Antioxidative and Antihypertensive Activities of Selected Malaysian ulam ( salad ) , Vegetables and Herbs

This study was conducted to investigate antioxidative and antihypertensive activities of selected Malaysian ulam (salad), vegetables and herbs. The aqueous extract of selected ulam (salad), vegetables and herbs were analysed for total phenolic content (TPC), antioxidant activities (2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical cation scavenging assay and ferric reducing antioxidant power assay (FRAP) and antihypertension activity (angiotensin converting enzyme (ACE) inhibitory activity assay). TPC analysis showed that Polygonum minus contains significantly (p<0.05) highest phenolic compound at 48.23 ± 0.17 mg GAE/g as compared to other plants. DPPH analysis showed that P. minus had significantly (p<0.05) highest percentage of radical scavenging activity at 79.09 ± 0.10% as compared to other plants. ABTS analysis showed that Sauropus androgynus had significantly (p<0.05) highest percentage of radical cation scavenging activity at 95.10 ± 0.26% as compared to other plants. FRAP analysis showed that P. minus had significantly (p<0.05) highest ferric reducing power at 63.61 ± 0.73 mmol Fe/g as compared to other samples. Murraya koenigii had the highest percentage of ACE inhibitory activity (91.20 ± 4.15%). Correlation analysis showed positive and significant (p<0.01) correlation between TPC and FRAP (r = 0.956), TPC and ABTS (r = 0.635), TPC and DPPH (r = 0.630) and TPC and ACE inhibitors (r = 0.645). This shows that Malaysian tropical plants especially P. minus are potential source of natural antioxidant and antihypertensive agents.


Introduction
Prevalence of mortality from non-communicable diseases (NCDs) such as cancer is increasing and it is at an alarming rate (WHO report, 2014).Reactive oxygen species (ROS) include free radicals such as superoxide anion radicals (O 2 -), hydroxyl radical (OH -) and non-radical species such as hydrogen peroxide (H 2 O 2 ) and singlet oxygen ( 1 O 2 ).ROS promote oxidative damage to biomolecules such as lipids, nucleic acids, proteins and carbohydrates that leads to ageing, cancer and other diseases (Campanella, Martini, Rita, & Tomassetti, 2006).The phenolic antioxidant in plant was reported to capture oxygen free radicals by donating a hydrogen atom or electron to the free radical (Antolovich, Prenzler, Kevin, & Danielle, 2002).
Malaysia is a tropical country with diverse species of flora and fauna due to the hot climate, high humidity and rain throughout the year.This climate led to the growth of various types of plant species including ulam (salad), vegetables and herbs (Mohd Faez et al., 2014).Ulam (salad) is defined as fresh or raw green salad that can been eaten with fermented sauce, herbs or spices as side dishes by the Malay community while vegetable refers to a plant that is often eaten either by blanching, boiled or stir-fried (Rukayah, 2000;Reihani & Azhar, 2012).In addition to ulam (salad) and vegetables, Malaysian foods are also rich with herbs such as turmeric, kesum and ginger (Huda-Faujan, Noriham, Norrakiah, & Babji, 2007).ethnic groups in Malaysia (Nurul Izzah et al., 2012).In addition, epidemiological studies show that consumption of vegetables can prevent the occurrence of diseases caused by oxidative stress such as cancer, hypertension, heart disease, and diabetes (Halvorsen, Carlsen, Philips, Bohn, & Holte, 2006).This is due to high antioxidant content in vegetables such as carotenoids, phenolic compounds, benzoic acid, flavonoids, proanthocyanin, stilbene, coumarins, lignans and lignin (Lindsay & Astley, 2002;Zheng & Wang, 2001).Phenolic compounds from plants are beneficial as an effective angiotensin I-converting enzyme (ACE) inhibitors that lowers the blood pressure (Nadin et al., 2013).
Several studies had been conducted to evaluate antioxidant activity of ulam (salad), vegetables and herbs (Faridah, Nordin, Israf, Khozirah, & Umi Kalsom, 2006;Huda-Faujan et al., 2007;Sivanesan & Hazeena, 2007;Pitchaon, Sirikarn, & Pitiporn, 2008;Krishna et al., 2010;Reihani & Azhar, 2012;Boonyadist et al., 2013;Shrawan et al., 2015).However, a sole method of evaluating antioxidant activities of plant extracts was not sufficient to identify all possible mechanisms characterising an antioxidant.Moreover, antihypertensive activity evaluation of ulam (salad), vegetables and other herbs are yet to be widely conducted.Therefore, the objective of the present work is to evaluate the antioxidant activity of several Malaysian ulam (salad), vegetables and herbs using three different methods and to evaluate the antihypertensive activity.

