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Pigment identification, nutritional composition, bioactivity, and in vitro cancer cellcytotoxicity of Rivina humilis L. berries, potential source of betalains
Mohammad Imtiyaj Khan, P.S.C. Sri Harsha, P. Giridhar, G.A. Ravishankar
PII: S0023-6438(12)00041-2
DOI: 10.1016/j.lwt.2012.01.025
Reference: YFSTL 2970
To appear in: LWT - Food Science and Technology
Received Date: 4 February 2011
Revised Date: 18 January 2012
Accepted Date: 19 January 2012
Please cite this article as: Khan, M.I., Sri Harsha, P.S.C., Giridhar, P., Ravishankar, G.A., Pigmentidentification, nutritional composition, bioactivity, and in vitro cancer cell cytotoxicity of Rivina humilisL. berries, potential source of betalains, LWT - Food Science and Technology (2012), doi: 10.1016/j.lwt.2012.01.025
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Pigment identification, nutritional composition, bioactivity, and in vitro cancer 1
cell cytotoxicity of Rivina humilis L. berries, potential source of betalains 2
Mohammad Imtiyaj Khan, P. S. C. Sri Harsha, P. Giridhar,† and G. A. Ravishankar 3
Plant Cell Biotechnology Department, Central Food Technological Research Institute (Constituent laboratory of 4
Council of Scientific and Industrial Research, New Delhi), Mysore 570 020, India. 5
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Running title: 12
Bioactive components of R. humilis berry 13
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†Address for correspondence: 20
Dr. P. Giridhar, Scientist, Plant Cell Biotechnology Department, 21
Central Food Technological Research Institute, Mysore 570 020, India. 22
Ph - +918212516501 Email – [email protected] 23
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A B S T R A C T 25
Ripened berries of Rivina humilis L. (pigeon berry) were investigated for pigment and 26
nutritional composition, in vitro bioactivities, and cancer cell cytotoxicity of its extracts. Ten 27
betalain pigments (total content − 0.35 g/100 g fresh weight, and 1.7 g/100 g dry weight) with 28
high level of betaxanthins were characterised. Carbohydrates (6.2 g), proteins (1.1 g), lipids (0.7 29
g), phenols (105.7 mg gallic acid equivalent), niacin (5.3 mg), and total tocopherols (0.77 mg) in 30
100 g fresh weight were observed. Antioxidant bioactivity of MeOH extract (maximum at 100 31
µg/mL) against OH· and β–carotene oxidation was studied. The extract (1000 µg/mL) effectively 32
protected kidney lipid peroxidation compared to butylated hydroxy anisole. Assays involving 33
betalains rich extract, and purified betacyanins and betaxanthins revealed EC50 against DPPH· 34
(51.0, 0.29, and 0.29 µg/mL), and reducing power (39.3, 2.79, and 1.34 µg/mL) which was 35
higher than that of gallic acid and ascorbic acid. In vitro cancer cell cytotoxicity was assessed 36
through MTT assay on HepG2 cells after exposing to betalains rich extract, betacyanins and 37
betaxanthins for 24 h; only betaxanthins exhibited cytotoxicity (EC50 12.0 µg/mL). After 48 h of 38
exposure, betacyanins and betaxanthins showed elevated cytotoxicity (EC50 17.5 and 2.0 µg/mL, 39
respectively). 40
Keywords: Rivina humilis L. (Phytolaccaceae), Betalains, Cytotoxicity, Bioactivity, Lipid 41
Peroxidation 42
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1. Introduction 47
Rivina humilis L. (Phytolaccaceae), commonly called as pigeon berry, is a wild herbaceous 48
bushy perennial plant found in colonies and in various types of shaded soils (Swarbrick, 1997), 49
standing up to a height of 120 cm (4 ft), and sometimes woody at base. It grows well in the 50
Caribbean and tropical America and is now widely naturalized in Indo-Malaysia and Pacific 51
(Swarbrick, 1997; Wealth of India, 2008). White flowers with a pink tinge grow on a spike 52
measuring 3–5 cm in length, which blooms from June to December. Leaves are usually widely 53
spaced, 4–12 cm long, 1.5–4 cm wide, both surfaces are glabrous or puberulent especially along 54
veins, petioles 1–3.5 cm long, usually puberulent along upper surface (Tseng, Wang, & Cheng, 55
2008). Berries (4 mm in diameter) are arranged in sub-globose inflorescence and pigmented with 56
betalains (Strack, Schmitt, Reznik, Boland, Grotjahn, & Wray, 1987) in various shades of 57
orange, red or purple. Recent report on dietary safety of R. humilis berries juice (Khan, Denny 58
Joseph, Muralidhara, Ramesh, Giridhar, & Ravishankar, 2011a) indicates that these berries could 59
be a prospective dietary or industrial source of betalains. 60
Betalains are nitrogenous pigments containing betalamic acid as chromophore. They are 61
exclusively deposited in 13 families under the order Caryophyllales (excluding caryophyllaceae 62
and the molluginaceae) in nature’s response to absence of anthocyanins in them. Two main 63
groups of betalains are red–violet betacyanins and yellow–orange betaxanthins. Betalamic acid 64
may condense spontaneously with various amino acids or amine derivatives to produce 65
betaxanthins, or with cyclic 3,4–dihydroxyphenylalanin (cyclo–DOPA), which may or may not 66
undergo glycosylation and further acylation to produce betacyanins. Betalains, especially 67
betaxanthins are linked with a higher free radical scavenging activity (Cai, Sun, & Corke, 2005; 68
