phytochemical screening, total phenolics, and antioxidant and … · 2020. 10. 12. ·...

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

Phytochemical Screening, Total Phenolics, and Antioxidant and Antibacterial Activities

of Selected Philippine Indigenous Fruits

*Corresponding Author: [email protected]

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.

Philippine Journal of Science149 (3-a): 697-710, October 2020ISSN 0031 - 7683Date Received: 19 Feb 2020

Mariam C. Recuenco*, James Russell P. De Luna, Nathalia G. Magallano, and Kevin C. Salamanez

INTRODUCTIONPlant-based foods, fruits and vegetables, are essential in the human diet because of their roles in sustaining life and health. They provide vital nutrients, carbohydrates, proteins, lipids, vitamins and minerals, fiber, and a wide variety of bioactive non-nutrient “phytochemicals” from plant secondary metabolism (Del Rio et al. 2013). The phenolics are one of the biggest groups of

phytochemicals that include compounds such as phenolic acids, flavonoids, stilbenes, coumarins, and tannins. These compounds have molecular structures characterized by the phenol moiety of aromatic hydrocarbons with hydroxyl groups (-OH). Many studies reported the potential health benefits of dietary phenolics from cocoa, coffee, red wine, tea, berries, citrus fruits, nuts, and vegetables (Del Rio et al. 2013). Evidence from in vitro studies suggested that phenolics exhibit anti-cancer cell proliferation, anti-inflammatory, and antimicrobial activities (Del Rio et al.

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2013). Moreover, some small-scale human intervention studies suggested that increased consumption of (poly)phenol-rich foods had beneficial effects on cardiovascular and neurocognitive health, and in the reduction of risks for certain cancers (Del Rio et al. 2013).

Studies on the phytochemical components and biological activities from fruits had been increasing in recent years. A number of studies had analyzed collections of fruits that included widely consumed and/or underutilized fruits in a certain country or locality (Leong and Shui 2002; Ikram et al. 2009; Rufino et al. 2010). We had recently reported on the total phenolics and β-carotene radical inhibition activities of 30 locally available fruits (Recuenco et al. 2016). With the diversity of fruit species in the Philippines, there are many more fruits that could be potential sources of bioactive phytochemicals. Ethnobotanical surveys such as those conducted by Chua-Barcelo (2014) and Santiago et al. (2014) can provide information on where indigenous fruits grow and thrive, and how locals use them for food and medicine.

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 are studies already conducted in other countries, it may be necessary to perform analysis on the locally grown fruits since phytochemical contents may be affected by cultivar, climate and location, and agronomic and harvest factors (Tiwari and Cummins 2013). This study is limited to using the edible portions as samples and having only one stage of maturity for each fruit (Table 1). To our knowledge, this may be the first report on the phenolic contents and antioxidant activities of Ficus ulmifolia (“as-is”), Ficus pseudopalma (“niyog-niyogan”), and Mangifera altissima (“paho”), which are endemic to the Philippines (Coronel et al. 2003; Ragasa et al. 2009). M. altissima mangoes are eaten fresh, ripe or unripe, pickled, or used in salads. M. altissima was included in the morphological characterization of five Mangifera spp. in the study of Coronel et al. (2003). Ficus pseudopalma young shoots and leaves are eaten and used as a medicinal plant in the Bicol region, but the use of the fruits may not be as common (Santiago et al. 2014). Terpenoids and sterols had been identified from the leaves of F. pseudopalma and F. ulmifolia (Ragasa et al. 2009; Santiago and Mayor 2014; De las Llagas et al. 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 the most studied for its phytochemical and antioxidant properties (Butkhup and Samappito 2011; Lizardo et al. 2015; Ngamlerst et al. 2019). Garcinia binucao or “batuan” fruits are commonly used as a souring agent in dishes, particularly in the Visayas (Quevedo et al. 2013).

There are a few studies done in the Philippines on this fruit (Quevedo et al. 2013; Ragasa et al. 2014b). Artocarpus altilis (“kamansi”) young fruits are cooked and consumed as a vegetable. A. altilis leaf dichloromethane extract was reported to contain several sterols, unsaturated fatty acids, lutein, and isoprenes (Ragasa et al. 2014a). Citrus hystrix or “kabuyaw” (kaffir lime) is a citrus fruit often used in Thailand as a flavorant (Panthong et al. 2013). While there were several studies on C. hystrix elsewhere (Panthong et al. 2013; Abirami et al. 2014; Seeka et al. 2016), there seemed to be no studies conducted on this fruit in the Philippines. Canarium ovatum or “pili” nuts and its by-products are of economic importance to the Philippines, with the Bicol region supplying 80% to the market (Pham and Dumandan 2015). Pham and Dumandan (2015) reported the lipid profiles of C. ovatum oil and pulp. Lastly, Rubus rosifolius or “sampinit” is a wild berry that is one of about 10 species of Rubus that could be found in the mountainous regions of the Philippines (Real 2016). Although Rubus was reported to be found in 39 provinces (Real 2016), there seems to be limited research on these locally grown berries.

