Phytochemical Analysis, Antioxidant, Antibacterial and Cytotoxicity Properties of Keys and Cores Part of Pandanus Tectorius Fruits

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

Yosie Andriani a,b,*, Nadiah Madihah Ramli a, Desy Fitrya Syamsumir a, Murni Nur Islamiah Kassim a, Jasmin Jaafar a, Nur Asniza Aziz a, Leni Marlina a, Noor Suryani Musa a, Habsah Mohamad a

a Institute of Marine Biotechnology, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia b Chemistry Department, Faculty of Mathematics and Natural Sciences, Universitas Bengkulu (UNIB), Bengkulu 38371, Indonesia

Received 10 September 2015; accepted 5 November 2015

KEYWORDS Abstract Pandanus tectorius fruits have been used as major food in Micronesia, but not fully Pandanus tectorius; exploited in South East Asia. In Malaysia and Indonesia, P. tectorius fruit is wasted and still not Phytochemical; utilized either as a source of food as well as for research. The aims of the present study were to Antioxidant; determine the phytochemical content, antibacterial and antioxidant activities, total phenolic content Antibacterial; (TPC) and cytotoxicity properties of cores and keys parts of P. tectorius fruits extracts against some Cytotoxicity normal (RAW and L-6) and cancer cell lines (HeLa, HepG2 and MCF-7). Samples were collected from Setiu Wetland, Terengganu, Malaysia. Extracts were obtained by successive extraction using hexane, ethyl acetate and methanol. The antibacterial activity was performed by disk diffusion method against gram positive (Bacillus subtilis and Staphylococcus aureus) and gram negative (Escherichia coli, and Pseudomonas aeruginosa) bacteria. Actives extract was continued for antibacterial kinetic study. The TPC and antioxidant were analyzed by Folin–Ciocalteu and DPPH free radical scavenging assay, respectively. Qualitative phytochemical analysis result conﬁrmed the presence of phenolics, ﬂavonoids, terpenoid, steroids, saponins and glycosides as chemical constituents in P. tectorius fruits extract. It was supported by high TPC content in this extracts. The ethyl acetate

extract from cores part (PEC) showed the highest antioxidant capacity (IC50 = 0.8 ± 0.20 mg/mL), while ethyl acetate extract from keys part (PEK) displayed highest antibacterial activity with inhibition zone of 10–15 mm, but less strong inhibition as compared to the investigated commercial antibiotics (ampicillin, penicillin, gentamycin, and tetracycline). Antibacterial kinetic study was

* Corresponding author at: Institute of Marine Biotechnology, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Tereng- ganu, Malaysia. Peer review under responsibility of King Saud University.

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http://dx.doi.org/10.1016/j.arabjc.2015.11.003 1878-5352 Ó 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

further proven that PEK has good antibacterial activity. Moreover, cytotoxicity study revealed that all

