Yields of various extracts of Calophyllum inophyllum.
Plant Part extracted Solvent Yield (g) % Yield
C. inophyllum Flower Hexane 6.73 1.64
Ethyl acetate 23.30 5.68
Methanol 241.46 58.89
Table 2
Total phenolic contents, total flavonoid contents, and total saponin contents of different extracts
Calophyllum inophyllum flower.
Solvent Total phenolic content*
(mg GAE/g CE)
Total flavonoid content*
(mg RU/g CE)
Total saponin content*
(mg ES/g CE)
Hexane 0.19 §0.02
c
0.91 §0.18
b
5.05 §0.79
a
Ethyl acetate 0.84 §0.07
a
1.48 §0.11
a
3.14 §0.55
b
Methanol 0.57 §0.05
b
1.03 §0.07
b
2.99 §0.41
b
* Each value is expressed as mean §standard deviation (SD) (n= 3). Values followed by a differ-
ent letter superscript in the same column are significantly different (p<0.05) and values having
the same letters are not statistically significant (p<0.05). GAE: gallic acid equivalent, CE: crude
extract.
L. Vittaya, C. Chalad, W. Ratsameepakai et al. South African Journal of Botany 154 (2023) 346355
348
To identify the phytochemical contents of C. inophyllum flower,
the separation of their components must first be considered based on
the polarity of the solvent used, following the principle of “like dis-
solves like”(Reichardt and Welton, 2011). Hexane is a non-polar sol-
vent with a polarity index of 0.1, which dissolves alkanes, fatty acids,
sterols, alkaloids, and some terpenoids. Ethyl acetate, a medium
polarity solvent with a polarity index of 4.4, extracts compounds
with intermediate polarity such as flavonoids and quinones. Metha-
nol is a high polarity solvent with a polarity index of 5.1 that can dis-
solve glycosides, polyphenols including tannins, and anthocyanins
(Feng et al., 2019; Sadek, 2002). In addition, the phytochemical con-
tents obtained with the three solvents might be influenced by factors
such as growing area, mineral availability, time of harvest, and stor-
age conditions before extraction (Figueiredo et al., 2008; Tiwari and
Cummins, 2013). It is therefore possible that the solvent extraction
method extracts bioactive compounds which might exhibit some
unexpected biological characteristics.
3.3. FTIR analysis of C. inophyllum flower extracts
The FTIR spectra of C. inophyllum flower extracts showed similar
profiles, except some differences between functional groups. All
spectra showed characteristic absorption bands between 3500 and
600 cm
1
(Fig. 1S). Tentative assignments of FTIR spectral data are
shown in Table 3. The results indicated that the hexane and ethyl ace-
tate extracts of C. inophyllum flower had more functional groups than
that of the methanol extract. The probable functional groups of com-
pounds in these extracts are alkanes, alcohol, carboxylic acid, aro-
matics, esters, aliphatic amine, and alkyl halides (Gilbert, 2017).
These results demonstrated the characteristic absorptions of OH,
C=O, CO, CH, and C=C groups among others, and support the find-
ings of previous works that mangrove plants are the rich sources of
various secondary metabolites of phenolic and flavonoid compounds
(NH and OH molecules) (Agoramoorthy et al., 2008;Glasenapp et
al., 2019;Rozirwan et al., 2022).
3.4. Chemical compositions
GCMS is frequently used to identify the constituents of volatile
compounds, long and branched chain hydrocarbons, alcohols, acids,
and esters. This work is the first report of GCMS profiling of hexane,
ethyl acetate, and methanol extracts of C. inophyllum flower. The
identified chemical compounds in terms of compound name, class,
molecular formula, molecular weight, and% peak area were presented
in Table 1S. A total of 97 different compounds were identified in the
chromatograms of the three crude extracts (Fig. 1). The GCMS pro-
files of hexane, ethyl acetate, and methanol contained signals of 54,
63, and 41 of the identified compounds, respectively. The compounds
were mostly classified as terpenes, fatty acid, and fatty acid deriva-
tives (Table 4). The major components of the hexane extract were
sesquiterpenoid (24.04%) and triterpenoid (13.50%). The main com-
ponent of the ethyl acetate extract was sesquiterpenoid (23.63%). On
the other hand, the major component of the methanol extract was
fatty acid and derivatives (27.30%). According to the literature survey,
the determination of composition existing in C. inophyllum flower by
GCMS is rarely. There is only the extraction by hydrodistillation
(Ojah et al., 2019,2020) stated that the 25 identified compounds
were found which is much less than that of our present work. The 63
identified compounds were found by hexane extraction. Sesquiterpe-
noid was the main component in hexane and ethyl acetate despite
derivative of fatty acid in methanol. Bioactive compounds like
a-copaene, a-muurolene, d-cadinene, cedrene, and 1-hexadecanol
were also detected in both hydrodistillation and solvent extraction.
