Carotenoid-Derived Flavor Precursors from Averrhoa carambola Fresh Fruit

The fruit of Averrhoa carambola L. (Oxalidaceae), commonly known as star fruit or carambola, is popular in tropical and subtropical regions. Carotenoid-derived components, mainly C13- and C15-norisoprenoids, contribute greatly to the flavor of star fruit. Previously reported norisoprenoids were tentatively identified by GC-MS analysis after enzymatic hydrolysis. To gain accurate information about glycosidically bound flavor precursors in star fruit, a phytochemical study was conducted, which led to the isolation of 16 carotenoid derivatives—One new C13-norisoprenoid glucoside, (5R,6S,7E,9R)-5,6,9-trihydroxy-7-megastigmene 9-O-β-d-glucoside (1); one new C15-norisoprenoid, (6S,7E,10S)-Δ9,15-10-hydroxyabscisic alcohol (11); and 14 known ones, of which 12 were in glucoside form. The structures of the two new compounds were elucidated on the basis of extensive spectroscopic data analysis and chemical reaction. Compound 11 was a rare C15-norisoprenoid with a double bond between C-9 and C-15, and its possible biogenetic pathway was proposed. The known compounds were identified by comparison of their mass and nuclear magnetic resonance (NMR) data with those reported in the literature. The structure identification of one new (1) and seven known (3–7, 9, and 10) C13-norisoprenoid glucosides from the genus Averrhoa for the first time enriches the knowledge of carotenoid-derived flavor precursors in star fruit.


Introduction
Averrhoa carambola L., belonging to the family Oxalidaceae, is widely cultivated in Southeast Asia, China, and India. Its fruit, commonly known as star fruit or carambola, is popular in tropical and subtropical regions and consumed mostly as fresh fruit [1]. The volatile components of star fruit have been extensively studied and approximately 200 aroma components have previously been reported [2][3][4]. Volatile carotenoid breakdown products play an important role in the flavor of star fruit. However, β-ionone and β-ionol were detected as the only two C 13 -norisoprenoids from star fruit until MacLeod and Ames [4] reported 14 C 13 -and C 15 -aroma compounds by using the simultaneous distillation/extraction (SDE) method. Star fruit extract was subjected to SDE treatment and almond glucosidase hydrolysis by Herderich et al. [5], which liberated 29 C 13 -aroma compounds, indicating that these carotenoid-derived flavor components are derived from non-volatile flavorless glycosidic precursors and that vigorous isolation techniques such as SDE have a high probability of liberating C 13 -aroma components. The C 13 -norisoprenoid aroma components of star fruit have been well studied for their attractive sensory qualities and low flavor thresholds. However, the structural information about their glycosidically bound precursors is deficient, and hitherto only three ionone peracetylated glycosides have been isolated from star fruit and structurally identified by mass and NMR measurements [6,7]. Therefore, there is a need to study the accurate structural information of the precursors of these carotenoid-derived components. In the present study, 10 C 13 -and 6 C 15 -norisoprenoid compounds including two new ones (1 and 11) were isolated from fresh star fruit (Figure 1), and their structures were elucidated by spectroscopic methods. These findings enrich the knowledge of carotenoid-derived flavor precursors in star fruit.
Molecules 2018, 23, x FOR PEER REVIEW 2 of 9 precursors and that vigorous isolation techniques such as SDE have a high probability of liberating C13-aroma components. The C13-norisoprenoid aroma components of star fruit have been well studied for their attractive sensory qualities and low flavor thresholds. However, the structural information about their glycosidically bound precursors is deficient, and hitherto only three ionone peracetylated glycosides have been isolated from star fruit and structurally identified by mass and NMR measurements [6,7]. Therefore, there is a need to study the accurate structural information of the precursors of these carotenoid-derived components. In the present study, 10 C13-and 6 C15norisoprenoid compounds including two new ones (1 and 11) were isolated from fresh star fruit (Figure 1), and their structures were elucidated by spectroscopic methods. These findings enrich the knowledge of carotenoid-derived flavor precursors in star fruit.
The new compound 11 was a rare C 15 carotenoid-derived norisoprenoid with a double bond between C-9 and C-15, and its possible biogenetic pathway was proposed, as shown in Figure 6. Compound 11 is likely generated from 9E-abscisic alcohol, the aglycone of compound 16, by oxidation to form an epoxide between C-9 and C-10, and then dehydration to form a double bond between C-9 and C-15 and a hydroxyl group at C-10 under acidic conditions.
In addition, the HMBC correlations from H-10 to C-8, δH 3.65 and 3.43 (1H each, dd, J = 11.4, 7.5 Hz, H2-11) to C-9 indicated a vicinal diol moiety connected to C-9. Further, the positive [α]D value and positive Cotton effect at 243 nm in the CD spectrum (Figure 4) determined the S absolute configuration of C-6 [13,14]. In order to determine the absolute configuration of C-10, the induced circular dichroism (ICD) spectrum by Mo2(OAc)4 was measured using Snatzke′s method [15], which exhibited a positive Cotton effect at 310 nm after the addition of dimolybdenum tetraacetate in DMSO solution ( Figure 4). According to the empirical rule, C-10 was determined to have an S absolute configuration. Therefore, compound 11 was identified as (6S,7E,10S)-Δ 9,15 -10-hydroxyabscisic alcohol.
The new compound 11 was a rare C15 carotenoid-derived norisoprenoid with a double bond between C-9 and C-15, and its possible biogenetic pathway was proposed, as shown in Figure 6. Compound 11 is likely generated from 9E-abscisic alcohol, the aglycone of compound 16, by oxidation to form an epoxide between C-9 and C-10, and then dehydration to form a double bond between C-9 and C-15 and a hydroxyl group at C-10 under acidic conditions. Glycosides perform accumulation, storage, and transport roles in aroma volatiles [27]. Herderich et al. [5] hydrolyzed star fruit extract with enzymes and identified 17 C13-norisoprenoids by GC-MS, including the aglycones of compounds 3-6, 9, and 10. The aglycone of the new compound 1 was elucidated as tobacco′s flavor by Wahlberg [8]. Compound 2 was previously reported as the flavor of quince [28] and purple passion fruit [29], and this is the first time that it was characterized as star fruit′s fragrance. In addition to compound 2, compounds 3-7, 9, and 10 were identified in the genus Averrhoa for the first time. The genin of compound 7 was not previously reported in nature. The peracetylated form of compound 8 was isolated from star fruit as an intact glycoconjugate flavor precursor [7].
Glycosides perform accumulation, storage, and transport roles in aroma volatiles [27]. Herderich et al. [5] hydrolyzed star fruit extract with enzymes and identified 17 C 13 -norisoprenoids by GC-MS, including the aglycones of compounds 3-6, 9, and 10. The aglycone of the new compound 1 was elucidated as tobacco s flavor by Wahlberg [8]. Compound 2 was previously reported as the flavor of quince [28] and purple passion fruit [29], and this is the first time that it was characterized as star fruit s fragrance. In addition to compound 2, compounds 3-7, 9, and 10 were identified in the genus Averrhoa for the first time. The genin of compound 7 was not previously reported in nature. The peracetylated form of compound 8 was isolated from star fruit as an intact glycoconjugate flavor precursor [7].

