Antioxidant and antibacterial activities in the fruit peel, flesh and seed of Ceri Terengganu (Lepisanthes alata Leenh.)

Malay cherry fruit or locally known as Ceri Terengganu (Lepisanthes alata Leenh.) is a local tropical exotic fruit and it is native to Malaysia. The Ceri Terengganu tree is widely distributed in the east coast of Peninsular Malaysia particularly in Terengganu, Pahang and Johor and commonly cultivated as an ornamental plant in the villages and gardens. A limited number of studies had been done on the proximate analysis and postharvest quality of Ceri Terengganu fruit, but the studies on the antioxidant and antibacterial activities of Ceri Terengganu fruit extract are still lacking. Hence, this study aimed to determine the antioxidant and antibacterial activity in the peel, flesh and seed extracts of Ceri Terengganu. The Ceri Terengganu was extracted using 60% ethanol and the total phenolic content (TPC), total flavonoid content (TFC), total monomeric anthocyanins, antioxidant and antibacterial activities were measured using standard methods. The results showed that the seed of Ceri Terengganu had the highest amount of TPC, TFC and antioxidant activity, followed by the peel and flesh extracts whilst the peel extract had the highest total monomeric anthocyanins content. Furthermore, all three extracts of Ceri Terengganu showed inhibition against selected pathogens tested. In conclusion, the seed of Ceri Terengganu possessed the greatest potential to be explored as a source of natural antioxidant and antibacterial agent in the food industry, and thus warrant further investigation.


Introduction
Malay cherry fruit or locally known as Ceri Terengganu or Pokok Johor (Lepisanthes alata Leenh.) is a local tropical exotic fruit and is native to Malaysia (Anuar et al., 2014;FRIM, 2019). The Ceri Terengganu fruit is a globose berry, arranged closely and attractively in big bunches formed by clusters containing about 20 fruits per cluster. Furthermore, the size of each fruit is about 2 cm to 3 cm in diameter and it is deep red with a pointed tip. Each of the fruit contains one to three seeds and its flesh is soft and tastes fairly sweet (FRIM, 2019). In addition, Ceri Terengganu is a non-seasonal fruit which can be found throughout the year. Unfortunately, Ceri Terengganu is not consumed by the locals but instead planted as an ornamental plant.
Several studies had been done using Ceri Terengganu fruit such as on the proximate analysis, total flavonoid content, antioxidants and antibacterial activities. A recent study by Anggraini et al. (2019) used different parts of Ceri Terengganu plant (whole mature fruit, young leaves and bark) and fruit (rind, flesh and seed) from Indonesia on the total phenolic content, total monomeric anthocyanin and antioxidant activity analysis. Another study by Rahmadi et al. (2016) proved that Ceri Terengganu fruit peel exerts antimicrobial effect against Escherichia coli and Staphylococcus aureus. However, none reported on the antibacterial activity of the seed and the methods of analysing the antioxidant activities varied with this study. In addition to that, these previous studies showed that, instead of being an ornamental plant in garden landscapes, the Ceri Terengganu fruit can be utilised for its natural antioxidant and antimicrobial activities to be used in the growing food industry. Hence, this study was aimed to determine the chemical properties, antioxidant and antibacterial activities in the peel, flesh and seed extracts of Ceri Terengganu.

Plant material and sample preparation
Fresh Ceri Terengganu fruits were harvested and collected from State Agriculture Complex's orchard located in Ajil Terengganu. The samples were freshly harvested based on the size uniformity and external colour. The collected Ceri Terengganu fruits were washed thoroughly under running tap water and dried with tissues. Each of the fruit parts (peel, flesh and seed) were then separated manually. The peel obtained was cut into small pieces while the flesh and seed obtained were homogenised separately in a waring commercial blender. Weighed portions of the peel, flesh and seed of the fruit samples were dried in the drying cabinet with a temperature of 60°C for 2 days until the moisture content reached approximately 10%. Then, the samples were grounded into a fine powder by using a commercial blender and was sieved through 250 µm laboratory test siever. The powder obtained was stored in airtight and amber containers at room temperature (25°C) until further analysis.

