Quali-Quantitative Profile of Native Carotenoids in Kumquat from Brazil by HPLC-DAD-APCI/MS

In this study the native carotenoids composition in kumquat (Fortunella margarita) (peel + pulp) from Brazil was determined for the first time by a HPLC-DAD-APCI/MS (high performance liquid chromatography-diode array detector-atmospheric pressure chemical ionization/mass spectrometry), methodology. Eleven carotenoids were successfully identified and quantified in kumquat: four carotenoids in the free form and seven carotenoids in the esterified form. β-citraurin-laurate was the carotenoid found in the highest content (607.33 µg/100 g fresh matter), followed by β-cryptoxanthin-laurate (552.59 µg/100 g). The different esterified forms of β-citraurin and β-cryptoxanthin represented 84.34% of the carotenoids found, which demonstrates the importance of esterification in natural fruits. β-carotene and free xanthophylls (β-cryptoxanthin, lutein and zeaxanthin) represented 5.50% and 14.96%, respectively, of total carotenoids in kumquat. The total carotenoid content of kumquat from Brazil was very high (2185.16 µg/100 g), suggesting that this fruit could contribute significantly to the intake of important bioactive compounds by the population.


Introduction
The citrus family is one of the first crops in the world, it is estimated that half of the marketed production comes from the Americas and 12% comes from the Mediterranean basin. Citrus cultivation is thought to date back at least 4000 years and is mainly from the Asiatic south-east territories. The estimated global citrus traffic for 2017-2018 was around 6 million tons. The most representative cultures were Citrus sinensis (61%), Citrus reticulata (22%), Citrus limon (11%) and Citrus paradisi (6%). In the Americas, the primacy of citrus production lies with Brazil followed by the United States. Sweet oranges are grown in Brazil, mainly in the state of São Paulo, over an area of about 584000 hectares, but also in the Amazonas area of northern Brazil, over about 2.7 hectares. [1].
The Citrus japonica, known by the common names of kincan (from Japanese kinkan) or cunquate (from Chinese kumquat), is a small citrus fruit of the Rutaceae family [2]. It has four major cultivated types, including Fortunella japonica, Fortunella margarita, Fortunella crassifolia, and Fortunella hindsii [3].
In eastern countries, this fruit is a part of the regular food habits of the population [4], but in Brazil it is considered exotic, in addition to being little known and commercialized. Among the Brazilian states, São Paulo has the largest production and commercialization of this fruit [5].

Collection and Preparation of the Samples
The fruits of kumquat (Fortunella margarita) ( Figure 1) were collected in the morning, in May 2017, in the rural area of Viçosa (latitude 20 • 44' 05" S and longitude 42 • 51' 27" W), Minas Gerais, Brazil. Samples were collected in four repetitions of approximately 1 kg each. The fruit maturation was determined according to Donadio et al. [27] and defined by the red-orange peel color and the characteristic smell. In addition, ripe fruits were considered as those obtained after their natural fall of the trees or fall after being lightly touched by the hands.
The species was identified with the help of taxonomists from the Universidade Federal de Viçosa Herbarium through the Angiosperm Phylogeny Group IV [28], where it has already been cataloged and registered in the Virtual Herbarium network with the following records: EAC 48987, HUCO 5197, HPL 8977 and SP 42766. The samples were transported from the harvest site to the laboratory protected in styrofoam boxes with blocks of ice, within two hours after collection. In the laboratory, the samples were selected for appearance, excluding those with any epidermis injury or mechanical damage due to transport. The fruits were removed from the seeds (peel + pulp) were homogenized in a food processor (RI 7625, Philips, São Paulo Brazil), lyophilized (Liotop-LP510, Liobras, São Carlos, Brazil) and stored in plastic containers with screw caps, covered with aluminum foil stored at −18 ± 1 • C until further analyses.  The species was identified with the help of taxonomists from the Universidade Federal de Viçosa Herbarium through the Angiosperm Phylogeny Group IV [28], where it has already been cataloged and registered in the Virtual Herbarium network with the following records: EAC 48987, HUCO 5197, HPL 8977 and SP 42766.
The samples were transported from the harvest site to the laboratory protected in styrofoam boxes with blocks of ice, within two hours after collection. In the laboratory, the samples were selected for appearance, excluding those with any epidermis injury or mechanical damage due to transport. The fruits were removed from the seeds (peel + pulp) were homogenized in a food processor (RI 7625, Philips, São Paulo Brazil), lyophilized (Liotop-LP510, Liobras, São Carlos, Brazil) and stored in plastic containers with screw caps, covered with aluminum foil stored at −18 ± 1 °C until further analyses.