General Experimental Procedures
ESI-MS spectra were measured on an MDS SCIEX API 2000 LC/MS/MS apparatus (Applied Biosystems Inc., Forster, CA, USA). The HR-ESI-MS spectrum of compound 1 was obtained on a Waters Xevo G2-XS QTOF mass spectrometer (Waters MS Technologies, Elstree, Hertfordshire, UK); a full MS scan was performed in the range of m/z 100-1500 Da, the capillary voltage was set at 2.5 kV, and the cone voltage was 40 V. Nitrogen gas was used for nebulizer and desolvation. The HR-ESI-MS spectrum of compound 11 was measured on a Bruker maXis mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany); a full MS scan was performed in the range of m/z 100-2000 Da, the capillary voltage was set at 4.5 kV, and the end plate offset voltage was −500 V. One-dimensional (1D) and two-dimensional (2D) NMR spectra were recorded on a Bruker DRX-500 NMR spectrometer at 25 • C using solvent residual peaks as references. The 1 H NMR spectra were run at 500.13 MHz proton frequency and the spectral width was 7500 Hz. The 13 C NMR spectra were run at 125.77 MHz spectrometer frequency and the spectral width was 28,850 Hz. HSQC and HMBC experiments were measured using gradient selected sequences with 512 transients and 2048 data points for each of the 128 increments. The spectral widths were set at 5100 Hz for 1 H and 27,500 Hz for 13 C in the HSQC experiment, and 5100 Hz for 1 H and 27,500 Hz for 13 C in the HMBC experiment. For the NOESY experiment, 128 transients were collected into 1024 data points for each of the 160 increments with a spectral width of 3597 Hz for both dimensions. Optical rotation and ultraviolet (UV) spectra were acquired on a 343 polarimeter and a Lambda 650 UV/Vis spectrophotometer (Perkin-Elmer, Waltham, MA, USA), respectively. CD spectra were recorded on a Chirascan circular dichroism spectrometer (Applied Photophysics Ltd., Surrey, UK). Silica gel (100-200 mesh) was from Qingdao Haiyang Chemical Co. (Shandong, China), Amberlite XAD-7HP macroporous resin was from Sigma-Aldrich (St. Louis, MO, USA), and Sephadex LH-20 was from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Authentic D-(+)-glucose and L-(−)-glucose were from Aladdin Industrial Corp. (Shanghai, China). L-Cystein methyl ester hydrochloride was from Shanghai Macklin Biochemical Co. (Shanghai, China). O-Tolylisothiocyanate was from Tokyo Chemical Industry Co. (Tokyo, Japan). Thin layer chromatography (TLC) was conducted on pre-coated silica gel HSGF 254 plates (Jiangyou Silica Gel Development Co., Yantai, China), and visualized by spraying 10% sulfuric acid in ethanol (v/v) followed by heating. Medium pressure liquid chromatography (MPLC) was performed on an EZ Purifier (Lisure Science, Suzhou, China) and the column used was a 400 mm × 25 mm inner diameter (i.d.) Chromatorex RP-18 SMB100, particle size 20-45 µm (Shanghai Lisui E-Tech Co., Shanghai, China). HPLC was conducted on a LC3000 set connected to a UV3000 scanning spectrophotometer detector (Beijing ChuangXin TongHeng Sci. and Tech. Co., Beijing, China) and the columns used were Cosmosil 5C18-MS-II (250 mm × 4.6 mm i.d., particle size 5 µm, Nacalai Tesque, Inc., Kyoto, Japan) for analysis and YMC-Pack ODS-A (250 mm × 20 mm i.d., particle size 5 µm, YMC Co., Kyoto, Japan) for preparation.