Gandia-Herrero, Escribano, & Garcia-Carmona, 2009). In addition, betalains have been shown to 69
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have various in vitro and in vivo biological activities (Kanner, Harel, & Granit, 2001; Gentile, 70
Tesoriere, Allegra, Livrea, & D’Alessio, 2004; Tesoriere, Allegra, Butera, & Livrea, 2004; 71
Siriwardhana, Shahidi & Jeon, 2006; Wu, Hsu, Chen, Chiu, Lin, & Ho, 2006; Sreekanth, 72
Arunasree, Roy, Reddy, Reddy, & Reddanna, 2007; Rebecca, Boyce, & Chandran, 2010). 73
Dietary components are required to be stable and bioavailable. Betalains are known to be 74
stable at pH 5, refrigerated temperature, and ascorbic acid protects them efficiently (Cai et al., 75
2005). Bioavailability of betalains is low (0.5−3.7%) but, it appears that bioavailability 76
varies with dietary source (Kanner et al., 2001; Tesoriere et al., 2004). In this regard, there is a 77
need for exploring betalains sources from which betalains may be absorbed efficiently in the 78
body. 79
Vegetables and fruits have been known for centuries to provide nutrition and health 80
benefits mainly due to the presence of phytochemicals such as alkaloids, pigments (chlorophylls, 81
carotenoids, anthocyanins, betalains, curcumin), polyphenols, flavonoids, proteins, 82
carbohydrates, etc. This investigation was conducted to identify pigments, study the nutritional 83
composition, in vitro bioactivity and cancer cell cytotoxicity of Rivina humilis L. (R. humilis) 84
berries with special emphasis on purified betalains, which could be used for food and 85
nutraceutical applications. 86
2. Materials and methods 87
2.1. Source of fruits 88
Ripened berries of Rivina humilis L. (red variety) were collected during September–89
November, 2009 from shady areas of the environs of CFTRI, Mysore (India) located 90
geographically between 12° 18′ 26″ north latitude and 76° 38′ 59″ east longitude. In the period, 91
the minimum and maximum temperatures were 20°C and 28°C, respectively, and rainfall was 92
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5−10 cm. Herbarium sheets of this plant were deposited at Herbarium Collection Centre (SKU–93
accession no. 11198), Sri Krishnadevaraya University, Anantapur, India. Physical characteristics 94
of the berries were recorded (Supplementary Table S1 and Supplementary Fig. S2). Berries were 95
stored at –20°C until use. 96
2.2. Chemicals 97
HPLC grade methanol (MeOH), acetone, isopropanol, ascorbic acid, reference standard 98
organic acids, and mineral solutions were obtained from Sisco Research Laboratory (Mumbai, 99
India). Amberlite IRA-400, Amberlite XAD 16, α-tocopherol, gallic acid, 2,2-dipyridyl and 2,2-100
diphenyl-1-picryhydrazyl radical (DPPH·), β–carotene, hydrogen peroxide, butylated hydroxy 101
anisole (BHA), linoleic acid, thiobarbituric acid (TBA), Dulbecco’s Modified Eagle Medium 102
(DMEM), fetal calf serum (FCS), antimycotic−antibiotic solution, and 3–(4,5–dimethylthiazol-2-103
yl)–2,5–diphenyltetrazolium bromide (MTT) were obtained from Sigma–Aldrich Co (St. Louis, 104
MO, USA). All other chemicals used were of analytical grade. For HPLC analysis, degassed and 105
0.22 µm membrane filtered triple distilled water was used. 106
2.3. Pigment identification of ripened R. humilis berries 107
Fresh berries were deseeded manually and pulverized using mortar and pestle in the 108
presence of sand particles. Betalains pigments were extracted using H2O, MeOH, and 109
MeOH/H2O (acidified with 50 mmol/L ascorbic acid) until the macerate was colourless. Solvents 110
were evaporated under reduced pressure using flash evaporator (R 205, M/s BUCHI 111
Labortechnik AG, Flawil, Switzerland). Quantification of betalains pigment was done by using 112
spectrophotometer (UV-160 A, Shimadzu Corporation, Kyoto, Japan) readings at 477 nm (for 113
betaxanthins) and 535 nm (for betacyanins) as reported (Castellanos-Santiago & Yahia, 2008). 114
Betaxanthins+betacyanins values have been presented as total betalains. 115
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Chromatographic analysis of betalains was carried out as reported (Castellanos-Santiago et 116
al., 2008) in Waters Alliance 2695 HPLC, equipped with an auto sampler and coupled with a 117
Waters 2696 photodiode array detector, using RP C18 column (SunfireTM, Waters Corporation, 118
Milford, MA, USA) of 250×4.6 mm i.d. and the absorbance was recorded at 477 nm and 535 nm. 119
Mass spectrum was acquired in a Q-TOF UltimaTM mass spectrometer set in positive electro-120
spray ionization (ESI–MS) interface (Waters Corporation, Manchester, UK). MS parameters 121
were set as: nitrogen gas flow rate, 12 L/min with nebulizing pressure (35 psi); electrospray 122
voltage, 3.5 kV; nebulizer temperature, 350 °C. 123
2.4. Determination of nutritional components of ripened R. humilis berries 124
Carbohydrates, reducing sugars, and cellulose were determined following standard methods 125
in ethanol (EtOH)/H2O (8/2, v/v) extract; proteins, and phenols (MeOH and EtOH/H 2O (8/2, 126
v/v) extracts) were estimated by using Folin’s reagent (Sadasivam & Manickam, 2008). Total 127
soluble solids (TSS) in juice was recorded using a digital refractometer RX-500 (Atago Co. Ltd., 128
Tokyo, Japan). 129
