Betalain and Phenolic Compositions, Antioxidant Activity of Tunisian Red Beet (Beta vulgaris L. conditiva) Roots and Stems Extracts

Abstract

In the present study, betalains content, phenolic composition, and antioxidant activity of different parts of red beet (Beta vulgaris L. conditiva) (i.e., roots and stems) were compared. Crude extract of root showed the highest betalain content with a maximum of 53 ± 4 mg betanin eq and 46 ± 3 mg vulgaxantin I eq g⁻¹ of extract stems showed higher total phenolic concentration than roots, ranging between 2.0 ± 0.4 and 14.6 ± 0.5 mg gallic acid eq⁻¹ of extract. Chemical composition was analyzed using LC-MS. Betalains (vulgaxanthin I, betanin, and isobetanin) and phenolics (gallic acid, ferulic acid, chlorogenic acid, caffeic acid, vanillic acid, syringic acid, ellagic acid myricetin, quercetin, rutin, kampferol) were identified in roots and stems. Betalain extract obtained from roots showed higher antioxidant activity than extract obtained from stems.

Keywords:

• Beta vulgaris L
• Roots
• Stems
• Betalains
• Phenolic compounds
• Antioxidant activity

INTRODUCTION

Fruits and vegetables have been found in many studies to protect against several chronic diseases associated with aging such as cancer, cardiovascular diseases, cataracts, brain, and immune dysfunction.^[1,^2] These natural protective effects have been attributed to various components, such as carotenoids, lycopenes, betalains, vitamins, polyphenols, and other phytochemicals.^[3,^4] Betalains, the red-violet betacyanins and yellow betaxanthins, are water soluble nitrogen-containing vacuolar pigments and replace the anthocyanins in flowers and fruits of the most families of the Caryophyllales.^[5,^6] A review of the literature has shown that data on the biological activity of betalains are rather scarce as compared to other natural colorants, such as carotenoids and anthocyanins. Recently, interest in these molecules has grown since their anti-radical and antioxidative activities were highlighted.^[7−9] Betalains are also used as additives in the food industries on account of their natural colorant properties, high solubility in water, and absence of toxicity.^[6,⁹,^10] Red beetroot is considered as the most important food product containing this class of colorant.^[11] Besides, red beets contain further health-promoting constituents such as phenolics (phenolic acids, phenolic acid esters, and flavonoids) and folic acid.^[12,^13] High antioxidant activities of betalain extracts from both beet roots and stems have been also reported.^[14−18] The objective of this study was to analyze the chemical composition of selected Tunisian red beet roots and stems extracts and to evaluate their antioxidative potential. The phenolic composition analysis was performed using a novel high-throughput chromatography-mass spectrometry (LC-MS) technique.

MATERIALS AND METHODS

Chemicals and Reagents

Standards of phenolic acids (gallic acid, ferulic acid, chlorogenic acid, caffeic acid, vanillic acid, syringic acid, ellagic acid) and of flavonoids (myricetin, quercetin, rutin, and kampferol), β-carotene, linoleic acid, 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH·), and Folin-Ciocalteu reagent were procured from Sigma-Aldrich Chemie (Steinheim, Germany). All reagents were of analytical grade.

Plant Material

The Red beets (Beta vulgaris L. var. conditiva) were obtained from local farmer in Tunisia during February 2010. Roots and stems were separated, washed, sliced, ground in a blender, and stored at –4°C until utilization.

Extraction

Betalains

An aqueous extraction was prepared from each part of plant, roots, and stems. Plant material (50 g) was homogenized with distillated water (250 mL) and macerated for three times (24 h × 3) at room temperature. The obtained crude extracts (CE) were centrifuged at 8000 rpm for 30 min, filtered, and evaporated at 40°C under reduced pressure until dry.

Phenolics

Residue extracts obtained previously were stirred with acetonitrile (3 × 40 mL × 80 min) to give an acetonitrile-soluble fraction (ASF) and an acetonitrile insoluble residue (AIR). The ASF were taken to dryness by rotary evaporator.