Extraction of Carotenoids
The carotenoid pigments were extracted from the lyophilized material (peel + pulp), according to the recommended procedures by Rodrigues-Amaya et al. [30]. Three grams of the edible portion of the samples (peel + pulp) were crushed by the use of a mortar and pestle, and a few drops of distilled water were added and extracted to color exhaustion with 20 mL of acetone (7 times) in an ultrasonic bath (Labsonic LBS 1-H22.5, Treviglio, Bergamo, Italy) for 10 min each time. Then, the extracts were individually centrifuged (Awel MF20-R, Multifunction Refrigerated Centrifuge, Blain, France) at 4000 rpm, 5 °C, for 10 min in order to withdraw clear solution on the top. The acetonic extracts were pooled together was concentrated to about 25 mL, in a rotary evaporator (Buchiheating bath B-491, Buchi, Milan, Italy) at temperature below 35 °C. The dry product was diluted with equal volumes (25 mL) of a mixture of ethyl ether and hexane (1:1) and distilled water (50 mL) and worked up with a separating funnel. The lipofilic phase, cleared by the hydrophilic impurities,

Extraction of Carotenoids
The carotenoid pigments were extracted from the lyophilized material (peel + pulp), according to the recommended procedures by Rodrigues-Amaya et al. [30]. Three grams of the edible portion of the samples (peel + pulp) were crushed by the use of a mortar and pestle, and a few drops of distilled water were added and extracted to color exhaustion with 20 mL of acetone (7 times) in an ultrasonic bath (Labsonic LBS 1-H22.5, Treviglio, Bergamo, Italy) for 10 min each time. Then, the extracts were individually centrifuged (Awel MF20-R, Multifunction Refrigerated Centrifuge, Blain, France) at 4000 rpm, 5 • C, for 10 min in order to withdraw clear solution on the top. The acetonic extracts were pooled together was concentrated to about 25 mL, in a rotary evaporator (Buchi-heating bath B-491, Buchi, Milan, Italy) at temperature below 35 • C. The dry product was diluted with equal volumes (25 mL) of a mixture of ethyl ether and hexane (1:1) and distilled water (50 mL) and worked up with a separating funnel. The lipofilic phase, cleared by the hydrophilic impurities, was evaporated to dryness using a rotary evaporator (Buchi-heating bath B-491 at 35 • C, and the residue was dissolved in 2 mL of MeOH/MTBE (1:1) and filtered in filter units (PTFE, 0.45µm, 13mm, Sigma Aldrich, Milan, Italy) prior to HPLC analysis. Samples were stored at −20 • C until they were analyzed.

Analysis of Carotenoids by HPLC-DAD-APCI-MS
The analysis was performed on an HPLC system (Shimadzu, Kyoto, Japan) equipped with a CBM-20A controller, two LC-20AD pumps, a DGU-20A3R deaerator, a SIL-20AC autosampler, a CTO

Identification and Quantification of Carotenoids
Carotenoids were identified by their UV-Vis spectra, MS spectra, elution order, comparison with the available standard and literature data.
The kumquat carotenoids quantification was performed from the analytical curves. External standards quantitative determination of each compound was performed using all reference materials listed in Section 2.1, in the concentration range from 5 to 50 µg/mL at six concentration levels. The results were obtained from the average of three determinations and the CV% was below 8% in all the LC measurements. The R coefficient for the calibration curves was always above 0.9962, with LOD and LOQ values of 0.07 and 0.22 ppm for β-carotene, 0.1 and 0.33 ppm for β-cryptoxanthin, 0.06 and 0.18 ppm for lutein, 0.08 and 0.3 ppm for zeaxanthin, and 0.12 and 0.24 ppm for physalein, respectively. Figure 2 shows the chromatographic profile of the carotenoid composition in not saponified kumquat fruits extracts. The identified compounds are shown in Table 1, together with the UV-Vis and MS spectra information. In Figure 3 are reported the UV-Vis (PDA) and mass spectrum of β-citraurin-laurate and β-citraurin-myristate, detected in the kumquat carotenoid extracts. It can be appreciated that the esterification does not affect the PDA spectra of β-citraurin.  Table 1.   Table 1.  The predominant carotenoids were β-citraurin-laurate and β-cryptoxanthin-laurate, both esterified with lauric acid. The chemical structures of these carotenoids are shown in Figure 4. Interestingly, no free β-citraurin was detected in the present study. Moreover, both the molecular ions [M] −• at respectively m/z 614 and m/z 642 relative to the β-citraurin-laurate and β-citraurin-myristate esters, obtained in the negative APCI mode, are also clearly shown in the same figure. Eleven different carotenoids were identified in kumquat from Brazil; four different β-citraurin esters and three β-cryptoxanthin esters were identified.