Plants
Plants were categorized into four groups, namely ulam (salad), local vegetables, legumes vegetables and herbs.The type of plants selected on each category and the specific parts used for this study were presented in Table 1.The plants selected were based on the types of ulam (salad), vegetables and herbs that were commonly consumed among Malaysians.All fresh samples were purchased from wet market in Bandar Baru Bangi, Selangor.

Sample Preparation and Extraction
The edible portion of each samples were separated and washed under running tap water.They were drained and air-dried.The samples were then chopped into small pieces and weighed.Next, the samples were stored at -80°C and freeze dried for 48 hours (Beta 2-8 LDplus, Christ, Germany).Upon freeze-drying, the samples were weighed and homogenized using a commercial blender (7011HS, Waring, U.S.A).The ground samples were stored in air-tight container at -20°C prior to extraction.The extract was prepared according to the method by Wong, Leong and Koh (2006) with some modifications.Five gram of ground sample was weighed and added with 250 mL of distilled water.This was followed by agitation of the mixture at 100 rpm using shaker (Innova 2000/2050, New Brunswick, Germany) for 24 hours at room temperature.The mixture was then filtered using filter paper No. 1 (Whatman, U.K) and lyophilised for 48 hours using freeze-dryer (Beta 2-8 LDplus, Christ, Germany).The dried samples were stored at -20°C for further analysis.The percentage of extract yield was calculated as below: % yield = Weight of sample extract Initial weight of the sample × 100

Determination of Total Phenolic Content (TPC)
TPC in the aqueous extract of plants was determined according to the Folin Ciocalteu method (Singleton & Rossi, 1965).For each sample, 1 mL of aqueous extract (5 mg/ mL) was transferred into test tube.Then 5 mL of 10 times diluted Folin Ciocalteu reagent were added and allowed to stand for 5 minutes.After 5 minutes, 4 mL of sodium carbonate was added and the mixture was incubated at room temperature for 2 hours in the dark.The absorbance was measured at 725 nm using a UV-VIS microplate reader (Epoch, Biotech, U.S.A).The standard curve was prepared using gallic acid at a series of concentrations of 0 to 100 ppm.The TPC was expressed as gallic acid equivalent per gram of dry weight (mg GAE/g).

Antioxidant Activity Assays
Antioxidant activity assays in the aqueous extract of plants was determined using three method.The first method was performed using DPPH scavenging test as described by Brand-Williams, Cuvelier and Berset (1995) with some modifications.For each sample, 500 µL of aqueous extract (500 µg/ mL) was mixed with 2.5 mL of 0.1 mM DPPH.The mixture was then incubated for 30 minutes in the dark.The control sample was prepared by mixing 500 µL of distilled water with 2.5 mL of 0.1 mM DPPH.After 30 minutes, absorbance was measured at 517 nm using UV-VIS microplate reader (Epoch, Biotech, U.S.A).Percentage of DPPH scavenging activity was calculated as below: where, A is the absorbance and A (control) is the absorbance of the reaction containing distilled water and 0.1 mM DPPH.
The second method used ABTS radical cation as described by Arnao, Cano and Acosta (2001) with some modifications.For each samples, 500 µL of aqueous extract (500 µg/ mL) were mixed with 2.5 mL of ABTS solution and incubated for 10 minutes in the dark.After 10 minutes, absorbance was measured at 734 nm using a UV-VIS microplate reader (Epoch, Biotech, U.S.A).Percentage of inhibition was calculated using the following formula: where, A is absorbance and A (control) is the absorbance of the reaction containing distilled water and ABTS solution.
The last antioxidant activity method was the ferric reducing power (FRAP) as described by Benzie and Strain (1996) with some modifications.For each samples, 500 µL of aqueous extract (500 µg/ mL) were mixed with 2.5 mL of FRAP reagent and allowed to react in the dark for 30 minutes.After 30 minutes, absorbance was measured at 593 nm using a UV-VIS microplate reader (Epoch, Biotech, U.S.A).Different concentrations of ferrous sulfate (0.1-1.0 mM) were prepared as standard.FRAP activity was calculated as a proportion of mM ferrous sulfate obtained from the standard curve of ferrous sulfate.The FRAP activity was expressed in mmol Fe 2+ /g.