Photometric Quantification of Betalains

Betacyanins and betaxanthins content of the extracts were determined spectrophotometrically (UVIKON XS; Biotech Instruments equipped with LabPower Junior program) following the Nilsson’s method. Pigment concentrations of liquid samples were determined as absorbance units of a 1% relative to the dry matter of the extract at 538 and 480 nm for betacyanins and betaxanthins, respectively. The betalains content (BLC) was calculated as: BLC [mg L⁻¹] = (A × DF × MW× 1000) / (ϵ × 1), where A is the absorption value, DF the dilution factor and 1 the path length (1 cm) of the cuvette. For the quantification of betacyanins (Bc) and betaxanthins (Bx), the molecular weights (MW) and molar extinction coefficients (ϵ) was respectively, 550 g mol⁻¹ and 60,000 L mol⁻¹cm⁻¹ in H₂O: λ = 538 nm for betanin, 339 g mol⁻¹ and 48,000 L mol⁻¹cm⁻¹ in H₂O: λ = 480 nm for vulgaxanthin I.

Betalains HPLC Analyses

All samples analyses were performed in an Agilent Technologies 1100 series liquid chromatographic equipped with a UV variable detector. The separation was performed with a C₁₈ (250 mm × 4.60 mm, 5 μm, waters) column using two eluents: acetonitrile (A) and phosphoric acid (0.20 M) (B). Complete separation of betalains was achieved within 35 min at room temperature with a flow rate of 1mL min⁻¹. The first 5 min was performed isocratically with 100% B, followed by linear gradient from 0–13% A in B in 30 min. Betalains were monitored at 480 and 538 nm for betaxanthins and betacyanins, respectively.

Identification of betalains compounds

The identification of the compounds was based on their molecular masses determined by high-performance liquid chromatography electrospray ionization-mass spectrometry (HPLC-ESI-MS) and comparison of UV-spectral characteristics. HPLC–MS analyses were carried out with an Agilent HPLC series 1100 instrument (Agilent, Waldbronn, Germany) equipped with UV visible absorbance detector in series with a Bruker Esquire ion trap mass spectrometer (Bremen, Germany). Specific mass data were obtained by applying the same solvent system as described above with the ESI source running in the positive ionization mode (range: m/z 100–1500). The optimal tuning parameters were found to be 4000 V for capillary and 5000 V for spray voltage at a source temperature of 365°C. Nitrogen was used as the dry gas at a flow rate of 8.00 L min⁻¹ and a pressure of 35 psi.

Colorimetric Determination of Total Phenols

The amount of total phenols in the extracts was determined according to a modification of the Folin-Ciocalteu method.^[19] Extracts (0.10 mL) were mixed with 0.50 mL of Folin–Ciocalteu reagent (1:10), 1.00 mL of distilled water and 1.50 mL of 20% sodium carbonate solution. The reaction time was 1 h at room temperature in darkness and the absorption of the solution was measured at 760 nm (Jenway 6505 UV/Vis. Spectrophotometer). The total phenolic content was expressed as mg gallic acid equivalents (GAE) g⁻¹ of extracts. The calibration curve range was 5–50 mg mL⁻¹.

UPLC Analyses and LTQ-Orbitrap Identification of Phenolic Compounds

Ultra performance liquid chromatography series performed by Accela 600 (Thermo Scientific, Germany) was used to separate phenolic components. Hypersil GOLD C18, 50 × 2.10 mm i.d., 1.90 μm column (Thermo Fisher Scientific, Germany) was used with a flow rate of 0.40 mL min⁻¹. The injection volume was 10 μL. The mobile phase consisted of eluent A containing 0.10% formic acid in water, and eluent B consisting of 0.10% formic acid in acetonitrile 98%. A linear gradient program started with 5–95% B and was kept for 5 min, followed by 2 min isocratically at 95% B.

Identification

The UPLC system was coupled to a LTQ Orbitrap XL Hybrid FTMS operating in Heated Electrospray Ionization mode. The Mass Spectrometer was operated in negative mode with masses scanned from 120–1000. MS settings were as follows: capillary temperature (270°C), vaporizer temperature (350°C), sheath and auxiliary gas pressures (35 and 10 arbitrary units). The ion source voltage was set to –4.00 kV, the capillary voltage to –37.00 V and the tube lens voltage to –204.11 V. Data acquisitions were carried out with Xcalibur QualBrowser software.

