Fresh leaves, barks, twigs and fruits of A. lacucha were collected from Laguboti, Simalungun, Sumatera Utara, Indonesia. The plant sample was successfully identified by the Indonesian Institute of Sciences with the serial number: 2027/IPH.1.01/If.07/VIII/2017. The materials used in the present study were: Ethanol 96%; methanol pro-analysis (Merck KGaA); methanol for high-performance liquid chromatography (HPLC; Merck KGaA); distilled water; 2,2-diphenyl 1-picrylhydrazyl (DPPH; Sigma-Aldrich; Merck KGaA); neocuproine (Sigma-Aldrich; Merck KGaA); copper(II) chloride (Merck KGaA); ascorbic acid (Merck KGaA); Folin-Ciocalteu reagent (Merck KGaA); quercetin (ROTH; gallic acid (Sigma-Aldrich; Merck KGaA); Mueller Hinton agar (MHA; HiMedia Laboratories, LLC); nutrient agar (Oxoid; Thermo Fisher Scientific, Inc.); Mueller Hinton broth (MHB; Oxoid); chloramphenicol disk (Oxoid); and dimethyl sulfoxide. Bacterial cultures used in the present study were all purchased from American Type Culture Collection and included: Pseudomonas aeruginosa (cat. no. 9027); Escherichia coli (cat. no. 8939); Salmonella typhi (cat. no. 6539); Methicillin resistant Staphylococcus aureus (MRSA; cat. no. BAA-44); Staphylococcus aureus (cat. no. 6538); Cutibacterium acnes (cat. no. 11827); Bacillus subtilis (cat. no. 6633); Staphylococcus epidermidis (cat. no. 12228); and Streptococcus mutans (cat. no. 25175). The instruments used in the present study were UV-Vis spectrophotometer DU 720 (Beckman Coulter, Inc.), HPLC UFLC Prominence (Shimadzu Corporation) and Laminar Air Flow (Telstar AV-100; Azbil Corporation).

The powdered leaves, barks, twigs and fruits of A. lacucha (300 g) were refluxed with 3 liters of 96% ethanol a total of three times. The filtrate was collected and evaporated at 50˚C for 5 h until a viscous fraction was produced.

Phytochemical screening was conducted using a standard method for alkaloids, phenols, flavonoids, tannins, coumarin, saponins and steroids/triterpenoids detections as previously described (23).

Gallic acid was used as a standard for TPC and the previously described method was followed (24). Briefly, 50 µl of the extract sample was added to 500 µl of 10% Folin-Ciocalteu reagent and 400 µl of 1 M Na₂CO₃, and then incubated at 25˚Cfor 15 min before the sample was measured with a UV-vis spectrophotometer at a wavelength of 765 nm. The TPC was expressed as g of gallic acid equivalents (GAE)/100 g extract (g GAE/100 g extract).

Quercetin was used as a standard for TFC and the previously described method was followed (25). Briefly, 100 µl of extract was added to 300 µl of methanol pro-analysis, 20 µl of 10% AlCl₃, 20 µl of sodium acetate and 560 µl of distilled water, and then incubated at 25˚C for 15 min and measured with a UV-vis spectrophotometer at a wavelength of 415 nm. TFC was expressed as g of quercetin equivalent (QE)/100 g of extract (g QE/100 g extract).

Ascorbic acid was used as a standard for antioxidant activity, following the previously described method (26) with modifications. The 12.5 µl of 10.000 µg/ml extract was added to 125 µl methanol pro-analysis and 750 µl of 50 µg/ml DPPH (purple powder in methanol pro-analysis), and then incubated at 25˚C for 30 min and the absorbance was measured with a UV-vis spectrophotometer at a wavelength of 517 nm. Antioxidant activity was expressed by the antioxidant capacity equivalent to ascorbic acid [mg ascorbic acid equivalent antioxidant capacity (AEAC)/g extract].

Cupric ion reducing antioxidant capacity (CUPRAC) method. Ascorbic acid was used as a standard for antioxidant activity, following the previously described method (27) with modifications. The 12.5 µl of 10.000 µg/ml extract was added to 237.5 µl of 77.08 mg/ml ammonium acetate and 750 µl of CUPRAC solution [mixture of 1,705 µg/ml CuCl₂ and 1,562 µg/ml neocuproine (in a 1:1 ratio)], and then incubated at 25˚C for 30 min and the absorbance was measured with a UV-vis spectrophotometer at a wavelength of 450 nm. Antioxidant activity was expressed by the antioxidant capacity equivalent to ascorbic acid (mg AEAC/g extract).

