Comprehensive Characterization of Phytochemical Composition, Membrane Permeability, and Antiproliferative Activity of Juglans nigra Polyphenols

The aim of our study was the detailed polyphenol profiling of Juglans nigra and the characterization of the membrane permeability and antiproliferative properties of its main phenolics. A total of 161 compounds were tentatively identified in J. nigra bark, leaf, and pericarp extracts by ultrahigh-performance liquid chromatography–high-resolution tandem mass spectrometry (UHPLC-HR-MS/MS). Eight compounds including myricetin-3-O-rhamnoside (86), quercetin-3-O-rhamnoside (106), quercetin-3-O-xyloside (74), juglone (141), 1,2,3,4-tetrahydro-7,8-dihydroxy-4-oxonaphthalen-1-yl-6-O-galloyl-glucoside (92), ellagic acid (143), gallic acid (14), and ethyl gallate (58) were isolated from J. nigra pericarp. The in vitro antiproliferative activity of the isolated compounds was investigated against three human cancer cell lines, confirming that juglone (141) inhibits cell proliferation in all of them, and has similar activity as the clinical standards. The permeability of the isolated compounds across biological membranes was evaluated by the parallel artificial membrane permeability assay (PAMPA). Both juglone (141) and ethyl-gallate (58) showed positive results in the blood–brain-barrier-specific PAMPA-BBB study. Juglone (141) also possesses logPe values which indicates that it may be able to cross both the GI and BBB membranes via passive diffusion.


Introduction
Black walnut (Juglans nigra L., Juglandaceae) is a tall deciduous tree with a typically dark grey or brownish bark that is deeply split into narrow ridges.It can be distinguished from the extensively cultivated English walnut (Juglans regia L.) based on the size and morphology of their leaves.The larger leaves of black walnut are composed of a higher number of lanceolate or ovate-lanceolate and serrate leaflets as compared to the common walnut [1].The tree is native to the central and eastern regions of North America and was introduced to Europe in the 16th or 17th centuries, where it now grows wild especially in Eastern and Central Europe.Black walnut is cultivated as an ornamental tree and for timber production, and its seeds are also consumed [2,3].
Kernel extracts from different black walnut cultivars showed antibacterial activity [32].Green husk extracts showed significant antifungal activity in female rats [33], while extracts prepared by supercritical fluid extraction have exerted antioxidant activity [34].The bark extract showed neuroprotective activity in a rat model of cerebral ischemia by normalizing mitochondrial function [35].J. nigra leaf extract has also shown antinociceptive activity in rats [36].Ho et al. also studied the anticancer activities of compounds from J. nigra kernels.Pentagalloyl-glucose and quercetin-3-O-glucoside inhibited the growth of A549 human lung adenocarcinoma cells.However, the authors did not isolate the constituents from the extract for unambiguous identification [37].
Nevertheless, the comprehensive screening of the phenolic profile of J. nigra has not been performed.Therefore, the aim of this study was the phytochemical screening of J. nigra leaf, bark, and pericarp samples.To extract a wide range of polyphenolic compounds, we aimed to prepare extracts with solvents of different polarity.For the identification of polyphenols ultrahigh-performance liquid chromatography coupled to diode-array detection and electrospray ionization tandem mass spectrometry (UHPLC-DAD-ESI-MS/MS) was used.Our further objective was to reveal the constituents of black walnut pericarp that might contribute to its biological effects.Therefore, we aimed to isolate representative components and investigate their in vitro antiproliferative activities in various human cancer cell lines.Additionally, we aimed to evaluate the compounds' ability to permeate biological membranes using parallel artificial membrane permeability assays for the gastrointestinal tract and the blood-brain barrier (PAMPA-GI and PAMPA-BBB).

