Introduction

Opuntia (subfamily: Opuntioideae; Family: Cactaceae) is a large genus of succulent shrubs, native of the new world, now widely grown in the warmer parts of the world, on account of their unique appearance and attractive flowers. They are commonly known as Prickly pears, because of their edible fruits. Recent data revealed the high content of some chemical constituents, which can give added value to this fruit on a nutritional and technological functionality basis. High levels of betalains, taurine, calcium, magnesium, and antioxidants are noteworthy. Another important compositional factor of prickly pear is the presence of pigments, which give particular attractiveness to fruit and products (Piga 2004). Betalains have a number of health properties. Infusions of betalains from the bracts of Bougainvillaea mixed with honey, for example, are used to treat coughs in some regions of Mexico. Some anticancer, antiviral and antioxidant activity has been attributed to betalains. The main focus of interest, however, has recently been on betalain pigments as natural antioxidants (Yahia and Castellanos-Santiago 2008). The purpose of the current review is to provide a systematic survey of the analytical techniques for the determination of betalain.

The most common connotation with pigmented flower petals and fruits is the attraction of animals both for pollination and seed dispersal. Anthocyanins mask the chlorophyll containing organelles and thereby protect chloroplasts against high light intensities to prevent photoinhibition (Stintzing and Carle 2004). Chalker-Scott (1999) suggested three functions of anthocyanins in plants, namely as absorbers of harmful radiation, as transport vehicles for monosaccharides and as osmotic adjusters during periods of drought and low temperature. The anthocyanins are a subgroup within the flavonoids characterized by a C6–C3–C6 skeleton.

Betalains are of great taxonomic significance in higher plants. The presence of betalains in members of the order Caryophyllales has been an important criterion for their classification. The presence of betalains and anthocyanins is mutually exclusive in the angiosperms. Betalains are water soluble nitrogenous chromoalkaloids and can be divided into two major structural groups, (1) The red to red-violet betacyanin (Latin Beta, beet and Greek kyanos, blue color) and (2) The yellow betaxanthins (Latin Beta and Greek xanthos, yellow). Betalains may function as osmolytes to uphold physiological processes, to stabilize subcellular structures, to reduce nitrogen toxicity and to be an excellent radical scavenger. Structurally, betacyanins are characterized by a cyclo-Dopa structure with additional substitutions through varying glycosylation and acylation patterns at C5 or C6 whereas the betaxanthins are condensation products of betalamic acid and various amino compounds. Betacyanins can be further classified by their chemical structures into four types: betanin-type, amaranthine-type, gomphrenin-type and bougainvillea-type (Stintzing and Carle 2004; Cai et al. 2005). Structures of betacyanins and betaxanthins found in the fruits of different Opuntia spices summarized in Fig. 1. The biosynthetic steps involved in betalain biosynthesis are summarized by (Strack et al. 2003).

Fig. 1
figure 1

Structures of betacyanins and betaxanthins found in prickly pear

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Numerous analytical methods have been designed and developed for the qualitative and quantitative determination of betalains in fruits of Opuntia species and are reviewed as follows.

Qualitative analysis

Chemical tests

Harborne (2007) reported chemical tests for the identification of betacyanins. Red color of betacyanin vanishes upon heating with 2 M HCl for 5 min at 100 °C and color changes to yellow by adding 2 M NaOH dropwise indicate presence of betacyanins.

Spectrophotometric method

Harborne (2007) reported visible spectrum of betacyanin in methanol-HCl give maximum absorbance in the range of 532–554 nm. Viloria-Matos et al. (2001) reported visible spectra of fruits of Opuntia boldinghii Br. et R., maximum absorbance at 537 nm at pH 6.1 which is similar to the earlier reported value of betacyanin (Bilyk 1979, 1981; Delgado-Vargas et al. 2000). Fernández-López and Almela (2001)extracted pigments from the prickly pear fruits (Opuntia ficus-indica), of reddish purple and yellow color, by homogenization of fruit flesh in methanol, with a ratio mass fruit (g)/solvent (ml) of 1:5 and two main pigments were obtained, which were identified as indicaxanthin (λmax 484 nm) and betanin (λmax 535 nm). The spectrophotometric analysis suggests that the external color of prickly pear fruits depends on the relative concentration of betacyanins (red pigments with maximum absorbance at around 535 nm) and betaxanthins (yellow pigments with maximum absorbance at around 480 nm) (Schliemann et al. 1996, 2000, 2001; Wybraniec et al. 2001; Fernández-López and Almela 2001; Cai and Corke 1999; Stintzing et al. 2003, 2005).

Chromatographic method

Thin layer chromatographic (TLC)

Harborne (2007) reported chromatography in 1 % aqueous HCl and n-butanol:acetic acid:water (BAW; 4:1:5) give high and very low Rf value, respectively.

High performance thin layer chromatographic (HPTLC)

Viloria-Matos et al. (2001) isolated and identified betacyanin from fruits of Opuntia boldinghii Br. et R. by HPTLC using two solvent systems (System I: isopropanol:ethanol:water:acetic acid 55:20:20:5; System II: isopropanol:ethanol:water:acetic acid 30:35:30:5) in one dimension. Results showed a major red fraction with a maximum absorbance at 537 nm which is similar to the reported value for betacyanin.