Angiotensin Converting Enzyme (ACE) Inhibitory Activity Assay
ACE inhibitory activity was determined following the method described by Ngo, Ryu and Kim (2014) with some modifications.A total of 50 µL of ACE enzyme (25 mU/ mL) was mixed with 50 µL of aqueous extract of plants (500 µg / mL) and incubated at 37°C for 10 minutes.Next, 150 mL Hippuryl-L-Histidyl-L-Leucine (HHL) (8.3 mM HHL in 50 mM sodium borate buffer containing 0.5 M NaCl, pH 8.3) was added and the mixture was incubated at 37°C for 30 minutes.Finally, the reaction was inhibited by adding 250 µL of 1 M HCl.The resulting hippuric acid was extracted with addition of 500 µL ethyl acetate.After centrifugation (3000 rpm, 10 minutes), 200 µL of the upper layer was transferred into the tube and dried at 90°C for 10 minutes.The hippuric acid was redissolved in 1 mL of distilled water and the absorbance was measured at 228 nm using a UV-VIS microplate reader (Epoch, Biotech, U.S.A).ACE inhibitory activity was calculated according to the following equation: where, A is absorbance and A (control) is the absorbance of the reaction containing distilled water and ACE enzyme (25 mU/ mL).

Statistical Analysis
The data was analysed using Statistical Packages for Social Science (SPSS) version 21.0.Analysis of variance (ANOVA) followed by Duncan's multiple range test was used to test the significant difference among the samples.Pearson correlation was used to test the association between the TPC and the antioxidant activity and ACE inhibitory activity.The results were reported as mean ± standard deviation.

Extraction Yield
The yield of plant aqueous extract was in the range of 5.03 to 12.42%, as presented in the Table 2. Moringa oleifera showed the highest extraction yield (12.42%) while P. minus was the lowest (5.03%).Extraction yield of M. oleifera was significantly higher (p <0.05) compared to the other plants.Values with the same lowercase within each column are not significantly different (p>0.05)

TPC Analysis
TPC of plant aqueous extract are also presented in Table 2; and it ranged from 2.46 mg to 48.23 mg GAE/g.P. minus showed the highest TPC (48.23 mg GAE/g extract) whereas the lowest TPC was observed in M. oleifera (2.46 mg GAE/g extract).TPC value for P. minus was significantly higher (p <0.05) compared to the other plants.

Correlation
The correlation between total phenolic content with antioxidant activity and ACE inhibitory activity was shown in Table 5.The results revealed that there was a strong correlation between TPC and FRAP (r = 0.956) followed by the TPC and ABTS (r = 0.635), and TPC with DPPH (r = 0.630).From Table 5, there was a positive correlation between TPC and ACE (r = 0.645).The antioxidant activity using DPPH, ABTS and FRAP assay in this study also showed a strong correlation.The highest correlation was observed between ABTS and FRAP assay (r = 0.651), while the lowest correlation was detected between DPPH and ABTS assay (r = 0.480).

