Philippine Journal of Science

149 (3-a): 697-710, October 2020

ISSN 0031 - 7683
Date Received: 19 Feb 2020

Phytochemical Screening, Total Phenolics,

and Antioxidant and Antibacterial Activities
of Selected Philippine Indigenous Fruits

Mariam C. Recuenco*, James Russell P. De Luna,

Nathalia G. Magallano, and Kevin C. Salamanez

Institute of Chemistry, College of Arts and Sciences

University of the Philippines Los Baños, College, Laguna 4031 Philippines

Nine Philippine indigenous fruits were screened for phytochemical constituents and assessed
for total phenolics and antioxidant and antibacterial activities. Qualitative tests revealed the
presence of alkaloids in Canarium ovatum, cardiac glycosides in Ficus pseudopalma and C.
ovatum, and terpenoids in Antidesma bunius and C. ovatum. Total phenolics were highest in
Garcinia binucao and Mangifera altissima with 758 and 694 mg gallic acid equivalent (GAE) / 100
g fresh matter (FM), respectively. The DPPH radical scavenging activities ranged from 82–516
mg ascorbic acid equivalent antioxidant activity (AEAC) /100 g FM, with M. altissima having
the highest value and followed by Rubus rosifolius (513 mg AEAC / 100 g FM). Ferric reducing
activities were highest for M. altissima and G. binucao with 111 mg and 121 mg ascorbic acid
equivalents (AAE) / 100 g FM, respectively. Phenolic and flavonoid contents were strongly and
positively correlated (P < 0.05). Moreover, phenolic contents may have significant contributions
to the observed radical scavenging and ferric reducing activities based on their strong positive
correlations (P < 0.05). For the antibacterial activities, extracts from Citrus hystrix and R.
rosifolius were the most effective against Escherichia coli (MIC80 = 1.70 mg GAE/mL), while
the F. pseudopalma extract was the most effective against Staphylococcus aureus (MIC80 = 0.56
mg GAE / mL). Present results showed that the selected indigenous fruits could be valuable
sources of phytochemicals, such as phenolics and flavonoids, with potential antioxidant and
antibacterial activities.

Keywords: antioxidant, antibacterial, phenolics, Philippine fruits, phytochemicals

INTRODUCTION phytochemicals that include compounds such as phenolic

acids, flavonoids, stilbenes, coumarins, and tannins. These
Plant-based foods, fruits and vegetables, are essential in compounds have molecular structures characterized by the
the human diet because of their roles in sustaining life phenol moiety of aromatic hydrocarbons with hydroxyl
and health. They provide vital nutrients, carbohydrates, groups (-OH). Many studies reported the potential health
proteins, lipids, vitamins and minerals, fiber, and a wide benefits of dietary phenolics from cocoa, coffee, red wine,
variety of bioactive non-nutrient “phytochemicals” tea, berries, citrus fruits, nuts, and vegetables (Del Rio
from plant secondary metabolism (Del Rio et al. et al. 2013). Evidence from in vitro studies suggested
2013). The phenolics are one of the biggest groups of that phenolics exhibit anti-cancer cell proliferation, anti-
*Corresponding Author: mcrecuenco@up.edu.ph inflammatory, and antimicrobial activities (Del Rio et al.

