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Simultaneous spectrophotometry determination of lycopene and beta-carotene concentrations in carotenoid mixtures of the extracts from tomatoes, papaya and orange juice.

Introduction

Carotenoids, the C40 tetraterpenoids derived from head-to-tail condensation of eight isoprenoid units, are notable for their wide distribution, structural diversity, and various functions. More than 600 carotenoids, excluding cis and trans isomers, have been isolated and characterized from natural sources (Pfander, 1987). Carotenoids have been credited with several beneficial effects on human health ranging from provitamin A activity to the enhancement of the immune response and reduction of the risk of degenerative diseases such as cancer, cardiovascular diseases, cataract, and macular degeneration (Olson 1999a; Astrog, 1997; Burri 1997; Mayne, 1996; Olson and Krinsky, 1995; Bendich, 1994; Krinsky, 1994; Gaziano and Hennekens, 1993). The action of carotenoids against chronic diseases has been mainly attributed to an antioxidant property, specifically, their ability to quench singlet oxygen and interact with free radicals (Palozza and Krinsky, 1992). However, other mechanisms, such as modulation of carcinogen metabolism, inhibition of cell proliferation, enhancement of cell differentiation and stimulation of cell-to-cell communication have been reported (Olson, 1999a; 1999b). The ability of carotenoids to quench singlet oxygen has been linked to the conjugated double bond system, the maximum efficiency being shown by carotenoids with nine or more conjugated double bonds (Foote et al., 1970). Lycopene was found to be twice more efficient than the dicyclic [beta]-carotene (Di Mascio et al., 1989), despite of both compounds possessing Hconjugated double bonds. The effects of lycopene on human health as an antioxidant protector against lung, stomach, and prostate cancers have attracted considerable interest (Giovannucci, 1999; Clinton, 1998; Sies and Stahl, 1998; Gerster, 1997; Stahl and Sies, 1996). Average daily dietary lycopene intake levels (assessed by means of food frequency questionnaire) were estimated to be 25.2 and 33.39 mg/day in the Canadian and Nigerian diets, respectively (Olajire et al., 2007; Rao et al., 1998).

Carotenoid analysis is inherently difficult and error prone. In developed countries, separation and quantification of carotenoids are usually carried out using high-performance liquid chromatography (HPLC) technique (Monge-Rojas and Campos, 2011; Barba et al., 2006). This method, although is reliable, accurate and time-consuming it requires extensive sample preparation, the use and disposal of hazardous organic solvents. It also requires highly trained personnel and expensive equipments. Novel chemical techniques that have been evaluated for direct determination of lycopene and other nutritionally important carotenoids include the application of optothermal and photothermal methods (Bicanic, 2011; Bicanic et al., 2005; 2004). These methods are gaining the popularity because of their simplicity. Other commonly used methods include resonance Raman spectroscopy (Bhosale et al., 2004), infrared (De Nardo et al., 2009; Halim et al., 2006) and near infrared spectroscopy (Baranska et al., 2006; Pedro and Ferreiram, 2005). Fourier transform infrared (FTIR) in combination with multivariate analysis offers a powerful and rapid technique for the analysis of agricultural and food products (Jha and Matsuoka, 2000). Conventional UV-visible spectrophotometric assays have also been shown to be simple, rapid, and inexpensive methods for measuring lycopene content in tomato and tomato products (Biswas et al., 2011; Davis et al., 2003), but absorbance interference from lycopene provides poor accuracy and overestimation of [beta]-carotene levels (Olives-Barba et al., 2006). However, in some developing nations with limited accessibility to HPLC, the quantification of lycopene and [beta]-carotene is often accomplished by means of the traditional ultraviolet-visible spectrophotometry. This method should be accurate enough, in that it has been shown that the content of lycopene in samples determined by the use of both experimental (calibration curve) and theoretical data (Beer-Lamberts law) shows a good agreement, with a relative error below 3% (Ravelo-Perez et al., 2008). Some published absorption coefficients values may contain significant levels of error or uncertainty (Britton, 1995), likewise different authors choose different absorption coefficients for same carotenoids (in the same solvents), thus accounting for a good part of the variations in analytical results. In some studies the spectrophotometer detector was set at 472 nm to quantify lycopene (Moraru and Lee, 2005; Sharma and Le Maguer, 1996) and at 436 nm (Lumpkin, 2005) while in others it was determined at 503 nm (Ravelo-Perez et al., 2008). Although the absorbance of lycopene at 503 nm is not the highest, it was so selected to avoid interferences from other carotenoids present in the samples. The values of [[lambda].sub.max] for carotenoids in hexane and petroleum ether are practically the same for diethyl ether, methanol, ethanol and acetonitrile, and 2-6 nm, 10-20 nm, 10-20 nm and 18-24 nm higher in acetone, chloroform, and n dichloromethane as well as in toluene, respectively (Britton, 1995). However, in a carotenoid mixture containing lycopene and [beta]-carotene, the choice of absorption wavelengths of 450 nm and 470 nm to quantify [beta]-carotene and lycopene, respectively may not reflect the true concentrations of each of the two carotenoids, since both absorb significantly at those two wavelength ranges, while the choice of 502 nm is perfect for lycopene determination. In the present work, the model equations for simultaneous determination of beta-carotene and lycopene concentrations in hexane layer extract of lycopene/[beta]-carotene mixtures have been derived. This was then used to quantify [beta]-carotene and lycopene in separate hexane extracts of the carotenoid mixtures from tomatoes, papaya and orange juice samples.