MATERIALS AND METHODS

Sample Collection and IdentificationSelected indigenous fruits (Table 1; Appendix Figure I) were obtained from the provinces of Laguna, Quezon, Batangas, and Negros Oriental. The maturity stage or ripeness of the fruits was indicated in Table 1. The samples (500–1000 g) were cleaned and refrigerated prior to extraction and evaluation. Samples were identified by Dr. Annalee S. Hadsall of the Museum of Natural History, University of the Philippines Los Baños.

Sample Preparation and ExtractionTwo (2.00) g edible portion of the fruit samples were extracted with 50 mL (80% v/v) aqueous methanol using a homogenizer. The homogenate was filtered and the filtrate was stored in amber-colored bottles at –20 °C. For the antibacterial assay, the methanolic fruit extracts were concentrated using a rotary evaporator and dried under a fume hood. The total phenolics of the concentrated extracts were determined using the Folin-Ciocalteu method (Singleton and Rossi 1965).

Moisture Content DeterminationFresh samples (2 g) in tared evaporating dishes were dried for 24 h in a hot air oven set at 50 °C. After 24 h, the dishes were placed in desiccators, cooled to room temperature, and weighed. These steps were repeated until the weights were constant. Moisture contents were calculated using the

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equation: % moisture: = [(initial weight – final weight) / initial weight] x 100%.

Qualitative Phytochemical Screening A few milliliters of the prepared extracts in 80% (v/v) methanol was subjected to qualitative tests for phenolics, alkaloids, cardiac glycosides, terpenoids, and saponins (Mandal et al. 2015). The tests done in duplicates were Folin-Ciocalteu test for phenolics, Wagner’s test for alkaloids, Keller-Killiani test for digitoxose (2,6 dideoxy-D-ribohexose) cardiac steroidal glycosides, Salkowski’s test for terpenoids, frothing test for saponins, lead acetate test for tannins, and alkaline reagent test for flavonoids.

Determination of Total Phenolic Content (TPC)The method was based on the procedure described by Singleton and Rossi (1965) with modifications. The methanolic extract (from the 2.00 g edible portion + 50 mL (80 % v/v) aqueous methanol), diluted 10-fold [10 µL methanolic extract + 90 µL 80% (v/v) methanol] was mixed with 500 µL distilled water and 250 µL of 10% Folin-Ciocalteu reagent. After 3 min, 500 µL of (20% w/v) Na2CO3 was added. The mixture was mixed and incubated at 40 °C for 40 min. In a 96-well microplate, 250 µL of the mixture was loaded and the absorbance was measured at 750 nm using Thermo Scientific Multi Scan Go (Thermo Fisher Scientific Inc.) microplate spectrophotometer. Gallic acid was used to prepare a calibration curve (0–31 mg/L, R2 = 0.9958). Results were expressed in mg GAE / 100 g FM and in mg GAE / 100 g dry matter (DM).

Determination of Total Flavonoid Content (TFC)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 water and 7.5 µL (5 % w/v) NaNO2. After 5 min, 7.5 µL of (10 % w/v) AlCl3 was added. After 5 min, 50 µL 1 M NaOH and 100 µL distilled water were added. The mixtures of fruit extracts and reagents were mixed and absorbances were read at 510 nm. Catechin was used to prepare a calibration curve (0–200 mg/L, R2 = 0.9954). Results were expressed in mg catechin equivalent (CE) / 100 g FM and in mg CE / 100 g DM.

Free Radical Scavenging Assay Using 2,2-diphenyl-1-picrylhydrazyl (DPPH)The assay was based on the method described by Brand-Williams et al. (1995) with modifications. Freshly prepared 0.1 mM DPPH in MeOH (150 µL) was added to 50 µL of diluted methanolic fruit extract (10-fold). The solution was incubated at room temperature and in the dark for 30 min. Absorbance at 515 nm was measured using a microplate reader. The % radical scavenging activity was calculated using the equation: % radical scavenging activity = [(A515 Control – A515 Sample) / A515 Control] x 100%, where A515 Control = absorbance of DPPH solutions without fruit extract at 0 min, while A515 Sample= absorbance of DPPH solutions with fruit extract after 30 min. Ascorbic acid was used as a standard. Results were expressed as mg AEAC / 100 g FM.

Table 1. Information on the selected Philippine indigenous fruits used in this study.