However, bioactive compounds of phytol, caryophyllene oxide, gera-
nylgeraniol, b-amyrin, eugenol, farnesol, a-amyrin palmitate, and
palmitic acid were only observed in this work. The majority of the
phytochemicals identified are known to exhibit various important
biological activities and many compounds exhibit pharmacological
activities. The molecular structures of some of these pharmacologi-
cally active compounds are shown in Fig. 2.
The relatively greater antibacterial activity of the hexane extract in
this study was correlated with the presence of a greater number of bio-
active compounds including phytol, caryophyllene oxide, geranylgera-
niol, b-amyrin, and eugenol. These compounds have been observed in
many medicinal plant extracts. Phytol is an important diterpenoid that
exhibits anticancer, antibacterial, and antioxidant activities (Saravanan
et al., 2015;Song and Cho, 2015). Caryophyllene oxide is a sesquiterpe-
noid oxide commonly applied as an antifeedant, insecticide, and as a
broad-spectrum antifungal in plant defense (Russo and Marcu, 2017).
Geranylgeraniol is a potential antibacterial inhibitor of Mycobacteruim
tuberculosis (Vik et al., 2007). The compound b-amyrin is known to
possess analgesic and antimicrobial properties (Sundur et al., 2014).
Table 3
Tentative functional group assignments of FTIR spectra of three different extracts of Calophyllum inophyllum flower.
Bond Functional group C. inophyllum
(Wavenumber, cm
1
)
Hexane Ethyl acetate Methanol
OH stretch in alcohol Alcohol, phenol 3438 3406 3420
sp
2
CH Asymmetric stretch
NH stretch
Alkene
Benzene
3140 3152 3155
CH stretch in CH
3
and CH
2
Alkane 2918 2928 2926
C·N stretch Alkyl nitriles 2362 2364 2364
C=O stretch Carbonyl in carboxylic acid, ester, aldehyde, ketone
Aromatic compound
1736 1708
C=O stretch in salts of carboxylic acid
NH bend in primary amines
NH bend in secondary amines
Unsaturated aldehyde, ketone
Conjugated and cyclic alkane
Amine
Amine
1604 1618 1610
CO stretch in salts of carboxylic acid
CF stretch
Aromatic
Methyl group/aliphatic alcohol
1400 1400 1400
NH bend in aromatic amines
CN stretch in primary and secondary aliphatic amine
Aromatic, ester, alkyl aryl ether
Aliphatic amine Alkyl halide
1254 1254
C–O stretch in –OCH
3
C–O stretch in secondary and tertiary alcohols
C–F stretch
Alcohol
Alcohol
Halogen compounds
1168 1096 1078
O–H out of plane bend in alcohols
O–H out of plane bend in cis- RCHHCHR
Alcohol
Alkene
720 ––
C–Cl stretch Alkyl halide 618 618 618
L. Vittaya, C. Chalad, W. Ratsameepakai et al. South African Journal of Botany 154 (2023) 346355
349
Eugenol is an essential oil component providing excellent antimicro-
bial activity against fungi and a wide range of Gram positive and Gram
negative bacteria (Marchese et al., 2017). Farnesol is found in the
essential oils of various plants, exhibiting potent anti-inflammatory
and anticancer effects, as well as alleviating allergic asthma (Jung et al.,
2018). a-amyrin palmitate was regarded as an effective agent against
arthritis (Arag~
ao et al., 2008). Additionally, n-hexadecanoic acid (pal-
mitic acid) and a-linolenic acid in ethyl acetate and methanol extracts
are known to possess various biological activities. Palmitic acid is a sat-
urated fatty acid found in almost all plant oils and microorganisms
(Senthilkumar et al., 2015). For example, it was isolated from Rhizo-
phora mucronata leaf and significant antibacterial activities were
observed (Joel and Bhimba, 2010). a-Linolenic acid is an efficient anti-
oxidant against oxidative DNA damage (Pal and Ghosh, 2012).