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2013). Moreover, some small-scale human intervention There are a few studies done in the Philippines on this fruit
studies suggested that increased consumption of (poly) (Quevedo et al. 2013; Ragasa et al. 2014b). Artocarpus
phenol-rich foods had beneficial effects on cardiovascular altilis (“kamansi”) young fruits are cooked and consumed
and neurocognitive health, and in the reduction of risks as a vegetable. A. altilis leaf dichloromethane extract was
for certain cancers (Del Rio et al. 2013). reported to contain several sterols, unsaturated fatty acids,
lutein, and isoprenes (Ragasa et al. 2014a). Citrus hystrix
Studies on the phytochemical components and biological or “kabuyaw” (kaffir lime) is a citrus fruit often used in
activities from fruits had been increasing in recent years. Thailand as a flavorant (Panthong et al. 2013). While there
A number of studies had analyzed collections of fruits that were several studies on C. hystrix elsewhere (Panthong et al.
included widely consumed and/or underutilized fruits in a 2013; Abirami et al. 2014; Seeka et al. 2016), there seemed
certain country or locality (Leong and Shui 2002; Ikram to be no studies conducted on this fruit in the Philippines.
et al. 2009; Rufino et al. 2010). We had recently reported Canarium ovatum or “pili” nuts and its by-products are of
on the total phenolics and β-carotene radical inhibition economic importance to the Philippines, with the Bicol
activities of 30 locally available fruits (Recuenco et al. region supplying 80% to the market (Pham and Dumandan
2016). With the diversity of fruit species in the Philippines, 2015). Pham and Dumandan (2015) reported the lipid
there are many more fruits that could be potential sources profiles of C. ovatum oil and pulp. Lastly, Rubus rosifolius
of bioactive phytochemicals. Ethnobotanical surveys such or “sampinit” is a wild berry that is one of about 10 species
as those conducted by Chua-Barcelo (2014) and Santiago of Rubus that could be found in the mountainous regions of
et al. (2014) can provide information on where indigenous the Philippines (Real 2016). Although Rubus was reported
fruits grow and thrive, and how locals use them for food to be found in 39 provinces (Real 2016), there seems to be
and medicine. limited research on these locally grown berries.
In this study, nine selected indigenous fruits (Table 1;
Appendix Figure I) were assessed for phytochemical
constituents using qualitative tests, total phenolics, and
antioxidant and antibacterial activities. Even though there MATERIALS AND METHODS
are studies already conducted in other countries, it may be
necessary to perform analysis on the locally grown fruits Sample Collection and Identification
since phytochemical contents may be affected by cultivar, Selected indigenous fruits (Table 1; Appendix Figure I)
climate and location, and agronomic and harvest factors were obtained from the provinces of Laguna, Quezon,
(Tiwari and Cummins 2013). This study is limited to using Batangas, and Negros Oriental. The maturity stage
the edible portions as samples and having only one stage of or ripeness of the fruits was indicated in Table 1. The
maturity for each fruit (Table 1). To our knowledge, this may samples (500–1000 g) were cleaned and refrigerated prior
be the first report on the phenolic contents and antioxidant to extraction and evaluation. Samples were identified by
activities of Ficus ulmifolia (“as-is”), Ficus pseudopalma Dr. Annalee S. Hadsall of the Museum of Natural History,
(“niyog-niyogan”), and Mangifera altissima (“paho”), University of the Philippines Los Baños.
which are endemic to the Philippines (Coronel et al. 2003;
Ragasa et al. 2009). M. altissima mangoes are eaten fresh,
Sample Preparation and Extraction
ripe or unripe, pickled, or used in salads. M. altissima
Two (2.00) g edible portion of the fruit samples were
was included in the morphological characterization of
extracted with 50 mL (80% v/v) aqueous methanol using
five Mangifera spp. in the study of Coronel et al. (2003).
a homogenizer. The homogenate was filtered and the
Ficus pseudopalma young shoots and leaves are eaten
filtrate was stored in amber-colored bottles at –20 °C. For
and used as a medicinal plant in the Bicol region, but the
the antibacterial assay, the methanolic fruit extracts were
use of the fruits may not be as common (Santiago et al.
concentrated using a rotary evaporator and dried under
2014). Terpenoids and sterols had been identified from
a fume hood. The total phenolics of the concentrated
the leaves of F. pseudopalma and F. ulmifolia (Ragasa et
extracts were determined using the Folin-Ciocalteu
al. 2009; Santiago and Mayor 2014; De las Llagas et al.
method (Singleton and Rossi 1965).
2014). Antidesma bunius (bignay) fruits are commonly
eaten fresh or prepared as preserves such as jams or jellies
or made into vinegars and wines. Bignay apparently is Moisture Content Determination
the most studied for its phytochemical and antioxidant Fresh samples (2 g) in tared evaporating dishes were dried
properties (Butkhup and Samappito 2011; Lizardo et for 24 h in a hot air oven set at 50 °C. After 24 h, the dishes
al. 2015; Ngamlerst et al. 2019). Garcinia binucao or were placed in desiccators, cooled to room temperature,
“batuan” fruits are commonly used as a souring agent in and weighed. These steps were repeated until the weights
dishes, particularly in the Visayas (Quevedo et al. 2013). were constant. Moisture contents were calculated using the

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Vol. 149 No. 3, October 2020 Activities of Philippine Fruits

Table 1. Information on the selected Philippine indigenous fruits used in this study.
Scientific name Local name Place of collection Part/s used and maturity
(Family)
Ficus ulmifolia Lam. “As-is” Tiaong, Quezon Whole fruit; ripe
(Moraceae)
Antidesma bunius (L.) Spreng “Bignay” Calauan, Laguna Whole fruit; ripe
(Phyllanthaceae)
Garcinia binucao (Blco.) Choisy “Binukaw,” “Batuan” Dumaguete City, Negros Flesh; ripe
(Clusiaceae) Oriental
Artocarpus altilis (Park.) Fosb. “Kamansi” Calauan, Laguna Strands and seeds; unripe
(Moraceae)
Citrus hystrix DC. “Kolong-kolong” Calauan, Laguna Pulp sac with juice; ripe
(Rutaceae)
Ficus pseudopalma Blco. “Niyog-niyogan” Calauan, Laguna Flesh and seeds; ripe
(Moraceae)
Mangifera altissima Blco. “Paho” San Juan, Batangas Flesh; unripe
(Anacardiaceae)
Canarium ovatum Engl. “Pili” Calauan, Laguna Pulp and nut (less brown sheath);
(Burseraceae) ripe
Rubus rosifolius Sm. “Sampinit” Dolores, Quezon Whole fruit; ripe
(Rosaceae)

