Phytochemical Characterization and Evaluation of the Antioxidant and Anti-Enzymatic Activity of Five Common Spices: Focus on Their Essential Oils and Spent Material Extractives

The essential oil industry of aromatic herbs and spices is currently producing a significant amount of by-products, such as the spent plant materials remaining after steam or hydrodistillation, that are simply discarded. The aim of this study was to comparatively investigate the phytochemical composition, antioxidant and multi-enzymatic inhibitory potential of the essential oils and spent plant material extractives obtained from cinnamon, cumin, clove, laurel, and black pepper. The essential oils were characterized by the presence of several phytochemical markers (cinnamaldehyde, cuminaldehyde, eugenol, eucalyptol, α-terpinene, limonene, β-caryophyllene or β-pinene). On the other hand, the LC-HRMS/MS profiling of the spent material extracts allowed the annotation of species specific and non-specific metabolites, such as organic acids, phenolic acids, flavonoids, proanthocyanidins, hydrolysable tannins, fatty acids, or piperamides. All samples exhibited very strong antioxidant effects, with the clove essential oil displaying the strongest radical scavenging (525.78 and 936.44 mg TE/g in DPPH and ABTS assays), reducing (2848.28 and 1927.98 mg TE/g in CUPRAC and FRAP), and total antioxidant capacity (68.19 mmol TE/g). With respect to the anti-acetylcholinesterase (0.73–2.95 mg GALAE/g), anti-butyrylcholinesterase (0–3.41 mg GALAE/g), anti-tyrosinase (0–76.86 mg KAE/g), anti-amylase and anti-glucosidase (both 0–1.00 mmol ACAE/g) assays, the spice samples showed a modest activity. Overall, our study reports that, not only the volatile fractions of common spices, but also their spent plant materials remaining after hydrodistillation can be regarded as rich sources of bioactive molecules with antioxidant and multi-enzymatic inhibitory effects.


LC-HRMS/MS Analysis of Extracts Obtained from Different Spices
The spice spent materials obtained after essential oil isolation were extracted with solvents with different polarities (hexane, dichloromethane, methanol, and 50% aqueous methanol) ( Table 1) and phytochemically profiled by LC-HRMS/MS (Table 3). For all samples, the highest extraction yields were provided by methanol, reinforcing the well-known ability of this solvent to efficiently solubilize both lipophilic and hydrophilic constituents [46].
With respect to the cumin extracts, eleven constituents were annotated; some of them (citric acid, coniferol-O-hexoside) were already noticed in the cinnamon extracts. In contrast, the flavonoid profile was slightly more complex, with seven luteolin and apigenin derivatives, such as free aglycons, O-hexosides, acetylhexosides or hexuronide-hexosides; additionally, two hydroxycinnamic acid derivatives were observed (Table 3). Previously, Ali et al. [48] reported around 24 constituents in a 70% aqueous ethanol cumin extract, whereas Vallverdú-Queralt et al. [47] described 34 different chemical entities in the 50% aqueous ethanol cumin extract; the most notable compounds belonged to the phenolic acids and flavonoids (flavanols, flavones, flavonols), which is in agreement with the results from the current study.
Except for citric acid, caffeoylquinic acid, gallic acid-O-hexoside, and a proanthocyanidin trimer, the remaining 11 compounds tentatively identified in the laurel extracts were derivatives of quercetin, isorhamnetin, and kaempferol (Table 3). Our data is in agreement with the study of Pacifico et al. [52] who documented the presence of 21 diverse flavonoids and proanthocyanidins in the methanolic laurel leaf extracts and that of Bourebaba et al. [53] in which 15 phenolic acids and flavonoids were labeled in the ethanol laurel leaf extracts. It is worth mentioning that three acylated kaempferol glycosides (La11-La13) were previously reported only by Fiorini et al. [54] in a 70% aqueous ethanol extract of laurel leaves.