A number of flavonoids were analyzed using the HPLC method. A reversed phase of HPLC method with a LiChrosper^® (Merck KGaA) 100 RP-C18 5 µm column (100 mm, 4 mm inner diameter), water (solution A) and methanol (solution B), and a linear gradient mode were designed in accordance with 40-60% solution B for 5 min, then linear gradient mode B to 70% on 10th min, linear gradient mode B to 40% on 15th min. The column temperature was 30˚C, the flow rate was set at 1 ml/min, the injection volume was 20 µl, and UV-vis detector was used at 360 nm.

Standard solutions. Standard solutions of luteolin 7-O-glucoside, rutin, quercetin, kaempferol and apigenin were prepared and analyzed with HPLC using 20 µl of each standard (50 µg/ml).

Extract sample preparation. For each sample, 100 mg of dry extract was dissolved in 10 ml of methanol for HPLC analysis. Subsequently, 20 µl of the solution was injected for HPLC.

Bacterial suspension (300 µl; ~1x10⁸ cells) was pipetted on a sterile Petri dish, before pouring 15 ml of MHA medium until homogenous and solid. The disk containing 15 µl of extract with 5, 10, 15 and 20% concentrations were placed on the media. DMSO was used as a negative control and chloramphenicol was used as a positive control. The plates were incubated at 37˚C for 18-24 h, before the clear zones were measured using a Vernier-caliper.

Microdilution. The microdilution was carried out by adding 100 µl MHB into each well of a 96-well sterile microplate with 100 µl extract in the first column until homogenous. From these wells in the first column, 100 µl sample was taken and added to the wells in the next column. The dilution was repeated as aforementioned until reaching the well with the lowest concentration that gave a clear zone of inhibition. Subsequently, 10 µl of bacterial suspension was added into each well after the absorbance on a UV-vis spectrophotometer at wavelength 625 nm was measured to be 0.08-0.13. The MHB was used as a negative control and chloramphenicol was used as a positive control. The microplate was incubated at 37˚C for 18-24 h and the turbidity level was observed in each well. After obtaining the minimum inhibitory concentration (MIC) value, non-turbid samples were streaked on MHA media and incubated at 37˚C for 24 h. Inhibited bacterial growth was observed to obtain the minimum bactericidal concentration (MBC).

Data were statistically analyzed using Minitab^® (version 21; https://dti.itb.ac.id/minitab/) software. Two-way analysis of variance (ANOVA), followed by Tukey's post hoc test, and Pearson correlation was used to analyze the collected data. P<0.05 was considered to indicate a statistically significant difference.

There were seven groups of compounds identified in the phytochemical screening, namely alkaloids, flavonoids, phenols, saponins, tannins, steroid/triterpenoid and coumarins, as presented in Table I. The four parts of the A. lacucha plant, namely leaves, barks, twigs and fruits positively contained flavonoids, phenols, saponins, tannins and coumarins. This result was similar to the phytochemical screening of the Artocarpus genus in the study by Buddhisuharto et al (28).

The total phenolic and flavonoid contents are presented in Table II. Based on the table, the highest TPC was observed in barks, while the highest TFC was obtained from leaves. Based on the statistical analysis using ANOVA-Tukey in Table II, a significant difference was revealed in the TPC values between: i) Leaves vs. barks and fruits; ii) barks vs. twigs and fruits; and iii) twigs vs. fruits, while no significant difference was revealed in leaves vs. twigs with P<0.05. A significant difference was also revealed in the TFC values between leaves vs. barks, twigs and fruits, while no significant difference was revealed between barks vs. twigs and fruits with P<0.05.

Folin-Ciocalteu reagent reduction by phenolic compounds under alkaline conditions is the basis for the Folin-Ciocalteu test, as a phosphomolybdic acid/phosphotungstic acid complex is reduced to produce a blue chromophore with a maximum absorption at 765 nm (29). This test is beneficial for determining the TPC, based on the simplicity, reproducibility and robustness of the test. However, this test is sensitive to pH, temperature and reaction time (30).

Phenolic compounds can scavenge reactive oxygen without causing other oxidative reactions. Thus, TPC evaluation is currently one of the standard tests for phytochemical studies to determine the antioxidant activity in an extract because 5,7,3',4'-hydroxy-substituted flavonoids are considered to have efficient radical scavenging capacity (31,32). This was in accordance with previous results on the direct association between phenolic and flavonoid contents on biological activity of plant extracts (33-36).

