Flavonoid compounds present in P. macrocarpa fruits were analyzed by using Reversed-Phase High Performance Liquid Chromatography (RP-HPLC). The flavonoid compounds have been identified according to their retention times and quantified according to their respective standard calibration curves (Figures 1, 2 and 3). Kaempferol, myricetin, naringin, and rutin were found as flavonoid compounds in pericarp of P. macrocarpa fruit (Table 1). The results also confirmed the presence of naringin and quercetin in mesocarp. Moreover, the seed of P. macrocarpa fruit contained only quercetin with the value of 0.452 mg/g dried weight (DW).

Among the flavonoids detected, the kaempferol content was significantly (p < 0.05) higher in the pericarp extract (76 μg/g DW). This value was lower than the kaempferol value in Chinese tea leaves (1.56–3.31 mg/g dried leaves) [12] but higher than in strawberries (8 μg/g dried-weight) [13]. This finding was supported by Zhang et al. [3] who isolated the kaempferol-3-O-β-d-glucoside from P. macrocarpa fruit.

Myricetin (59.90 μg/mg DW) located at the pericarp has a lower content compared with blackcurrant (71 μg/mg DW) but higher than blueberry (26 μg/mg DW) [13]. The amount of naringin present at the pericarp and mesocarp (39.00 and 1.90 μg/mg DW) was found to be lower than that of citrus (3.26 mg/g DW) [14]. Furthermore, rutin was detected in pericarp with a value of 17.80 μg/mg DW and it was found to be lower than citrus with a value of 3.26 mg/g DW [14] and Amaranthus viridis (58.2 μg/mg DW) [15]. Table 1 shows the concentration of quercetin present in mesocarp and seed with the values of 31.80 and 42.80 μg/mg DW, respectively. These values were found to be lower than the quercetin that is found in onion and garlic which is reported by Crozier et al. [16] with value 201 and 227 μg/mg DW.

The results obtained from antimicrobial assay are presented in Table 2 at a concentration of 0.3 mg/disc. According to Table 2, the extracts of P. macrocarpa fruit showed variable ranges of antimicrobial activities against 8 bacteria (Gram-positive and Gram-negative).

In general, most of the extracts indicated weak to moderate inhibitory activities against the bacteria tested. The extract of P. macrocarpa fruits at 0.3 mg/disc showed variable inhibitory activity against all bacteria with inhibition zone diameters ranging from 0.93–2.33 cm, as shown in Table 1. The microorganisms responses were different to the different extracts obtain from various parts of P. macrocarpa friut. Pericarp of P. macrocarpa fruit showed high inhibition on Gram-positive and Gram-negative bacteria compared to mesocarp, seed and kanamycin (1 μg/disc). In this study, it was shown that all the extracts could give higher inhibition to Gram-positive bacteria compared to Gram-negative bacteria. These results are in agreement with Othman [17] that Gram-negative microorganisms are typically more resistant to antimicrobial agents than Gram-positive bacteria. This has long been explained by the presence of an outer-membrane permeability barrier in Gram-negative bacteria, which limits access of the antimicrobial agents to their targets in the bacterial cells.

Furthermore, all the extracts assayed indicate no activity to weak inhibitory activities against all fungi. Based on Table 1, the antifungal activity of extracts from different parts, pericarp and mesocarp showed no antifungal activity against all fungi tested but seed extract at the concentration of 0.3 mg/well showed antifungal activity against Aspergilus. niger with inhibition 1.87 cm. This inhibition was low compared to Amphotericin B (25 μg/well). Interestingly, the tested bacteria and fungi showed different sensitivities to the different extracts of P. macrocarpa fruit. These results might be due to the presence of phytochemical in seeds such as phorbol esters. Borris et al. [18] mentioned that Phaleria sp. contain phorbol esters which are present in the seed of the fruit. Saetae and Suntornsuk stated that phorbol esters were found to be responsible for fungal growth inhibition in the crude extract of Jatropha curcas seed cake.

