Tropical Agricultural Research

Volume 30 Number 3

Editor-in-Chief Dr. U.W.A. Vitharana

Postgraduate Institute of Agriculture University of Peradeniya Peradeniya, Sri Lanka

May, 2019 Editor-in-Chief Dr. U.W.A. Vitharana B.Sc.(Peradeniya, Sri Lanka), M.Sc., Ph.D. (Ghent, Belgium) Department of Soil Science, Faculty of Agriculture, University of Peradeniya, Peradeniya, Sri Lanka.

Editorial Advisory Board Prof. P. Abeygunawardena, Former Professor, Texas A & M University, U.S.A Prof. B.F.A. Basnayake, University of Peradeniya, Sri Lanka Prof. W.A.J.M. De Costa, University of Peradeniya, Sri Lanka Prof. D.M De Costa, University of Peradeniya, Sri Lanka Dr. H.M.V.G. Herath, University of Peradeniya, Sri Lanka Prof. A. Hetherington, University of Bristol, United Kingdom Dr. W. Jenner, Centre for Agriculture and Biosciences International, Switzerland Prof. D. Kumaragamage, University of Winnipeg Winnipeg, Canada Dr. B.E.P. Mendis, University of Peradeniya, Sri Lanka Prof. N. Mukhopadhyay, University of Connecticut, USA Prof. D. Pavelkova, Tomas Bata University in Zlin, Czech Republic Prof. R. Pegg, University of Georgia, USA Dr. B.L. Peiris, University of Peradeniya, Sri Lanka Dr. N. Sanderatne, Postgraduate Institute of Agriculture, University of Peradeniya Dr. U.W.A. Vitharana, University of Peradeniya, Sri Lanka Dr. S. H. Wani, Michigan State University, USA Prof. W.A.D.P. Wanigasundera, University of Peradeniya, Sri Lanka Prof. M.P.B. Wijayagunawardhana, University of Peradeniya, Sri Lanka

Editorial Board Prof. D.U. Ahn, Iowa State University, USA Prof. J.M.R.S. Bandara, University Brunei Darussalam, Brunei Dr. R.S. Dharmakeerthi, University of Peradeniya, Sri Lanka Prof. D. Banks, Duke University, USA Prof. G.M. Hettiarachchi, University of Kansas, USA Dr. N. S. Hettiarachchy, University of Arkansas, USA Prof. V. Hurry, Swedish University of Agricultural Sciences, Sweden Prof. K. Kawamoto, Saitama University, Japan Prof. S. Kodithuwakku, University of Peradeniya, Sri Lanka Dr. J. Lam, Memorial University, Canada Prof. W.M.T. Madhujith, University of Peradeniya, Sri Lanka Prof. B. Marambe, University of Peradeniya, Sri Lanka Prof. J. Prohens, Universitat Politecnica de Valencia, Spain Prof. G.L.L.P. Silva, University of Peradeniya, Sri Lanka Prof. R.P. de Silva, University of Peradeniya, Sri Lanka Prof. C. Sivayoganathan, Emeritus Professor, University of Peradeniya, Sri Lanka Dr. R. Sulaiman, Centre for Research on Innovation and Science Policy (CRISP), India Prof. R.O. Thattil, Emeritus Professor, University of Peradeniya, Sri Lanka Prof. J. Weerahewa, University of Peradeniya, Sri Lanka Prof. S. Wijesundara, National Institute of Fundamental Studies, Sri Lanka Prof. C. Wilson, Queensland University of Brisbane, Australia

ISSN: 1016 – 1422 Abstracting and Indexing: The Journal is indexed by CAB Abstracts

The journal ‘Tropical Agricultural Research’ publishes the papers accepted upon rigorous reviewing process. In the past, the journal ‘Tropical Agricultural Research’ carried scholarly articles submitted to the annual congress. Moving a step further, the editorial board welcomes submissions throughout the year. Further, submissions can be made throughout the online submission system available at the official website of the PGIA. Over the years, the journal has evolved as an international journal which is now abstracted and indexed in CAB Abstracts. This issue of volume 30 of the journal contains an ensemble of scholarly articles related to agriculture and allied fields those were presented at the 30th Annual Congress of the Postgraduate Institute of Agriculture.

I extend my sincere thanks for the guidance and support rendered by the Director/PGIA, the members of the Editorial Board, reviewers and the authors who submitted their research work for publication.

Dr. U.W.A. Vitharana Editor-in-Chief May, 2019

CONTENTS

Research Articles

Antioxidant Potential of Selected Underutilized Fruit Crop Species Grown in Sri Lanka 1 M.A.L.N. Mallawaarachchi, W.M.T. Madhujith and D.K.N.G. Pushpakumara

Millet Phenolics as Natural Antioxidants in Food Model Systems and Human LDL/VLDL 13 Cholesterol in vitro K.D.D. Kumari, W.M.T. Madhujith and G.A.P. Chandrasekara

Preliminary Evaluation of Probiotic Potential of Yeasts Isolated from Bovine Milk and Curd of Sri 27 Lanka D.U. Rajawardana, I.G.N. Hewajulige, C.M. Nanayakkara, S.K.M.R.A. Athurupana and T. Madhujith

Identification of Phosphorus Efficient Rice Cultivars under Low P Nutrition through Hydroponic 43 based Screening D.S. Kekulandara, P.C.G. Bandaranayake, D.N. Sirisena, W.L.G. Samarasinghe and L.D.B. Suriyagoda

Dynamics of Nitrifiers in Soils of Intensively Vegetable Cultivated Areas in Sri Lanka 55 K.K.K. Nawarathna, W.S. Dandeniya, R.S. Dharmakeerthi and P. Weerasinghe

Performance of Macrobrachium rosenbergii in Perennial Reservoirs: A Comparative Assessment of 69 Fisheries in Five Perennial Reservoirs in the Northern Province of Sri Lanka R. Rajeevan, U. Edirisinghe and A.R.S.B. Athauda

Short Communications

Effect of Seaweed Extract (Kappaphycus alvarezii) on the Growth, Yield and Nutrient uptake of 81 Leafy Vegetable Amaranthus polygamous S. Senthuran, B.L.W.K. Balasooriya, S.J. Arasakesary and N. Gnanavelrajah

Mineral Contents of Sri Lankan Rice Varieties as Affected by Inorganic Fertilization 89 H.M.A.J. Herath, G.A.P. Chandrasekara, U. Pulenthiraj, C.M.N.R. Chandrasekara and D.G.N.G. Wijesinghe

Determination of Optimum Nitrogen Concentrations in Hydroponics for Tomato Grown in Coir 97 Medium in Tropical Greenhouse H.R.U.T. Erabadupitiya, W.A.P. Weerakkody and K.A. Nandasena

Impact of Glass Ceiling on Women Career Development in Non-state Banking Sector in Colombo 105 U. K. S. M. Uduwella and M.W.A.P. Jayatilaka

Author Guide 109

Author Index 113

i Tropical Agricultural Research Vol. 30 (3): 1 – 12 (2018)

Antioxidant Potential of Selected Underutilized Fruit Crop Species Grown in Sri Lanka

M.A.L.N. Mallawaarachchi, W.M.T. Madhujith1* and D.K.N.G. Pushpakumara2

Postgraduate Institute of Agriculture University of Peradeniya Sri Lanka

ABSTRACT: Lyophilized aqueous extracts of four underutilized fruit species namely Diospyros discolor (Velvet apple), Pouteria campechiana (Lavulu/Canistel), Phylanthus acidus (Mal-Nelli/Star gooseberry) and Phyllanthus emblica (Nelli/Indian gooseberry) were investigated for the antioxidant potential (AP) by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) assay and ferrous reducing antioxidant power (FRAP) assay. Total phenolic content (TPC) and total monomeric anthocyanin content (TMAC) were determined by Folin-Ciocalteu’s colorimetric assay and pH differential method, respectively. Vitamin C (VitC) content of fresh fruit was evaluated titrimertically and expressed as mg of ascorbic acid in 100 g of fresh weight (FW). The TPC and TMAC were expressed as mg of gallic acid equivalents (GAE)/100g FW and mg of cyanodin-3-glucoside (C3G)/100g FW. The measured parameters differed significantly among four fruit species. The values ranged between 84.42 – 1939.70 mg GAE/100g FW, 10.41 – 55.64 mg C3G/100g FW, 0.067 – 310.63 mg FW/ml, 9 – 81.29%, 238.25 – 2891.57 2+ Fe mol/100g FW and 17.12 – 523.14 mg/100g FW for TPC, TMAC, IC50, RSA, FRAP and VitC, respectively. Phyllanthus emblica possessed highest values in all parameters while Phyllanthus acidus showed the lowest except in TPC. The lowest TPC was observed in Diospyros discolor. The extract of Pouteria campechiana also showed considerable amount of TPC (640 mg C3G/100g FW), RSA (76%) and VitC of 53 mg/ 100g. The results revealed that these underutilized fruit crops can be used as sources of natural antioxidants and vitamin C.