The antioxidant activity is presented in Table III. The highest antioxidant activity using the DPPH method was revealed in barks, while the highest antioxidant activity using the CUPRAC method was revealed in twigs.

From the DPPH method in Table III, a significant difference (P<0.05) was revealed in: i) Leaves vs. barks, twigs and fruits; ii) barks vs. twigs and fruits; and iii) twigs vs. fruits. In the CUPRAC method, a significant difference (P<0.05) was revealed in fruits vs. leaves, barks and twigs, while no significant difference was revealed in leaves vs. barks and twigs nor in barks vs. twigs.

DPPH is a water-insoluble and stable compound, and the latter two properties of DPPH are responsible for its use as a radical model in various antioxidant studies (37-39). DPPH can accept electrons or hydrogen radicals to become a stable molecule (38,39). DPPH has a purple color (at 515-517 nm in an alcohol solution), but can turn to a yellow color when the counterparts, N radicals, are reduced (37-39). Thus, the higher the antioxidant activity, the stronger the reagent decolorization. The DPPH method is simple, quick to perform, cost-effective and efficient, thereby becoming the most commonly used method for antioxidant screening of constituents, mixtures, extracts or biological matrices (38,39).

Additionally, the CUPRAC method was originally used to determine the total antioxidant activity and has been modified for various measurement methods (such as calculating the IC₅₀ or the antioxidant capacity in an antioxidant). Antioxidant activity is based on the reduction of Cu²⁺ to Cu⁺. Similar to other tests, ligands are used to form cuprum-ligand complexes to facilitate absorbance measurements. Neocuproine (2,9-pimethyl-1,10 phenanthroline) is a ligand commonly used in the CUPRAC test (35). This method has been applied to various matrices containing lipophilic and hydrophilic antioxidants (39,40).

L-ascorbic acid, also known as vitamin C, is a water-soluble antioxidant that has a role as an electron donor, which can neutralize and reduce ROS, protecting the cells from free radical damage (41,42). As an antioxidant, ascorbic acid can directly react with free radicals, either with or without enzyme catalysts, and form mono-dehydroascorbic and/or dehydroascorbic acids (19).

The correlation results of total phenolics, total flavonoids and antioxidant activity are presented in Table IV. Schober et al (43) categorized the interpretation of correlation based on the value obtained as: i) If the correlation value is 0.00-0.09 the parameters are in a negligible correlation; ii) a correlation value of 0.10-0.39 is a weak correlation; iii) a correlation value of 0.40-0.69 is a moderate correlation; iv) a correlation value of 0.70-0.89 is a strong correlation; and v) a correlation value of 0.90-1.00 is an extremely strong correlation. From Table IV, the correlation of the TPC with DPPH and CUPRAC in leaves is extremely strong, while the TFC with DPPH and CUPRAC in leaves is strongly correlated. The correlation of the TPC with DPPH and CUPRAC as well as the TFC with the DPPH method in barks is extremely strong, while the TFC with the CUPRAC method in barks was strongly correlated. An extremely strong correlation of the TPC with DPPH and CUPRAC was revealed in twigs, while the TFC with DPPH in twigs was strongly correlated and with CUPRAC in twigs was extremely strong. In fruits, extremely strong correlations of the TPC and TFC was revealed with the DPPH method, while a strong correlation was revealed with the CUPRAC method. Gaweł-Bęben et al (44) considered that the high content of phenolic compounds in plant extracts was often correlated with their significant antioxidant activity.

Several flavonoids identified using HPLC are presented in Table V. The standards were three major flavonols (namely, kaempferol, rutin and quercetin) and two major flavones (apigenin and luteolin) in plants (45). The Artocarpus genus was reported to contain kaempferol (46), rutin (47,48), quercetin (46,48), luteolin and apigenin (49). The highest levels of luteolin-7-glucoside, quercetin, kaempferol and apigenin were revealed in leaves, while the highest levels of rutin was revealed in barks. Luteolin-7-O-glucoside was undetected in barks, rutin was undetected in leaves, kaempferol was undetected in barks and twigs, and apigenin was undetected in fruits. This may occur due to low levels in each part, and thus remained undetected.

It is important to ensure all analytes have separated completely; however, it is not always easy to achieve, especially in the case of natural products (50-52), which are known to contain >50 different types of phenols and flavonoids. In the present case, finding a gradient method capable of achieving complete separation for all constituents is almost impossible since a number of these phenolic compounds are similar in chemical composition and polarity (53). However, in the present study, orientation (a search for a suitable system and mobile phase) was carried out to determine the appropriate analyte separation method so that well separated analytes were obtained when the levels of several flavonoid compounds present in the sample were determined.