Quantification

Quantitative analyses were assessed with the external standard method. Standard calibration curves were established by plotting the height of peaks against seven different concentrations of polyphenolic compounds (varying from 5 ppb to 500 ppb), obtaining regression coefficients above 0.996 in all cases.

Antioxidant Activity (AA) by the β-Carotene Bleaching Method

The AA of samples was evaluated by the β-carotene-linoleic acid model system according to the modified literature procedure.^[20,21] β-Carotene (0.20 mg), 20 mg of linoleic acid and 200 mg of Tween 20 were mixed in 0.50 mL chloroform and the solvent evaporated under vacuum. The resulting mixture was diluted with 50 mL of distilled water. To the 4.00 mL of this emulsion, 0.20 mL of test samples in ethanol was added. BHT was used for comparative purposes. A solution with 0.20 mL of ethanol and 4.00 mL of the above emulsion was used as control. The tubes were covered with aluminum foil and were maintained at 50°C in a water bath. Absorbance was taken at zero time (t = 0) and after every 15 min. Measurement of absorbance was determined at 470 nm and continued until the color of β-carotene disappeared in the control reaction (t = 120 min). The AA of extracts was determined as percent inhibition (PI) relative to control sample: AA (%) = [(A_A(120)−A_{C (120)} )/(A_C(0) - A_{C (120)})] × 100, where A_A(120) is the absorbance of the antioxidant at 120 min, A_{C (120)} is the absorbance of the control at 120 min, and A_C(0) is the absorbance of the control at 0 min.

Free Radical Scavenging Activity Using DPPH Radical

To evaluate the free radical scavenging activity, the samples were allowed to react with a stable free radical, 2,2-diphenyl-1-picryl hydrazyl radical (DPPH·).^[22] Different concentrations of CE, ASF, and AIR (2.00 mL) were added to 2.00 mL DPPH· solution in ethanol (10⁻⁴ mol L⁻¹). The reduction of (DPPH·) was followed by monitoring the decrease in absorbance at 517 nm until the reaction reached a steady state. The reaction time was 30 min. The percent DPPH decolorization of the sample was calculated by the equation: PI = [1-(A_sample/A_control)] × 100, where A_control is the absorbance of the control; A_sample is the absorbance of reaction mixture. The amount of sample needed to decrease the initial DPPH concentration by 50%, EC₅₀, was calculated graphically.

Statistical Analysis

All data are presented as mean ± standard deviation of triplicate analyzes. For comparisons between samples, data were analyzed by ANOVA and Tukey’s multiple comparison test (Statgraphics Centurion XVI). A probability of 5% or less was accepted as statistically significant.

RESULTS AND DISCUSSION

Pigment Extraction

Betalains are water soluble pigments. A water extraction is a simple, highly efficient and low cost method for crude betalain extraction and promotes better stability for the pigments.^[23,^24] The comparison between roots and stems showed that betalains concentrations were significantly organ-dependent (p < 0.05) (Fig. 1). The pigments were distributed mostly in roots and were about 53 ± 4 mg betanin equivalent g⁻¹ of extract and 46 ± 3 mg vulgaxanthin I equivalent g⁻¹ of extract, while, betalains concentrations in stems were only 11.0 ± 0.5 mg betanin equivalent g⁻¹ of extract and 10.4 ± 0.8 mg vulgaxanthin I equivalent g⁻¹ of extract. Previous studies have discussed betacyanin accumulation against damaging UV radiation in the ice plant (Mesembryanthemum crystallinum L.).^[25,26] Less clear, however, was the coloration of fruit growing underground, such as the red beet which was thought to be related to increased pathogen resistance and to improve viral defense. These assumptions were supported by a study on red beets where highest betacyanin concentrations were found in the peel followed by crown and flesh.^[27]

Figure 1  Betalain contents of CE and AIR of Beta vulgaris conditiva roots and stems. Results were presented as means ± SD values from three independent experiments (level of significance p < 0.05).

