Carotenoids and Carotenoid Esters of Red and Yellow - American

Article pubs.acs.org/JAFC

Carotenoids and Carotenoid Esters of Red and Yellow Physalis (Physalis alkekengi L. and P. pubescens L.) Fruits and Calyces Xin Wen,†,‡,§,∥ Judith Hempel,† Ralf M. Schweiggert,† Yuanying Ni,*,‡,§,∥ and Reinhold Carle†,⊥ †

Institute of Food Science and Biotechnology, University of Hohenheim, 70599 Stuttgart, Germany College of Food Science and Nutritional Engineering, China Agricultural University, 100083 Beijing, China § National Engineering Research Center for Fruit and Vegetable Processing, 100083 Beijing, China ∥ Key Laboratory of Fruit and Vegetable Processing, Ministry of Agriculture, 100083 Beijing, China ⊥ Biological Science Department, King Abdulaziz University, P.O. Box 80257, 21589 Jeddah, Saudi Arabia ‡

ABSTRACT: Carotenoid profiles of fruits and calyces of red (Physalis alkekengi L.) and yellow (P. pubescens L.) Physalis were characterized by HPLC-DAD-APCI-MSn. Altogether 69 carotenoids were detected in red Physalis, thereof, 45 were identified. In yellow Physalis, 40 carotenoids were detected and 33 were identified. Zeaxanthin esters with various fatty acids were found to be the most abundant carotenoids in red Physalis, accounting for 51−63% of total carotenoids, followed by β-cryptoxanthin esters (16−24%). In yellow Physalis, mainly free carotenoids such as lutein and β-carotene were found. Total carotenoid contents ranged between 19.8 and 21.6 mg/100 g fresh red Physalis fruits and 1.28−1.38 mg/100 g fresh yellow Physalis fruits, demonstrating that Physalis fruits are rich sources of dietary carotenoids. Yellow Physalis calyces contained only 153−306 μg carotenoids/g dry weight, while those of red Physalis contained substantially higher amounts (14.6−17.6 mg/g dry weight), thus possibly exhibiting great potential as a natural source for commercial zeaxanthin extraction. KEYWORDS: Physalis alkekengi L., Physalis pubescens L., zeaxanthin, lutein, β-carotene, xanthophyll esters, provitamin A

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INTRODUCTION Physalis are herbaceous plants belonging to the Solanaceae, comprising approximately 120 species worldwide, which are widely distributed in tropical and temperate regions of America and temperate regions of Europe and Asia.1 The formation of the large papery husk inflated from the calyx, completely enveloping the fruit, is a notable feature of Physalis, with color ranging from greenish to yellow to tangerine and sometimes flushed red or purple.2 In China, the five species Physalis alkekengi L, P. angulate L., P. minima L., P. peruviana L., and P. pubescens L. are found.3 Among them, Physalis alkekengi L. and P. pubescens L. are two well-known and widely cultivated species, especially in northeastern China. P. alkekengi, also known as Chinese lantern, is a rhizomatous and perennial herb with red-orange fruits and fruiting calyces, being the only native Eurasian species of the genus Physalis. It has long been grown throughout China, Japan, and Europe.2 P. pubescens is a weedy annual herb native to the Americas with yellow fruits and calyces.2 Their calyces contain various bioactive secondary metabolites, mainly withanolides (especially physalins), flavonoids, alkaloids, phenylpropanoids, and terpenoids, which are commonly used in Traditional Chinese Medicine due to their anti-inflammatory, antipyretic, diuretic, antitussive, and antitumor effects.4 Their fruits are aromatic, juicy, sweet, and a little acidic, rich in minerals, vitamin C, fibers, polyphenols, essential amino acids, and carotenoids.5 In China, they are usually consumed as fresh fruits, juice, pulp, and jam. However, most recent research is focused on the physalins, flavonoids, and alkaloids in the calyces, leaves, stems, and cuticles.1,4,6 So far, studies about carotenoids in both fruits and calyces are scarce.5,7−10 © 2017 American Chemical Society

Consumption of carotenoids from food has numerous health benefits to humans. The most prominent bioactivity of socalled provitamin A carotenoids like β-carotene, α-carotene, γcarotene, and β-cryptoxanthin is their well-known metabolic conversion to vitamin A, since vitamin A deficiency is still a worldwide nutritional problem, especially in developing countries. Besides vitamin A supply, carotenoids have shown potentially beneficial effects for the human immune system and for putatively delaying or even preventing chronic diseases, such as cardiovascular disease and cancers.11 Furthermore, zeaxanthin and lutein, two xanthophylls referred to as human macular pigments, are found to play a protective role in the eye, reducing the risk of eye disease, in particular, age-related macular degeneration (AMD). Recent studies highlighted their role in human cognitive function as well, which supported the important contribution of zeaxanthin and lutein to both visual and cognitive health throughout human lifespan.12 Lutein is normally ingested with green leafy vegetables or commercially produced by marigold flowers, while naturally zeaxanthin-rich vegetables, fruits, and animal products are scarce. Recently, characterizations of carotenoids in goji berries13 and orange paprika14 have revealed both mentioned fruits to be rich in zeaxanthin. Nevertheless, the identification of more sources of dietary or commercially extractable zeaxanthin is still urgent. P. alkekengi fruits and calyces with bright red color were reported to be rich sources of xanthophylls,9 mainly zeaxanthin Received: Revised: Accepted: Published: 6140

May 31, 2017 July 5, 2017 July 11, 2017 July 11, 2017 DOI: 10.1021/acs.jafc.7b02514 J. Agric. Food Chem. 2017, 65, 6140−6151

Article

Journal of Agricultural and Food Chemistry

Figure 1. HPLC separation of carotenoids and chlorophylls from red Physalis fruits (a) and calyces (b) monitored at 450 nm. The chromatogram was taken from samples originating from the province Liaoning (LN). The enlarged chromatograms of fruits (a′) and calyces (b′) show details. For peak assignment see Table 1.

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esters (6.39 mg zeaxanthin/100 g fresh fruits, 54.89 mg zeaxanthin/100g fresh calyces),7 followed by β-cryptoxanthin esters (no data about fruits, 3.92 mg/g dry calyces).15 However, detailed studies of carotenoid profiles and quantitative data are limited. Although not investigating the complex carotenoid profile in detail, EI Sheikha et al.5 described P. pubescens fruits and juice to contain 69.55 μg/g fresh weight (FW) and 70 μg/ mL total carotenoids, respectively, which was comparable with the total carotenoids of papaya (30.79−76.90 μg/g FW).16 Therefore, our study aimed at obtaining information on the genuine profiles and quantitative data of carotenoids and carotenoid esters from differently colored Physalis fruits and calyces by HPLC-DAD-MSn, particularly targeting the identification of raw materials for the extraction of nutritionally relevant carotenoids like zeaxanthin. In addition, fundamental insights into the nutritional and medicinal potential of Physalis should be provided, to fill the knowledge gap between empirical use of the plant in Traditional Chinese Medicine and earlier literature reports.

MATERIALS AND METHODS

Sample Material and Reagents. Fresh red Physalis fruits (P. alkekengi var. f ranchetii (Mast.) Makino) and yellow Physalis fruits (P. pubescens) with calyces were collected from three different provinces (Liaoning, Jilin, and Heilongjiang) in China. All fruits were harvested at full maturity stage, i.e., the yellow ones in July 2016 and the red ones in September 2016. Calyces and fruits were separated manually and freeze-dried. Subsequently, samples were homogenized, packed into aluminum bags, and stored at −80 °C until carotenoid analyses. Authentic standards of (all-E)-violaxanthin, (all-E)-neoxanthin, (all-E)antheraxanthin, (all-E)-zeaxanthin, (all-E)-lutein, (all-E)-α-carotene, (all-E)-β-carotene, (all-E)-β-cryptoxanthin, (all-E)-zeinoxanthin, (allE)-mutatoxanthin, (all-E)-zeaxanthin dipalmitate were purchased from CaroteNature (Ostermundigen, Switzerland). All reagents were of analytical or HPLC grade. Ultrapure water was used throughout. Carotenoid Extraction. Prior to extraction, the seeds were removed from the fruit powder by sieving. An aliquot of 200 ± 10 mg of red Physalis fruit, 20 ± 2 mg of red Physalis calyces, 500 ± 10 mg of yellow Physalis fruit, or 200 ± 10 mg of yellow Physalis calyces was combined with 50 mg of CaCO3 and 3 mL of extraction solvent (methanol/ethyl acetate/petroleum ether, 1:1:1, v/v/v), containing 0.1 g/L BHA and BHT. The extraction was performed according to the ultrasonication-based extraction method of Hempel et al.13 All 6141