Lastly, the LC-HRMS/MS metabolite profiling of the black pepper extracts revealed a totally different chemical composition, as compared to other four spices, with 26 out of a total of 30 tentatively labeled compounds represented by piperamides (Table 3). Among them, piperine, piperlonguminine, pellitorine, pipernonaline, retrofractamide B, guineensine, and N-isobutyldodecadienamide are some of the most notable congeners. A similar chemical composition was documented in previous literature data for different black pepper extracts [55][56][57].
To sum up, the LC-HRMS/MS analysis of the extracts obtained with solvents with different polarities from the plant materials after hydrodistillation (usually discarded as waste products by the essential oil industry) revealed the presence of a diverse range of bioactive phytochemicals. Nevertheless, the adequate extraction solvent should be selected considering the category of phytochemicals as well as the bioactivities that are targeted; for instance, our data (Table 3) showed that cinnamon, cumin, clove, and laurel contained numerous polar flavonoids and phenolics, which were present almost exclusively in the methanol and 50% aqueous methanol extracts, whereas black pepper was rich in non-polar piperamides, easily extractible with hexane, dichloromethane, or methanol. Overall, as compared to previously literature data, the LC-HRMS/MS analysis revealed a lesser phenolic profile of the investigated spices; such differences could be due not only to the extraction conditions (processed/spent vs. unprocessed material), but also to plant phenotype, pedoclimatic conditions, harvesting, and storage conditions.

Total Phenolic and Flavonoid Content of Extracts Obtained from Different Spices
The spice residue extracts obtained after the essential oil isolation were next evaluated with respect to their total phenolic content (TPC) and total flavonoid content (TFC) ( Table 4). In the cinnamon extracts, TPC varied between 14.00 mg gallic acid equivalents (GAE)/g extract (CiD) and 92.90 mg GAE/g (CiMW), whereas TFC ranged from 1.76 mg rutin equivalents (RE)/g extract (CiMW) and 9.83 mg RE/g [58]. According to the existing literature data, total phenolics levels retrieved in our samples are significantly lower than those previously reported in the 50% aqueous acetone, 80% aqueous methanol, or aqueous cinnamon extracts (148-309 mg GAE/g) [59,60]. Cumin extracts displayed TPC from 7.86 (CuH) to 30.77 mg GAE/g (CuMW), with a significantly higher TFC, especially for the polar extracts (50.68 mg RE/g in CuM and 66.25 mg RE/g in CuMW); these two extracts also showed the highest flavonoid levels from all analyzed spices (Table 4). A similar trend was previously observed by Gupta [61] with a reported TPC of 12.15 mg GAE/g and 46.56 mg quercetin equivalents/g in a methanol cumin extract. On the other hand, Rebey et al. [62] showed TPC and TFC between 1.09 and 18.60 mg GAE/g and 0.52-5.91 mg catechin equivalents/g, respectively, in cumin extracts obtained with solvents with different polarities (ethanol, methanol, acetone, water, 80% acetone, 80% methanol, 80% ethanol); generally, the hydro-organic extracts showed superior TPC and TFC, which is in agreement with the results from our study.
All four clove extracts displayed the highest TPC (90.67-103.76 mg GAE/g) of all analyzed spices; in contrast, TFC was significantly lower, especially in the non-polar extracts (1.43 mg RE/g in ClH and 5.89 mg RE/g in ClD), suggesting that the contribution to TPC values is not due to the presence of flavonoids. Previous reports showed even higher TPC for various aqueous, ethanol, or methanol clove extracts (212.85-425.04 mg GAE/g) [59,61], whereas the levels of flavonoids (4.70-17.5 mg quercetin equivalents/g) in polar clove extracts [63] were significantly lower than the one reported in the current study (41.71 mg RE/g in ClMW and 31.00 mg RE/g in ClM). With respect to the laurel extracts, TPC varied from 22.49 (LaH) to 75.53 mg GAE/g (LaMW), whilst TFC varied from 0.50 (LaH) to 27.33 mg RE/g (LaM). Previous data revealed a high variability of the phenolics profile, with TPC ranging from 30.73 to 135.47 mg GAE/g for laurel extracts obtained with polar solvents (methanol/ethanol-water) under different extraction conditions (heat-reflux, ultrasound-assisted, microwave-assisted, mechanochemical-assisted) [64,65]. Lastly, black pepper extracts showed a homogenous TPC trend, with amounts between 31.50 (BpH) and 37.01 mg GAE/g (BbMW); a significantly lower TFC, ranging from 2.31 (BpMW) and 6.97 mg RE/g (BpM), was noticed. These results are in agreement with previous literature reports; for instance, different black pepper methanolic extracts displayed total phenolics and flavonoids levels between 36.71 and 58.90 mg GAE/g and 5.42 and 18.37 mg RE/g, respectively [56,66].
