3.3. Analysis of Non-Polar Extract by GC-MSAs listed in Table 1, alkanes, alkenes, naphthoquinones, terpenes, sterols, and fatty acids were the main compounds. The most prevailing compounds were γ-sitosterol (2.47–33.8%), vitamin E (7.57–15.98%), β-tocopherol (1.43–2.92%), lupeol (1.04–2.28%), juglone (0.12–3.66%), and fatty acids (oleic acid, linoleic acid, palmitic acid, stearic acid); the other compounds always being present at less than 2%.In walnut husk extracts, alkanes with different chain lengths (n-C9 to n-C28) were identified (Table 1). Short-chain alkanes typically originate from algae and bacteria [27], but they were also found in husk non-polar extract (Table 1). Seabra et al. [4] identified nine alkanes (tetradecane, tetracontane, docosane, pentacosane, pentatriacontane, heptacosane, eicosane, heptatriacontane, heneicosane) in the husk high-pressure supercritical extracts. However, in our study, more components, especially short-chain alkanes (heptane, octane, nonane, decane, undecane, etc.), were detected. Alkenes are another important type of hydrocarbons and the 1,19-eicosadiene was actually detected in non-polar extract. The presence of 1,19-eicosadiene was also reported by Seabra et al. [4]. The 1,4-naphthalenedione, 5-hydroxy- or juglone is a strong antioxidant and the main compound found in all parts of the walnut fruit [28].The terpene compounds such as dl-limonene and β-limonene are the characteristic aroma components of walnut fruits and were detected in the husks [17]. Tocopherols have antioxidant activities and play a key role in the oxidative reactions of unsaturated fatty acids in foods and biological systems. α-Tocopherol and γ-tocopherol are the main vitamin E components [29,30]. Two tocopherol homologs (α- and γ-tocopherol) were found in the extracts. These compounds are liposoluble metabolites that are found in many plants [29]. The γ-sitosterol and lupeol are sterols with strong antifungal, antibacterial, and antispasmodic activities that were detected. In addition, different fatty acids and other compounds were found. In summary, the non-polar extract was mainly composed of volatile compounds which are probably related to the odor of walnut [17]. 3.5. Analysis of Polar Extract by UHPLC-PDA-HRMS/MSThe green walnut husk hydroethanolic extracts were analyzed by UHPLC-PDA-HRMS/MS, and their UV282 chromatograms are shown in Figure 1. Twelve of the main compounds observed in UV282 were identified thanks to their exact masses (parent ions seen in mono- or diprotonated forms) and their high-resolution MS2 fragmentation patterns. In detail, five of the twelve compounds of interest (namely minoxidil, myricitrin, quercetin 4’-glucoside, taxifolin and quercetin pentoside) were identified by automatic search in the high-resolution mass spectral library “mzCloud”. In addition, three compounds (namely catechin, abscisic acid and salicylate glucuronide) were automatically identified thanks to the “Chemspider” mass database. Finally, three other compounds of interest (namely neochlorogenic acid, taxifolin 7-glucoside and gallic acid) were identified manually because they were not found in the various databases queried. The name, formula, calculated molecular weight (Cal. MW), exact m/z values of parent ions, and retention time (RT) are shown in Table 3. To the best of our knowledge, four compounds are reported for the first time: taxifolin 7-glucoside, minoxidil, abscisic acid, and salicylate glucuronide, although others have been previously identified in walnut waste [21,28,32].Flavonoids, as the largest group of natural polyphenolic compounds, exist in free or glycoside forms. The basal backbone of flavonoids is constituted of a benzene ring A linked to a gamma-benzopyrone (an oxidized heterocyclic C ring substituted in C2 by benzene B ring) by a three carbon bridge. Based on the location of the unsaturated bonds or substitution and the number of OH groups in the gamma-benzopyrone structure, flavonoids are divided into flavon, flavanol, flavonol, and flavanonol subgroups [39]. Cianidanol (m/z 291.09, RT = 11.77 min) is an antioxidant plant metabolite and belongs to the