Extraction, Profiling, and Characterization of Phytosterols and Triterpenoids from Pili (Canarium ovatum Engl.) Pulp Oil Exhibiting Antioxidant and Antibacterial Properties

Pili (Canarium ovatum Engl.), an indigenous tree found in the Philippines, is highly regarded for its fruit due to its high economic value. During processing, the pulp is often discarded as waste but contains considerable amounts of oil and bioactive minor lipid components. The present study explored the antioxidant and antibacterial properties of saponified diethyl ether extract of pili pulp oil and related this activity to the nature of compounds present in the extract through GCMS. The extract indicated the elution of 18 major compounds which are mostly cyclic triterpenic (α-and β-amyrin, lupenone, and β-amyrone) and phytosterol (β-sitosterol, brassicasterol, and stigmasterol) class of compounds. Characterization of the bioactivity of the extract showed high antioxidant activities measured by DPPH radical scavenging (EC50: 74.45 ± 1.29 μg/mL) and lipid peroxidation inhibition (EC50: 3.02 ± 0.06 μg/mL) activities that were comparable with that of α-tocopherol. Moreover, an observed bactericidal activity was demonstrated by the extract against E. coli and S. typhi with MIC values of 40 and 35 μg/mL, respectively. The observed bioactivity of the pili pulp oil extract can be attributed to these compounds which may provide desirable health benefits.


Introduction
Canarium ovatum Engl., locally known as pili, is an indigenous tree commonly found in the Philippines which is cultivated for its edible fruit [1]. Pili nut kernel is the most valuable part of the fruit due to its high economic value owing to its increasing competitiveness in the global confectioneries market [2]. In pili nut processing, its pulp is often discarded as waste, but it contains an appreciable amount of oil and important minor lipid species such as carotenoids, phytosterols, and tocopherols [3,4]. Tese nutritionally benefcial minor lipid compounds have gained considerable interest, particularly in their bioactivity which plays an important role in the development of high-value products.
Antioxidants and antimicrobial agents play a signifcant role in the food sector primarily because bacterial growth and lipid oxidation are the main factors that determine food quality loss and shelf-life reduction. Oftentimes, synthetic additives such as BHA/BHT are commonly added to food products to inhibit the process of lipid oxidation and microbial growth and to extend their shelf-life. However, a shift to naturally derived compounds is seen and increasingly being sought by many companies due to possible adverse efects associated with long-term intake of synthetic compounds [5,6]. Phytosterols, including other cyclic triterpenes which constitute the majority of unsaponifable fractions of seeds oils, are known to have several bioactive properties linked to various implications on human health, including anti-infammatory, antioxidative, antimicrobial, cholesterol-lowering, and anticarcinogenic activities [7][8][9][10][11][12]. On the other hand, triterpenoid extracts that are rich in lupeol, betulinic acid, and amyrin have been shown to inhibit the growth of foodborne pathogenic bacteria particularly the methicillin-resistantS. aureus, E. faecalis, and P. aeruginosa are studied by Amoussa et al. [13] and Nzogong et al. [10]. Phenolic compounds have been shown to fuse with extracellular soluble proteins of the microbial cell wall resulting in the suppression of microbial growth and/or oxidative damage [14].
Hence, the present study explored the potential of minor lipid components in pili pulp oil as a source of phytosterols and cyclic triterpenoids with antioxidant and antibacterial properties. Te interest in these naturally derived compounds is not only due to their biological activity but also to maximize the economic potential of pili pulp oil.

Sample Preparation and Oil Extraction.
Pili fruits were manually depulped by blanching in lukewarm water for about 15-20 min. Te pulps were then collected and dried in a convection-type oven at 70°C overnight or until moisture content reached about less than 3%. Extraction of oil was carried out by using n-hexane at 1 : 4 ratio of dried pulp weight (g) to solvent volume (mL). After 12 h of extraction at room temperature with constant agitation, the oil was recovered by solvent evaporation by using a rotary evaporator.

Saponifcation and Fractionation.
Te unsaponifable fraction of oil was obtained by saponifcation following the method of Almeida et al. [15] with some modifcations. A 0.3 g of the oil sample was saponifed by using 10 mL 3% w/v ethanolic potassium hydroxide at 50°C for 3 h. Ten, the solution was cooled by adding 10 mL of distilled water. Subsequent fractionation of the phytosterol and triterpenoids was carried out by repeated liquid-liquid extraction by using 10 mL diethyl ether three times. Te organic layers were then combined, washed twice with 10 mL of distilled water, and dried over anhydrous sodium sulfate. Te saponifed diethyl ether extract (SDEE) of pili pulp oil was then collected upon fltration and solvent evaporation under a stream of nitrogen gas.