Overall, our data suggest that the spent plant materials after essential oil isolation can be confidently used for the efficient recovery of phenolics and flavonoids; a high extractability of these categories of phytochemicals could be a good indicator for a wide range of biological activities with putative applications in the management of diverse chronic diseases. Data are presented as mean ± standard deviation (SD) of three determinations; different superscript letters within columns indicate significant differences in the tested extracts for the same species (p < 0.05). Abbreviations: BpD-black pepper dichloromethane extract; BpH-black pepper hexane extract; BpM-black pepper methanol extract; BpMW-black pepper 50% aqueous methanol extract; CiD-cinnamon dichloromethane extract; CiHcinnamon hexane extract; CiM-cinnamon methanol extract; CiMW-cinnamon 50% aqueous methanol extract; ClD-clove dichloromethane extract; ClH-clove hexane extract; ClM-clove methanol extract; ClMW-clove 50% aqueous methanol extract; CuD-cumin dichloromethane extract; CuH-cumin hexane extract; CuM-cumin methanol extract; CuMW-cumin 50% aqueous methanol extract; GAE-gallic acid equivalents; LaD-laurel dichloromethane extract; LaH-laurel hexane extract; LaM-laurel methanol extract; LaMW-laurel 50% aqueous methanol extract; RE-rutin equivalents; TFC-total flavonoid content; TPC-total phenolic content.

Antioxidant Activity of Extracts and Essential Oils Obtained from Different Spices
The antioxidant properties of spices are largely known; besides their common use as food preservatives, spices display additional health benefits in the prevention and complementary treatment of oxidative stress-related disorders (e.g., cardiovascular diseases, neurodegenerative diseases, gastro-intestinal disorders, and cancers) [1,67]. Polyphenols and terpenoids are considered the main antioxidant compounds from spices that act as radical scavenging, reducing power, and metal chelating agents [3]. To evaluate the antioxidant capacity of the essential oils and their spent plant material extracts, several in vitro methods were undertaken, namely, 1,1'-diphenyl-2-picrylhydrazyl (DPPH) assay, 2,2 -azino-bis(3-ethylbenzothiazoline) 6-sulfonic acid (ABTS) assay, cupric ion reducing antioxidant capacity (CUPRAC) assay, ferric ion reducing antioxidant power (FRAP) assay, ferrous ion chelating ability (MCA) assay, and phosphomolybdenum assay (PBD). The obtained results are summarized in Table 5. By using an integrative approach, we report herein for the first time the antioxidant effects of spent spice material extracts derived after essential oil hydrodistillation from cinnamon, cumin, clove, laurel, and black pepper. In order to illustrate that the spent spice material is a valuable source of phytochemicals, we compared our results with literature data on unprocessed plant material. Still, even though several studies assessed the antioxidant potential of investigated spices, a direct comparison between the results obtained in the present study and literature data is hampered as a consequence of the implementation of different protocols and various ways of expressing the results.
