International Food Research Journal 24(4): 1763-1770 (August 2017)

Journal homepage: http://www.ifrj.upm.edu.my

Heavy metal and microbiological profiles of defatted pili (Canarium ovatum,

Engl.) pulp meal residue and acute oral toxicity of its ethanolic
extract in mice
1,2*
Arenas, E.H. and 2Trinidad, T.P.
1
Department of Food Technology, College of Education, University of Santo Tomas, España,
Manila, 1015, Philippines
2
The Graduate School, University of Santo Tomas, España, Manila, 1015, Philippines

Article history Abstract

Received: 17 May 2016
Received in revised form:
Canarium ovatum, Engl. (Burseraceae) or Pili is a valued indigenous fruit tree crop in the
4 July 2016
Accepted: 11 August 2016 Philippines. The defatted pili pulp meal, a mixture of fruit peel and pulp that remains after
pili pulp oil extraction, may be considered a functional ingredient because of its high dietary
fiber and phytonutrient content. This study provides initial information on the microbiological
and heavy metal contents of defatted pili pulp meal residue and preliminary toxicity
Keywords report of its ethanolic extract. Ground lyophilized samples were subjected to heavy metal
and microbiological analyses. An ethanolic extract of the plant material was prepared for
Canarium ovatum
phytochemical screening and acute oral toxicity testing. Results showed that both heavy
Pili
Heavy metals
metals and microbiological profiles of defatted pili meal residue pass the criteria for botanical
Phytochemicals ingredients set by relevant regulatory agencies. Phytochemical screening showed the presence
Acute oral toxicity of important bioactive compounds namely, flavonoids, tannins, anthraquinones, indoles,
alkaloids, sterols, and terpenes. In acute oral toxicity, no mortalities were recorded over a 14-
day experimental period. Neither significant gross and histopathological findings in liver and
kidneys were detected. The LD50 of ethanolic extract in mice was estimated to be greater than
5 g/kg body weight. In conclusion, the pili pulp mixture may not present any potential public
health risk when used as a component for food and drug products.
© All Rights Reserved

Introduction is the solid waste of such processing. Remnants

consist of a mixture of fruit peels and fibrous pulp.
Canarium ovatum, Engl. (Burseraceae) is a Typically, this by-product is turned as livestock feed
valued indigenous fruit tree crop in the Philippines. and compost. Recent experiments carried out in our
It is a deciduous tree measuring about 20-25 meters laboratory have shown that this agro-industrial waste
in height and 40-50 centimeters in diameter. Locally is a good source of dietary fiber and phytonutrients.
known as pili, this plant is abundantly found in the As a potential functional ingredient for the food
Bicol region of Southern Luzon, particularly in the and pharmaceutical industries, the safety of this
provinces of Sorsogon, Albay and Camarines Sur food-derived material needs to be evaluated. No
(Coronel et al., 1983). It thrives in the primary and toxicological studies have been done to determine
secondary forests of low to medium elevations (Orwa any possible adverse health effects. Heavy metal
et al., 2009). accumulation in the edible and non-edible parts of
Pili is cultivated chiefly for its kernels. The pili fruit is likewise unknown. Because defatted pili
Philippines holds monopoly over processed pili pulp meal residue has not yet found its way into the
products in the global market (Coronel et al., 1983). human food chain, microbial quality is ignored. The
Aside from pili nut confections, pili pulp oil is one present study provides initial information on the
of the highly acknowledged pili commodities. This microbiological and heavy metal contents of defatted
oil is utilized for culinary and cosmetic products. pili pulp meal residue and preliminary toxicity report
Rural folks have attested to the curative properties of its ethanolic extract.
of pili oil against skin diseases. It is also utilized for
the treatment of worms in livestock such as pigs and Materials and Methods
chicken (Tacio, 2010).
Oil is obtained by manual extraction using a Plant material
mechanical pulp press. Defatted pili pulp meal residue Defatted pili pulp meal residue was collected