GCMS Profling.
Profling of SDEE was performed on a Shimadzu GCMS-QP2020 equipped with a Shimadzu AOC-20i Plus auto-injector (Shimadzu Corp., Kyoto, Japan) under electron impact ionization at 70 eV. Separation of components was carried out in SH-Rxi-5Sil MS capillary column with dimensions of 30 m × 0.25 mm ID × 0.25 μm flm thickness (Shimadzu Corp., Kyoto, Japan). Te initial oven temperature was held at 190°C for 1 min then raised to 300°C at 15°C/min and kept constant for 10 min. Helium was used as the carrier gas with a constant fow rate of 1 mL/min. Te injector, MS ion source, and MS interface temperatures were set at 310, 230, and 280°C, respectively. A sample injection of 1 μL was performed in a split mode of 10 : 1 and peaks were detected in full scan acquisition mode from m/z 50 to 500. Identifcation of the individual components was performed by NIST mass spectral library on the basis of the mass fragment and e/z values of each component. Te relative concentration of each peak was computed based on the total ion count.

DPPH Radical Scavenging Activity.
Te scavenging activity of SDEE against DPPH radical was evaluated by using the method of [16] with some modifcations. Briefy, 3.8 mL of 0.07 mM DPPH solution in chloroform was mixed with 0.2 mL of the sample with varying concentrations. Te mixture was incubated in the dark at room temperature for 30 min. Chloroform and α-tocopherol were used as a blank and positive control, respectively. Te absorbance of the resulting mixture was measured at 516 nm and the percent radical scavenging activity was calculated based on the following equation: Te EC 50 value is defned as the concentration of sample required to scavenge 50% of DPPH radical under the assayed conditions.

Lipid Peroxidation Inhibition Activity.
A mixture of 0.1 mL of 0.1-10 μg/mL of SDEE (in chloroform) and 0.5 mL of 10% egg yolk homogenate (w/v in distilled water) was mixed in a screwcapped tube and the volume was made up to 1.0 mL by adding distilled water. Ten, 0.05 mL 0.07 M FeSO 4 (in distilled water) was added, and the mixture was incubated at 37°C for 30 min to induce lipid peroxidation. A 0.05 mL of 20% (w/v) trichloroacetic acid solution (TCA) in distilled water and 1.0 mL 0.1% (w/v) thiobarbituric acid solution (TBA) in distilled water was added to the mixture, vortexed, and heated in a water bath at 80°C for 30 min. After cooling, 3 mL of butanol was added, and the mixture was centrifuged at 3000 rpm for 5 min. Te absorbance of the upper layer was measured against 3 mL butanol at 512 nm. Chloroform and α-tocopherol were used as blank and positive controls, respectively. Te lipid peroxidation inhibition activity was calculated by using the following equation:  [17]. Briefy, a suspension of the test microorganism standardized to 0.5 McFarland with approximately 1.5 × 10 8 CFU/mL was uniformly spread onto individual solid media plates of Muller-Hinton agar by using a sterile cotton swab. Disks of 6 mm in diameter were impregnated, until saturation, with 0.1 mg/mL of the extract. Te disks were then allowed to dry placing in the inoculated agar. Chloramphenicol and chloroform served as positive and negative controls, respectively. Te inoculated plates were incubated at 37°C for 18-24 h. Antimicrobial activity was evaluated by measuring the zone of inhibition against the test organisms.

Minimum Inhibitory Concentration Assay.
Te MIC of the extract exhibiting sensitivity to the tested microorganism based on the disk difusion assay was determined following the methods outlined by Elof [18] with some modifcations. First, a stock solution of the extract (1 mg/mL) was prepared in Meuller Hinton broth supplemented with 0.02% Tween 80. Te solution was then sonicated for 30 s and vortex homogenized for 2 min to obtain a stable emulsion. Serial dilutions of the extract in broth were prepared in microtubes of 1 mL with a concentration range from 0.1 to 100 μg/mL. Ten, 295 μL of each dilution was transferred into a 96-well microplate. A 5 μL of test bacterial suspension (1.0 × 10 6 CFU/mL) was inoculated to obtain a fnal concentration of 1.67 × 10 4 CFU/mL and a fnal volume of 300 μL per well. Te inoculum (positive control) and culture medium (negative control) were put into the frst column of the microplate, and the chloramphenicol antibiotic control ranging from 0.5 to 75 μg/mL was in the fnal column. Each plate was wrapped loosely with cling flm to prevent dehydration during incubation for 18-24 h at 37°C. Subsequently, 10 μL of bacterial growth indicator, resazurin at 6.75 mg/mL, was added to wells, which were then incubated for 30 min at 37°C. Te lowest concentration of the extract that visually showed no growth was determined as MIC.

Statistical Analysis.
All experiments and measurements were carried out in triplicate. Te data were presented as mean ± SEM and were analyzed by using oneway ANOVA. Means were compared using Tukey's post hoc comparison test by using R software (version 4.2.1, R Foundation for Statistical Computing, Vienna, Austria). Signifcant diferences between means were determined at P < 0.05.

