Phytochemical analysis, antioxidant, antibacterial and cytotoxicity properties of keys and cores part of Pandanus tectorius fruits

2.1 Materials

The sample of P. tectorius fruits was collected from the same location of beach area in Setiu Wetland, Terengganu, Malaysia. The sample (30 kg) was collected from March to May 2013. After collection, P. tectorius fruits were divided into two parts, keys (13 kg) and cores (4 kg) (Fig. 1). The samples were cut into small pieces, dried using freeze dryer and then grinded to be a powder (keys = 2.6 kg, cores = 500 g). Antioxidant and cytotoxicity properties were analyzed by DPPH free radical scavenging and MTT using 96 well plate by Elisa plate reader, respectively. Mueller–Hintonagar (MHA) was used for the screening of antibacterial activity followed by kinetic study using 96 wells plate method. All other chemicals and reagents were of analytical reagent grade and purchased from either Sigma or Merck.

2.2 Extraction of samples

The extracts from keys and core part were obtained by successive extraction (slightly modified from Andriani et al., 2011). Samples was extracted sequently using n-hexane, ethyl acetate and methanol to yield hexane crude extract (PHF), ethyl acetate crude extract from keys (PEK) and cores (PEC) part, and methanol crude extract from keys (PMK) and cores (PMC) part. Hexane crude extract from keys and core part was combined due to the same TLC pattern (data not shown) and labeled as PHF. The ground sample was soaked in the solvents and then extracted until colorless at room temperature for 24 h. The each extract was evaporated under reduced pressure to give PHF (15 g), PEK (8.9 g), PEC (2.7 g), PMK (386.5 g) and PMC (108 g) crude extracts. The extracts were analyzed on phytochemical analysis, TPC, and their bioactivities by antioxidant, antibacterial and cytotoxicity properties against five cell lines (RAW, L-6, HepG2, MCF-7, and HeLa).

2.3 Phytochemical screening

Phytochemical analysis of phenolics, tannins, flavonoids, terpenoids, alkaloids, steroids, saponins and glycoside were adapted from Yadav and Agarwala (2011) as follows.

Test for phenols and tannins. 2 mL of 2% Ferric chloride solution was added to the crude extracts. A blue-green to black color indicates the presence of phenols and tannis.

Test for flavonoid. Crude extract was mixed with 2 mL of 2% sodium hydroxide solution. An intense yellow color was formed turned to colorless on addition of few drops of diluted acid indicated the presence of flavonoids.

Test for steroids. Crude extract was mixed with 2 mL of chloroform and concentrated H₂SO₄ was added sidewise of test tube. A red color produced in the lower chloroform layer indicated the presence of steroids.

Test for terpenoids. Crude extract was dissolved in 2 mL of chloroform and evaporated to dryness. Then 2 mL of concentrated sulfuric acid was added and heated for about 2 min. A grayish color of mixture indicated the presence of terpenoids.

Test for alkaloids. Crude extract was mixed with 2 mL of 1% HCl and heated gently. Mayer’s and Wagner’s reagent were then added to the mixture. Turbidity precipitate formed shows the positive result for the presence of alkaloids.

Test for saponins. Crude extract was diluted with 5 mL of distilled water in a test tube. The mixture was shaken vigorously. The formation of stable foam indicated the presence of saponins.

Test for glycosides. Crude extract was mixed with 2 mL of glacial acetic acid containing 1–2 drops of 2% ferric chloride solution. The mixture was then poured into another test tube containing 2 mL of concentrated sulfuric acid carefully. A brown ring interphase shows the positive result for the presence of cardiac glycosides.