*Corresponding author.
Email: [email protected], [email protected]
1764 Arenas, E.H. and Trinidad, T.P./IFRJ 24(4): 1763-1770

from a pili pulp oil manufacturer in Sorsogon City, Preparation of plant extract
Philippines. Fruit material was lyophilized and Ground sample of lyophilized defatted pili
pulverized into fine particles using a household pulp meal residue (400 grams) was initially soaked
grinder. Samples were kept in air-tight containers and overnight in 2.5L of ethanol with occasional stirring
stored at -20oC until use. at ambient temperature. The immersion process was
Fruit specimens used in pili pulp oil production repeated twice with fresh solvent to ensure complete
were also obtained from the same manufacturer. extraction. Each mixture was filtered through a cotton
The botanical identity of the plant material was plug. Filtrates were combined and concentrated
authenticated by the curator of the University of under reduced pressure at 40oC. Residue remaining
Santo Tomas Herbarium, Manila, Philippines. after evaporation was weighed and stored in a clean
glass container at 4oC until use. This crude ethanolic
Microbiological analysis extract was used directly in phytochemical and acute
Assays were performed following the protocols toxicity studies.
described in the Bacteriological Analytical Manual
(BAM) by U.S. Food and Drug Administration. Phytochemical screening
Aliquots from serial dilutions were aseptically Qualitative phytochemical analysis was done to
transferred onto appropriate selective media. Total detect presence of bioactive compounds according
yeast and mold count was determined on Potato to standard methods described by Aguinaldo et al.,
Dextrose Agar (PDA) plates (Tournas et al., 2001). (2005). Thin layer chromatographic (TLC) profiling
Total aerobic bacteria count was conducted using was carried out on pre coated silica gel 60 F254 sheets
Plate Count Agar (PCA) (Maturin and Peeler, 2001). (Merck, Germany). Chromatogram was developed
Total coliforms and E.coli were enumerated using using ethyl acetate as solvent and visualized under
the Most Probable Number (MPN) technique on visible and ultraviolet light (UV254 nm and UV366
lactose broth. Positive tubes were subcultured to nm). Various spray reagents were used to characterize
Brilliant Green Lactose Bile (BGLB) and E. coli the class of compounds present in the extract.
(EC) broths. EC tubes producing gas were finally
streaked onto Eosine Methylene Blue (EMB) agar to Acute oral toxicity
obtain discrete colonies (Feng et al., 2002). Isolation Female ICR mice between 5 and 6 weeks of age,
of Staphylococcus aureus was performed on Baird- weighing 29-32 grams were employed in the study.
Parker (BP) agar (Bennett and Lancette, 2001). All were purchased from the Industrial Technology
For Salmonella detection, Rappaport Vassiliadis Development Institute - Standards and Testing
(RV) and Tetrathionate (TT) broths were used as Division (Taguig, Philippines). Experiment was
enrichment media. Samples from these enrichment conducted in an accredited animal house facility by
cultures were subsequently streaked onto Hektoen trained personnel. The animals were housed in cages
enteric (HE), Bismuth Sulfite (BS) and Xylose Lysine and kept under standard environmental conditions
Dexoxycholate (XLD) agar plates (Andrews et al., of temperature (21 + 2oC), relative humidity (30-
2007). All experiments were carried out in duplicate. 70%) and light (12-h light-dark cycle). They were
fed with a standard diet and provided with water ad
Heavy metals analysis libitum throughout the course of the experiment.
Sampling and digestion procedures were The animals were acclimatized for 7 days before
performed based on AOAC methods (2012). treatment and were fasted overnight prior to dosing.
Appropriate blanks were prepared and analysed Experimental protocols were in accordance with the
simultaneously. Determination of arsenic was carried Code of Practice for the Care and Use of Laboratory
out by atomic fluorescence spectroscopy (AFS). Animals of the Philippine Association for Laboratory
Mercury content was quantified by atomic absorption Animal Science (PALAS, 2002) and approved by the
spectroscopy (AAS) – cold vapour technique. Levels local Institutional Animal Care and Use Committee
of cadmium and lead were analysed by inductively (IACUC).
coupled plasma- atomic absorption spectroscopy Acute oral toxicity procedure was executed as
(ICP-AAS). Elements were measured against a series per Organization of Economic Cooperation and
of working standards of different concentrations. Development (OECD) 401 guidelines (OECD,
Recovery rates of metals ranged from 94% to 111%. 1993). Animals were randomly distributed to control
Coefficient correlation (r) values of calibration plots and treated groups; each composed of 10 mice. The
were between 0.998 to 0.999. Results obtained from control group was given distilled water alone while
triplicate readings were expressed in ug/g. the treated group received a single dose of the test
 Arenas, E.H. and Trinidad, T.P./IFRJ 24(4): 1763-1770 1765