of extracts did not have any cytotoxic activity against all selected cell lines (IC50 value > 30 lg/mL). Result founded that both keys and core parts of P. tectorius fruits were found to be rich by phenolics content and potent as antioxidant and antibacterial agents. Since it is not toxic against selected cell lines, the mechanism study on antiinﬂammatory, antidiabetic, antiatherosclerosis could be carried out. Ó 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction terpenes (phytosteroid mixture), eudesmin, kobusin, desmin and hydro- furan (Jong and Chau, 1998), while, physcion, daucosterol, b-sitosterol, The therapeutic potentials of plant and animal origin are being used camphosterol, palmitic acid and steric acid in rhizomes have been from the ancient times by the simple process without the isolation of reported by Venkatesh et al. (2012), and its root extracts were rich the pure compounds. The pharmacological action of crude drug is antioxidant compound from lignans, benzofuran derivate, a-terpineol, determined by the nature of its constituents. Thus, the plant species b-carotene, b-sitosterol, vitamin C, tangerine, germacrene-B, and vani- can be considered as a contributor for definite physiological effects dine (Adkar and Bhaskar, 2014). Moreover, the P. tectorius fruit extract from a huge number of compounds for example alkaloids, terpenoids, was also rich by antioxidant compound such as, caffeoylquinic acids flavonoids, glycosides and phenolics. These chemical compounds are (Zhang et al., 2013a; Liu et al., 2013), vitamins such as vitamins C, E mostly responsible for the desired beneficial properties as plants are and b-carotene (Englberger et al., 2003, 2006, 2009), Isopentenyl, known as a source of secondary metabolites that come with a variety dimethylallyl acetates and cinnamates (Vahirua-Lechat et al., 1996), of structural arrangements and properties with interesting biological and these all could be correlated with its biological potency. activities (De Fatima et al., 2002). Therefore, plants continue to be The biological potencies of P. tectorius were reported as anti- major source of medicine, as they have throughout human history inflammatory (Chiang et al., 2005), antioxidant and anticancer (Prince and Prabakaran, 2011).Pandanus tectorius, locally named as (Prajapati et al., 2003; Zhang et al., 2013b), antitumor (Mani et al., Pandan laut also known as others botanical name of P. odoratissimus, 2008), antiviral (Nahmias et al., 2008), antidiabetic (Kumara et al., P. fascicularis, and P. amaryllifolius is one of mangrove coastal plant 2012), cholesterol-lowering activity (Lee et al., 1999; Kurowska and from Pandanaceae family comprises of about 700 species distributed Manthey, 2004), hypolipidemia (Zhang et al., 2013a; Liu et al., 2013). worldwide in the subtropical and tropical regions include Malaysia Moreover, the antihyperglycemic and antihyperlipidemic effects of caf- and Indonesia (Adkar and Bhaskar, 2014). Referring to Thomson feoylquinic acids-rich P. tectorius fruit extract were also investigated by et al. (2006), the unique composite edible fruits (Fig. 1a) are made Wu et al. (2014). They proved that this extract increases insulin sensitiv- up of individual pieces called ‘‘keys” (Fig. 1b) attached to a fibrous ity and regulates hepatic glucose and lipid metabolism in diabetic db/db core (Fig. 1c). The term ‘‘bunch” refers to the entire composite fruit mice. Many researcher were focused on P. tectorius leaves, while the (including keys and the core). The inner parts of the keys are chewed researches on its fruit are still limited. Research related to bioactivity and sucked for their sweet pulp (Englberger et al., 2009). of keys and core part of this fruits was separately uninvestigated. Thus, The P. tectorius especially its leaves is traditionally recommended by this study was conducted to evaluate phytochemicals content, antibac- the Indian Ayurvedic medicines for treatment of many diseases (Adkar terial and antioxidant activities, total phenolic contents and cytotoxic- and Bhaskar, 2014). The therapeutic effect of natural substance is ity properties of keys and cores part of P. tectorius fruits against normal directly related to their chemical constituent contents. The pandanus oils (RAW, L-6) and cancer cell lines (HepG2, MCF-7, and HeLa). have been used traditionally for earache, headache, arthritis, debility, giddiness, laxative, rheumatism, small pox, and spams (Kumar and 2. Materials and methods Sanjeeva, 2011). Its leaves are rich with essential oils contained, such a as ether (37.7%), terpene-4-ol (18.6%), -terpineol (8.3%), 2- 2.1. Materials phenylethyl alcohol (7.5%), benzyl benzoate (11%), viridine (8.8%), and germacrene (8.3%) together with small amount of benzyl salicylate, benzyl acetate, and benzyl alcohol. Furthermore, P. tectorius leaves are The sample of P. tectorius fruits was collected from the same used in treatments for cold/flu, hepatitis, dysuria, asthma, boils, and can- location of beach area in Setiu Wetland, Terengganu, cer (Meilleur et al., 1997). It was rich by phenolic compounds such as Malaysia. The sample (30 kg) was collected from March to alkaloids, flavonoids, saponins (Kumar and Sanjeeva, 2011), phenols May 2013. After collection, P. tectorius fruits were divided into (vanillin, benzoic acid methyl ester, and a new benzofuran derivative), two parts, keys (13 kg) and cores (4 kg) (Fig. 1). The samples

Figure 1 Pandanus tectorius fruits (a), its keys (b) and core parts (c).