The removal of the colorless ASF did not affect the betalains levels (Fig. 1). According to Kujala et al.,^[13] the acetonitrilic fraction consisted in colorless phenol acids and flavonoids. Previous studies have reported that colorless polyphenols are frequently found in association with pigments in the vacuoles of the colored cells of higher plant organs.^[28] This complexation represents the main color-stabilizing mechanism in plants. In fact, its presence increases the color intensity.^[29,30]

The HPLC analyses showed clearly the presence of three main compounds, in both extracts, identified as follow: One betaxanthin at 480 nm and two betacyanins at 538 nm (Fig. 2). While studying these chromatograms, by peak area comparison, the content of the different betalains compounds seemed to be higher in red beet root than in stems extract.

Figure 2  HPLC-UV chromatograms of major compounds in betalain crude extracts from (a) roots and (b) stems at 538 nm and 480 nm. 1: vulgaxanthins I; 2: betanin; 3: isobetanin.

The identification of these common pigments requires standards, identical to each pigment under consideration. The lack of commercially available standards entailed the use of other methodologies.^[31] Numerous analytical techniques including HPLC-MS, and HPLC-MS/MS have been applied for the separation and detection of the individual compounds of a mixture.^[13,³²,^33] Therefore, the individual betalains patterns were thoroughly assessed in this work by using HPLC–MS analyses. Individual pigments were identified according to specific mass spectrometric, UV–spectral and retention time characteristics reported elsewhere^[13,27,^34] and presented in Table 1. These results were similar to earlier studies reporting that vulgaxanthin I, betanin, and isobetanin were the major betalain components of red beet.^[13]

Table 1  Retention time and MS data on the positive ionization mode of major compounds detected at 480 and 538 nm in red beet roots and stems crude extracts

Peaks^a	λ (nm)	Rt^b (min)	[M _ H]^+^c	Identification	Bases^d
1	480	4.05	340	Vulgaxanthin I	MS, UV
2	538	31.40	551	Betanin	MS, UV
3	538	33.25	551	Isobetanin	MS, UV

^aPeak numbers refer to HPLC analysis in Figures 2a and 2b.

^bRt, retention time, refers to HPLC analysis in Figures 2a and 2b.

^c[M _ H]⁺, molecular ion in the positive ionization mode.

^dMS, mass spectrum and UV, ultraviolet spectrum.

Phenolic Content

The Folin-Ciocalteu method is widely used to evaluate the total content of phenols.^[35] The concentration of these compounds in the different extracts: CE, ASF, and AIR is shown in Fig. 3. Results obtained in the present study revealed that B. vulgaris L. extracts had appreciable amounts of phenolic compounds. Stems had a significantly higher total phenolic content than roots in all samples (p < 0.05). Previous studies have shown that the developmental stage of the plant might affect biosynthetic pathways of phenolic compounds, the total phenolic and flavonoid contents.^[36,^37] It has also been shown that the biosynthesis of polyphenol is accelerated by light exposure and serves as a filtration mechanism against UV-B radiation.^[38]

Table 2  Phenol composition and content of acetonitrile fractions

				Concentration (mg/g dry extract)
Peaks	Rt^a(min)	[M−H]^−(m/z)^b	Identification	Roots	Stems
1	0.36	169	Gallic acid	Tr	0.1
2	0.46	193	Ferulic acid	0.8	0.9
3	1.56	353	Chlorogenic acid	Tr	0.2
4	1.60	179	Caffeic acid	0.35	Tr
5	1.63	167	Vanillic acid	1.7	1.3
6	1.93	197	Syringetic acid	0.7	3.2
7	2.06	609	Rutin	ND	1.2
8	2.10	301	Quercetin	3,0	4.7
9	2.67	317	Myricetin	2,0	3.2
10	2.76	285	Kampferol	3.6	1.6
11	7.02	301	Ellagic acid	1.6	Tr

^aRt, retention time refers to UPLC analysis.

