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Evaluation of the antioxidant and anticancer activities of Canarium ovatum

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 Int. J. Biosci. 2017

International Journal of Biosciences | IJB |

ISSN: 2220-6655 (Print) 2222-5234 (Online)
http://www.innspub.net
Vol. 11, No. 3, p. 247-256, 2017

RESEARCH PAPER OPEN ACCESS

Evaluation of the antioxidant and anticancer activities of

Canarium ovatum (Burseraceae) pulp extracts
Lilibeth A. Cajuday*, Daile Meek S. Membreve, Jocelyn E. Serrano

Biology Department College of Science, Bicol University Legazpi City, Philippines

Key words: Antiangiogenic, Anticancer, Antioxidant, Canarium Ovatum, Cytotoxicity, Phytochemical screening.

http://dx.doi.org/10.12692/ijb/11.3.247-256 Article published on September 30, 2017

Abstract
The study evaluated the antioxidant and anticancer activities of Canarium ovatum pulp. Mechanical and
ethanolic extractions were done to prepare the oil (COPO) and ethanolic (COPE) extracts from the C. ovatum
pulp. The extracts were used to screen for the biological activities and phytochemical components. The
antioxidant level was evaluated using the DPPH (2, 2-diphenyl-1-picrylhydrazyl) and FRAP (ferric reducing
ability of plasma) assays. The CAM (chorioallantoic membrane) and sea urchin fertilization assays were used to
test for anticancer activity and cytotoxicity. Results of the DPPH and FRAP assays showed high reducing power
and free radical scavenging activities of COPE compared to COPO and ascorbic acid. CAM assay revealed a
significant inhibition (p=0.05) in blood vessel formation in all the COPE-treated samples only compared to the
vehicle control. In the sea urchin fertilization assay, the inhibitory effect of COPE was observed in the medium
(1mg/ml) and high (10mg/ml) treatments where there were significant reductions (p=0.05) in the percentage of
fertilized egg compared with the control. The lowest concentration (0.1mg/ml) of COPE was inactive in this
assay. The computed IC50 for COPE was 0.369mg/mL. Phytochemical screening of the different extracts revealed
the presence of bioactive constituents such as alkaloids, flavonoids, glycosides, saponins, sterols, tannins and
triterpenes. Further studies be done to validate the results and elucidate mechanism of action of the bioactive
components present in the extracts.
* Corresponding Author: Lilibeth A. Cajuday  [email protected]