equation: % moisture: = [(initial weight – final weight) / Determination of Total Flavonoid Content (TFC)
initial weight] x 100%. The procedure was modified from Zhishen et al. (1999). In
a 96-well microplate, 25 µL of the prepared fruit extract in
80% (v/v) methanol was combined with 100 µL distilled
Qualitative Phytochemical Screening
water and 7.5 µL (5 % w/v) NaNO2. After 5 min, 7.5
A few milliliters of the prepared extracts in 80% (v/v)
µL of (10 % w/v) AlCl3 was added. After 5 min, 50 µL
methanol was subjected to qualitative tests for phenolics,
1 M NaOH and 100 µL distilled water were added. The
alkaloids, cardiac glycosides, terpenoids, and saponins
mixtures of fruit extracts and reagents were mixed and
(Mandal et al. 2015). The tests done in duplicates were
absorbances were read at 510 nm. Catechin was used to
Folin-Ciocalteu test for phenolics, Wagner’s test for
prepare a calibration curve (0–200 mg/L, R2 = 0.9954).
alkaloids, Keller-Killiani test for digitoxose (2,6 dideoxy-
Results were expressed in mg catechin equivalent (CE) /
D-ribohexose) cardiac steroidal glycosides, Salkowski’s
100 g FM and in mg CE / 100 g DM.
test for terpenoids, frothing test for saponins, lead acetate
test for tannins, and alkaline reagent test for flavonoids.
Free Radical Scavenging Assay Using 2,2-diphenyl-
1-picrylhydrazyl (DPPH)
Determination of Total Phenolic Content (TPC)
The assay was based on the method described by Brand-
The method was based on the procedure described by
Williams et al. (1995) with modifications. Freshly
Singleton and Rossi (1965) with modifications. The
prepared 0.1 mM DPPH in MeOH (150 µL) was added
methanolic extract (from the 2.00 g edible portion + 50
to 50 µL of diluted methanolic fruit extract (10-fold). The
mL (80 % v/v) aqueous methanol), diluted 10-fold [10
solution was incubated at room temperature and in the dark
µL methanolic extract + 90 µL 80% (v/v) methanol] was
for 30 min. Absorbance at 515 nm was measured using
mixed with 500 µL distilled water and 250 µL of 10%
a microplate reader. The % radical scavenging activity
Folin-Ciocalteu reagent. After 3 min, 500 µL of (20% w/v)
was calculated using the equation: % radical scavenging
Na2CO3 was added. The mixture was mixed and incubated
activity = [(A515 Control – A515 Sample) / A515 Control] x 100%,
at 40 °C for 40 min. In a 96-well microplate, 250 µL of the
where A515 Control = absorbance of DPPH solutions without
mixture was loaded and the absorbance was measured at
fruit extract at 0 min, while A515 Sample= absorbance of
750 nm using Thermo Scientific Multi Scan Go (Thermo
DPPH solutions with fruit extract after 30 min. Ascorbic
Fisher Scientific Inc.) microplate spectrophotometer.
acid was used as a standard. Results were expressed as
Gallic acid was used to prepare a calibration curve (0–31
mg AEAC / 100 g FM.
mg/L, R2 = 0.9958). Results were expressed in mg GAE
/ 100 g FM and in mg GAE / 100 g dry matter (DM).

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Reducing Power Ability (RPA) Assay corrects for the color or turbidity of the fruit extract; and
The assay was based on the procedure by Oyaizu (1986) sample (NB, bacteria + fruit extract). The experiment
with modifications. A 50-µL aliquot of the methanolic was performed in triplicates for every concentration.
fruit extract, 250 µL of 0.2 M phosphate buffer pH 6.6, The mixtures were incubated at 37 °C for 20 h. After
and 250 µL of (1 % w/v) potassium ferricyanide were incubation, the optical densities (OD) at 600 nm were
mixed. The mixture was incubated at 50 °C for 20 min. measured. Percentage growth inhibition was calculated
Afterward, 250 µL (10 % w/v) trichloroacetic acid was as % Inhibition = [ODNegative – (ODSample- ODVehicle)]/
added and the mixture was centrifuged at 3,000 rpm for (ODNegative– ODPositive) x 100 %. Dose-response curves
10 min. In a 96-well microplate, 50 µL supernatant was were plotted. The MIC80 in mg GAE/mL growth medium
added with 50 µL distilled water and 100 µL (1 % w/v) determined from the curve was the concentration that
ferric chloride. The absorbance was measured at 700 nm. inhibited 80% of bacterial growth.
Ascorbic acid was used to prepare a calibration curve
(0–100 mg/L, R2 = 0.9988). Results were expressed as
Statistical Analysis
mg AAE / 100 g FM.
Results were expressed as mean ± standard deviation of
three replicates. Pearson correlation tests and the Tukey
Minimum Inhibitory Concentration (MIC80) and Dunnett post-tests to determine significant differences
Determination by Broth Microdilution Method in the group means were performed using GraphPad Prism
The broth microdilution method was performed based on (GraphPad Software, Inc.) with statistical significance set
Wiegand et al. (2008). The (80% v/v) aqueous methanolic at P < 0.05. The MIC80 for the antibacterial assays were
fruit extracts were concentrated using a rotary evaporator. determined from dose-response curves plotted in Sigma
To make stock solutions, extracts were dissolved in DMSO Plot 12.0.
(10% v/v of final volume), filter sterilized through a 0.22-
μm syringe filter, and diluted with sterile nutrient broth
(NB). The phenolic contents of the stock solutions were 32
mg GAE / mL, 64 mg GAE / mL, or 128 mg GAE / mL. RESULTS AND DISCUSSION
The assays were performed in 96-well microplates in the
manner prescribed by Wiegand et al. (2008). Wells were Phytochemical Screening
filled with 100 μL of sterile NB. At least 10 concentrations Qualitative tests could provide simple and rapid ways of
(from 0 until 16 mg GAE / mL, 32 mg GAE / mL, or 64 detecting certain phytochemical families in plant samples.
mg GAE / mL) were prepared through serial dilution: Results indicated that the selected indigenous fruits
adding 100 μL extract to the first well, mixing the 200 may contain a wide variety of phytochemicals (Table
μL mixture, and transferring 100 μL to the next well, etc. 2). All extracts were positive for phenolics, flavonoids,
Ten (10) μL of Escherichia coli or Staphylococcus aureus and tannins – indicating that these groups were the
suspension (approx. 108 CFU/mL) was added to each well. most widespread in plants. Alkaloids were detected in
Specific wells were designated: negative control (NB + C. ovatum pulp and C. ovatum nut. Cardiac glycosides
bacteria); positive control (NB, bacteria + 0.25 mg/mL were detected in F. pseudopalma, C. ovatum pulp, and
ampicillin); vehicle control (NB + fruit extract), which

Table 2. Phytochemical screening of the selected Philippine indigenous fruits.