DOI: 10.1021/acs.jafc.7b02514 J. Agric. Food Chem. 2017, 65, 6140−6151

Article

Journal of Agricultural and Food Chemistry samples were extracted in triplicate. Prior to HPLC analyses, the dried extracts were dissolved in a mixture of tert-butyl methyl ether (tBME) and methanol (1:1, v/v) and filtered (0.45 μm PTFE membrane) into amber vials. HPLC-DAD-APCI-MSn Analyses. Carotenoid identification and quantification were carried out on a series 1100 HPLC (Agilent, Waldbronn, Germany) with a G1315B diode array detector (DAD) serially interfaced with a Bruker Esquire 3000+ ion trap mass spectrometer (Bruker, Bremen, Germany) fitted with an atmospheric pressure chemical ionization (APCI) source operated in both positive and negative mode. All MS parameters were set according to the method of Schweiggert et al.17 The HPLC separation was performed on a YMC C30 reversed phase column (250 mm × 4.6 mm i.d., 3 μm particle size; YMC Europe, Dinslaken, Germany) protected by a YMC C30 guard column of the same material and the following separation conditions. Regarding red Physalis samples, the mobile phase consisted of methanol/tBME/water (80:18:2, v/v/v) as eluent A and methanol/ tBME/water (8:90:2, v/v/v) as eluent B, both containing 0.4 g/L ammonium acetate. The elution gradient was as follows: from 0% to 25% B in 8 min, from 25% B to 35% B in 12 min, isocratic at 35% B for 5 min, from 35% B to 50% B in 10 min, from 50% B to 100% B in 10 min, isocratic at 100% B for 5 min, from 100% B to 0% B in 5 min, and isocratic at 0% B for 5 min. Total run time was 60 min at a flow rate of 1 mL/min. Column temperature was 40 °C. Regarding yellow Physalis samples, the mobile phase consisted of methanol/water (90:10, v/v) as eluent A and methanol/tBME/water (20:78:2, v/v/v) as eluent B, both containing 0.4 g/L ammonium acetate. The elution gradient was as follows: from 0% to 30% B in 5 min, from 30% B to 60% B in 55 min, from 60% B to 80% B in 25 min, from 80% B to 100% B in 5 min, isocratic at 100% B for 5 min, from 100% B to 0% B in 5 min, and isocratic at 0% B for 5 min. Total run time was 105 min at a flow rate of 0.85 mL/min. Column temperature was 30 °C. Carotenoids were monitored at 450 nm, and additional UV/vis spectra were recorded in the range of 200−700 nm. Compounds were identified by comparing retention times, UV/vis absorption, and mass spectra to those of authentic standards. When standards were unavailable, compounds were identified by comparing their UV/vis absorption and mass spectra with previously published data.13,17−27 (all-E)-violaxanthin, (all-E)-antheraxanthin, (all-E)-zeaxanthin, (all-E)lutein, (all-E)-β-carotene, (all-E)-β-cryptoxanthin, and (all-E)-zeinoxanthin standards were used to quantitate free carotenoids and corresponding esters by linear external calibration. Additionally, an approximated quantitation of neoxanthin, luteoxanthin, and their esters was carried out using the violaxanthin calibration. Mutatoxanthin ester levels were estimated by the antheraxanthin calibration. Levels of α-carotene, ζ-carotene, phytofluene, and phytoene were approximated using the β-carotene calibration. For red Physalis, levels of 5,8-epoxy-β-cryptoxanthin ester and all unidentified carotenoids were estimated by the zeaxanthin calibration. For yellow Physalis, levels of lutein 5,6-epoxide were approximated by the lutein calibration, while those of 5,6-epoxy-β-carotene and all unknown carotenoids were estimated by the β-carotene calibration. Molecular weight correction factors were used when necessary, representing the ratio of the molecular weight of the compound to be quantitated and that of the corresponding standards. Retinol activity equivalents (RAE) were calculated by considering 12 μg of (all-E)-β-carotene and 24 μg of other provitamin A carotenoids as 1 μg of retinol, i.e., 1 RAE.28 Statistical Analysis. All samples were analyzed in triplicate, and data were reported as the mean ± standard deviation (SD). ANOVA and Duncan’s multiple range tests were carried out using SAS University Edition (SAS Institute, Cary, NC) at a significance level of P < 0.05.

alkekengi L.) were found to be similar. Red Physalis fruits from different Chinese provinces consistently exhibited qualitatively identical carotenoid profiles, only differing in their content of individual carotenoids (Table 3). The same applied to the respective red-colored Physalis calyces (Table 3). A total of 69 different carotenoids and carotenoid esters were detected in red Physalis fruits and calyces by HPLC-DAD-MSn. Thereof, 36 were tentatively identified, and 9 were unequivocally identified by authentic standards. Among them, only 10 carotenoids were previously reported to be present in P. alkekengi fruits and calyces.7−9 In addition to carotenoids, minor amounts of chlorophyll b (peak 4), chlorophyll a (peak 8), and pheophytin a (peak 20) were also tentatively identified by comparing spectroscopic and mass spectrometric data with the literature.13,29 As illustrated in Figure 1a,b, compounds 27, 43, 55, and 59 represented the major carotenoids in both red Physalis fruits and calyces. Compound 59 was assigned to (all-E)-zeaxanthin dipalmitate by comparing its UV/vis spectra and MS data with those of an authentic standard. Similarly, compounds 58 and 60 showed mass spectra identical with that of compound 59, but their UV/vis absorption spectra were slightly hypsochromically shifted and exhibited an additional band at 338 nm as compared to that of compound 59. Thus, they were identified as Z-isomers of the (all-E)-zeaxanthin.19 Furthermore, comparing their DB/DII ratios of 0.45 and 0.09 with literature data,23 compound 58 and 60 were identified more precisely as (15Z)and (9Z)-zeaxanthin dipalmitate, respectively. Another major zeaxanthin ester, compound 27, was assigned to zeaxanthin palmitate due to its characteristic UV/vis spectrum, its molecular ion [M]− at m/z 806, its protonated molecular ion [M + H]+ at m/z 807 and its in source-fragment [M + H − 256 (palmitic acid)]+ at m/z 551. Occurring only in traces, compound 32 showed identical fragments as compound 27 but different UV/vis absorption maxima at 404, 426, and 452 nm. Thus, compound 32 was tentatively assigned to 5,8-epoxyβ-cryptoxanthin palmitate based on comparing our analytical data to previously reported elution orders, UV/vis spectra, and MS spectra.30,31 The identification of the major compounds 27 and 59 was in full agreement with previous reports on carotenoid composition in P. alkekengi fruits and calyces.7,9,15 Compound 55a was found to coelute with compound 55b (Figure 1b and b′). As shown in Figure 1a, the mentioned two compounds were earlier considered one single peak and, thus, have been identified as zeaxanthin myristate palmitate in previous reports.7,9 In our study, compound 55a was assigned to zeaxanthin myristate palmitate, due to its UV/vis spectrum, its molecular ion [M]− at m/z 1016, its protonated molecule [M + H]+ at m/z 1017, and three typical in source-fragments, i.e., [M + H − 228 (myristic acid)]+ at m/z 789, [M + H − 256 (palmitic acid)]+ at m/z 761 and [M + H − 228 − 256]+ at m/ z 533, being in agreement with previous reports.7,9 The coeluting compound 55b was tentatively assigned to lutein dipalmitate, being present at higher levels in calyces than in fruits (Figure 1a′,b′). Compound 55b revealed a molecular ion [M]− at m/z 1044, predominant in source-fragments [M + H − 256]+ at m/z 789 and [M + H − 256 − 256]+ at m/z 533, thus being assigned to lutein dipalmitate. In addition, a protonated molecule [M + H]+ was absent, confirming the presence of a lutein derivative.25 Besides the major zexanthin esters mentioned above, compounds 24, 49, 53, and 61 were identified as zeaxanthin myristate, zeaxanthin dimyristate, zeaxanthin palmitate palmitoleate, and zeaxanthin palmitate

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RESULTS AND DISCUSSION Identification of Carotenoids in Red Physalis Fruits and Calyces. As illustrated in Figure 1a,b, qualitative carotenoid profiles of fruits and calyces of red Physalis (P. 6142