Keywords: Antioxidant potential, total anthocyanin content, total phenolic content, underutilized fruits crop species, Vitamin C

INTRODUCTION

Reactive oxygen species (ROS) are considered as harmful intermediates produced during oxygen metabolism in biological systems. Excess ROS in the body can lead to cumulative damage in proteins, lipids, and DNA, resulting in the condition termed as oxidative stress (Dudonne et al., 2009). Antioxidants can effectively mitigate the oxidative damage in biological systems by delaying or inhibiting the oxidation process caused by ROS (Shofian et al., 2011). Numerous epidemiological studies have reported that the regular consumption of fruits and vegetables is associated with the reduction of chronic diseases which are directly

1 Department of Food science and Technology, Faculty of Agriculture, University of Peradeniya, Sri Lanka 2 Department of Crop science, Faculty of Agriculture, University of Peradeniya, Sri Lanka * Corresponding author: [email protected]

Mallawaarachchi et al. linked with the oxidative stress. The increased intake of natural antioxidants, particularly the antioxidative compounds present in fruits and vegetables contribute to the antioxidant capacity of plasma and these constituents are reported to mitigate the damage caused by the oxidative stress (Lie et al., 2005; Oviasogie et al., 2009; Vidhan et al., 2010; Bopitiya and Madhujith, 2012).

In nutritional sciences and medicine, there is much interest on analysis of vitamins such as C, E and A as they are widely reported to have antioxidant activities via multiple mechanisms (Rutkowski and Grzegorczyk, 2007). Vitamin C, reduces the risk of arteriosclerosis, cardiovascular diseases and some forms of cancer (Jacob, 1996; Lee and Kader, 2000). Vitamin C is also known to have many biological functions in collagen formation, absorption of inorganic iron, reduction of plasma cholesterol level, inhibition of nitrosoamine formation, enhancement of the immune system and reaction with singlet oxygen and other free radicals (Lee and Kader, 2000). Both vitamin A and E are lipid soluble. Vitamin A is important for normal vision, growth, ageing and reproduction (Adeolu and Enesi, 2013). Vitamin E considered as a chain breaking antioxidant and reported that regular intakes are correlated with a reduced risk of cardiovascular diseases (Flome and Traber, 1999) Therefore, these plant extracts can be used as potential candidates to isolate natural antioxidants.Wide varieties of fruit crop species are available in Sri Lanka. Besides major fruit crops, there is a large number of fruit species that remain underutilized. As a result, their nutritive, medicinal and therapeutic values are little known to date.

The commonly grown two members of the family Euphorbiaceae, namely Phyllanthus emblica L. (Nelli/Indian gooseberry) and Phyllanthus acidus L. (Rata-Nelli/Star gooseberry) are cultivated as backyard fruit species in Sri Lanka. The fruits of P. emblica eaten in fresh form are considered as highly nutritious and therapeutic due to its high amount of Vitamin C, antioxidant and polyphenol content (Pushpakumara and Heenkenda 2007). It is reported to use in prevention and management of haemorrhage, anaemia, colic, acute leprosy, fits, insanity, jaundice, cough, hiccough, indigestion, dyspepsia, asthma and other diseases in traditional medicine (Jayaweera, 1981). P. acidus fruits may be eaten fresh after sprinkled with salt, processed into pickle and sweetened dried fruits. In Malaysia, ripe and unripe fruits are cooked and served as a relish, or made into a thick syrup or sweet preserve (Lim, 2012a). Ramasamy et al. (2011) reported that leaves of P. acidus perform mild cytotoxicity on human breast cancer cell line (MCF7), epidermal carcinoma of cervix cell line (CaSki), ovarian cancer cell line (KO3) and colon cancer cell line (HT29). However, these fruit crops have been neglected in Sri Lanka and their uses have not been properly exploited (Pushpakumara and Heenkenda 2007).

Pouteria campechiana (Kunth) Baehni. (Sapotaceae), Canistel locally known as Lavulu mainly grown in home gardens of wet and intermediate zones of Sri Lanka. Due to its compact crown and glossy leaves, the tree is used for landscaping as well. The fruit is consumed as a dessert fruit or pickled with salt and pepper. The dehydrated pulp can be used as natural food colorant especially in baked products. Lanerolle et al. (2008) reported that P. campechiana fruit pulp is a rich source of pro-vitamin A carotenoids.

Diospyros discolor Willd. a member of family Ebenaceae commonly known as velvet apple, normally grown in the home gardens of wet and intermediate zones of Sri Lanka as ornamental and road side shade tree. It has been reported that fruits, leaves and bark of D. discolor is used in traditional medicine to cure diarrhoea, cough, fever and dysentery. The ripe fruit is peeled and eaten fresh or used in salad or stew or fried like a vegetable (Lim, 2012b). According to Lee et al. (2006) the leaves of D. discolor possesses higher antioxidant activity. However to

2 Antioxidant Potential of Fruit Crop Species the best of our knowledge, there are no previous reports on antioxidant potential of P. campechiana and D. discolor fruit.

Present study was carried out to evaluate the antioxidant potential, total phenolic, total anthocyanin and vitamin C contents of four underutilized fruit crop species namely, Phyllanthus acidus, Phyllanthus emblica, Pouteria campechiana and Diospyros discolor.

METHODOLOGY

Chemicals and regents

The gallic acid, Folin-Ciocalteu’s phenol reagent, sodium carbonate, 2,2-azinobis (3- ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-Azobis (2- amidinopropane) dihydrochloride (AAPH), 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH), 2, 4, 6-tris (2-pyridyl)-1, 3, 5-triazine (TPTZ) and 2,6-dichlorophenolindophenol, were purchased from Sigma, USA. All other chemicals used were of analytical grade.

Sample preparation

Fully matured fruits of P. acidus (Star gooseberry), P. emblica (Indian gooseberry) and fully ripe fruits of P. campechiana (Canistel) and D. discolor (Velvet apple) (Figure 1) were collected from the plants at mid fruiting stage from the home gardens in the Bandarawela region belonging to the upcountry intermediate zone (IU3), Sri Lanka and transported to the Regional Agriculture Research and Development Centre, Bandarawela under cold conditions. The samples were sorted for physical and pest damages, washed with running tap water followed by distilled water and drained to remove the surface water. Hundred grams of each fruit was taken after removing seeds and outer skin of P. campechiana and D. discolor. Then homogenized to prepare the composite sample. Six samples from each fruit were used to prepare the composite sample. For the evaluation of total phenolic content total monomeric anthocyanin content and antioxidant potential, 40 g of homogenates of each species were extracted with 120 ml of distilled water by keeping on reciprocating shaker at 450 rpm for 90 min at room temperature followed by centrifugation at 4,500 rpm for 10 min. The supernatants were collected separately and the extraction was repeated twice with residues, supernatants were combined and lyophilized at -50 °C, 24 mbar for 96 h and subsequently stored at •20 °C until further analysis.

To determine total phenolic content (TPC), ferric reducing antioxidant power (FRAP), total antioxidant capacity (TAC) and total monomeric anthocyanin content (TMAC), twenty milligrams of each lyophilized samples were dissolved in 3 ml of methanol, filtered using whatman number one filter papers, the filtrates were volume up in the 5 ml volumetric flasks with distilled water to have final concentration of 4 mg/ml. For the DPPH assay, 50 mg of lyophilized samples were taken followed same procedure then six different concentrations ranging between 0.5 mg/ml and 10 mg/ml were prepared.

Determination of total phenolic content

The TPC was measured using the method described by Yu et al. (2002) with slight modifications. Briefly, 100 μl of 2 N Folin-Ciocalteu regent and 1.58 ml of distilled water were added to 20 μl of each sample, vortexed, incubated at room temperature for 8 minutes, subsequently 300 μl of 0.7 M sodium carbonate was added, incubated for 30 minutes at room

3 Mallawaarachchi et al. temperature, and absorbance was measured at 765 nm by using a Helios Omega – UV – VIS spectrophotometer. Different concentrations of gallic acid (0 - 1 mg /ml) were used to construct the standard curve and the results were expressed as mg gallic acid equivalents in 100 g of fruit in fresh weight (mg GAE/100 g FW).

(a) (b)

Figure 1. Fruits of Selected Species (a - D. discolor fruit, b - P. acidus fruit, c - P. emblica fruit, and d - P. campechiana fruit)

Determination of antioxidant potential

Ferric reducing antioxidant power assay

The assay was adapted from Benzie and Strain (1996) with minor changes. The FRAPreagent was freshly prepared by mixing acetate buffer (150 mM, pH 3.6), TPTZ solution (5 mM TPTZ 0 in 40 mM HCl), FeCl36H2O (10 mM) in a ratio of 10:1:1. The regent was pre-heated at 37 C in a water bath for 10 min. To perform the assay, 1.5 ml of FRAP reagent and 0.05 ml sample (4 mg/ml) were mixed and vortexed for 30 s, absorbance was measured at 593 nm, using the FRAP working solution as a blank after a lapse of 4 min. The antioxidant potential of samples were determined using a linear regression equation (Y= 6.723x, r2= 0.9389) obtained from the standard curve plotted using FeSO4 7H2O (0 - 5 mM) and results were expressed as M of Fe 2+/100 g fruits in fresh weight (FW).