flavanol (flavanol-3-ol) family. Cianidanol or (+)-catechin is the (+)-enantiomer of catechin. In flavanols, the ketone group in the ring C (C4) does not exist. They have two OH groups in positions 5 and 7 in the benzene A ring, one in position 3 in the heterocycle C ring, and two in positions 4’ and 5’ in the B ring. The daughter ion at m/z = 139 indicates the presence of an A ring in the cianidanol structure [13,40]. Myricetin (m/z = 465.10, RT = 17.01 min), quercetin-4’-glucoside (m/z = 465.10, RT = 17.18 min) and quercetin-3-O-pentoside (m/z = 435.09, RT = 18.22 min) are flavonols. In this family, two OH groups in positions 5 and 7 of the benzene A ring and one in position 4 in the C ring are present [39]. For myricetin, three OH groups (positions 3’, 4’ and 5’) are substituted in ring B. Quercetin 4’-glucoside is quercetin (two OH groups in positions 3’, 4’ in ring B) with a beta-D-glucosyl residue attached in position 4’ [13]. Its intense daughter ion at m/z = 303.0 [M+H-C6H12O6] indicates the loss of glucose, and the presence of the ion moiety of quercetin [33]. Furthermore, the neutral loss of 133 (daughter ion [M+H-133]+) is linked to the departure of the pentoside moiety from the quercetin-3-O-pentoside. This compound was also reported in previous works in the septum of walnut [38].Taxifolin (m/z = 305.07, RT = 17.40 min) or 5,7,3′,4′-flavan-on-ol belongs to the flavanonols. The daughter ion at m/z = 287.1 reveals the loss of a water molecule as reported by Sheng et al. [33]. Taxifolin is the dehydrogenated form of quercetin, and due to the absence of a double bond on carbon 2 and 3 in ring B, it has less antioxidant properties than quercetin [41]. Thanks to the daughter ion [M+H-C6H12O6] seen at m/z = 305.1, the compound associated to peak 2 was clearly identified as taxifolin 7-glucoside. The peaks 1 and 11 could be identified as neochlorogenic acid (5-O-caffeoylquinic acid) and gallic acid derivative, respectively. Neochlorogenic acid is a natural phenolic acid and is structurally constituted of a cyclitol carboxylic acid and a cinnamate ester. Gallic acid is a trihydroxybenzoic acid and hydroxy groups are present in positions 3, 4, and 5. These compounds were identified through their parent ions reported in walnut husk by Medic et al. [28] and Sheng et al. [33]. 1-Salicylate glucuronide (m/z = 315.07, RT = 15.99 min), a glucuronide conjugate of salicylic acid, is also a phenolic acid identified in husk extracts. The minoxidil (m/z = 210.14, RT = 13.30 min) structure is a pyrimidine-2,4-diamine 3-oxide that is substituted by a piperidin-1-yl group at position 6. Well known as a vasodilator, hair-growth stimulator and antihypertensive agent [42], this compound was identified for the first time in walnut husk, thanks to our work. (±)-(2E)-Abscisic acid or dormin (m/z = 265.14, RT = 13.34 min) is an abscission-accelerating plant growth substance and is found in different parts of plants [43].The semi-quantitative analysis of the compounds in green walnut husk extracts was evaluated. Most of the identified compounds exist in all walnut husk extracts. The Saman variety, which was dried after storage and was brown in color, showed the greatest differences from the other samples and did not contain taxifolin 7-glucoside, or quercetin pentoside. In this sample, most compounds including catechin, taxifolin, and minoxidil were at their minimum level. Taxifolin and catechin were the highest in Reha 4; abscisic acid and salicylate glucuronide in the brown Saman sample and salicylate glucuronide and catechin were the highest in other samples (Figure 1). 