Article

Journal of Agricultural and Food Chemistry Table 1. Carotenoids from Red Physalis Fruits and Calycesa retention time (min) peak no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16a 16b 17 18 19a 19b 20 21 22 23a 23b 24 25 26 27 28 29 30 31a 31b 32

fruits

6.8 7.0 7.2 7.7 8.6 9.3 10.1 10.2 10.5 11.1 11.5 11.8 12.5 12.5 13.1 13.6 14.3 14.3 14.6 14.9 16.5 16.5 16.9 17.2 18.2 19.2 19.8 21.0 21.3 21.6

calyces 5.0 5.5 6.2 6.8 7.0 7.2 7.7 8.6 9.3 10.1 10.2 10.5 11.1 11.5 11.8 12.5 12.5 13.1 13.6 14.3 14.3 14.6 14.9 15.6 16.5 16.5 16.9 17.2 18.2 19.2 19.8 21.0 21.3

21.9

21.6 21.9

33

22.4

22.4

34 35a 35b 36a 36b 37 38

23.2 23.6

23.2

24.6 24.6 25.0 25.6

23.6 24.6 24.6 25.0

25.9

39 40 41 42 43 44a 44b 45

26.2 26.5 27.0 28.0 28.6 28.6 29.0

26.5 27.0 28.0 28.6 28.6 29.0

46a

29.6

29.6

46b

29.6

identity b

(all-E)-violaxanthin luteoxanthin (all-E)-antheraxanthinb chlorophyll b (all-E)-luteinb (15Z)-zeaxanthinc (all-E)-zeaxanthinb chlorophyll a n.i. n.i. n.i. (all-E)-zeinoxanthinb n.i. (all-E)-β-cryptoxanthinb phytoene phytofluene isomer 1 violaxanthin palmitate n.i. phytofluene isomer 2 n.i. n.i. pheophytin a (13Z)-β-carotened (all-E)-α-caroteneb n.i. ζ-carotene zeaxanthin myristate (all-E)-β-caroteneb lutein 3-O-palmitate zeaxanthin palmitate n.i. n.i. n.i. n.i. n.i. 5,8-epoxy-β-cryptoxanthin palmitate violaxanthin myristate palmitate neoxanthin dipalmitate n.i. n.i. β-cryptoxanthin myristate zeinoxanthin palmitate violaxanthin dipalmitate β-cryptoxanthin palmitate isomere antheraxanthin dimyristate n.i. n.i. luteoxanthin dipalmitate β-cryptoxanthin palmitate n.i. n.i. antheraxanthin myristate palmitate lutein 3-O-myristate-3′-Opalmitoleate auroxanthin dipalmitate

UV/vis absorption maxima λmax (nm)

[M]− m/z

[M + H]+ m/z

418/440/470 400/422/448 424/446/472 466/600/650 422/446/472 338/422/442/470 426/450/476 432/618/664 420/446/472 336/420/444/470 336/420/444/470 426/446/472 418/440/470 426/450/476 276/286/296 330/348/368 418/440/470 400/422/448 330/348/368 380/402/426 420/446/472 410/610/666 338/418/444/468 426/446/474 422/446/472 378/400/426 426/450/476 428/452/478 422/446/472 426/450/476 426/450/476 414/436/466 418/440/470 416/440/468 404/426/452 404/426/452

600 600 584 906 568 568 568 892 n.r. n.r. n.r. 552 n.d. 552 544 542 838 n.r. 542 n.r. n.r. 870 536 536 n.r. 540 778 536 806 806 n.r. n.r. n.d. n.r. n.r. 806

601 601 585 907 569 569 569 893 n.r. n.r. n.r. 553 n.d. 553 545 543 839 n.r. 543 n.r. n.r. 871 537 537 n.r. 541 779 537 n.d. 807 n.r. n.r. 1021 n.r. n.r. 807

[601]: [601]: [585]: [907]: [569]: [569]: [569]: [893]:

418/440/470

n.d.

1049

[1049]: 1031, 821, 803, 793, 775, 565, 547

414/436/466 402/422/450 418/440/472 426/450/476 426/446/472 418/440/470 338/422/444/470

1076 n.r. n.r. 762 790 n.d. 790

1077 n.r. n.r. 763 791 1077 791

[1077]: 1059, 821, 803, 785, 711, 565, 547, 529

424/446/472 414/436/466 402/422/448 400/422/448 426/450/476 404/428/454 428/450/478 424/446/474

1004 n.r. n.r. n.d. 790 n.r. n.r. 1032

1005 n.r. n.r. 1077 791 n.r. n.r. 1033

422/446/472

n.d.

n.d.

1015, 805, 787, 777, 759, 567, 549f [585]: 567, 549, 531 959, 761, 787, 533f

380/402/426

1076

1077

1059, 967, 821, 803, 583, 565f

6143

HPLC/APCI(+)MSn m/z 583 583 567, 549, 493 629 551 551 551 615, 583, 555

[553]: 535 793, 583f [553]: 535

[839]: 821, 803, 583, 565

[537]: 347, 281, 255 [537]: 481, 399

[779]: [537]: [789]: [807]:

761, 445, 751, 789,

687, 431, 697, 715,

669, 281, 685, 551,

551, 533 255 533, 477 533

[1021]: 1003, 803, 785, 711, 565, 547, 529

789, 551f

[763]: 671, 535 [791]: 699, 535 [1077]: 1059, 985, 967, 821,803, 729, 565, 547 [791]: 699, 685, 535 [1005]: 987, 777, 759, 549, 531

[1077]: 1059, 821, 803, 565 [791]: 699, 685, 535, 443


Article

Journal of Agricultural and Food Chemistry Table 1. continued retention time (min) peak no.

fruits

calyces

47 48 49 50

31.0 32.2 33.2 33.7

31.0 32.2 33.2 33.7

51

34.3

34.3

52a 52b 53

34.5 34.5 34.9

34.5 34.5 34.9

54a

35.2

54b

35.2

35.2

55a

36.0

36.0

55b 56 57 58

36.2 36.8 37.2 38.0

36.2 36.8 37.2 38.0

59 60 61

38.4 39.6 40.6

38.4 39.6 40.6

identity n.i. antheraxanthin dipalmitate zeaxanthin dimyristate mutatoxanthin palmitoleate palmitate isomer mutatoxanthin palmitoleate palmitate isomer n.i. n.i. zeaxanthin palmitoleate palmitate mutatoxanthin dipalmitate lutein 3-O-palmitate-3′O-oleate zeaxanthin myristate palmitate lutein dipalmitate n.i. n.i. (15Z)-zeaxanthin dipalmitatec (all-E)-zeaxanthin dipalmitate (9Z)-zeaxanthin dipalmitatec zeaxanthin palmitate stearate


[M]− m/z

[M + H]+ m/z

330/414/436/464 424/446/474 428/450/478 404/428/454

n.r. 1060 988 1058

n.r. 1061 989 1059

404/428/454

1058

1059

428/450/478 404/428/454 428/450/478

n.r. n.r. 1042

n.r. n.r. 1043

404/428/454

1060

1061

422/446/472

n.d.

n.d.

426/450/478

1016

1017

422/446/472 428/450/478 428/450/478 338/422/444/470

1044 n.r. n.r. 1044

n.d. n.r. n.r. 1045

[789]: 697, 533, 477, 411

426/450/478 338/424/446/472 426/450/478

1044 1044 1072

1045 1045 1073

[1045]: 953, 789, 697, 533 [1045]: 953, 789, 533 [1073]: 981, 817, 789, 715, 533, 441

HPLC/APCI(+)MSn m/z [1061]: 1043, 969, 805, 787, 713, 549, 531 [989]: 897, 761, 669, 533 919, 805, 803, 787, 713, 567, 549f [585]: 567, 549, 531 967, 949, 805, 803, 787, 567, 549f [585]: 567, 549, 531

[1043]: 789, 787, 697, 533 1043, 805, 787, 549f [585]: 567, 549, 531 789, 815, 533f [789]: 771, 697, 551, 533, 477, 411 [1017]: 789, 761, 697, 669, 533

[1045]: 953, 789, 533

a

n.i., not identified; n.d., not detected; n.r., not reported due to ambiguous mass signal. bIdentified by authentic standards. cDB/DII ratio of (15Z)zeaxanthin and (9Z)-zeaxanthin were 0.45 and 0.09, respectively. dDB/DII ratio of (13Z)-β-carotene was 0.42. eDB/DII ratio of the Z-isomer of βcryptoxanthin was 0.45 fPreliminary MS-data obtained from MS1 due to low signal intensity.

