Phytochemical content and antioxidant activity of selected wild ulam/vegetables consumed by indigenous Jakun community in Taman Negara Johor Endau Rompin (TNJER), Malaysia

This study aimed to investigate the phytochemical content and antioxidant activity of selected wild ulam/vegetables consumed by indigenous Jakun Community in Taman Negara Johor Endau Rompin (TNJER), Malaysia. Five ulam/vegetables were collected from TNJER, Malaysia namely Dillenia suffruticosa (pucuk simpuh), Amischotolype griffithii (tebu gogok), Paederia foetida (pucuk sekentut), Gnetum gnemon (pucuk empeng), and Barringtonia macrostachya (pucuk putat). Total phenolic content (using Folin-Ciocalteu) and total flavonoid content (using aluminium chloride colorimetric), as well as antioxidant activity using 2,2-diphenylpicrylhydrazyl (DPPH) and 2,2'-azino-bis(3 -ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays were determined spectrophotometrically. For the phytochemical study, D. suffruticosa showed the highest value of total phenolic content (93.87±0.10 μg gallic acid equivalent/mL) while B. macrostachya displayed the highest value of total flavonoid content (115.06±21.54 μg rutin equivalent/mL) as compared to all samples tested. For the antioxidant activity, G. gnemon showed the lowest EC50 value in DPPH assay with the value of 3.92±1.52 μg ascorbic acid/mL, indicated the strongest antioxidant potential. Meanwhile, D. suffruticosa showed the highest scavenging activity using ABTS assay (EC50 value, 7.93 μg/mL). In conclusion, ulam/vegetables of TNJER are rich in phytochemicals and antioxidant which can be a great potential for use in complementary and alternative medicines as well as functional food applications.


Introduction
Being a tropical country, Malaysia is well-known for its richness in the biodiversity of many indigenous ulam/ vegetables which grow naturally in the wild (Aralas et al., 2009). In Malaysia, the consumption of raw ulam/ vegetable is considered as a common traditional healthy diet by many people due to their health-promoting properties (Chan et al., 2014). Majority of Malay restaurants in this country also serve ulam as a side dish to be eaten with rice (staple food) and sometimes, ulam may also be blanched, sautéed, curried or fried (Husain et al., 2004;Chan et al., 2014). Historically, ulam is used for many years in folk medicines either to prevent or cure the diseases which make them to receive great attention in spite of their flavourful popular side dishes (Reihani and Azhar, 2014). Currently, over 120 species of traditional vegetables from various plant families in Malaysia have been identified and regarded as ulam (Aralas et al., 2009). Recent research has identified a vast variety of antioxidant from wild ulam/vegetables throughout the world especially Malaysia (Reihani and Azhar, 2014). Phytochemical (from plants) elements in the diet play a major role in contributing to human health (Nahak et al., 2014). Consumption of ulam/vegetables are good for health as they lower the risk of getting diseases such as cancer, diabetes, cardiovascular diseases, Alzheimer's disease and atherosclerosis due to presence of various forms of phytochemicals and antioxidants; polyphenol compounds including flavonoids and total phenolic compounds (Abdul Karim et al., 2014;Ahmad et al., 2014). In general, vegetables are considered to be among the top four common daily diets by Malaysians as they offer various health benefits to humans (Norimah et al., 2008)

Sample extraction
Sample of 0.1 g was mixed with 25 mL of 80% methanol. The mixture was agitated on an orbital shaker set at 200 rpm for 2 hrs followed by filtration by using Whatman filter paper and the supernatant was stored at -20°C in a freezer. The samples were extracted for phytochemical and antioxidant activity analysis. Each test was done in triplicate to ensure the accuracy of the results.

Total phenolic content
Total phenolic content was determined using Folin-Ciocalteu reagent done by Velioglu et al. (1998) with some modifications. The absorbance values were obtained spectrophotometrically at 750 nm against zero absorbance (T60 Aurora Oasis Scientific, Inc., China). Gallic acid was used as a standard and the results were expressed as µg gallic acid equivalents/ml (µg GAE/ mL).

Total flavonoid content
Total flavonoid was measured using aluminium chloride colorimetric method (Zhishen et al., 1999). The absorbance was measured spectrophotometrically at 510 nm against zero absorbance (T60 Aurora Oasis Scientific, Inc., China). Rutin was used as a standard and results were expressed as µg rutin equivalent/ml (µg RE/ mL).

Statistical analysis
Statistical analysis was done using Analysis of Variance (ANOVA) and the results were expressed as mean ± standard deviation. In ANOVA, the differences between the mean values were considered significantly different when the value of p<0.05 was obtained.