Qualitative Analysis of Juglans nigra Polyphenols by UHPLC-DAD-ESI-MS/MS
UHPLC-DAD-ESI-MS/MS was used to evaluate the phenolic profile of the black walnut leaf, bark, and pericarp extracts.We performed the tentative characterization of 161 compounds: we compared the retention times, UV spectra, and mass spectra of the detected constituents with the literature data; the results are presented in Table 1.A representative UHPLC-DAD chromatogram of J. nigra pericarp ethyl acetate extract is shown in Figure 1, while chromatograms of all extracts (chloroform, ethyl acetate, and methanol extracts of leaf, bark, and pericarp samples) are shown in Supplementary Figures S1-S3.a Abbreviations: JnLC: J. nigra leaf chloroform extract; JnLE: J. nigra leaf ethyl acetate extract; JnLM: J. nigra leaf methanol extract; JnBC: J. nigra bark chloroform extract; JnBE: J. nigra bark ethyl acetate extract; JnBM: J. nigra bark methanol extract; JnPC: J. nigra pericarp chloroform extract; JnPE: J. nigra pericarp ethyl acetate extract; JnPM: J. nigra pericarp methanol extract; +: present in the extract; HHDP: hexahydroxydiphenoyl; DHHDP: dehydrohexahydroxydiphenoyl; TFA: trifluoroacetic acid; HCOOH: formic acid; b Compared to a reference substance; c Reported for the first time in J. nigra.Compound numbers refer to Table 1.Compound numbers refer to Table 1.

Characterization and Analysis of Catechin Derivatives
Catechin and epicatechin derivates exhibited a typical fragment ion in their mass spectra at m/z 289 during their mass spectrometric fragmentation.Compound 93 also presented this fragment which arose from the loss of a 152 Da galloyl moiety (m/z 441 → 289); therefore, 93 was characterized as catechin-gallate or epicatechin-gallate [60].The [M−H] − ion of the catechin dimer 21 showed a mass-to-charge ratio of m/z 575, 2 Da less than the B-type procyanidin dimers, due to the additional C-O-C linkage that occurs in A-type procyanidins.Hence, compound 21 was identified as an A-type procyanidin dimer [45,60].

Characterization and Analysis of Gallic Acid Derivates
Gallic acid derivatives presented a diagnostic fragment ion at m/z 169 corresponding to the deprotonated molecular ion of gallic acid.An additional fragment ion at m/z 125 was also formed by the cleavage of the carboxyl group from gallic acid [41].In gallic acid derivates conjugated with sugars, the loss of 162 Da denotes a hexose moiety and the loss of 132 Da a pentose moiety.Therefore the [M−H] − ion at m/z 331 indicates a monogalloylhexose isomer (3, 4, 5, 7, 10, 11, 12, 15, 35), and the [M−H] − ion at m/z 301 corresponds to galloyl-pentose (34) [38].
Gallotannins consist of a polyol (usually a glucose) core which is esterified through its hydroxyl groups by galloyl units to form polymers.Gallotannins also present the aforementioned fragment ions of gallic acid at m/z 169 and 125 as well as typical neutral losses of 170, 152, and 134 Da equaling to gallic acid, a galloyl moiety, and a galloyl moiety losing a water molecule, respectively.Trigalloyl-hexose isomers ( Therefore, 69, 107, and 111 were characterized as galloyl-methylgallic acid isomers [42]. Compound 88 produced a [M−H] − ion at m/z 463 (ellagic acid+162 Da) and a prominent fragment ion at m/z 301 (ellagic acid residue); therefore, it was presumed to be ellagic acid-O-hexoside.Compound 95 was a galloyl-quinic acid derivate.The fragment ion at m/z 343 in its MS/MS spectrum implied the presence of a gallic acid moiety linked to a dehydrated quinic acid (169 + 174 Da) [38].