cryptoxanthin and particularly zeinoxanthin was corroborated by their appearance in saponified extracts. For the first time, we detected several lutein esters in red Physalis fruits and calyces. As lutein is a structural isomer of zeaxanthin, its esters showed MS spectra similar to zeaxanthin esters with minor deviations. Lutein is an asymmetric dihydroxyl xanthophyll presenting one β- and one ε-ring on its ends. Consequently, it will undergo retro-Diels−Alder (RDA) fragmentation of the ε-ring to produce [M + H − 56]+ or eliminate the ε-ring to present [M + H − 122]+, which can be used to distinguish lutein from zeaxanthin, in addition to their slightly distinct UV/vis absorption spectra.19,24,32 By these means, compound 26 was assigned to lutein palmitate due to its UV/vis spectrum being identical with lutein standard, its molecular ion [M]− at m/z 806, in source-fragments [M + H − 18]+ at m/z 789, [M + H − 56]+ at m/z 751, [M + H − 18 − 92]+ at m/z 697, [M + H − 122]+ at m/z 685, [M + H − 18− 256 (palmitic acid)]+ at m/z 533, and [M + H − 18 − 56 − 256]+ at m/z 477. Additionally, the fact that the predominant in source-fragment ion is produced by neutral loss of the fatty acid or hydroxyl group at position 3′ of lutein (at its ε-ring) allowed the differentiation of lutein ester regioisomers.19,25,27,33 Therefore, compound 26 was further identified as lutein 3-Opalmitate due to high intensity of [M + H − 18]+ at m/z 789. Similarly, compounds 46a and 54b were also assigned to lutein esters (Table 1). Compounds 39, 45, and 48 were identified as antheraxanthin esters due to their UV/vis absorption spectra being identical with the antheraxanthin standard. Compound 48 was assigned to antheraxanthin dipalmitate based on its molecular ion [M]−

stearate, respectively. While zeaxanthin dimyristate (peak 49) was reported earlier,9 compounds 24, 53, and 61 were detected for the first time in red Physalis fruits and calyces. Compound 43, another major peak found in extracts from both red Physalis fruit and calyces, was identified as βcryptoxanthin palmitate due to its characteristic UV/vis spectrum, its molecular ion [M]− at m/z 790, its protonated molecule ion [M + H]+ at m/z 791, and typical in sourcefragments [M + H − 92 (toluene)]+ at m/z 699, [M + H − 106 (xylene)]+ at m/z 685, and [M + H − 256 (palmitic acid)]+ at m/z 535, being in agreement with previous reports.7,9,15 On the basis of a mass spectrum identical to that of compound 43 and a typical “cis-peak” at 338 nm in the slightly hypsochromically shifted UV/vis absorption spectrum, compound 38 was tentatively identified to be a Z-isomer of β-cryptoxanthin palmitate. Its DB/DII ratio was 0.45, not allowing a clear assignment of the positioning of the Z-configured double bond, as the DB/DII ratios of (15Z)- and (13Z)-β-cryptoxanthin were reported to be 0.47 and 0.43.23 Further β-cryptoxanthin ester was found. Our mass signals eluting at peak 36 revealed a protonated molecule [M + H]+ at m/z 763 and a typical in source-fragment [M + H − 228 (myristic acid)]+ at m/z 535. Noteworthy, at the same elution time, a less intense signal of a second protonated molecule [M + H]+ at m/z 791 was also observed. Simultaneously, the observed UV/vis absorption spectra at that elution time indicated the putative presence of both β-cryptoxanthin and zeinoxanthin. Thus, under consideration of all available analytical data, compounds 36a and 36b was tentatively identified as β-cryptoxanthin myristate and zeinoxanthin palmitate, respectively. The presence of both β6144


Article

Journal of Agricultural and Food Chemistry Table 2. Carotenoids from Yellow Physalis Fruits and Calycesa retention time (min) peak no.

fruits

calyces

identity

34 35 36 37

68.1 70.1 71.2 72.7

38 39 40 41 42

74.1 78.6 80.0 81.3 84.3

43

84.5

44

87.2

87.2

pheophorbide b pheophorbide a n.i. n.i. pheophorbide a isomer (all-E)-violaxanthinb (all-E)-neoxanthinb lutein-5,6-epoxide (15Z)-luteinc (13Z)-luteinc (all-E)-luteinb chlorophyll b (all-E)-zeaxanthinb phytoene isomer 1 n.i. n.i. chlorophyll a (all-E)-zeinoxanthinb (all-E)-β-cryptoxanthinb phytoene isomer 2 5,6-epoxy-β-carotene phytofluene isomer 1 pheophytin b neoxanthin sterate phytofluene isomer 2 (15Z)-β-carotened pheophytin a (13Z)-β-carotened ζ-carotene (all-E)-α-caroteneb n.i. lutein 3-O-palmitoleate (all-E)-β-caroteneb (9Z)-β-carotened violaxanthin butyrate myristate violaxanthin dimyristate neoxanthin dimyristate n.i. violaxanthin myristate palmitate β-cryptoxanthin myristate β-cryptoxanthin palmitate n.i. lutein dimyristate lutein 3-O-myristate-3′O-palmitate lutein 3-O-palmitate-3′O-myristate lutein dipalmitate

45

89.6

89.6

zeaxanthin dipalmitate

1 2 3 4 5 6 7 8 9 10 11 12 13 14a 14b 15 16 17 18 19 20 21 22 23 24 25 26 27a 27b 28 29 30 31 32 33

11.7 12.1 12.8 13.0 14.8 17.8 18.7 19.5 21.1

27.9 32.2 36.1 37.4 38.8 40.5 41.5 42.5 45.2 46.5 46.9 48.3 48.3 50.7 52.1 53.0 55.3 58.1 61.8

10.2 11.1 11.7 12.1 12.7 12.8 13.0 14.8 17.8 18.7 19.5 21.1 21.4 23.6 23.6 26.4 27.9 32.2 36.1 37.4 38.8 41.5

46.5 46.9 48.3 50.7 52.1 53.0 55.3 58.1

74.1 78.6 80.0 81.3 84.3


[M]− m/z

[M + H]+ m/z

438/600/654 410/610/666 418/442/470 400/422/450 410/610/666 418/440/470 414/436/466 416/440/468 330/418/438/465 330/418/438/466 422/446/472 464/600/648 428/452/478 272/282/294 420/440/468 420/442/470 432/618/664 426/446/474 428/452/478 276/286/296 422/446/472 332/348/368 436/600/656 414/436/466 332/348/368 338/420/446/468 408/610/666 338/422/446/468 380/402/426 426/446/474 426/446/474 422/446/472 428/452/478 338/422/446/472 418/440/470

n.r. n.r. 600 600 n.r. 600 600 584 568 568 568 906 568 544 568 568 892 552 552 544 552 542 884 866 542 536 870 536 540 536 706 n.d. 536 536 880

n.r. n.r. 601 601 n.r. 601 601 585 569 569 569 907 569 545 569 569 893 553 553 545 553 543 885 867 543 537 871 537 541 537 707 787 537 537 881

418/440/470 414/436/466 420/442/472 418/440/470

1020 1020 n.r. 1048

1021 1021 n.r. 1049

428/452/478 428/452/478 422/446/474 422/446/474 422/446/474

762 790 n.r. 988 1016

763 791 n.r. n.d. n.d.

422/446/474

1016

n.d.

422/446/474

1044

n.d.

428/452/478

1044

1045

HPLC/APCI(+)MSn m/z

[601]: 583, 509 [601]: 583 [601]: [601]: [585]: [551]: [551]: [551]: [907]: [569]:

583, 583, 567, 513, 513, 533, 629, 551

565, 565, 505 495 495 513, 597,

509, 491 547, 509, 491

[569]: [569]: [893]: [553]: [553]:

551 551 615, 583, 555 535 535

495, 477 569, 541

[553]: 535 [885]: 607, 547 [867]: 849, 831, 583, 565 [537]: 445, 255 [871]: 593, 533 [537]: 445, 255 [537]: [707]: [787]: [537]: [537]: [881]:

481 535 769, 445, 445, 863,

551, 431, 431, 793,

533 255, 243, 203, 189 347, 255 775, 671, 565, 547

[1021]: 1003, 911, 793, 775, 547 [1021]: 1003, 911, 793, 775, 547 [1049]: 1031, 821, 803, 793, 565, 547 [763]: 535 [791]: 699, 535 [761]: 743, 687, 551, 533, 477, 441, 411 761, 789, 533e [761]: 551, 533 789, 761, 533e [789]: 551, 533 953, 789, 551, 533e [789]: 551, 533 [1045]: 789, 551, 533

a

n.i., not identified; n.d., not detected; n.r., not reported due to ambiguous mass signal. bIdentified by authentic standards. cDB/DII ratio of (15Z)lutein and (13Z)-lutein were 0.51 and 0.43, respectively. dDB/DII ratio of (15Z)-β-carotene, (13Z)-β-carotene, and (9Z)-β-carotene were 0.40, 0.43 and 0.10, respectively. ePreliminary MS-data obtained from MS1 due to low signal intensity.

and [M + H − 256 − 256]+ at m/z 549, which has also previously been observed in P. alkekengi fruits and calyces by

at m/z 1060, protonated molecule [M + H]+ at m/z 1061, in source-fragments [M + H − 256 (palmitic acid)]+ at m/z 805 6145


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Figure 2. HPLC separation of carotenoids and chlorophylls from yellow Physalis fruits (a) and calyces (b) monitored at 450 nm. The chromatogram was taken from samples originating from the province Liaoning (LN). The enlarged chromatograms of fruits (a′) and calyces (b′) show details. For peak assignment see Table 2.

