A cyclic dipeptide from the Chilean hazelnut cotyledons (Gevuina avellana Mol., Proteaceae)

The Chilean hazelnut (Gevuina avellana Mol., Proteaceae) is a southern South American nut consumed as a snack and included in different preparations of traditional Chilean cuisine. Recently we described the fatty acid profile, oxylipins, phenolic compounds, as well as the antioxidant capacity. The main compounds of the phenolic-enriched extract were only tentatively identified by spectrometric means. In the present work, we describe the isolation and full characterization of a cyclic dipeptide cyclo(Arg-Trp) and other compounds from the phenolic enriched extracts of the G. avellana cotyledons. Compounds were isolated by means of counter-current chromatography and structures were established by spectroscopic and spectrometric methods. This is the first report on small peptides in G. avellana and adds evidence on the possible beneficial effects of this nut in human health.

The phenolic-enriched extract (PEE) was prepared as described in Pino-Ramos et al. 2 . In addition, a sample of air-dried raw cotyledons (914 g) was powdered and extracted two times with aqueous sulfuric acid (pH 2-3) (4 L) under sonication (20 min each time). The aqueous solution was filtered, defatted with PE (3×500 mL), basified with 25% NH 4 OH to pH 10 and extracted three times with EtOAc (3×1 L) to afford the basic extract. Some 6.5 g of solubles were obtained. Most of the fraction consisted of saccharose, filtered off after standing overnight in MeOH:water 1:1.
HpLc-DAD Analysis. The PEE, acid-base extraction and fractions from G. avellana were analyzed by HPLC coupled to a diode array detector (DAD) using a Shimadzu equipment (Shimadzu Corporation, Kyoto, Japan), as previously described 2 . An Inertsil ODS-3 RP-18 column was used for all analysis (GL Sciences Inc., Tokyo, Japan; 250 mm ×4.6 mm, 5 µm). Separation was carried out using MeOH (solvent A) and H 2 O:formic acid (solvent B, 99.9:0.1 v/v). The chromatographic conditions were: 5% A: 95% B from 0 to 2 min, 20% A: 80% B at 8 min; 52% A: 48% B at 40 min; 75% A: 25% B at 45 min; 100% A from 50 to 55 min and return to 5% A and 95% B at 60 min; flow rate: 0.8 mL/min, T° 30 °C. The chromatograms were monitored at 254, 280 and 330 nm and the UV/visible spectra were recorded from 200 to 600 nm.
Counter-current Chromatography. A J-type Quattro MK5 Lab Prep (AECS, Wales, UK) counter-current chromatography (CCC) equipment was used. The equipment characteristics were described in Mieres-Castro et al. 6 . The biphasic solvent systems evaluated consisted of TBME/1-BuOH/ACN/H 2 O in different proportions, acidified with 0.1% of trifluoroacetic acid. The partition coefficients (K D ) were determined as follows: 5 mg of the sample was dissolved in 4 mL of each one of the pre-equilibrated biphasic solvent system assayed (1:1 v/v). The mixture was dissolved, agitated in vortex and left to equilibrate for 2 min. When two clear phases were observed, 1 mL of each phase was taken, evaporated under reduced pressure, suspended in 0.5 mL of the mobile phase of the HPLC solvents and injected into the HPLC-DAD system described above. The K D was calculated as the ratio of peak areas of target compounds in the upper phase, divided into the peak areas found in the lower phase (head-to-tail mode) 7 , by dividing the area of the main compounds found in the upper phase by the area found in the inferior phase in the HPLC-DAD system described above. All separations were carried using the organic upper phase as the stationary phase. The CCC separation was repeated two times with 657 and 573 mg of the PEE, respectively. Temperature was set at 30 °C, flow rate of 4 mL/min and revolution speed of 650 rpm. Fractions were collected with a Gilson FC 203B (Middleton, WI, USA) set at 1.0 min/tube. To end the CCC, rotation was stopped and the column content was pushed out of the system using 600 mL of a mixture MeOH:H 2 O (6:4, v/v).
The components of each fraction were visualized by means of thin-layer chromatography (TLC) using Alugram ® plates (Macherey-Nagel GmbH & Co, Düren, Germany). The mobile phase consisted of EtOAc:formic acid:H 2 O (7:1.5:1, v/v/v), and the plate was revealed with p-anisaldehyde-sulfuric acid. Fractions with similar TLC patterns were combined and taken to dryness under reduced pressure. The fraction was freeze-dried, and the resulting solid was used for spectroscopic and spectrometric characterization. nMR, MS and iR Analysis. The NMR spectra were recorded on a Bruker Avance 400 spectrometer (Bruker, Rheinstetten, Germany) at 400 MHz for 1 H and 100 MHz for 13 C in CD 3 OD. Chemical shifts are given in ppm with residual methanol as the internal standard. The HR-ESI-MS-QTOF analyses were carried out using a Micromass Q-TOF instrument (Manchester, UK). The samples were infused directly using a syringe pump (Harvard Apparatus, Holliston, MA, USA) at a flow rate of 10 µL/min. The mass spectra were measured in the positive ion mode. The infrared (IR) spectra were measured using a Nexus Nicolet 470 Fourier-Transform IR (FT-IR) transmission spectrophotometer (Thermo Nicolet Corp, Madison, WI, USA).