3.7. Total Antioxidant Activity by DPPH AssayAs can be seen in Figure 2a, the DPPH scavenging activity logically increases with the concentration of husk powder. As DPPH IC50 is the concentration required for 50% inhibition of DPPH free radical, therefore, lower values indicate higher antioxidant activity. The IC50 of samples was in the range 146.8–249.3 µg/mL of husk. These results are in accordance with the total phenolic content previously measured in each sample. The green husk varieties had different antioxidant activities, green Saman and Chandler had the highest and lowest, respectively. Furthermore, the Saman sample was examined in two colors, green and brown, and a significant difference in IC50 was observed depending on the color. The results obtained in the DPPH assay were always related to the peak intensity of the phenolic compounds. Oliveira et al. [7] measured the EC50 in the aqueous walnut husk extracts of different cultivars and the values were found between 350 and 590 µg/mL. Similarly, the EC50 of the walnut husk extracted by different solvents was reported in the range 330–720 µg/mL [8]. In another study, the obtained IC50 values of methanolic extracts, depending on the cultivar, ranged from 141 to 2890 μg/mL [45]. As mentioned before, the most important problem encountered when using common methods such as DPPH is the impossibility to accurately compare the results of different studies with each other. Therefore, in the following, the PAOT technology was used to reliably measure the antioxidant power. 3.8. Total Antioxidant Power by PAOT TechnologyFigure 3 shows the PAOT liquid technology scale for ingredients and the total antioxidant activity of various varieties of dried green walnut husk powder. According to the PAOT scores, ingredient samples with a value below 250 are not labeled as antioxidants and samples with a value greater than 1000 are stated as very effective products in the same way as the reference molecules (Figure 3a). The average PAOT score/g dw of husk samples was in the range 256.52 ± 5.89 to 746.88 ± 6.96 (Figure 3b). According to the PAOT liquid technology scale for ingredients, two varieties were rated as effective, and five were rated as moderately effective. The brown variant of Saman was labeled as a product with weak effectiveness. It should be noted that these results are based on the dry powder and without any purification or isolation steps, so the green walnut husk can certainly be categorized as a strong antioxidant.In the case of the green-colored powders, Saman showed the greatest antioxidant power (746.88 ± 6.96), while Chandler was the weakest. Between the two types of Saman, the brown showed a low antioxidant power compared to the green (about 40% less). This result could be due to the oxidation mechanism. Given that the only distinction between green and brown Saman powder was the length of storage, probably the freezing and quick drying of the sample are crucial to obtain a product with strong antioxidant properties. The antioxidant power was consistent with the results obtained for the total phenolic compounds and DPPH assay (except for Aytak). In samples with a high phenolic content, the antioxidant power was high, as expected. In addition, the chemical composition was probably another main reason for the difference in the antioxidant power of the extracts. In the green Saman sample, the peak areas of the antioxidant compounds were the highest, and in the brown-colored Saman, lower peak areas were observed. In brown Saman, the amount of abscisic acid, which has no antioxidant activity, had the highest peak intensity, which is probably the reason for its low PAOT score. Additionally, in other samples, the relationship between the peak intensity of the phenolic compounds and the antioxidant power was obvious. According to Figure 1, there was a clear difference in the peak area of catechin (with antioxidant capacity), which was very low in Saman brown. The differences in peak area observed for salicylate glucuronide, quercetin 4′-glucoside, quercetin pentoside and taxifolin 7-glucoside most certainly explain the differences in the measured antioxidant power. The flavonoid structure, with an OH group on the 3′ and 4′ carbons in ring B and an OH group in ring C, is probably crucial for inhibiting free radicals. Due to the presence of hydroxyl groups in ring A, the antioxidant property and free radical inhibition increase significantly. The double bond in carbon 2 and 3, which is conjugated with the carbonyl group, is responsible for electron distribution in the B ring and is the factor of increasing the antioxidant property. Taxifolin lacks a double bond on carbon 2 and 3, so its antioxidant property is lower than quercetin. The presence of a hydroxyl group on carbon 3 in ring C increases the antioxidant property. Therefore, glycosylation in this position reduces the antioxidant property [13,39,41]. 