Scientific name(Family)

Local name Place of collection Part/s used and maturity

Ficus ulmifolia Lam. (Moraceae)

“As-is” Tiaong, Quezon Whole fruit; ripe

Antidesma bunius (L.) Spreng (Phyllanthaceae)

“Bignay” Calauan, Laguna Whole fruit; ripe

Garcinia binucao (Blco.) Choisy (Clusiaceae)

“Binukaw,” “Batuan” Dumaguete City, Negros Oriental

Flesh; ripe

Artocarpus altilis (Park.) Fosb. (Moraceae)

“Kamansi” Calauan, Laguna Strands and seeds; unripe

Citrus hystrix DC. (Rutaceae)

“Kolong-kolong” Calauan, Laguna Pulp sac with juice; ripe

Ficus pseudopalma Blco. (Moraceae)

“Niyog-niyogan” Calauan, Laguna Flesh and seeds; ripe

Mangifera altissima Blco. (Anacardiaceae)

“Paho” San Juan, Batangas Flesh; unripe

Canarium ovatum Engl. (Burseraceae)

“Pili” Calauan, Laguna Pulp and nut (less brown sheath); ripe

Rubus rosifolius Sm. (Rosaceae)

“Sampinit” Dolores, Quezon Whole fruit; ripe



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Reducing Power Ability (RPA) AssayThe assay was based on the procedure by Oyaizu (1986) with modifications. A 50-µL aliquot of the methanolic fruit extract, 250 µL of 0.2 M phosphate buffer pH 6.6, and 250 µL of (1 % w/v) potassium ferricyanide were mixed. The mixture was incubated at 50 °C for 20 min. Afterward, 250 µL (10 % w/v) trichloroacetic acid was added and the mixture was centrifuged at 3,000 rpm for 10 min. In a 96-well microplate, 50 µL supernatant was added with 50 µL distilled water and 100 µL (1 % w/v) ferric chloride. The absorbance was measured at 700 nm. Ascorbic acid was used to prepare a calibration curve (0–100 mg/L, R2 = 0.9988). Results were expressed as mg AAE / 100 g FM.

Minimum Inhibitory Concentration (MIC80) Determination by Broth Microdilution MethodThe broth microdilution method was performed based on Wiegand et al. (2008). The (80% v/v) aqueous methanolic fruit extracts were concentrated using a rotary evaporator. To make stock solutions, extracts were dissolved in DMSO (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. The assays were performed in 96-well microplates in the manner prescribed by Wiegand et al. (2008). Wells were filled with 100 μL of sterile NB. At least 10 concentrations (from 0 until 16 mg GAE / mL, 32 mg GAE / mL, or 64 mg GAE / mL) were prepared through serial dilution: adding 100 μL extract to the first well, mixing the 200 μL mixture, and transferring 100 μL to the next well, etc. Ten (10) μL of Escherichia coli or Staphylococcus aureus suspension (approx. 108 CFU/mL) was added to each well. Specific wells were designated: negative control (NB + bacteria); positive control (NB, bacteria + 0.25 mg/mL ampicillin); vehicle control (NB + fruit extract), which

corrects for the color or turbidity of the fruit extract; and sample (NB, bacteria + fruit extract). The experiment was performed in triplicates for every concentration. The mixtures were incubated at 37 °C for 20 h. After incubation, the optical densities (OD) at 600 nm were measured. Percentage growth inhibition was calculated as % Inhibition = [ODNegative – (ODSample- ODVehicle)]/ (ODNegative– ODPositive) x 100 %. Dose-response curves were plotted. The MIC80 in mg GAE/mL growth medium determined from the curve was the concentration that inhibited 80% of bacterial growth.

Statistical Analysis Results were expressed as mean ± standard deviation of three replicates. Pearson correlation tests and the Tukey and Dunnett post-tests to determine significant differences in the group means were performed using GraphPad Prism (GraphPad Software, Inc.) with statistical significance set at P < 0.05. The MIC80 for the antibacterial assays were determined from dose-response curves plotted in Sigma Plot 12.0.

RESULTS AND DISCUSSION

Phytochemical Screening Qualitative tests could provide simple and rapid ways of detecting certain phytochemical families in plant samples. Results indicated that the selected indigenous fruits may contain a wide variety of phytochemicals (Table 2). All extracts were positive for phenolics, flavonoids, and tannins – indicating that these groups were the most widespread in plants. Alkaloids were detected in C. ovatum pulp and C. ovatum nut. Cardiac glycosides were detected in F. pseudopalma, C. ovatum pulp, and

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

Fruit Phenolics Flavonoids Tannins Alkaloids Cardiac glycosides

Terpenoids Saponins

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. ovatum pulp, and R. rosifolius. Saponins were detected in all the fruits except in G. binucao and A. altilis. These present results could provide useful information prior to more focused studies on specific phytochemical groups in these plants.

