Plant-derived peptides rubiscolin-6, soymorphin-6 and their c -terminal amide derivatives: Pharmacokinetic properties and biological activity

The aim of this work is to investigate the pharmacokinetic properties, antinociceptive and antioxidant activities of rubiscolin-6, soymorphin-6 and their C -terminal amides; The four peptides were synthesized following Fmoc-SPPS strategy to give the ﬁ nal peptides in excellent overall yields and purity following analytical RP-HPLC analysis. None of them shows antioxidant activity and α -tyrosinase inhibition in vitro . All compounds are able to activate G-protein coupled receptor at the δ -opioid receptor (DOR) at 100 μ M concentration however, rubiscolin- 6-amide exhibits signi ﬁ cative antinociceptive e ﬀ ect after i.c.v. administration in the tail ﬂ ick test (TF) and s.c. administration in the formalin test (FT). Rubiscolin-6 shows the best in vitro intestinal bioavailability in CaCo2 cell monolayer and stability to the brush border exopeptidases in the apical compartment. In silico experiments show the interaction of rubiscolin-6 and rubiscolin-6 amide at the binding cavity of DOR compared with the crystallographic ligand TIPP-NH 2 .


Introduction
Exogenous peptides endowed with opioid activity deriving from natural proteins, acting on plants and animals are called exorphins (Zioudrou, Streaty, & Klee, 1979). Some peptides derived from plant proteins might show beneficial effect for plant suppressing escape reaction of insects (Yang et al., 2001;Zioudrou et al., 1979). In general, such bioactive peptides may exert several pharmacological activity as receptor's ligands, enzymes inhibitors, peptides modulating transport, anti-microbial peptides and antioxidant and radical scavenger. The affinity of those peptides for opioid receptors are much smaller than the endogenous ones, however some of them exhibit central and peripheral effects after oral administration (Kaneko et al., 2012;Yoshikawa, its C-terminal amide.
The aim of this project is to investigate the biological and pharmacokinetic properties of opioid plant-derived peptides soymorphin-6, rubiscolin-6 and their novel C-terminal amides derivatives.

Chemicals and reagents
Chemicals and reagents were purchased by VWR (MI, Italy) and Merck (MI, Italy), amino acids from GLS Shanghai (China). Final crude peptides were purified on C18 prep RP-HPLC at 216, 235, 254, and 275 nm (Waters C18 4.6 mm × 150 mm); flow rate of 7 mL/min; gradient eluent of H 2 O/ACN-0.1% TFA, from 5% ACN to 90% ACN in 32 min. Pure peptides were identified by 1 H NMR at 25°C on a 300 MHz Varian Oxford spectrometer, DMSO-d 6 as solvent and chemical shifts in parts per million (δ) downfield from the internal standard TMS. LCQ Finnigan-Mat mass spectrometer (San Jose, CA) equipped with ESI-spray source and ion trap analyzer was used at capillary temperature of 200°C, spray voltage at 4.00 kV and nitrogen and helium as sheath and auxiliary gas respectively.

Peptides and chemical characterization
Soymorphin-6, Rubiscolin-6 and their C-terminal amide analogues were prepared following the well-established Fmoc-solid phase peptide synthesis (Fmoc-SPPS) and were purified by reverse-phase high-performance liquid chromatography (RP-HPLC) (Poli et al., 2019;. The purity of four peptides was checked by analytical RP-HPLC at 236, 268 and 214 nm (C18-bonded 4.6 mm × 150 mm), flow rate of 1 mL/min, using a gradient of H 2 O/ ACN-0.1% TFA from 5% to 95% ACN in 26 min, and was found to be ≥95%. Peptide structures were identified by 1 H NMR spectrometry and LRMS (see SI).