Carotenoids Qualitative Profile of Brazilian Kumquat
The predominant carotenoids were β-citraurin-laurate and β-cryptoxanthin-laurate, both esterified with lauric acid. The chemical structures of these carotenoids are shown in Figure 4. Interestingly, no free β-citraurin was detected in the present study. The predominant carotenoids were β-citraurin-laurate and β-cryptoxanthin-laurate, both esterified with lauric acid. The chemical structures of these carotenoids are shown in Figure 4. Interestingly, no free β-citraurin was detected in the present study. Shirra et al. [9] detected only 4 carotenoids in kumquat from Italy without saponification (βcarotene, β-cryptoxanthin, lutein and zeaxanthin), which were also detected in the present study. In contrast to our study, Agos et al. [18] reported only β-citraurin and β-cryptoxanthin in the free form in kumquat from Hungary. However, these researchers used saponification after the extraction process and did not quantify the carotenoid esters. Saponification may result in destruction or Shirra et al. [9] detected only 4 carotenoids in kumquat from Italy without saponification (β-carotene, β-cryptoxanthin, lutein and zeaxanthin), which were also detected in the present study. In contrast to our study, Agos et al. [18] reported only β-citraurin and β-cryptoxanthin in the free form in kumquat from Hungary. However, these researchers used saponification after the extraction process and did not quantify the carotenoid esters. Saponification may result in destruction or structural transformation of carotenoids [31]. Huyskens et al. [19] studied the qualitative composition of kumquat carotenoids from Israel by thin-layer chromatography and reported several carotenoids, including β-citraurin, β-cryptoxanthin, lutein, β-carotene and zeaxanthin which were, also determined in the present study. In addition, Huyskens et al. [19] reported that violaxanthin was the predominant component in kumquat from Israel, whereas in our study β-citraurin-laurate was the major component.
Esterification with saturated fatty acids improves the stability of xanthophylls such as β-citraurin and β-cryptoxanthin against heat and UV light, but does not affect their antioxidant activity [32]. During the storage and processing of the fruits the xanthophyll esters were more stable than the free xanthophyll [33]. The pigment β-citraurin is responsible for the citrus reddish color, it derives from of β-cryptoxanthin or zeaxanthin and accumulates in some citrus varieties [34,35]. In these fruits, the accumulation of β-citraurin is not a common event, it is observed only in the flavedos of some varieties during fruit ripening [34]. Frutita, a tropical fruit from Panama, showed a very high content of β-citraurin [36].
Recent studies have shown that β-cryptoxanthin and β-citraurin esterified with lauric acid, myristic acid and palmitic acid are found in the Mandarin Satsuma, [35].
The dietary intake of β-cryptoxanthin has been shown to prevent and reduce some pathologies such as: cancer, diabetes and rheumatism due to its antioxidant activity [37][38][39]. Breithaupt et al. [40] have verified that in chili, papaya, peach and persimmon, β-cryptoxanthin is mainly esterified with saturated fatty acids. Furthermore, the bioavailability of β-cryptoxanthin esters is comparable to the non-esterified form, since fatty acids can be effectively hydrolyzed by β-cryptoxanthin esters before intestinal absorption in the human body [40]. In several citrus varieties, the esterified form β-citraurin is found [35]. As already observed in another study [41], we have found β-cryptoxanthin and β-citraurin in both free and esterified forms.

Carotenoids Quantitative Profile of Brazilian Kumquat
In the present study, β-citraurin-laurate was the carotenoid found in the highest content in kumquat from Brazil (607.33 µg/100 g fresh matter, representing 27.80% of total carotenoids), followed by β-cryptoxanthin-laurate (552.59 µg/100 g fresh matter, representing 25.31% of total carotenoids). Thus, β-citraurin-laurate and β-cryptoxanthin-laurate represented 53.11% of the total carotenoids found. The different forms of β-citraurin (4 components esterified with fatty acids) and β-cryptoxanthin (1 component in free form and 3 in esterified form) represented 84.34% of the carotenoids found in kumquat from Brazil. Forms of β-citraurin and β-cryptoxanthin esterified with fatty acids accounted for 79.54% of the total carotenoids in kumquat. These results show the importance of the study of the intact carotenoids composition in different matrices (Table 2). β-carotene was found in smaller amounts (120.19 µg/100 g fresh matter), representing 5.50% of total carotenoids in kumquat, as well as free xanthophylls (β-cryptoxanthin, lutein and zeaxanthin), which represented 14.96% of total carotenoids (326.96 µg/100 g) ( Table 2). β-carotene and β-cryptoxanthin possess provitamin A activity, playing a key role in human health [42]. Differently from our results, Wang et al. [20] reported that β-cryptoxanthin was the major carotenoid present in kumquat cultivated in Taiwan, while Schirra et al. [9] found lutein to be the major carotenoid present in kumquat from Italy.