All investigated extracts displayed variable radical scavenging abilities, as assessed by the DPPH and ABTS assays. Thus, cinnamon extracts showed significant radical scavenging effects, with CiMW exhibiting the highest activity in the DPPH test and CiEO in the ABTS assay (210.00 and 839.94 mg Trolox equivalents (TE)/g, respectively). The polar extracts (CiMW and CiM) were the most effective as scavenging agents, while the nonpolar extracts (CiH and CiD) were significantly less active (Table 5). A similar trend was observed by Saranya et al. [68], when the anti-DPPH radical effects of cinnamon extracts decreased in the following order: methanol (IC 50 = 11.9 µg/mL) > chloroform (IC 50 = 166.3 µg/mL) > hexane (IC 50 = 689.2 µg/mL). Regarding the cumin extracts, CuMW displayed the highest DPPH and ABTS radical scavenging potency, with values of 46.39 and 77.14 mg TE/g, respectively. The non-polar extracts were less potent, while CuEO showed no activity in the DPPH assay. All clove extracts were significantly effective as radical scavengers, with ClEO exhibiting the highest activity towards DPPH and ABTS radicals among investigated spices (values of 525.78 and 936.44 mg TE/g, respectively) ( Table 5). In addition, the spent material extracts were shown to exhibit anti-radical effects, with values ranging from 355.23 (ClD) to 488.45 mg TE/g (ClMW) in the DPPH test, and from 669.32 (ClD) to 770.12 mg TE/g (ClMW) in the ABTS assay. Previously, clove essential oil was reported to scavenge DPPH and ABTS radicals in a similar extent (EC 50 values of 0.40 and 0.42 mg/mL, respectively) to an ethanol extract (EC 50 values of 0.41 and 0.43 mg/mL, respectively) [69]. The DPPH scavenging ability of laurel extracts varied between 2.08 mg TE/g (LaH) and 194.14 mg TE/g (LaMW), while LaEO exhibited the highest activity in the ABTS assay (507.33 mg TE/g). Speroni et al. [70] documented the radical quenching capacity of methanol and chloroform laurel extracts (DPPH: IC 50 of 38 and 155 µg/mL; ABTS: 0.65 and 0.21 mmol TE/g). Within the black pepper extracts, the polar extracts BpMW and BpM were the most effective in scavenging synthetic radicals (DPPH: 45.41 and 32.41 mg TE/g; ABTS: 76.59 and 49.88 mg TE/g), followed by the non-polar extracts, BpH and BpD. In contrast, BpEO was less active compared to other extracts in the two assays (Table 5). In our previous study, we reported a similar radical quenching ability for the methanol extracts derived from Piper sp. (63.67-82.44 mg TE/g in DPPH assay and 61.63-77.60 mg TE/g in ABTS assay) [56].
The reducing potential, as assessed by CUPRAC and FRAP methods, varied largely between the investigated extracts. In the case of cinnamon extracts, their ability to reduce transition metals displayed a similar pattern and decreased in the following order: CiEO > CiMW > CiM > CiD > CiH, with the highest potential at 762.89 mg TE/g and 834.77 mg TE/g in the CUPRAC and FRAP assays, respectively. Moreover, the polar extracts showed greater electron donor capacity as compared to the non-polar extracts (Table 5). Similarly, previous reports on cinnamon extracts demonstrated their power reducing ability, with methanol extracts being more potent ferric ion reducers (0.74 molar ascorbic acid equivalents (MAAE)/g) when compared to non-polar solvent extractives, e.g., hexane (0.28 MAAE/g) [68]. Among the cumin extracts, the most effective reducing agents were found to be CuMW (118.66 mg TE/g) and CuEO (87.71 mg TE/g) in the CUPRAC and FRAP assays, respectively. The reducing potential of cumin essential oil and methanol extract were previously assessed by Einafshar et al. [71] using the FRAP method (459.4 and 18.47 mmol Fe 2+ /g, respectively). Clove extracts displayed a noticeable reducing power, with ClEO being the most active among all investigated spices (2848.28 mg TE/g and 1927.98 mg TE/g in the