Phytochemical analysis of phenolics, tannins, flavonoids, ter- Antioxidant capacity was obtained by DPPH free radical scav- penoids, alkaloids, steroids, saponins and glycoside were enging assay (adopted from Kumaran and Karunakaran, adapted from Yadav and Agarwala (2011) as follows. 2006) using the quercetin as a positive control and DMSO as Test for phenols and tannins. 2 mL of 2% Ferric chloride negative control. Crude extract was prepared in varying solution was added to the crude extracts. A blue-green to black concentrations by twofold serial dilution in DMSO with color indicates the presence of phenols and tannis. concentrations of 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156 mg/mL Test for flavonoid. Crude extract was mixed with 2 mL of in 96 well plates. 200 ll of methanolic DPPH solution 2% sodium hydroxide solution. An intense yellow color was (6 10 5 M) was added into all wells and the mixture was formed turned to colorless on addition of few drops of diluted incubated for 30 min at room temperature. The absorbance acid indicated the presence of flavonoids. was measured at 517 nm using Elisa reader (Multiskan ascent, Test for steroids. Crude extract was mixed with 2 mL of Thermo electron corporation). Free radical scavenging activity chloroform and concentrated H SO was added sidewise of 2 4 was determined according to the equation: test tube. A red color produced in the lower chloroform layer indicated the presence of steroids. Ac As Free radical scavenging activity ð%Þ¼ 100% Test for terpenoids. Crude extract was dissolved in 2 mL of Ac chloroform and evaporated to dryness. Then 2 mL of concen- where As is the absorbance of sample. Ac is the absorbance of trated sulfuric acid was added and heated for about 2 min. A negative control. grayish color of mixture indicated the presence of terpenoids. Test for alkaloids. Crude extract was mixed with 2 mL of 2.6. Cytotoxicity property 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. Cytotoxicity properties of samples against five cell lines (L-6, Test for saponins. Crude extract was diluted with 5 mL of RAW, HepG2, HeLa, and MCF-7) were determined by distilled water in a test tube. The mixture was shaken vigor- MTT assay (modified from Mosmann, 1983). Each cell was ° ously. The formation of stable foam indicated the presence maintained on culture flask at 37 C under 5% CO2 incubator of saponins. for few days or until cells confluence more than 80% to Test for glycosides. Crude extract was mixed with 2 mL of continue for the MTT assay. The cell lines were trypsinized glacial acetic acid containing 1–2 drops of 2% ferric chloride and counted as stated in Table 1. l solution. The mixture was then poured into another test tube The cell seeded 100 L aliquot into 96-well plate. Plate was ° containing 2 mL of concentrated sulfuric acid carefully. A incubated in 37 C and 5% CO2 incubator for 24 h. After 24 h,