DPPH radical scavenging assay

The 2, 2-diphenyl-1-picrylhydrazyl hydrate (DPPH) free radical scavenging activity of fruit crop species was carried out according to the method described by Su et al. (2007) with some modifications. Six different concentrations ranged between 0.5 mg/ml and 10 mg/ml of each lyophilized extract (0.2 ml) was mixed with 1.8 ml of 0.1 mM methanolic DPPH radical. The absorbance was read at 517 nm using Helios Omega – UV – VIS spectrophotometer after

4 Antioxidant Potential of Fruit Crop Species leaving the mixture for an hour in the dark at room temperature. The procedure was repeated six times for each concentration. The control was prepared by adding 0.2 ml of methanol into 1.8 ml of DPPH radical. Using following formula, the radical scavenging activity (RSA) was calculated as percentage of discoloration of DPPH radical.

RSA% = {1- (A sample/A control)} * 100 where; A sample is the absorbance of the sample at 517 nm and A control is the absorbance of the control at 517 nm.

The results were expressed as IC50 values that denote the concentration of the sample required (mg of FW/ml) to scavenge 50% of DPPH radicals, was derived from RSA vs Concentration plot of each sample.

Determination of total antioxidant capacity

The TAC was determined using ABTS radical scavenging capacity assay. The ABTS radical cation (ABTS•+) was generated by reacting 2,2- azino-bis (2-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, 2.5 mM in PBS at pH 7.4) with 2,2'- azino-bis (2- methylpropanimidamide) dihydrochloride (AAPH, 2 mM in PBS at pH 7.4) in to 1ː 1 ratio. The mixture was kept in a water bath at 60 ºC until color developed which possess the absorbance between 0.3 - 0.5 at 734 nm. To 40 l of each sample, 1.96 ml of stock was added and absorbance was measured over six minutes at one minute interval at 734 nm (Zhou and Yu, 2004), the RSA was calculated as percentage inhibition of ABTS+ radical.

Determination of total monomeric anthocyanin content

The pH differential method, previously reported by Lee et al. (2005) was used with slight modifications to determine the total monomeric anthocyanin content of the freeze dried extracts. In brief, two separate reaction medium were prepared with 1.80 ml of, potassium chloride buffer (pH 1) and sodium acetate buffer (pH 4.5) with 0.2 ml of extract (4 mg/ml) for each. Reaction medium was kept for 15 min at room temperature followed by absorbance was measured at 520 nm and 700 nm wave lengths using Helios Omega – UV – VIS spectrophotometer. The TMAC was calculated as follows and expressed as mg of Cyanidin- 3-glucoside in 100 g of Fresh sample. A x MW x DF x 103 C3G = Ɛ x 1 A = Absorbance (A 520 –A 700) pH1 - (A 520 –A 700) pH4.5 MW = Molecular weight C3G (449.2g) DF = Dilution factor 1 = Path length (cm) Ɛ = Molar extinction co-efficient (26 900)

Determination of the vitamin C content

Determination of vitamin C content was performed on the basis of content of L- Ascorbic acid (AA), by using the titrimetric method described by Omale and Ugwu (2011). Accurately weighed (5 g) of fresh sample was macerated with 0.25% Oxalic acid solution, filtered, filtrate

5 Mallawaarachchi et al. was transferred to a 50 ml volumetric flask volume up to the mark with 0.25% oxalic acid and 10 ml of it taken for the titration with standardized 2,6 Dichlorophenolindophenol dye solution.

Statistical analysis

Data, obtained by six replicates were statistically analyzed using SAS 9.1 statistical software. Analysis of variance and least significant difference tests were conducted to identify mean differences. Statistical significance was declared at p = 0.05. To evaluate the relationship between methods used, linear regression and correlation analysis of the values were performed using MS Office Excel software.

RESULTS AND DISCUSSION

Total phenolic content

Polyphenolic compounds are commonly found in both edible and inedible plants, and reported to have multiple biological effects, including antioxidant activity (Kähkönen et al., 1999; Wojdylo et al., 2007). The TPC of selected fruit species was evaluated by Folin-ciocaltures’ colorimetric method, varied widely among selected fruit species ranging from 84.42 mg GAE/100 FW in D. discolor to 1,939.70 mg GAE/100 g FW in P. emblica (Table 1). All aqueous extracts tested, contained high TPC compared to commonly consumed fruit species. The TPC of pineapple, mango, papaya, apple, and berries were 47.9, 56.0, 57.6, 11.9, and 28.7 mg GAE/100g, respectively (Kriengsak et al., 2006; Bopitiya and Madhujith 2012). While Silva and Sirasa (2018) reported 80% (v/v) methanolic extracts of TPC of P. emblica was 915.7 mg GAE/100 g FW, which was lower than that of our results. The water extracts of P. emblica prepared by boiling for 5 min, reported to have 295.94 mg GAE/g of dry weight (Jayathilake et al., 2016). The methanolic extracts of different parts (bark, fruits and leaves) of D. discolor reported to contain 9.16, 5.95 and 5.65 mg GAE/g of extract, respectively (Das et al., 2010). Ethanolic extracts of oven dried fruits of P. acidus are reported to have 4.26 mg GAE/g (Zulaikha et al., 2017). It was found that the yield in total phenolic compounds depends on the method and the choice of solvent (Goli et al., 2005). The TPC also varies with the growing season, soil and climatic factors, stage of maturity of the plants (Wang and Zheng, 2001).

Total monomeric anthocyanin content

Anthocyanins considered as one of the major bioactive compounds among six classes of flavonoids (flavonols, flavanones, isoflavones, flavan-3-ois, flavones and anthocyanins) (Haminuik et al., 2012). There is increasing interest in the anthocyanin content of food and neutraceuticals because of possible health benefits (Lee et al., 2005). In this study, pH differential method was adapted to measure TMAC and calculations were done based on the cyaniding-3-glucoside (C3G), results were expressed as C3G equivalents in mg/100 g FW. According to the results presented in Table 1, the highest (55.64 mg C3G/ 100 g FW) TMAC was observed in P. emblica followed by P. campechiana, D. discolor and P. acidus. To the best of our knowledge this is the first report of TMAC of these fruit species in Sri Lanka.

Vitamin C content (Vit C)

The vitamin C content was expressed as mg of ascorbic acid (AA) in 100 g of fruits in fresh weight. The results summarized in Table 1, revealed that P. emblica had the highest AA and

6 Antioxidant Potential of Fruit Crop Species

P. acidus had the lowest. The value for the AA of P. emblica (523.14 mg AA/100 g FW) was differed with the observation of Silva and Sirasa (2018). Lee and Kader, 2000 stated that nutritional composition of a fruit type at harvest can vary widely depending on cultivar, maturity, climate, soil type, and fertility.

Vitamin C (AA) is an important dietary antioxidant in humans; it acts as highly effective antioxidant (Padayatty et al., 2003). In view of its antioxidant property, ascorbic acid and its derivatives are widely used as preservatives in food industry. Ascorbic acid plays an important role in the maintenance of collagen, which represents about one third of the total body protein.

Table 1. Total Phenolic Content (TPC), Total monomeric anthocyanin content (TMAC) and Vitamin C content (VitC) of selected fruit species

Fruit Species TPC TMAC VitC

P. emblica 1,939.70 ± 0.81a 55.64± 13.79 a 523.14± 2.24 a P. campechina 640.01 ± 0.48b 29.68± 4.33 b 53.03± 1.04 b P. acidus 112.89 ± 0.26c 10.41± 3.59 c 17.12± 0.76 d D. discolor 84.42 ± 0.09d 13.14± 3.29 c 39.15± 0.78 c Data are presented as Mean ±SD (n = 6) Values with different letters are significantly different at p < 0.5 TPC - mg GAE/100g fruit in fresh weight (FW), TMAC - mg C3G/100g fruit in FW and VitC - mg AA/100g fruits in FW

Antioxidant potential of fruit extracts

Antioxidant potential (AP) of plant extracts can be evaluated by using ability of scavenging of free radicals, as they deactivated or stabilized by the antioxidants before they cause oxidative damage towards cellular structures (Lee et al., 2014). Therefore, AP of lyophilized water extracts were determined by their ability to scavenge DPPH• and ABTS•+ radicals and reducing power of Ferric to Ferrous using three different assays of DPPH, ABTS and FRAP respectively. In DPPH, results were expressed as concentration (mg/ml) of fresh sample needed for inhibit 50% of radicals (IC50 value) in the reaction medium, which was obtained from the graphs developed from RSA values of different concentrations of each sample (0.5 – 10 mg/ml) and higher IC50 values denote lower AP. The IC50 b values of four extracts ranged from 0.067 to 310.63 mg FW/ml. According to the IC50 b values presented in the Table 2, the highest AP was obtained in P. emblica followed by P. acidus, P. campechiana and D. discolor. According to the IC50 value of less than 1 mg/ml is categorized as a fruit with extremely high antioxidant potential (Safaa et al., 2010, Bopitiya and Madhujith, 2012). Therefore, P. Emblica fruits can be categorized as extremely high antioxidative fruits. In ABTS assay, results are presented as RSA values, which denote the percentage of inhibiting of ABTS•+ by the individual sample (4 mg/ml) over six minutes. As described in the Figure 2, among the selected fruit extracts, the highest total antioxidant capacity being observed in P. emblica (81.29%), while least was recorded in P. acidus (9%). The extracts of D. discolor and P. campechiana were also possessed considerably higher RSA values of 71.17% and 76.14%, respectively. But interestingly within first minute of the reaction, more than 50% of the ABTS cation radical was inhibit by the fruit extract of P. emblica and D. discolor and completed the reaction within four minutes. In contrast, P. acidus started to react with ABTS•+ after first minute and showed very slow reaction rate, finally reached to 9% of RSA within six minutes. Nevertheless, the

7 Mallawaarachchi et al. extracts of P. campechiana initially showed slow activity, reached the second highest RSA value of 76.14% after six minutes.