Table 1. Mean microbial counts of defatted pili pulp meal residue and standard limits

a
Food and Drug Administration Philippines limits for herbal food products – plant materials that will
undergo pre-treatment;
b
American Herbal Products Association limits for dried unprocessed herbs for use as ingredients in dietary
supplements
c
World Health Organization limits for raw herbal material intended for further processing
d
United States Pharmacopeia limits for dried or powdered botanicals
*
Enterobacterial Count
**
As total coliforms (cfu/g)
NA – Not Assigned

extract. Doses were administered to animals by oral the level of microorganism in a product (Maturin and
gavage using a gastric feeding tube attached to a Peeler, 2001). Oftentimes, APCs are used to gauge
1-ml syringe. Food and water were withheld for the if HACCP (Hazard Analysis Critical Control Point)
next 3-4 hours. Observations were carried out at 30, plans are carried out effectively in a food plant (Hong
60, 120, 180 and 240 minutes post administration et al., 2008). Results for APC did not exceed the limit
and daily for a period of 14 days. Body weight and for herbal products imposed by regulatory agencies.
mortality were recorded. Behavioural and clinical Total yeast and mold count (TYMC) is a test to
manifestations of toxicity were likewise carefully measure fungal load. TYMC level of defatted pili
monitored. On day 15, all animals were sacrificed pulp meal residue complied with the stated microbial
and subjected to gross necropsy. specifications.
Liver and kidneys were collected for The coliform group consists of the genus
histopathological examinations. Following 18 hours Escherichia, Citrobacter, Enterobacter, and
of fixation in 10% neutral-buffered formalin, organs Klebsiella. But unlike other coliforms, Escherichia
were embedded in paraffin. Sections of 5µm were coli is more specific to indicate fecal contamination
cut and stained with hematoxylin and eosin for (Gerba, 2008). Microbial examination of defatted pili
microscopic evaluation. pulp meal residue showed that coliform organisms
were present in the product. However, the number
Statistical analysis of coliform bacteria was relatively few. The absence
Experimental results were expressed as mean of E.coli indicates that the product conformed to
+ standard error of mean (SEM). Student’s t-test the microbiological standards. According to the set
was applied to determine difference between body microbial guidelines on herbal products, Salmonella
weights in mice. P values < 0.05 were deemed spp and Staphyloccocus aureus should not exist.
significant. Statistical analysis was performed using In this study, the tested material adhered to this
SPSS version 20. requirement since both pathogens were not detected.

Results Heavy metals analysis

Results revealed that defatted pili pulp meal
Microbiological analysis residue is relatively free of mercury and contains
The average microbial concentrations from very low residual levels of arsenic, cadmium and
defatted pili pulp meal residue are presented in lead. A look at the data shows that the concentration
Table 1. The microbial criteria for herbal materials of all analysed metals falls within the tolerable limits
developed by various regulatory bodies are likewise established by national and international regulatory
shown in Table1. Aerobic plate count (APC) shows authorities (Table 2). This implies that the use of this
1766 Arenas, E.H. and Trinidad, T.P./IFRJ 24(4): 1763-1770

Table 2. Mean concentration of heavy metals in defatted pili pulp meal residue
compare with published standards

Values are mean + SEM, n =3

a
Food and Drug Administration Philippines maximum limits for herbal food products – dried
plants;
b
United States Food and Drug Administration permissible limits in herbal drugs
c
World Health Organization permissible limits for herbal ingredients
d
United States Pharmacopeia limits for nutritional supplements
e
Association of South East Asian Nations maximum limits for traditional medicines and
health supplements
*
None-detected at the method detection limit of 0.10 µg/g for Mercury

Table 3. Phytochemical compounds in ethanolic extract of Canarium ovatum, Engl.