247 Cajuday et al.

 Int. J. Biosci. 2017

Introduction scavenging or antiradical activity (Tirzitis and

Canarium ovatum Engl., indigenous in the Bartosz, 2010). The FRAP assay is a simple and rapid
Philippines and commonly known as pili is a nut colorimetric assay which is based on reducing ferric
producing tree of the family Burseraceae ion, where antioxidants are the reducing agent
(Brown,1954). It is known to have high economic (Rabeta and Faraniza, 2013).
value due to its edible kernel nut which has many
uses (Coronel, 1996). Whereas, the pili pulp is usually Plants have been the most significant source of
discarded off as a waste or consumed as vegetable bioactive compounds currently used for cancer
dish. Studies conducted on Canarium ovatum treatment. In many countries around the world,
focused on the phytochemical components of its oil medicinal plants constitute a common alternative for
(Kakuda et al., 2000; Madamba et al., 1991, Pham et al., cancer treatment (Tascilar et al., 2006). Currently,
1998). However, the biological activities of C. ovatum there are 2 plant-derived compounds being tested in
are not well documented. To date what are known are clinical trials that have been shown to exhibit
the antioxidant property of its nuts and leaves (Zarinah anticancer effects with lesser toxicity than
et al., 2014; del Rosario, 2009) and its promising conventional drugs: flavopiridol, isolated from the
antimutagenic property: decreasing the number of Indian tree Dysoxylum binectariferum, and
micronucleated polychromatic erythrocytes of ICR mice meisoindigo, isolated from the Chinese plant
(Chichioco-Hernandez and Paguigan, 2009). Recently, Indigofera tinctoria (Saklani and Kutty, 2008;
18 known terpenoids have been isolated from the Solowey et al., 2014).
methanol extract of C. ovatum resin (Kikuchi et al.,
2012) while the dichloromethane extracts of the leaves There are several methods to test for the anticancer
afforded triterpenes and acylglycerols (Ragasa et activity and cytotoxic potential of a natural product.
al.,2015). Previous studies on related species of C. Angiogenesis, growth of new network of blood
ovatum showed compounds with various biological vessels, is one of the hallmarks of cancer. Currently,
activities which includes antimicrobial, antioxidant, several synthetic angiogenesis inhibitors have been
antitumor, anti-inflammatory, anti-diabetic, produced, but few studies have looked into natural
hepatoprotective and analgesic property (Mogana and sources of these compounds. The chick chorioallantoic
Wiart, 2011; Sharkirin et al., 2012). membrane (CAM) assay has been widely used to study
angiogenesis (Richardson and Singh, 2003) tumor cell
Since plants are known to be a source of antioxidants, invasion and metastasis (Tufan and Satiroglu-Tufan,
the list of plants with antioxidant activity is 2005; Deryugina and Quigley, 2008). The control of
increasing. Recently, Canarium album, C. proliferation of cancer cells without harmful side
odontophyllum and C. patentinervium; related effects to normal cells of the patient remains a major
species of C. ovatum, have been reported to exhibit goal in the continuing search for improved methods of
antioxidant activities (Mogana and Wiart, 2011). treating cancer (Bojo et al., 2010). The sea urchin
Antioxidant capacity describes the ability of redox fertilization assay (Semenova et al., 2006) is a practical
molecules to scavenge free radicals in the biological and popular cell viability tests to evaluate the cytotoxic
systems (Floegel et al., 2011). DPPH (2,2-diphenyl-1- potential of a compound.
picrylhydrazyl) and FRAP(ferric reducing ability of
plasma) assays are antioxidant assays that are based For a long period of time, plants have been a valuable
on electron transfer and involves reduction of colored source of natural products for maintaining human
oxidants (Floegel et al., 2011). The scavenging activity health, especially in the last decade with more
against DPPH free radical has been extensively used intensive studies on natural products (Selmavohan et
to evaluate antioxidant activity of plant extracts. It al., 2012). Thus, it is important to identify new
characterizes the ability of compounds to react with natural sources of safer bioactive components such as
free radicals, giving information on the radical those found in pili pulp. In the Bicol Region, the pili

248 Cajuday et al.

 Int. J. Biosci. 2017

plant has a great potential for development not only Determination of antioxidant activity
as a major export crop but also as a possible source of The antioxidant activity of different extracts of C.
useful compounds with medicinal value. Literature ovatum pulp were investigated using FRAP and
search revealed limited information on the biological DPPH. For FRAP assay, the antioxidant activity was
activities of the plant. Available data present the evaluated using DetectX® Assay Kit. Briefly, 20µL of
antioxidant action of the roasted pili nut oil by DPPH extracts were allowed to react with FRAP solution for
assay (24.66% at a concentration of 140ug/ml) thirty minutes in the dark condition. Readings of the
(Zarinah et al., 2014) and angiosuppressive effect of the colored product at 595 nm absorbance were then
pili leaves using CAM assay (Chan and Cajuday, 2013). taken using a microplate reader. Standard aqueous
Thus, this study aims to provide information on the solution of ferrous sulphate was used for the
medicinal potential of pili pulp. Knowledge of these calibration curve and results were expressed in µM.
biological activities is significant to its utilization and the DPPH assay was done according to the method of
possible introduction to industrial and pharmaceuticals Mensor et al. (2001) with minor modifications. One
production. Investigating the bioactivities of pili pulp ml of 0.3mM DPPH ethanol solution was added to
may provide information regarding its alternative use 2.5ml of different of different concentrations and was
and additional economic value. allowed to react at room temperature. Ethanol
(1.0mL) plus plant extract solution (2.5ml) was used
Materials and methods as a blank while DPPH solution plus ethanol was used
Pili pulp collection and extraction as a negative control. After 30 minutes, the
Canarium ovatum (pili) fruits were obtained from the absorbance values were measured at 518 nm and
breeding farm of Pili Research and Technology calculated into percentage antioxidant activity (%
Development Center (PRTDC, Albay Philippines) and scavenging) based on the formula below (Rajesh and
transported to Bicol University College of Science Natvar, 2011):
Laboratory for the extraction process. In the 𝐴𝑏𝑠 𝑆𝑎𝑚𝑝𝑙𝑒−𝐴𝑏𝑠 𝐵𝑙𝑎𝑛𝑘 𝑥 100
% 𝑆𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 =
𝐴𝑏𝑠 𝐶𝑜𝑛𝑡𝑟𝑜𝑙
laboratory, the pili fruits were decorticated to obtain
the pulp. For the ethanolic extraction of the pili pulp,
the collected pulp samples were washed, air- dried Test for anticancer activity