a-copaene and a-muurolene in the hexane and ethyl acetate extracts
were also reported to for antioxidant, antiparasitic, and antimicrobial
activities (Karakaya et al., 2016;Mennai et al., 2021). Similarly,
b-caryophyllene, gand d-cadinene in all three extracts are valuable
bioactive compounds presenting anticancer, antioxidant, and antimi-
crobial activities (Dahham et al., 2015;Kundu et al., 2013;Mennai
et al., 2021).
The GCMS data confirmed that the selection of solvent with the
appropriate polarity is one of the main factors affecting the extraction
process. There is a possible interaction between each secondary
metabolite and plant components such as proteins, lipids, and carbo-
hydrates. These interactions can result in the formation of complexes
that are quite difficult to dissolve. The polarity of the solvent used
also affects the solubility of compounds. These results were in good
agreement with the work of Wakeel et al. (2019), which showed that
different parts of the woad plant, containing different levels of sec-
ondary metabolites with specific polarities, require extraction with
specific solvents of suitable polarity. Solvent and solute polarity could
be one of the reasons for the higher antimicrobial activity exhibited
by the hexane extract of C. inophyllum flower compared to that of
ethyl acetate and methanol extracts as stated earlier.
Fig. 1. GCMS Chromatograms of hexane (a), ethyl acetate (b), and methanol (c) extracts of Calophyllum inophyllum flower.
Table 4
Classification of identified phytochemicals in Calophyllum inophyllum flower
extracts.
Class C. inophyllum extracts (%)
Hexane Ethyl acetate Methanol
Number of identified compounds 54 63 41
Total identified compound contents 85.79 82.39 45.62
Diterpenoid
Monoterpenoid
Sesquiterpenoid
Triterpenoid
Quinone and hydroquinone lipid
Benzene and substituted derivative
Carbonyl compound
Cinnamic acid and derivative
Coumarin and derivative
Epoxide
Fatty acid and derivative
Glycerolipid
Organoheterocyclic compound
Organooxygen compound
Phenol
Pyran
Saturated hydrocarbon
Unsaturated hydrocarbon
Steroid
8.34
-
24.04
13.50
0.32
2.56
4.42
-
4.59
4.03
3.31
-
4.51
-
0.25
-
11.01
1.78
3.13
1.67
1.35
23.63
0.91
0.66
4.31
10.53
0.37
11.49
0.96
6.89
0.16
5.48
1.25
1.24
-
4.49
1.63
5.37
0.37
-
4.55
-
-
3.80
0.41
1.61
-
0.38
27.30
1.24
-
-
4.28
1.12
0.56
-
-
350
L. Vittaya, C. Chalad, W. Ratsameepakai et al. South African Journal of Botany 154 (2023) 346355
3.5. Antibacterial activity
Three different C. inophyllum flower extracts were evaluated for
growth inhibitory activity against seven bacteria. Three strains were
Gram positive bacteria (B. cereus, S. aureus,andS. epidermidis) and four
were Gram negative bacteria (E. coli, S. thyphi, K. pneumoniae,andP.
aeruginosa). Photographs of the results of the disk diffusion assay are
included in Fig. 2S. All extracts showed antibacterial activity against all
pathogenic bacteria. The antibacterial activity of the extracts against
the Gram positive bacteria was similar to their antibacterial activity
against the Gram negative bacteria, except for K. pneumoniae where
the inhibition zones produced by hexane, ethyl acetate, and methanol
exhibited significant differences (p<0.05) (Table 5).
Fig. 3 shows the effects of extracting solvents of C. inophyllum
flower on the growth of seven bacteria, indicating that hexane and
ethyl acetate extracts strongly inhibited the growth of S. typhi, B.
Fig. 2. The molecular structure of some compounds identified in hexane, ethyl acetate, and methanol extracts of Calophyllum inophyllum flower.
Table 5
Antibacterial activity of Calophyllum inophyllum flower extracts against seven pathogenic bacteria strains.