Total Phenolic and Flavonoid Contents In fruits, the phenolic contents can be indicators of developmental stages and responses to environmental factors, such as light and temperature (Macheix et al. 2018). Phenolics have various roles in growth, ripening, abscission of plant organs, lignification, as metabolic effectors, as protectors of cell structures, and in resistance to biological stresses. Phenolic content can affect fruit quality such as color, flavor, and aroma (Macheix et al. 2018). Numerous studies on dietary polyphenols suggested protective effects against chronic diseases based on evidence from controlled human intervention studies (Del Rio et al. 2013).

Table 3 shows the TPCs of the selected fruits. The TPC ranged from 191–758 mg GAE/100 g FM basis, with the highest from G. binucao. The fruits could be classified into categories: low (< 100 mg GAE / 100 g FM), medium (100–500 mg GAE / 100 g FM), and high (> 500 mg GAE / 100 g FM) (Rufino et al. 2010). G. binucao, M. altissima, F. ulmifolia, F. pseudopalma, and C. ovatum pulp belong to the high group; the rest – R. rosifolius, A. bunius, C. hystrix, A. altilis, and C. ovatum nut – belong to the medium group.

The TFCs are shown in Table 3. The TFC values ranged from 132–421 mg CE / 100 g FM, with the highest from F. pseudopalma. The fruits could be classified into categories: low (< 50 mg CE / 100 g FM), medium (50–250 mg CE / 100 g FM) and high (> 250 mg CE / 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, A. altilis, A. bunius, M. altissima, C. ovatum nut, and C. hystrix.

Present results agreed with past studies indicating that some of the fruits may contain medium to high levels of phenolics and flavonoids. The TPC of the unripe M. altissima (“paho”) was within the range reported for mature mango varieties, M. foetida and M. odorata (Ikram et al. 2009). The TPC of ripe A. bunius was close to the reported levels from ripe fruits (Recuenco et al. 2016; Ngamlerst et al. 2019). The TPC from the pulp and juice of ripe C. hystrix may be higher compared to the reported TPC from C. hystrix juice (Abirami et al. 2014). Campbell et al. (2017) also reported high levels of phenolics from Rubus. Furthermore, Campbell et al. (2017) identified ellagic acid as the predominant phenolic and detected considerable quantities of p-hydroxybenzoic acid, caffeic acid, quercetin, kaempferol, and catechin.

In some fruits, the degree of maturity could affect the phenolic content and the observed antioxidant activities (Butkhup and Samappito 2011; Palafox-Carlos et al. 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 %moisture

Total phenolics* (mg GAE / 100 g FM)

Total flavonoids* (mg CE / 100g FM)

Radical scavenging* (mg AEAC / 100 g FM)

Ferric reducing power * (mg AAE / 100 g FM)

pH of extract

E. coli MIC80, (mg GAE / mL)

S. aureus MIC80 (mg GAE / 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 on the effects of degree of ripeness on the phytochemical content and various bioactivities, future studies could focus on one fruit species similar to the studies of Butkhup and Samappito (2011) and Palafox-Carlos et al. (2012).

Antioxidant Activities of the Fruit ExtractsDietary phenolics or polyphenols are recognized for their possible roles in preventing chronic diseases such as cancers and cardiovascular diseases (Del Rio et al. 2013). These diseases could develop due to oxidative stress from increased levels of reactive oxygen species and free radicals that may damage cellular molecules such as DNA, lipids, and proteins (Del Rio et al. 2013). Phenolics and flavonoids could prevent such reactions by acting as antioxidants due to their capacity to act as hydrogen donors, reducing agents, and singlet oxygen quenchers.

Present results showed that all the fruit extracts exhibited antioxidant and reducing activities toward the DPPH radical and ferric species, respectively (Table 3). The DPPH radical scavenging values ranged from 82.0–516.0 mg AEAC / 100 g FM. When arranged in increasing activity, the order is: C. ovatum nut < C. hystrix < A. bunius < A. altilis < C. ovatum-pulp < G. binucao < F. ulmifolia < F. pseudopalma < R. rosifolius < M. altissima. For the ferric RPA, values ranged from 31–121 mg AAE / 100 g FM. The arrangement in order of increasing RPA is: C. ovatum nut < A. altilis < C. hystrix < A. bunius < R. rosifolius < C. ovatum pulp < F. pseudopalma < F. ulmifolia < M. altissima < G. binucao.