^b[M _ H]⁻, molecular ion in the negative ionization mode. ND: not detected; Tr: trace.

Figure 3  Total phenolic contents of CE, ASF, and AIR of red beet roots and stems. Bars indicate standard deviations (level of significance p < 0.05). CE: crude extract; ASF: acetonitrile soluble fraction; AIR: acetonitrile insoluble residue; BHT: synthetic antioxidant; GAE: gallic acid equivalent.

The amounts of total phenolics decreased after fractionation with acetonitrile. Highest concentrations were obtained in the ASF. The amounts of phenols of ASF for roots and stems were about 6.6 ± 0.7 and 10.4 ± 0.5 mg GAE g⁻¹ of extract, respectively. Results obtained, for ASF, from UPLC-MS analyses revealed the presence of five phenolic acids (ferulic, vanillic, syringic, ellagic, and caffeic), three flavonoids (quercetin, kampferol, and myricetin) for roots and six phenolic acids (gallic, vanillic, chlorogenic, ferulic, caffeic, syringic), four flavonoids (myricetin, quercetin, rutin, and kampferol) for stems (Table 2). These compounds were identified according to their retention times and spectral characteristics of their peaks against those of standards. The results showed differences in the phenolic profiles of the studied fractions. Notably, gallic, chlorogenic, and rutin were not present in the ASF obtained from root extract, but was found in the ASF of stem. Furthermore, it was shown that ASF had substantially higher concentrations of the identified phenolic compounds with a considerable amount of syringic (3.2 mg g⁻¹ dry extract), ferulic (0.9 mg g⁻¹ dry extract), vanillic (1.3 mg g⁻¹ dry extract) acids, myricetin (3.2 mg g⁻¹ dry extract), and quercetin (4.6 mg g⁻¹ dry extract). In contrast, the ellagic acid was only detected in the root acetonitrile soluble fraction (1.6 mg g⁻¹ dry extract). This is a potentially interesting finding, since it is well known that ellagic acid is a biologically active phenolic acid that interacts synergistically with many other active substances.^[39] In this respect, it can be suggested that the observed difference in phenolic content and composition of the different parts of red beets affects their biological activities.

Table 3  Antioxidant activity of samples and BHT assessed by β-carotene bleaching method

	AA (%)^a
Extracts	Roots	Stems
CE	93 ± 3	48 ± 1
ASF	32 ± 2	35 ± 1
AIR	61 ± 2	34 ± 2
BHT	56 ± 1

Results expressed as means ± SD from triplicate measurements (level of significance p < 0.05).

^aAA: antioxidant activity expressed as percentage of inhibition of β-carotene; CE: crude extract; ASF: acetonitrile soluble fraction; AIR: acetonitrile insoluble residue; BHT: synthetic antioxidant.

AA by the β-Carotene Bleaching Method

The comparative β-carotene bleaching rates of the control, BHT (standard), water CE, and acetonitrile soluble fractions and insoluble residues (ASF and AIR) of different parts of B. vulgaris L. are shown in Fig. 4. The decrease in absorbance was due to the oxidation of linoleic acid and β-carotene emulsion. All studied CE and fractions showed appreciable AA. The mean AA percentage (AA%), based on the β-carotene bleaching rate of aqueous CE and roots and stems ASF are presented in Table 3. The tested CE showed significantly greater AA than BHT (AA% = 56 ± 1). The highest AA was observed for red beetroots in CE (AA = 93 ± 3%) which was two times greater than that of the extract of stems CE (AA = 48 ± 1%). The significant differences in phenolic and betalains concentrations found in the extracts were consistent with the observed differences in their antioxidant activities. Indeed, the high BLC of the roots extract was correlated with an increase of inhibition (%) of β-carotene decolorization (r = 0.870). After removing phenolic fraction from betalains CEs, the AA decreased (Table 3). This could be explained by possible existence of synergistic effects between phenolic compounds and betalains, leading to significantly increase in biological activities of betalain containing extracts.