Berries were dried at 40–45°C overnight and the reduction in weight was calculated as 130
moisture content according to the official method Da 2a-48 (AOCS, 2003). 131
Lipids were extracted using hexane at 40°C for 8 h from dried (moisture-free) berries 132
following Soxhlet extraction method Ba 3-38 (AOCS, 2003). 133
Acid insoluble crude fiber analysis was done according to the method described (Sadasivam 134
& Manickam, 2008). 135
Energetic value (kcal/100 g deseeded fruit) was calculated as, 136
Energetic value= 4 × proteins (g) + 9 × lipids (g) + 4 × carbohydrates (g). 137
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Fatty acid methyl esters (FAMEs) were prepared by treating the extracted oil, from R. 138
humilis berries, with methanolic KOH (2 mol equivalent/L) and analyzed as reported (Khan, 139
Asha, Bhat, & Khatoon, 2008). FAME was identified by using reference standard methyl esters. 140
Iodine value was determined from fatty acid composition by using official method Cd 1c–85 141
(AOCS, 2003). 142
Atherogenicity and thrombogenicity indices were calculated following the method described 143
by Ulbricht, & Southgate (1991). Atherogenicity Index (AI) = [%C12:0 + 4× %C14:0 + 144
%C16:0] / Σ% unsaturated fatty acids. Thrombogenicity index (TI) = (% C18:0 + %C16:0 + 145
%C14:0) / 0.5 × (Σ%Monounsaturated fatty acids + Σ% polyunsaturated fatty acids). 146
Niacin was determined in sulphuric acid extract of ripened berries following the standard 147
procedure (Sadasivam et al., 2008) wherein the extract reacts with cyanogen bromide in presence 148
of aniline to form colored complex (λmax 420 nm). 149
For HPLC determination of ascorbic acid, fresh ripened berries were extracted with H2O. 150
The extract was prepared as described earlier (Brause, Woollard, & Indyk, 2003) and 151
chromatographed as reported (Khan, Sri Harsha, Giridhar, & Ravishankar, 2011b). 152
Tocopherols from ripened berries were extracted with acetone. The extract was dried and 153
resuspended in HPLC eluate and chromatographed as described (Rogers, Rice, Nicolosi, 154
Carpenetr, McClelland, & Romanczyk Jr., 1993). The isomers of tocopherol and tocotrienol 155
were calculated based on α–tocopherol content following the official method Ce 8-89 (AOCS, 156
2003). 157
Organic acids in R. humilis deseeded berries (0.5 g) were extracted in water (20 mL), 158
centrifuged at 5000 × g, filtered through 0.45 µm membrane under vacuum, and purified passing 159
through Amberlite IRA-400 to remove sugar and neutral compounds (Marconi, Floridi, & 160
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Montanari, 2007). The purified extract was analysed by using LC 20 AD (M/s Shimadzu 161
Corporation, Kyoto, Japan) under the following conditions: column, µBondapak C18 (300 × 3.9 162
mm i.d., 10 µm pore size) (Waters Corporation, Manchester, UK); mobile phase, 163
Water/MeOH/TFA (97.7/2.2/0.1, v/v/v); flow rate, 0.7 mL/min; UV detection at 210 nm. 164
Elements content in R. humilis berries was estimated as reported (Khan et al., 2011b) by 165
using atomic absorption flame emission spectroscopy (Model AA-670IF, M/s Shimadzu 166
Corporation, Kyoto, Japan) with graphite furnace attachment. 167
2.5. Sample preparation for in vitro bioactivity, and cancer cell cytotoxicity assays 168
MeOH, MeOH/H2O (6/4, v/v) and MeOH/H2O (6/4, containing 50 mmol/L ascorbic acid) 169
extracts of R. humilis berries were used for in vitro bioactivity and cancer cell cytotoxicity 170
assays. The acidified MeOH/H2O extract was further processed to remove pectic substances and 171
purified by passing through Amberlite XAD 16 column (20×2.5 mm i.d.) as reported (Stintzing 172
et al., 2005). Betacyanins (70% pure) and betaxanthins (95% pure) fractions were concentrated 173
under reduced pressure. Considering the molecular weight and molar extinction co–efficient of 174
betanin [MW, 550 g/mol; ε, 60,000 L/(mol. cm)], and vulgaxanthin I [(MW, 339 g/mol; ε, 175
48,000 L/(mol.cm)], betacyanins and betaxanthins concentrations were quantified by using 176
spectrophotometer readings at 535 nm and 477 nm, respectively, in the equation reported 177
earlier (Castellanos-Santiago et al., 2008). In crude extract (red colour), reading at 477 nm 178
was about 2 times (200%) that of 535 nm. However, after partial purification, betacyanins 179
fraction had only 30% betaxanthins. Its colour was red-violet whereas in betaxanthins 180
fraction, there was only 5% betacyanins. This fraction was yellow in colour having light 181
red tinge. The purified betacyanins and betaxanthins were used for in vitro bioactivity and 182
cancer cell cytotoxicity assays. 183
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2.6. Bioactivity assays 184
Hydroxyl radical scavenging activity of R. humilis berries crude MeOH extract (100-1000 185
µg/mL) was tested as reported (Khan et al., 2011b). The assay involved measurement of the pink 186
colour complex formed by TBA reactive substances by monitoring absorbance at 535 nm against 187
a reagent blank. Gallic acid (10-500 µg/mL) was used as standard antioxidant. Hydroxyl radical 188
scavenging (%) was calculated by using the following formula, 189
Hydroxyl radical scavenging activity (%) = [1− (Absorbancesample/ Absorbanceblank)] × 100. 190