The observed abilities to scavenge DPPH radicals by extracts from A. bunius, A altilis, C. hystrix, G. binucao, and R. rosifolius agreed with previous reports (Butkhup and Samappito 2011; Abirami et al. 2014; Barcelo 2015; Jalal et al. 2015; Oliveira et al. 2016; Campbell et al. 2017; Soifoini et al. 2018). However, numerical 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 2013). Here, we could only account for the contributions of phenolics and flavonoids to the observed activities. The phytochemical screening also provided some insights about other possible contributing components. Ascorbic acid, tocopherols, and carotenoids – not quantified in this study – may also have significant contributions (Hassimotto et al. 2005; Moon and Shibamoto 2009). Antioxidant activities may be due to combinations of synergistic and antagonistic interactions between different phytochemical components (Hassimotto et al. 2005). Future studies should consider quantifying various compounds and performing additional assays for a more comprehensive measure of the antioxidant and/or metal-reducing activities.

Antibacterial Activities of the Fruit ExtractsPolyphenols could have antagonistic effects on the growth of foodborne pathogenic or food-spoiling bacterial strains (Bouarab-Chibane et al. 2019). All fruit extracts exhibited 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 mg GAE/mL. The MIC80 values against S. aureus were generally lower compared to the values against E. coli except for F. ulmifolia, A. bunius, and A. altilis. This may suggest that S.aureus was more sensitive compared to E. coli. The most effective against S. aureus were extracts from C. hystrix and F. pseudopalma (MIC80 < 1.00 mg GAE / mL). Against E. coli, the most effective were extracts from C. hystrix and R. rosifolius (MIC80 values ~ 1.70 mg GAE / mL). For R. rosifolius, the MIC80 value was within the range from a previous report (Oliveira et al. 2016).

Present results from the broth microdilution assay showed that as the concentrations of the fruit extracts increase, the antibacterial activities also increase. The Gram-negative bacteria, E. coli, were shown to be more resistant while the Gram-positive bacteria, S. aureus, were more susceptible to inhibition by the fruit extracts – similar to those observed in Shan et al. (2007). Inhibition of bacterial growth could be due to fruit extract components that attack the bacterial cell wall and cell membrane and cause leakage and coagulation of cytoplasmic components (Shan et al. 2007).

We would like to believe that potential antibiotic compounds may be present from the fruit samples tested, especially from those which exhibited the lowest MIC80 values – C. hystrix, R. rosifolius, and F. pseudopalma. Since the assays conducted here were simple and used only two bacterial species, we suggest using more test organisms to identify specific targets of potential antibiotic compounds from these fruits.

Correlation of Total Phenolics with Antioxidant and Antibacterial ActivitiesTo determine whether the antioxidant, ferric reducing power, and antibacterial activities of the fruit extracts could be attributed to their phenolic contents, pairwise correlation analyses were performed. The results are presented in Table 4.

There was a strong positive and significant correlation between total phenolics and total flavonoids (DM basis with Pearson’s r = 0.7232, P < 0.05). This suggests that flavonoids contribute significantly to the TPCs of the fruit samples. Such strong positive correlations had also been observed in previous studies (Barreto et al. 2009; Recuenco et al. 2016).



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The pairwise analyses of the total phenolics with the radical scavenging activities and reducing power abilities all gave strong and statistically significant correlations with Pearson’s r values 0.6641–1 (P < 0.05). Similarly, pairwise analysis of the total flavonoids (DM) with the radical scavenging activities also gave a strong and statistically significant correlation with Pearson’s r value ~ 0.79 (P < 0.05). These positive correlations were similar to the findings of Barreto et al. (2009). These may suggest that phenolics and flavonoids contribute directly to the observed radical scavenging and ferric reducing activities of the fruit extracts. However, some studies found no correlation or inverse correlation between phenolics and antioxidant activities (Hassimotto et al. 2005; Ikram et al. 2009). Ikram et al. (2009) suggested that the antioxidant activities of plant extracts could not be attributed solely to the phenolic content. In the future, other components that might be capable of exerting antioxidant and reducing activities such as ascorbic acid, carotenoids, tocopherols, minerals, and proteins should also be considered (Ikram et al. 2009).

The correlations of phenolic contents to the antibacterial activities (as MIC80 values) were found to be not statistically significant (Table 4), different from the strong correlations reported in Shan et al. (2007). This could indicate that phenolic contents may have

little to no influence on the observed antibacterial activities. Although phenolics had been reported to exert antimicrobial activities, relationships between the structural features and their interactions with bacteria are not well-established (Bouarab-Chibane et al. 2019). The toxic effects on bacteria could be due to their ability to modify the integrity of the cell wall, change the permeability of the cell membranes, bind to enzymes, coagulate cell content, modify cellular pH, chelate iron, and affect DNA and RNA synthesis (Bouarab-Chibane et al. 2019). However, Bouarab-Chibane et al. (2019) suggested that the effects of phenolics could range from growth stimulation to antibacterial activities. Due to the limitations of our methods, we could not identify specific components involved in the observed antibacterial activities. Further studies are necessary to identify components and to elucidate mechanisms involved in the antibacterial activities of the fruit extracts.