and soaked in 95% ethanol in 1:3 ratio (w/v) for 48 Chorioallantoic membrane (CAM) assay

hours. Thereafter, the mixture was filtered and A total of fifty six (56) pieces of fertilized duck eggs

evaporated to dryness using a rotary evaporator. The (Anas platyrynchos L) were used for the assay. The

dried Canarium ovatum pulp ethanolic (COPE) eggs were obtained from a local breeder in Polangui

extract was weighed and set aside for use in the Albay and transported to Bicol University College of

different assays. Mechanical extraction of the pili pulp Science Laboratory for random sorting and

to obtain the oil extract was done according to the Pili incubation prior to treatment with varying
Oil Extraction Guide of Department of Science and concentrations of COPE and COPO extracts. Day 0
Technology-V (Asuncion, 2006). Briefly, pili pulp eggs were wiped clean, randomly sorted into 8 groups
samples were washed, air-dried and soaked in (n=7) and incubated at 37.5C with a constant
distilled water at 1:1 ratio for 48 hours. Thereafter, humidity. At day 3, the eggs were administered with
the concentrate was filtered and the filtrate set aside 0.2mL of the 25, 50, and 75% COPO and 0.1, 1, and
in a glass cylinder until the oily component occupies 10mg/ml COPE. For the control groups, corn oil and
the topmost layer. Then the oil was separated from distilled water were used respectively. After 7 days of
the aqueous layer by gentle aspiration. The oil incubation, the eggs were harvested and a window
aspirate was then heated under 40°C until the was made for each egg by cutting the upper side of the
aqueous liquid extract evaporated and served as the shell to expose the developing embryo contained in
C. ovatum pulp oil extract (COPO). The obtained the CAM. The proliferation of the extra embryonic
extracts were set aside for use in the different assays. blood vessels for the treatment groups was compared

249 Cajuday et al.

 Int. J. Biosci. 2017

with the control group in terms of the number of a hydrogen atom (Duan et al., 2007) using FRAP assay
branch point, primary blood vessel (PBV), secondary was investigated. It showed that C. ovatum pili pulp oil
blood vessel (SBV), and tertiary blood vessel (TBV). (COPO) had lower antioxidant capacity (133µM)
whereas the COPE (542.56µM) showed significantly
Sea urchin fertilization assay higher value in comparison to ascorbic acid (258.98
The sea urchins (Tripneustes gratilla) collected from µM), a known potent antioxidant (Fig. 1).
Albay Gulf were induced to spawn by injection of 2 ml
A similar result was also obtained using DPPH assay
0.5M KCl in the laboratory. Prior to the co-culture of
wherein COPE demonstrated the highest scavenging
the male and female gametes, 2ml of the different
activities against DPPH free radical (Table 1). The
concentrations of COPE (0.1, 1.0, 10mg/ml) were first
scavenging activities of the extract is significantly
added into the well-plate with 3ml filtered seawater.
higher compared to ascorbic acid. Free radical
Then, 0.02ml of sperm suspension was added in each
scavenging activity is the most important mechanism
well. After 10min exposure, 0.2ml of egg suspension
by which antioxidants inhibits lipid peroxidation (Hu
was added. The eggs were allowed 20 min for
et al., 2014). Lipid peroxidation is the result of the
fertilization before fixation with 10% formalin.
excessive accumulation of ROS due to the altered
Subsamples of eggs from each treatment were
balance between ROS generation and elimination in
subsequently evaluated for fertilization success by
organism (Hu et al., 2014). However, negative values
noting the presence or absence of fertilization
were obtained for COPO extract in the same assay.
membrane. One hundred eggs or zygotes were
Yet, this does not indicate that COPO does not have
counted for each concentration of test substance to
obtain the percentage of normal cells. antioxidant activity since FRAP assay has shown that
COPO also exhibited antioxidant activity (Fig. 1).