Structural Characterization of the Isolated Compounds
In order to unambiguously identify their structures, three flavonol-O-glycosides (74, 86, 106), one tetralone-glycoside (92), one naphthoquinone (141), two gallic acid derivatives (14, 58), and ellagic acid (143) were isolated by C 18 flash chromatography followed by multiple successive C 18 (semi-)preparative HPLC separations.The structures of the compounds were elucidated by HR-ESI-MS analyses, by comparing their retention times and mass spectrometric fragmentation with those of standard substances.Additional 1D and 2D NMR experiments were performed in the case of compound 92. Figure 2 presents the structures of the isolated constituents and Table 2 summarizes their high-resolution mass spectrometric data.Based on our LC-MS analyses, we proposed a trihydroxy-tetralone skeleton with an attached galloyl-hexose moiety for compound 92.The assumed structure was proven by NMR spectroscopy. 1 H and 13 C spectrum were in good agreement with the literature data [79].In the aromatic region of the 1 H NMR spectrum, three distinct signals were identified.The singlet at 6.99 ppm representing two hydrogens was attributed to the two aromatic protons of gallic acid, while the two coupled doublets at 7.15 ppm and 6.81 ppm representing 1-1 protons were identified as the aromatic protons of the 1,2,3,4-tetrahydro-7,8-dihydroxy-4-oxonaphthalen substructure.In the aliphatic part of the spectrum, the signals of a β-glucopyranose ring were identified, along with two pairs of methylene protons and the methine proton of the naphthalene derivative between 3.0 and 2.0 ppm and at 5.22 ppm, respectively.The linkage of gallic acid to the C-6 position of the glucopyranose ring was proved by three-bond correlation between the carbonyl carbon of gallic acid and the H-6 of glucose.Similarly, an intensive three-bond correlation was identified between H-1 of glucose and C-1 of the naphthalene moiety.The indicative signal of the ketone was also found in the 13 C spectrum at 206.1 ppm.Accordingly, 92 was identified as 1,2,3,4-tetrahydro-7,8-dihydroxy-4-oxonaphthalen-1-yl-6-O-[(3,4,5trihydroxyphenyl)carbonyl]-β-D-glucopyranoside.Atom numbering for the chemical shift assignment as well as 1 H and 13 C NMR spectra of compound 92 are presented in Supplementary Figures S4-S6.

Determination of the In Vitro Antiproliferative Activity
For the evaluation of the in vitro antiproliferative activity of the compounds, the cell viability was determined by resazurin (Alamar Blue) assay on MDA-MB 231 breast carcinoma, A2058 melanoma, and HT-29 colon carcinoma cell culture.The control wells were treated only with serum-free medium.The IC 50 is the micromolar (µM) compound concentration required for 50% inhibition of the cells' viability carefully calculated from dose-response curves (Table 3).
Compounds 141 and 14 induce cytostasis on MDA-MB 231 cells with IC 50 of 9.9 and 49.8 µM, respectively.The other compounds have no in vitro effect on these cells.On the melanoma culture, compound 141 and compound 14 showed a fair antiproliferative effect (IC 50 = 15.5 and 57.2 µM).Other compounds had no or limited inhibiting activity.Compounds 143, 14, and 141 revealed IC 50 values of 12.1, 0.53, and 71.2 µM, respectively, on HT-29 cell culture.
To determine the selectivity, we assessed the compounds on the Vero E6 non-tumorous kidney cells from African green monkeys (C.sabaeus), and their cytostatic activity was determined (Table 3).Compounds 106, 86, 141, 92, 14, and 58 showed cytostatic activity on the Vero E6 cells with IC 50 values of 15,9, 3.7, 3.7, 61.1, 49.9, and 82.1 µM, respectively.Compounds 143 and 74 were not cytostatic on the Vero E6 cells.We also determined the IC 50 value for daunomycin and tamoxifen, which are highly active, as suggested by the IC 50 values value, but very toxic and not selective compounds.As positive controls, we applied daunomycin and tamoxifen as clinically used reference compounds.Daunomycin (isolated from Streptomyces peucetius [80]) is among the first cytostatic compounds and the most employed, active on different tumors (e.g., leukemia, lymphoma, breast, and lung cancers) as an anthracycline aminoglycoside agent.A 50% inhibition in all cell lines is demonstrated, reported here at 0.2-1.1 µM concentration (IC 50 value).Daunomycin is a DNA intercalating molecule [81] and is not a selective compound with many side effects (partly due to its low IC 50 value).Tamoxifen treats estrogen-receptorpositive breast cancers as well as preventing the incidence of breast cancer in high-risk populations.Structural analysis showed tamoxifen is a non-steroidal antiestrogen used, and its metabolites bind DNA via hydrophobic and hydrophilic interactions [82].