2.4 Total phenolic content (TPC) (Binsi et al., 2013)

The modified Folin–Ciocalteu assay was used for the determination of the total phenol content. 2.0 M Folin–Ciocalteu phenol reagent, gallic acid (Sigma, Germany) and anhydrous sodium carbonate (Sigma, USA) were reagent for this method. For the preparation of calibration curve, 0.1 mL aliquots of 0.3, 0.105, 0.075, 0.024, 80 mg/mL ethanolic gallic acid solutions were mixed with 7 mL of distilled water and 0.5 mL Folin–Ciocalteu reagent was put into test tube. The mixture was shaken well and incubated for 8 min at room temperature. Then, 1.5 mL of 1.85 M sodium carbonate (Na₂CO₃) and 0.9 mL of distilled water were added to the mixture. The mixtures were incubated for 2 h in the dark room at room temperature and the absorbance for were measured using spectrophotometer at 765 nm. Same procedure was done for test material with just replacing gallic acid with the crude extract tested. The total phenolic content of the extract was determined from the standard curve of gallic acid and expressed as mg/g gallic acid equivalent (GAE) using standard curve: $$y=2.002x+0.067\text{;}{R}^2=0.999$$ where y is the absorbance at 765 nm and x is the total phenolic content of crude extract.

2.5 Antioxidant

Antioxidant capacity was obtained by DPPH free radical scavenging assay (adopted from Kumaran and Karunakaran, 2006) using the quercetin as a positive control and DMSO as negative control. Crude extract was prepared in varying concentrations by twofold serial dilution in DMSO with concentrations of 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156 mg/mL in 96 well plates. 200 μl of methanolic DPPH solution (6 × 10⁻⁵ M) was added into all wells and the mixture was incubated for 30 min at room temperature. The absorbance was measured at 517 nm using Elisa reader (Multiskan ascent, Thermo electron corporation). Free radical scavenging activity was determined according to the equation: $$\text{Free radical scavenging activity}(\%)=\frac{A_c-A_s}{A_c}\times 100\%$$ where A_s is the absorbance of sample. A_c is the absorbance of negative control.

2.6 Cytotoxicity property

Cytotoxicity properties of samples against five cell lines (L-6, RAW, HepG2, HeLa, and MCF-7) were determined by MTT assay (modified from Mosmann, 1983). Each cell was maintained on culture flask at 37 °C under 5% CO₂ incubator for few days or until cells confluence more than 80% to continue for the MTT assay. The cell lines were trypsinized and counted as stated in Table 1.

The cell seeded 100 μL aliquot into 96-well plate. Plate was incubated in 37 °C and 5% CO₂ incubator for 24 h. After 24 h, cultured medium was discarded and 100 μL of the crude extract diluted culture medium with different concentrations (first concentration sample for all cell lines (MCF-7, L-6, HepG2, HeLa and RAW) was prepared as 60 μg/mL, continued by twofold dilution of each. Positive control was prepared with concentration same as sample. 96-well plate was incubated for 72 h. MTT was dissolved in PBS (5 mg/mL) and then 20 μl of MTT was loaded into each well and incubated for another 4 h. All the solutions in each well were discarded before adding 100 μl dimethyl sulfoxide (DMSO) into each well, and then incubated another 10 min before analysis. Test plate was shake and absorbance was measured by enzyme linked immunosorbent assay (ELISA) reader at 571 nm. Percentage of cell viability was calculated by using the following: $$\frac{\mathit{abs}\mathit{sample}}{\mathit{abs}\mathit{blank}}\times 100$$

A graph was plotted and 50% inhibition concentration (IC₅₀) calculated. According to Andriani et al. (2011), the criteria of nontoxic activity for the IC₅₀ value of the sample if it is yielded were more than 30 μg/mL.

2.7 Antibacterial activity

Antibacterial activity was carried out by disk diffusion method (modified from Bauer et al., 1966). The aqueous methanolic extract of fruits which are key and core of P. tectorius fruits was prepared by dissolving 10 mg crude in 1 mL of dimethyl sulfoxide (DMSO). Samples (20 μL) were loaded onto each Whatman No. 1 filter paper disks (Ø, 6 mm) and dried in laminar flow to remove the solvent of stock solution. Separately, the four types of target bacteria suspension (the concentration of the bacteria is 0.5 McFarland standards) were spread evenly by using cotton swab onto the Mueller Hilton agar (MHA). Then, the disks were located on the surface of the previously inoculated agar. Four antibiotics were used as control, namely ampicillin, penicillin, gentamycin, and tetracycline. The plates were inverted and incubated for 24 h at 37 °C. Clear inhibition zones around the disks indicated the presence of antimicrobial activity. The diameter for inhibition zone (mm) of the crudes and antibiotics was measured and compared. The actives crude was continued for kinetic study to verify the antibacterial ability.