Table 2. DPPH, ABTS Radical Scavenging Activity and FRAP of Fruit Extracts

Fruit species DPPH Radical Scavenging ABTS FRAP Value Activity RSA% IC50 a IC50 b P. emblica 0.01 ± 0.00d 0.067 ± 0.00c 81.29 ± 2.42a 2,891.57 ± 4.95a P. campechina 8.97 ± 0.25a 130.49 ± 3.64b 76.14 ± 0.16b 1,941.54 ± D. discolor 7.19 ± 0.11b 310.63 ± 4.74a 71.17 ± 0.42c 16.44b P. acidus 5.60 ± 0.40c 124.14± 8.84b 9.00 ± 0.48d 405.35 ± 19.10c 238.25 ± 56.05d Data are presented as Mean ±SD (n = 6) Values with different letters are significantly different at p < 0.5 2+ RSA – lyophilized sample of 4 mg/ml (over six minutes), FRAP - M of Fe /100 g of fruit in fresh weight, IC 50 a - mg of lyophilized sample/ml, IC 50 b – mg of fruit in fresh weight/ml

The FRAP assay was carried out to investigate the ability of selected fruit extracts to reduce ferric ion (Fe3+) to ferrous ion (Fe2+). It quantifies the reducing power of extract which is an integral attribute of such compounds (Bopitiya and Madhujith, 2012). The highest and lowest reducing powers were observed in P. emblica and P. acidus, respectively. The extracts of P. campechiana also showed higher reducing property with 1,941.54 Fe2+M/100 g FW (Table 2). In this study both P. emblica and P. campechiana fruit extracts showed higher reducing power than the commonly consumed fruits such as mango and banana as observed by Silva and Sirasa (2018).

100

60 P. emblica

nging Activity% P. campechiana 40 D. discolor 20 P. acidus

Radical Radical Scave 0 0 2 4 6 8 -20 Time (Minutes)

Figure 2. ABTS+ radical scavenging activity of fruit crop species

Use of a single method to determine antioxidant capacity is insufficient, thus adoption of different assays and model systems provide a better insight into the actual activity of the extracts (Bopitiya and Madhujith, 2012). The results summarized in the Table 2, illustrates antioxidant potential of selected fruit extracts are significant at p=0.05 except in IC50 in FW basis. It may be due to different extraction yield of individual fruit species. Based on the results, P. emblica exhibited the highest TPC, TMAC, VitC and AP.

8 Antioxidant Potential of Fruit Crop Species

Correlation analysis

The Table 3 illustrates the correlation between tested parameters with R2 values. The DPPH, FRAP, ABTS, TMAC and VitC showed strong positive correlation with TPC. The least correlation, but positive (R2 = 0.0009) was observed between DPPH and ABTS assays, while other four methods performed were possessed more than 0.5 R2 values. This poor correlation may be due to different attributes of phenolic compounds. Also the reaction time of the ABTS assay is only six minutes much shorter than that of DPPH (Surveswaran et al., 2007).

Table 3. Correlation between TPC, DPPH, ABTS, FRAP, TMAC and VitC

R2 TPC DPPH FRAP ABTS TMAC

TPC - - - - - DPPH 0.6509 - - - - FRAP 0.8803 0.5591 - - - ABTS 0.9500 0.0009 0.4691 - - TMAC 0.9784 0.5889 0.9533 0.4120 - VitC 0.9384 0.5989 0.6781 0.1811 0.8533

CONCLUSIONS

The results revealed that water extracts of the fruit crops tested have potential antioxidant activity and vitamin C content. The extract of P. emlica exhibited significantly higher antioxidant potential, vitamin C, TPC and TMAC than other fruit crops. This study highlighted significance of selected underutilized fruit species as cheap sources of natural antioxidants and Vitamin C. Further studies to identify individual phenolic compounds and also in vivo studies to understand their mechanism of action are therefore suggested.

ACKNOLEDGEMENT

The authors acknowledge Postgraduate Institute of Agriculture, University of Peradeniya for their financial support through Research Facilitation Fund (RFF).

REFERENCES

Adeolu, A.T. and Enesi, D.O. (2013). Assessment of proximate, mineral, vitamin and phytochemical composition of plantain (Musa paradisiaca) bract – an agricultural waste. Int. J. Plant Sci. 4(7), 192-197.

Benzie, I.F.F. and Strain, J.J. (1996). Ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal. Bio-chem. 239(1), 70–76.

Bopitiya, D. and Madhujith, T. (2012). Antioxidant potential of pomegranate (Punica granatum L.) cultivars grown in Sri Lanka. Trop. Agric. Res. 24(1), 71–81.

9 Mallawaarachchi et al.

Das, S.C., Hamid, K., Bulbul, I.J., Sultana S. and Islam, S. (2010). In Vitro Antioxidant Activity of Different Parts of the Plant Diospyros discolor. Res. J. of Agric. and Biol. Sci. 6(4), 472-475.

Dudonne S., Vitrac, X., Coutiere, P., Woillez, M. and Merillon, J.M. (2009). Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of Industrial interest using DPPH, ABTS, FRAP, SOD and ORAC assays. J. Agric. Food Chem. 57(5), 1768-1774.

Flohe, R.B and Traber M.G. (1999). Vitamin Eː function and metabolism. The FASEB J. 13, 1145-1155.

Goli, A.H, Barzegar, M. and Sahari, M.A. (2005). Antioxidant activity and total phenolic compounds of pistachio (Pistachia vera) hull extracts. Food Chem. 92, 521–525.

Haminiuk, C.W.I., Maciel, G.M., Plata-Oviedo, M.S.V. and Peralta, R.M. (2012). Invited review Phenolic compounds in fruits – an overview. Int. J. Food Sci. and Tech. 47(10), 2023- 2044.

Jacob, A.R. (1996). Chapter 01ː Introduction. pp. 1-16. In Harris, J.R. (Ed.) Subcellular Biochemistry, Ascorbic Acid: Biochemistry and Biomedical Cell Biology, vol. 25. Plenum, New York, NY, USA.

Jayathilake, C., Rizliya, V. and Liyanage, R. (2016). Antioxidant and free radical scavenging capacity of extensively used medicinal plants in Sri Lanka. Procedia Food Sci. 6, 123-126.

Jayaweera, D.M.A. (1981). Medicinal plants (Indigenous and exotic) used in Ceylon. Part II. The National Science Foundation, Sri Lanka. pp. 228-229.

Kahkonen, M. P., Hopia, A. I., Vuorela, H. J., Rauha, J.-P., Pihlaja, K., Kujala, T. S. (1999). Antioxidant activity of plant extracts containing phenolic compounds. J. Agri. and Food Chem. 47, 3954–3962.

Kriengsak, T., Unaroj, B., Kevin, C., Luis, C. and David, H.B. (2006). Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts. J. Food Com. and Analysis. 19, 669-675.

Lanerolle, M.S.De, Priyadharshani, A.M.B., sumithraarachchi, D.B. and Jansz E.R. (2008). The carotenoids of Pouteria campechiana (Sinhalaː ratalawalu). J. of the National. Science Foundation, Sri Lanka. 36(1), 95-98.

Lee, S.K. and Kader, A.A. (2000). Preharvest and postharvest factors influencing vitamin C content of horticultural crops. Postharvest Bio. and Tech. 20, 20-220.

Lee, J., Robert, W.D. and Ronald, E.W. (2005). Determination of total monomeric anthocyanin pigment content of fruit juices, beverages, natural colorants, and wines by the pH differential method: collaborative study. J. AOAC Int. 88(5), 1269-1278.

10 Antioxidant Potential of Fruit Crop Species

Lee, M.H., Jiang, C.B., Juan, S.H., Lin, R.D. and Hou, W.C. (2006). Antioxidant and heme oxygenase-1 (HO-1)-induced effects of selected Taiwanese plants. Fitoterapia. 77(2), 109– 115.

Lee, S., Mediani, A., Nur Ashikin, A., Azliana, A. and Abas, F. (2014). Antioxidant and α- glucosidase inhibitory activities of the leaf and stem of selected traditional medicinal plants. Int. Food Res. J. 21(1), 165-172.

Lie-Fen, S., Jieh-Hen, T., Je-Hsin, C., Chih-Yang, C. and Chiu-Ping, L. (2005). Antioxidant properties of extracts from medicinal plants popularly used in Taiwan. Int. J. Applied Sci. and Eng. 3(3), 195 - 202.

Lim, T.K. (2012a). Phyllanthaceaeː Phyllanthus acidus in Edible Medicinal and non-medicinal plants, Vol 4, Fruits, Springer Science+Business Media. London, pp. 252- 257.