Parallel Artificial Membrane Permeability Assay (PAMPA)
Among the compounds investigated, only juglone (141) had a logP e value greater than −5.0 (−4.41 ± 0.10) in the PAMPA-GI experiments; meanwhile, in the PAMPA-BBB studies, juglone (141) and ethyl gallate (58) possessed logP e values greater than −6.0 (−4.11 ± 0.19 and −5.77 ± 0.31, respectively) (Table 4).Accordingly, these constituents can be considered to have good membrane penetration ability [83]; thus, they can be expected to be absorbed in the gastrointestinal tract and cross the blood-brain barrier by transcellular passive diffusion.However, it should be noted that, for both juglone (141) and ethyl gallate (58), additional minor compounds (comprising <10% in the case of juglone (141) and <5% for ethyl gallate (58) based on UHPLC-DAD) appeared in the aqueous solutions prepared for the PAMPA experiments, indicating that some kind of chemical reactions had occurred.These are most likely to be the reduction of juglone (141) to hydrojuglone and the ester hydrolysis reaction of ethyl gallate (58).Although such small changes in compound concentration are not expected to significantly affect the results, given the accuracy of the PAMPA, they should be treated with caution.
Compounds 14, 74 86, 92, and 106 were not detected in the acceptor phase of the PAMPA models; meanwhile, the calculated logP e value for ellagic acid (143) was less than −6.0 in both assays.This indicates that these compounds are unable to traverse the lipid membranes of the gastrointestinal tract (GIT) and the blood-brain barrier (BBB) via passive diffusion.Among these, myricetin-3-O-rhamnoside ( 86) and quercetin-3-O-xyloside (74) showed significant degree of degradation in the pH 7.4 buffer after 4 h of incubation at 37 • C [84].Nonetheless, the negative results of the PAMPA-BBB experiments can be considered valid, as both compounds are unlikely to cross the model membranes via passive diffusion due to their high molar mass (>430 g/mol) and polarity (clogP < 1).

Discussion
We have performed a comprehensive phytochemical screening of J. nigra leaf, bark, and pericarp samples.In the present study, we tentatively characterized 161 phenolic compounds in J. nigra extracts by UHPLC-MS/MS.In line with the literature data, we detected flavonoids, phenolcarboxylic acids, ellagitannins, and naphthoquinones.Additionally, we described several gallotannins, flavonoids, juglone and tetralone derivatives, and linear and cyclic diarylheptanoids in J. nigra for the first time.We observed prominent differences between the composition of the different plant parts.Flavonol glycosides were dominant in the leaf and bark extracts, flavanone and chalcone glycosides occurred only in the bark, while gallotannins and ellagitannins prevailed in pericarp samples.Naphthoquinones and tetralones were present in all parts of the plant.The most prevalent constituents of the samples were hydrojuglone-O-hexoside (75), myricitrin (86), and quercitrin (106).
Although some of its constituents, characteristically the naphthoquinones and phenolic acid derivatives, have been known to exert cytotoxic actions [8,[85][86][87], biological activities and pharmacokinetic properties of black walnut polyphenols have not been characterized yet.Therefore, we have also performed an in vitro antiproliferative assay with a representative compound set in human cancer cell cultures.Juglone (141) demonstrated similar cytostatic activity on MDA-MB 231 and A2058 cells as the clinical standards.Gallic acid (14) was one order of magnitude less effective but had moderate selectivity.Further research is needed to investigate the mechanism of action regarding the promising new cytostatic candidates.It is important to note that their effect is in a similar concentration range as the natural compound vermelhotin (from fungal endophytes origin), which causes 50% inhibition at 12-20 µM concentrations depending on cell culture [88].
As shown by our previous results, only a handful of natural products may be able to cross biological membranes of the gastrointestinal tract or the blood-brain barrier via passive diffusion in the PAMPA experiments [84,89].In this study, we demonstrated that most of the major constituents of black walnut pericarp cannot be considered to have satisfactory membrane penetration ability.Based on the results of the PAMPA experiments, only juglone (141) and ethyl gallate (58) are able to cross the membranes of the GIT and the BBB by passive diffusion.Juglone (141) showed both good membrane permeability and cytostatic activity.Furthermore, based on our PAMPA-BBB results, it can be expected to reach the central nervous system (CNS).Therefore, as suggested by other studies as well [90,91], it may be a suitable candidate for future research into antitumor agents targeting the CNS.
Gallic acid (14) exhibited moderate but not selective cytostatic activity and poor membrane permeability in the PAMPA model.This latter result is in accordance with those of previous studies [92,93], which indicated that it is moderately absorbed in the gastrointestinal tract via paracellular transport.Based on our results and the net negative surface charge of endothelial cells in the blood-brain barrier, refusing to accept negatively charged compounds [94], transcellular passive diffusion of gallic acid ( 14) across the BBB is very unlikely.The results taken together suggest that gallic acid (14), in contrast to juglone (141), is not an ideal candidate for research into the therapy of central nervous system tumors.However, given its moderate absorption rate and cytostatic activity on the HT-29 colon carcinoma cell line, further studies in this direction would be worthwhile.
Ellagic acid (143) exerted moderate and selective cytostatic effect on the HT-29 colon cancer cell line.Considering this, the poor-moderate transcellular membrane penetration ability of compound 143 in the PAMPA-GI model, which is also in agreement with the literature data [85], may even be beneficial.The remaining isolated compounds (86, 92, 106, 74) had no effect on the tumor cell lines, nor did they show good permeability in the PAMPA experiments.
In vitro antioxidant, anti-inflammatory, and anticancer activities of black walnut and its constituents have been shown.However, in a human study, consumption of black walnuts did not cause any significant difference in LDL antioxidant capacity [24].Several factors might play a role in this negative result, with the possible inability of the antioxidative constituents to be absorbed in the gastrointestinal tract being one.On the other hand, black walnut bark extract and juglone showed in vivo neuroprotective effects in cerebral ischemia in rats [35].
According to our results, compounds showing the ability to cross biological membranes (e.g., 141, 58) and/or antiproliferative activity (e.g., 141, 14) presumably contribute to the biological activities of the J. nigra extracts.However, the role of the other components cannot be neglected, as they may be converted in the human body to metabolites with better bioavailability.Furthermore, due to the artificial nature of the membrane used in the PAMPA, only passive transport mechanisms can occur, and active transport cannot be studied.It should be noted that results might also be influenced by the accuracy of the analytical method that was applied to quantify the compounds.In some cases (e.g., for 14 and 74), inter-day accuracy was outside ±20% at the lowest concentration level, partly due to phenolic compounds often being susceptible to decomposition.Therefore, further studies aimed at investigating the biological activity and pharmacokinetic properties of J. nigra constituents would be of great interest.