Weller et al.7 Similarly, compound 39 and 45 were identified as antheraxanthin dimyristate and antheraxanthin myristate palmitate, respectively. To date, we only found compound 39 in red Physalis calyces, being devoid in the fruits. Additionally, all three antheraxanthin diesters uniformly produced extra fragments [M + H − 18] +, corresponding to the neutral losses of water, [M + H − fatty acid −18]+ and [M + H − fatty acid − fatty acid − 18]+ (Table 1). Such eliminations of water were not detected with zeaxanthin diesters, being in accordance with the observation of Petry et al.25 The loss of water from the antheraxanthin ion was attributed to be related with the fragmentation of its 5,6-epoxy group. Mutatoxanthin esters were also detected in both fruits and calyces of the red Physalis species. Since mutatoxanthin is a structural isomer of antheraxanthin, its esters showed MS spectra similar to antheraxanthin esters. However, they exerted different UV/vis absorption maxima at 404, 428, 454 nm. On the basis of these observations, compound 54a was identified as mutatoxanthin dipalmitate, while compounds 50 and 51 were both tentatively assigned as mutatoxanthin palmitoleate palmitate isomers.

Compound 37 was assigned to violaxanthin dipalmitate due to its UV/vis absorption maxima at 418, 440, 470 nm identical with the violaxanthin standard, its protonated molecule [M + H]+ at m/z 1077, its in source-fragment [M + H − 256 (palmitic acid)]+ at m/z 821, [M + H − 256 − 256]+ at m/z 565, and additional fragments of higher intensity [M + H − 18]+ at m/z 1059, [M + H − 256 − 18]+ at m/z 803 and [M + H −2 × 256 − 18]+ at m/z 547, corresponding to losses of water from epoxy groups.21,24,34 On the basis of our findings, compounds 16b and 33 were identified as violaxanthin esters (Table 1). Similarly, compound 42 was tentatively identified as luteoxanthin dipalmitate with λmax at 400, 422, 448 nm, while compound 46b was tentatively assigned to auroxanthin dipalmitate with λmax at 380, 402, 426 nm. Compound 34 was identified as neoxanthin dipalmitate due to its neoxanthinlike UV/vis spectrum and characteristic mass spectra. Although carotenoid esters accounted for a large proportion of total carotenoids of red Physalis fruits and calyces, several free carotenoids were detected. (all-E)-violaxanthin (peak 1), (all-E)-antheraxanthin (peak 3), (all-E)-lutein (peak 5), (all-E)zeaxanthin (peak 7), (all-E)-zeinoxanthin (peak 12), (all-E)-β6146


Article


m/z 505.21 Compound 20 was tentatively assigned to 5,6epoxy-β-carotene due to its characteristic UV/vis absorption spectra, its molecular ion [M]− at m/z 552, its protonated molecule [M + H]+ at m/z 553, and its in source-fragments [M + H − 18]+ at m/z 535, being in agreement with the literature data.20,26,30 Apart from the above-mentioned free carotenoids, a total of 13 carotenoid esters was detected in yellow Physalis fruits and calyces, accounting for a small proportion of total carotenoids, and the proportion of esters was especially low in the calyces. Among these esterified carotenoids, lutein esters were distinctly prevailing. Particularly, compounds 30, 41, 42, 43, and 44 were identified as lutein 3-O-palmitoleate, lutein dimyristate, lutein 3-O-myristate-3′-O-palmitate, lutein 3-O-palmitate-3′-O-myristate, and lutein dipalmitate, respectively, according to the characteristic MS fragmentation pattern mentioned above when analyzing the lutein esters of red Physalis. Small amounts of βcryptoxanthin esters (compounds 38 and 39) and zeaxanthin dipalmitate (compound 45) were also observed in both fruits and calyces of yellow Physalis, while violaxanthin esters (compounds 33, 34, and 37) and neoxanthin esters (compounds 23 and 35) were only detected in fruits, according to the aforementioned principles regarding carotenoid ester identification in red Physalis (Table 2). Quantitation of Carotenoids in Physalis Fruits and Calyces. Apart from their completely different carotenoid profiles, total carotenoid contents of red Physalis fruits ranged from 19.8 to 21.6 mg/100 g FW, being more than 14 times higher than those of yellow Physalis fruits (1.28−1.38 mg/100 g FW). Additionally, carotenoid esters accounted for 94−96% of total carotenoids in red Physalis fruit, while their proportion was only 22−27% in yellow Physalis fruits. Zeaxanthin esters (56−63% of total carotenoids) were the most abundant esters in red Physalis fruits, followed by β-cryptoxanthin esters (18− 24%), antherxanthin esters (4−6%), violaxanthin esters (2− 4%), along with small amounts of lutein esters, luteoxanthin esters, mutatoxanthin esters, neoxanthin esters, auroxanthin esters, and zeinoxanthin esters. A comparable proportion of βcryptoxanthin esters (22.5%) and zeaxanthin esters (69−70%) was reported earlier by Deineka et al.9 and Weller and Breithaupt.7 When comparing with other zeaxanthin-rich fruits and vegetables,7 the total carotenoids (TC) (19.8−21.6 mg/100 g FW or 140.2−172.0 mg/100 g DW) and total zeaxanthin contents (TZ) (12.0−13.0 mg/100 g FW or 84.7−103.5 mg/ 100 g DW) of red Physalis fruits are comparable to those of orange paprika (TC, 109.69−190.43 mg/100 g DW; TZ, 85.06−151.39 mg/100 g DW),14 much higher than those of sea buckthorn (TC, 53.1−96.7 mg/100 g DW; TZ, 19.3−42.4 mg/ 100 g DW),36 but much lower than those of goji berries (TC, 44.4 mg/100 g FW; TZ, 38.8 mg/100 g FW).13 Regarding carotenoids in yellow P. pubescens fruits, free carotenoids prevailed, comprising 36−40% of (all-E)-βcarotene, 4−8% of (all-E)-lutein, 3−4% of (13Z)-β-carotene and ζ-carotene, and 2−3% of (9Z)-β-carotene, along with other minor amounts of some free carotenoids. Among the carotenoid esters (22−27% of total carotenoids) in yellow Physalis fruits, lutein esters accounted for 12−17% of total carotenoids, followed by violaxanthin esters (4−6%), βcryptoxanthin esters (3−4%), neoxanthin esters (2%), and small proportions of zeaxanthin esters. β-carotene was found to be the prevailing carotenoid of other yellow Physalis species as well, including goldenberry (Physalis peruviana L.),38 P. angulate