Phytochemical Screening Table 1. DPPH free radical scavenging activity of C.

The different extracts were submitted to Industrial ovatum pulp extracts.
Technology Development Institute (ITDI)
Extract Treatment % Scavenging
Department of Science and Technology (Taguig, activity
Metro Manila) for qualitative phytochemical analysis. COPE 0.1 mg/ml 30.30±0.211
1 mg/ml 190.55±6.41
10 mg/ml 1406.34±275.83*
Statistical Analysis COPO 25% -143.63±52.31
Results were expressed as the mean ± standard error 50% -10.12±25.23
75% -134.23±81.06
of means. Comparisons of means were analyzed by
100% -102.19±65.49
one-way analysis of variance (ANOVA) to determine Ascorbic acid 100% 30.62±8.63
inter group differences. If the results of the ANOVA *significantly different compared to all extract-treatments;
were significant (p≤0.05), Tukey’s honestly **Significantly different compared to 25%, 75%, 100% COPO
significant difference (HSD) was applied to the data
to compare the treated groups with control group
using SPSS software. The IC50 value of the extract to
inhibit fertilization in sea urchin was computed using
the IC50 Tool Kit (http://ic50.tk/)

Results and discussion

Test for antioxidant activity
Various plant extracts have been investigated as Fig. 1. Antioxidant capacity of C. ovatum extracts by
potential sources of antioxidant compounds. In this reducing the amount of FeCl2. Values are expressed
study the reducing power, which exerts antioxidant as means ±SEM, P<0.05. Different letters indicate
action by breaking the free radical chain and donating significantly different from each other (p≤0.05).

250 Cajuday et al.

 Int. J. Biosci. 2017

The high antioxidant capacity of C. ovatum pulp vessels of the embryo in all the treated groups
shown in this study is in agreement with the result of compared to control group (Table 2). However,
del Rosario (2009) wherein the pulp of C. ovatum has indications of blood vessel obstructions such as ghost
revealed good antioxidant property; exhibiting an vessel formation, hyperemia and petechial
antioxidant activity that ranged from 70.83% to hemorrhage were observed in the vascular area of
87.50% inhibition in lipid peroxidation assay, 50% and 75% COPO treated samples (Fig. 2). On the
reducing power ranges from 91.02 to 96.50% and has other hand, the chorioallantoic membrane (CAM)
shown 60.56% to 85.2% radical scavenging activity in angiogenesis of the embryos administered with
deoxyribose system assay. In that study, it was also Canarium ovatum ethanolic extract (COPE) recorded
reported that the pulp contains phenolic compounds significant decreases in blood vessel count compared to
such as cyanidin and ferulic acid. Phenolic the blood vessels of the embryos given only with distilled
compounds are recognized to exhibit antioxidant water (Table 3). Both the medium (1mg/ml) and high
properties, a high statistical correlation between free (10mg/ml) doses of COPE significantly inhibited the
radical scavenging activity and total phenolic content growth of primary blood vessels. The medium dose (1
has been accounted by earlier works (Hu et al., 2014, mg/ml) of COPE consistently minimized the growth of
Maurya et al., 2014). secondary and tertiary blood vessels as well as the
branch point number. Results were significantly
Test for Anticancer Activity different compared to the control group. Similar results
The Canarium ovatum pulp oil (COPO) did not cause were observed by Chan and Cajuday (2013), using the
significant decreases in the growth of the blood aqueous leaf extract of C. ovatum.