Solvents and Chemicals
Chloroform, ethyl acetate, and methanol of reagent grade as well as HPLC-grade methanol and acetonitrile were purchased from Molar Chemicals Kft.(Halásztelek, Hungary).The HPLC-grade acetic acid and formic acid were delivered by Sigma-Aldrich (Budapest, Hungary).High-purity water was gained by a Millipore Direct Q5 Water Purification System (Billerica, MA, USA).

Plant Material and Sample Preparation
Pericarp samples of Juglans nigra were collected in Hungary, in the Fiumei Road Cemetery (Budapest, October 2021); later, the leaves and bark were collected at the same place (Budapest, June 2022).Authenticated samples and herbarium specimens are deposited at the Herbarium of the Department of Pharmacognosy, Semmelweis University, Budapest, Hungary.
For the analytical studies, the dried and milled samples (1 g) were extracted in an ultrasonic bath (Bandelin Sonorex Digitec DT 1028, Berlin, Germany) with chloroform, ethyl acetate, and methanol, consecutively (3 × 20 mL for all solvents, 30 min each), at room temperature.The extracts were distilled to dryness with a rotary evaporator (Büchi Rotavapor R-200, Flawil, Switzerland) at 40 • C. The samples were suspended in HPLC-grade methanol and filtered through Phenomenex RC 15 mm 0.2 µm syringe filters (Torrance, CA, USA).For the isolation of the main constituents, the pericarp samples (649 g) were prepared in the same way, except for the volume of the solvents (2 L) and the extraction time (2 h).

