Total Phenolic and Flavonoid Contents
Phenolics are one group of larger secondary metabolites which are synthesized by plants and are utilized as UV, wounding and infection protectant in plants. Phenolics have been indicated to have several biological activities such as antioxidant, antimutagenic, anticarcinogenic, anti-inflammatory and antimicrobial activities in human .
As presented in Table 1, Mesocarp of P. macrocarpa fruit showed the highest phenolic content (60.5 ± 0.18 mg GAE/g DW) followed by pericarp and seed with a value of 59.16 ± 0.037 and 47.70 ± 1.036 mg GAE/g DW respectively. The total flavonoid content of pericarp was found to be higher (161.3 ± 1.58 mg rutin equivalent/g DW) than mesocarp and seed with the values of 131.74 ± 1.665 and 35.99 ± 2.471 mg rutin equivalent/g DW respectively.
From the results obtained, the total flavonoids contents was higher compared to the previous results which were reported by Rohyami  who reported the total flavonoids content from dry fruit (without seed) of P. macrocarpa (22.33 mg rutin equivalent/g DW) extracted by soxhlet using methanol as solvent.
Antioxidant Assay for P. macrocarpa Extracts
Total Antioxidant Activity Assay
The FTC method was used to measure the peroxide level during the initial stage of lipid oxidation. Peroxides are formed during the linoleic acid oxidation, which react with Fe2+ to form Fe3+. The latter ions form a complex with SCN- ion and this complex has a maximum absorbance at 500 nm . The individual activity of different parts of P. macrocarpa fruit extract by the FTC method (Figure 1) showed low absorbance values compared to negative control, which indicated high levels of antioxidant activity, as shown in Figure 2. The levels of antioxidant activity of the samples tested were lower compared to BHT (butyl hydroxyl toluene). Figure 2 shows the antioxidant activity of the tested samples measured by using TBA on the last day where the absorbance of negative control decreased. Results show a somewhat different pattern from that of the FTC method, where pericarp showed high antioxidant activity compared to mesocarp and seed. Pericarp extract showed no significant difference as compared to BHT as a standard antioxidant, which indicated the appreciable antioxidant activity of pericarp in retarding the linoleic acid oxidation. However the mesocarp and seed extract showed significant (P < 0.01) lower activities as compared to the BHT. The differences in antioxidant activities observed here could be due to numerous factors, including the different mechanisms involved in the FTC and TBA methods, secondary metabolites in the sample, the antioxidant mechanisms exhibited by the compounds and possibly, due to the synergistic effects of different compounds. However, the antioxidant activity of P. macrocarpa fruit might be attributed to the presence of flavonoid compounds presented in different part of the fruits. Hendra et al.  investigated the presence of flavonoid compound in different parts of P. macrocarpa fruit, and pericarp was found to contain several flavonoids such as kaempferol, myricetin, naringin, and rutin. Although the naringin and quercetin were found in mesocarp and quercetin in the seed extract.
Antioxidant Activity of P. macrocarpa Fruit
Antioxidant is defined as a substance which significantly delays or inhibits oxidation process. The antioxidant activity is measured indirectly by determining the inhibition rate of oxidation processes in the presence of an antioxidant . DPPH, an organic stable radical in its crystalline form and in solution, is widely used to determine the antiradical activity of a given compound or extract. The antioxidant activity of a given compound or extract is also often associated with its radical-scavenging activity [18, 19]. Figure 3 shows the free radical scavenging activity of pericarp, mesocarp, seed extracts of P. macrocarpa fruit and BHT at different concentrations. The results showed that pericarp gave the highest scavenging activity which was 71.97% while the lowest was seed extract which was 54.44% at concentration of 300 μg/ml.
Furthermore, the ability of extracts to reduce iron (III) to iron (II) was determined and compared to butylated hydrotoluene (BHT) which are known to be strong reducing agents as shown on Figure 4. The result shows that the extracts could reduce iron in a dose dependent manner in pericarp, mesocarp and seed with values of 92.45%, 78.78%, 66.40% respectively.
Figure 5 shows the NO scavenging activity of all the tested samples. All samples exhibited NO scavenging activity in a dose-dependent manner. The corresponding IC50 values for NO scavenging activity are presented in Table 2. All extracts showed IC50 values between 200-400 μg/ml which indicated moderate NO-scavenging activity. The NO scavenging values were categorized according to Tsai et al  and Oskoueian et al.. The IC50 concentration (Table 2) showed significant differences in DPPH, FRAP and nitric oxide scavenging activity among the extracts obtained from different parts.
The antioxidant activity of P. macrocarpa fruit might be due to the presence of phenolic and flavonoid compounds since Hendra et al. reported the presence of kaempferol, myricetin, naringin, quercetin, and rutin as the major flavonoids present in P. macrocarpa fruit. The correlation between flavonoids and their antioxidant activity might be due to the presence of a 3-hydroxyl group in the heterocyclic ring while additional hydroxyl or methoxyl groups at positions 3,5 and 7 of rings A and C seem to be less important . This statement is in accordance with Amic et al.  who investigated 29 flavonoids for free radical scavenging activity followed with analysis by using quatitative structure-activity relationship (QSAR) software. The results showed that the developed structure-antiradical activity indicated that highly active flavonoids possess a 3'4'-dihydroxy occupied B ring and/or 3-OH group.