Some studies associated the observed antibacterial activities of plant extracts to the presence of phytochemicals and to the pH of their tissues (Friedman and Jürgens 2000). Accordingly, we determined the pH of the fruit extracts (Table 3) and analyzed correlations with phenolics and antioxidant and antibacterial activities (Table 4).

Fruit acidity, as measured by pH and/or titratable acidity, is mainly due to organic acids such as citric and malic acids (Tomotake et al. 2006). Here, the strong negative correlation (r = –0.711, P < 0.05) between TPC (DM basis) and the pH of the fruit extracts may indicate an inverse relationship, i.e. the higher level of phenolics may be related to more acidity of the extract. Phenolics may contribute to fruit acidity due to the weakly acidic properties of the phenol group, and also due to carboxylic acid groups found in phenolic acids like hydroxybenzoic acids and hydroxycinnamic acids. However, the extent of contributions to the pH – as well as factors affecting changes in pH such as changes in maturity – could not be determined from our data.

The correlations between the pH of the extracts and the MIC80 values were found to be strongly positive (against E. coli, r = 0.679, P < 0.05), suggesting a strong direct relationship. The low MIC80 values (high antibacterial activities) may have been strongly influenced by the low pH of the extracts – especially observed from the most acidic fruits, G. binucao, C. hystrix, M altissima, and R. rosifolius. In citrus fruits, lemon and lime, citric acid was said to be responsible for the observed antibacterial activity (Tomotake et al. 2006). Moreover, the lower pH in plant extracts may be necessary for maintaining the stability and antioxidant and antibacterial activities of some naturally-occurring phenolics (Friedman and Jürgens 2000). Further investigation is needed to establish

Table 4. Pairwise correlation analysis among phenolics and antioxidant and antibacterial activities of selected Philippine indigenous fruits.

Correlation pairs Pearson’s r P-value (two-tailed)

I. Phenolics FM

Paired with:A. Flavonoids FM 0.614 0.059ns

B. % radical scavenging activity

0.739 0.015*

C. Ferric RPA 1.000 < 0.0001***

D. pH of extract –0.444 0.198ns

E. MIC80 vs. E. coli –0.294 0.409ns

F. MIC80 vs. S. aureus –0.501 0.140ns

II. Phenolics DM

Paired with:A. Flavonoids DM 0.721 0.019*

B. % radical scavenging activity

0.664 0.036*

C. Ferric RPA 0.849 0.002*

D. pH of extract –0.711 0.021*

E. MIC80 vs. E. coli –0.607 0.062ns

F. MIC80 vs. S. aureus –0.560 0.092ns

*Significant correlations at *P < 0.05, ***P < 0.001; ns – not significant; FM – fresh matter; DM – dry matter.



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whether there are any synergistic interactions between the major organic acids and the phenolics associated with the antibacterial activities of the selected fruits.

CONCLUSIONThe data obtained from the present study suggest that the selected Philippine indigenous fruits contain a variety of phytochemicals, good quantities of phenolics and flavonoids, and antioxidant and antibacterial species. Therefore, more detailed studies that identify specific compounds and bioactivities, and those that focus on structure-function relationships may provide a better understanding of how the phytochemicals from these fruits act in biological systems. Finally, future studies that will explore aspects related to nutrition, horticulture, and processing may help boost the utilization of these minor and underutilized fruits.

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APPENDIX

Notes on the Phytochemical Screening of Selected Philippine Indigenous FruitsAlkaloids perform functions related to protection from parasites, and from insects and animals (Cordell et al. 2001). Alkaloids had found applications in medicine and have a wide variety of physiological effects. The positive reaction of C. ovatum’s nut and pulp extracts was in agreement with the study of Salvador-Membreve et al. (2018). Some alkaloid compounds had been isolated from the roots of C. hystrix (Panthong et al. 2013). However, our test failed to detect alkaloids from the edible portion of C. hystrix.

The Keller-Kiliani test detects cardiac glycosides containing the sugar digitoxose. Three fruit samples gave positive results: F. pseudopalma, C. ovatum (pulp), and R. rosifolius. Our result for C. ovatum pulp agreed with that of Cajuday et al. (2017), while the result for R. rosifolius differed from that of Campbell et al. (2017).