Isolation of Compounds from J. nigra Pericarp
For the isolation of the main constituents, J. nigra pericarp sample was collected in the Fiumei Road Cemetery (Budapest, October 2021).Similarly to the analytical samples, the lyophilized pericarp was extracted in an ultrasonic bath with chloroform, ethyl acetate, and methanol successively (3 × 2 L for all solvents, 2 h each).
The chloroform extract was evaporated to dryness under reduced pressure at 40 • C and suspended in methanol (final concentration: 0.1 g/mL).The extract was then fractionated by flash chromatography (CombiFlash NextGen 300+, Teledyne Isco, Lincoln, NE, USA), using a RediSep Rf Gold Silica gel column (40 g, Teledyne Isco) as stationary phase.Eluent A was acetone, eluent B was n-hexane.The following gradient elution was applied: 0% B (0.0-3.0 min), 0-100% B (3.0-43.0min), 100% B (43.0-60.0min), and a flow rate of 40 mL/min.The separation process was repeated two more times to use up all the extract.Fractions of 16 mL each were collected and fractionated further by semi-preparative and preparative HPLC.
The quantity of the isolated substances was as follows: gallic acid ( 14

NMR Conditions
NMR spectra were recorded in DMSO-d 6 at room temperature on a Varian Mercury 400 spectrometer (Varian Inc., Palo Alto, CA, USA) equipped with ATB PFG probe head using standard 5 mm Wilmad ® NMR tubes (Merck, Budapest, Hungary).The pulse programs were taken from the vendors software library (VnmrJ 3.1).As chemical shift reference, the residual solvent signals were used (DMSO-d 6 at 2.500 ppm in 1 H and 39.520 in 13 C).

Quantitative UHPLC-DAD Conditions
Quantities of the isolated compounds in the PAMPA experiments (14, 58, 74, 86, 92, 106, 141, and 143) were determined by UHPLC-DAD.The Juglans extracts were analyzed by an ACQUITY UPLC H-Class PLUS System equipped with a quaternary solvent delivery pump (QSM), an auto-sampler manager (FTN), a column compartment (CM), and a photodiode array (PDA) detector (Waters Corporation).An Acquity BEH C18 column (100 × 2.1 mm i.d., 1.7 µm; Waters Corporation) maintained at 30 • C was used as stationary phase.Eluent A was 0.3% acetic acid in water and eluent B was acetonitrile, the following gradient elution was applied (flow rate: 0.3 mL/min): 5.0-100.0%B (0.0-11.0 min), 100.0-5.0%B (11.0-11.5 min), 5% B (11.5-15.0min).The injection volume was 5 µL and chromatograms were recorded at 200-400 nm.The chromatograms acquired at the UV absorption maxima of each compound were used for data evaluation.Quantitation was performed by the external standard method.Stock solutions containing 10 mM of the isolated compounds (14, 58, 143, 74, 86, 106, 141, 92) in HPLC-grade methanol were prepared.For the preparation of the calibration curve, stock solutions were diluted with 50% methanol of HPLC-grade and 50% distilled water, to yield solutions with concentrations of 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 µM.Each standard solution was prepared in triplicate and injected once.Standard solutions were stored at 4 • C before injection.Linearity curves were constructed by plotting peak areas against corresponding concentrations.Slope, intercept, and correlation coefficient were determined by least squares polynomial regression analysis.Limits of detection (LOD) and quantitation (LOQ) were determined at signal-to-noise (S/N) ratios 3 and 10, respectively.The selectivity of the method was evaluated by analyzing blank samples (HPLC-grade methanol).The linearity regression equations, correlation coefficients (r 2 ), linearity ranges, and LOD and LOQ values of the method are shown in Supplementary Table S1.
(BioTek, Winooski, VT); at λ ex = 530/30 and λ em = 610/10 nm.The cytostatic effect (%) was calculated with a high degree of precision using the following Equation (3): cytostatic effect (%) = 1 − Fluorescence intensity treated Fluorescence intensity control × 100 The cytostasis values were expressed in the percentage of untreated control.The 50 percent inhibitory concentration (IC 50 ) was determined with a rigorous approach by fitting a sigmoid curve on the data points using Microcal™ Origin2021 software and then calculating X values at Y = 50.These values were expressed in micromolar units, providing a precise measure of the compound's effectiveness.