Fruit Phenolics Flavonoids Tannins Alkaloids Cardiac Terpenoids Saponins
glycosides
Ficus ulmifolia + + + – – – +
Antidesmabunius + + + – – + +
Garcinia binucao + + + – – – –
Artocarpus altilis + + + – – – –
Citrus hystrix + + + – – – +
Ficus pseudopalma + + + – + – +
Mangifera altissima + + + – – – +
Canarium ovatum nut + + + + – – +
Canarium ovatum pulp + + + + + + +
Rubus rosifolius + + + – + + +
The symbols + and – indicate the detection (+) or non-detection (–) of a phytochemical constituent.

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R. rosifolius. Terpenoids were detected in A. bunius, C. The TFCs are shown in Table 3. The TFC values ranged
ovatum pulp, and R. rosifolius. Saponins were detected from 132–421 mg CE / 100 g FM, with the highest
in all the fruits except in G. binucao and A. altilis. These from F. pseudopalma. The fruits could be classified
present results could provide useful information prior to into categories: low (< 50 mg CE / 100 g FM), medium
more focused studies on specific phytochemical groups (50–250 mg CE / 100 g FM) and high (> 250 mg CE /
in these plants. 100 g FM) (Rufino et al. 2010). The high group includes
F. pseudopalma, F. ulmifolia, C. ovatum pulp, and G.
binucao, while the medium group includes R. rosifolius,
Total Phenolic and Flavonoid Contents
A. altilis, A. bunius, M. altissima, C. ovatum nut, and
In fruits, the phenolic contents can be indicators of
C. hystrix.
developmental stages and responses to environmental
factors, such as light and temperature (Macheix et al. Present results agreed with past studies indicating that
2018). Phenolics have various roles in growth, ripening, some of the fruits may contain medium to high levels
abscission of plant organs, lignification, as metabolic of phenolics and flavonoids. The TPC of the unripe M.
effectors, as protectors of cell structures, and in resistance altissima (“paho”) was within the range reported for
to biological stresses. Phenolic content can affect fruit mature mango varieties, M. foetida and M. odorata
quality such as color, flavor, and aroma (Macheix et (Ikram et al. 2009). The TPC of ripe A. bunius was close
al. 2018). Numerous studies on dietary polyphenols to the reported levels from ripe fruits (Recuenco et al.
suggested protective effects against chronic diseases based 2016; Ngamlerst et al. 2019). The TPC from the pulp
on evidence from controlled human intervention studies and juice of ripe C. hystrix may be higher compared to
(Del Rio et al. 2013). the reported TPC from C. hystrix juice (Abirami et al.
2014). Campbell et al. (2017) also reported high levels
Table 3 shows the TPCs of the selected fruits. The TPC
of phenolics from Rubus. Furthermore, Campbell et
ranged from 191–758 mg GAE/100 g FM basis, with the
al. (2017) identified ellagic acid as the predominant
highest from G. binucao. The fruits could be classified
phenolic and detected considerable quantities of
into categories: low (< 100 mg GAE / 100 g FM), medium
p-hydroxybenzoic acid, caffeic acid, quercetin,
(100–500 mg GAE / 100 g FM), and high (> 500 mg
kaempferol, and catechin.
GAE / 100 g FM) (Rufino et al. 2010). G. binucao, M.
altissima, F. ulmifolia, F. pseudopalma, and C. ovatum In some fruits, the degree of maturity could affect the
pulp belong to the high group; the rest – R. rosifolius, A. phenolic content and the observed antioxidant activities
bunius, C. hystrix, A. altilis, and C. ovatum nut – belong (Butkhup and Samappito 2011; Palafox-Carlos et al.
to the medium group. 2012). This study focused on only one stage of maturity

Table 3. Total phenolics, total flavonoids, and the radical scavenging, ferric reducing, and antibacterial activities of selected Philippine
indigenous fruits.
Sample % Total phenolics* Total Radical Ferric pH of E. coli S. aureus
moisture (mg GAE / 100 flavonoids* scavenging* reducing extract MIC80, MIC80
g FM) (mg CE / (mg AEAC / power * (mg (mg GAE
100g FM) 100 g FM) (mg AAE / GAE / / mL)
100 g FM) mL)
Ficus ulmifolia 71.1 ± 0.7 608 ± 22a 362 ± 7a 482.7 ± 4.2a 97.3 ± 3.5a 5 8.13 12.88
Antidesma. bunius 83.8 ± 0.4 278 ± 10b 161 ± 7b 361.0 ± 5.8a 44.8 ± 1.6b 6 2.00 8.71
Garcinia binucao 84.1 ± 0.6 758 ± 22c 312 ± 9c 479.7 ± 4.0a 121.1 ± 3.5c 3 2.00 1.33
Artocarpus altilis 77.6 ± 0.3 234 ± 5d 162 ± 7b 396.4 ± 5.9a 37.8 ± 0.9b 7 10.00 26.30
Citrus hystrix 83.6 ± 0.2 242 ± 4d 132 ± 7d 359.7 ± 2.7a 39.1 ± 0.6b 3 1.70 0.88
Ficus Pseudopalma 76.4 ± 0.4 530 ± 3e 421 ± 5e 497.0 ± 3.7b 84.9 ± 0.5d 7 2.40 0.56
Mangifera altissima 86.2 ± 0.2 694 ± 13f 161 ± 3b 516.0 ± 0.7b 110.8 ± 2.1e 3 2.24 1.66
Canarium ovatum nut 29.6 ± 0.8 191 ± 4g 144 ± 5d 82.0 ± 6.1a 31.1 ± 0.6b 7 15.50 13.34
Canarium ovatum pulp 64.3 ± 0.3 519 ± 6e 347 ± 4a 469.3 ± 3.3a 83.1 ± 1.0d 7 16.60 5.89
Rubus rosifolius 84.0 ± 0.6 475 ± 5h 231 ± 4f 512.5 ± 4.0b 76.2 ± 0.8d 3 1.70 1.00
*All measurements are reported as mean ± SD (n = 3). When followed by the same superscript, it would indicate that the means do not vary significantly. Legend:
GAE – gallic acid equivalent; CE – catechin equivalent; AEAC – ascorbic acid equivalent antioxidant capacity; AAE – ascorbic acid equivalent; FM – fresh matter;
MIC80 – minimum inhibitory concentration of extract that inhibits bacterial growth by 80%.