An aliquot of MeOH (0.2 mL) as blank or R. humilis berries crude MeOH extracts (100-191
1000 µg/mL) or butyl hydroxy anisole (BHA) (100−1000 µg/mL) were tested for antioxidant 192
activity following β−carotene linoleate model assay described earlier (Singh, Murthy, & 193
Jayaprakasha, 2002). Each experiment was carried out in triplicate. The absorbance values 194
having less than 10% deviation were considered for result computation. 195
Protection against lipid peroxidation (LPO) was analysed in cell-free preparations of albino 196
rat (Wistar strain) brain and kidney homogenates following the procedure of Murthy 197
Jayaprakasha, & Singh (2002). Different concentrations (100, 500, and 1000 µg/mL) of R. 198
humilis berries crude MeOH extract were tested. Lipid peroxidation inhibition was calculated as, 199
Lipid peroxidation inhibition (%) = [1– (Absorbancesample./ Absorbanceblank)] × 100. 200
Antioxidant activity of R. humilis berries crude extract (10–160 µg/mL), betacyanins (0.05–8 201
µg/mL), and betaxanthins (0.05–8 µg/mL) fractions were analysed after incubating them for 20 202
min at ambient temperature (25°C −27°C) with methanolic solution of DPPH⋅⋅⋅⋅ (100 µmol/L) in 2 203
mL reaction volume. The colour change was measured at 517 nm (Khan et al., 2011b). Gallic 204
acid (0.16–7.2 µg/mL), and ascorbic acid (2.5–40 µg/mL) were used as standard antioxidants. 205
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Activity (%) = [1– (Absorbancesample./ Absorbancecontrol)] × 100. The concentration which 206
showed 50% activity was represented as effective concentration (EC50). 207
For reducing power assay, R. humilis berry crude extract (20–320 µg/mL), betacyanins (0.8–208
6.4 µg/mL), betaxanthins (0.8–6.4 µg/mL), gallic acid (5–80 µg/mL), and ascorbic acid (50-500 209
µg/mL) in deionised water were mixed with 2.5 mL of phosphate buffer (0.2 mol/L, pH 6.6), 2.5 210
mL of potassium ferricyanide [K3 Fe (CN)6] (1 g/100 mL H2O), and then the mixture was 211
incubated for 30 min at 50°C. Afterwards, 2.5 mL of trichloroacetic acid (10 g/100 mL H2O) 212
was added, centrifuged at 1500 × g for 10 min. Upper layer (2.5 mL) solution was taken, mixed 213
with 2.5 mL of water, and 0.5 mL ferric chloride (0.1 g/100 mL H2O). The colour developed 214
was read at 700 nm (Oyaizu, 1986). The effective concentration (EC50) giving 0.5 absorbance 215
was derived from the graph of absorbance (700 nm) against concentration. 216
2.7. Cell culture and MTT assay for cancer cell cytotoxicity 217
HepG2 cells (obtained from NCCS, Pune, India) were grown in a humidified chamber with 218
CO2/air (5/95, v/v) and subcultured as monolayer in DMEM supplemented with heat-inactivated 219
FCS (10 mL/100 mL DMEM) and antibiotic−antimycotic solution (1 mL/100 mL DMEM) . 220
Cells were seeded in a 96 well micro−titer plate at a concentration of 5×103 cells/well in a final 221
volume of 100 µL culture medium and left for 24 h at 37°C. The cells were treated with R. 222
humilis berry crude extract, betacyanins, and betaxanthins for 24 and 48 h. Cell survival (%) was 223
determined using standard MTT assay described by Mossman (1983). Percent cell survival was 224
calculated using the formula: (Absorbance of treated group/ Absorbance of untreated group) × 225
100. The concentration which showed 50% survival was represented as EC50. 226
2.8. Statistical analysis 227
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Three parallel experiments were carried out for all the analyses and results are presented in 228
mean ± S.D. Microcal Origin 6.0 software (M/s Microcal Software Inc., Northampton, MA, 229
USA) was used to determine EC50. Two-way ANOVA test was carried out by using Microsoft 230
Excel programme of Windows 7 software. P ≤ 0.05 was considered significant. 231
3. Results and discussion 232
3.1. Pigments of R. humilis berries 233
Betalains pigments were identified by using spectral characteristics as listed in Table 1 and 234
Fig. 1. The identified betalains have been earlier reported in different systems (Cai et al., 2005, 235
Castellanos-Santiago et al., 2008, Stintzing et al., 2005) including R. humilis berries (Strack et 236
al., 1987), except that in the current method betalamic acid was not detected. It could be due to 237
the run time (40 min) or the mobile phase employed for separation (Castellanos-Santiago et al., 238
2008). 239
Betalains contents in H2O, MeOH, MeOH/H2O (6/4, acidified with 50 mmol/L ascorbic 240
acid) extracts of R. humilis berries are presented in Table 2a. On fresh weight, 241
betaxanthins:betacyanins ratio was about 1.5 in water and MeOH extracts, and 1.3 in acidified 242
MeOH/H2O extract (data not presented), whereas on dry weight, the ratio increased to 243
approximately 10 in H2O extract, 15 in MeOH extract, and 16 in acidified MeOH/H2O extract 244
(data not presented). The reported composition of betacyanins (58−82 mg/100 g fresh weight 245
(fw)) and betaxanthins (35−48 mg/100 g fw) in red beet (Gasztonyi, Daood, Hajos, & Biacs, 246
2001) was less than that of R. humilis berries betalains composition and the latter had more 247
betaxanthins than betacyanins. The highest level of betalains in peel and pulp of Opuntia ficus-248
indica fruits was approximately 114 mg/100 g fw (Stintzing et al., 2005). However, in another 249