Solvent extraction
The extraction of the samples was done according to Anzian et al. (2017) and Ghasemzadeh et al. (2015). Briefly, 10 g samples powder of each part (seeds, flesh and peel) from Ceri Terengganu fruit was mixed with 100 mL of 60% ethanol aqueous solution for 2 hrs at 65°C using water bath shaker to maintain the temperature. Each sample was then centrifuged at 4000 x g for 10 mins (Liang et al., 2012). The suspension was filtered through Whatman No. 1 filter paper in a filter funnel. The filtrate was pooled and the solvent was evaporated and concentrated using rotary vacuum evaporator at 40°C to obtain a final volume of concentrate crude extract (Anzian et al., 2017). The filtrate was then stored at -20°C .

Total phenolic content (TPC)
The total phenolic contents of different parts of Ceri Terengganu fruit extract was measured using the Folin-Ciocalteu method (Rakitzis, 1975) with some modifications. Briefly, an aliquot of 1 mL sample solution was mixed with 1.5 mL of 7.5% sodium carbonate and 1 mL of 0.1M Folin-Ciocalteu reagent. After incubation at room temperature for 30 mins in the dark, the absorbance of the reaction mixture was measured at 765nm against reagent blank. Gallic acid was used to obtain a standard curve. The results were presented in mg gallic acid equivalent (GAE)/100 g of sample on a dry weight basis (DW).

Total flavonoid content (TFC)
The total flavonoids content of different parts of Ceri Terengganu fruit extract was determined by the aluminium chloride colourimetric method described by Makris et al. (2007) with slight modification. An aliquot of 1 mL sample solution was mixed with 4 mL of distilled water in a tube. 0.3 mL of 5% sodium nitrite (NaNO 2 ) was then added and allowed to react for 5 mins. Next, 0.3 mL of10 % aluminium chloride (AlCl 3 ) was added and the mixture was allowed to stand for further 5 mins before adding 2 mL of 1 M sodium hydroxide (NaOH) and 2.4 mL of distilled water to the reaction mixture. The absorbance was measured at 510 nm against a blank of distilled water. Quercetin was used as a standard compound for the quantification of total flavonoids. All values were expressed as mg of quercetin equivalent (QE)/100 g of sample on a dry weight basis (DW).

Total anthocyanin content (TAC)
Total anthocyanin content of different parts of Ceri Terengganu fruit extract was measured according to the pH differential method by using two buffer systems. The potassium chloride buffer solution (0.025M, pH= 1) and sodium acetate trihydrate (0.4 M, pH = 4.5) was prepared. Briefly, 1mL test sample extracts were diluted with 3mL of the corresponding buffer for 15 mins. The absorbance of each solution was measured at 512nm and 700nm (for haze correction) against a blank cell filled with distilled water (Moldovan et al., 2016). The concentration (mg/L) of total monomeric anthocyanin for each extract was calculated according to the following formula and expressed as Cy-3-glc equivalents: Where MW is the molecular weight (g/mol) = 449.2 g/ mol for Cy-3-glc, DF is the dilution factor (1 mL sample is diluted to 4 mL, DF= 4); and is the extinction coefficient (L cm -1 mol -1 ) = 26,900 for Cy-3-glc, where L (pathlength in cm) = 1 while 1000 = conversion factor from gram to milligram and A was the nett absorbance.

Total ascorbic acid content (TAA)
The ascorbic acid concentration was measured based FULL PAPER on the reduction of the dye 2,6dichlorophenolindophenol (DCPIP) by ascorbic acid described by Fattahi et al. (2011) with slight modification. Briefly, 0.5 g of samples were mixed in 3 mL metaphosphoric acid (1%). Then, 0.5 mL of DCPIP was added to the supernatant and measured at 520 nm spectrophotometrically. Ascorbic acid standard curve was prepared. All values were expressed as mg of ascorbic acid equivalent (AA)/100 g of sample on a dry weight basis (DW).
2.6 Antioxidant activity analysis 2.6.1 DPPH free radical scavenging capacity The DPPH scavenging activity of different parts of Ceri Terengganu fruit extract was determined as described by Liang et al. (2012) with some modification. Briefly, 2 mL of sample solution was added to 2 mL of the DPPH solution (0.1 mM). The mixture was shaken and incubated for 30 mins at room temperature in the dark. The absorbance of the resulting solution was measured at 517 nm against a blank. Butylated hydroxytoluene (BHT), Vitamin C and Vitamin E were used as standard antioxidant. The radical-scavenging activity was calculated as a percentage of inhibition using the equation below: Where A 0 is the absorbance of the blank sample and As is the absorbance in the presence of the different test samples.