In vitro antioxidant activity
For antioxidant capacity, different test systems, including radical quenching (DPPH and ABTS), reducing power (CUPRAC and FRAP), were used. The methods details were described in our earlier paper (Grochowski et al., 2017;Zengin et al., 2019). Trolox equivalents were selected as standards and compounds abilities were expressed as equivalents of trolox (mg TE/g). The significance of differences (p < 0.05) among the peptides was calculated using the parametric One-way ANOVA test together with Tukey's test (GraphPad Prism 6.0).
Kojic acid was used as a standard inhibitor and the results were expressed as kojic acid equivalents (mg KAE/g) the significance of differences (p < 0.05) among the peptides was calculated using the parametric One-way ANOVA test together with Tukey's test (GraphPad Prism 6.0). endomorphin-1 (EM-1) were dissolved in dimethyl sulfoxide (DMSO) at the concentration of 10 mM. The stock solutions were kept at −20°C until use.

Cells
CHO cells lines permanently co-expressing μ-receptors with the Cterminally modified Gα qi5 and δ-receptors with the C-terminally modified Gα qG66Di5 , were used. Details regarding the generation of these cells have been previously described (Camarda et al., 2009;Lambert & Rainbow, 2013).
Cells were cultured in culture medium consisting of Dulbecco's modified Eagle's medium (DMEM) / HAMS F12 (1:1) supplemented with 10% fetal bovine serum (FBS), penicillin (100 IU/mL), streptomycin (100 mg/mL), geneticin (G418; 200 µg/mL) and hygromycin B (100 µg/mL). Cell cultures were kept at 37°C in 5% CO 2 / humidified air. When confluence was reached (3-4 days), cells were sub-cultured as required using trypsin / EDTA and used for experimentation. Cells were seeded at a density of 50,000 cells / well into 96-well black, clearbottom plates. After 24 h incubation the cells were loaded with Hank's Balanced Salt Solution (HBSS) supplemented with 2.5 mM probenecid, 3 µM of the calcium sensitive fluorescent dye Fluo-4 AM, 0.01% pluronic acid and 20 mM HEPES (pH 7.4) for 30 min at 37°C. Afterwards the loading solution was aspirated and a washing step with 100 µL/well of HBSS, HEPES (20 mM, pH 7.4), 2.5 mM probenecid and 500 µM Brilliant Black was carried out. Subsequently 100 µL/well of the same buffer was added. After placing cell culture and compound plates into the FlexStation II (Molecular Devices, Sunnyvale, CA, USA), changes in fluorescence of the cell-loaded calcium sensitive dye Fluor-4 AM were measured. On-line additions were carried out in a volume of 50 μL/well.

Data analysis and terminology
All data were analyzed using Graph Pad Prism 6.0 (La Jolla, CA, USA). Data are expressed as mean ± sem of n experiments performed in duplicate. In calcium mobilization studies, agonist effects were expressed as maximum change in percent over the baseline fluorescence. Baseline fluorescence was measured in wells treated with vehicle. Agonist potency was expressed as pEC 50 , which is the negative logarithm to base 10 of the agonist molar concentration that produces 50% of the maximal possible effect of that agonist. Concentration response curve to agonists were fitted with the four-parameter logistic non-linear regression model: 2.6. In vivo antinociceptive assays 2.6.1. Animals CD-1 male mice (Charles River, Italy) weighing 25-30 g were used in all experiments. Before the experimental sessions, the mice were maintained in colony, housed in cages (7 mice per cage) under standard light/dark cycle (from 7:00 AM to 7:00 PM), temperature (21 ± 1°C) and relative humidity (60 ± 10%) for at least 1 week. Food and water were available ad libitum. The research protocol was approved by the Service for Biotechnology and Animal Welfare of the Istituto Superiore di Sanità and authorized by the Italian Ministry of Health, according to Legislative Decree 26/14, which implemented the European Directive 2010/63/UE on the protection of laboratory animals in Italy. Animal welfare was routinely checked by veterinarians from the Service for Biotechnology and Animal Welfare.