report, in nine types of cactus pear fruits the betaxanthins, and betacyanins ranged from 0.3−19, 250
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and 0.2−30 mg/100 g fw (Chavez-Santoscoy, Gutierrez-Uribe, & Serna-Saldíva, 2009), 251
respectively. In case of Amaranthaceae family, the level of betalains was 8−136 mg/100 g fw 252
(Cai et al., 2005). 253
3.2. Nutritional components of ripened R. humilis berries 254
Carbohydrates, proteins, reducing sugars, non-reducing sugars, cellulose, and moisture 255
content in 100 g fresh weight, and lipids and fiber contents in 100 g dry weight are shown in 256
Table 2a. Due to low lipids content, the energetic value (35.5 kcal/100 g) of the berries was also 257
low. Similar studies on nutritional composition have been reported in other systems (Barros, 258
Carvalho, & Ferreira, 2010). Phenols content in MeOH extract was less than that of EtOH/H 2O 259
(8/2, v/v) extract, probably due to better solubility of R. humilis berry phenols in the latter 260
solvent (Table 2a). Among the betalains rich fruits, prickly pear fruits have been reported to 261
contain phenols in the range 0.022−0.226 mg GAE/g (Chavez-Santoscoy et al., 2009). 262
Fatty acid composition of lipids extracted from dry berries of R. humilis is presented in 263
Table 2b. Palmitic acid was major saturated fatty acid (SFA) whereas oleic and linoleic acids 264
were major unsaturated fatty acids. It was observed that the content of SFAs, monounsaturated 265
fatty acids, and polyunsaturated fatty acids were 56 g, 25 g, and 18 g in 100 g lipids, 266
respectively. The ratio of SFA: Mono-unsaturated fatty acids: Poly-unsaturated fatty acid was 267
2.2:1:0.7. For balanced fat intake, the proposed ratio is approximately 1:1:1 in India (RDA, 268
2010) however, it varies from nation to nation as recommended by FAO/WHO expert 269
consultation on fats and fatty acids in human nutrition, 2008. The fatty acid composition was 270
comparable to some of widely used edible oils (Khan et al., 2008). 271
Atherogenicity and thrombogenicity indices indicate the propensity of a diet or fat to 272
influence the incidence of coronary heart disease. AI (0.97) and TI (2.0) of lipids extracted from 273
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dry R. humilis berries are presented in Table 2b. Oils with low (≤ 1) AI and TI values have been 274
considered good for health (Ulbricht et al., 1991). British diet was assessed to have an AI of 0.93 275
and TI of 1.2, whereas oils rich in saturated fats such as coconut oil and palm oil have been 276
reported to have AI of 13.6 and 0.88, and TI of 6.18 and 1.7, respectively (Ulbricht et al., 1991). 277
Vitamins such as niacin (5.3 mg), ascorbic acid, and tocopherols (0.8 mg) were quantified 278
in 100 g fw as presented in Table 2c. Niacin content in 125 g of unfortified cereals has been 279
reported to be 5–7 mg, whereas 100 g of peanuts contain 10 mg niacin as listed in USDA Food 280
Composition Database (available at www.nal.usda.gov/fnic/foodcomp/niacin.html, accessed on 281
April 15, 2010). Niacin has been recommended at 16 mg and 14 mg/day for male and female, 282
respectively, because of its direct or indirect involvement in various biological functions 283
including DNA repair, cancer prevention, fighting reactive oxygen species (Hageman, & 284
Stierum, 2001). Tocopherols content was comparable to that of recently reported exotic berry 285
Tinospora cordifolia (Khan et al., 2011b). Tocopherols are known to protect bio-membranes 286
from lipid peroxidation that causes cell lysis resulting in many disease conditions. 287
Organic acid profile in R. humilis berries showed that tartaric acid content was the highest 288
whereas malic acid was the least (Table 2d). Organic acid contents of some commercially 289
important berries have been reported (Kafkas, Kosar, Turemis, & Baser, 2006; Milivojevic, 290
Maksimovic, & Nikolic, 2009). Compared to these reports, contents of citric, oxalic and 291
tartaric acid in R. humilis berries (in the present study) appear to be high. Tartaric acid has 292
not been reported to accumulate more than any other acid. 293
Element composition of deseeded R. humilis berries is shown in Table 2e. Among the 294
microelements analysed in the deseeded fruit, iron was high, whereas copper and zinc were in 295
the typical range (<2 mg/100 g) of berries (Cunningham, Milligan, & Trevisan, 2001). Whereas 296
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macroelement potassium content was high, sodium and magnesium were in the usual range 297
(20−100 mg/100 g) of berries (Cunningham et al., 2001). High levels of potassium, and 298
magnesium are used for energy and sport drinks to uphold mineral pool during periods of 299
physical exhaustion, whereas low levels of sodium are preferred for preventing high blood 300
pressure (Munoz de Chavez, 1995). Considering the high content of potassium, the fruit can be 301
used for potassium fortification in food formulations. 302
3.3. Bioactivity of pigment rich extracts from R. humilis berries 303
OH· is one of the reactive oxygen species in vivo which are metabolic products that cause 304
damage to biomolecules including proteins, lipids, and DNA leading to metabolic dysfunctions. 305