2.8 Statistical analysis

All the experiments were conducted in triplicate and the data are presented as mean values ± standard deviation.

3.1 Phytochemical analysis

Phytochemical studies were carried out on P. tectorius fruits that indicated the presence of phenolics, flavonoids, steroids, terpenoids, saponins and glycoside chemical constituents (Table 2). Phenolic, flavonoid, and steroid compounds were detected in methanol and ethyl acetate extract of keys and core parts (PMK, PMC, PEK and PEC). Terpenoid and saponin were founded only in hexane crude (PHF) and PMK, respectively. Besides glycoside, PMK and PMC also contained protein and carbohydrate (data not shown). Phytochemical property of samples had correlation with their bioactivity properties. Some studies have reported those chemicals constituents (Table 2) as antioxidant and antibacterial agents (Dahija et al., 2014; Mohammed et al., 2014; Medini et al., 2014; Lunga et al., 2014; Ali et al., 2002). Moreover, only alkaloid did not exist in P. tectorius fruit extracts compared to phytochemical study by Kusuma et al. (2012). They have reported that the active chemical constituents from this plant were phenolics, flavonoids, alkaloids, glycosides, carbohydrate, steroids and triterpenoid. In addition, Kumar and Sanjeeva, 2011 reported the presence of alkaloids, carbohydrates, proteins, steroids, sterols, phenols, tannins, terpenes, flavonoids, gums and mucilage, saponins, and glycosides in the P. tectorius leaves.

3.2 Antioxidant property

A quantitative analysis using radical scavenging DPPH assay was followed by standard protocol of Gadov et al. (1997). Quercetin has been used as standard to compare antioxidant activity of plant extracts. The result obtained from the antioxidant activity is shown in Fig. 2. It shows that only PHF showed antioxidant activity less than 50% (low antioxidant activity) compared to others. The PEK, PMK, PEC and PMC had antioxidant activity more than 50% and IC₅₀ value of less than 2 mg/mL, except PMK with IC₅₀ value of 8 mg/mL. They are considered have strong antioxidant activity compare to standard quercetin with IC₅₀ value is 0.2 mg/mL.

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Figure 2

Antioxidant activity of keys and core part of P. tectorius fruits extracts. PHF: Pandanus hexane fruit extract, PEK: Pandanus ethyl acetate keys extract, PMK: Pandanus methanol keys extract, PEC: Pandanus ethyl acetate core extract, PMC: Pandanus methanol core extract Q = Quercetin. Each value in the graph is expressed as means ± standard deviations.

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Ghasemzadeh et al., 2010 reported that high total phenolics and flavonoids content increase antioxidant activity and there was a linear correlation between phenolics or flavonoids content and antioxidant activity. Correlated with the phytochemical property in this study, phenolics, flavonoids and steroids were major chemical constituents in these extract that are responsible for their antioxidant activity. Steroidal glycoside was reported to have antioxidant activity. Steroids were found to be important hormone regulator which possesses oxytocic, anti-inflammatory, antioxidant, and anti-asthmatic, liver detoxifying actions and help to normalize sticky blood (Okwu and Ohenhen, 2010). Moreover, previous study reported that phenolics and flavonoids compounds from P. tectorius fruits such as ethyl caffeate, dihydroconiferyl alcohol and tangeretin have been isolated from the P. tectorius fruits (Zhang et al. (2012)). Two new phenolic compounds, pandanusphenol A and pandanusphenol B were isolated by Zhang et al. (2013b). In addition, Jong and Chau (1998) published that pinoresinol and 3,4-bis(4-hydroxy-3-methoxybenzyl) tetrahydrofuran are phenolic compounds isolated from methanol extract of P. tectorius were showed moderate and strong antioxidant activity, respectively. The P. tectorius fruit extract was also rich with vitamins such as C, E and β-carotene (Englberger et al., 2003, 2006, 2009), isopentenyl, dimethylallyl acetates and cinnamates (Vahirua-Lechat et al., 1996), and these all could be correlated with its bioactivities property.