Total flavonoid content (TFC)
The results for the total flavonoid content of wild ulam/vegetables in this study ranged from 24.90±5.30 to 115.06±21.54 µg rutin equivalent/mL. Pucuk putat showed the highest total flavonoid content with the value of 115.06±21.54 µg rutin equivalent/mL, followed by pucuk empeng, pucuk simpuh, pucuk sekentut and tebu

Discussion
The health-promoting effect of vegetables, fruits, whole grains, and nuts may be contributed by polyphenols and antioxidant constituents (Pandey and Rizvi, 2009). For instance, phenolic compounds are secondary metabolites commonly found in plants such as vegetables, fruits and spices (Chua et al., 2014). They are natural antioxidants and play a role as scavengers of free radicals and hence, they form an essential part of the human diet as they may contribute directly to antioxidative action in the body (Duh et al., 1999;Shahidi and Ambigaipalan, 2015). It is believed that their radical scavenging ability is due to their hydroxyl groups (Hatano et al., 1989). The level of total phenolic contents in wild ulam/vegetables varied from 17.08±1.02 to 93.87±0.10 µg gallic acid equivalent/mL. Dillenia suffruticosa (pucuk simpuh) demonstrated the highest phenolic content with the value of 93.87±0.10 µg gallic acid equivalent/mL. Previous study demonstrated that Cosmos caudatus (ulam raja) had the highest amount of total phenols as compared to other vegetables such as Polyscias pinnata, Pluchea indica, Nothopanax scutellarius, Talinum triangulare, Pilea melastomoides and Etlingera elatior (Andarwulan et al., 2010).
Flavonoids are the most diverse group of natural compounds which possess a broad spectrum of chemical and biological properties (Prasad et al., 2010). Not only for the antioxidant activity, but they also have the properties as anti-carcinogenic and anti-arteriosclerosis (Wong et al., 2006). In this study, the total flavonoid content of wild ulam/vegetables varied from 24.90±5.30 to 115.06±21.54 µg rutin equivalent/mL. The previous study reported that the flavonoid content of the vegetables ranged from 0.3 to 143 mg/100 g fresh weight, with the highest value was found in Sauropus androgynus (L) Merr or known as cekur manis in Malay. This might be due to the presence of quercetin and kaempferol (Andarwulan et al., 2010). Other factors such as climate and cultivar were also identified as the main factors affecting the structure of phenolics and other bioactive compounds .
Antioxidant inhibits the cellular damage by its known mechanism, free radical scavenging. DPPH free radical scavenging is one of the methods used to evaluate antioxidant activities with a very limited period of time compared to other methods. The EC 50 value was determined to better quantify and determine the radical scavenging activity in the samples. Gnetum gnemon or pucuk empeng showed the lowest EC 50 value with 3.92±1.52 µg/mL as shown in Table 1. Therefore, it possessed the strongest DPPH radical scavenging activity. There was also significant difference for all samples for 80% methanolic extract (p< 0.05). The previous study also reported that the stick of G. gnemon extracted with methanol gave the highest scavenging activity with a value of 38.679 ± 0.745% (Wazir et al., 2011). Other study demonstrated that C. caudatus (ulam raja) extract inhibited linoleic acid oxidation which resulted in greatest DPPH free radical scavenging activity (Andarwulan et al., 2010) that the extract with a high amount of polyphenol content was able to exhibit high antioxidant activity Reihani and Azhar, 2012). In this study, the extract of pucuk empeng corresponded with this suggestion but not in pucuk simpuh since it showed a relatively low antioxidant activity but higher total phenolic content. Poor specificity of the total phenolic content assay might be the reason for this disagreement (Singleton et al., 1999;Escarpa and González, 2001). Besides, the number of phenolic compounds present may affect its respond towards Folin-Ciocalteu reagent (Singleton et al., 1999).
ABTS radical scavenging assay is a method used to study the antioxidant potential and the results varied from that of DPPH radical scavenging activity method. In this assay, the highest scavenging activity was found in pucuk simpuh with the value of 7.93±0.02 µg ascorbic acid/mL. However, the results from ABTS assay showed that pucuk simpuh had the highest scavenging activity and it was varied from DPPH method whereby pucuk empeng had the highest scavenging activity. The difference in the trend of the results might be due to the different mechanism of action in both methods.
Phytochemicals such as the phenolic and flavonoid compounds contributed to the antioxidant activity in the wild ulam/vegetables extracts when correlation analysis was performed. Some correlations were observed between antioxidant activity and phytochemical content for several samples. From this analysis, DPPH free radical scavenging activity of pucuk putat was highest, moderately positively correlated with the phenolic content of the extracts (r = 0.615) while tebu gogok showed the lowest positive correlation with total phenolic content (r = 0.264). The results were in agreement with the previous studies which showed a strong correlation between the total phenolic content and antioxidant activity (Deighton et al., 2000;Martínez et al., 2012).
Meanwhile, total flavonoid content of pucuk putat showed the strongest positive correlation with antioxidant activity in all samples tested (r = 0.999), which was in agreement with an earlier study which also reported that antioxidant activity was closely related to the phenolic and flavonoid content (Sabli et al., 2012;Ling et al., 2015). Furthermore, ABTS radical scavenging of the extracts of pucuk putat was positively correlated to the total phenolic (r = 0.982) and pucuk empeng showed the lowest correlation with total phenolic content (r = 0.480). However, total flavonoid content of the pucuk simpuh extract was highly correlated with ABTS free radical scavenging (r = 0.987), while pucuk putat showed lowest correlation with ABTS free radical scavenging (r = 0.718).

Conclusion
Wild ulam/vegetables in Taman Negara Johor Endau Rompin (TNJER) are rich in phytochemicals and antioxidant which can be a great potential for use in complementary and alternative medicines as well as functional food applications. Chan, E.W.C., Tan, Y.P., Chin, S.J., Gan, L.Y., Kang, K.X., Fong, C.H., Chang, H.Q. and How, Y.C.