Endogenous enzymic and non-enzymic antioxidants scavenge this radical through cascades of 306
reactions to render it harmless. Exogenous or dietary antioxidants have been recommended to 307
improve the radical scavenging potential of body. OH· radical scavenging activity of R. humilis 308
berries MeOH extract is shown in Fig. 2a. The most effective one among the tested 309
concentrations was 100 µg/mL that showed 46% activity. There was no increase in the activity 310
when the extract concentration was increased from 500 µg/mL to 1000 µg/mL. Gallic acid, 311
standard antioxidant, showed EC50 value of 119 µg/mL. The observation that betalains exhibit 312
low hydroxyl radical scavenging activity has been also reported earlier in betalains rich cactus 313
pear fruit extract that exhibited only 65% activity at 500 µg/mL (Siriwardhana et al., 2006). This 314
could be attributed to the low pH employed for the quantification of thiobarbituric acid reactive 315
substances in this method, because betalains are better radical scavengers at basic pH (Gandia-316
Herrero et al., 2009). 317
β−Carotene linoleate bleaching antioxidant capacity is based on the principle that linoleic 318
acid, an unsaturated fatty acid, gets oxidized to peroxides in the presence of oxygenated water. 319
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The products formed initiate β-carotene oxidation causing discoloration the pigment. 320
Antioxidants prevent the oxidation and hence, antioxidant activity is proportional to the colour of 321
β-carotene. Fig. 2b shows the antioxidant activity of MeOH extract of R. humilis berries. 322
Linoleate peroxidation inhibition by red beet betalains was investigated earlier (Kanner et al., 323
2001) and it was observed that betalains (< 1 µmol/L) were more efficient than α-tocopherol (5 324
µmol/L) and catechin (1.2 µmol/L). Not only betalains, MeOH extracts of pomegranate peel 325
have been shown to inhibit LPO and the activity was ascribed to phenols present in it (Murthy et 326
al., 2002). It was observed that 100 µg/mL of R. humilis berry MeOH extract was more efficient 327
in preventing β-carotene discoloration than BHA (100 µg/mL). 328
Fig. 2c shows the protection against LPO by MeOH extract of R. humilis berries and 329
standard antioxidant BHA. MeOH extract of R. humilis berries did not show significant 330
protection whereas the most effective concentration of BHA that inhibited brain LPO was 100 331
µg/mL. We observed that brain LPO inhibition activity increased when the extract concentration 332
was increased from 100 µg/mL to 500 µg/mL, however at 1000 µg/mL the inhibition activity 333
was negative. The reason for this anomaly could not be understood. Contrastingly, MeOH extract 334
provided significant protection (∼ 55%) only at 1000 µµµµg/mL against LPO of kidney tissues, 335
which was comparable to that of BHA. Betalains have been shown to inhibit lipid peroxidation 336
in vivo (Tesoriere et al., 2004). In addition to betalains, phenols in MeOH extracts of 337
pomegranate peel inhibited LPO by scavenging free radicals (Murthy et al., 2002). 338
Antiradical activity of R. humilis berry extracts against DPPH· is shown in Fig. 3a. EC50 339
values of MeOH/H2O, and MeOH/H2O acidified with 50 mmol/L ascorbic acid were 81.4 µg/mL 340
and 51 µg/mL, respectively, and both showed dose dependent activity. DPPH· scavenging 341
activity (EC50) of betalains rich cactus pear fruit was reported to be ~200 µg/mL (Siriwardhana 342
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et al., 2006). The antioxidant activity (EC50) of red pitaya peel and flesh extracts against DPPH⋅ 343
was 22.4 ± 0.29 and 118 ± 4.12 µmol vitamin C equivalents/g, respectively (Wu et al., 2006). 344
While phenols were found to contribute the most towards antioxidant activity, strong correlation 345
between phenols and betalains contents was also observed (Stintzing et al., 2005). Antiradical 346
activity of purified betacyanins (EC50–0.29 µg/mL) and betaxanthins (EC50–0.11 µg/mL) (Fig. 347
3b) was much higher than that of commercial important fruits such as mango, strawberry, guava, 348
etc (Corral-Aguayo, Yahia, Carrillo-Lopez, & Gonzalez-Aguilar, 2008). It was also observed in 349
this study that ascorbic acid (EC50–8.3 µg/mL) and gallic acid (EC50–2.3 µg/mL) exhibited less 350
scavenging activity against DPPH·. This result was in agreement with earlier report (Cai et al., 351
2005) that betaxanthins and betacyanins are more potent antioxidants than ascorbic, and ferulic 352
acid. The intrinsic radical scavenging activity of betalains is due to presence of an electronic 353
resonance system which acts as electron acceptor. In addition, presence of one or two phenolic 354
hydroxy groups in the structure of betaxanthins has been shown to enhance the free radical 355
scavenging activity (Gandia-Herrero et al., 2009). 356
Reduction of ferric to ferrous by antioxidants resulting in prussian blue colour formation 357
indicates reducing power. Ferric reducing power of R. humilis berry extracts is presented in Fig. 358
3c. EC50 values of the extracts were 99.4 µg/mL, and 39.3 µg/mL, respectively. MeOH/H2O 359
acidified with 50 mmol/L ascorbic acid exhibited higher efficiency due to presence of ascorbic 360