High performance liquid chromatographic (HPLC)

HPLC is an excellent means in the analysis of betalains. The most common support is C18-derivatized silica providing adequate efficiency and retention of betacyanins as well as their sufficient resolution on conventional stationary phases. Because betacyanins exist in aqueous solution in different ionisation forms at changed pH values, the use of typical acidic eluents with or without buffers is a useful factor governing their separation (Schliemann et al. 1996, 2000, 2001; Wybraniec et al. 2001, 2006). Fernández-López and Almela (2001) separated and identified betalain pigments from methanolic extract of two cultivars of prickly pear (Opuntia ficus-indica) fruits using reversed-phase high performance liquid chromatography and photodiode array detector. The chromatographic separation program consisted of a 30 min linear gradient elution from solvent A (1 % acetic acid in water) to 12 % solvent B (1 % acetic acid in acetonitrile) with a flow of 1 ml/min. The chromatographic pattern of the methanolic extract showed two major peaks with a retention time of 16.2 min at 484 nm and 17.4 min at 535 nm, identified as indicaxanthin and betanin, respectively. Fernández-López et al. (2002) also analyzed presence of betalains using method proposed by Fernández-López and Almela (2001) from the fruits of Opuntia stricta, Opuntia undulata and Opuntia ficus-indica and found HPLC patterns of betalains with retention time at 16.8 min (λmax 484 nm), 19.6 min, and 22.8 min (λmax 537 nm) assigned to indicaxanthin, betanin and isobetanin, respectively. Stintzing et al. (2003) separated betalains from Opuntia ficus-indica cv. ‘Rossa’ and cv. ‘Gialla’ using aqueous 0.2 % trifluoroacetic acid and 10 % formic acid solutions at a ratio of 65/35 (v/v) as eluent A, and a mixture of 100 % acetonitrile and 10 % aqueous formic acid (80/20,v/v) as eluent B. After 15 min of isocratic elution with 100 % A, a linear gradient was followed from 0 % B to 20 % B in 60 min. Betaxanthins was monitored at 470 nm and betacyanins at 538 nm, respectively. Stintzing et al. (2006b) developed a process for the production of both juice concentrates and powders from Opuntia ficus-indica fruits of the cultivar ‘Gialla’ at laboratory and pilot plant-scale, respectively. Since betalains are regarded as thermolabile compounds, alternative processes for juice concentration and preservation, including cross-flow microfiltration and freeze drying, considered. HPLC–DAD peak separation was achieved using mobile phase A (1 % v/v formic acid in water) and B (Aqueous MeCN, 80:20 MeCN/H2O, v/v). Starting isocratically with 100 % A for 2 min, a linear gradient was followed from 0 to 20 % B in 60 min and then from 20 to 100 % B in 5 min. Pigment retentions of the major betaxanthins and betacyanins were determined at 10.4 min (Histidine-betaxanthin), 16.3 min (Glutamine-betaxanthin), 29.2 min (GABA-betaxanthin), 29.9 min (Isoproline-betaxanthin), 31.2 min (Proline-betaxanthin) at 470 nm and 36.8 min (betanin) and 40.7 min (isobetanin) at 538 nm.

Wybraniec (2006, 2008) reported the effect of tetraalkylammonium salts on retention of betacyanins and decarboxylated betacyanins in ion-pair reversed-phase HPLC and investigated chromatographic acyl migration in betacyanin and their decarboxylated derivatives. Identification of betalains from the fruits of 10 Mexican prickly pear cultivars by HPLC–DAD analysis was performed by Yahia and Castellanos-Santiago (2008) using water (eluent A) and methanol (eluent B) mixture at a flow rate of 1 ml/min. Betalains were separated starting isocratically with 100 % A in 10 min followed by a linear gradient from 0 % B to 30 % B in 30 min, and finally a linear gradient from 30 % B to 100 % B in 20 min, before re-equilibration to the starting conditions. Betaxanthins and betacyanins were monitored at 482 and 535 nm, respectively. Several solvent systems were used for betalain analysis; the best results were obtained in water/methanol system than other methods, acetic acid in water/acetic acid in acetonitrile or phosphoric acid solution buffer. Table 1 shows the qualitative data of betalains by HPLC.

Table 1 Qualitative analysis of betalains by HPLC
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Liquid chromatography–Mass spectroscopy (LC–MS)

The use of mass spectrometry (MS) coupled to HPLC complements the use of photodiode-array detectors (PAD) and permits immediate identification of components of a mixture and characterization of an extract in terms of its chemical composition. MS provides molecular weight and structural information of the chromatographic bands so that fully-resolved peaks are not required, thus shortening chromatographic runs and reducing sample preparation while ensuring high sensitivity and selectivity. This technique is commonly used in investigations on betalain pigments (Schliemann et al. 1996, 2000, 2001; Wybraniec et al. 2001). Fernández-López et al. (2002) screened the presence of betalain pigments in fruits of Opuntia stricta, Opuntia undulata and Opuntia ficus-indica, also Yahia and Castellanos-Santiago (2008) identified betalains from the fruits of 10 Mexican prickly pear cultivars by HPLC and ESI–MS, qualitative data summarized in Table 2.