+: present; -: absent

product is unlikely to be associated with heavy metal of vitamin E, cyanidin and ferulic acid in pili.
toxicity. These findings are consistent with previous
reports conducted on other Canarium species. The
Phytochemical screening fruit of Canarium odontophyllum was found to
Ethanolic extraction resulted to a green thin fluid be a rich source of phenolic acids, flavonoids and
with reddish-orange upper layer. The extractive yield anthocyanins (Azrina et al., 2010; Shakirin et al.,
from defatted pili pulp meal residue was 65.1 grams 2010; Chew et al., 2012; Khoo et al., 2013; Ali-Hassan
(16.28%). The pH and specific gravity values of the et al., 2013). Several of these phenolic constituents
crude extract were 6.57 and 0.9264, respectively. have been isolated and identified (Khoo et al., 2012;
TLC analysis revealed the occurrence of various Mokiran et al., 2014). Tannins and flavonoids were
secondary metabolites as summarized in Table 3. detected in different solvent extracts of Canarium
The absence of steroids, cardenolides, coumarins, schweinfurthii fruit pulp (Shaba et al., 2013; Wahab
anthrone and sugars was recorded. On the other hand, et al., 2015). Appreciable amount of tannins in the
pili extract contains a wealth of polyphenol compounds mesocarp of Canarium schweinfurthii has likewise
such as flavonoids, tannins and anthraquinones. been determined by Nyam et al. (2014) and Ehirim et
The results conformed to an unpublished work of al. (2015). Meanwhile, studies on Canarium album
Del Rosario (2007) on antioxidant components of fruit showed high phenolic and flavonoid contents
Canarium ovatum. According to this report, pili (Guo et al., 2008; Liu et al., 2008). The structures of
extracts tested positive on phenolic compounds. some phenolic compounds isolated from Canarium
Total phenolic and flavonoid contents were greater album fruit have been elucidated by He and Xia
in the pulp and peel extracts than in the nut. Further (2007), He et al. (2008), He and Xia (2008), He et al.
analysis of these phenolic compounds using TLC, (2009), Xiang et al. (2010) and Xiang et al. (2012).
UV/IR spectroscopy and HPLC revealed the presence Pili extract also showed positive results for
 Arenas, E.H. and Trinidad, T.P./IFRJ 24(4): 1763-1770 1767

Discussion

Defatted pili pulp meal residue represents a

considerable proportion of the raw material used in
pili pulp oil manufacture. Phytochemical analysis
confirmed that this fruit residue is a valuable source
of bioactive substances which have been reported to
exert multiple biological actions to promote human
health. Many flavonoids have been reported to have
antioxidative, hepatoprotective, anti-inflammatory,
antibacterial, and anticancer activities, whereas
Figure 1. Final body weight gain in mice. Data are mean some possess antiviral activities (Kumar and Pandey,
+ SEM (n=10). Bars labelled with same letter are not 2013). Tannins have shown cardioprotective effects
significantly different at P < 0.05 level. and function as vasodilator, anti-carcinogenic, anti-
allergic, anti-inflammatory, antibacterial, and antiviral
indoles, alkaloids, sterols and terpenes. These agents (Rosales-Castro et al., 2014). Anthraquinones
results are in accordance to literature data reporting have been recognized to exert diuretic, cathartic,
alkaloid contents of Canarium schweinfurthii fruits vasorelaxing, anticancer, anti-inflammatory,
(Shaba et al., 2013; Ehirim et al., 2015; Wahab et antimicrobial and phytoestrogen activities (Chien
al., 2015). Earlier studies by Prasad et al. (2011) et al., 2015). Alkaloids are known to exhibit a
and Ali-Hassan et al. (2013) revealed that Canarium broad spectrum of biological effects such as anti-
odontophyllum fruit contains lutein and ß-carotene. cholinergic, antihypertensive, emetic, antitussigen,
Carotene concentration of peel was higher compared antitumor, diuretic, sym-pathomimetic, antiviral,
to pulp and seeds. Xiang et al. (2012) has isolated miorelaxant, hypnoanalgesic, antidepressant,
a triterpenoid saponin from the n-butanol fraction of antimicrobial and anti-inflammatory activities
Canarium album dried fruits. (Aberoumand, 2012). Terpenes have potent action
against malaria, cancer, inflammation and a variety
Acute oral toxicity of infectious diseases caused by viruses and bacteria
Ethanolic extract of Canarium ovatum fruit (Wang et al., 2005). Phytosterols block cholesterol
residue caused no deaths in mice within the 14-day absorption and offer protection against oxidation,
study period. Single oral administration of the test cancer and inflammation (Bartnikowsk, 2009).
extract did not result in any observable toxic effect. Bacteria, fungi and viruses represent a wide
Normal body weight gains were recorded during the variety of microbiological contaminants that may
test period. In both groups, increase in body weight be associated with medicinal plants (Kunle et al.,
before and after treatment was significant (p < 0.05). 2012). Microbial contamination of plant materials
As shown in Figure 1, body weight gain after 14 days takes place in all stages of horticultural production
was not statistically different (p > 0.05) between the namely growth, harvesting and postharvest handling
control and treated groups. (Vidovic et al., 2013). Aerobic bacteria and fungi
Gross necropsy performed at termination often found in soil make up most of the natural
revealed no abnormal findings in the vital organs of microflora in plants. Additional contaminants like
all animals. Histopathological examination of liver Escherichia coli or Salmonella spp. may arise as a
and kidneys in the control and treatment groups did result of faulty harvesting cleaning, drying, handling,
not display significant morphological changes. and storage techniques (Kunle et al., 2012).
Findings indicate that the acute LD50, if any, of Applying the criteria mentioned in Table 1,
Canarium ovatum extract is in excess of 5g/kg body the microbiological quality of defatted pili pulp
weight. Following the Hodge and Sterner (1943) meal residue was found to be satisfactory. From
toxicity scale, such substance can be classified as a microbiological point of view, it is deemed safe,
practically nontoxic. Similarly, Kennedy et al. (1986) provided further processing treatments (i.e. boiling,
stated that any substance with LD50 value greater steeping etc.) are applied prior to consumption.
than 5000mg/kg via oral route may be regarded as The said plant material was not free from microbial
practically nontoxic. contamination. Observed numbers of APC, TYMC
and coliforms provide evidence of contamination
at one or more points along the production line.
Introduction of microbial impurities in plant foods
1768 Arenas, E.H. and Trinidad, T.P./IFRJ 24(4): 1763-1770