acid that can contribute through regeneration of betalains and/or cause an additive effect in the 361
reducing power. EC50 value of reducing power of betalain rich red dragon fruit was reported 362
(Rebecca et al., 2010) to be 62.5 mg/mL, which was by far lower compared to that of R. humilis 363
berries. Fig. 3d presents the reducing power (EC50) of betcyanins (2.79 µg/mL) and betaxanthins 364
(1.34 µg/mL). Standard antioxidants such as ascorbic (EC50–160 µµµµg/mL) and gallic acid 365
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(EC50–7.9 µµµµg/mL) were used for comparision. Ferric reducing power assay is carried out at 366
physiological pH in which betaxanthins have been reported to have higher activity (Gandia-367
Herrero et al., 2009). Iron is a major redox metal that can contribute to oxidative stress in the 368
cellular level leading to various physiological dysfunctions. Due to efficient ferric reducing 369
power of betalains and phenols rich R. humilis berries, the berries can be investigated further 370
systematically to ascertain the quantum of contribution in protecting cells from metal-induced 371
oxidative stress. 372
3.4. In vitro cancer cell cytotoxicity 373
In vitro cancer cell cytotoxicity studies were carried out through MTT assay after exposing 374
HepG2 cells to betalains rich R. humilis berry crude extract, and purified betacyanins and 375
betaxanthins from R. humilis berries. The results are shown in Fig. 4. No cytotoxicity was 376
observed after 24 h exposure of the cells to R. humilis berry crude extract and betacyanins, 377
however betaxanthins exhibited cytotoxicity (EC50–12.0 µg/mL) (Fig. 4a). After 48 h of 378
exposure, R. humilis berry crude extract did not cause cell death (Fig. 4b), whereas betacyanins 379
(EC50–17.5 µg/mL) (Fig. 4c) and betaxanthins (EC50–2.0 µg/mL) (Fig. 4d) showed increased 380
cytotoxicity. Earlier report on betalains from prickly pear fruit showed no cytotoxicity of betanin 381
after 24 h exposure to in vitro model of endothelial cells, whereas indicaxanthin was toxic 382
(Gentile et al., 2004). The report also showed that betalains protected endothelium from 383
cytokine−induced redox state alterations through the inhibition of inter−cellular cell adhesion 384
molecule–1 (ICAM–1) expression. In another study, red pitaya betanin (EC50 ∼150.0 µg/mL) 385
was shown to have strong inhibition of the growth of B16−F10 melanoma cancer cells and the 386
inhibition activity was attributed to molecular structural effects similar to that of flavonoids (Wu 387
et al., 2006). Recently, antiproliferative activity (EC50) of betanin was observed at ∼22 µg/mL 388
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against human chronic myeloid leukemia cell line (K562) and it was attributed to the release of 389
cytochrome C into the cytosol, poly (ADP) ribose polymerase (PARP) cleavage, down regulation 390
Bcl-2, and reduction in the membrane potentials (Sreekanth et al., 2007). 391
4. Conclusions 392
Pigments, phenols and nutrients composition of R. humilis berries were characterised in this 393
study and antioxidant and bioactivities of the extracts were assessed. High betalains content 394
predominantly betaxanthins, various bioactivities exhibited by the extracts and purified 395
betacyanins and betaxanthins indicate the usefulness of this berry. Interestingly, it appears that 396
bioavailability of betalains varies with dietary source, and hence the current study holds 397
promise for further investigations into bioavailability aspects of betalains. This is first report on 398
characterisation of R. humilis berry and its potential antioxidant activities and bioactivities. 399
5. Acknowledgements 400
Authors thank Department of Biotechnology, Government of India, New Delhi for financial 401
assistance. The author, Mohammad Imtiyaj Khan, is grateful to CSIR, New Delhi for providing 402
Senior Research Fellowship. Part of this paper was presented in 6th International Congress on 403
Pigments in Food, Budapest, Hungary, June 20–24, 2010. 404
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511
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513
514
515
516
517
518
519
520
521
522
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Tables 523
Table 1. Betalains identified in the water extract of dry pulp of ripened Rivina humilis berries. 524
525
526
527
528
529
530
531
532
533
534
535
tR- retention time, Bx- betaxanthin. aQualitative only. Trivial names of betalains are provided in parenthesis. 536
537
538
539
540
541
542
543
544
545
546
547
Peak tR min Betalainsa λmax [M+H] +
1 4.87 Proline-Bx (Indicaxanthin) 482 309
2 5.08 DOPA-Bx (Dopaxanthin) 470 391, 357, 311
3 5.50 Glutamic acid-Bx (Vulgaxanthin I) 471 391, 341
4 5.97 Glutamine-Bx 470, 275 374, 341
5 7.62 Aspartic acid-Bx 458 391, 281, 238
6 8.23 Hydroxynorvaline-Bx (Humilixanthin) 477 327, 309
7 8.53 Betanin 535 551, 389
8 8.87 Betanidin 535 551, 389
9 9.60 Tyrosine-Bx 474 375, 347
10 10.75 Dopamine-Bx 458 347, 303
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Table 2a. Pigment and nutritional components of ripened R. humilis berries. 548
Components Content (g/ 100 g fw)
Pigment
H2O extract 0.35 ± 0.03a
(1.7 ± 0.09a)a
MeOH extract 0.29 ± 0.02b
(1.0 ± 0.1c)a
MeOH/H2O (6/4, 50