3.3 Total phenolic content

Result showed that high total phenolic content (TPC) was obtained from sample with the high antioxidant activity (Fig. 3). PEC extract was showed to have higher antioxidant activity and also higher TPC content, followed by PMC, PEK, PMK and PHF. Strong correlation between TPC and antioxidant properties was shown in Fig. 4, with coefficient correlation (R²) of 0.7499. There was a claim that antioxidant activity of extracts should be proportioned with its total phenolic content (Lee et al., 2008). Various studies of its correlation were also reported (El-Sayed Saleh, 2009; Amarowicz et al., 2004; Lee et al., 2008).

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Figure 3

Total phenolic content of P. tectorius fruits extracts. PHF: Pandanus hexane fruit extract, PEK: Pandanus ethyl acetate keys extract, PMK: Pandanus methanol keys extract, PEC: Pandanus ethyl acetate core extract, PMC: Pandanus methanol core extract. Each value in the graph is expressed as means ± standard deviations.

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Figure 4

Correlation between antioxidant and total phenolic content of Pandanus tectorius fruit extract. PHF: Pandanus hexane fruit extract, PEK: Pandanus ethyl acetate keys extract, PMK: Pandanus methanol keys extract, PEC: Pandanus ethyl acetate core extract, PMC: Pandanus methanol core extract.

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The phytochemical property in this study showed that phenolics and flavonoids were major chemical constituents in these extracts that could be contributed on their TPC property. Other factors than total phenolics can play in the antioxidant activity of the samples, could have effect on R² value. Kahkonen et al. (1994) reported that different phenolic compounds have different responses in Folin–Ciocalteu and the molecular antioxidant respond of phenolic compounds varied remarkably, depending on their chemical structure. Interference of other chemical components present in the extract may also be contributed to the correlation.

Plant phenolics included phenolics acids, flavonoids, tannins and the less common stilbenes and lignans (Day and Mumper, 2010). Many researcher have become more interested in phenolics due to their potent antioxidant properties, their credible property for the prevention or treatment of various diseases (Manach et al., 2004) that include cancer (Zhang et al., 2008), antidiabetic (You et al., 2012; Sergent et al., 2012), antiatherosclerosis (Andriani et al., 2015), anti-inflammatory (Sergent et al., 2010) and anticancer (Day and Mumper, 2010). The P. tectorius fruit extract was rich in caffeoylquinic acids (Zhang et al., 2013a; Liu et al., 2013), vitamins such as vitamins C, E and β-carotene (Englberger et al., 2003, 2006, 2009), isopentenyl, dimethylallyl acetate and cinnamate (Vahirua-Lechat et al., 1996), and these all could be correlated with its biological potential.

3.4 Cytotoxicity properties

Fig. 5 shows that keys and core parts of the P. tectorius fruits were founded to have no cytotoxic against normal (L-6 and RAW) and cancer (MCF-7, HeLa, and HepG2) cell lines. According to Andriani et al. (2011) cytotoxicity property of sample with the IC₅₀ value more than 30 μg/mL was considered as noncytotoxic activity. The 50% inhibitory activity (IC₅₀) value of all extracts toward RAW was obtained more than 30 μg/mL. However, the reduction of cell viability in RAW cells produced by PEK and PMK seems substantial, even at concentrations lower than 30 μg/mL. In low concentration (<5 μg/mL) PEK and PMK seem enhance the RAW cells viability, and then cell viability was reduced by increasing the PMK concentration. Higher concentration of extracts could be toxic effect for Raw cells viability. Low concentration of all extracts (less than its IC₅₀ value, ±40 μg/mL) could be used for further study. Further study might be needed to observe the cytotoxicity effect on cell morphology to confirm this activity. The behavior of the concentration–response curves, in particular for L-6, MCF-7, HeLa and HepG2 cells, being extracts active at low concentrations and then approaching to a plateau in most cases, appears unusual. It could be effect of many different compounds in the extracts may cause the pharmacological interactions.