CUPRAC and FRAP assays, respectively). Nonetheless, the spent spice material extracts exhibited significant reducing potential, that varied between 753.39 (ClD) and 978.08 mg TE/g (ClMW) in the CUPRAC assay, and from 876.30 (ClM) to 1209.92 mg TE/g (ClH) in the FRAP assay. Compared to clove, laurel extracts were less active as transition metal reductants (Table 5). Their reducing power varied with a similar pattern in both assays, increasing from 28.58 (LaH) to 416.67 mg TE/g (LaEO) in the CUPRAC test, and between 14.11 (LaH) and 435.97 mg TE/g (LaEO) in the FRAP test. Besides, the polar fractions were more active compared to non-polar extracts, displaying up to a 10-fold and 14-fold activity increase in the CUPRAC and FRAP assays, respectively (Table 5). In accordance to our study, Carlsen et al. [72] reported superior reducing effects for clove compared to laurel 50% methanol extracts as evaluated by FRAP method (273.3 vs. 27.8 mmol TE/100 g). The reducing properties of black pepper extracts varied from 51.22 (BpM) to 176.06 mg TE/g (BpEO) in the CUPRAC assay, while in the FRAP assay activity values from 14.11 (LaH) to 435.97 mg TE/g (LaEO) were noticed. In our previous study on Piper species, the methanolic extracts were shown to be active in both CUPRAC and FRAP assays, their reducing capacity varying from 37.36 to 140.52 mg TE/g, and from 16.05 to 77.00 mg TE/g, respectively [56]. Moreover, a comprehensive report on antioxidant effects of 39 spices revealed the reducing potency (as evaluated by CUPRAC method) of methanol extracts obtained from cinnamon (53.65 mg TE/g), cumin (1.71 mg TE/g), clove (54.47 mg TE/g), laurel (11.34 mg TE/g), and black pepper (2.09 mg TE/g) [73].
To summarize, the spent plant material extracts of the studied spices exhibited significant antioxidant properties, with different degrees of activity. It is noteworthy to mention the remarkable antioxidant effects of cinnamon and clove extracts, as revealed by the radical scavenging capacity and reducing power assays (Table 5). In accordance with our study, Assefa et al. [73] highlighted the clove and cinnamon extracts among 39 common spices as notable sources of antioxidants able to prevent the deleterious effects induced by oxidative stress. As depicted in Figure 1, significant correlations between TPC and free radical scavenging activity, reducing power, and total antioxidant capacity were established (r > 0.8). Similarly, several studies have demonstrated a significant correlation between antioxidant properties of spices and their total amount of phenolic compounds [47,73,79]. No relevant correlation was found between TFC and the observed antioxidant effects (r < 0.37). Therefore, we can assume that phenolic compounds are the main contributors to the overall antioxidant activity of investigated extracts. In addition, TPC and TFC did not correlate with metal-chelating activity, thus other constituents might be responsible for the chelation capacity of the spent spice materials.

Anti-Enzymatic Activity of Extracts and Essential Oils Obtained from Different Spices
For a better overview on the potential involvement of cinnamon, cumin, clove, laurel, and black pepper in the management of different chronic diseases, we next investigated the in vitro anti-enzymatic activity of the essential oils as well as of their spent spice material extracts ( Table 6). As target enzymes, AChE, BChE, tyrosinase, amylase, and glucosidase were selected, since they are considered to be directly linked to the etiopathogeny of Alzheimer's disease, skin hyperpigmentation, or type II diabetes mellitus [56]. To the best of our knowledge, there are no previous comprehensive studies assessing the multienzymatic properties of these five spices. Similar to the antioxidant assays, we compared our results with literature data on unprocessed plant material.