Materials and Methods

Tomato fruit samples (Ibadan-local cultivar bought from a farmland near Wazobia Market, Ogbomoso, Nigeria) were randomly selected and packed into nylon bags and quickly taken into the laboratory, then they were rinsed with distilled water and left to drain for some minutes. About 500 g (15-25 fruits) of fresh tomatoes was chopped, blended and then homogenized in a laboratory homogenizer. Conventional solvent extraction methods (Perkins-Veazie et al., 2001; Sadler et al., 1990) were employed for carotenoid extraction. Approximately 10g of the tomato serum was subjected to extraction with hexane, methanol and acetone (2:1:1) containing 2.5% butylated hydroxytoluene (BHT). The extract was treated with distilled water, methanol and 20 % KOH/methanol (1:1:1) to saponify any triglyceride present. The extract was then washed with distilled water and re-dissolved in hexane. The hexane extracts were scanned in the visible light wavelength range of 400-750 nm using HE[lambda]I0S[alpha] UV-visible spectrophotometer (in a 1 cm path length quartz cuvette blanked with n-hexane) and the maximum absorbances were observed at 450, 472 and 502 nm, respectively for the lycopene - [beta]-carotene hexane layer mixture. The mixture was diluted with n-hexane using dilution factors of 2, 3, 6, 8 and 16, respectively to check for result consistencies and subsequent absorbances were measured. Samples of papaya and orange juice extracted in hexane were also subjected to carotenoid analysis. The molar extinction coefficient of 172,000 L/mol/cm at 502 nm was used to estimate lycopene concentration, using the Beer-Lamberts law (Ravelo-Perez et al., 2008; Zechmeister and Polgar, 1943).

