*3.4. Cell Culture and Cytotoxicity Assessment*

The human glioblastoma U-87 cell line and human fibroblasts (HF) were kindly provided by the Bank of Human and Animal Continuous Cell Lines-CEINGE Biotecnologie Avanzate – Napoli - Italy. The U-87 cell line was cultured in Dulbecco's medium (DMEM) supplemented with 10% of heat-inactivated Fetal Bovine Serum (FBS) and 1% l-glutamine (Sigma-Aldrich, St. Louis. MO, USA); HF were cultured in cultured in Dulbecco's medium (DMEM) supplemented with 20% of heat-inactivated Fetal Bovine Serum (FBS) and 1% l-glutamine (Sigma-Aldrich). Both cells were grown in a 5% CO<sup>2</sup> humidified incubator, at 37 ◦C. For treatments, the cells were incubated with LnHS at different concentrations in serum-free fresh medium for different incubation times.

#### 3.4.1. MTT Cell Viability Assay

U-87 and HF cells were seeded in 10% FBS-containing medium in a 96-well plate at the density of 3 <sup>×</sup> <sup>10</sup><sup>3</sup> cells/well. The day after, the cells were treated as above described. Cell viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) assay. The next day, cells were treated as above described and, after incubation times, the assay was performed as previously described [55].

#### 3.4.2. LDH Release Assay

The lactate dehydrogenase (LDH) leakage assay is a colorimetric test useful for quantifying cell death and lysis through the measurement of LDH released from the cytosol of damaged cells, which was into supernatant. The test was carried out as previously reported [56].

### 3.4.3. Colony Forming Assay

For colony forming assays, U-87 cells (1 <sup>×</sup> <sup>10</sup><sup>3</sup> ) were seeded per well of a 6-well plate and maintained for 10 days with medium changed every other day. Colonies were stained using crystal violet (Sigma-Aldrich) at room temperature for 20 min and washed repeatedly in water. Colonies were counted manually using a light microscope as previously described [57].

### 3.4.4. Comet Assay

DNA breakage was evaluated using a comet assay kit (Trevigen, Gaithersburg, MD, USA). Three independently reproduced experiments were performed. To determine DNA strand breakage, U-87 cells and HF were plated at 1 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL in a 24-well plate and left to attach for 24 h. The next day, cells were treated with LnHS (0.2, 2.5, 5, 25, 50 µg/mL) for 24 h. Comet slides were stained with diluted SYBR green and examined under an automated robotic epifluorescence microscope with an excitation filter (510 to 550 nm). One hundred cells were analyzed per slide. Experiments were performed in duplicate. Untreated cells served as negative controls.

#### 3.4.5. Wound Healing Assay

U-87 cells and HF were seeded at a density of 3 <sup>×</sup> <sup>10</sup><sup>5</sup> cells in a 6-well plate in complete culture media and grown to confluence. The day after, cells were treated with 4 µg/mL of mitomycin (Sigma-Aldrich) for 2 h to inhibit cell proliferation and then a wound was inflicted using a tip. After washing with phosphate-buffered saline (PBS), cells were incubated as above mentioned in comparison to untreated cells. The scratch wound was observed and photographed at different time points using an inverted-phase-contrast microscope (TS100 fluorescence microscope and video camera, Nikon, Tokyo, Japan). Three measurements per scratch were performed (two replicates/condition, experiments performed in duplicate).

### 3.4.6. RNA Extraction and Real Time Quantitative PCR

U-87 and HF cells, after 12 h starvation, were treated in 0% FBS medium as above described. After incubation, total RNA was isolated from using TRIzol (Invitrogen, Carlsbad, CA, USA) and Real-time PCR was performed as previously described using GAPDH as housekeeping gene [58]. The primers for Sirt1, Sirt2, IL-6, IL-10 and GAPDH are available on request. The experiments were performed two times in triplicate.

#### 3.4.7. Preparation of Cell Extracts and Western Blotting Analysis

U-87 cells and HF cells, after 12-h starvation, were treated in 0% FBS medium as above described. After incubation, total proteins, extracted in RIPA buffer, were quantified by Bradford's method (Bio-Rad, Hercules, CA, USA). Total proteins were subjected to electrophoresis and transferred to PVDF membranes; the membranes were incubated with the following antibodies according to the manufacturer's instructions: E-cadherin and GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA), p-AKT, ULK-1, Bcl-2, Beclin 1 (Cell Signaling Technology, ZA Leiden, Netherlands). The blots were developed by ECL (Amersham Biosciences, Piscataway, NJ, USA) and analyzed by densitometry as previously described [58]. Each sample was tested three times in duplicate.