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per fruit species (Table 1). For a detailed understanding Antibacterial Activities of the Fruit Extracts
on the effects of degree of ripeness on the phytochemical Polyphenols could have antagonistic effects on the growth
content and various bioactivities, future studies could of foodborne pathogenic or food-spoiling bacterial strains
focus on one fruit species similar to the studies of Butkhup (Bouarab-Chibane et al. 2019). All fruit extracts exhibited
and Samappito (2011) and Palafox-Carlos et al. (2012). antibacterial activities against E. coli, a Gram-negative
bacterium, and S. aureus, a Gram-positive bacterium
(Table 3). The MIC80 values ranged from 0.56–26.30
Antioxidant Activities of the Fruit Extracts
mg GAE/mL. The MIC80 values against S. aureus were
Dietary phenolics or polyphenols are recognized for
generally lower compared to the values against E. coli
their possible roles in preventing chronic diseases such
except for F. ulmifolia, A. bunius, and A. altilis. This may
as cancers and cardiovascular diseases (Del Rio et al.
suggest that S.aureus was more sensitive compared to E.
2013). These diseases could develop due to oxidative
coli. The most effective against S. aureus were extracts
stress from increased levels of reactive oxygen species and
from C. hystrix and F. pseudopalma (MIC80 < 1.00 mg
free radicals that may damage cellular molecules such as
GAE / mL). Against E. coli, the most effective were
DNA, lipids, and proteins (Del Rio et al. 2013). Phenolics
extracts from C. hystrix and R. rosifolius (MIC80 values
and flavonoids could prevent such reactions by acting
~ 1.70 mg GAE / mL). For R. rosifolius, the MIC80 value
as antioxidants due to their capacity to act as hydrogen
was within the range from a previous report (Oliveira et
donors, reducing agents, and singlet oxygen quenchers.
al. 2016).
Present results showed that all the fruit extracts exhibited
Present results from the broth microdilution assay showed
antioxidant and reducing activities toward the DPPH
that as the concentrations of the fruit extracts increase,
radical and ferric species, respectively (Table 3). The
the antibacterial activities also increase. The Gram-
DPPH radical scavenging values ranged from 82.0–516.0
negative bacteria, E. coli, were shown to be more resistant
mg AEAC / 100 g FM. When arranged in increasing
while the Gram-positive bacteria, S. aureus, were more
activity, the order is: C. ovatum nut < C. hystrix < A.
susceptible to inhibition by the fruit extracts – similar
bunius < A. altilis < C. ovatum-pulp < G. binucao < F.
to those observed in Shan et al. (2007). Inhibition of
ulmifolia < F. pseudopalma < R. rosifolius < M. altissima.
bacterial growth could be due to fruit extract components
For the ferric RPA, values ranged from 31–121 mg AAE
that attack the bacterial cell wall and cell membrane and
/ 100 g FM. The arrangement in order of increasing RPA
cause leakage and coagulation of cytoplasmic components
is: C. ovatum nut < A. altilis < C. hystrix < A. bunius <
(Shan et al. 2007).
R. rosifolius < C. ovatum pulp < F. pseudopalma < F.
ulmifolia < M. altissima < G. binucao. We would like to believe that potential antibiotic
compounds may be present from the fruit samples tested,
The observed abilities to scavenge DPPH radicals by
especially from those which exhibited the lowest MIC80
extracts from A. bunius, A altilis, C. hystrix, G. binucao,
values – C. hystrix, R. rosifolius, and F. pseudopalma.
and R. rosifolius agreed with previous reports (Butkhup
Since the assays conducted here were simple and used
and Samappito 2011; Abirami et al. 2014; Barcelo
only two bacterial species, we suggest using more test
2015; Jalal et al. 2015; Oliveira et al. 2016; Campbell
organisms to identify specific targets of potential antibiotic
et al. 2017; Soifoini et al. 2018). However, numerical
compounds from these fruits.
values differed likely due to differences in experimental
conditions and other factors related to how the fruits
were grown, harvested, and stored (Tiwari and Cummins Correlation of Total Phenolics with Antioxidant and
2013). Here, we could only account for the contributions Antibacterial Activities
of phenolics and flavonoids to the observed activities. To determine whether the antioxidant, ferric reducing
The phytochemical screening also provided some insights power, and antibacterial activities of the fruit extracts
about other possible contributing components. Ascorbic could be attributed to their phenolic contents, pairwise
acid, tocopherols, and carotenoids – not quantified in correlation analyses were performed. The results are
this study – may also have significant contributions presented in Table 4.
(Hassimotto et al. 2005; Moon and Shibamoto 2009).
Antioxidant activities may be due to combinations There was a strong positive and significant correlation
of synergistic and antagonistic interactions between between total phenolics and total flavonoids (DM basis
different phytochemical components (Hassimotto et al. with Pearson’s r = 0.7232, P < 0.05). This suggests that
2005). Future studies should consider quantifying various flavonoids contribute significantly to the TPCs of the
compounds and performing additional assays for a more fruit samples. Such strong positive correlations had also
comprehensive measure of the antioxidant and/or metal- been observed in previous studies (Barreto et al. 2009;
reducing activities. Recuenco et al. 2016).