Terpenoids are considered the most diverse group of plant secondary metabolites with at least 40,000 structures identified (Tholl 2015). Volatile or semi-volatile, low molecular weight isoprenes, mono-, sesqui-, and diterpenoids in plants serve as attractants of pollinators and as repellents against herbivores (Tholl 2015). Nonvolatile terpenoids in the roots may be involved in defense against competing plants and for signaling root development (Tholl 2015). The positive results for A. bunius fruit, C. ovatum pulp, and R. rosifolius were in agreement with previous reports (Ragasa et al. 2015; Campbell et al. 2017). C. ovatum nut oil was reported to contain terpenoid derivatives (Pham and Dumandan 2015). However, our screening failed to detect terpenoids in C. ovatum nut. The negative results for A. altilis and G. binucao also did not agree with previous reports (Amarasinghe et al. 2008; Ragasa et al. 2014b).

Saponins are a group of amphipathic molecules that contain a glycoside moiety and a triterpene or steroid moiety. They may serve as antifeedants against animals and protection against pathogens such as microbes and fungi (Hussain et al. 2019). Saponins were detected in all samples, except in G. binucao and in A. altilis. For R. rosifolius, our positive result differed from the negative result reported by Campbell et al. (2017).

Different plant parts may vary in their phytochemical content. Here, where only the edible portions were used, some phytochemical families were not detected. Some previous studies using the same plant species had detected and/or identified phytochemical compounds from another part such as leaves, roots, and peel. Ragasa et al. (2009) identified terpenoids and sterols from the leaves

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, flavonols, and glycosides (Seeka et al. 2016).

Comments on the Selected Antioxidant and Antibacterial AssaysIt was recommended that multiple assays be used to evaluate the antioxidant activities of plant extracts (Moon and Shibamoto 2009). Palafox-Carlos et al. (2012) stated that the best combination of antioxidant assays for different fruits was Trolox equivalent antioxidant capacity or ferric reducing antioxidant power (FRAP), and the DPPH assay. Here, we chose the DPPH assay, and the reducing power ability (RPA) assay as an alternative to the FRAP assay. The DPPH assay’s mechanism could be a mix of hydrogen atom transfer and single electron transfer (SET) (Prior et al. 2005). The FRAP assay’s mechanism could be SET, where a donor transfers an electron to the ferric ion in complex with the probe 2,4,6-tripyridyl-s-triazine (Prior et al. 2005). The RPA assay has the same key reaction as FRAP but it is monitored through the formation of the green to blue-colored ferric ferrocyanide complex, Fe(III)4 [(Fe(II)(CN)6)]3.

At present, there is no standardized assay or universal method to assess for antioxidant activity (Palafox-Carlos et al. 2012), and there seemed to be no strict rules regarding combinations and manner of expressing results (Moon and Shibamoto 2009). In the DPPH assay, some ways of expressing the results are % inhibition or % quenching (Molyneux 2004), IC50 or EC50 (Brand-Williams et al. 1995), and AEAC (Leong and Shui 2002). Results as IC50 or EC50 had a drawback of having to relate the lower values to higher antioxidant activity (Molyneux 2004). We chose to report the DPPH activities in terms of equivalence with AEAC, similar to the study of Leong and Shui (2002). Since the exact composition of the fruit extracts was not known, using AEAC allows some means of relating the structures, reaction mechanisms, and stoichiometries to the observed radical scavenging activities (Molyneux 2004).

In this study, the broth microdilution assay was employed instead of the disk diffusion assay for the antibacterial assay. The disk diffusion assay may not always be suited when testing plant extracts due to polarity differences of phytochemical components in the agar media (Klančnik et al. 2010). To allow better distribution of both polar and nonpolar components, we chose liquid media over solid or semi-solid agar media. Furthermore, the broth microdilution assay was reported to be more sensitive than agar diffusion methods and may be more suitable in determining quantitatively the antimicrobial activity of plant extracts (Klančnik et al. 2010).



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CAJUDAY LA, MEMBREVE, DMS, SERRANO JE. 2017. Evaluation of the antioxidant and anticancer activities of Canarium ovatum (Burseraceae) pulp extracts. Int J Biosci 11(3): 247–256.

CORDELL GA, QUINN-BEATTIE ML, FARNSWORTH NR. 2001. The potential of alkaloids in drug discovery. Phytother Res 15(3): 183–205.

HUSSAIN M, DEBNATH B, QASIM M, BAMISILE BS, ISLAM W, HAMEED MS, WANG L, QIU D. 2019. Role of saponins in plant defense against specialist herbivores. Molecules 24(11): 2067.

KLANČNIK A, PISKERNIK S, JERŠEK B, MOŽINA SS. 2010. Evaluation of diffusion and dilution methods to determine the antibacterial activity of plant extracts. J Microbiol Methods 81(2): 121–126.

MOLYNEUX P. 2004. The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating anti-oxidant activity. Songklanakarin J Sci Technol 26(2): 211–219.

OBICO JJA, RAGRAGIO EM. 2014. A survey of plants used as repellents against hematophagous insects by the Ayta people of Porac, Pampanga province, Philip-pines. Philipp Sci Lett 7(1): 179–186.