The anti-AChE effects of cinnamon extracts varied between 1.14 mg galanthamine equivalents (GALAE)/g (CiD) and 2.01 mg GALAE/g [58], whereas slightly better anti-BChE properties were noticed, especially for CiM extract (Table 6). Interestingly, CiEO was somehow active against AChE, but inactive against BChE, which could indicate a potential selectivity against the two enzymes. Previously, an ethanol cinnamon extract (200 µg/mL) was shown to exhibit 54.30% and 66.43% inhibition against AChE and BChE, respectively [80]. Regarding cumin extracts, the non-polar extracts (CuH and CuD) showed the highest AChE and BChE inhibitory activities, with the other three samples being inactive, especially against BChE. Furthermore, CuH displayed the highest anti-AChE effects from all tested spices (2.95 mg GALAE/g). Mahnashi et al. [81] showed considerable inhibitory activities against AChE and BChE with IC 50 values of 198 and 37 µg/mL, respectively. The anti-cholinesterase activity of clove extracts varied in the following order: ClEO > ClH > ClD > ClM > ClMW, with the highest inhibitory capacity at 2.77 and 2.97 mg GALAE/g in AChE and BChE assays, respectively (Table 6). In a previous study, IC 50 values of 49.73-61.50 and 88.14-103.53 µg/mL were reported for a clove methanol extract and essential oil in AChE and BChE assays, respectively [82]. The non-polar LaH extract exhibited the highest anti-BChE effects from all analyzed spices (3.41 mg GALAE/g); in contrast, LaEO displayed very low BChE inhibitory properties (0.41 mg GALAE/g). With respect to the anti-AChE activity, the effects varied from 1.04 (LaMW) to 2.47 mg GALAE/g (LaH). Ferreira et al. [83] showed that the laurel essential oil, aqueous, and ethanol extracts produced AChE inhibitions of 51.3% 19.9%, and 48.4%, respectively, at 0.5 mg/mL; there was no information about their BChE inhibitory properties. Lastly, BpD exhibited the highest anti-AChE ability (2.55 mg GALAE/g), whereas the highest anti-BChE effects (2.46 mg GALAE/g) were noticed for BpMW. In a previous study, Luca et al. [56] showed that the AChE and BChE inhibitory activity of various pepper samples varied from 0 to 2.35 mg GALAE/g and 0.60 to 3.11 mg GALAE/g, respectively.
When assessing tyrosinase inhibition (Table 6), it was noticed that the polar CiMW and CiM extracts clearly displayed the most potent effects (66.45 and 61.56 mg kojic acid equivalents (KAE)/g, respectively), which were 2.5-3.6-fold higher than the remaining cinnamon samples. Previously, Tamfu et al. [80] showed that an ethanol cinnamon extract (200 µg/mL) exhibited a 45.37% inhibition against tyrosinase. Cumin extracts exerted a comparable anti-tyrosinase activity, with the highest effects also noticed in the polar extracts CuM (58.05 mg KAE/g) and CuMW (63.12 mg KAE/g). Mukherjee et al. [84] and Gholamhoseinian and Razmi [85] reported tyrosinase inhibitions of 52% and 64.17% for methanol cumin extracts at 1.14 and 1 mg/mL, respectively. The tyrosinase inhibitory properties of clove extracts varied between 20.79 (ClH) and 59.23 mg KAE/g (ClMW), with ClEO showing an inhibition of only 38.16 mg KAE/g. The anti-tyrosinase abilities of clove have been scarcely reported; for instance, Ahmed et al. [86] compared the activity of different clove extracts; with respect to the IC 50 values, the following order was observed: essential oil (12.1 µg/mL) > hexane extract (17.4 µg/mL) > methanol extract (30.1 µg/mL) > ethanol extract (65.3 µg/mL). Within the series of laurel extracts, LaEO, LaM, and LaMW showed anti-tyrosinase effects to different extents (22.48-43.38 mg KAE/g), whereas the non-polar extracts were inactive. Deniz et al. [87] noticed that the 80% ethanol extracts of laurel exhibited a low tyrosinase inhibition (34.09% at 666 µg/mL). With respect to the black pepper extracts, it was observed that the non-polar extracts (BpH and BpD) had the most potent anti-tyrosinase abilities; in contrast, BpEO was the weakest tyrosinase inhibitor (Table 6). Previously, it was reported that black pepper extracts were able to significantly down-regulate mushroom tyrosinase (monophenolase and diphenolase) as well as murine tyrosinase [56].