2.7. Antibacterial activity 3. Results and discussion

Antibacterial activity was carried out by disk diffusion method 3.1. Phytochemical analysis (modified from Bauer et al., 1966). The aqueous methanolic extract of fruits which are key and core of P. tectorius fruits Phytochemical studies were carried out on P. tectorius fruits was prepared by dissolving 10 mg crude in 1 mL of dimethyl sul- that indicated the presence of phenolics, flavonoids, steroids, l foxide (DMSO). Samples (20 L) were loaded onto each What- terpenoids, saponins and glycoside chemical constituents man No. 1 filter paper disks (Ø, 6 mm) and dried in laminar (Table 2). Phenolic, flavonoid, and steroid compounds were flow to remove the solvent of stock solution. Separately, the four detected in methanol and ethyl acetate extract of keys and core types of target bacteria suspension (the concentration of the bac- parts (PMK, PMC, PEK and PEC). Terpenoid and saponin teria is 0.5 McFarland standards) were spread evenly by using were founded only in hexane crude (PHF) and PMK, respec- cotton swab onto the Mueller Hilton agar (MHA). Then, the tively. Besides glycoside, PMK and PMC also contained pro- disks were located on the surface of the previously inoculated tein and carbohydrate (data not shown). Phytochemical agar. Four antibiotics were used as control, namely ampicillin, property of samples had correlation with their bioactivity penicillin, gentamycin, and tetracycline. The plates were inverted properties. Some studies have reported those chemicals con- ° and incubated for 24 h at 37 C. Clear inhibition zones around stituents (Table 2) as antioxidant and antibacterial agents the disks indicated the presence of antimicrobial activity. The (Dahija et al., 2014; Mohammed et al., 2014; Medini et al., diameter for inhibition zone (mm) of the crudes and antibiotics 2014; Lunga et al., 2014; Ali et al., 2002). Moreover, only was measured and compared. The actives crude was continued alkaloid did not exist in P. tectorius fruit extracts compared for kinetic study to verify the antibacterial ability. to phytochemical study by Kusuma et al. (2012). They have reported that the active chemical constituents from this 2.7.1. Kinetic study of antibacterial activity plant were phenolics, flavonoids, alkaloids, glycosides, Two sets of study were conducted which are for bacteria only carbohydrate, steroids and triterpenoid. In addition, Kumar and bacteria with samples modified from Kareem et al. (2008). and Sanjeeva, 2011 reported the presence of alkaloids, Extract with the highest antibacterial activity was chosen for this carbohydrates, proteins, steroids, sterols, phenols, tannins, ter- study. Four target bacteria were suspended in Mueller Hinton

Table 1 Culture medium and cell concentration of each cell lines for cytotoxicity assay. Kind of cell lines Name of cell lines Culture medium Cell concentration Normal cell lines L6 (rat muscle cell) DMEM + 10% fetal bovine serum 2 104 mL cell/well RAW 264.7 (macrophage DMEM + 2 mM glutamine + 10% FBS/FCS (FBS) 2 104 mL cell/well from blood) Cancer cell lines HepG2 (human liver MEM + 10% fetal bovine serum + 1% sodium pyruvate + 2 104 mL cell/well carcinoma cells) 1% non-essential amino acid + 1% penicillin streptomycin Hela (cervical cancer) DMEM + 10% fetal bovine serum + 1% penicillin streptomycin 1 105 mL cell/well MCF7 human breast cancer) MEM + 10% fetal bovine serum + 1% sodium pyruvate + 1 105 mL cell/well 1% non-essential amino acid + 1% penicillin streptomycin