cryptoxanthin (peak 14), (all-E)-α-carotene (peak 22), and (all-E)-β-carotene (peak 25) were identified by comparison of their retention times and UV/vis and MS spectra with those of authentic standards. Compound 2 was identified as luteoxanthin comparing data of its protonated molecule [M + H]+ at m/z 601, UV/vis absorption maximum at 400, 422, 448 nm, and elution order on column YMC C30 (250 mm × 4.6 mm, 3 μm) with those found in the literature.26,30,35 Compound 6 was assigned to (15Z)-zeaxanthin, because it displayed a MS spectrum similar to (all-E)-zeaxanthin, exhibiting a DB/DII ratio of 0.45.23 Compound 21 was identified as (13Z)-β-carotene according to its molecular ion [M]− at m/z 536, its protonated molecule [M + H]+ at m/z 537, and a typical UV/vis spectrum with a characteristic DB/DII ratio of 0.42.17,23 Compounds 15, 16a, 18, and 23b were tentatively identified as phytoene, two phytofluene isomers, and ζ-carotene by comparing their elution order and UV/vis and MS spectra with those reported previously.17,23,30,35 Identification of Carotenoids in Yellow Physalis Fruits and Calyces. Yellow Physalis (P. pubescens L.) fruits and calyces displayed a completely different carotenoid composition as compared to its red-colored counterpart P. alkekengi, mainly containing free carotenoids instead of carotenoid esters (Table 2). Furthermore, yellow Physalis fruits showed significant differences in individual carotenoid contents with their calyces (Figure 2 and Table 4). However, yellow Physalis fruits and calyces from different regions exhibited generally similar carotenoid profiles. Therefore, chromatograms of yellow Physalis fruits (YF-LN) and calyces (YC-LN) from the Liaoning province are exemplarily illustrated (Figure 2a,b). A total of 40 carotenoids was detected in yellow Physalis fruits and calyces. Among them, 25 carotenoids were tentatively identified, and 8 carotenoids were identified with the aid of corresponding commercial reference compounds. In addition, chlorophyll a (peak 16), chlorophyll b (peak 12), and their corresponding derivatives (peak 1, 2, 5, 22, and 26) were also tentatively identified by comparing their UV/vis absorption maxima at 660 and 644 nm including the MS spectra of previous reports.13,29,36,37 (all-E)-violaxanthin (peak 6), (all-E)antheraxanthin (peak 7), (all-E)-lutein (peak 11), (all-E)zeaxanthin (peak 13), (all-E)-zeinoxanthin (peak 17), (all-E)-βcryptoxanthin (peak 18), (all-E)-α-carotene (peak 28), and (all-E)-β-carotene (peak 31) were identified by comparison of their retention times and UV/vis and MS spectra with those of authentic standards. Among them, (all-E)-zeaxanthin was only detected in yellow Physalis calyces, being absent in yellow Physalis fruits. Compounds 9 and 10 were assigned to (15Z)lutein and (13Z)-lutein, respectively, due to their characteristic UV/vis absorption spectra (cis-peak at 330 nm with DB/DII ratios 0.51 and 0.43, respectively.23 Similarly, compounds 25, 27a, and 32 were identified as (15Z)-β-carotene, (13Z)-βcarotene, and (9Z)-β-carotene, exhibiting DB/DII ratios of 0.40, 0.43, and 0.10, respectively.23 Compound 14a, 19, 21, 24, and 27b were tentatively identified as phytoene isomer 1, phytoene isomer 2, phytofluene isomer 1, phytofluene isomer 2, and ζcarotene, respectively, according to their elution order and UV/ vis and MS spectra.17,23,25,30 However, phytofluene and ζcarotene were only observed in yellow Physalis fruits, not in their calyces. Compound 8 was tentatively identified as lutein5,6-epoxide based on its UV/vis absorption maxima at 416, 440, 468 nm, its molecular ion [M]− at m/z 584, its protonated molecule [M + H]+ at m/z 585, and characteristic in sourcefragments [M + H − 18]+ at m/z 567 and [M + H − 80]+ at 6147


Article

Journal of Agricultural and Food Chemistry Table 3. Contents of Major Carotenoids from Red Physalis Fruits and Calycesa red Physalis fruits (μg/100 g FM) peak no. 1 2 3 5 6 7 12 14 15 16a 16b 18 21 22 23b 24 25 26 27 32 33 34 36 37 38 39 42 43 45 46 48 49 50 51 53 54 55a

compounds (all-E)-violaxanthin luteoxanthin (all-E)-antheraxanthin (all-E)-lutein (15Z)-zeaxanthin (all-E)-zeaxanthin (all-E)-zeinoxanthin (all-E)-β-cryptoxanthin phytoene phytofluene isomer 1 violaxanthin palmitate phytofluene isomer 2 (13Z)-β-carotene (all-E)-α-carotene ζ-carotene zeaxanthin myristate (all-E)-β-carotene lutein 3-O-palmitate zeaxanthin palmitate 5,8-epoxy-β-cryptoxanthin palmitate violaxanthin myristate palmitate neoxanthin dipalmitate β-cryptoxanthin myristate +zeinoxanthin palmitated violaxanthin dipalmitate β-cryptoxanthin palmitate isomer antherxanthin dimyristate luteoxanthin dipalmitate β-cryptoxanthin palmitate antheraxanthin myristate palmitate lutein 3-O-myristate-3′-Opalmitoleate + auroxanthin dipalmitateb antheraxanthin dipalmitate zeaxanthin dimyristate mutatoxanthin palmitoleate palmitate isomer mutatoxanthin palmitoleate palmitate isomer zeaxanthin palmitoleate palmitate mutatoxanthin dipalmitate +lutein 3-O-palmitate-3′-Ooleatec zeaxanthin myristate palmitate lutein dipalmitate (15Z)-zeaxanthin dipalmitate (all-E)-zeaxanthin dipalmitate (9Z)-zeaxanthin dipalmitate zeaxanthin palmitate stearate free carotenoids carotenoid esters unidentified carotenoids carotenoids

55b 58 59 60 61 total total total total RAEd

RF-LN

RF-JL

red Physalis calyces (μg/g DM)

RF-HLJ

RC-LN

RC-JL 6±1B tr. 16 ± 2 B 43 ± 5 B 6±1B 166 ± 14 5±1A 56 ± 6 B 75 ± 8 A 54 ± 6 A 26 ± 4 A 17 ± 1 A 15 ± 1 B 12 ± 1 A 29 ± 4 B 20 ± 2 B 106 ± 11 290 ± 38 868 ± 68 24 ± 3 A

RC-HLJ

16 ± 1 ab tr. 110 ± 5 a n.q. 52 ± 3 a 5±1b 6±0b 23 ± 4 a n.q. 21 ± 1 a

18 ± 2 a tr. 73 ± 4 b n.q. 41 ± 2 ab 8±1a 8±1a 18 ± 5 ab n.q. 16 ± 0 b

14 ± 1 b tr. 74 ± 2 b n.q. 29 ± 10 b 5±1b 5±0c 13 ± 4 b n.q. 12 ± 2 c

20 ± 2 a 56 ± 2 a 124 ± 4 a 149 ± 1 a 679 ± 12 a tr.

15 ± 2 b 41 ± 1 b 83 ± 6 b 158 ± 5 a 497 ± 17 c tr.

13 ± 1 b 30 ± 2 c 83 ± 1 b 123 ± 10 b 560 ± 18 b tr.

10 ± 2 A tr. 25 ± 2 A 66 ± 5 A 8±1A 231 ± 16 A 5±0A 66 ± 2 A 46 ± 2 B 36 ± 1 B 33 ± 3 A 12 ± 1 B 22 ± 1 A 14 ± 1 A 42 ± 3 A 26 ± 1 A 146 ± 4 A 319 ± 5 A 814 ± 15 A 28 ± 3 A

104 ± 11 a

131 ± 29 a

92 ± 32 a

43 ± 4 AB

51 ± 7 A

33 ± 4 B

68 ± 7 a 256 ± 27 b

66 ± 9 a 319 ± 9 a

75 ± 9 a 220 ± 1 c

50 ± 6 A 75 ± 6 A

46 ± 4 A 80 ± 10 A

49 ± 5 A 40 ± 5 B

508 ± 82 a 267 ± 30 a

462 ± 112 a 269 ± 4 a

454 ± 147 a 240 ± 20 a

339 ± 35 A

349 ± 51 A

361 ± 43 A

119 ± 20 a 4334 ± 60 a 91 ± 10 a

129 ± 38 a 3588 ± 88 b 115 ± 15 a

157 ± 29 a 3758 ± 135 b 84 ± 16 b

218 ± 13 A 64 ± 14 A 2835 ± 70 A 50 ± 7 B

241 ± 27 A 79 ± 10 A 3132 ± 250 A 70 ± 8 A

155 ± 21 B 71 ± 7 A 2725 ± 285 A 38 ± 3 B

132 ± 6 a

116 ± 1 b

109 ± 11 b

65 ± 14 A

70 ± 12 A

62 ± 9 A

1021 ± 219 a 378 ± 12 b 91 ± 2 b

1053 ± 176 a 539 ± 5 a 100 ± 2 a

993 ± 324 a 328 ± 11 c 68 ± 3 c

865 ± 147 A 102 ± 10 A 151 ± 20 A

970 ± 170 A 102 ± 6 A 141 ± 16 A

1011 ± 134 A 45 ± 5 B 75 ± 8 B

44 ± 8 a

55 ± 31 a

72 ± 38 a

tr.

tr.

tr.