Results and Discussion

Figure 1A-B represent typical overlap spectra of the hexane extract of the lycopene/[beta]-carotenoid mixture in tomatoes with absorption maxima at 450 nm, 472 nm and 502 nm. Most carotenoids exhibit absorbance maxima at three wavelengths, resulting in a three-peak spectrum. As the number of conjugated double bonds increases, the [[lambda].sub.max] shifts to longer wavelengths. Thus, the most unsaturated acyclic carotenoid, lycopene, with 11 conjugated double bonds is red and absorbs at the longest wavelengths ([[lambda].sub.max] at 443, 471, 502 nm) (Rodriguez-Amaya and Kimura, 2004). Cyclization results in steric hindrance between the methyl group at C-5 of the ring and the hydrogen atom at C-8 of the polyene chain. This hindrance takes the it electrons of the ring double bond out of plane with respect to those of the chain, causing a hypsochromic shift (displacement of [[lambda].sub.max] to shorter wavelength), a hypochromic effect (decrease in absorbance) and loss of fine structure (spectrum with less defined peaks). Thus, the dicyclic molecule, [beta]-carotene, is yellow-orange and despite possessing the same number of conjugated double bonds as lycopene, exhibits absorption peaks at 450 and 472 nm and a mere shoulder at 425 nm (Rodriguez-Amaya and Kimura, 2004). This is so because both carotenoids absorb substantially in the overlapping wavelength ranges. As already stated that the choice of 502 nm to quantify lycopene is alright despite the fact that this wavelength value is not equal to [[lambda].sub.max] (Ravelo-Perez et al., 2008). The correlations of the absorbances of the mixture at 450 nm and 502 nm relative to those at 472 nm, respectively (Fig. 2A-B) is very high ([R.sup.2] = 0.999 in both cases). This makes quantification of these carotenoids in mixture more ambiguous. Concentrations of lycopene and [beta]-carotene in the mixture were calculated (Table 1) based on the assumption that the absorbances at 450 nm and at 502 nm are exclusively, for [beta]-carotene and lycopene respectively. This is to be re-calculated using the proposed model and will then be compared henceforth. However, analytes such as norfloxacin, ofloxacin and lomerfloxacin in their mixture have been determined simultaneously using chemometric method (Huang et al., 2009) and other analytes such as chlorpromazine, perphenazine and acetopromazine were quantified by kinetic wavelength-pair method (Carreto et al., 1997). Here, lycopene and [beta]-carotene concentrations are determined by partial least squares calibration (Skoog et al., 2000). This involves determination of the absorbances of several solutions at the wavelength at which analytes, lycopene and [beta]-carotene, absorb. The wavelengths were carefully chosen such that the molar absorptivity of one component is much larger than that of the second component. Therefore, absorbances at 450 nm and 502 nm were used where the molar absorptivities of [beta]-carotene are 1.39 x [10.sup.5] and 2.63 x [10.sup.4] L/mol/cm, respectively and those of lycopene being 1.16 x [10.sup.5] and 1.72 x [10.sup.5] L/mol/cm, respectively. According to the law of Lamberts and Beer, the absorbance at 450 nm and 502 nm (in a 1 cm pathlength quartz cuvette) of the carotenoid mixture of lycopene and [beta]-carotene can be expre as follows:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (1)

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (2)

where, [A.sub.450] and [A.sub.502] are the absorbances (at 1 cm cell path-length) of the lycopene/ [beta]-carotene mixture in the hexane extract at 450 nm and 502 nm, respectively; [lycopene] and [[beta]-carotene] are molar concentrations of lycopene and [beta]-carotene, resspectively; [[epsilon].sup.450.sub.[beta]-carotene] and [[epsilon].sup.450.sub.[beta]-carotene] the molar absorptivities of lycopene and [beta]-carotene at 450 nm while [[epsilon].sup.502.sub.[beta]-carotene] and [[epsilon].sup.502.sub.[beta]-carotene] represent the respective molar absorptivities of lycopene and [beta]-carotene at 502 nm.

Solving equations (1) and (2) simultaneously, the molar concentration of lycopene from equation (1) could be expressed as:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (3)

Substituting equation (3) into equation (2), the concentration of [beta]-carotene can be calculated as:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (4)

But the values [[epsilon].sup.450.sub.[beta]-carotene], [[epsilon].sup.450.sub.[beta]-carotene], [[epsilon].sup.502.sub.[beta]-carotene] and [[epsilon].sup.502.sub.[beta]-carotene] are known to be 1.39 x [10.sup.5], 1.16 x [10.sup.5],2.63 x [10.sup.4] and 1.72 x [10.sup.5] L/mol/cm, respectively (Clinton, 1998; Du et al, 1998; Krinsky et al, 1990; Zechmeister and Polgar, 1943),therefore:

[[beta]-carotene] = 1.483 [A.sub.450] - [A.sub.502] / 1.798 x [10.sup.5] (5)

[Lycopene] = [A.sub.450] - 1.39 x [10.sup.5] [[beta]-carotene] / 1.16 x [10.sup.5] [approximately equal to] [A.sub.502] / 1.72 x [10.sup.5] (6)

Equations (5) and (6) were employed to calculate the concentrations of lycopene and [beta]-carotene, respectively and the results are as presented in Table 2. It was clearly shown that the use of the absorbances at 450 nm to exclusively determine [beta]-carotene concentration is extremely prone to error. This is so because the relative accuracy of the predicted [beta]-carotene concentration using the modeled equations compared with what could have been reported when its concentration was calculated from their absorbance values at 450 nm, is extremely low (an average of 26.78%). However, it could be inferred that absorbance at 502 nm could be exclusively attributed to lycopene, since an average relative accuracy of more than 95% was obtained. This is in agreement with other study (Fish et al., 2002) which suggests that constituent carotenoids other than lycopene will contribute to the absorbance at 503 nm up to about 4% for fresh red tomatoes, 2% for red-fleshed watermelon and 6% for pink grapefruit. This also corroborated the findings that the lycopene contents in different varieties of tomatoes, analyzed by UV-visible spectrophotometry at 502 nm and HPLC methods, are quite similar (Laleye et al., 2010).