Please cite this article in press as: Andriani, Y. et al., Phytochemical analysis, antioxidant, antibacterial and cytotoxicity properties of keys and cores part of Pandanus tectorius fruitsPandanus tectorius fruits –>. Arabian Journal of Chemistry (2015), http://dx.doi.org/10.1016/j.arabjc.2015.11.003 Keys and cores part of Pandanus tectorius fruits 5 penes, flavonoids, gums and mucilage, saponins, and glyco- 100 sides in the P. tectorius leaves. PHF 80 PEK PMK 3.2. Antioxidant property PEC 60 PMC Q A quantitative analysis using radical scavenging DPPH assay 40 was followed by standard protocol of Gadov et al. (1997). 20 Quercetin has been used as standard to compare antioxidant activity of plant extracts. The result obtained from the antiox- of antioxidant Percentage 0 idant activity is shown in Fig. 2. It shows that only PHF 024681012 showed antioxidant activity less than 50% (low antioxidant Concentration of sample (mg/mL) activity) compared to others. The PEK, PMK, PEC and Figure 2 Antioxidant activity of keys and core part of P. PMC had antioxidant activity more than 50% and IC value 50 tectorius fruits extracts. PHF: Pandanus hexane fruit extract, of less than 2 mg/mL, except PMK with IC value of 8 mg/ 50 PEK: Pandanus ethyl acetate keys extract, PMK: Pandanus mL. They are considered have strong antioxidant activity com- methanol keys extract, PEC: Pandanus ethyl acetate core extract, pare to standard quercetin with IC value is 0.2 mg/mL. 50 PMC: Pandanus methanol core extract Q = Quercetin. Each Ghasemzadeh et al., 2010 reported that high total phenolics value in the graph is expressed as means ± standard deviations. and flavonoids content increase antioxidant activity and there was a linear correlation between phenolics or flavonoids content and antioxidant activity. Correlated with the phytochemical activity and also higher TPC content, followed by PMC, property in this study, phenolics, flavonoids and steroids were PEK, PMK and PHF. Strong correlation between TPC and major chemical constituents in these extract that are responsible antioxidant properties was shown in Fig. 4, with coefficient for their antioxidant activity. Steroidal glycoside was reported correlation (R2) of 0.7499. There was a claim that antioxidant to have antioxidant activity. Steroids were found to be important activity of extracts should be proportioned with its total phe- hormone regulator which possesses oxytocic, anti-inflammatory, nolic content (Lee et al., 2008). Various studies of its correla- antioxidant, and anti-asthmatic, liver detoxifying actions and tion were also reported (El-Sayed Saleh, 2009; Amarowicz help to normalize sticky blood (Okwu and Ohenhen, 2010). et al., 2004; Lee et al., 2008). Moreover, previous study reported that phenolics and flavonoids The phytochemical property in this study showed that phe- compounds from P. tectorius fruits such as ethyl caffeate, dihy- nolics and flavonoids were major chemical constituents in droconiferyl alcohol and tangeretin have been isolated from the these extracts that could be contributed on their TPC property. P. tectorius fruits (Zhang et al. (2012)). Two new phenolic com- Other factors than total phenolics can play in the antioxidant pounds, pandanusphenol A and pandanusphenol B were isolated activity of the samples, could have effect on R2 value. by Zhang et al. (2013b).Inaddition,Jong and Chau (1998) pub- Kahkonen et al. (1994) reported that different phenolic com- lished that pinoresinol and 3,4-bis(4-hydroxy-3-methoxybenzyl) pounds have different responses in Folin–Ciocalteu and the tetrahydrofuran are phenolic compounds isolated from methanol molecular antioxidant respond of phenolic compounds varied extract of P. tectorius were showed moderate and strong antioxi- remarkably, depending on their chemical structure. Interfer- dant activity, respectively. The P. tectorius fruit extract was also ence of other chemical components present in the extract b rich with vitamins such as C, E and -carotene (Englberger may also be contributed to the correlation. et al., 2003, 2006, 2009), isopentenyl, dimethylallyl acetates and Plant phenolics included phenolics acids, flavonoids, tannins cinnamates (Vahirua-Lechat et al., 1996), and these all could be and the less common stilbenes and lignans (Day and Mumper, correlated with its bioactivities property. 2010). Many researcher have become more interested in phenolics due to their potent antioxidant properties, their credible 3.3. Total phenolic content property for the prevention or treatment of various diseases (Manach et al., 2004) that include cancer (Zhang et al., 2008), Result showed that high total phenolic content (TPC) was antidiabetic (You et al., 2012; Sergent et al., 2012), obtained from sample with the high antioxidant activity antiatherosclerosis (Andriani et al., 2015), anti-inflammatory (Fig. 3). PEC extract was showed to have higher antioxidant (Sergent et al., 2010) and anticancer (Day and Mumper,

Table 2 Phytochemical analysis of P. tectorius fruits. Sample Phytochemical test Phenol Flavonoid Steroid Terpenoid Alkaloid Saponin Glycoside PHF + ++ PEK + + + PEC + + + PMK + + + ++ PMC + + + + 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.