Lim, T.K. (2012b). Ebinaceaeː Diospyros blancoi in Edible Medicinal and non-medicinal plants, Vol 2, Fruits, Springer Science+Business Media. London, UK. pp. 421-424.

Omale J. and Ugwu C. E. (2011). Comparative studies on the protein and mineral composition of some selected Nigerian vegetables. African J. Food Sci. 5(1), 22–25.

Oviasogie, P.O., Okoro, D. and Ndiokwere, C.L. (2009). Determination of total phenolic amounts of some edible fruits and vegetables. African J. Biotech. 8(12), 2819 -2820.

Padayatty, S., Katz, A., Wang,Y., Eck, P., Lee, J., Chen, S., Corpe, C., Dutta, A., Dutta, S. and Levine, M. (2003). Vitamin C as an antioxidant: evaluation of its role in disease prevention. J. Am. Clin. Nutr. 22(1), 18-35.

Pushpakumara, D.K.N.G. and Heenkenda, H.M.S. (2007). Chapter 06: Nelli (Amla) (Phyllanthus embilica L.). pp. 180-221. In Pushpakumara, D.K.N.G., Gunasena, H.P.M., Singh, V.P. (Ed.) Underutilized Fruit Trees in Sri Lanka Volume 1. World Agro-forestry Centre, South Asia Regional office, New Delhi, India; National Multipurpose Tree Species Research Network of Sri Lanka; Sri Lanka Council for Agricultural Research Policy, Asian Centre for Underutilized Crops, Sri Lanka.

Ramasamy, S., Wahab, N.A., Abidin, N.Z. and Manickam, S. (2011). Cytotoxicity evaluation of five Malaysian Phyllanthaceae species on various human cancer cell lines. J. Med. Plant Res. 5(11), 2267-2273 .

Safaa, Y. Q., Abo-khatwa, A.N. and Lahwa, M.A.B. (2010). Screening of antioxidant activity and phenolic content of selected food items cited in the Holly Quran. Europ. J. Biol. Sci. 2(1), 40-51.

Shofian, N.M., Hamid, A.A., Osman, A., Saari, N., Anwar, F., Dek, M. S. P. and Hairuddin, M.R. (2011). Effect of freeze-drying on the antioxidant compounds and antioxidant activity of selected tropical fruits. Int. J. Mol. Sci. 12, 4678-4692.

Silva, K.D.R.R. and Sirasa M.S.F. (2018). Antioxidant properties of selected fruit cultivars grown in Sri Lanka. Food Chem. 238, 203-208.

11 Mallawaarachchi et al.

Su, L., Yin, J.J., Charles, D., Zhou, K., Moore, J. and Yu, L. (2007). Total Phenolic contents, chelating capacities and radical-scavenging properties of black peppercorn, nutmeg, hrosewhip, cinnamon and oregano leaf. J. Food Chem. 100(3), 990-997.

Surveswaran, S., Cai, Y.Z., Cork, H. and Sun, M. (2007). Evaluation of natural phenolic antioxidant from 133 Indian medicinal plants. Food Chem. 102, 938-953.

Vidhan, J., DerMarderosian, A. and John, R.P. (2010). Anthocyanins and polyphenol oxidase from dried arils of pomegranate (Punica granatum L.). J. Food Chem. 118(1), 11-16.

Wang, S.Y and Zheng, W. (2001). Effect of plant growth temperature on antioxidant capacity in strawberry, J. Agric. Food Chem. 49, 4977–4982.

Wojdyło, A., Oszmianski, J. and Czemerys R. (2007). Antioxidant activity and phenolic compounds in 32 selected herbs. Food Chem. 105, 940-949.

Yu, L., Perret, J. and Davy, B., Wilson, J. and Melby, C.L. (2002). Antioxidant properties of cereal products. J. Food Sci. 67(7), 2600-2603.

Zhou, K. and Yu, L. (2004). Antioxidant properties of bean extracts from Trego wheat grown at different locations. J. Agri. Food Chem. 52(5), 1112-1117.

Zulaikha, A.G.S., Mediani, A., Khoo, L.W., Lee, S.Y., Leong, S.W. and Abas, F. (2017). Effect of different drying methods and solvent ratios on biological activities of Phyllanthus acidus extracts. Int. Food Res. J. 24(1), 114-120.

12 Tropical Agricultural Research Vol. 30 (3): 13 – 26 (2018)

Millet Phenolics as Natural Antioxidants in Food Model Systems and Human LDL/VLDL Cholesterol in vitro

K.D.D. Kumari, W.M.T. Madhujith1 and G.A.P. Chandrasekara2*

Postgraduate Institute of Agriculture University of Peradeniya Sri Lanka

ABSTRACT: Dehulled grain flour of finger millet (Eleusine coracana), proso millet (Panicum miliaceum) and foxtail millet (Setaria italica) and phenolic extracts of millet hulls were evaluated for ability to inhibit lipid oxidation in several food model systems, namely cooked comminuted pork and fish, roasted peanut butter and gingelly oil. Food samples were kept for 14 days with added millet hull extracts and dehulled grain flours. The percentage inhibition of production of thiobarbituric acid reactive substances (TBARS) during storage was determined. Inhibitory activities of phenolic extracts of finger millet dehulled grain and finger millet foods, namely, Rotti, Pittu, Halapa, Thalapa and porridge, against human very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) oxidation were determined by measuring the production level of conjugated dienes (CD) in vitro. Finger millet had higher phenolic content and antioxidant activities compared to the respective proso and foxtail millet samples. Finger millet hull extracts exhibited the highest inhibition of lipid peroxidation in food model systems. The maximum percentage inhibition of TBARS in pork, fish, and peanut with added finger millet hull extracts were observed at days 3, 7, 5, and 14, respectively. The percentage inihibition of TBARS in cooked pork and fish with added millet hull extracts ranged from 4.4 to 12.8% and 63 to 77%, respectively at the end of the second week. Millet grains and desolventized millet phenolic extracts can act as natural sources of antioxidants at different degrees in pork, fish, peanut and gingelly oil to prevent lipid oxidation during storage.

Keywords: Lipid oxidation inhibition, millet, natural antioxidants, meat, gingelly oil, roasted peanut butter

INTRODUCTION

Lipid oxidation products formed in foods lead to the development of off-flavours and affect nutritional and sensory qualities. Further, they act as atherogenic agents in addition to their mutagenic and carcinogenic properties (Shahidi et al., 2012). Polyunsaturated fatty acids (PUFA) in foods are susceptible to oxidation during processing and storage (Shahidi et al., 2012). A number of factors affects the rate of lipid oxidation in foods. Special measures are taken during processing and storage to prevent food lipid oxidation and addition of antioxidants is a common practice among others. Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butyl hydroquinone (TBHQ) are most widely used synthetic antioxidants in food industry (Shahidi and Zhong, 2005). There are several controversies in

1 Department of Food science and Technology, Faculty of Agriculture, University of Peradeniya, Sri Lanka 2 Department of Applied Nutrition, Wayamba University of Sri Lanka, Makandura, Gonawila, Sri Lanka * Corresponding author: [email protected] Kumari et al. the use of synthetic antioxidants as health concerns have been raised on their addition in foods (Shahidi and Zhong, 2005). Food additives are subjected to the most stringent toxicological testing procedures, and only a few synthetic antioxidants have been used in foods so far. There is a growing interest of using natural antioxidants in foods. The natural antioxidants include phenolic compounds, tocopherols, and flavonoids (Artajo et al., 2006). Oxidized Low-density lipoproteins (LDLs) have been detected in atherosclerotic vessels and they appear to play a major role in the disease process (Esterbauer et al., 1992; Steinberg, 1992). Naturally occurring antioxidants in the diet may play a role in inhibiting the oxidative modification of LDL and hence they might act as anti-atherosclerotic agents (Shahidi et al., 2012)

Millets are recognized as potential future crops due to their nutrient contents similar to other major cereals and rich non-nutrient profiles, especially phenolic compounds (Shahidi and Chandrasekara, 2014; Kumari et al., 2017(. Previous work on millets have proven that phenolic compounds present in millets are bioaccessible and may act as antioxidants within the human body to protect against oxidative stress (Chandrasekara and Shahidi, 2012a). The aim of the present work was to evaluate the ability of millet phenolic compounds to prevent the oxidation of lipids present in food model systems namely, pork, fish, peanut, and gingelly oil and to determine the ability of millet phenolics to prevent the oxidation of human LDL cholesterol in vitro.

METHODOLOGY

Samples of finger millet (Elusine coracana) variety “Oshada”, local variety of proso millet (Panicum miliacium) and foxtail millet (Setaria italica) grown in Sri Lanka were used in this study. All grain materials were obtained from the Department of Agriculture, Sri Lanka. Folin- Ciocalteu’s reagent was purchased from Research Lab Fine Chem Industries, Mumbai, India. Vanillin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2 azinobis (3-ethylbenzothiazoline-6- sulfonate diammonium salt) (ABTS), 2,2-azobis (2-methylpropionamidine) dihydrocholride (AAPH), ferrous chloride, sodium chloride, butylated hydroxyanisole (BHA), ferulic acid, trolox, catechin, and ethanol were purchased from Sigma-Aldrich, St Louis, USA. Sodium carbonate, ferric chloride, were purchased from Thomas Baker (Chemicals) Limited, Bombay, India. Aluminium chloride, trichloroacetic acid (TCA), and potassium phosphate dibasic were purchased from Techno Pharm Chem, India. Sodium hydroxide, and potassium phosphate monobasic were purchased from Loba Chem Pvt Ltd, India. 3-(2-pyridyl)-5,6-diphenyl-1,2,4- triazine-4,4-disulfonic acid sodium salt (Ferrozine) were purchased from SERVA Electrophoresis GmbH, Heildberg, Germany. Ethylene diamine tetra acetic acid tri sodium salt (Na3EDTA) was purchased from Needham Market Sufflock, England. 2, 2, 4- trimethylpentane and 1,1,3,3-tetramethoxypropane were purchased from Sigma-Aldrich, St Louis, USA.