Results and Discussion
Counter-current chromatography (CCC) was used to isolate compounds present in the PEE from G. avellana cotyledons. CCC is a method based on the partition of analytes between two immiscible liquid phases to obtain a suitable separation, avoiding a solid support and irreversible adsorption 7 (Fig. 2a), while compound e was found in tubes 186-200 (744-800 ml, 57 mg) (Fig. 2b).  (Table 2) showed characteristic signals of aromatic H from a heterocycle at δ 7.55 d (7.6), 7.32 d (7.6), 7.08 t (7.6, 7.2), 7.00 t (7.6, 7.2) and 7.09 (s) ppm, a pair of dd at δ 3.15 and δ 3.37 ppm and a H at δ 4.75 dd, suggesting a tryptophan derivative. Additional signals at δ 3.36 (m) and three CH 2 m at δ 3.00, 2.21 and 1.79 ppm, indicated a second amino acid unit. The 13 C-NMR spectrum showed 17 signals, including two carbonyls at δ 173.99 and 173.77 ppm, eight aromatic sp 2 carbons from the tryptophan and two C doublets at δ 48.17 and 53.26 ppm, suggesting a diketopiperazine formed from tryptophan and a second amino acid. The sp 2 C at 157.26 ppm supports the presence of the amino acid arginine. Correlation spectra allowed the identification of the compound as the dipeptide cyclo(Arg-Trp), in agreement with the proposed structure and with literature data. The NMR data and structure of the compound are summarized in Table 2. The cyclic dipeptide cyclo(Arg-Trp) was previously described by NMR and chiral spectroscopy studies on the absolute configuration and conformation of cyclic dipeptides 8 .
Cyclic dipeptides are 2,5-diketopiperazines that are found as natural products in a several sources, including bacteria, yeast, fungi, plants and mammals 9 . They can also be formed during chemical and thermal processing of food products 10 , including the roasting process 11,12 . Cyclic dipeptides contribute to the final taste of food, increasing astringency, saltines, bitterness and metallic flavors 9 . In addition, they have shown several bioactivities, including antibacterial, antifungal and antitumoral effects 13 . The recent review of Borthwick and Da Costa 9 list the occurrence of cyclic dipeptides in food and beverages. To the best of our knowledge, this is the first time that this compound is reported in Gevuina avellana cotyledons. The Cyclo(Arg-Trp) was isolated from roasted cotyledons of G. avellana; however, its presence was confirmed also in the acid-base extraction of the cotyledons.
In summary, tryptophan and a cyclic dipeptide were identified from G. avellana cotyledons. Arginine, aspartic acid, leucine and tryptophan were the main amino acids in the hydrolysate of Chilean hazelnut 5 . Tryptophan is an essential amino acid occurring in most of the peptides in G. avellana cotyledons and plays a relevant role on cognition and mood, with positive effects in memory and attention 14 . In addition, Trp is a key amino acid  www.nature.com/scientificreports www.nature.com/scientificreports/ in the biosynthesis of serotonin and melatonin, as well as kynurenin and their derivatives 15 . The relevance of tryptophan-rich foods on nutrition and psychiatric symptoms has been recognized 16 . The relation of Trp with depression and obesity 17 , food craving 18 , food intake and microbiota 19 has been also studied. The content of serotonin in several nuts, including Macadamia nuts, was recently determined but no information is available on the serotonin content in Chilean hazelnuts 20 . Additional studies are needed to fully disclose the possible beneficial effects of the G. avellana nuts in human health.