Regarding the amylase and glucosidase inhibition, the five spice extracts displayed weak inhibitory properties; generally, they exhibited slightly better anti-glucosidase effects than anti-amylase effects. From all tested samples, LaEO presented the most notable amylase inhibition (1.29 mg acarbose equivalents (ACAE)/g) followed by BpH and BpD (1.00 and 0.99 mg ACAE/g, respectively); CiEO, CuEO, and BpEO were inactive against the same enzyme. From all essential oils, only ClEO was able to inhibit glucosidase (1.00 mg ACAE/g); nonetheless, the spice residue extracts showed lower anti-glucosidase effects (max. 0.88-0.89 mg ACAE/g in CiH, CiD, ClM, and LaM) ( Table 6). The modulation of the two anti-diabetic enzymes by different spices was found to be in agreement with our data; for example, several cinnamon aqueous extracts were shown to inhibit amylase, maltase, and sucrose, with IC 50 values of 1.23-1.77, 0.58-1.96, and 0.42-2.96 mg/mL, respectively [88], whereas a cumin ethanol extract displayed inhibitory effects against glucosidase of 45% at 100 µg/mL [89]. An 80% aqueous acetone clove extract was reported to exhibit anti-glucoside and anti-amylase effects, with IC 50 values of 145.07 and 497.27 µg/mL, respectively [90], whilst the clove essential oil displayed IC 50 values of 71.94 and 88.89 µg/mL against the same two enzymes [91]. Moreover, a laurel extract exerted a 47.26% inhibition against glucosidase at 200 µg/mL [92], whereas different black pepper extracts showed weak anti-glucosidase effects of 0.84-1.22 mmol ACAE/g [56].

Plant Materials
Dried plant materials (cinnamon barks, cumin fruits, clove buds, laurel leaves, and black pepper fruits) were purchased from pharmacies and local markets and their botanical identity was confirmed by one of the authors (A.T.). Voucher specimens (Ci 1/2020, Cu 2/2020, Cl 3/2020, La 4/2020, Bp 5/2020) were deposited in the Department of Pharmacognosy, Faculty of Pharmacy, "Grigore T. Popa" University of Medicine and Pharmacy Iasi, Romania.

Essential Oil Isolation
The powdered plant materials (50 g each) were subjected to hydrodistillation for 3 h in a Clevenger-type apparatus. EOs were dried over anhydrous sodium sulfate and stored in dark glass tubes at a temperature of 4 • C until further analysis.

Preparation of Spent Plant Material Extracts
The spent plant materials (after essential oil isolation) were dried at 40 • C (48 h) and further extracted using solvents of different polarities (hexane, dichloromethane, methanol, and 50% aqueous methanol) by ultra-sonication (3 cycles of 30 min each, at room temperature). The obtained extracts were evaporated to dryness under vacuum (with the yields provided in Table 1) and kept at −20 • C until subsequent analysis.

GC-MS and GC-FID Analysis
An Agilent 6890N gas chromatograph [93] coupled with a mass spectrometer (MS) detector (Agilent model 5975 inert XL) and a flame ionization detector (FID) was used to assess the qualitative and quantitative profile of essential oils isolated from investigated spices. TRB-5MS capillary columns (30 m length, 0.25 mm internal diameter, 0.25 µm film thickness) were used for each detector. The analysis was conducted according to a previously described method [94]. Retention indices were calculated for the individual compounds using a standard mixture of C8-C20 n-alkanes. The compounds from the essential oils were identified by comparison of their mass spectra with those from the NIST 11 Mass Spectra Library, and their retention indices with literature data (NIST Chemistry WebBook; [94,95]). The relative percentages of individual compounds were obtained from the FID peak areas without using correction factors.