Acid Hydrolysis
Acid hydrolysis was conducted following our previously reported procedures [30]. In brief, compound 1 (1 mg) was dissolved in 5 mL of 2 M aqueous hydrochloride and refluxed at 95 • C for 4 h. After removal of the solution under vacuum, 5 mL of water was added and partitioned with 5 mL of ethyl acetate three times. The aqueous layer was concentrated to dryness to yield a residue. The residue, authentic D-glucose, and L-glucose were individually dissolved in 1 mL of pyridine containing 1 mg/mL L-cystein methyl ester hydrochloride. After each solution was heated at 60 • C for 1 h, 2 mL of O-tolylisothiocyanate was added, heated at 60 • C for 1 h, and then concentrated to dryness. Each residue was dissolved in 1 mL of methanol and filtrated and analyzed by an Agilent Infinity 1260 HPLC at the wavelength of 254 nm and a 40 • C oven temperature. The column used was a Cosmosil 5C18-MS-II with acetonitrile/water/acetic acid (v/v, 22:78:0.1) as the mobile phase at a flow rate of 0.8 mL/min for 60 min, followed by washing with 90% acetonitrile/water (v/v).

Induced CD Spectrum by Mo 2 (OAc) 4
According to the published procedure [15], about 1:1 diol-to-molybdenum mixtures were prepared using 0.66 mg/mL of compound 11. The first CD spectrum was recorded soon after mixing and its evolution monitored for 30 min. The sign of the diagnostic band at 310 nm correlated with the absolute configuration of the diol moiety.

Conclusions
The fresh fruit of Averrhoa carambola (star fruit) possesses a fascinating and unique flavor, and carotenoid-derived C 13 -and C 15 -norisoprenoids contribute greatly to the flavor of star fruit.
However, the exact structural information about the glycosidically bound precursors in star fruit was deficient. Our study on fresh star fruit led to the isolation of two new (1 and 11) and 14 known carotenoid-derived C 13 -and C 15 -norisoprenoids, of which 12 were in glucoside form. In addition to the two new compounds, compound 2 and seven known C 13 -norisoprenoid glucosides (3-7, 9, and 10) were identified from the genus Averrhoa for the first time. The new compound 11 was a rare C 15 carotenoid-derived norisoprenoid with a terminal double bond between C-9 and C-15. In view of the previous reports of the aglycones of compounds 1, 3, 7, and 8 and compound 2 itself as volatile flavor components in star fruit [6] or other fruits [8,28,29], it could be concluded that some of the 12 C 13 -and C 15 -norisoprenoid glucosides were carotenoid-derived flavor precursors in star fruit.