2010). The P. tectorius fruit extract was rich in caffeoylquinic 120 acids (Zhang et al., 2013a; Liu et al., 2013), vitamins such as vita- 100 R² = 0.7499 mins C, E and b-carotene (Englberger et al., 2003, 2006, 2009), PEK isopentenyl, dimethylallyl acetate and cinnamate (Vahirua- 80 PEC PMC Lechat et al., 1996), and these all could be correlated with its bio- 60 logical potential. PMK 40 activity (%) activity PHF 3.4. Cytotoxicity properties 20 0

DPPH fre radical scavenging 0 50 100 150 200 Fig. 5 shows that keys and core parts of the P. tectorius fruits were founded to have no cytotoxic against normal (L-6 and GAE value (µg/g) RAW) and cancer (MCF-7, HeLa, and HepG2) cell lines. Figure 4 Correlation between antioxidant and total phenolic According to Andriani et al. (2011) cytotoxicity property of content of Pandanus tectorius fruit extract. PHF: Pandanus hexane l sample with the IC50 value more than 30 g/mL was consid- fruit extract, PEK: Pandanus ethyl acetate keys extract, PMK: ered as noncytotoxic activity. The 50% inhibitory activity Pandanus methanol keys extract, PEC: Pandanus ethyl acetate (IC50) value of all extracts toward RAW was obtained more core extract, PMC: Pandanus methanol core extract. than 30 lg/mL. However, the reduction of cell viability in RAW cells produced by PEK and PMK seems substantial, even at concentrations lower than 30 lg/mL. In low concentra- cytotoxicity property against HeLa and MCF-7 cell lines. It tion (<5 lg/mL) PEK and PMK seem enhance the RAW cells could be antagonistic effect between chemical constituents in viability, and then cell viability was reduced by increasing the P. tectorius fruits which can block anticancer potency or cyto- PMK concentration. Higher concentration of extracts could be toxic effect of extracts against both cancer cell lines. Antagonis- toxic effect for Raw cells viability. Low concentration of all tic study of polyphenolic compound was reported by Golden extracts (less than its IC50 value, ±40 lg/mL) could be used et al. (2009). They reported that polyphenol, epigallocatechin for further study. Further study might be needed to observe gallate (EGCG) which is given as anticarcinogenic and antitu- the cytotoxicity effect on cell morphology to confirm this activ- mor effects individually can block the anticancer effect of estab- ity. The behavior of the concentration–response curves, in par- lished cancer treatments such as bortezomib (BZM). The severe ticular for L-6, MCF-7, HeLa and HepG2 cells, being extracts antagonistic effect of EGCG appeared to require the presence of active at low concentrations and then approaching to a plateau the boronic acid moiety in BZM. Thus, characterization of in most cases, appears unusual. It could be effect of many dif- chemical compounds needed that could contribute to antioxi- ferent compounds in the extracts may cause the pharmacolog- dant and cytotoxicity is needed to confirm their cytotoxicity ical interactions. property against cancer cell lines. Evaluation of anticancer by phenolic and flavonoids compounds was widely reported (Ren et al., 2003; Yao et al., 2011; 3.5. Antibacterial activity Fresco et al., 2010). Recent studies have reported that polyphenolic compounds such as flavonoids have been shown to possess The highest antibacterial activities were demonstrated by PEK a variety of biological activities at nontoxic concentrations in against Escherichia coli, Pseudomonas aeruginosa, Staphylococ- organisms. The role of dietary flavonoids in cancer treatment cus aureus and Bacillus Subtilis with the range of inhibition and prevention is extensively discussed and promising it as anti- zone that was 10–15 mm, followed by PHF, PEC, PMK and cancer agents (Ren et al., 2003). In our current study, P. tectorius PMC (Table 3). Almost all of extracts showed antibacterial fruits which rich content of phenolics and flavonoids con- activity against all selected bacteria. Phytochemicals analysis stituents and showed high antioxidant property were not shown showed the presence of phenolics, flavonoids, steroids, triterpenoids and saponins, (Table 2)inP. tectorius fruits extracts, 200 and these compounds were could be contributed on their 180 antibacterial activity. Phenolics, flavonoids, and steroids in 160 PEK possibility have a role as an antibacterial agent. Many 140 studies have reported of phenolics and flavonoids (Dahija 120 et al., 2014; Mohammed et al., 2014; Medini et al., 2014), ster- 100 80 oidal saponin and triterpenoids saponin (Lunga et al., 2014), 60 and steroidal glycoside (Ali et al., 2002) possessed antibacterial 40 activity. The PEK was chosen for further kinetic study since it µg GAE/g sample 20 is the most active extract than others. 0 PHF PEK PMK PEC PMC 3.5.1. Kinetic study of antibacterial activity Samples Fig. 6a shows the effect of the PEK extract on growth pattern Figure 3 Total phenolic content of P. tectorius fruits extracts. of four tested bacteria. Observations were made every 1 h in PHF: Pandanus hexane fruit extract, PEK: Pandanus ethyl acetate 24 h period after they all were given by extract. Extract began keys extract, PMK: Pandanus methanol keys extract, PEC: showing activity and inhibits the growth of bacteria since one Pandanus ethyl acetate core extract, PMC: Pandanus methanol hour observation. The B. subtilis, S. aureus, E. coli, and core extract. Each value in the graph is expressed as means P. aeruginosa began to die at 1, 8, 9, and 15 h after the addition ± standard deviations. of the extract, respectively. Compared to the control without