ABTS radical scavenging assay
ABTS assay of different parts of Ceri Terengganu fruit extract was determined according to the method of Fattahi et al. (2011) and Rajurkar and Hande (2011) with some modifications. ABTS radical cation (ABTS •+ ) was prepared by mixing 100mL of potassium persulfate solution (2.45 mM) and 100 mL of ABTS + solution (7 mM) in dark for 24 hrs. The ABTS solution was diluted with 95% ethanol to an absorption value of 0.70±0.02 at 734nm. Briefly, 0.5 mL of sample extract was added to 4 mL ABTS •+ radical cation solution for 5 mins before the absorbance being measured at 734 nm. Trolox was used as standard antioxidant. The ABTS scavenging effect was calculated as a percentage of ABTS •+ discoloration using the equation below: Where A c is the absorbance of the control and As is the absorbance of the sample plus ABTS radical after 5 mins incubation.

Preparation of inoculum and inoculation of microorganisms on MHA agar
Preparation of the inoculum was prepared using the method suggested by Gebreyohannes et al. (2013). Firstly, the inoculum was standardized by matching the turbidity with a 0.5 McFarland standard (No. 1) in Mueller-Hinton Broth (MHB). The MHA agar was then labelled and divided into five zones (peel, flesh, seed, positive control and negative control). The MHA agar plate surface was then inoculated by spreading 100 µL of the microbial inoculum over the entire agar surface using cotton swab was dried at room temperature for about 3 to 5 mins.

Preparation of wells and incubation of MHA agar plates
Preparation of wells followed the method used in the previous study by Balouiri et al. (2016). Firstly, 4 wells were punched aseptically with a sterile tip (8 mm) on each zone of MHA agar. 50 l of each sample extract was added into the wells at its respective zone (peel, flesh and seed). Ampicillin, oxytetracycline and chloramphenicol antibiotic disc was served as a positive control and were placed in the respective zone. Sterile distilled water served as negative control. The MHA agar plates were incubated at 37°C for 18 to 24 hrs. The diameter of the resulting zones of inhibition was measured.

Statistical analysis
All treatments and analysis were carried out in triplicates. All results were reported as mean standard deviation. One-way ANOVA was carried out to determine the significant difference. The differences between means were determined using Fisher's least significant difference (LSD) test with the degree of significant (p< 0.05).

Total phenolic content (TPC)
The results obtained in Figure 1 there was no significant difference in the total phenolic content between peel and seed of Ceri Terengganu (p<0.05) in which the seed (1.00 mg GAE/100 g) extract exhibited the highest total phenolic content followed by the peel (0.92 mg GAE/100 g) and the flesh (0.32 mg GAE/100 g). The findings of this study were in agreement with the previous study by Anggraini et al. (2019). The high total phenolic content obtained by Anggraini et al. (2019) was primarily due to the different solvent used. The use of 100% methanol in Anggraini et al. (2019) study could enable the extraction of low molecular weight phenolic compound due to the high polarity index of methanol compared to ethanol. Additionally, methanol was effective at extracting compounds with higher antioxidant potential (Abarca-Vargas et al., 2016). However, in this study, aqueous ethanol was used to extract these compounds primarily due to the ethanol is safer and cheaper as compared to methanol if this study is going to be scaled up.
In other comparisons, the TPC obtained in all three extracts were low as compared to those obtained from grape pomace ethanolic extract (MohdMaidin et al., 2018) which showed 2100mg GAE/100 g of TPC values. The reason for this was possibly due to the grape pomace is actually consist of the whole fruit including its seed, peel, flesh and stalk at one extract. Whereas in this study, the TPC values of the different parts were determined, respectively.