Parallel Artificial Membrane Permeability Assay (PAMPA)
A parallel artificial membrane permeability assay (PAMPA) was used to determine the effective permeability (P e ) for the isolated compounds 14, 58, 74, 86, 92, 106, 141, and 143.Stock solutions (10 mM in methanol) were diluted with the defined buffer (pH 7.4 for the PAMPA-BBB and pH 6.8 for the PAMPA-GI assays) to obtain the donor solutions (composition: 594.0 µL buffer + 6.0 µL stock solution).The buffers were prepared as follows: pH = 6.8: 20.2 g Na 2 HPO 4 • 7H 2 O and 3.4 g NaH 2 PO 4 • H 2 O dissolved in distilled water to achieve the final volume of 1000.0 mL, pH adjustment with 0.5 M NaOH or 0.5 M HCl; pH = 7.4: one PBS tablet (Phosphate Buffered Saline, pH 7.4; Sigma Aldrich) dissolved in 200.0 mL distilled water.Donor solutions were filtered through Phenex-RC 15 mm, 0.2 µm syringe filters (Gen-Lab Ltd., Budapest, Hungary).
For the PAMPA-BBB test, 5 µL of porcine polar brain lipid extract (PBLE) solution (16.0 mg PBLE + 8.0 mg cholesterol dissolved in 600.0 µL n-dodecane) was applied for each well of the 96-well polycarbonate-based filter donor plates (top plate) (Multiscreen™-IP, MAIPN4510, pore size 0.45 µm; Merck).For the PAMPA-GI assay, the wells of the top plate were coated with 5 µL of the mixture of 16.0 mg phosphatidylcholine + 8.0 mg cholesterol dissolved in 600.0 µL n-dodecane.A measure of 150.0 µL of aliquots of the filtrated donor solutions were placed on the membrane.The 96-well PTFE acceptor plates (bottom plates) (Multiscreen Acceptor Plate, MSSACCEPTOR; Merck), were filled with 300.0 µL buffer solution (0.01 M PBS buffer, pH 7.4).The donor plate was placed upon the acceptor plate, and both plates were incubated together at 37 • C for 4 h in a Heidolph Titramax 1000 Vibrating platform shaker (Heidolph, Schwabach, Germany).
After incubation, sandwich plates were separated and the concentrations of each compound in the starting donor solution and in the acceptor and donor wells were determined in triplicate by UHPLC-DAD method described above.UV spectra and chromatograms were recorded at 200-400 nm and the chromatograms acquired at the UV absorption maxima of each compound were used for data evaluation.The effective permeability and the membrane retention in the PAMPA-BBB and the PAMPA GI experiments were calculated by Equations ( 4)- (7), respectively [100]: ) where P e is the effective permeability coefficient (cm/s), A is the filter area (0.24 cm 2 ), V D and V A are the volumes in the donor (0.15 cm 3 ) and acceptor phases (0.30 cm 3 ), t is the incubation time (s), τ SS is the time (s) to reach steady-state (240 s), C D (t) is the concentration (mol/cm 3 ) of the compound in the donor phase at time t, C D (0) is the concentration (mol/cm 3 ) of the compound in the donor phase at time 0, and MR is the estimated membrane retention factor (the estimated mole fraction of solute lost to the membrane), r a is the sink asymmetry ratio (gradient-pH-induced), defined as: All experiments were performed in three triplicates on three consecutive days (n = 9), caffeine standard was used as positive, while rutin as negative control.ClogP values were calculated by ChemAxon Marvin 22.3.
representative UHPLC-DAD chromatogram of J. nigra pericarp ethyl acetate extract is shown in Figure 1, while chromatograms of all extracts (chloroform, ethyl acetate, and methanol extracts of leaf, bark, and pericarp samples) are shown in Supplementary Figures S1-S3.

Figure 2 .
Figure 2. Compounds isolated from the pericarp of J. nigra.

Table 1 .
UHPLC-MS/MS data and tentative characterization of constituents from Juglans nigra leaf, bark, and pericarp extracts.

Table 2 .
HR-MS data of the isolated constituents of J. nigra pericarp.

Table 3 .
The in vitro cytostatic activity of the compounds on different cell cultures.

Table 4 .
Results of the PAMPA experiments expressed as logP e values (n = 9).
Abbreviations: n.d.: not detected in the acceptor phase; PAMPA-GI: parallel artificial membrane permeability assay for the gastrointestinal tract; PAMPA-BBB: parallel artificial membrane permeability assay for the blood-brain barrier.