100 100 PHF PHF PEK 75 PEK 75 PMK PMK PEC PEC 50 50 PMC PMC

25 25 Viability of Viability cells (%) Viability of Viability cells (%) 0 0 0 20406080 0 20406080 Concentration of samples (µg/mL) Concentrations of samples (µg/mL) (a) RAW (b) L-6

250 175 150 200 PHF PHF 125 PEK PEK 150 PMK 100 PMK PEC PEC 75 100 PMC PMC 50 50 25 Viability of cells (%) Viability ofViability cells (%) 0 0 0 20406080 020406080 Concentrations of samples (µg/mL) Concentrations of samples (µg/mL) (c) HepG2 (d) HeLa

100

50 PHF PEK 25 PMK PEC PMC Viability of cells (%) of cells Viability 0 0 204060 Concentrations of samples (µg/mL) (e) MCF-7

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.

Table 3 Zone of inhibition for antibacterial activity of P. tectorius fruits extracts. Samples Inhibition zone (mm) E. coli (gram negative) P. aeruginosa (gram negative) S. aureus (gram positive) B. subtilis (gram positive) PHF 10 ± 0.21 10 ± 0.12 9 ± 0.08 10 ± 0.10 PEK 10 ± 0.13 14 ± 0.15 15 ± 0.11 15 ± 0.13 PEC 0 7 ± 0.11 10 ± 0.11 8 ± 0.08 PMK 13 ± 0.11 0 11 ± 0.08 0 PMC 0 0 0 7 ± 0.11 Ampicillin 19 ± 0.12 20 ± 0.20 19 ± 0.20 19 ± 0.10 Penicillin 13 ± 0.20 15 ± 0.12 18 ± 0.15 19 ± 0.10 Gentamycin 15 ± 0.11 17 ± 0.21 18 ± 0.15 15 ± 0.08 Tetracycline 27 ± 0.08 20 ± 0.12 31 ± 0.11 29 ± 0.15 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.

0.15 and Jasmin Jaafar did antibacterial experiment. Sample preparation and processing was done by Nur Asniza Aziz. 0.12 All co-authors reviewed and discussed the paper. 0.09 E. coli P. a e r u g i n o sa 0.06 Acknowledgments S. aureus B. subtilis 0.03 The authors wish to thank the Ministry of Higher Education

Absorbance at 630 nm (MOHE) – Malaysia for the fund provided under the Funda- 0 0 3 6 9 12 15 18 21 24 mental Research Grant Scheme (FRGS) Fasa I/2014 (Vote Time of observation (hours) No. 59346). Appreciation goes to Institute of Marine Biotech- nology, University Malaysia Terengganu, Kuala Terengganu, (a) Malaysia, for the facilities in doing research.

0.6

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