Antibacterial activity, as an inhibition zone diameter, is presented in Table VI and the MIC and MBC values are presented in Table VII. From these results, A. lacucha has an increased antibacterial effect against Gram-positive bacteria, namely MRSA, S. aureus, C. acnes, B. subtilis, S. epidermidis and S. mutans. This was in line with previous studies, which reported that Artocarpus integra (47,54) and Artocarpus altilis (55) had antibacterial activity and demonstrated a higher activity on Gram-positive bacteria compared with Gram-negative bacteria.

This may be due to the different constituents and organization of the cell membrane (56). Gram-negative bacteria have an outer phospholipid membrane called the lipopolysaccharide component structure. Therefore, the cell wall has a reduced permeability to antimicrobial agents. Gram-positive bacteria are more sensitive to antimicrobial agents, as they only have an outer and ineffective peptidoglycan layer. Therefore, Gram-negative bacteria have a more complex outer layer compared with Gram-positive bacteria, which is capable of resisting the antimicrobial agents to a greater extent compared with Gram-positive bacteria (57).

However, an inhibition diameter for the fruit extract was obtained with disc diffusion and MICs were revealed for Gram-negative bacteria, namely P. aeruginosa, E. coli and S. typhi. High concentrations of 2,250 and 3,000 µg/disk fruit extract produced small inhibition diameter zones of 6.80±0.40 mm and 7.4±0.00 mm against P. aeruginosa, respectively; 7.43±0.05 mm and 7.46±0.05 against E. coli, respectively; and 6.40±0.00 and 7.40±0.00 against S. typhi, respectively. As a positive control, chloramphenicol provided antibacterial activity based on the inhibition zone diameter and MIC against all nine species of bacteria. Chloramphenicol is a broad-spectrum antibiotic that can be used for Gram-positive and Gram-negative bacteria, although it is most commonly used for treating S. typhi bacterial infection.

Plants are a source of a number of important components, such as phenolic compounds that can scavenge free radicals and reduce oxidative stress (58-60). Compounds belonging to the phenolic group that provide antioxidant effects are flavonoids, phenolic acids, lignans and stilbenes. The properties of these compounds are used by plants as defense mechanisms against the adverse effects of UV radiation, temperature and mechanical damage, as well as releasing important chemical defenses through their specific physiological actions against herbivores and insects (60-65). In addition, phenolic compounds can prevent adverse changes in living organisms by reacting directly with fatty acid oxidation products. They also prevent deterioration of organoleptic and sensory characteristics in food products (66-68).

Plants have been demonstrated to produce phenolics in response to oxidative stress (69,70), including flavonoids. Flavonoids are a well-known class of phenolic compounds with antioxidant properties (71). In addition to being antioxidants, flavonoids also function as scavenger chemical controls due to ROS exposure. This dual role applies to the large number of free OH-groups, especially 3-OH and the higher reactivity of flavonoid is observed in hydroxyl groups with oxidants (65,72,73). Currently, it is considered that phenolics and flavonoids function to promote health as anti-inflammatory and antioxidant agents, thus protecting the body against chronic diseases (74-76). In addition, these secondary metabolites also provide protection to plants against abiotic and biotic stresses (77,78).

Free radicals are compounds or molecules that have one or more unpaired electrons in its outermost orbit, causing them to become very reactive in order to find bonds, such as by attacking and binding to the electrons of molecules (17,18,79).

Antioxidants are substances from food or in the body that can delay, control or prevent oxidative stress at low concentrations, thus reducing the deterioration of food and the spread of degenerative diseases in the body. Antioxidants that fit this definition include free radical scavengers, singlet oxygen quenchers, inactivators of peroxides and other ROS, metal ion chelators, secondary oxidation product quenchers, and pro-oxidative enzyme inhibitors (80). Currently, the use of antioxidant compounds has been growing rapidly along with an increasing value of free radical activity against several degenerative diseases such as cancer, cardiovascular disease, atherosclerosis and aging (22,81).

Antioxidants obtained from food are useful to eliminate excessive ROS in the body. Antioxidants will be utilized to protect biomolecules from being oxidized. Thus, it is important to investigate whether there is a correlation between the strong antioxidants and oxidative stress level (82,83). Phenolic compounds are abundant in plants and exhibit antimicrobial (84-86) and antioxidant (34) properties, thus can be applied for further usage in pharmaceutics, cosmetics and natural food preservatives (86,87,88).