Discussions
The plant's active compounds usually occur in low concentration, therefore, an extraction technique is needed in order to obtain the required extract with high yield and minimal changes to the functional properties.(Quispe Candori, Foglio, Rosa, & Meireles, 2008).Based on our findings, percentage of extraction yield was higher from the leguminous parts compared to the leafy parts.A previous study by Pitchaon et al. (2008) also showed that better extraction yield could be achieved from nuts and fruits (1.3-4.5%)than leaves and flowers (0.7-2.5%).Besides, a study of Lee et al. (2011) reported that the extraction yield of P. tetragonolobus (8.53%) was higher compared with S. androgynus (5.20%).Sample particle size, extraction temperature and extraction ratio of solvent to samples are some of the factors that influence percentage of extraction (Herodez, Hadolin, Skerget, & Knez, 2003).
Antioxidants are capable of either delaying or inhibiting the oxidation process that occurs due to presence of free radicals (Pisoschi & Negulescu, 2011).Free radicals promote lipid, DNA, protein and carbohydrate damage that cause various diseases such as cancer, atherosclerosis and rheumatoid arthritis (Aruoma, 1994).Antioxidant acts with free radicals by donating one electrons to free radical atom that neutralizes the effects of oxidative damage (Valko et al., 2007).Epidemiological studies have shown that intake of natural antioxidant like phenolics from vegetables and fruits were associated with lower risk of cancer (Temple, 2000;Wolfe & Liu, 2003).The aqueous extract of all analyzed 16 plants contained varying amount of TPC.Naczk and Shahidi (2006) reported that extraction method using more polar solvent, longer extraction time and larger sample to solvent ratio will result in higher amount of phenolic compounds.In addition, cultivars and climate are also among the other factors that influence the phenolic structure and bioactive components in plants (Bolling, Dolnikowski, Blumberg, & Chen, 2010).P. minus contained the highest TPC among the studied samples.Huda-Faujan et al. (2007) also reported the highest contents of the TPC in P. minus (44.35 mg TAE/100g).P. minus is a herb that is often used as a seasoning for preparation of fish, noodles and other Malay cuisine (Rukayah, 2000).P. minus was reported to have several medicinal properties such as antimicrobial activity (Uyub, Nwachukwu, Azlan, & Fariza, 2010), cytotoxic activity towards human cervical carcinoma (Mackeen et al., 1997), antioxidant activity (Huda-Faujan et al., 2007) and anticancer activity (Abdullah, Mavaddat, & Mohd Ali, 2013).
In the present study, determination of antioxidant capacity was done using DPPH, ABTS and FRAP.According to Frankel and Meyer (2000), just one assay method cannot identify all possible mechanisms characterising an antioxidant activity.ABTS method is based on the ability of antioxidant molecules to inhibit the radical cation ABTS that change the color of blue-green complex (Antolovich et al., 2000;Wootton-Beard, Aislin, & Lisa, 2011).On the other hand, DPPH assay relies on DPPH itself as the stable free radical to determine antioxidant activity in natural compounds.The assay will be determined based on the colour change of purplish DPPH into a yellowish compound of α, α-diphenyl-β-picrylhydrazine (Pisoschi & Negulescu, 2011).FRAP is a method based on reaction of sample extract that results in colour change from colourless Fe (III) to the blue colour complex of Fe (II) (Antolovich et al., 2000;Wootton-Beard et al., 2011).P. minus showed the highest DPPH free radical scavenging activity and ferric reducing activity.Maizura, Aminah and Wan Aida (2011) also reported the highest DPPH free radical scavenging activity of P. minus extract (82.6 ± 0.7%) compared to Zingiber officinale (79.0 ± 0.6%) and C. longa (64.6 ± 2.4%).On the other hand, S. androgynus extract resulted in the highest ABTS radical cation scavenging activity.Similarly, S. androgynus extract was also reported to potray the highest ABTS inhibitory activity in another separate study conducted by Andarwulan, Ratna, Diny, Bradley and Hanny (2010).ABTS method showed higher values due to it ABTS