Total flavonoid content (TFC)
From Figure 1(B), there was a significant difference in the total flavonoid content of different parts of Ceri Terengganu (p<0.05) in which the seed exhibited the highest (2.5 mg QE/100 g) total flavonoid content followed by the peel (1.77 mg QE/100 g) and flesh (0.46 mg QE/100 g). Similar findings have been reported by Gokgoz and Pekgoz (2017) using black grapes (Vitis vinifera L.) where the seed had higher total flavonoid content than the flesh. The differences between the different parts could possibly contribute by the different composition of the seed of Ceri Terengganu. Petkova and Antova (2015) stated that the main composition of melon seed included sterols, tocopherols and also fatty acids. Although the composition of the Ceri Terengganu seed was not determined in this study, the differences in TFC values could be explained by these lipids.
Moreover, the seed which is the most important part of the fruit contains the most amount of nutrients which are needed for the germination process. The seed coat/ hull composed of epidermis, hypodermis, chlorenchyma, palisade, parenchyma and endothelial cells, all of which contain most organelles such as vacuoles and cell walls, thus containing high amounts of phenolics in both the soluble and insoluble-bound forms (Shahidi and Yeo, 2016). This might have explained the highest total phenolic acid content and total flavonoid content presented in the seed of the Ceri Terengganu fruit.

Total anthocyanin content (TAC)
From Figure 2(A), the total anthocyanin content of the peel exhibited significantly higher total anthocyanin content compared to seed and flesh. The finding of this analysis was in agreement with the previous study by Anggraini et al. (2019). These results could be clearly observed in the colour of its peel where it exerted the brightest red colour, followed by the seed and finally flesh which had a soft red colour. Besides that, according to Gao and Mazza (1995), dark-coloured cherries have a total anthocyanin content ranging from 82 to 297 mg/100 g whereas light-coloured cherries have a total anthocyanin content ranging from 2 to 41 mg/100 g which accounts for the major phenolic content in cherries.
Furthermore, the colour of the cherry fruit is determined by the concentration and distribution of different anthocyanins in the skin (Esti et al., 2002;Gonçalves et al., 2007). The few major anthocyanins found in cherries were 3-rutinoside and 3-glucoside of cyanidin whereas minor anthocyanins include 3rutinoside and 3-glucoside of peonidin as well as pelargonidin 3-rutinoside (Robards et al., 1999). In addition, the anthocyanin content may vary between fruits even if they were of the same type. This could be due to the external and internal factors such as genetic and agronomic factors, intensity and type of light, temperature, processing, handling and storage (Kayesh et al., 2013).

Total ascorbic acid content (TAA)
From Figure 2(B), the peel showed the highest total ascorbic acid content followed by the flesh and seed. There was no significant difference between total ascorbic acid in the peel, flesh and seed. The ascorbic acid content of Ceri Terengganu fruit was previously studied by Anuar et al. (2014) and the finding was the ascorbic acid content of Ceri Terengganu fruit decreased significantly from green to red stage. The study also showed that green Ceri Terengganu fruit contained the highest ascorbic acid content of 6.33 mg/100 g while the ascorbic acid content decreased to 5.36 mg/100 g as it matures towards the red stage.
In addition, the major organic acid present in Ceri Terengganu may not be ascorbic acid but instead malic acid, which was not measured in this study (Esti et al., 2002;Bernalte et al., 2003). Supporting that, Gundogdu and Bilge (2012) also found that the content of malic acid in cherries was identified to be higher in content compared to other organic acids. These organic acids might cause an effect on the antioxidant and antimicrobial activities tested further. Figure 3(A) showed the radical scavenging activity of different extracts of Ceri Terengganu. The results showed that the seed extract exhibited the highest (83.9%) antioxidant capacity as compared to that of the peel (83.2%) and flesh (52.4%). There was no significant difference between the antioxidant capacity of the peel and seed. The finding of this analysis was in agreement with the previous study by Anggraini et al. (2019). In their study, the sample extracted using ethanol showed that the result of the seed of the Ceri Terengganu fruit had the highest DPPH radical scavenging activity, followed by the peel and the flesh.