mmol/L ascorbic acid)
extract
0.3 ± 0.03b
(1.7 ± 0.08a)a
Carbohydrates 6.3 ± 0.64
Proteins 2.6 ± 0.24
Total soluble solidsb 15.0 ± 0.3
Reducing sugars 3.5 ± 0.2
Non-reducing sugarsc 2.7 ± 0.4
Cellulose 0.4 ± 0.4
Moisture 82.7 ± 2.5
Lipidsa 0.7 ± 0.1
Crude fibrea 3.0 ± 0.4
Energetic valued 35.5
Phenols (mg GAE)
80% EtOH extract 105.7 ± 1.6
MeOH extract 90.2 ± 2.7
fw− fresh weight, GAE−gallic acid equivalent. aValues are based on dry weight. bTotal soluble solids is expressed in 549
°Brix at 25°C. cNon-reducing sugar was calculated by subtracting reducing sugars from total carbohydrates. dValue 550
is in kilocalorie/100 g. Values are mean ± S.D., n=3. Different alphabets in the same column indicates significant 551
difference at P ≤ 0.05. 552
553
554
555
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Table 2b. Fatty acid composition of lipids extracted from ripened Rivina humilis berries. 556
Fatty acid Content (g/100 g dw)
Lauric (C12:0) 1.1 ± 0.03
Myristic (C14:0) 2.7 ± 0.2
Palmitic (C16:0) 30.3 ± 1.3
Palmitoleic (C16:1) 2.4 ± 0.08
Stearic (C18:0) 9.7 ± 0.2
Oleic (C18:1) 23.0 ± 1.1
Linoleic (C18:2) 18.1 ± 0.5
Linolenic (C18:3) ND
Arachidic (C20:0) 12.6 ± 0.7
Total saturated fat 56.5 ± 1.8
Total unsaturated fat 43.5 ± 2.1
Atherogenicity index 0.97
Thrombogenicity index 2.0
dw−−−− dry weight, ND– not detected. Fatty acid composition values correspond to the mean of relative peak area (%) 557
of GLC chromatograms of three consecutive injections. Fatty acids were identified by comparing the retention times 558
of the peaks with standard methyl esters. Values are mean ± S.D. (n=3). 559
560
561
562
563
564
565
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Table 2c. Vitamins content of ripened Rivina humilis berries. 566
Vitamins Content (mg/100 g fw)
Niacin 5.3 ± 1.0
Ascorbic acid tr
Total tocopherols 0.8 ± 0.1
α-T ND
β & γ-T ND
δ-T ND
α-T3 0.006 ± 0.002
β & γ-T3 0.8 ± 0.1
δ-T3 0.002 ± 0.001
fw− fresh weight, T- Tocopherol, T3- tocotrienols, tr- traces, ND- not detected. Values are mean ± S.D. of three 567
analyses. 568
569
570
571
572
573
574
575
576
577
578
579
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Table 2d. Organic acid profile of ripened R. humilis berries. 580
581
582
583
584
585
586
587
588
589
590 fw− fresh weight. Values are mean ± S.D. of three analyses. 591
592
593
594
595
596
597
598
599
600
601
602
603
Organic acids Content (mg/100 g fw)
Oxalic 25.4 ± 1.2
Lactic 11.1 ± 0.4
Tartaric 110.4 ± 2.6
Malic 0.5 ± 0.05
Acetic 4.1 ± 0.1
Citric 37.3 ± 0.8
Succinic 7.9 ± 2.8
Total organic acids 196.7
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Table 2e. Elemental composition (mg/100 g dry weight) of Rivina humilis deseeded berries 604
Sample Fe Cu Zn Mg K Na
Deseeded fruit 12.2 ± 1.7 0.3 ± 0.14 1.2 ± 0.2 42.7 ± 1.1 845 ± 169 38.4 ± 3.7
All values are mean ± S.D. of three analyses except for zinc and potassium which were analyzed four times 605
independently. 606
607
Figures 608
609
Fig. 1. HPLC profile of betacyanins (535 nm) and betaxanthins (477 nm) in ripened berries of R. 610
humilis. Major peaks were identified by comparing with beetroot betalains, earlier report on R. 611
humilis fruit, and confirmed by MS. Peak identification data is given in Table 1. 612
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613
614
Fig. 2. Hydroxyl radical scavenging activity (a), β-carotene linoleate model antioxidant activity 615
(b), and inhibition of lipid peroxidation (c) of methanol extract of R. humilis berries. Values are 616
b a
c
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mean ± S.D, n=3. MeOH (�), Gallic acid (�), BHA ( ), effect of BHA (�), MeOH extract (�) 617
on brain sample, effect of BHA (�), MeOH extract (�) on kidney sample. 618
619
620
a b
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621
Fig. 3. DPPH· scavenging activity of R. humilis berry crude extracts (a), betacyanins (Bc) and 622
betaxanthins (Bx) fractions (b) of Rivina humilis berry. Reducing power of crude extracts (c), 623
betacyanins and betaxanthins fractions (d) of Rivina humilis berries. Values are mean ± S.D. 624
(n=3) significant at P<0.01. MeOH/H2O (6/4, v/v) containing ascorbic acid (50 mmol/L) (�), 625
MeOH/H2O (6/4, v/v) (), betacyanins (�), betaxanthins (�). 626
c d
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627
628
Fig. 4. Cytotoxity studies on HepG2 cell line after exposing the cells for 24 h to R. humilis berry 629
crude MeOH/H2O (6/4, acidified with 50 mmol/L) extract, betacyanins and betaxanthins 630
fractions (a), after 48 h incubation with R. humilis berry crude MeOH/H2O (6/4, acidified with 631
50 mmol/L) extract (b), betacyanins (c), and betaxanthins (d). Rivina humilis berry crude extract 632
(�), betacyanins (�), betaxanthins (�). Values are mean ± S.D. (n=3) significant at P < 0.01. 633
a b
c d
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Research Highlights
� Ten betalains were identified in R. humilis berries (total content − 0.35% fw).
� Nutrient composition including vitamins and phenols were determined.
� Methanol extract showed strong DPPH· and OH· scavenging activity and reducing power.
� MeOH extract protected β–carotene oxidation and lipid peroxidation.
� Crude extract and betacyanins were not cytotoxic, whereas betaxanthins were toxic.
