Canarium Ovatum, Engl. (Burseraceae) Or Pili Is a Valued Indigenous Fruit Tree Crop in the 4 July 2016 Accepted: 11 August 2016 Philippines
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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. 1Department of Food Technology, College of Education, University of Santo Tomas, España, Manila, 1015, Philippines 2The 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