Antimicrobial activities observed in this study might be due to the presence of flavonoid compounds. Extracts of various medicinal plants containing phenolic and flavonoids have been previously reported to possess antimicrobial activity [19,20]. Vaquero et al. [21] investigated the properties of gallic, caffeic, vanillic acid, rutin, and quercetin of different wine against pathogenic microorganisms. Escherichia coli were the most sensitive bacterium and Flavobacterium sp. was resistant against all phenolic compounds tested. The flavonoid analyses (Table 1) revealed the presence of kaempferol, myricetin, naringin, quercetin and rutin in P. macrocarpa fruit. The presence of these compounds might contribute to antimicrobial activity of P. macrocarpa fruit since Cushnie and Lamb [11] reported that kaempferol, myricetin, naringin, quercetin and rutin have antimicrobial activity against human pathogenic microorganisms with some mechanisms of action such as inhibition of nucleic acid synthesis, cytoplasmic membrane function and energy metabolisms. The antimicrobial activity of the extracts of P. macrocarpa fruit might be due to one of the mechanisms of action mentioned above. Antimicrobial activity from pericarp, mesocarp, and seed of P. macrocarpa fruit might be due to the presence of kaempferol, myricetin, naringin, quercetin and rutin. Teffo et al. [22] isolated four kaempferols from Dodonaea viscosa Jacq. var. angustifolia leaf extracts and applied them to some human phatogenic bacteria. The results showed that all isolated kaempferols could inhibit growth rate of Staphylococcus aureus, Enterococcus faecalis, E. coli and Pseudomonas aeruginosa. In addition, Demetzos et al. [23] investigated the antimicrobial activity of myricetin and its derivate and the results showed that they could inhibit Gram-positive bacteria compared to Gram-negative. Furthermore, Li, Xu and Mandalari et al. [24,25] mentioned that quercetin and naringin have antimicrobial activity.

The results of the present study are quite encouraging as almost all of extracts exhibited antimicrobial activity against most of the pathogens. However, the antimicrobial activity varies widely, depending on the microorganism.

The fruits of P. macorcarpa were obtained from Faculty of Mathematic and Natural Sciences, University of Riau, Riau province, Indonesia. The plant species was identified by the laboratory of Plant Taxonomy staff at Herbarium Bogoriense, Bogor, Indonesia. The voucher specimen (SA1611/2008) was deposited at Herbarium Bogoriense, Bogor, Indonesia. The fruits were washed and separated into pericarp, mesocarp and seed. Those parts were air-dried for 7 days and kept for further analyses.

The extractions of P. macrocarpa were carried out based on Crozier et al. [16] with some modification. Air-dried powders of each part of P. macrocarpa (0.5 g) were weighed and placed into a 100 mL conical flask. 40 mL of methanol was added, followed by 10 mL and 6 M HCL solution. The mixture was stirred using a magnetic stirrer. The mixture was placed in a sample flask (250 mL), attached to reflux for 2 h at 90 °C, then the mixture was filtered using Whatman No.1 filter paper (Whatman, England), and taken to dryness by using a vacuumed Rotary Evaporator (Buchii, Switzerland) at 40 °C.

The flavonoid compounds of different parts of P. macrocarpa fruit were quantitatively measured by a reversed-phase HPLC technique based on the method described by Crozier et al. [16]. Flavonoid compounds standards consisted of quercetin, rutin, myricetin, kaempferol, naringin, apigenin, and luteolin. an aliquot of sample extract was loaded on a high-performance liquid chromatography (HPLC) Agilent-1200 series instrument equipped with a UV-Vis photodiode array (DAD) detector, binary pump, vacuum degasser, auto sampler and analytical column (Nova-Pak^® C18 60Å 4 μm 3.9 × 150 mm, Waters, NANPA, MA, USA). The mobile phase comprises deionized water and acetonitrile. The pH of water was adjusted to 2.5 with trifluoroacetic acid. The flavonoid compounds were detected at 365 nm. The column was equilibrated by 100% solvent A (15% acetonitrile) then the ratio of solvent B (35% acetonitrile) was increased to 100% in 20 min. This ratio was maintained for the rest of the analysis with a flow rate of 1 mL/min.