Solvent Zone of inhibition diameter (mm)
Gram positive Gram negative
B. cereus S. aureus S. epidermidis E. coli S. typhi K. pneumoniae P. aeruginosa
Hexane 15.07§1.03
b
14.95§0.29
b
8.48§0.51
b
8.54§0.51
b
16.63§0.79
b
8.44§0.31
b
13.92§3.18
b
Ethyl acetate 14.46§0.21
b
14.81§0.85
b
8.61§0.58
b
8.25§0.60
b
14.35§0.94
c
7.70§0.39
c
12.01§2.74
b
Methanol 14.09§0.75
b
13.66§0.22
c
8.58§0.30
b
7.68§0.65
b
16.62§0.35
b
6.85§0.25
d
12.55§2.18
b
Gentamycin 23.15§0.12
a
23.91§0.06
a
22.01§10.34
a
20.74§0.04
a
26.14§0.04
a
18.69§0.15
a
23.41§0.17
a
Data shown as means §SD from triplicate analysis.
Different lowercase superscripts in a column denote significant (p<0.05) differences in means §SD values.
351
L. Vittaya, C. Chalad, W. Ratsameepakai et al. South African Journal of Botany 154 (2023) 346355
cereus, and S. aureus (p<0.05). Methanol extract was only strongly
active against S. typhi (p<0.05). Notably, all three extracts were
most active against S. typhi. The stronger resistance of the Gram nega-
tive bacteria E. coli and K. pneumoniae to the extracts could be attrib-
uted to the complexity of the double membrane expressed by
lipoprotein and lipopolysaccharide. The complexity of membrane
structure presents a barrier to antibacterial substances compared to
that of the single membrane structure of Gram positive bacteria S.
epidermidis (Song and Cho, 2015;Yitayeh and Wassihun, 2022).
The MICs of the C. inophyllum flower extracts ranged from 0.098 to
12.5 mg/mL against Gram positive bacteria and from 6.25 to 50 mg/
mL against Gram negative bacteria (Table 6). All extracts showed
strong activity against B. cereus with low MIC values of <0.098, 0.39
mg/mL and 0.098 mg/mL for hexane, ethyl acetate, and methanol,
respectively. The mechanism of antibacterial activity is influenced by
the molecular weight of the compounds involved. This affects the dif-
fusion through agar, causing the difference in sensitivity exhibited by
Gram positive and Gram negative species. Sensitivity differences are
related to the chemical composition of the extracts (Russo and Marcu,
2017).
3.6. Relationship between the amount of TPC, TFC and TSC with
antibacterial activities
Table 7 shows the correlation between the amount of phytochem-
ical components with antibacterial activities. It appears that the phe-
nolic content was positively correlated with flavonoid (r= 0.876; p<
0.01) and saponin (r= 0.794; p<0.05) but no correlation was
observed between flavonoid and saponin. The antibacterial activities
against almost all bacteria strains were not significantly correlated
with phenolic, flavonoid, and saponin contents, except for the flavo-
noid content against S. typhi (r=0.777; p<0.05). Saponin content
was positively correlated with K. pneumoniae (r= 0.697; p<0.05). It
was possible that the discrepancies in antibacterial activity involved
other specific phenolic, flavonoid, and saponin compounds in each
extract or other phytochemical components, such as alkaloid, terpe-
noid, anthraquinone (Arulkumar et al., 2020;Singamoorthy et al.,
2021). In this work, such classes of sesquiterpenoid, triterpenoid,
fatty acid and derivatives, saturated hydrocarbon, carbonyl com-
pound, coumarin and derivatives, and saturated hydrocarbon were
founded in C. inophyllum flower which may be supported the antibac-
terial activity. Similarly, Vittaya et al. (2022) reported no significant
or a negative correlation between the phenolic and flavonoid con-
tents and their antibacterial activity against Derris indica.
4. Conclusion
To the best of our knowledge, this is the first report on the chemi-
cal profiles of bioactive compounds that can be extracted from C. ino-
phyllum flower using hexane, ethyl acetate, and methanol.