#### *3.5. Statistical Analysis*

Experiments were performed three times with replicate samples, except where otherwise indicated. Data are expressed as mean ± SD (standard deviation). The means were compared using analysis of variance (ANOVA) plus Bonferroni's *t*-test. A *p*-value of < 0.05 was considered to indicate a statistically significant result.

#### **4. Conclusions**

The recovery of bioactive compounds (pure or in their mixture form) from hempseed meal could be the driving force for developing new hemp seed-based goods, in which the processing of hemp fruits waste is valuable for fully exploiting the innumerable advantages of this crop [59,60]. In this context, high resolution negative tandem mass spectrometric techniques could be a useful tool in the structure elucidation of hemp seeds compounds such as phenylamides and lignanamides.

The main phenylamides in hemp seeds are hydroxycinnamoyl amides in which the amine moiety was octopamine or tyramine. They are readily differentiable based on their TOF-MS<sup>2</sup> spectra as the octopamine conjugate promptly lost a water molecule and the base peak corresponds to a hydrocinnamoyl moiety. Caffeoyl and feruloyl-derived phenyldihydronaphthalene lignanamides are distinguishable by a characteristic loss of 110 and 124 Da, whereas phenylcoumaran lignandiamide were characterized by a first direct tyramine moiety. Moreover, isocyanic acid (HNCO) + *p*-hydroxystyrene represent a common neutral loss for all the identified lignanamides. The presence of an α,β-unsaturated function favors a facile CO-Cα cleavage, and low fragment ions give additional structural information on the investigated lignanamides. Cytotoxicity assessments of LnHS on the U-87 glioblastoma cell line and on human fibroblasts provided new insight into the molecular effects of this lignanamide extract. Indeed, even if further studies are necessary, our data strongly suggest that LnSH negatively and specifically regulates U-87 cell line survival and migration then reinforcing the need to fully analyze its biochemical behavior, and lignanamides purified therefrom. Indeed, the compounds' purification will be addressed in order to deeply investigate their quantitation in hemp seeds products from the different cultivars available on the market, as well as to achieve a clearer picture of their already promising anti-cancer activity. In this context, the ability of LnHS (and pure compounds therefrom) to cross the blood brain barrier (BBB) is aimed to be promptly pursued. This is in line with several recent evidences which highlight the ability of dietary polyphenols, and their known physiologically relevant metabolites, to enter the brain endothelium, cross the BBB and to impact brain health and cognition [61–63]. The BBB-crossing feature in phenylamides and lignanamides could be improved

by the intrinsic amide function occurrence. In fact, recent studies showed that the introduction of an amide function was a strategy to enhance BBB transport of antineoplastic drug [64], whereas the development of *N*-acetylcysteine amide (NACA) preserved *N*-acetylcysteine antioxidant ability improving its permeability through cell membranes [65]. Furthermore, considering the identified compounds mainly as polyamine derivatives, it was recently shown that tyramine analogue in *Gingko biloba* extract were identified among compounds able to cross BBB [66].