To check for the correctness of the lycopene concentration at 472 nm, equation 1 may be re-written as:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (7)

Assuming [[epsilon].sup.472.sub.[beta]-carotene] to be unknown, it can be calculated thus:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (8)

Substituting the value of [A.sub.472] and [[epsilon].sup.472.sub.[beta]-carotene] L/mol/cm (Du et al., 1998) as well as the concentrations of lycopene and [beta]-carotene of the stock solution (solution of dilution factor of 1, that is 6.876 and 2.175 [micro]M [[epsilon].sup.472.sub.[beta]-carotene] value of 1.82x105 L/mol/cm was obtained as against a value of 1.86 x 105 L/mol/cm previously reported by Clinton (1998). This gives a relative accuracy (ratio of predicted value to the true value) of about 98%.

The absorption spectra of the hexane extracts samples of papaya and orange juice Fig. 3A-B are shown in Equations (5) and (6) were then tested on the absorbance results of the hexane extract of papaya and orange juice samples to calculate the respective amounts of [beta]-carotene and lycopene in the lycopene/[beta]-carotene mixture. The results are presented in Table 3. The results with other fruit or fruit product apart from tomatoes confirm that the [beta]-carotene concentrations are higher than that of lycopene. This is evident from the spectra shown in Fig. 3A-B. High relative accuracy values of the predicted [beta]-carotene concentration relative to that computed exclusively at 450 nm of 61.42% and 82.45% were observed for papaya and orange juice, respectively in contrast with an average of 26.78% observed for tomato fruits. Furthermore, the relative accuracies (82.95% and 62.39%) of the predicted lycopene relative to that computed exclusively at 502 nm observed for papaya and orange juice, respectively are lower than an average of 95.55% obtained for tomatoes. This may be attributed to lycopene/ [beta]-carotene concentration ratio in the sample under investigation or/and the presence of appreciable amounts of other hydrocarbon carotenoids in papaya and orange. It is hereby recommended that the predicted equations in this work be employed in the determination of concentrations of lycopene and [beta]-carotene in the hexane extract of lycopene/ [beta]-carotene mixture and the results can be compared with those from HPLC analysis, to check for precision and accuracy.

Conclusion

A simple and inexpensive spectrophotometric equation model for the simultaneous determination of lycopene and [beta]-carotene concentrations in a carotenoid mixture is proposed. This could be useful to analysis in some developing nations where UV-visible spectrophotometry is available but accessibility to HPLC is minimal. The equations proposed here worked best if the percentage composition of other hydrocarbon carotenoids (relative to lycopene and [beta]-carotene) in the hexane extract is minimal.

Acknowledgement

The authors acknowledge the support of German Academic Exchange Service (DAAD), Germany for the grant for the purchase of small equipments and materials offered to the corresponding author. The research aid to young researchers by the Faculty of Pure and Applied Sciences, LAUTECH, Ogbomoso is also acknowledged.

References

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Misbaudeen Abdul-Hammed *, Isah Adewale Bello and Sunday Olusegun Oladoye

Department of Pure and Applied Chemistry, Ladoke Akintola University of Technology, Ogbomoso, Nigeria

(received February 2, 2012; revised June 26, 2012; accepted June 27, 2012)

* Author for correspondence; E-mail: misbaulhameed@ yahoo.co.uk

Table 1. The ideal molar concetrations of lycopene and
[beta]-carotene in tomatoes, (Ibadan-local cultivars) in case
that absorbances at 450nm and 502 nm are exclusively due to
[beta]-carotene and lycopene, respectively

Diltution                              (a) [beta]-Carotene
factors      Absorbances (nm)          concentration [+ or -] SD
             450 nm    472     502     ([microM])

1            1.100     1.487   1.240   7.929 [+ or -] 0.202
2            0.550     0.720   0.615   3.965 [+ or -] 0.156
3            0.453     0.620   0.516   3.265 [+ or -] 0.162
6            0.225     0.305   0.250   1.622 [+ or -] 0.224
8            0.133     0.191   0.152   0.959 [+ or -] 0.102
16           0.068     0.109   0.080   0.490 [+ or -] 0.126