Sample preparation

Millet whole grains were dehulled and hulls were separated from grains. Finger millet grains were dehulled using a rice polishing machine (Rice husker and polisher PM 500, Satake Engineering Co Ltd, Japan). Foxtail millets and proso millets were dehulled using rice milling machine (Rice machine, Satake Engineering Co Ltd, Japan). Dehulled grains, and hulls were separately used for the extraction of phenolics. Samples were ground using an electric grinder and sieved (0.038 seive opening; As 200 test seiver, Retsch, Germany(. Samples were defatted using hexane (1:5 w/v, 2 min, two times) at ambient temperature. Defatted samples packed in

14 Millet Phenolics as Natural Antioxidants polythene pouches were stored at -80 ºC until used within a week for extraction of phenolic compounds.

Extraction of soluble phenolic compounds

Soluble phenolic compounds were extracted from dehulled grains and hulls of millets. Defatted meal (5 g) was mixed with 100 mL of 80% (v/v) aqueous ethanol in a capped conical flask and placed in a shaking water bath at 50°C, stirring at 175 rpm speed for 40 minutes. The extraction procedure was repeated two more times. Combined supernatants were evaporated in rotary evaporator at 40ºC at 125 rpm. Concentrated samples were freeze dried at -55ºC, and 12 x 10- 3 mbar. Lyophilized crude phenolic extracts were stored at -80ºC until used for further analysis. During all stages, extracts were protected from light by covering with aluminum foil.

Determination of oxidative stability in comminuted pork model

The samples were prepared to determine the oxidative stability of dehulled millet flour and soluble phenolic extracts of millet hull according to Chandrasekara and Shahidi (2012b). In brief, ground pork (80g) was mixed with 20 g deionized water in a glass jar and mixed with testing materials, namely dehulled millet flour (2%), hull phenolic extracts (0.2%), ferulic acid (0.02%) or BHA (0.02%) followed by thoroughly mixing with a glass rod. A control without added compounds was maintained under same experimental conditions. Capped glass jars were cooked in a water bath at 80 ± 2 ºC for 40 minutes while stirring with a glass rod at every 5 minutes. Samples were cooled to room temperature and were transferred to polythene bags. Bags were stored in a refrigerator at 4 ºC and samples were drawn on days 0, 3, 5, 7 and 14 for the analysis of thiobarbituric acid reactive substances (TBARS). The experiment of each test material was carried out in three replications.

Determination of oxidative stability in fish model

The oxidative stability of dehulled millet grains and soluble phenolic extracts in the inhibition of TBARS production in cooked Tuna fish (ground boneless flesh) was determined as previously explained (Wijeratne et al., 2006). The experimental procedure was similar to the pork model explained in the previous section.

Determination of oxidative stability in peanut model

The effect of dehulled millet grain and soluble phenolic extracts in the inhibition of TBARS production in roasted ground peanut was determined according to Wijeratne et al. (2006). Dehulled millet flour (2%), phenolic extracts of hulls (0.2%), ferulic acid (0.02%) and BHA (0.02%) were added to 100 g of ground peanut and thoroughly mixed with a glass rod in a glass jar. A separate control sample without extracts was also prepared. Samples were mixed thoroughly and transferred into polythene bags. Samples were stored in room temperature for 14 days. Samples were drawn on days 0, 3, 5, 7 and 14 for analysis of TBARS.

Determination of oxidative stability in gingelly oil

The efficacy of soluble millet extracts to inhibit the oxidation of gingelly oil was measured according to the method explained by Chandrasekara and Shahidi, (2011b). Hull phenolic extracts (1%), ferulic acid (1%) and BHA (1%) were added separately into 100 mL glass jars, and then 2 g of gingelly oil sample was added following vortexing thoroughly for 2 minutes. Each treatment was prepared in triplicate and jars were kept in the oven at 60ºC. Samples for

15 Kumari et al. the analysis of TBARS were drawn on 0, 6, 12, 24, 36 and 48 hours and were analyzed for the inhibition percentage of TBARS and the production of conjugated dienes (CD). Control samples of oil without test materials were maintained under identical experimental conditions.

Inhibition percentage of TBARS

The inhibition percentage of TBARS was determined according to the method explained by Chandrasekara and Shahidi, (2012b). In this, 2 g from each fish, meat, or peanut sample was weighed in a 50 mL centrifuge tube. Then 5 mL of 10% (w/v) solution of trichloroacetic acid (TCA) were added and vortexed at high speed for 2 minutes. Thiobarbituric acid (TBA) solution (0.02 M, 5 mL) was added to each centrifuge tube and vortexed for 30 seconds. The samples were subsequently centrifuged at 3000 g for 10 minutes and the supernatants were filtered through a Whatman No.3 paper. Filtrates were heated in a boiling water bath for 45 minutes and cooled to room temperature on an ice bath. The absorbance of the pink coloured chromogen was measured at 532 nm (UV-VIS Spectrophotometer, Labomed Inc, USA). Standard curve was prepared using 1,1,3,3-tetramethoxypropane as a precursor of the malondialdehyde (MDA). The percentage inhibition of TBARS formation was calculated as follows. Inhibition percentage = {(TBARS control TBARS sample) / TBARS control} x 100, where TBARS control = TBARS formed in the control and TBARS sample = TBARS formed in the sample. TBARS values of oil samples were determined according to the standard AOCS method. In brief the oil (50 – 100 mg) was weighed into a 25 mL volumetric flask and made up to volume with 1-butanol. Then 5 mL of this solution was transferred into a screw capped test tube with added 5 mL of freshly prepared 2-TBA reagent (500 mg of TBA in 250 mL 1- butanol). Contents were thoroughly mixed and heated in a water bath at 95ºC for 2 hours. Samples were cooled in an ice bath and the absorbance values were measured at 532 nm. Standard curve was prepared using 1,1,3,3-tetramethoxypropane as a precursor of the malondialdehyde (MDA). The percentage inhibition of TBARS formation was calculated as follows. Inhibition percentage = {(TBARS control TBARS sample) / TBARS control} x 100, where TBARS control = TBARS formed in the control and TBARS sample = TBARS formed in the sample.

Determination of conjugated diene (CD) in gingelly oil sample

The CD contents of oil samples were determined according to the method explained by Shahidi and Zhong (2005). In this a sample of gingelly oil (0.02-0.03 g) was weighed into a 25 mL volumetric flask, and made up to the mark with 2,2,4-trimethylpentane. The solution was thoroughly mixed and the absorbance was measured at 234 nm. Pure 2, 2, 4- trimethylpentane was used as a reference. CD values were calculated using the following equation; CD=Absorbance of solution at 234 nm / C*L, where C= concentration of oil in g per 100 mL, L=length of the cuvette (cm(.

Inhibition of copper-mediated human LDL and VLDL oxidation

Inhibitory activities of phenolic extracts of finger millet dehulled grain and finger millet foods (Rotti, Pittu, Halapa, Thalapa and Porridge) against human VLDL and LDL oxidation were determined by measuring conjugated dienes (CD) produced in the system using the method described by Chandrasekara and Shahidi, (2012b). LDL and VLDLs from human serum were isolated using HDL precipitating reagents. Pellets (LDL/VLDL precipitate) were obtained by decanting the supernatant (HDL) and suspended in phosphate buffer to obtain diluted cholesterol solution of 0.1mg/mL protein concentration in PBS (pH 7.4, 0.15M NaCl). Protein concentrations of lipoproteins were determined using the biuret reagent test. The diluted

16 Millet Phenolics as Natural Antioxidants solution (0.8 mL) was mixed with 100 μL of soluble extract of foods (0.5 mg / mL in PBS) in an eppendorf tube. Oxidation of LDL was initiated by adding 0.1 mL of 100 μM CuSO4 solution in distilled water. The mixture was incubated at 37ºC for 24 hours. The initial absorbance (t=0) was read at 234 nm immediately after mixing and CD hydroperoxides formed were measured at 0, 3, 6, 12 and 24-hour intervals. The concentration of CD formed was calculated using the molar extinction coefficient of 29500M-1cm-1.

Statistical analysis

All experiments were carried out in triplicates and data were reported as mean ± standard deviation. The differences of mean values among samples were determined by one-way analysis of variance (ANOVA) followed by Tukeys Honestly Significant Difference (HSD) multiple rank tests at p = 0.05, significance level. All statistical analysis was performed by SPSS version 16 (SPSS Inc., Chicago, IL).