PRIOR RL, WU X, SCHAICH K. 2005. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J Agric Food Chem 53(10): 4290–4302.

RAGASA CY, TORRES OB, GUTIERREZ JMP, KRIS-TIANSEN HPBC, SHEN CC. 2015. Triterpenes and acylglycerols from Canarium ovatum. J Appl Pharm Sci 5(4): 94–100.

SALVADOR-MEMBREVE DM, CAJUDAY LA, SER-RANO JE, BALDO DEB. 2018. Immunomodulatory properties of ethanol extract of Canarium ovatum Burseraceae pulp. Trop J Pharm Res 17(8): 1565–1569.

THOLL D. 2015. Biosynthesis and biological functions of terpenoids in plants. Adv Biochem Eng Biotechnol 148: 63–106.

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

Scientific name, local name (family)

Place of collection Plant part; information reported Reference

Ficus ulmifolia Lam.“As-is”(Moraceae)

Philippines Leaves; terpenoids and sterols Ragasa et al. (2009)

Pampanga, Phil. Leaves and stems; ethnobotanical survey and insect repellant properties

Obico and Ragrario (2014)

Antidesma bunius (L.) Spreng“Bignay”(Phyllanthaceae)

Thailand Fruits; physicochemical properties, polyphenols, antiradical activity

Butkhup and Samappito (2011)

Philippines Fruits; phenolic content, antioxidant and antimicrobial properties

Lizardo et al. (2015)

Thailand Fruits; anthocyanidin, polyphenol, flavonoid contents, animal feeding studies

Ngamlerst et al. (2019)

Garcinia binucao (Blco.) Choisy“Binukaw,” “Batuan” (Clusiaceae)

Leyte, Phil. Fruits; proximate and mineral composition, physicochemical properties, vitamin A, C, tannin content, sensory properties

Quevedo et al. (2013)

Iloilo, Phil. Fruits; sterols and triglycerides Ragasa et al. (2014b)

Benguet, Phil. Fruits; phytochemical screening, total phenolics, flavonoids, radical scavenging activities

Barcelo (2015)



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Artocarpus altilis (Park.) Fosb.“Kamansi” (Moraceae)

Sri Lanka Fruit; identification of compounds, e.g. stilbenes, flavones, prenylated flavanols, benzofurans

Amarasinghe et al. (2008)

Malaysia Whole fruit, pulp; total phenolics, flavonoids, antioxidant, antimicrobial activities

Jalal et al. (2015)

Comoros Fruits; phenolic compounds, antioxidant activity, vitamin C, lipids, fiber, carbohydrates

Soifoini et al. (2018)

Citrus hystrix DC.“Kolong-kolong” (Rutaceae)

Thailand Roots; isolation and identification of coumarins, benzenoid and alkaloids, antioxidant, anti-HIV and antibacterial activities

Panthong et al. (2013)

India Fruit juice; total phenolics, tannins, flavonoids, antioxidant and enzyme inhibitory activities

Abirami et al. (2014)

Thailand Peels; isolation and identification 16 compounds including coumarin-flavanol-glucoside conjugates, anti-cholinesterase activity

Seeka et al. (2016)

Ficus pseudopalma Blco.“Niyog-niyogan” (Moraceae)

Camarines Sur, Phil. Leaves; ethnobotanical survey and nutritional composition

Santiago et al. (2014)

Camarines Sur, Phil. Leaves; triterpenes Santiago and Mayor (2014)

Laguna, Phil. Leaves; cytotoxicity and apoptotic activities

De las Llagas et al. (2014)

Mangifera altissima Blco.“Paho” (Anacardiaceae)

Philippines Morphological characterization Coronel et al. (2003)

Canarium ovatum Engl.“Pili” (Burseraceae)

Philippines Leaves, stem, mesocarp, kernel; chemical constituents

Ragasa et al. (2015)

Philippines Fruits; fatty acid composition, carotenoids, tocopherols, sterol contents

Pham and Dumandan (2015)

Philippines Pomace; fate of polyphenols after in vitro digestion

Arenas and Trinidad (2017)

Rubus rosifolius Sm.“Sampinit” (Rosaceae)

Jamaica Fruits; anthocyanins isolation and identification, lipid peroxidation, COX, tumor cell proliferation inhibitory assays

Bowen-Forbes et al. (2010)

Brazil Fruits; phenolic content, nutritional composition, antioxidant and antimicrobial activity.

Oliveira et al. (2016)

Jamaica Fruits; phytochemical screening, phenolics, antioxidant and enzyme inhibitory assays

Campbell et al. (2017)



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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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phytochemical screening, total phenolics, and antioxidant and … · 2020. 10. 12. ·...

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