Table 2. Comparison on the Growth of Blood Vessels in the Different Treatments of COPO.
Treatment PBVns SBVns TBVns BPN ns
Corn oil 2.78±0.22 16.56 ±1.17 86.44 ± 6.17 107.67 ± 8.74
25 % COPO 2.55±0.17 14.78 ±1.83 85.00 ± 6.90 102.00 ±7.44
50 %COPO 2.44± 0.29 15.11 ±1.44 76.00 ± 5.83 92.89 ± 7.53
75 % COPO 2.11± 0.20 12.11 ±1.47 77.77 ± 5.21 91.78 ± 6.24
Values are expressed as means ± standard error of the mean (SEM). n=7. COPO= C. ovatum pulp oil; PBV=
primary blood vessel ; SBV= secondary blood vessel; TBV= tertiary blood vessel; BPN= Branch point number.

Table 3. Comparison on the Growth of Blood Vessels in the Different Treatments of COPE.
Treatment PBV SBV TBV BPN
Distilled water 3.00±.3651 18.00±1.632 102.83±8.235 121.50±9.351
0.1 mg/ml COPE 2.08±.0833 13.83±0.542 78.00±4.647 91.83±5.166
1 mg/ml COPE 1.33±.4216** 10.00±2.25** 52.66±18.478** 75.83±17.654*
10 mg/ml COPE 1.66±.3333* 14.16±0.542 83.16±4.415 97.33±4.835
Values are expressed as means means ± standard error of the mean (SEM). n=7.COPE= C. ovatum pulp ethanolic
extract; PBV= primary blood vessel; SBV= secondary blood vessel; TBV= tertiary blood vessel; BPN= Branch
point number*p≤0.05, **p≤0.01 significantly different with control.

The antiangiogenic activity of the COPE is further endothelial growth factor receptor (EGFR) (Mojzis et
manifested through morphological examinations of al., 2008; Dai et al., 2013) and triterpenes via
the vascular area of the blood vessels (Fig. 3). VEGFR2-mediated Jak2–STAT3 signaling pathway
Indications of blood vessel obstruction (e.g. ghost (Dong et al., 2010; Zhu et al., 2016). Kaempferol, a
vessel formation, hyperemia and petechial dietary flavonoid is effective in reducing vascular
hemorrhage) were observed in all the COPE-treated endothelial growth factor (VEGF) expression in
samples. The data presented herein, confirm the ovarian cancer cells. It enhances the effect of cisplatin
presence of angiosuppressive components in the pulp through downregulation of cMyc in promoting
ethanolic extract. Specifically, flavonoids that can act apoptosis of ovarian cancer cells (Luo et al., 2010).
as angiogenic inhibitor by regulating the expression of Recently, triterpenes isolated from a Chinese Herbal
VEGF, matrix metalloproteinases (MMPs) and Medicine Actinidia chinensis Planch exhibited anti-

251 Cajuday et al.

 Int. J. Biosci. 2017

tumor action via inhibition of tumor angiogenesis (1mg/ml) and high (10 mg/ml) treatments where
using human umbilical vein endothelial cells there were significant reductions in the percentage of
(HUVEC) (Zhu et al., 2016). Interestingly, fertilized egg compared with the control. The lowest
phytochemical analysis of COPE reported the concentration (0.1mg/ml) of COPE was inactive in
presence of flavonoids and triterpenes which are not this assay. Fertilization was inhibited by 42% and
present in the COPO. 38% in the gametes exposed to 10 mg/ml and 1
mg/ml COPE respectively with the highest
concentration as the most active in terms of %
fertilization inhibition, indicating the presence of
possible cytotoxic compounds. The computed IC50 for
COPE is 0.369mg/mL (Fig. 5).