Figure 4  Absorbance changes of β-carotene at 470 nm in the presence of (a) roots and (b) stem extracts and BHT at 200 ppm. Values are means ± SD (n = 3) (level of significance p < 0.05). CE: crude extract; ASF: acetonitrile soluble fraction; AIR: acetonitrile insoluble residue; Control: blank assay prepared without antioxidant adding to β-carotene-linoleic acid emulsion.

DPPH Radical-Scavenging Activity

The DPPH· scavenging activities of extracts of different parts of B. vulgaris L. are shown in Fig. 5. All the extracts were significantly (p < 0.05) able to scavenge DPPH radical in a dose-dependent manner. The crude betalain extract from roots showed the highest DPPH radical scavenging activity of 97 ± 1% at the maximal concentration (200 μg mL⁻¹) whereas stems showed 85 ± 4% inhibition at the same concentration. These data were comparable with those obtained by Wootton-Beard et al.^[40] who reported that the juice of red beetroot displayed the strongest radical scavenging ability in both the DPPH^• and ABTS^•+ assays and the highest reducing capacity as measured by FRAP among 23 commercially available vegetable juices.

Figure 5  DPPH scavenging effect of (a) roots and (b) stems crude extracts (CE), acetonitrile soluble fractions (ASF), and acetonitrile insoluble residues (AIR) at different concentrations. Values are means ± SD (n = 3) (level of significance p < 0.05). PI: percent inhibition.

The EC₅₀ values of various parts were compared with BHT (Table 4). As can be seen, the studied crude root extracts were significantly more effective than BHT. EC₅₀ values of 5.0 ± 1.00 and 19.0 ± 1 μg mL⁻¹ were obtained for crude root extract and BHT, respectively. After acetonitrile fractionation, a decrease of DPPH radical scavenging activity was observed. Indeed, The EC₅₀ which is negatively related to the AA increased. Nevertheless, the antiradical activity of AIR of roots remained higher than the synthetic antioxidant (EC₅₀ = 10.0 ± 1.4 μg mL⁻¹).

Table 4  EC₅₀ value of samples and BHT

	EC50 (μg mL^−1)^a
Extracts	CE	ASF	AIR	BHT
Roots	5 ± 1	102 ± 6	10 ± 1
				19 ± 1
Stems	47 ± 3	170 ± 5	176 ± 5

Results expressed as means values ± SD of three measurements (level of significance p < 0.05).

^aEC₅₀, DPPH free radical scavenging activity expressed as efficient concentration (EC) values required to decrease the initial DPPH concentration by 50%; CE: crude extract; ASF: acetonitrile soluble fraction; AIR: acetonitrile insoluble residue; BHT: synthetic antioxidant.

The difference in the radical scavenging activity of the different red beet parts was attributed to the difference in betalains and total phenol concentration. Indeed, the observed increase of EC₅₀ was associated with decreased concentrations of betalains in root extracts (r = –0.999). Previous studies have reported that both betacyanins and betaxanthins showed a great antiradical activity at various pH values.^[18] Their chemical structure (Fig. 2) are likely to stabilize radicals due to the presence of aromatic amino compounds moieties.^[9] In another study, the relationship between the chemical structure and activity of the betalains was investigated. The finding revealed that the free radical scavenging activity usually increased with the number of hydroxyl/imino-groups, and depended on the position of the hydroxyl groups and glycosylation of aglycones in the betalains molecules.^[24]

CONCLUSION

This study revealed that there were significant differences in the distribution of betalains and phenolic compounds in the different B. vulgaris L. parts leading to a difference in their AA. A high correlation between BLC and AA was observed suggesting that the betalains might become a useful source of both natural antioxidants and natural colorants. The removal of phenolic fraction from CE decreased the AA which could be explained by possible existence of synergistic effects between phenolic compounds and betalains.

ACKNOWLEDGMENTS

The authors are grateful to Mr. Nicolas Mabon from “Faculté Universitaire des Sciences Agronomiques de Gembloux,” Belgium for his technical assistance for the HPLC analyses. The EC does not share responsibility for the content of the article.

FUNDING

This work was supported by the FP7 RegPot project FCUB ERA GA No. 256716.