Tail flick test
The tail flick test was used to determinate antinociceptive response induced by a thermal stimulus and was performed as described earlier . Tail flick apparatus (Ugo Basile, Varese, Italy) consists of an infrared radiant light source (100 W, 15 V bulb) targeted on a photocell utilizing an aluminum parabolic mirror. During the trials, the mice were gently hand restrained with a glove. Radiant heat was targeted 5-6 cm from the tip of the tail, and the latency (s) of the tail withdrawal recorded. The measurement was disconnected if the latency crossed the cutoff time. A cutoff time of 15 s was imposed, and data were expressed as time course of the percentage of maximum effect (% MPE) = (post drug latency/baseline latency)/(cutoff time baseline latency) × 100. In all experiments, the baseline was calculated as the mean of three readings recorded before testing at intervals of 10 min, and the time course of latency was determined 10-120 min after compound treatment. Compounds were freshly diluted in saline containing 0.1% v/v DMSO, and were injected at 10 µg/10 µL for intracerebroventricular (i.c.v.) administrations, as previously reported (Mollica et al., 2013;Piekielna-Ciesielska et al., 2018).

Formalin test
The method utilized was comparable to the one previously described (Mollica et al., 2011).
Subcutaneous injection of 20 μL of a 1% solution of formalin in saline into the dorsal surface of the mouse hind paw evoked nociceptive behavioural responses, such as licking, biting the injected paw, or both, which are considered indices of nociception. The nociceptive response showed a biphasic trend: an early phase, occurring from 0 to 10 min after formalin injection, produced by the direct stimulation of peripheral nociceptors, and a late prolonged phase, occurring from 15 to 40 min, which reflected the response to inflammatory pain. During the test, the mouse was placed in a Plexiglas observation cage (30 × 14 × 12 cm), 1 h before the formalin administration and allowed to acclimatize to the testing environment. Compounds were solubilized in saline (0.9% NaCl in distilled water) and DMSO in the ratio DMSO:saline 1:3 (v/v) and were administered subcutaneously in the dorsal surface of the right hind paw of the mouse using a microsyringe with a 27-gauge needle 15 min before formalin injection at the dose of 100 µg/20 µL. Then, the total time the animal spent licking or biting its paw in the early and late phase was recorded.

Statistical analysis
Statistically significant differences between groups in the tail flick test and formalin test were measured with a two-way analysis of variance (2-ANOVA) followed by Tukey's or Sidak's post-hoc comparisons. GraphPad Prism 6.0 software (San Diego, CA, USA) was used to analyze the data. Data were considered statistically significant when a value of p < 0.05 was performed.
GI digestion was distinguished into salivary, gastric and duodenal A. Stefanucci, et al. Journal of Functional Foods 73 (2020) 104154 digestive steps. For the salivary digestion, an aliquot of each peptide (1.0 mg) was mixed with 6 mL of artificial saliva composed of: KCl (89.6 g/L), KSCN (20 g/L), NaH 2 PO 4 (88.8 g/L), Na 2 SO 4 (57.0 g/L), NaCl (175.3 g/L), NaHCO 3 (84.7 g/L), urea (25.0 g/L) and 290 mg of αamylase. The pH of the solution was adjusted to 6.8 with 0.1 M HCl. The mixture was introduced in a plastic bag containing 10 mL of water and homogenized in a Stomacher 80 Microbiomaster (Seward, Worthing, UK) for 3 min. Immediately, 0.5 g of pepsin (14,800 U) dissolved in HCl 0.1 N was added, the pH was adjusted to 2.0 with 6 M HCl, and then incubated at 37°C in a Polymax 1040 orbital shaker (250 rpm) (Heidolph, Schwabach, Germany) for 2 h. After the gastric digestion, the pancreatic digestion was simulated as follows: the pH was increased to 6.5 with 0.5 M NaHCO 3 and then 5 mL of a mixture pancreatin (8.0 mg/mL) and bile salts (50.0 mg/mL) (1:1; v/v), dissolved in 20 mL of water, were added and incubated at 37°C in an orbital shaker (250 rpm) for 2 h. Intestinal digested sample was freezedried and stored at −80°C until further analysis.