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Table 4. Pairwise correlation analysis among phenolics and little to no influence on the observed antibacterial
antioxidant and antibacterial activities of selected Philippine activities. Although phenolics had been reported to
indigenous fruits. exert antimicrobial activities, relationships between the
Correlation pairs Pearson’s r P-value (two- structural features and their interactions with bacteria
tailed) are not well-established (Bouarab-Chibane et al. 2019).
I. Phenolics FM The toxic effects on bacteria could be due to their ability
Paired with:
to modify the integrity of the cell wall, change the
A. Flavonoids FM 0.614 0.059ns permeability of the cell membranes, bind to enzymes,
B. % radical scavenging 0.739 0.015*
coagulate cell content, modify cellular pH, chelate
activity iron, and affect DNA and RNA synthesis (Bouarab-
C. Ferric RPA 1.000 < 0.0001***
Chibane et al. 2019). However, Bouarab-Chibane
et al. (2019) suggested that the effects of phenolics
D. pH of extract –0.444 0.198ns
could range from growth stimulation to antibacterial
E. MIC80 vs. E. coli –0.294 0.409ns activities. Due to the limitations of our methods, we
F. MIC80 vs. S. aureus –0.501 0.140ns could not identify specific components involved in
II. Phenolics DM the observed antibacterial activities. Further studies
are necessary to identify components and to elucidate
Paired with:
A. Flavonoids DM 0.721 0.019* mechanisms involved in the antibacterial activities of
the fruit extracts.
B. % radical scavenging 0.664 0.036*
activity
Some studies associated the observed antibacterial
C. Ferric RPA 0.849 0.002* activities of plant extracts to the presence of phytochemicals
D. pH of extract –0.711 0.021* and to the pH of their tissues (Friedman and Jürgens 2000).
E. MIC80 vs. E. coli –0.607 0.062ns Accordingly, we determined the pH of the fruit extracts
(Table 3) and analyzed correlations with phenolics and
F. MIC80 vs. S. aureus –0.560 0.092ns
antioxidant and antibacterial activities (Table 4).
*Significant correlations at *P < 0.05, ***P < 0.001; ns – not significant; FM –
fresh matter; DM – dry matter. Fruit acidity, as measured by pH and/or titratable acidity,
is mainly due to organic acids such as citric and malic
The pairwise analyses of the total phenolics with the acids (Tomotake et al. 2006). Here, the strong negative
radical scavenging activities and reducing power abilities correlation (r = –0.711, P < 0.05) between TPC (DM
all gave strong and statistically significant correlations basis) and the pH of the fruit extracts may indicate an
with Pearson’s r values 0.6641–1 (P < 0.05). Similarly, inverse relationship, i.e. the higher level of phenolics
pairwise analysis of the total flavonoids (DM) with the may be related to more acidity of the extract. Phenolics
radical scavenging activities also gave a strong and may contribute to fruit acidity due to the weakly acidic
statistically significant correlation with Pearson’s r properties of the phenol group, and also due to carboxylic
value ~ 0.79 (P < 0.05). These positive correlations were acid groups found in phenolic acids like hydroxybenzoic
similar to the findings of Barreto et al. (2009). These may acids and hydroxycinnamic acids. However, the extent
suggest that phenolics and flavonoids contribute directly of contributions to the pH – as well as factors affecting
to the observed radical scavenging and ferric reducing changes in pH such as changes in maturity – could not be
activities of the fruit extracts. However, some studies determined from our data.
found no correlation or inverse correlation between
phenolics and antioxidant activities (Hassimotto et al. The correlations between the pH of the extracts and the
2005; Ikram et al. 2009). Ikram et al. (2009) suggested MIC80 values were found to be strongly positive (against
that the antioxidant activities of plant extracts could not E. coli, r = 0.679, P < 0.05), suggesting a strong direct
be attributed solely to the phenolic content. In the future, relationship. The low MIC80 values (high antibacterial
other components that might be capable of exerting activities) may have been strongly influenced by the low
antioxidant and reducing activities such as ascorbic acid, pH of the extracts – especially observed from the most
carotenoids, tocopherols, minerals, and proteins should acidic fruits, G. binucao, C. hystrix, M altissima, and
also be considered (Ikram et al. 2009). R. rosifolius. In citrus fruits, lemon and lime, citric acid
was said to be responsible for the observed antibacterial
The correlations of phenolic contents to the antibacterial activity (Tomotake et al. 2006). Moreover, the lower
activities (as MIC 80 values) were found to be not pH in plant extracts may be necessary for maintaining
statistically significant (Table 4), different from the the stability and antioxidant and antibacterial activities
strong correlations reported in Shan et al. (2007). of some naturally-occurring phenolics (Friedman and
This could indicate that phenolic contents may have Jürgens 2000). Further investigation is needed to establish

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Vol. 149 No. 3, October 2020 Activities of Philippine Fruits

whether there are any synergistic interactions between the BRAND-WILLIAMS W, CUVELIER ME, BERSET C.
major organic acids and the phenolics associated with the 1995. Use of a free radical method to evaluate anti-
antibacterial activities of the selected fruits. oxidant activity. LWT-Food Sci Technol 28(1): 25–30.
BUTKHUP L, SAMAPPITO S. 2011. Changes in physico-
chemical properties, polyphenol compounds and an-
tiradical activity during development and ripening of
CONCLUSION maoluang (Antidesma bunius L. Spreng) fruits. J Fruit
The data obtained from the present study suggest that the Ornam Plant Res 19(1): 85–99.
selected Philippine indigenous fruits contain a variety
CAMPBELL TF, MCKENZIE J, MURRAY J, DELGO-
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DA R, BOWEN-FORBES CS. 2017. Rubus rosifolius
flavonoids, and antioxidant and antibacterial species.
varieties as antioxidant and potential chemopreventive
Therefore, more detailed studies that identify specific
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understanding of how the phytochemicals from these edible wild fruits in Benguet, Cordillera administra-
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APPENDIX of F. pseudopalma and F. ulmifolia by employing NMR