may originate from different factors such as poor the test extract above 5 g/kg does not cause structural
worker hygiene, inadequate equipment sanitation, damage to these organs in mice. Single oral dose of
the use of contaminated irrigation or process water this plant extract appears to be non-toxic as seen in
and application of biosolids or manure as fertilizers this in vivo animal study.
(Beuchat, 1996). A closer examination of agricultural
and manufacturing practices is needed in order to Conclusion
come up with effective strategies towards reduction
of microbial contamination of defatted pili pulp meal In conclusion, the pili pulp mixture may not
residue. present any potential public health risk when used as
Food crops supply energy and essential nutrients a component for food and drug products. Although
in the human diet. Plant species, as well as different microbial quality was adequate to guarantee
plant parts, vary widely in their ability to accumulate safety, there is a call for stringent implementation
heavy metals (Ul Islam et al., 2007; Naser et al., of proper agricultural and sanitation practices in
2011). Consumption of contaminated food is a order to minimize microbiological contamination.
major route of heavy metal exposure in humans and Further studies on chronic toxicity, carcinogenicity,
ingestion of toxic elements can lead to serious illness mutagenicity and genotoxicity, are necessary to
or death. Arsenic, cadmium, lead and mercury are gain a deeper insight about the safety of this plant
the most lethal and widespread heavy metals (Morais material. The present study is the first attempt to
et al., 2012) that are commonly associated to major provide key evidence for the safe use of defatted pili
human health problems and poisoning (Hutton pulp meal residue as ingredient in food and medicinal
1987; Karayil et al., 2013). High cadmium intake preparations.
damages the kidneys that can cause profound renal
dysfunction (Bernard, 2008). Mercury is known Acknowledgements
to have deleterious effects on the central nervous
system leading to behavioural changes and cognitive This work was funded by Department of Science
disorders. In addition, mercury is correlated with and Technology – Science Education Institute
cardiotoxicity (Azevedo et al., 2012). Arsenic, a (DOST-SEI). Authors are grateful to Dr. Mario A.
recognized human carcinogen, is mostly linked Tan of UST - Chemistry Department and Dr. Cynthia
to skin and lung cancers. Elevated dose of arsenic M. Nalo-Ochona of DOST – ITDI Standards Testing
results to skin lesions, cardiovascular, neurological Division for technical assistance.
and endocrine disorders (Guha Mazumder, 2008;
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