3.9. In Vitro Antimicrobial PropertiesThe antibacterial properties of the husk extracts and positive controls (streptomycin and penicillin) are presented in Table 4. A ratio MBC/MIC (minimum bactericidal concentration/minimum inhibitory concentration) less than or equal to four is considered bactericidal, while a ratio MBC/MIC greater than four is considered bacteriostatic. Since the MIC to MBC ratio (1–2) for all bacterial collections was less than 4, all extracts of walnut husk exhibit bactericidal activity. The lowest and highest MIC were obtained for E. coli (0.5–1 mg/mL) and P. aeruginosa (4–7 mg/mL), respectively. The antibacterial properties of the extracts were probably related to the compounds in the husk of walnut. In the non-polar part, compounds such as monoterpenes, tocopherols, sterols and juglone and in the polar part, phenolic acids, have strong antipyretic, analgesic, anti-inflammatory, antimicrobial, and antioxidant properties [4]. The antibacterial activity of walnut husks was also investigated in previous studies and different results were reported. Vieira et al. [23] evaluated the antimicrobial properties of the hydroethanolic extract of walnut husks on Gram-negative (E. coli, K. pneumoniae, M. morganii, P. mirabilis, and P. aeruginosa) and Gram-positive (E. fecalis, L. monocytogenes, and methicillin-resistant S aureus) bacteria. The MIC of husk extract was 5 mg/mL for S. aureus, 10 mg/mL for L. monocytogenes, and E. coli and 20 mg/mL for K. pneumoniae, M. morganii, P. mirabilis, and E. fecalis. The extract showed no inhibition effect on P. aeruginosa (MIC > 20 mg/mL) [23]. Fernández-Agulló et al. [8] also reported antibacterial potential against B. cereus (20 mg/mL), and a weak potential against P. aeruginosa and E. coli (100 mg/mL). In the study of Oliveira et al. [7], the amounts of MIC against S. aureus (0.1 mg/mL), Bacillus cereus (0.1–1 mg/mL), Bacillus subtilis (0.1–10 mg/mL) and P. aeruginosa (100 mg/mL) for different Portuguese cultivars of green walnut husks were lower, which showed higher antibacterial properties. In general, it can be stated that the selected variety, the agricultural conditions, and the protocol used for the antibacterial measurement have an effect on the antibacterial properties of the extract of green walnut husk [23]. As is clear in Table 4, all the varieties had good bacterial characteristics, even a bactericidal effect on P. Aeruginosa was seen. 3.10. Anticancer ActivitySince the green Saman variety had the highest total phenolic, antioxidant, and antibacterial properties in all the analyses, the anticancer activity of its extract at different concentrations (0.05, 0.5, 5, 50, and 500 µg/mL) against the human liver tumor cancer cell line (HepG2) over 24, 48, and 72 h was investigated; the results are shown in Figure 4. The toxicity effect of walnut husk extract on HepG2 was dependent on concentration and time and increased from 1.03% at 0.05 µg/mL in 24 h to 69.23% at 500 µg/mL in 72 h. Therefore, only 30.77% of the cells were able to survive (viability%), which showed the high antitumor effect of green walnut husk extract. The amount of IC50 was also calculated, based on the drawing of the curve and the equation of the line, and it was equal to 94.80, 68.81, 28.21 µg/mL of husk over 24, 48 and 72 h, respectively. Vieira et al. [23] reported that, at 24 µg/mL concentration of hydroethanolic husk extract, the total HepG2 cell growth reduced by 50%. (GI50) [23]. Other studies have been conducted on the antitumor effect of pure extracted compounds from husks, such as juglone and terpenes, instead of the whole extract against various human cancer cell lines, including MCF-7, HCT-116, HeLa, K562, Raji, and THP-1 and high cytotoxic potential has been reported [6]. However, Soto-Maldonado et al. [26] stated that the toxicity of the final extract against the HL-60 cells was higher than that of the pure compound (juglone).