Diltution    (b) Lycopene
factors      concentration [+ or -] SD
             ([micro]M)

1            7.209 [+ or -] 0.175
2            3.576 [+ or -] 0.129
3            3.000 [+ or -] 0.173
6            1.453 [+ or -] 0.032
8            0.884 [+ or -] 0.205
16           0.465 [+ or -] 0.092

(a) = Calculated from Beer-Lamberts Law using the absorbances at
450 nm and molar absorptivity of 1.39x [10.sup.5] L mol/cm/ (Du
et al., 1998); (b) = Calculated from Beer-Lamberts Law using the
absorbances at 502 nm and molar absorptivity of 1.72 x [10.sup.5]
L mol/cm (Markovic et al., 2006); The results are the means of
triplicate with reported standard deviation (SD).

Table 2. Predicted concentrations of lycopene and [beta]-carotene
in the hexane extract from tomatoes (Ibadan-local cultivar)

                      Absorbances (nm)
Dilution              450      472       502
factors

1                     1.100    1.487     1.240
2                     0.550    0.720     0.615
3                     0.453    0.620     0.516
6                     0.225    0.305     0.250
8                     0.133    0.191     0.152
16                    0.068    0.109     0.080
Average percentages   --        --         --

                      (a) [beta]-Carotene      (b) Lycopene
Dilution              concentration            concentration
factors               [+ or -] SD ([micro]M)   [+ or -] SD ([micro]M)

1                     2.175 [+ or -] 0.127     6.876 [+ or -] 0.212
2                     1.116 [+ or -] 0.062     3.404 [+ or -] 0.156
3                     0.870 [+ or -] 0.152     2.866 [+ or -] 0.124
6                     0.466 [+ or -] 0.094     1.381 [+ or -] 0.136
8                     0.250 [+ or -] 0.022     0.847 [+ or -] 0.087
16                    0.116 [+ or -] 0.013     0.448 [+ or -] 0.056
Average percentages   --                       --

                        (c) Relative accuracy
Dilution              [beta]-carotene   Lycopene
factors               at 450 nm         at 502 nm

1                     27.431            95.377
2                     28.150            95.201
3                     26.644            95.533
6                     28.733            95.013
8                     26.057            95.845
16                    23.666            96.320
Average percentages   26.780            95.548

(a) = Calculated using equation (5); (b) = Calculated using
equation (6); The results are the means of triplicate analysis with
reported standard deviation (SD); (c) = Relative accuracy means the
ratio of the predicted concentrations of lycopene and [beta]-carotene
when the modeled equations were used relative to what could have
been reported when their concentrations were calculated from their
absorbance values at 502 nm and 450 nm, the wavelengths assumed to
be attributed exclusively to the respective carotenoids in their
mixtures.

Table 3. Predicted concentrations of lycopene and [beta]-carotene in
papaya and orange juice

                                  (b) [beta]-Carotene
(a) Sample     Absorbances (nm)   concentration
               450      502       ([micro]M)

Papaya         0.585    0.401     2.590 [+ or -] 0.132
Orange juice   0.123    0.051     0.731 [+ or -] 0.092

               (c) Lycopene           [beta]-Carotene
(a) Sample     concentration          concentration
               ([micro]M)             ([micro]g/g)

Papaya         1.934 [+ or -] 0.124   27.765 [+ or -] 1.415
Orange juice   0.185 [+ or -] 0.022   7.836 [+ or -] 0.986

               Lycopene
(a) Sample     concentration
               ([micro]g/g)

Papaya         20.732 [+ or -] 1.329
Orange juice   1.983 [+ or -] 0.236

(a) = Sample weight is 10g and the volume of hexane extract is 200 mL;
(b) = calculated using equation (5); (c) = calculated using
equation (6); The results are the means of triplicate analysis with
reported standard deviation (SD).
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Author:Abdul-Hammed, Misbaudeen; Bello, Isah Adewale; Oladoye, Sunday Olusegun
Publication:Pakistan Journal of Scientific and Industrial Research Series B: Biological Sciences
Article Type:Report
Geographic Code:6NIGR
Date:Jul 1, 2013
Words:4777
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