In vitro intestinal stability and transepithelial transport studies
The assay was performed as previously described by Tenore, Campiglia, Giannetti, and Novellino (2015).

Molecular modeling study
Molecular modeling study was performed on rubiscolin-6 and rubiscolin-6 C-amide using Maestro 2017, following the well-established procedure reported by Stefanucci et al. (2020).

Antioxidant and tyrosinase inhibition activities of synthetic peptides
The antioxidant abilities of the synthetic peptides were investigated by DPPH, ABTS, FRAP and CUPRAC methods. The results are in Table 1. The highest DPPH radical scavenging ability was found for rubiscolin-6 C-amide, followed by soymorphin, rubiscolin-6 and soymorphin-6 C-amide, while the best ABTS radical ability was recorded by soymophin-6 C-amide. In FRAP assay, rubiscolin-6 C-amide showed the highest potential with the value of 5.79 mg TE/g. However, there was no significant differences in FRAP values among rubiscolin-6, rubiscolin-6 C-amide and soymorphin-6 C-amide. As shown in Table 1, rubiscolin-6 exhibited the best tyrosinase inhibition ability with a value of 24.51 mg KAE/g, followed by soymorphin-6 C-amide and rubiscolin-6 C-amide. Soymorphin-6 is not active on tyrosinase.

Calcium mobilization assay
In these experiments we evaluated the capability of the compounds to activate the μand δ-human recombinant receptors permanently transfected in CHO cells. In such cells the expression of a chimeric G protein that forces the opioid receptors to couple with the calcium pathway allows measuring receptor activation with an automated calcium mobilization assay. Dermorphin and DPDPE were used as standard ligands for μand δ-receptors, respectively. In CHO μ cells the standard agonist dermorphin evoked a robust concentration-dependent stimulation of calcium release displaying high potency (pEC 50 of 8.06) and maximal effects (254 ± 18% over the basal values). EM-1 mimicked the stimulatory effect of dermorphin showing similar maximal effects but 3-fold lower potency (pEC 50 of 7.45). Rubiscolin-6 C-amide, Soymorphin-6 and Soymorphin-6 C-amide stimulated calcium mobilization only at micromolar concentrations eliciting incomplete concentration response curves, while Rubiscolin-6 was completely inactive. The concentration response curves obtained with these ligands in CHO μ cells are displayed in Fig. S1 (see SI) and their pharmacological parameters summarized in Table 2. In CHO δ cells the standard agonist DPDPE evoked a robust concentration-dependent stimulation of calcium release displaying high potency (pEC 50 of 7.51) and maximal effects (223 ± 17% over the basal values). All the other compounds showed an incomplete concentration response curve, being active only at micromolar concentrations. The concentration response curves obtained with these ligands in CHO δ cells are displayed in Fig. S2 (see SI) and their pharmacological parameters summarized in Table 2.

Antinociceptive effects of Rubiscolin-6 C-amide and Soymorphin-6 Camide
The antinociceptive effect of Rubiscolin-6 and Rubiscolin-6 C-amide are reported in Fig. 1. In the tail flick test, Rubiscolin-6 induced a significant antinociceptive effect 15 min after administration, followed by a rapid decrease, which becomes similar to that observed in vehicletreated animals from 60 to 120 min after administration. On the contrary Rubiscolin-6 C-amide induced a robust and significant antinociceptive effect, which is observed 15 min later and which light decreased up to 120 min after administration (Fig. 1). In the formalin test, Rubiscolin-6 was not able to change the nociceptive effect of formalin, both in the early and in the late phase of the test. As previously observed in the tail flick test, Rubiscolin-6 C-amide provoked an antinociceptive effect since it reduced formalin-induced nociception in both test phases (Fig. 1). In Fig. 2 are reported the effects of Soymorphin-6 and Soymorphin-6 C-amide in the tail flick and formalin tests. Soymorphin-6 and Soymorphin-6 C-amide induced similar effects in these tests. Both peptides exerted significant antinociceptive effect 15 and 30 min after the administration, followed by a gradual and similar decrease in the antinociception. In the formalin test, both peptides did not change the nociceptive effects of formalin in the early phase of the test. In the late phase of the formalin test, Soymorphin-6 induced a light increase and Soymorphin-6 C-amide a light decrease of the nociceptive effect provoked by formalin, but both effects were not statistically significant in comparison with vehicle-treated animals.