radical cation's that is reactive towards most antioxidants and it is soluble in aqueous solvent (Martysiak-Zurowska & Wenta, 2012).However, O. javanica and S. grandiflora showed higher value using DPPH method maybe due to some compounds react very rapidly with DPPH (Martysiak-Zurowska & Wenta, 2012).Generally, the extracts with high amounts of phenolic content showed high antioxidant activity (Bolling et al., 2010).The reduction potential of Fe (III) is contributed by the phenolic components in the extracts which donated the hydrogen and reduced the Fe (III)-TPTZ to Fe (II)-TPTZ complex (Shimada, Fujikawa, Yahara, & Nakamura, 1992).ACE inhibitory activity was conducted via ethyl acetate extraction and spectrophotometric assay of hippuric acid (Cushman & Cheung, 1971).ACE inhibitory activity was only detected in aqueous extract of C. longa, P. speciosa, G. gynandra, C. asiatica, C. caudatus, P. minus, C. longa and M. koenigii.TPC analysis indicated high phenolic content in the aqueous extract of these plants.A study conducted by Dong, Xu, Liang, Head and Bennett (2011) showed that antioxidants such as polyphenols extracted from the sample can act as an ACE inhibitor.C. caudatus is the most preferred ulam by Malaysian community while P. minus, C. hystrix and M. koenigii are a herb that is often used in dishes such as laksa, tom yam and curry stew.They also believe that these herbs have medicinal properties such as anticancer and antihypertensive (Rukayah, 2000;Wan Hassan & Mustaffa, 2010;Jamilah, Abdul Kadir Gedi, Suhaila, & Md. Zaidul, 2011).
Correlation analysis was conducted to examine the relationship between two variables.Previous studies reported that phenolics compounds in vegetables and herbs significantly contributed to their antioxidant properties (Wong et al., 2006;Huda-Faujan et al. 2007;Maizura et al., 2011).Strong correlation between TPC and FRAP (r = 0.956), TPC and ABTS (r = 0.635) and TPC with DPPH (r = 0.630) was observed among all the 16 plants.It showed that, the higher total phenolics content of plants extracts resulted in higher antioxidant activity.A previous study by Wan-Ibrahim, Sidik and Kuppusamy (2012) also indicated a strong correlation between TPC and FRAP (r = 0.887) and TPC with DPPH (r = 0.984) among the 20 plants analyzed.Rice-Evans, Miller and Paganga (1997) stated that the phenolic compound acts as a reducing agent, hydrogen donors and oxygen trappers which plays an important role in determining the antioxidant potential.Whereas, high DPPH free radical scavenging activity was contributed by low molecular content.Positive correlation was also observed between TPC and ACE (r = 0.645).Nadin et al. (2013) reported that ACE inhibition mechanism involves the interaction of three phenolic groups of gallic acid, phenolic acids and flavonoids and other flavonoids with ACE active group.Positive correlations between DPPH, ABTS and FRAP assays were observed.The highest correlation was between ABTS and FRAP (r = 0.615) and the lowest correlation was between DPPH and ABTS (r=0.481).The high correlation between ABTS and FRAP implies that ABTS radical cation react rapidly with antioxidants (Martysiak-Zurowska & Wenta, 2012) and the FRAP technique showed high reproducibility (Thaipong et al., 2006).While the low correlation between DPPH and ABTS implies that the reaction of DPPH with most antioxidants is slower compared with ABTS radical cation as reported by Martysiak-Zurowska and Wenta (2012).

Conclusions
In conclusion, P. minus showed the highest total phenolic content (48.23 mg GAE/g) and antioxidant activity (DPPH: 79.09 ± 0.1%, FRAP: 63.61 ± 0.73 mmol Fe 2+ /g) among all the aqueous extract.Whereas, the highest ACE inhibitory activity was detected in M. koenigii extract (91.20 ± 4.15%).The findings from our study attracts interests on one of the selected Malaysian plants, P. minus as a promising source of antioxidants and antihypertensive.

Table 1 .
List of selected Malaysian ulam (salad), vegetables and herbs

Table 2 .
Extraction yield and total phenolic content of selected Malaysian ulam (salad), vegetables and herbs

Table 4 .
ACE inhibitory activities of selected Malaysian ulam (salad), vegetables and herbs