622 ± 31 a

526 ± 16 b

538 ± 47 b

216 ± 20 A

222 ± 21 A

227 ± 19 A

115 ± 44 a

157 ± 44 a

129 ± 31 a

42 ± 11 A

59 ± 15 A

40 ± 5 A

1701 ± 198 b

2111 ± 42 a

1435 ± 175 b

536 ± 83 B

749 ± 72 A

276 ± 22 C

269 ± 15 a 204 ± 59 a 9150 ± 2274 a 85 ± 5 a 19 ± 3 a 354 ± 14 a 20480 ± 2706 a 754 ± 58 a 21592 ± 2695 a 147 ± 1 a

365 ± 58 a 169 ± 30 a 8854 ± 216 a 67 ± 1 b 19 ± 1 a 261 ± 8 b 19924 ± 593 a 788 ± 53 a 20974 ± 575 a 122 ± 3 b

343 ± 67 a 199 ± 56 a 8762 ± 2286 a 64 ± 7 b tr. 234 ± 10 c 18848 ± 3088 a 715 ± 56 a 19798 ± 3047 a 125 ± 3 b

788 ± 62 A 65 ± 8 A 6631 ± 1634 A 43 ± 6 B 27 ± 4 B 728 ± 32 A 14422 ± 1986 A 632 ± 49 AB 15782 ± 1979 A

771 ± 85 A 86 ± 14 A 7760 ± 1546 A 55 ± 3 A 38 ± 6 A 604 ± 59 B 16298 ± 2024 A 736 ± 52 A 17638 ± 2073 A

593 ± 65 B 69 ± 11 A 6559 ± 898 A 43 ± 5 B 14 ± 1 C 456 ± 64 C 13604 ± 1010 A 581 ± 62 B 14642 ± 1048 A

B

B A A

tr. tr. 14 ± 3 B 33 ± 5 C 6±1B 155 ± 26 B 2±0B 43 ± 6 C 38 ± 5 B 25 ± 2 C 25 ± 4 A 12 ± 2 B 11 ± 1 C 8±1B 27 ± 3 B 11 ± 2 C 82 ± 11 C 200 ± 24 B 868 ± 101 A 14 ± 2 B

a Different lowercase letters within a row indicate significant differences of carotenoid contents of red Physalis fruits (P < 0.05). Different uppercase letters within a row indicate significant differences of carotenoid contents of red Physalis calyces (P < 0.05). tr., trace amount (3 < S/N < 10); n.q.,

6148


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Journal of Agricultural and Food Chemistry Table 3. continued

not quantifiable (S/N < 3). bCarotenoid contents were quantitated by lutein 3-O-myristate-3′-O-palmitoleate. cIn the case of red Physalis fruits, carotenoid contents were quantitated by lutein 3-O-palmitate-3′-O-oleate, while red Physalis calyces did not contain mutatoxanthin dipalmitate. dβcryptoxanthin myristate was not included into summarizing RAE due to coelution with zeinoxanthin palmitate.

Table 4. Contents of Major Carotenoids from Yellow Physalis Fruits and Calycesa yellow Physalis fruits (μg/100 g FM) peak no.

identity

6 (all-E)-violaxanthin 7 (all-E)-neoxanthin 8 lutein-5,6-epoxide 9 (15Z)-lutein 10 (13Z)-lutein 11 (all-E)-lutein 13 (all-E)-zeaxanthin 14a phytoene isomer 1 17 (all-E)-zeinoxanthin 18 (all-E)-β-cryptoxanthin 19 phytoene isomer 2 20 5,6-epoxy-β-carotene 21 phytofluene isomer 1 23 neoxanthin sterate 24 phytofluene isomer 2 25 (15Z)-β-carotene 27 (13Z)-β-carotene+ζ-caroteneb,c 28 (all-E)-α-carotene 30 lutein 3-O-palmitoleate 31 (all-E)-β-carotene 32 (9Z)-β-carotene 33 violaxanthin butyrate myristate 34 violaxanthin dimyristate 35 neoxanthin dimyristate 37 violaxanthin myristate palmitate 38 β-cryptoxanthin myristate 39 β-cryptoxanthin palmitate 41 lutein dimyristate 42 lutein 3-O-myristate-3′-O-palmitate 43 lutein 3-O-palmitate-3′-O-myristate 44 lutein dipalmitate 45 zeaxanthin dipalmitate total unknown carotenoids total free carotenoids total carotenoid esters total carotenoids RAE

YF-LN

YF-JL

YF-HLJ

9±1b 4 ± 1 ab 12 ± 2 a 3±1a 6±2a 103 ± 6 a

12 ± 1 a 4±1a 7±2b 2±1a 4 ± 1 ab 104 ± 5 a

8±1c 3±0b 5±1b 2±0a 3±0b 54 ± 2 b

9±0b 13 ± 1 a 63 ± 3 a 11 ± 2 a 37 ± 1 a 14 ± 1 b 15 ± 1 a 6±2a 44 ± 3 a 10 ± 0 a 20 ± 2 a 526 ± 13 a 34 ± 1 a 30 ± 5 a 18 ± 2 a 14 ± 2 a 16 ± 1 a 24 ± 1 a 21 ± 2 a 79 ± 3 c 23 ± 1 a 29 ± 1 b 20 ± 1 a 12 ± 0 a 152 ± 2 a 905 ± 34 a 320 ± 10 b 1377 ± 24 a 48 ± 1 a

13 ± 1 a 15 ± 1 a 34 ± 1 c 9±2a 21 ± 1 b 16 ± 2 ab 7±1b 5±2a 42 ± 2 a 9±0a 25 ± 1 a 485 ± 20 b 35 ± 2 a 32 ± 2 a 17 ± 2 a 12 ± 2 a 12 ± 2 b 23 ± 1 a 22 ± 1 a 113 ± 4 a 26 ± 1 a 35 ± 1 a 18 ± 1 ab 11 ± 1 a 163 ± 7 a 808 ± 32 b 364 ± 14 a 1335 ± 54 a 45 ± 2 b

13 ± 1 a 13 ± 2 a 39 ± 1 b 9±1a 20 ± 1 b 17 ± 1 a 8±1b 7±1a 49 ± 4 a 8±2a 24 ± 4 a 488 ± 11 b 37 ± 2 a 36 ± 3 a 18 ± 2 a 13 ± 1 a 15 ± 1 ab 26 ± 2 a 20 ± 2 a 95 ± 4 b 25 ± 2 a 33 ± 1 a 18 ± 1 b 5±0b 174 ± 32 a 765 ± 24 b 344 ± 22 ab 1283 ± 78 a 45 ± 1 b

yellow Physalis calyces (μg/g DM) YC-LN

YC-JL

YC-HLJ

1±0B tr. 1±0B 5±1B 3±0B 95 ± 7 B 3±0B 2±0A 1±0B 2±0B 1±0B tr.

1±0A 1±0 1±0A 8±0A 4±0A 203 ± 10 A 5±0A 1±0C 1±0A 3±0A 1±0A tr.

tr. tr. 1±0B 4±0B 2±0C 103 ± 3 B 3±0B 2±0B 1±0C 2±0C 1±0C tr.

1±0B 2±0B 1±0B 2±0B 17 ± 2 B 3±0B

2±0A 3±0A 2±0A 3±0A 32 ± 2 A 5±0A

1±0B 1±0C 1±0B 1±0C 13 ± 1 C 2±0C

tr. tr. tr. tr.

tr. 1±0 tr. 1±0

tr. tr. tr. tr.

tr. tr. 16 ± 1 B 137 ± 8 B 4±1B 156 ± 8 B

1±0 tr. 27 ± 1 A 274 ± 14 A 6±0A 306 ± 14 A

tr. tr. 15 ± 1 B 136 ± 4 B 3±0C 153 ± 5 B

a

Different lowercase letters within a row indicate significant differences of carotenoid contents of yellow Physalis fruits (P < 0.05). Different uppercase letters within a row indicate significant differences of carotenoid contents of yellow Physalis calyces (P < 0.05). bYellow Physalis fruits contains both (13Z)-β-carotene and ζ-carotene, while yellow Physalis calyces contains only (13Z)-β-carotene and the carotenoid contents were all quantitated by β-carotene. cFor yellow Physalis fruits, (13Z)-β-carotene was not included into summarizing RAE due to coelution with ζ-carotene.

yellow Physalis fruits, coming close to those of mango (54 μg/ 100 g FW).41 Red Physalis calyces are popular as a decoration because of their lantern-shaped appearance and their stable red color, being imparted by 14.6−17.6 mg carotenoids/g DW. These findings were in accordance with previous reports (15.45 mg/g DW).15 Its total zeaxanthin contents (8.3−10.1 mg/g DW) were about 10 times higher than those of red Physalis fruits (0.85−1.04 mg/g DW). Its total carotenoid content (14.6−17.6 mg/g DW) was even more than 2 times higher than those of the well-known lutein-rich marigold petals (17−570 mg/100 g DW).42 Therefore, red Physalis calyces may represent a