DPPH free radical scavenging capacity
Moreover, the results obtained in this study showed that the antioxidant capacity of peel and seed was significantly higher than that of BHT (58.2%) and vitamin E (42.7%) but was similar to that of vitamin C (88.2%). This showed that the peel and seed had a comparable scavenging property as compared to synthetic antioxidants. Interestingly, from Figure 2(B), there was no significant difference between total ascorbic acid in the peel, flesh and seed but the total antioxidant content was significantly higher in the peel and seed. Therefore, it can be said that the high antioxidant capacity of peel and seed of Ceri Terengganu was not contributed by vitamin C (ascorbic acid) but could possibly be due to the actions of other antioxidants such as phenolic and flavonoid as depicted in the total phenolic and flavonoid content shown in Figure 1(A) and 1(B). In addition, Deighton et al. (2000) have also stated that ascorbic acid only made a minor contribution to the total antioxidant capacity. antioxidant activity where both tests use strongly coloured stable radical compounds (Holtz, 2009). Owing to the same principles, similar trends of antioxidant activity results were obtained in this study hence, further strengthened the results obtained by both tests. Therefore, a Pearson correlation test was done to understand the correlation between each test and the results was tabulated in Table 1.

ABTS radical scavenging assay
From Table 1, DPPH and ABTS were highly correlated with each other with a correlation coefficient of 0.933. Another point to note was that both DPPH and ABTS was highly correlated with TPC and TFC (both with more than 0.9). By referring to the results obtained for TPC and TFC in Figure 1(A) and 1(B), both tests showed that the peel and seed of Ceri Terengganu contained high total phenolic content and total flavonoid content. These results might possibly explain the high antioxidant activity of the peel and seed observed in ABTS and DPPH. This correlation between antioxidant capacity and phenolics was also reported in previous studies. González-Gómez et al. (2010) pointed out that the content of flavonoids was correlated with the antioxidant capacity in the sweet cherry extract.
Similarly, Vangdal and Slimestad (2006) also found a correlation between the antioxidant capacity of sweet cherry fruits and the content of phenolic compounds and anthocyanin present in these fruits (Lima et al., 2002). Table 2 shows the antibacterial activity of different parts of Ceri Terengganu against selected foodborne pathogens. The results obtained showed inhibition of all peel, flesh and seed extracts against S. aureus, B. cereus and B. subtilis. However, no inhibition was observed against L. monocytogenes, E. coli, P. aeruginosa and S. enterica ser. Typhimurium. Additionally, the seed extract showed the largest zone of inhibition against S. aureus, B. cereus and B. subtilis followed by the peel and the flesh extracts. This indicated that the seed of Ceri Terengganu had the highest antimicrobial activity among all parts.