Fig. 2. CAM angiogenesis of the duck embryos given

with (A) corn oil and COPO at (B)25%, (C)50%, and
(D) 75% concentrations. With indication of blood
vessel obstructions forming ghost vessels (green
arrow), hyperemia (blue arrow) and petechial
hemorrhage (yellow arrow). 10X.
Fig. 3. CAM angiogenesis of the duck embryos given
The cytotoxicity of the different concentrations of the with (A) distilled water and COPE at (B) 0.1 mg/ml
COPE extracts on sea urchin eggs was manifested in (C) 1 mg/ml, and (D) 10 mg/ml concentrations. With
terms of inhibition of fertilization. However, only indication of blood vessel obstructions forming ghost
moderate cytotoxicity was observed in the results. Fig. vessels (green arrow), hyperemia (blue arrow) and
4 shows the inhibitory effect of COPE in the medium petechial hemorrhage (yellow arrow). 10X.

Fig. 4. Percent fertilization of the sea urchin eggs exposed to different concentrations of COPE compared with control.
Values are expressed as mean ± SEM. Different letters indicate significantly different from each other (p≤0.05).

252 Cajuday et al.

 Int. J. Biosci. 2017

Fig. 5. Fertilization inhibition curve. IC50 computed by online curve- fitting using IC50 Tool Kit.

The effect of COPE on sea urchin fertilization may Phytochemical analysis of the COPE extract revealed the
indicate an activity related to inhibition of cellular presence of bioactive components such as: alkaloids,
motility. Fertilization is an ideal system in which to flavonoids, tannins, triterpenes, glycosides and
study the specific effects of inhibitors of cellular saponins. Previous work reported the ability of steroidal
motility. In the sea urchin sperm, fertility depends glycosides, triterpenes and saponins to inhibit
upon motility and capacity to undergo the acrosome development in sea urchin eggs (Rahman, 1995) and
reaction upon encountering a specific ligand derived more recently, a potent sperm motility-inhibiting activity
from the egg's jelly coat (Schatten et al., 1982). of bioflavonoids was isolated from Alstonia macrophylla
leaf extract (Chattopadhyay et al., 2005).
In the sea urchin egg, microfilament-mediated
motility in the fertilization cone and egg cortex Phytochemical screening
appears required for sperm incorporation (Schuel et
Phytochemical analysis has revealed various compounds
al., 1991). Previous works support that sperm
such as alkaloids, flavonoids, glycosides, saponins,
incorporation is sensitive to microfilament inhibitors
sterol, tannins and triterpenes. Both the aqueous and
and the pronuclear migrations are prevented by
ethanolic extracts contain the mentioned compounds
microtubule inhibitors.
except for sterols while the oil also contained all except
flavonoids and triterpenes. This result is comparable
Much earlier studies using sea urchin fertilization
with the previously reported phytochemical content of
assay reported strong cytotoxicity of taxol due to
related species of C. ovatum. Flavonoids, tannins,
inhibition of microtubule depolymerization in vitro
saponins and terpenes were found in different plant
and in vivo on sea urchin eggs during fertilization
parts of C. schweinfurthii, C. album and C. boivinii
(Schiff et al., 1980) and cannabinoids by preventing
(Mogana et al., 2011).
the initiation of acrosome reaction thereby reducing
the fertilizing capacity of sperm (Schuel et al., 1991).
Conclusion

Sea urchin fertilization was also inhibited by plant The findings of the study elucidated the medicinal
lectins that bind to the egg and sperm of the sea value of Canarium ovatum. The pili pulp is a very
urchin (Macedo et al., 2007). Recently, the leaf good source of natural antioxidants. It contains
extract of Carica papaya which contains flavonoids, bioactive components with angiosuppressive activity.
alkaloids, phenolic compounds and cynogenetic Further explorations should be conducted to establish
compounds exhibited antimitotic activity on cell its role as a possible treatment for cancer and stress-
proliferation of sea urchin embryos (Gutierrez, 2016). related diseases.

253 Cajuday et al.

 Int. J. Biosci. 2017

Acknowledgment Del Rosario OM. 2009. Antioxidant components of

This work was financially supported by Bicol Pili. Institute of Food Science and Technology,
University Research and Development Center University of the Philippines Los Baños Laguna.
(BURDC) and College of Science (BUCS).
Deryugina EI, Quigley JP. 2008. Chick embryo
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