Depending on the type of solvent used, the yield of C. inophyllum
flower extract was in an increasing order of methanol, ethyl acetate,
and hexane. The total phenolic and flavonoid contents were signifi-
cantly correlated and the maximum content of both were obtained
from extraction using ethyl acetate. The highest content of saponin
was observed when hexane was used in extraction. All extracts inhib-
ited the growth of Gram positive and Gram negative bacteria, espe-
cially S. typhi. Moreover, the chemical composition in the extracts
was rich in sesquiterpenoid, triterpenoid, fatty acid and fatty acid
derivatives. The extracts could be developed for food and pharmaceu-
tical applications. To realize the potential of this plant, further study
is needed to investigate its toxicity and the environmentally friendly
extraction method should be developed. LC-MS analysis can also be
used as an alternative technique to study the correlation between
chemical profiles and the biological properties of the investigated
extracts.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
This work was supported by the Faculty of Science and Fisheries
Technology, Rajamangala University of Technology Srivijaya, Trang
campus. The authors would like to thank the Department of National
Parks, Wildlife and Plant Conservation, Thailand, who supplied the
specimen voucher of Calophyllum inophyllum and the National Center
for Genetic Engineering and Biotechnology (BIOTECH), Thailand who
supplied pathogenic bacteria. We would also like to thank academi-
cian Thomas Duncan Coyne for proofing and editing the English of
the manuscript.
Supplementary materials
Supplementary material associated with this article can be found,
in the online version, at doi:10.1016/j.sajb.2023.01.052.
Fig. 3. Effects of extracting solvents of Calophyllum inophyllum flower on the growth of bacteria strains. Results are expressed as means §SD (n= 3), significant (p<0.05) differen-
ces. Different lowercase superscripts denote significant (p<0.05) differences in means §SD values.
352
L. Vittaya, C. Chalad, W. Ratsameepakai et al. South African Journal of Botany 154 (2023) 346355
Table 6
Minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of Calophyllum inophyllum flower extracts.
Solvent MIC and MBC (mg/mL)
Gram positive Gram negative
B. cereus S. aureus S. epidermidis E. coli S. typhi K. pneumoniae P. aeruginosa
MIC MBC R MIC MBC R MIC MBC R MIC MBC R MIC MBC R MIC MBC R MIC MBC R
Hexane <0.098 3.12 <31.8 12.5 50 4 12.5 25 2 25 >50 >2 12.5 >100 8 50 >100 >225>100 >4
Ethyl acetate 0.39 25 64 6.25 >50 >8 6.25 25 4 6.25 50 8 12.5 >100 8 12.5 100 8 6.25 100 16
Methanol 0.098 3.12 31.8 6.25 6.25 1 6.25 50 8 12.50 >50 4 12.5 >100 8 25 >100 >4 12.5 >100 8
Values are expressed as means from quadruplicate determination (n= 4).
R values were calculated from MBC/MIC (R h4.00 indicates bactericidal extract, R i4.00 indicates bacteriostatic extract).
Table 7
Correlation coefficient (r) between phytochemical compositions and seven pathogenic bacteria of Calophyllum inophyllum flower.
Variables TPC TFC TSC B. cereus S. aureus S. epidermidis E. coli S. typhi K. pneu P. aeruginosa
TPC 1 0.876
**
0.794* 0.371 0.078 0.103 0.178 0.649 0.551 0.446
TFC 1 0.441 0.352 0.330 0.181 0.181 0.777* 0.243 0.607
TSC 1 0.198 0.458 0.068 0.324 0.195 0.697* 0.265
B. cereus 1 0.188 0.606 0.269 0.262 0.490 0.472
S. aureus 10.107 0.539 0.123 0.522 0.259
S. epidermidis 1 0.313 0.331 0.112 0.419
E. coli 10.132 0.531 0.492
S. typhi 10.124 0.375
K. pneu 1 0.375
P. aeruginosa 1
TPC = total phenolic content; TFC = total flavonoid content; TSC = total saponin content.
*, ** = significant different from 0 at * p<0.05 and ** p<0.01, respectively; ns = non-significant (p>0.05). Data shown are mean §SD. Pearson’s
correlation coefficient (r) between each chemical composition (n= 9) and bacteria. * p<0.05, ** p<0.01.
353
L. Vittaya, C. Chalad, W. Ratsameepakai et al. South African Journal of Botany 154 (2023) 346355
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