Table 2 Qualitative data of betalains in prickly pear (Opuntia spp.) fruit by HPLC–ESI–MS
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Nuclear magnetic resonance (NMR)

Unambiguous betalain structures can only be elucidated by nuclear magnetic resonance (NMR) measurements, requiring tedious isolation and solid experimental set up (Strack et al. 2003; Stintzing and Carle 2007). Stintzing et al. (2004) analyzed betacyanin pigments by LC–NMR and 2D NMR spectroscopy from red–purple pitaya (Hylocereus polyrhizus (Weber) Britton and Rose) at neutral pH (Table 3). Wybraniec et al. (2006) elucidated decarboxylated betanin, phyllocactin and hylocerenin of purple pitaya (Hylocereus polyrhizus) fruits by 1H and 13C NMR spectroscopy summarized in Tables 4, 5 and 6, respectively. Betaxanthin structure was elucideted based on 1H NMR data (Trezzini and Zr 1991) and Stintzing et al. (2006b) has reported 13C NMR data of two betaxanthins (indicaxanthin and miraxanthin) by applying only slightly acidic conditions.

Table 3 1H and 13C NMR data of betacyanins; D2O, ref δ = 4.7 ppm; 500 MHz
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Table 4 1H (600 MHz, D2O) and 13C (150 MHz, D2O) NMR data of decarboxylated betanin
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Table 5 1H (600 MHz, D2O) and 13C (150 MHz, D2O) NMR data of decarboxylated phyllocactin
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Table 6 1H (600 MHz, D2O) and 13C (150 MHz, D2O) NMR data of decarboxylated hylocerenin
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Quantitative analysis

Spectrophotometric method

The most convenient way to quantify betalains is spectrophotometric method. First, Nilsson (1970) established a method to quantify pigments in beetroot. The total contents of betacyanins and betaxanthins were determined using the formula reported by Cai et al. (2005); Nilsson (1970); Chethana et al. (2007); Fernández-López and Almela (2001); Fernández-López et al. (2002). Their molar absorptivity (ε) values were 5.66 × 104 (amaranthin, \( E_{{1 {\text{cm}}}}^{1\% } \) 536 nm = 779), 6.16 × 104 (betanin, \( E_{{1 {\text{cm}}}}^{1\% } \) 536 nm = 1120), and 5.06 × 104 (gomphrenin I, \( E_{{1 {\text{cm}}}}^{1\% } \) 540 nm = 920). The mean molar absorptivity (ε) value for betaxanthins is 4.80 × 104. Another formula for determination of betalain content was described by (Cai and Corke 1999; Stintzing et al. 2003, 2005): [BLC [mg/l] = (A × DF × MW × 1000)/(e × 1)], where A is the absorption value at the absorption maximum, DF the dilution factor and 1 the pathlength (1 cm) of the cuvette. For quantification of betacyanins and betaxanthins, the molecular weights (MW) and molar extinction coefficients (ε) of betanin (MW = 550 g/mol; ε = 60,000 l/mol cm in H2O; λ = 538 nm) and indicaxanthin (MW = 308 g/mol; ε = 48,000 l/mol cm in H2O; λ = 480 nm) were applied, respectively. Stintzing et al. (2005, 2006a) developed a process for production of cactus pear juice and fruit powders. Quantitative and qualitative color changes during processing were monitored by analysing juice samples after each processing step in terms of CIEL*C*h° and betalain contents. Table 7 summarizes spectrophotometric quantification of betalains.

Table 7 Spectrophotometric quantification of betalains in prickly pear
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Yahia and Castellanos-Santiago (2008) extracted the pigments using two solvents, McIlvaine buffer (pH 6.5, citrate–phosphate) and water from the fruits of 10 Mexican prickly pear Cultivars. The betalain content (BC) was calculated according to literature with a slight modification; BC [mg/g]) [(A(DF)(MW)Vd/εLWd)], where A is the absorption value at the absorption maximum of 535 and 483 nm for betacyanins and betaxanthins, respectively, DF is the dilution factor, Vd is the dried pulp solution volume (ml), Wd is the dried pulp weight (g), and L is the path-length (1 cm) of the cuvette. In all cases, water extracted the highest level of pigments. Spectrophotometric quantification of betalains summarized in Table 8.

Table 8 Spectrophotometric quantification of betalains in the fruits of 10 Mexican prickly pear cultivars
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Conclusion

From the present review, analysis of betalains from the prickly pear carried out easily using visible spectrophotometer and HPLC. Presently, analysis of phytochemical constituents mostly carried out using HPTLC, but in case of betalains still such method is not available. Estimation of betalains carried out by spectrophotometer which is more reliable and economic. Though various analytical methods are reported, but still more focus is required towards HPLC and HPTLC with marker’s evidence.