LC-HRMS/MS Analysis
The LC-HRMS/MS was performed on an Agilent 1200 HPLC system (Agilent Technologies, Palo Alto, CA, USA) equipped with a binary pump (G1312C), column thermostat (G1316A), auto-sampler (G1329B), and accurate-mass quadrupole-time-of-flight MS detector (G6530B). The separations were carried out on a Phenomenex Gemini C18 column (2 × 100 mm, 3 µm), with phase A (0.1% formic acid in water) and phase B (0.1% formic acid in acetonitrile); the mobile phase gradient was as follows: 10-60% B (0-45 min) and 90% B (46-50 min); flow rate 0.2 mL/min; injection volume 2 µL. The following MS parameters were set: electrospray ionization source (ESI); full-scan high-resolution accurate-mass acquisition mode; negative and positive ionization modes; m/z range 50-1000; N 2 flow rate 12 L/min, vaporizer temperature 350 • C, nebulizer pressure 40 psi, capillary voltage 4000 V, skimmer 65 V, fragmentor 140 V, and collision-induced dissociation (CID) energy 40 V. Data acquisition and analysis were achieved with MassHunter Workstation Data Acquisition 8.0 and Qualitative Navigator 8.0, respectively. The assignment of the peaks observed in the base peak chromatograms (BPC) of the different spice extracts was performed by comparing the spectrometric data with previous literature data reporting on the LC-MS analysis of similar constituents or online databases (METLIN, KNApSacK, PubChem, NIST Chemistry WebBook).

TPC, TFC, Antioxidant and Anti-Enzymatic Assays
TPC and TFC were determined according to previously described methods [96,97] and expressed as mg GAE/g (TPC) and mg RE/g (TFC). DPPH, ABTS, CUPRAC, and FRAP were performed as in [96,97], with the results presented as mg TE/g. MCA and PBD were carried out as described in [96,97], with the data provided as mg EDTAE/g (MCA) and mmol TE/g (PBD). AChE, BChE, tyrosinase, amylase, and glucosidase inhibition methods were detailed in [96,97]. The anti-enzymatic activities were expressed as mg GALAE/g in AChE and BChE assays, mg KAE/g d.w. in tyrosinase assay and mmol ACAE/g d.w. in amylase and glucosidase assays.

Statistical Analysis
All the experiments were performed in three replicates, with the results presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) with Turkey's post hoc test was conducted; p < 0.05 was considered statistically significant. The correlation analysis between TPC, TFC, and biological activities was reported as Pearson's coefficients, calculated using R software (v. 3.6.2).

Conclusions
The GC-MS and GC-FID analyses of the essential oils obtained from five common spices pointed out several phytochemical markers: cinnamaldehyde in cinnamon; cuminaldehyde and safranal in cumin; eugenol in clove; eucalyptol in laurel; and α-terpinene, limonene, β-caryophyllene, and β-pinene in black pepper. On the other hand, the LC-HRMS/MS profiling of the spent plant materials extracted with different polarity solvents revealed a complex phytochemical composition of the same spices: organic acids, phenolic acids, proanthocyanidins, and oxygenated fatty acids in cinnamon; flavonoids (free aglycons or glycosides of luteolin and apigenin) in cumin; flavonoids (free aglycons or glycosides of quercetin, isorhamnetin, luteolin, chrysoeriol, cirsiliol, cirsimaritin) and hydrolysable tannins in clove; proanthocyanidins and flavonoids (free aglycons and glycosides of quercetin, isorhamnetin and kaempferol) in laurel; and piperamides (piperine, piperlonguminine, pellitorine, pipernonaline, retrofractamide B, guineensine and N-isobutyl-dodecadienamide) in black pepper.
The comparative assessment of the antioxidant potential of the essential oils and spent spice material extracts indicated a very strong activity for all samples, as depicted from the significantly high values obtained in radical scavenging (ABTS, DPPH), metal chelating (MCA), reducing (CUPRAC, FRAP), and PBD tests. Interestingly, significant correlations between TPC and antioxidant activity of spent extracts were observed, with no relevant correlations between their TFC and the antioxidant effects. Lastly, the anti-enzymatic assays unveiled that the investigated spices exhibited a mild modulation of several key enzymes (cholinesterases, tyrosinase, glucosidase, and amylase) targeted in the management of chronic diseases, such as Alzheimer's disease, type 2 diabetes mellitus, or skin disorders.
Overall, our study shows that, not only the volatile fractions of common spices, such as cinnamon, cumin, clove, laurel, and black pepper, but also their spent plant materials remained after hydrodistillation, and often discarded by the essential oil industry, can be further considered as rich sources of exploitable bioactive molecules endowed with antioxidant and multi-enzymatic inhibitory activities.