**Supplementary Materials:** The following are available online: Figure S1. Simplified extraction and fractionation scheme of cryo-crushed hempseeds. Figure S2. Morphological changes in SH-SY5Y cells treated with LnHS fraction in respect to untreated cells. Representative images were acquired by Inverted Phase Contrast Brightfield Zeiss Primo Vert Microscope. Figure S3. A) TOF-MS/MS spectrum; B) proposed fragmentation pathway of the [M − H]<sup>−</sup> ion; C) UV-DAD spectrum for compound **2**. In B panel, the theoretical *m*/*z* value is reported below each structure. Figure S4. A) Extracted ion chromatogram (XIC) of the [M − H]<sup>−</sup> ion at *m*/*z* 312.124 ±0.025; TOF-MS/MS spectra of B) compound 8, and C) compound **17**. In the grey panel the tentatively proposed fragmentation pathway of the [M − H]<sup>−</sup> ions. Representative UV-DAD spectrum, acquired under peak **17**, is reported in panel D. Figure S5. A) TOF-MS spectrum for compound **11**; B) TOF MS/MS spectrum of the [M − H]<sup>−</sup> ion; C) UV-DAD spectrum. Figure S6. A) TOF-MS/MS spectrum and B) proposed fragmentation pathway of the [M − H]<sup>−</sup> ion for compound **33**; theoretical *m*/*z* values are reported below each structure. Figure S7. Compound 16 A) UV-DAD spectrum; B) TOF-MS/MS spectrum (in grey panel TOF-MS spectrum is reported. C) Proposed fragmentation pathway of the [M − H]<sup>−</sup> ion (theoretical *m*/*z* values are reported below each structure). Figure S8. Proposed fragmentation pathway of the [M − H]<sup>−</sup> ion relative to cannabisin B isomers (theoretical *m*/*z* values are reported below each structure). Figure S9. A) Proposed fragmentation pathway of the [M − H]<sup>−</sup> ion of the compound **28** (theoretical *m*/*z* values are reported below each structure); B) UV-DAD, and C) TOF-MS/MS spectra. Figure S10. TOF-MS/MS spectra of compounds **32**, **36** and **38** (A, B, and C, respectively). Proposed fragmentation pathway of the [M − H]<sup>−</sup> ion of the compound **32** is reported in grey panel, whereas that of **36** and **38** is in light green panel (theoretical *m*/*z* values are reported below each structure). Figure S11. TOF-MS/MS spectra of compounds **4** and **6** (A, and B, respectively). Proposed fragmentation pathway of the [M − H]<sup>−</sup> ion of both the compounds are reported in grey panels (theoretical *m*/*z* values are reported below each structure). Figure S12. TOF-MS/MS spectrum (A) and proposed fragmentation pathway of the [M − H]<sup>−</sup> ion of compound **13** (theoretical *m*/*z* values are reported below each structure). Figure S13. TOF-MS/MS and proposed fragmentation pathway of the [M − H]<sup>−</sup> ion for compound **26**. The theoretical *m*/*z* value is reported below each structure. Figure S14. TOF-MS/MS spectra of compounds A) **27**, B) **29**, C) **37**, and D) **39**. Figure S15. A) TOF-MS/MS spectrum of compound **30**, and B) proposed fragmentation pathway of its [M − H]<sup>−</sup> ion (theoretical *m*/*z* values are reported below each structure). Figure S16. Proposed fragmentation pathway of the [M − H]<sup>−</sup> ion for compounds **31** and **34**. Figure S17. Flavanol glycosides in LnHS hempseed fraction. Figure S18. TOF-MS/MS spectra of quercetin derivatives A) **3**, B) **7** and C) **18**. Figure S19. A) Extracted ion chromatogram (XIC) of the [M − H]<sup>−</sup> ion at *m*/*z* 417.083 ±0.025; TOF-MS/MS spectra of B) compound **9**, and C) compound **10**. Figure S20. TOF-MS/MS spectra of kaempferol derivatives A) **12**, and B) **14**. Figure S21. A) Relative content of each class of the tentatively identified compounds: HAAs – hydroxycinnamoyl amides; LnAs – lignanamides; Fls – Flavonols; B) relative content of lignanamides sharing a common [M − H]<sup>−</sup> ion in LnHS fraction. Figure S22. A) mRNA expression of Sirt1 and Sirt2 by real-time; B) mRNA expression of IL6 and IL10 by real-time in U-87 and HF cells after 24 and 48 h exposure times.

**Author Contributions:** Conceptualization, S.P. (Severina Pacifico); methodology, E.N., S.P. (Simona Piccolella), M.F., M.T.P. and G.C.; formal analysis, S.P. (Simona Piccolella), M.M., M.F., M.T.P., and G.C.; data curation, E.N., M.F., S.P. (Severina Pacifico); writing—original draft preparation, S.P. (Severina Pacifico) and E.N.; writing—review and editing, M.F, A.D., S.P. (Severina Pacifico); supervision, A.D and S.P. (Severina Pacifico). All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** This research was supported by grant L.R. n. 5/2017. DRD n. 92 del 21.06.2018 ("Progetto per la Rivalutazione Olistica della canapa oltre il PIL", PROHEMPIL PROJECT) from the Campania Region (Italy).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Sample Availability:** Samples of the compounds are available from the authors.

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