Oil Extraction, Fractionation, and GCMS Profling of the Diethyl Ether Unsaponifable Extract of Pili Pulp Oil.
Extraction of oil revealed 23.92 ± 0.21% yield, expressed in dry weight basis of pulp. Te unsaponifable fraction of oil obtained from diethyl ether extraction showed 1.0294 ± 0.0501%. Tese values were slightly lower than our previous fndings [3] which can be attributed to the varietal diferences of pili fruit used, for which this was also observed in the report of Tugay et al. [4] with 0.47 to 0.53% UM content depending on the fruit's variety. Analysis of the chromatogram obtained from the SDEE extract indicated the elution of 18 major compounds which are mostly triterpenic and phytosterol class of compounds ( Figure 1, Table 1).
Amyrin, both α-and β-isomers, are generally found in diferent plant part extracts such as in C. tramdenum bark which contains 0.03 mg/g β-amyrin [19]. Areal parts of M. barteri and S. longifolia also contain high amounts of α-amyrin as reported by Ce et al. [20] and Saeidnia et al. [21]. Other than plant parts, amyrin along with other sterols were also detected in oil samples such as in olive, faxseed, and camellia seed oils [22,23].

Antioxidant Activity.
Te antioxidant activity of the SDEE was analyzed in terms of scavenging activity against DPPH radical and the ability to inhibit lipid peroxidation in the egg yolk system. As shown in Table 2, SDEE showed comparable EC 50 values of 3.02 μg/mL with that of α-tocopherol (2.92 μg/mL), a known antioxidant compound, though a moderate activity was observed in terms of its DPPH radical scavenging activity with a slightly higher EC 50 value of 74.45 μg/mL in SDEE as compared to 65.91 μg/mL in control.
Terpenoids and sterols, in general, have been shown to exert antioxidant properties owing to their hydroxyl group that participates via hydrogen atom transfer or single electron transfer mechanism in quenching reactive oxygen species [24,25]. It has been demonstrated that phytosterol components such as campesterol, β-sitosterol, and stigmasterol have been shown to exhibit scavenging activities against DPPH radical and provide hepato-and neuroprotection of hydrogen peroxide-induced oxidative stress levels [25,26]. Isomers of amyrin isolated from Myrcianthes pungens leaves revealed high antioxidant activity with antioxidant protection equivalent to the Trolox of 137 and 129% for α-and β-amyrin, respectively, at 500 μg/mL [27].

Antibacterial Activity.
Te antibacterial potential of SDEE against 5 common food pathogens is shown in Table 3. Te extract was found to exhibit signifcant inhibitory activity against E. coli and S. typhi with 10.3 and 11.7 mm zone of inhibition, respectively, at 0.1 mg/mL. Te MIC showed that 40 and 35 μg/mL of the extract were able to inhibit the growth of E. coli and S. typhi, respectively. Te extract was not active or did not show antibacterial activity in S. aureus, B. cereus, and P. aeruginosa at 0.1 mg/mL. Te antibacterial activity of SDEE can be attributed to the high amyrin content which was also observed by Abdel-Raouf et al. [28] in several algal extracts containing β-amyrin exerting antibacterial activities against the 5 pathogens tested in this study. In a separate study, hexane extract of Manilkara subsericea fruit which is mostly α-andβ-amyrin acetate showed a MIC value of 250 μg/mL against S. aureus [29]. Aside from amyrin, Alawode et al. [30] reported that phytosterols, specifcally stigmasterol and β-sitosterol that  Table 1 for peak identities.  were isolated from Icacina trichantha, showed high antimicrobial properties against B. subtilis, E. coli, and C. albicans. Te regulatory activity of amyrin including other triterpenoids in disrupting pathways responsible for cell division and protein synthesis, as well as destabilization of bacterial cell membrane and inhibition of cell growth are some of the plausible mechanisms of action of these compounds [31]. Furthermore, these compounds also cause disorganizing efects on cardiolipin-rich domains present in the membrane of E. coli as demonstrated by Broniatowski et al. [32].

Conclusion
Extraction, saponifcation, and diethyl ether fractionation of the unsaponifable matter of pili pulp oil showed strong antioxidant properties as measured by its scavenging activity against DPPH radical and lipid peroxidation inhibition activities and are found to be comparable to α-tocopherol, a known antioxidant compound. Te extract also exerts antibacterial activities against E. coli and S. typhi which are bacterial pathogens of concern, especially in food preparation. GCMS profling revealed 18 compounds majority of which are cyclic triterpenes and phytosterols such as α-and β-amyrin, lupenone, β-sitosterol, brassicasterol, and stigmasterol. Our results suggest that these bioactive compounds are responsible for the observed bioactivities which may provide desirable health benefts.

Data Availability
Datasets related to this article will be made available upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest. Means ± SEM (n � 3) in a row without a common superscript letter difer (P < 0.05) as analyzed by one-way ANOVA and the TUKEY HSD test SDEE-saponifed diethyl ether extract of pili pulp oil.