spectroscopy. C. hystrix peel ethyl acetate extract was
reported to contain a wide variety of coumarins, terpenes,
Notes on the Phytochemical Screening of Selected
flavonols, and glycosides (Seeka et al. 2016).
Philippine Indigenous Fruits
Alkaloids perform functions related to protection from
parasites, and from insects and animals (Cordell et al. Comments on the Selected Antioxidant and
2001). Alkaloids had found applications in medicine and Antibacterial Assays
have a wide variety of physiological effects. The positive It was recommended that multiple assays be used to
reaction of C. ovatum’s nut and pulp extracts was in evaluate the antioxidant activities of plant extracts (Moon
agreement with the study of Salvador-Membreve et al. and Shibamoto 2009). Palafox-Carlos et al. (2012)
(2018). Some alkaloid compounds had been isolated from stated that the best combination of antioxidant assays for
the roots of C. hystrix (Panthong et al. 2013). However, different fruits was Trolox equivalent antioxidant capacity
our test failed to detect alkaloids from the edible portion or ferric reducing antioxidant power (FRAP), and the
of C. hystrix. DPPH assay. Here, we chose the DPPH assay, and the
reducing power ability (RPA) assay as an alternative to
The Keller-Kiliani test detects cardiac glycosides the FRAP assay. The DPPH assay’s mechanism could be a
containing the sugar digitoxose. Three fruit samples gave mix of hydrogen atom transfer and single electron transfer
positive results: F. pseudopalma, C. ovatum (pulp), and R. (SET) (Prior et al. 2005). The FRAP assay’s mechanism
rosifolius. Our result for C. ovatum pulp agreed with that could be SET, where a donor transfers an electron to the
of Cajuday et al. (2017), while the result for R. rosifolius ferric ion in complex with the probe 2,4,6-tripyridyl-s-
differed from that of Campbell et al. (2017). triazine (Prior et al. 2005). The RPA assay has the same
Terpenoids are considered the most diverse group of plant key reaction as FRAP but it is monitored through the
secondary metabolites with at least 40,000 structures formation of the green to blue-colored ferric ferrocyanide
identified (Tholl 2015). Volatile or semi-volatile, complex, Fe(III)4 [(Fe(II)(CN)6)]3.
low molecular weight isoprenes, mono-, sesqui-, and At present, there is no standardized assay or universal
diterpenoids in plants serve as attractants of pollinators method to assess for antioxidant activity (Palafox-
and as repellents against herbivores (Tholl 2015). Carlos et al. 2012), and there seemed to be no strict
Nonvolatile terpenoids in the roots may be involved in rules regarding combinations and manner of expressing
defense against competing plants and for signaling root results (Moon and Shibamoto 2009). In the DPPH assay,
development (Tholl 2015). The positive results for A. some ways of expressing the results are % inhibition or
bunius fruit, C. ovatum pulp, and R. rosifolius were in % quenching (Molyneux 2004), IC50 or EC50 (Brand-
agreement with previous reports (Ragasa et al. 2015; Williams et al. 1995), and AEAC (Leong and Shui 2002).
Campbell et al. 2017). C. ovatum nut oil was reported Results as IC50 or EC50 had a drawback of having to relate
to contain terpenoid derivatives (Pham and Dumandan the lower values to higher antioxidant activity (Molyneux
2015). However, our screening failed to detect terpenoids 2004). We chose to report the DPPH activities in terms
in C. ovatum nut. The negative results for A. altilis and of equivalence with AEAC, similar to the study of Leong
G. binucao also did not agree with previous reports and Shui (2002). Since the exact composition of the
(Amarasinghe et al. 2008; Ragasa et al. 2014b). fruit extracts was not known, using AEAC allows some
Saponins are a group of amphipathic molecules that means of relating the structures, reaction mechanisms,
contain a glycoside moiety and a triterpene or steroid and stoichiometries to the observed radical scavenging
moiety. They may serve as antifeedants against animals activities (Molyneux 2004).
and protection against pathogens such as microbes and In this study, the broth microdilution assay was employed
fungi (Hussain et al. 2019). Saponins were detected in instead of the disk diffusion assay for the antibacterial
all samples, except in G. binucao and in A. altilis. For R. assay. The disk diffusion assay may not always be suited
rosifolius, our positive result differed from the negative when testing plant extracts due to polarity differences of
result reported by Campbell et al. (2017). phytochemical components in the agar media (Klančnik
Different plant parts may vary in their phytochemical et al. 2010). To allow better distribution of both polar
content. Here, where only the edible portions were and nonpolar components, we chose liquid media over
used, some phytochemical families were not detected. solid or semi-solid agar media. Furthermore, the broth
Some previous studies using the same plant species had microdilution assay was reported to be more sensitive
detected and/or identified phytochemical compounds from than agar diffusion methods and may be more suitable in
another part such as leaves, roots, and peel. Ragasa et al. determining quantitatively the antimicrobial activity of
(2009) identified terpenoids and sterols from the leaves plant extracts (Klančnik et al. 2010).