L.,30 P. ixocarpa Brot., and P. philadelphica Lam.39 Among these studies, only De Rosso and Mercadante30 reported the detailed carotenoid composition of Physalis angulate. The completely different carotenoid profiles in red and yellow Physalis imply the predominant carotenoid biosynthetic pathways to be different, although belonging to the same genus. Both red and yellow Physalis fruits contained provitamin A carotenoids, namely, β-carotene, α-carotene, and β-cryptoxanthin and its esters. As shown in Table 3 and 4, the RAE of red Physalis fruits reached 121.6−147.4 μg/100 g FW, being comparable with that reported for papaya (132−166 μg/100 g FW).40 RAE levels of 44.8−48.2 μg/100 g FW were found for 6149


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(3) Institute of Botany of the Chinese Academy of Sciences. Solanaceae. In Chinese Flora (Zhongguo Zhiwu Zhi); Science Press: Beijing, China, 1978; pp 50−59. (4) Chen, L. X.; Xia, G. Y.; Liu, Q. Y.; Xie, Y. Y.; Qiu, F. Chemical constituents from the calyces of Physalis alkekengi var. franchetii. Biochem. Syst. Ecol. 2014, 54, 31−35. (5) El Sheikha, A.; Zaki, M.; Bakr, A.; El Habashy, M.; Montet, D. Physico-chemical properties and biochemical composition of Physalis (Physalis pubescens L.) fruits. Food 2008, 2 (2), 124−130. (6) Kranjc, E.; Albreht, A.; Vovk, I.; Makuc, D.; Plavec, J. Nontargeted chromatographic analyses of cuticular wax flavonoids from Physalis alkekengi L. J. Chromatogr. A 2016, 1437, 95−106. (7) Weller, P.; Breithaupt, D. E. Identification and quantification of zeaxanthin esters in plants using liquid chromatography-mass spectrometry. J. Agric. Food Chem. 2003, 51 (24), 7044−7049. (8) Pintea, A.; Varga, A.; Stepnowski, P.; Socaciu, C.; Culea, M.; Diehl, H. A. Chromatographic analysis of carotenol fatty acid esters in Physalis alkekengi and Hippophae rhamnoides. Phytochem. Anal. 2005, 16 (3), 188−195. (9) Deineka, V. I.; Sorokopudov, V. N.; Deineka, L. A.; Tret’yakov, M. Y.; Fesenko, V. V. Studies of Physalis alkekengi L. fruits as a source of xanthophylls. Pharm. Chem. J. 2008, 42 (2), 87−88. (10) Huang, Z.; Yang, M. J.; Ma, Q.; Liu, S. F. Supercritical CO2 extraction of Chinese lantern: Experimental and OEC modeling. Sep. Purif. Technol. 2016, 159, 23−34. (11) Schweiggert, R. M.; Carle, R. Carotenoid deposition in plant and animal foods and its impact on bioavailability. Crit. Rev. Food Sci. Nutr. 2017, 57 (9), 1807−1830. (12) Johnson, E. J. Role of lutein and zeaxanthin in visual and cognitive function throughout the lifespan. Nutr. Rev. 2014, 72 (9), 605−612. (13) Hempel, J.; Schädle, C. N.; Sprenger, J.; Heller, A.; Carle, R.; Schweiggert, R. M. Ultrastructural deposition forms and bioaccessibility of carotenoids and carotenoid esters from goji berries (Lycium barbarum L.). Food Chem. 2017, 218, 525−533. (14) Kim, J. S.; An, C. G.; Park, J. S.; Lim, Y. P.; Kim, S. Carotenoid profiling from 27 types of paprika (Capsicum annuum L.) with different colors, shapes, and cultivation methods. Food Chem. 2016, 201, 64−71. (15) Pintea, A.; Diehl, H. A.; Momeu, C.; Aberle, L.; Socaciu, C. Incorporation of carotenoid esters into liposomes. Biophys. Chem. 2005, 118 (1), 7−14. (16) Schweiggert, R. M.; Steingass, C. B.; Esquivel, P.; Carle, R. Chemical and morphological characterization of Costa Rican papaya (Carica papaya L.) hybrids and lines with particular focus on their genuine carotenoid profiles. J. Agric. Food Chem. 2012, 60 (10), 2577− 2585. (17) Schweiggert, R. M.; Vargas, E.; Conrad, J.; Hempel, J.; Gras, C. C.; Ziegler, J. U.; Mayer, A.; Jiménez, V.; Esquivel, P.; Carle, R. Carotenoids, carotenoid esters, and anthocyanins of yellow-, orange-, and red-peeled cashew apples (Anacardium occidentale L.). Food Chem. 2016, 200, 274−282. (18) Breithaupt, D. E.; Wirt, U.; Bamedi, A. Differentiation between lutein monoester regioisomers and detection of lutein diesters from marigold flowers (Tagetes erecta L.) and several fruits by liquid chromatography-mass spectrometry. J. Agric. Food Chem. 2002, 50 (1), 66−70. (19) Britton, G. UV/Visible Spectroscopy. In Carotenoids, Vol. 1B: Spectroscopy; Britton, G., Liaaen-Jensen, S., Pfander, H., Eds.; Birkhäuser Verlag: Basel, Switzerland, 1995; pp 13−62. (20) Chacón-Ordóñez, T.; Schweiggert, R. M.; Bosy-Westphal, A.; Jiménez, V. M.; Carle, R.; Esquivel, P. Carotenoids and carotenoid esters of orange- and yellow-fleshed mamey sapote (Pouteria sapota (Jacq.) H.E. Moore & Stearn) fruit and their post-prandial absorption in humans. Food Chem. 2017, 221, 673−682. (21) Delgado-Pelayo, R.; Gallardo-Guerrero, L.; Hornero-Ménndez, D. Carotenoid composition of strawberry tree (Arbutus unedo L.) fruits. Food Chem. 2016, 199, 165−175. (22) Hempel, J.; Amrehn, E.; Quesada, S.; Esquivel, P.; Jiménez, V. M.; Heller, A.; Carle, R.; Schweiggert, R. M. Lipid-dissolved γ-

potential source of natural carotenoids, especially zeaxanthin esters. Considering the effects of growing locations, red Physalis fruits and calyces from different provinces did not show significant difference (P > 0.05) regarding total carotenoid contents and the major carotenoid zeaxanthin dipalmitate, although some other minor individual carotenoids showed significant differences (P < 0.05) (Table 3). Carotenoid profiles of yellow Physalis calyces showed some differences to those of yellow Physalis fruits (Figure 2). For instance, (all-E)-lutein was the major carotenoid in yellow Physalis calyces, accounting for 60−67%. In contrast, (all-E)-βcarotene was the major carotenoid in the fruits, accounting for 36−40% of total carotenoid. Furthermore, it was found that the total carotenoid contents of yellow Physalis fruits were widely independent from the growing location (P > 0.05), although contents of most individual carotenoids showed significant differences (P < 0.05). Regarding yellow Physalis calyces, those from Jilin province (YC-JL) showed significantly (P < 0.05) higher total carotenoid and individual carotenoid contents than those from the other provinces (Table 4). In conclusion, large qualitative and quantitative differences in carotenoids profiles of red and yellow Physalis (P. alkekengi L. and P. pubescens L., respectively) were observed. Carotenoid esters appeared to be predominant in red Physalis, while free carotenoids prevailed in yellow Physalis. Red Physalis fruit showed to be a valuable dietary source of these nutritionally relevant carotenoids. Although yellow Physalis fruit showed a much lower carotenoid content than the red ones, they may still be considered as good sources of provitamin A carotenoids like β-carotene and the nutritionally relevant lutein. Thus, their bioavailability should be determined to confirm their efficient absorption in humans. Additionally, because of their strikingly high levels of zeaxanthin (8.3−10.1 mg/g DW), red Physalis calyces might serve for the extraction of zeaxanthin to be used in commercial food supplements.

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AUTHOR INFORMATION

Corresponding Author

*Phone/fax: +86 10 62737514. E-mail: [email protected]. ORCID

Yuanying Ni: 0000-0002-7998-3155 Funding

One of the authors (X.W.) gratefully acknowledges a scholarship from China Scholarship Council (CSC, Grant 201606350121). Notes

The authors declare the following competing financial interest(s): The authors declare no competing financial interest. During the execution of the study, one of the authors (R.M.S.) has left the University of Hohenheim to take up a position at a manufacturer of carotenoid supplements (DSM Nutritional Products, Kaiseraugst, Switzerland).

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