In vitro intestinal bioaccessibility
Results shown in Table 3 clearly indicate a quite high susceptibility of the peptides to the in vitro gastrointestinal environment. Specifically, their amide derivatives are the least resistant to the chemical and enzymatic conditions of the simulated digestive protocol. The intestinal stability and bioavailability of peptides were evaluated by using single layers of CaCo2 cells as a model of absorption in the small intestine. HPLC analysis highlighted very moderate hydrolysis of Rubiscolin-6 and Soymorphin-6 (less than 10% and 15%, respectively) in the apical solution by the brush border exopeptidases after 120 min incubation and regardless of the peptide concentration (Fig. S3, see SI). Differently, their respective amide derivatives revealed an important susceptibility to enzymatic degradation, showing average hydrolysis rates of 74% and 65% for Rubiscolin-6 C-amide and Soymorphin-6 C-amide, respectively. HPLC analyses of basolateral solution revealed that all peptides were absorbed intact through CaCo2 monolayer, with a concentration-dependent transport following a saturable pattern described by a linear curve (Fig. 3). Interestingly, the actual amount of Rubiscolin-6 and Soymorphin-6 transepithelially transported was about 10%, thus higher than what generally reported for different size (3-17 aminoacid units) and polarity peptides transported from CaCo2 monolayer apical to basolateral side (Okumu, Pauletti, Vander Velde, Siahaan, & Borchardt, 1997;Regazzo et al., 2010;Satake et al., 2002).

Docking study
In silico molecular modeling approach was applied to investigate the relationship between the bioactivity found in vitro and in vivo by rubiscolin-6 and its C-terminal amide and their interactions at the δopioid receptor (DOR). The docking of the two peptides was performed on the crystal structure of the DOR (4RWD) downloaded from the PDB database and the so obtained poses have been compared with crystallographic ligands. The receptor was prepared for the docking experiments as previously reported  with the PrepWizard module embedded in Maestro 2017 (Schrödinger Release, 2016). The missing side chains were added, and all the co-crystallized molecules were removed except for the opioid ligand TIPP-NH 2 and the water molecules 1303 and 101 which are fundamental for the correct activation of the receptor (Kaserer, Lantero, Schmidhammer, Spetea, & Schuster, 2016). These water molecules are necessary for a correct and predictable docking procedure (Kaserer et al., 2016;Stefanucci et al., , 2020. Thus in this model is present a water network connecting the ligand to a key residue of Histidine 278. After the preparation steps, the docking was performed by using the software Gold 6.0 with a docking procedure previously validated by our group . The fitness functions GOLDSC-ORE was employed for these experiments basing on our previous studies . The two water molecules were set to "toggle and spin", in order to allow the program to automatically decide whether the water molecules should be included during the docking and to optimize their orientation. An area of 10 Å around the co-crystallized ligand was defined as the binding site.  (Camarda et al., 2009). In the tail flick test, compounds were administered i.c.v. at the dose of 10 μg/10 μL; in the formalin test, compounds were administered s.c., in the dorsal surface of the mouse hind paw, at the dose of 100 μg/20 μL, 15 min before formalin. V is for vehicle-treated animals. ** is for P < 0.01, *** is for P < 0.001, **** is for P < 0.0001 vs V;°is for P < 0.05,°°is for P < 0.01,°°°is for P < 0.001 vs R. N = 7.
The best poses found are reported in Fig. 4; At the DOR, both rubiscolin-6 and rubiscolin-6 C-amide showed a similar interaction behavior, by assuming a convergent conformation and by establishing similar interactions to the key residues Asp128, His278 and water network.
Indeed, rubiscolin-6 is able to bind to the accessory residue Arg192, whereas this interaction is missing in its amide derivative, which preferentially bind the residue Leu200. Both of them lack to interact with Trp284, which is involved in a key interaction present in the crystallographic ligand; on the other hand they show the three main interactions to the water 101, Asp128 and His278 through the water network, which are responsible for the opioid activity found.
However, both peptides weakly bind to DOR in our calcium mobilization assay and are capable of stimulating the activation of G protein coupled to DOR at high doses (Table 2). This binding behavior falling in the micromolar range, could be explained by the lacking of those additional interactions found for TIPP-NH 2 (Trp284) and by the non-simultaneous presence of the interactions to Leu200 and Arg192, which could justify the low potency of rubiscolin-6 and rubiscolin-6 C-amide. The interactions found for both peptides, together with those of the crystallographic ligand have been reported in Table 4.