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Figure 5

Cytotoxicity property of P. tectorius fruits extracts from keys and cores parts against RAW, L-6, HepG2, HeLa, and MCF-7 cell lines. PHF: Pandanus hexane fruit extract, PEK: Pandanus ethyl acetate keys extract, PMK: Pandanus methanol keys extract, PEC: Pandanus ethyl acetate core extract, PMC: Pandanus methanol core extract. Each value in the graph is expressed as means ± standard deviations.

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Evaluation of anticancer by phenolic and flavonoids compounds was widely reported (Ren et al., 2003; Yao et al., 2011; Fresco et al., 2010). Recent studies have reported that polyphenolic compounds such as flavonoids have been shown to possess a variety of biological activities at nontoxic concentrations in organisms. The role of dietary flavonoids in cancer treatment and prevention is extensively discussed and promising it as anticancer agents (Ren et al., 2003). In our current study, P. tectorius fruits which rich content of phenolics and flavonoids constituents and showed high antioxidant property were not shown cytotoxicity property against HeLa and MCF-7 cell lines. It could be antagonistic effect between chemical constituents in P. tectorius fruits which can block anticancer potency or cytotoxic effect of extracts against both cancer cell lines. Antagonistic study of polyphenolic compound was reported by Golden et al. (2009). They reported that polyphenol, epigallocatechin gallate (EGCG) which is given as anticarcinogenic and antitumor effects individually can block the anticancer effect of established cancer treatments such as bortezomib (BZM). The severe antagonistic effect of EGCG appeared to require the presence of the boronic acid moiety in BZM. Thus, characterization of chemical compounds needed that could contribute to antioxidant and cytotoxicity is needed to confirm their cytotoxicity property against cancer cell lines.

3.5 Antibacterial activity

The highest antibacterial activities were demonstrated by PEK against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus Subtilis with the range of inhibition zone that was 10–15 mm, followed by PHF, PEC, PMK and PMC (Table 3). Almost all of extracts showed antibacterial activity against all selected bacteria. Phytochemicals analysis showed the presence of phenolics, flavonoids, steroids, triterpenoids and saponins, (Table 2) in P. tectorius fruits extracts, and these compounds were could be contributed on their antibacterial activity. Phenolics, flavonoids, and steroids in PEK possibility have a role as an antibacterial agent. Many studies have reported of phenolics and flavonoids (Dahija et al., 2014; Mohammed et al., 2014; Medini et al., 2014), steroidal saponin and triterpenoids saponin (Lunga et al., 2014), and steroidal glycoside (Ali et al., 2002) possessed antibacterial activity. The PEK was chosen for further kinetic study since it is the most active extract than others.

3.5.1

3.5.1 Kinetic study of antibacterial activity

Fig. 6a shows the effect of the PEK extract on growth pattern of four tested bacteria. Observations were made every 1 h in 24 h period after they all were given by extract. Extract began showing activity and inhibits the growth of bacteria since one hour observation. The B. subtilis, S. aureus, E. coli, and P. aeruginosa began to die at 1, 8, 9, and 15 h after the addition of the extract, respectively. Compared to the control without extract, it appears that all of the bacteria were still in the growth phase (exponential) at that time (1–15 h of observation, Fig. 6b). Results clearly demonstrated that PEK extracts of the P. tectorius fruits have antibacterial activity against gram positive (B. subtilis and S. aureus) and gram negative (E. coli, and P. aeruginosa).

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Figure 6

The effect of pandanus ethyl acetate keys (PEK) extract from P. tectorius fruits on growth pattern against selected bacteria (a) compared to control without extract (b). Each value in the graph is expressed as means ± standard deviations.

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