RESULTS AND DISCUSSION

The ability of the lipid containing foods to act against oxidation is expressed as oxidative stability of foods. TBARS values measure the level of lipid oxidation. Table 1 and 2 show the effect of the millet extracts, ferulic acid and BHA on the inhibition of TBARS on pork and fish models. In communited pork model BHA showed the highest inhibition of TBARS in the range of 80 to 94 % from day 0 to end of the second week, compared to ferulic acid and millet extracts. The inhibition percentage of TBARS in pork model with added millet extracts ranged from 1 to 40% from day 0 and end of second week. In addition, the ability of TBARS production inhibition decreased with time in pork model with added millet flour or extracts. This could be due to the reduction of antioxidant power in millet extracts with the storage time. The inhibition percentage of TBARS in the fish model with added BHA and millet flour/extracts were 62 and 83% and 3 and 57%, respectively on day 0 and end of second week.

Generally, the highest inhibition percentage was observed on day 0 and day 14 in pork and fish models respectively, with added millet flour/extract. Millet grain hull extracts showed high TBARS production inhibition which ranged from 56 to 63% in fish model at the end of second week . Further, the greater effect of the millet hull extract on inhibiting the production of TBARS could be due to the high phenolic content and the antioxidant activities in addition to the presence of other compounds such as, vitamin C, E, with antioxidant activities in hull extracts compared to those of dehulled grain. Previous studies also reported the ability of plant extracts to prevent the oxidation of lipids in meat (Rhee et al., 1996; Karwowska and + Dolatowski, 2007). The phenolics present in natural plant extracts have strong H donating activity. In the present study also the phenolic extracts of millet showed high antioxidant activity against lipid oxidation of the meat and fish samples. These results are in agreement with several other studies that reported natural antioxidants from plant sources could prevent the lipid oxidation of meat and meat products (Yu et al., 2002).

Table 3 shows the the effect of the millet flour/extracts, ferulic acid and BHA on production inhibition of TBARS on roasted peanut model. BHA showed the highest inhibition percentage of TBARS at the day 0 compared to ferulic acid and millet flour/extracts. Peanut sample with added proso millet dehulled grain flour showed the highest ability to prevent the production of TBARS at the end of second, third and fourth weeks. Tea catechins, as a polyphenolic flavonoid to prevent the lipid oxidation in green tea has been demonstrated in a variety of food systems (McCarthy, 2001; Nissen, 2004; Mitsumoto, 2005). Moreover, the use of millet

17 Kumari et al. phenolic extracts shows antioxidant effects similar or better than those of BHA. Polyphenolic extracts are excellent electron and proton donors, and their intermediate radicals are quite stable due to electronic delocalization phenomenon as well as the lack of position attackable by oxygen (Rhee et al., 2001). Therefore, millets can be used in peanut butter preparations to improve the quality of the product.

Table 4 shows the effect of the millet flour/extracts, ferulic acid and BHA on production inhibition of TBARS on gingelly oil model. Among those phenolic extracts of millet showed higher ability to prevent the production of TBARS reflecting their higher antioxidant activity for protection of gingelly oil against oxidation compared to ferulic acid and BHA.The highest TBARS inhibition was showed at 72 hours by phenolic extracts of finger millet hull. Compared to BHA, phenolic extracts of millet dehulled grains and hulls showed increased duration of oxidative stability in oil samples. Table 5 shows the production of CD in gingelly oil model system. Compared with the control millet hull extract added oil samples showed significantly low CD levels at 36, 48 and 72 hours. During the 0 to 24 hours of time period there were not any significant difference in the production of CD in oil samples with added millet hull extracts.

Results of TBARS and CD in gingelly oil model showed that phenolic extracts of millets have the ability to delay the oxidation of gingelly oil during storage. Previous studies showed that natural antioxidants from plant sources have an effect to increase the oxidative stability in oil samples (Kamali et al., 2011). Further, they showed that addition of cinnamon extracts increased the oxidative stability of sunflower oil (Kamali et al., 2011). The antioxidant potential of plant extracts is a result of not only the presence of the active phenolic compounds but also of other components present in the system. Figure 1 shows the ability of phenolic extracts of finger millet dehulled grain and finger millet foods (Rotti, Pittu, Halapa, Thalapa and Porridge) to prevent the oxidation of human VLDL and LDL in vitro. Pittu had the highest ability to prevent the production of conjugated dienes in oxidation of human VLDL and LDLs in vitro while Thalapa shows the lowest ability.

The phenolic compounds which are present in finger millet foods should have the ability to chelate cupric ions and scavenge the formed peroxyl radicals to prevent the oxidation of VLDL and LDL cholesterol in vitro (Decker et al., 2001). According to Chandrasekara and Shahidi (2011b) soluble phenolic extracts of pearl millet hulls and dehulled grain at a low concentration (0.125 mg/mL) showed 1.6 and 3.9 times higher inhibition, respectively, of LDL oxidation than that at high concentration (0.5 mg/mL). Authors explained that there is a possibility that phenolic compounds at high concentrations may complex with protein moieties of the LDL cholesterol molecules, which makes them unavailable to inhibit oxidation of cholesterol. Previous studies have shown that phenolic compounds can inhibit protein oxidation by binding of the proteins to form complexes with protein molecules (Siebert et al., 1996; Riedl and Hagerman, 2001). Therefore, the low ability of finger millet foods with high phenolic content to inhibit the oxidation of LDL and VLDL cholesterol in vitro might be due to the interactions of proteins and phenolic compounds at high concentrations of phenolics.

18 Tropical Agricultural Research Vol. 30 (3): 13 – 26 (2018)

Table 1. Inhibition percentage of thiobarbeturic acid reactive substances (TBARS) at different time periods in the pork model system

Inhibition percentage of TBARS Day 0 Day 3 Day 5 Day Day 14 7 BHA 80.09 ± 0.11a1 93.66 ± 0.38a2 86.94 ± 0.69a3 88.98 ± 1.01a3 79.47 ± 0.39a1 Ferulic acid 14.39 ± 0.05b1 23.94 ± 0.03b2 19.65 ± 2.20b3 27.34 ± 2.06b4 3.67± 1.25b5 Finger millet dehulled grain flour 4.60 ± 0.61c1 18.34 ± 0.07c2 19.20 ± 1.30b2 16.76 ± 3.28c3 8.18 ± 0.63b4 Foxtail millet dehulled grain flour 10.89 ± 0.05d1 9.45 ± 0.21d1 21.07 ± 2.13b2 24.72 ± 2.43b3 12.85 ± 0.82c4 Proso millet dehulled grain flour 30.53 ± 1.78e1 10.41 ± 0.10e2 0.64 ± 0.04c3 13.40 ± 1.30c4 8.49 ± 0.04d5 Finger millet hull extract 38.06 ± 0.10e1 25.25 ± 0.83b2 18.20 ± 0.07b3 20.24 ± 1.81b4 5.09 ± 0.82e5 Foxtail millet hull extract 4.20 ± 0.45c1 18.18 ± 0.03c2 7.94 ± 0.22d3 23.58 ± 3.25b4 6.62 ± 1.02e5 Proso millet hull extract 39.71 ± 0.79e1 10.93 ± 0.83e2 15.63 ± 1.63e3 13.19 ± 0.22d4 4.51 ± 0.70e5 Superscripts with the same letters in each column and same numbers in each row are not significantly different

Table 2. Inhibition percentage of thiobarbeturic acid reactive substances (TBARS) at different time periods in fish model system

Inhibition percentage of TBARS Day 0 Day 3 Day 5 Day 7 Day 14

BHA 81.73 ± 2.99a1 83.33 ± 1.39a1 80.63 ± 1.26a1 77.87 ± 2.54a2 61.88 ± 1.86a2 b1 b1 b2 b3 b4 Ferulic acid 18.27 ± 2.09 18.13 ± 0.62 5.66 ± 0.19 20.55 ± 0.35 4.65 ± 0.10 Finger millet dehulled grain flour 22.36 ± 1.25c1 3.37 ± 0.68c2 18.93 ± 0.88c3 3.82 ± 1.62c2 5.08 ± 0.18 c4 Foxtail millet dehulled grain flour 38.03 ± 0.58d1 5.21 ± 0.16d2 16.92 ± 1.07d3 7.16 ± 1.20d4 2.61 ± 0.34d5

Proso millet dehulled grain flour 34.46 ± 1.68e1 3.91 ± 0.16c2 18.49 ± 1.24c3 8.78 ± 5.08e4 6.10 ± 0.19e5 Finger millet hull extract 33.38 ± 6.22e1 9.12 ± 0.16e2 17.48 ± 1.53e3 30.70 ± 5.06f4 56.10 ± 0.04f5 f1 f2 e3 g4 g5 Foxtail millet hull extract 46.35 ± 5.23 16.83 ± 4.55 17.61 ± 3.60 21.61 ± 2.22 63.13 ± 0.14 Proso millet hull extract 49.72 ± 5.07g1 8.85 ± 3.08g2 21.51 ± 1.00f3 30.57 ± 1.73f4 56.10 ± 0.19f5

Superscripts with the same letters in each column and same numbers in each row are not significantly different Kumari et al.