Discussion
Exorphins from plant proteins and their synthetic derivatives could exhibit a plethora of biological activity acting as modulating agents, antioxidants and anti-microbial compounds on diverse human targets such as receptors, cell's membrane and enzymes. Thus, such peptides contain certain desirable functional properties that could make them suitable in several formulated food systems.
In the present paper, we detected antioxidant properties by using different methods (DPPH, ABTS, CUPRAC and FRAP) (Kim, Lee, Lee, & Lee, 2002;Mareček et al., 2017). Generally, the tested peptides exhibit low antioxidant abilities. Rubiscolin-6 C-amide shows the best DPPH scavenging ability while soymorphin-6 C-amide exhibits the best ABTS scavenging ability. Reducing abilities of the tested peptides were evaluated by CUPRAC and FRAP assays, which indicate low electrondonating abilities for all of them. This could be due to the presence of the hydrophobic amino acids such as proline, glycine and methionine in the peptides that can contribute to the reduction abilities because of high electron density (Nwachukwu & Aluko, 2019). Literature reports that tyrosine and phenylalanine containing oligopeptides and long sequence peptides could present tyrosinase inhibitory activity (Pillaiyar, Manickam, & Namasivayam, 2017). Tyrosinase is one key enzyme in the synthesis of melanin, which is an important pigment for protection of the eye and skin from sunlight (Schurink, van Berkel, Wichers, & Boeriu, 2007). Thus novel and safe tyrosinase inhibitors are gaining interest among scientific research topics due to their importance in the prevention of hyperpigmentation problems such as melisma and age spots (Zolghadri et al., 2019). Furthermore, recent results indicate that opioids play a key role in skin homeostasis by modulating keratinocyte differentiation, wound healing and inflammation (Bigliardi-Qi et al., In the tail flick test, compounds were administered i.c.v. at the dose of 10 μg/10 μL; in the formalin test, compounds were administered s.c., in the dorsal surface of the mouse hind paw, at the dose of 100 μg/20 μL, 15 min before formalin. V is for vehicle-treated animals. ** is for P < 0.01, **** is for P < 0.0001 vs V. N = 7. Values are the means ± SD (n = 5; P < 0.01). 2006; Schmelz & Paus, 2007); β-endorphin and MOR are expressed in human epidermal melanocyte (EM) cells and they are closely associated to melanosomes. Kauser and co-workers (Kauser, Schallreuter, Thody, Gummer, & Tobin, 2003) demonstrated that they up-regulate melanocyte dendricity, proliferation, pigmentation and hair growth (Furkert et al., 1997;Tobin & Kauser, 2005) via melanocortin-1 receptor (MC-1R) independent mechanism, thus the discovery of a peptide with exogenous opioid-like structure able to inhibit tyrosinase enzyme could be useful to better understand the relationship between hyperpigmentation process and opioid peptide system. All our peptides exhibit moderate tyrosinase inhibitory effect with the only exception of soymorphin-6 which is inactive; among them rubiscolin-6 shows the best value. Calcium mobilization assay data reveal that all compounds are able to stimulate the DOR and MOR at 100 μM concentration, however rubiscolin-6 is inactive at MOR according to literature data (Hirata et al., 2007). Overall, detailed pharmacological features of rubiscolin-6, soymorphin-6, and their C-amide derivatives could not be investigated due to their low potency in the present calcium mobilization assay. In vivo experiments confirm the previously reported antinociceptive effects of rubiscolin-6 in the experimental model of acute pain in laboratory animals (Yang et al., 2001(Yang et al., , 2003.