The direct reaction that occurs between DPPH and antioxidants has been used to measure the antioxidant activity (50) and high percentage of DPPH radical-scavenging compound indicates the excellent antioxidant activity (36). In the present study, the antioxidant capacity using DPPH and CUPRAC methods indicates that an increased antioxidant activity will produce an increased antibacterial activity. However, A. lacucha is known to have antibacterial activity, thus a further study is necessary to determine the mechanism and location of the antibacterial action.

Antioxidant compounds in plants can act as antibacterials in several ways, such as by inhibiting oxidation in several areas of the cell, damaging the membrane structure leading to cell nutrient leakage and blocking the binding sites of DNA gyrase for DNA coiling, thus negatively affecting the growth of the cell (89).

By reviewing the literature, Kuete (90) classified the MIC values to indicate a significant activity if <100 µg/ml, to have good activity if 100-625 µg/ml and to have low activity if >625 µg/ml. In the present study, low antibacterial activity was revealed with the MIC value of 0.16% (barks against MRSA) and the results of other extracts demonstrated low antibacterial activity based on their MIC values. Similar observations for MIC values with minor variations have also been observed in other studies (54-57,90-94). However, Ouchari et al (95) reported that the antimicrobial inhibition zones were grouped into four categories, namely weak (<5 mm), moderate (5-10 mm), strong (10-20 mm) and highly strong (>20-30 mm) antimicrobial activities. In the present study, the inhibition zone was classified as moderate to strong (95) for the bark and twig extracts against MRSA, S. aureus, C. acnes, B. subtilis, S. epidermidis and S. mutans at concentrations starting from 750 µg/disc, as well as for the fruit extract against P. aeruginosa, E. coli, S. typhi, MRSA, S. aureus, C. acnes, B. subtilis, S. epidermidis and S. mutans at concentrations starting from 2,250 µg/disc. This was based on the results of the disc diffusion method, and thus the bark and twig extracts can be considered as a potential for further investigation and development as an antibacterial agent. However, the disc diffusion method has limitations, as polar compounds diffuse from the disk to the agar medium easily, while semi-polar and non-polar compounds will remain undiffused (96).

Based on the mechanism of action to inhibit the growth of microorganisms, Khameneh et al (97), Radji (98), Kapoor (99) and Reygaert (100) stated that antibacterials can be classified as: First, cell wall synthesis inhibitors. The bacterial cell wall is important for maintaining the structure of bacterial cells. Substances that can damage the cell wall will lyse the cell wall and affect the shape and structure of the cell, thereby killing the bacterial cell (97-100). Second, cell membrane disruptors. The cell membrane functions to regulate nutrients and metabolites inside and outside the cell (97-100). A number of antibacterials can disrupt the cell membrane and negatively affect the growth of the bacterial cell including lipopeptides (100) such as polymyxins (97). Third, nucleic acid biosynthesis disruptors. The process of DNA replication in bacterial cells is an important cycle in the cell. A number of antibacterials can interfere with nucleic acid metabolism, thus affecting the entire growth phase of bacteria, including quinolons (97,99,100) such as ciprofloxacin (97). Fourth, protein synthesis inhibitors. In protein synthesis, the DNA is transcribed into mRNA and the mRNA is translated continuously into protein (97-100). Antibacterials can inhibit these processes, so that protein synthesis will be disrupted, such as macrolides, aminoglycosides, tetracyclins, chloramphenicol and oxazolidinones (97,99,100).

In various studies it has been reported that A. lacucha has pharmacological activities, such as acting as an antimicrobial (101), as a treatment for skin ailments (102), antiglycation (103) and traditionally this plant has been used by communities in Thailand for tapeworm infections (5), as an anti-inflammatory, to relieve stomachaches, headaches and fevers (6,101). In North Sumatera, Indonesia, the fruit is used as seasoning in food. Lakoochins A and B, the isolated compounds from A. lacucha, have antimycobacterial activity (5) and the isolated compound cathecin has antinociceptive activity (6). It has also been reported by Islam et al (6) that A. lacucha extracts demonstrated a low toxicity profile in acute toxicity tests. In a previous study, it has also been concluded that leaves extract from this plant have a strong antioxidant activity and the potential to inhibit bacterial growth (104).

In conclusion, A. lacucha has the potential to be developed as a natural antioxidant with improved antibacterial properties against Gram-positive bacteria compared with Gram-negative bacteria. The present study indicated that a number of the compounds in A. lacucha, namely from flavonoids, support the antioxidant and antibacterial activities. Therefore, further investigations should be carried out in the Artocarpus genus in order to potentially find new natural resources.