Table 3. Inhibition percentage of thiobarbeturic acid reactive substances (TBARS) at different time periods in peanut model system

Inhibition percentage of thiobarbeturic acid reactive substances (TBARS) Day 0 Day 7 Day 14 Day 21 Day 28 BHA 63.71 ± 0.09a1 23.81 ±3.23a2 43.86 ±1.25a3 24.83 ±4.04a2 25.75 ±3.21a2 Ferulic acid 59.72 ±0.46b1 23.05 ±0.14a2 37.14 ±2.13b2 22.43 ±0.06b2 25.87 ±0.22b2 Finger millet dehulled grain flour 57.61 ±0.58c1 24.37 ±0.13b2 45.45 ±3.62c3 29.67 ±5.87c3 34.13 ±0.35c4 Foxtail millet dehulled grain flour 51.75 ±4.21d1 22.08 ±0.13c2 37.28 ±1.35d3 32.98 ±0.22d3 32.52 ±0.17d3 Proso millet dehulled grain flour 49.13 ±1.46e1 27.96 ±0.07d2 45.85 ±1.31e3 33.16 ±0.43e4 37.91 ±1.09e3 Finger millet hull extract 51.89 ±0.05d1 32.67 ±0.09e2 28.15 ±0.10f3 13.00 ±0.10f4 19.97 ±0.11f5 Foxtail millet hull extract 55.15 ±0.99f1 31.49 ±1.77f2 27.55 ±0.13g3 6.87 ±1.02g4 7.47 ±0.27g5 Proso millet hull extract 33.54 ±2.65g1 28.26 ±0.20g2 28.19 ±0.15h2 9.72 ±1.11h3 2.52 ±1.09h4 Superscripts with the same letters in each column and same numbers in each row are not significantly different

Table 4. Inhibition percentage of thiobarbeturic acid reactive substances (TBARS) at different time periods in gingelly oil model system

Inhibition percentage of TBARS 0 hours 6 hours 12 hours 24 hours 36 hours 72 hours BHA 12.88 ± 3.30a1 4.90 ±0.90a2 5.00 ±1.89a2 6.26 ±1.15a3 9.90 ±2.03a4 18.71 ±1.90a5 Ferulic acid 21.46 ±3.40b1 1.96 ±0.90b2 20.71 ±1.89b1 3.68 ±1.94b3 9.20 ±0.77b4 53.00 ±2.31b5 Finger millet hull extract 28.03 ±2.00c1 13.33 ±0.68c2 32.38 ±2.89c3 23.57 ±1.39c4 41.84 ±1.17c5 61.87 ±1.25c6 Foxtail millet hull extract 20.20 ±1.10d1 10.59 ±1.18d2 19.29 ±0.71b1 32.04 ±2.53d3 8.68 ±0.80e4 56.12 ±1.90d5 Proso millet hull extract 11.62 ±1.20e1 14.12 ±2.04e2 37.86 ±0.71d3 29.47 ±2.61e4 43.23 ±2.03f5 54.68 ±0.72e6 Superscripts with the same letters in each column and same numbers in each row are not significantly different

20 Millet Phenolics as Natural Antioxidants

Table 5. Production of conjugated dienes (CD) in gingerlly oil model system

Time (hr) 0 06 12 24 36 48 72 Control 9.3 ±1.0a1 11.2 ±2.1a2 12.3 ±0.5a2 12.7 ±0.5a2 13.5 ±1.1a3 11.7 ±0.2a2 14.0 ±1.4a3 BHA 8.2 ±1.2a1 11.9 ±1.2a2 12.2 ±0.7a3 13.7 ±0.4b2 11.9 ±0.9b3 11.3 ±0.3a2 12.5 ±1.3b3 Ferulic acid 9.1 ±1.0a1 11.7 ±1.1a2 11.7 ±0.7a2 11.9 ±1.2a2 13.6 ±0.7a3 12.4 ±0.4a4 13.9 ±1.0b3 Finger millet hull extract 8.3 ±0.8a1 10.9 ±0.9a2 11.5 ±1.2a3 12.8 ±1.1a1 13.8 ±0.1a4 9.6 ±0.5b5 9.8 ±0.5c5

Foxtail millet hull extract 8.1 ±0.2a1 10.2 ±0.8a2 11.7 ±1.1a1 12.7 ±0.9a3 11.80 ±0.5b1 9.4 ±0.4b2 12.4 ±0.8b3 Proso millet hull extract 8.5 ±1.3a1 11.9 ±0.4a2 12.4 ±1.6a2 11.8 ±1.3a2 12.1 ±0.4b2 11.4 ±1.2a2 12.9 ±0.2b2

Superscripts with the same letters in each column and same numbers in each row are not significantly different

21 Tropical Agricultural Research Vol. 30 (3): 13 – 26 (2018)

Figure 1. The production of CD in human LDL and VLDLs samples in vitro with added synthetic antioxidants and phenolic extracts of finger millet foods

Previous studies showed that plant phenolic compounds have potential to prevent the oxidation of LDL cholesterols in vitro. Phenolic compounds found in wine are potent antioxidants in inhibiting LDL oxidation in vitro (Teissedre et al., 1996; Meyer et al., 1997). According to Heinonen et al. (1998) phenolic extracts of berries, namely black berries, red raspberries, sweet cherries, blueberries, and strawberries demonstrated inhibition of human LDL oxidation. Therefore, further in vivo research is warranted to reveal the effect of millet foods consumption to prevent and control or reduce the burden of cardio vascular disease among humans. The oxidized LDL can increase the risk of initiating vascular lesions in blood vessels leading to atheroma formation. Millet Phenolics as Natural Antioxidants

CONCLUSIONS

Millet grains and millet phenolic extracts act as natural sources of antioxidants in comminuted pork and fish, peanut butter and gingelly oil to prevent lipid oxidation during storage. Millet hull phenolics are potential sources of natural antioxidants which can be utilized in food industry replacing synthetic antioxidants. Further, it is worthwhile to explore the possibility of using millet hull phenolics as potential raw materials for the production of nutraceuticals to attenuate LDL cholesterol oxidation.

ACKNOLEDGEMENT

This research was supported by the National Research Council of Sri Lanka (NRC 12-096).

REFERENCES

Benzie, I.F.F. and Strain, J.J. (1996). Ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal. Bio-chem. 239(1), 70–76.

Bopitiya, D. and Madhujith, T. (2012). Antioxidant Potential of Pomegranate (Punica granatum L.) Cultivars Grown in Sri Lanka. Trop. Agric. Res. 24(1), 71–81.

Das, S.C., Hamid, K., Bulbul, I.J., Sultana S. and Islam, S. (2010). In Vitro Antioxidant Activity of Different Parts of the Plant Diospyros discolor. Res. J. of Agric. and Biol. Sci. 6(4), 472-475.

Flohe, R.B and Traber M.G. (1999). Vitamin Eː function and metabolism. The FASEB J. 13, 1145-1155.

Goli, A.H, Barzegar, M. and Sahari, M.A. (2005). Antioxidant activity and total phenolic compounds of pistachio (Pistachia vera) hull extracts. Food Chem. 92, 521–525.

Haminiuk, C.W.I., Maciel, G.M., Plata-Oviedo, M.S.V. and Peralta, R.M. (2012). Invited review Phenolic compounds in fruits – an overview. Int. J. Food Sci. and Tech. 47(10), 2023- 2044.

Jacob, A.R. (1996). Chapter 01ː Introduction. pp. 1-16. In Harris, J.R. (Ed.) Subcellular Biochemistry, Ascorbic Acid: Biochemistry and Biomedical Cell Biology, vol. 25. Plenum, New York, NY, USA.

23 Kumari et al.

Jayathilake, C., Rizliya, V. and Liyanage, R. (2016). Antioxidant and free radical scavenging capacity of extensively used medicinal plants in Sri Lanka. Procedia Food Sci. 6, 123-126.

Jayaweera, D.M.A. (1981). Medicinal plants (Indigenous and exotic) used in Ceylon. Part II. The National Science Foundation, Sri Lanka. pp. 228-229.

Lee, S.K. and Kader, A.A. (2000). Preharvest and postharvest factors influencing vitamin C content of horticultural crops. Postharvest Bio. and Tech. 20, 20-220.

Lee, M.H., Jiang, C.B., Juan, S.H., Lin, R.D. and Hou, W.C. (2006). Antioxidant and heme oxygenase-1 (HO-1)-induced effects of selected Taiwanese plants. Fitoterapia. 77(2), 109– 115.

Lim, T.K. (2012a). Phyllanthaceaeː Phylanthus acidus in Edible Medicinal and non-medicinal plants, Vol 4, Fruits, Springer Science+Business Media. London, pp. 252- 257.

Lim, T.K. (2012b). Ebinaceaeː Diospyros blancoi in Edible Medicinal and non-medicinal plants, Vol 2, Fruits, Springer Science+Business Media. London, UK. pp. 421-424.

Omale J. and Ugwu C. E. (2011). Comparative studies on the protein and mineral composition of some selected Nigerian vegetables. African J. Food Sci. 5(1), 22–25.

Oviasogie, P.O., Okoro, D. and Ndiokwere, C.L. (2009). Determination of total phenolic amounts of some edible fruits and vegetables. African J. Biotech. 8(12), 2819 -2820.

24 Millet Phenolics as Natural Antioxidants