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Additional References OBICO JJA, RAGRAGIO EM. 2014. A survey of plants

ARENAS EH, TRINIDAD TP. 2017. Fate of polyphenols used as repellents against hematophagous insects by
in pili (Canarium ovatum Engl.) pomace after in vitro the Ayta people of Porac, Pampanga province, Philip-
simulated digestion. Asian Pac J Trop Biomed 7(1): pines. Philipp Sci Lett 7(1): 179–186.
53–58.
PRIOR RL, WU X, SCHAICH K. 2005. Standardized
CAJUDAY LA, MEMBREVE, DMS, SERRANO JE. methods for the determination of antioxidant capacity
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activities of Canarium ovatum (Burseraceae) pulp Food Chem 53(10): 4290–4302.
extracts. Int J Biosci 11(3): 247–256.
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CORDELL GA, QUINN-BEATTIE ML, FARNSWORTH TIANSEN HPBC, SHEN CC. 2015. Triterpenes and
NR. 2001. The potential of alkaloids in drug discovery. acylglycerols from Canarium ovatum. J Appl Pharm
Phytother Res 15(3): 183–205. Sci 5(4): 94–100.
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ISLAM W, HAMEED MS, WANG L, QIU D. 2019. RANO JE, BALDO DEB. 2018. Immunomodulatory
Role of saponins in plant defense against specialist properties of ethanol extract of Canarium ovatum
herbivores. Molecules 24(11): 2067. Burseraceae pulp. Trop J Pharm Res 17(8): 1565–1569.
KLANČNIK A, PISKERNIK S, JERŠEK B, MOŽINA THOLL D. 2015. Biosynthesis and biological functions
SS. 2010. Evaluation of diffusion and dilution methods of terpenoids in plants. Adv Biochem Eng Biotechnol
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MOLYNEUX P. 2004. The use of the stable free radical
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oxidant activity. Songklanakarin J Sci Technol 26(2):
211–219.

Appendix Table I. Previous studies on the selected Philippine indigenous fruits.

Scientific name, local name Place of collection Plant part; information reported Reference
(family)
Ficus ulmifolia Lam. Philippines Leaves; terpenoids and sterols Ragasa et al. (2009)
“As-is”
Pampanga, Phil. Leaves and stems; ethnobotanical survey Obico and Ragrario (2014)
(Moraceae)
and insect repellant properties
Antidesma bunius (L.) Spreng Thailand Fruits; physicochemical properties, Butkhup and Samappito (2011)
“Bignay” polyphenols, antiradical activity
(Phyllanthaceae)
Philippines Fruits; phenolic content, antioxidant and Lizardo et al. (2015)
antimicrobial properties
Thailand Fruits; anthocyanidin, polyphenol, Ngamlerst et al. (2019)
flavonoid contents, animal feeding studies
Garcinia binucao (Blco.) Choisy Leyte, Phil. Fruits; proximate and mineral Quevedo et al. (2013)
“Binukaw,” “Batuan” composition, physicochemical properties,
(Clusiaceae) vitamin A, C, tannin content, sensory
properties
Iloilo, Phil. Fruits; sterols and triglycerides Ragasa et al. (2014b)

Benguet, Phil. Fruits; phytochemical screening, total Barcelo (2015)

phenolics, flavonoids, radical scavenging
activities

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Artocarpus altilis (Park.) Fosb. Sri Lanka Fruit; identification of compounds, e.g. Amarasinghe et al. (2008)
“Kamansi” stilbenes, flavones, prenylated flavanols,
(Moraceae) benzofurans

Malaysia Whole fruit, pulp; total phenolics, Jalal et al. (2015)

flavonoids, antioxidant, antimicrobial
activities
Comoros Fruits; phenolic compounds, antioxidant Soifoini et al. (2018)
activity, vitamin C, lipids, fiber,
carbohydrates
Citrus hystrix DC. Thailand Roots; isolation and identification of Panthong et al. (2013)
“Kolong-kolong” coumarins, benzenoid and alkaloids,
(Rutaceae) antioxidant, anti-HIV and antibacterial
activities
India Fruit juice; total phenolics, tannins, Abirami et al. (2014)
flavonoids, antioxidant and enzyme
inhibitory activities
Thailand Peels; isolation and identification 16 Seeka et al. (2016)
compounds including coumarin-flavanol-
glucoside conjugates, anti-cholinesterase
activity
Ficus pseudopalma Blco. Camarines Sur, Phil. Leaves; ethnobotanical survey and Santiago et al. (2014)
“Niyog-niyogan” nutritional composition
(Moraceae)
Camarines Sur, Phil. Leaves; triterpenes Santiago and Mayor (2014)

Laguna, Phil. Leaves; cytotoxicity and apoptotic De las Llagas et al. (2014)
activities
Mangifera altissima Blco. Philippines Morphological characterization Coronel et al. (2003)
“Paho”
(Anacardiaceae)
Canarium ovatum Engl. Philippines Leaves, stem, mesocarp, kernel; chemical Ragasa et al. (2015)
“Pili” constituents
(Burseraceae)
Philippines Fruits; fatty acid composition, carotenoids, Pham and Dumandan (2015)
tocopherols, sterol contents
Philippines Pomace; fate of polyphenols after in vitro Arenas and Trinidad (2017)
digestion
Rubus rosifolius Sm. Jamaica Fruits; anthocyanins isolation and Bowen-Forbes et al. (2010)
“Sampinit” identification, lipid peroxidation, COX,
(Rosaceae) tumor cell proliferation inhibitory assays
Brazil Fruits; phenolic content, nutritional Oliveira et al. (2016)
composition, antioxidant and antimicrobial
activity.
Jamaica Fruits; phytochemical screening, Campbell et al. (2017)
phenolics, antioxidant and enzyme
inhibitory assays

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Appendix Figure 1. Philippine indigenous fruits used in this study. The fruits were collected
from Laguna, Quezon, Batangas, and Negros provinces in the Philippines.

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