During inflammation, the ultimate phase of a series of signaling events, macrophages induce the expression of pro-inflammatory genes such as inducible nitric oxide synthase (iNOS). This enzyme is up-regulated by secretion of pro-inflammatory cytokines, and produces NO from L-arginine. The regulation of NO production is therefore an important target for inflammatory disease [21, 24, 25].
Anti-inflammatory activity was assessed using LPS/IFN-γ stimulated RAW 264.7 macrophages and NO production quantification using the Griess reagent. The cytotoxic effect of the extract was evaluated on macrophages using MTT to ensure that the anti-inflammatory activity was not due to cytotoxicity effect from the extract.
From Figure 6(a, c, e), all the extracts showed NO inhibitory effect in a dose-dependent manner. Mesocarp showed highest NO inhibition compared to pericarp and seed with values of 69.5 ± 1.4%, 63.4 ± 2.7%, 38.1 ± 1.2% respectively. According to Kim et al.  classification, the percentage of NO inhibition from plant extract represent it's anti inflammatory potential therefore pericarp and mesocarp extract could be considered as moderate and seed extract as an week anti-inflammatory agent. As shown in Figure 6(b, d, f), pericarp and mesocarp showed percentage of cell viability more than 90% for all concentration except for seed extract. Seed extract with concentration more than 6.25 μg/ml showed percentage of cell viability dropped significantly.
The production of NO in positive control (L-NAME) was lower than all the extracts tested in this study. The lowest cell viability of the pericarp and mesocarp in RAW 264.7 cell line ranged from 90-95% while the seed extract showed 62.47% cell viability.
According to the results obtained from anti-inflammatory assay, P. macrocarpa extract appeared to be potent as anti-inflammatory agent and to the best of our knowledge this is the first report on anti inflammatory activity of P. macrocarpa fruit. The ability of P. macrocarpa fruit as anti-inflammatory agent might be due to the presence of phenolic and flavonoid compounds or other phytochemicals such as terpenoid compound which could play a role as anti-inflammatory agents. In vitro studies have confirmed that the flavonoids were able to inhibits nitric oxide production and the expression of iNOS but their strength depends on their structure or subclass of flavonoids . Oskoueian et al  reported the role of phenolic compounds in antioxidant activity and their ability to act as free radical and NO scavengers, leading to the formation of phenoxyl radicals. Recently, Kazlowska et al. suggested that the inhibition of iNOS in the RAW 264.7 cell is due to the NO suppressing action of flavonoid and phenolic compounds such as rutin and cathecol.
The results of cytotoxic activity of samples tested are presented in table 3. From the results obtained, all the extracts could inhibit all cancer cells and normal human hepatocyte cells. Based on Boyd , a plant extract is usually regarded as interesting for in vitro cytotoxic activity when IC50 < 100 μg/ml. Based on this study, all the extracts showed interesting in vitro cytotoxic activity to all cancer cells with various IC50. Jonville et al  mentioned that the definition of promising activity was reserved for extracts with IC50 values of less than 50 μg/ml and that further investigation regarding to isolation compounds and drug mechanisms were needed. From table 3, all the extracts show promising activity toward in vitro cytotoxic activity against MCF-7 and HeLa cell lines with IC50 between 25.5 - 40.8 μg/ml. However, when applied to HT-29 cell line, only seed extract showed promising in vitro cytotoxic activity.
Furthermore, Tamoxifen as positive control showed the lowest IC50 value as compared to other extracts. From table 3, there were no significant differences of cytotoxic activity between tamoxifen and seed for HT-29 and MCF-7 cell lines. When tamoxifen was compared to pericarp and mesocarp, pericarp showed significantdifferences of cytotoxic activity for all cancer cells and mesocarp showed no significant difference with tamoxifen activity only for MCF-7 cell lines.
Since it is known that different cell lines might exhibit different sensitivities while treated with different plant extracts therefore the use of more than one cell line seems necessary for the comprehensive plant extract anti cancer activity screening. Cell type cytotoxic specificity of plant extracts is likely to be due to the presence of different classes of compounds in the extract .
Phaleria macrocarpa potency as an anticancer agent has been known empirically for generations, and its stem, fruit, seed or leaf boiled water extract have been used by people in Indonesia . Faried et al. have isolated gallic acid from fruits of P. macrocarpa and shown that it selectively induces cancer cell death in various cancer cells, such as human esophageal cancer (TE-2), gastric cancer (MKN-28), colon cancer (HT-29), breast cancer (MCF-7), cervix cancer (CaSki), and malignant brain tumor (CGNH-89 and CGNH-PM). The results demonstrated a significant inhibition of cell proliferation in a series of cancer cells. The cytotoxic activity results presented in this report (Table 3) are in agreement with Faried et al. although further investigations on compounds responsible for cytotoxic effects in this fruit are required.