Antimicrobial activity analysis
The difference in the inhibitory effect of different parts of Ceri Terengganu against different bacteria can be explained by the structural differences in the bacteria. From the results obtained, Ceri Terengganu was effective against most gram-positive bacteria but not against L. monocytogenes. In addition, all the three extracts of Ceri Terengganu had no inhibitory effect against all gram-negative bacteria tested in this study. These differences in sensitivity between gram-positive and gram-negative bacteria to Ceri Terengganu extract was probably being attributed to the structural and compositional differences in membranes between the two groups (Lambert et al., 2001). Gram-negative bacteria have an outer membrane that served as an impermeable barrier for many small molecules while gram-positive bacteria were generally lacked outer membrane making gram-positive bacteria more prone to antimicrobial agents (Wendakoon et al., 2012). The barrier formed surrounding gram negative bacteria contains lipopolysaccharides which protects the bacteria membrane from antimicrobial agents (Cui et al., 2012;Nohynek et al., 2006).
Although L. monocytogenes is a gram positive bacteria, its resistance towards Ceri Terengganu extract can be explained by the formation of biofilm making the bacteria become less susceptible to antimicrobial agents (Olaimat et al., 2018). The biofilm promotes the formation of persister cells with improved efflux pump activity which are responsible for removing harmful materials from bacteria such as antimicrobial agents. Besides that, the biofilm may also provide an environment for the transfer of resistant genes across bacteria (Walsh et al., 2001;Wilson et al., 2018). This clearly explained the antimicrobial test results obtained using Ceri Terengganu. Furthermore, the difference in antimicrobial effect portrayed by each part of Ceri Terengganu fruit was due to the phytochemical content present in the fruit. Phytochemicals are bioactive non-nutrient plant compounds that occur naturally in plants (Diep et al., 2014;Huang et al., 2016). Phytochemicals present in Ceri Terengganu might possibly possess hydrophobic characteristics which enable them to embed into lipid components of bacterial cell membrane and mitochondria, thus resulting in the leakage of intracellular material (Carson et al., 2002). Moreover, the phytochemicals can also affect the enzymatic action in the bacteria thus blocking their virulence (Ankri and Mirelman, 1999). This can be done by affecting the structure like flagella that can inhibit bacterial adhesion (Burt et al., 2007). In addition, polyphenols which are a class of phytochemical was found to demonstrate the antimicrobial activity by interacting with the bacteria cell membrane. Inouye et al. (2001) stated that the polyphenols were more effective against gram positive bacteria compared to gram negative bacteria mainly due to the difference in composition in the cell membrane of both bacteria type. Besides that, the presence of an organic acid such as malic acid that presented in the Ceri Terengganu fruit also acted as an antimicrobial barrier against the microbes. This acid was often used in the food industry as preservative agents, attributing their antimicrobial efficacy to the pH changes of the treated media (Joshi et al., 2012) and might help in the antimicrobial efficiency by attacking the cell walls, cell membranes, metabolic enzymes, protein synthesis systems and the genetic material of microorganisms and thus preventing them to grow (Tripathi and Dubey, 2004).
On the other hand, the seed extract of Ceri Terengganu fruit showed the highest antimicrobial activity compared to the flesh and the peel. This was possibly due to the seed possessed the highest amount of phytochemical compounds as compared to other parts of the fruit. Joshi et al. (2012)  the fruit against pathogenic agents by penetrating the cell membrane of microorganisms, causing cell lysis. Moreover, flavones, flavonoids and flavonols were also some of the effective antimicrobial substances against a wide range of microorganisms due to their ability to complex microbial cell walls (Takahashi et al., 1995;Cowan, 1999;Zhao et al., 2001). Furthermore, Lambert et al. (2001) have stated that the higher the phenolic content, the higher the antimicrobial effect which could possibly explain the high antimicrobial effect of the Ceri Terengganu seed against the microbes.

Conclusion
In conclusion, the bioactive compound of different parts of Ceri Terengganu (Lepisanthes alata Leenh.) fruit extract has been determined. The Ceri Terengganu seed exhibited the highest total phenolic content and total flavonoid content among all parts of Ceri Terengganu fruit followed by the peel and the flesh. In addition, the seed of Ceri Terengganu expressed the highest antioxidant capacity in DPPH free radical scavenging capacity test and ABTS assay followed by the peel and the flesh. The antioxidant activity of the Ceri Terengganu seed was found to be higher than synthetic antioxidant (BHT). Hence, Ceri Terengganu seed would be a promising source of natural antioxidant replacing the synthetic antioxidants in the food industry. Furthermore, the peel, seed and flesh of Ceri Terengganu fruit showed inhibition against most gram positive bacteria such as S. aureus, B. cereus and B. subtilis with the seed showing the highest antimicrobial activity followed by the peel and flesh but there was no inhibition for L. monocytogenes, E. coli, P. aeruginosa and S. enterica ser. Typhimurium. Therefore, the Ceri Terengganu fruit extract could be a choice of natural antimicrobial agent that can be used in the food industry. The seed particularly should be studied in detail for its composition, including its lipid content, that might have contributed to its excellent bioactivities showed in this study. Finally, the seed of Ceri Terengganu possessed a great potential to be explored as a source of natural antioxidant and antimicrobial agent in the food industry, and thus warrant further investigation.