Rubiscolin-6 was able to increase the nociceptive threshold to thermal stimuli after supraspinal administration, but was ineffective after subcutaneous administration in an experimental paradigm of chemical-induced nociception as the formalin test. Rubiscolin-6 Camide centrally administered demonstrates a strong antinociceptive effect higher than the parent compound, and it is effective after subcutaneous administration. To the best of our knowledge no data are available on the in vivo effects of Soymorphin-6, thus these results are novel and firstly described. After central administration in the tail flick test, both soymorphin-6 and its derivative soymorphin-6 C-amide induce a robust antinociceptive effect until 30 min after the administration but both peptides are not able to change the behavioral response to chemical-induced nociception in the formalin test. All peptides were absorbed intact through CaCo2 monolayer however, rubiscolin-6 Camide exhibits the most interesting in vivo biological profile, which prompt us to further investigate its effect after oral administration.
The carrier-mediated transport systems involving the H + -coupled PepT1 transporter, may be excluded since they are active and saturable symporters specific for intestinal absorption of charged di-and tripeptides (Brandsch, Knütter, & Bosse-Doenecke, 2008). The average polar properties of these peptides would also exclude a possible passive transcellular diffusion since a vescicular-mediated internalization, the main mechanism involved, would imply absorption by apical cell membrane through hydrophobic interactions (Knipp, Velde, Siahaan, & Borchardt, 1997). The low level of degradation of rubiscolin-6 and soymorphin-6 in transepithelial transfer indicates that passive transcellular diffusion could be partially involved in their transport. In fact, the amino acids ability to join hydrogen bonds with lipid phosphates of cell membranes, has been reported as a key physicochemical feature helping the translocation process via transcytosis route (Pauletti et al., 1996).
All together, these data support the possible transport of our peptides via paracellular route. Notheworthy, the passive paracellular transport via tight junctions has been recognized for the absorption of water-soluble low molecular weight and short-chain peptides positively charged due to the average negative charge of tight junctions (Salamat-Miller & Johnston, 2005). Finally, the docking study on rubiscolin-6 and its C-terminal amide into DOR gave an insight on their binding mode and justifies the low potency and efficacy in vitro, comparing to the DOR crystallographic ligand TIPP-NH 2 . These results are in line with the information previously obtained with CoMFA and CoMSIA models applied to rubiscolin-6 analogues (Caballero, Saavedra, Fernandez, & Gonzalez-Nilo, 2007), and represent the first temptative to expand our knowledge on the mechanism of action of these natural compounds mediated by DOR contribution.
In conclusion, we found that rubiscolin-6 C-terminal amide is able to exert antinociceptive effect after i.c.v. and s.c. administrations in the formalin test, at 100 μmol to stimulate opioid receptors in calcium mobilization assay. This let us suppose an activity at the central level and periphery (Cassell et al., 2019) by δ-opioid receptor modulation in light of previous studies, our docking prediction and a good pharmacological profile in terms of safety and peripheral use due to a low intestinal absorption. The potential applications of this novel peptide derived from plant involve its use as food supplement, various biotechnological products and processes.

Ethics statement
The research protocol for in vivo tests was approved by the Service for Biotechnology and Animal Welfare of the Istituto Superiore di