Neuroprotective Effect of Yucca schidigera Roezl ex Ortgies Bark Phenolic Fractions, Yuccaol B and Gloriosaol A on Scopolamine-Induced Memory Deficits in Zebrafish

Y. schidigera contains a number of unusual polyphenols, derivatives of resveratrol and naringenin, called spiro-flavostilbenoids, which have potent in vitro anti-inflammatory, antioxidant, and moderate cholinesterase inhibitory activities. To date, these compounds have not been tested in vivo for the treatment of neurodegenerative diseases. The aim of the present study was to evaluate the effects of both single spiro-flavostilbenoids (yuccaol B and gloriosaol A) and phenolic fractions derived from Y. schidigera bark on scopolamine-induced anxiety and memory process deterioration using a Danio rerio model. Detailed phytochemical analysis of the studied fractions was carried out using different chromatographic techniques and Nuclear Magnetic Resonance (NMR). The novel tank diving test was used as a method to measure zebrafish anxiety, whereas spatial working memory function was assessed in Y-maze. In addition, acetylcholinesterase/butyrylcholinesterase (AChE/BChE) and 15-lipooxygenase (15-LOX) inhibition tests were performed in vitro. All pure compounds and fractions under study exerted anxiolytic and procognitive action. Moreover, strong anti-oxidant capacity was observed, whereas weak inhibition towards cholinesterases was found. Thus, we may conclude that the observed behavioral effects are complex and result rather from inhibition of oxidative stress processes and influence on cholinergic muscarinic receptors (both 15-LOX and scopolamine assays) than effects on cholinesterases. Y. schidigera is a source of substances with desirable properties in the prevention and treatment of neurodegenerative diseases.


Introduction
Yucca schidigera Roezl ex Ortgies (Mojave/Mohave yucca) of the Asparagaceae family is native to the southwestern United States, Mexico and grows in hot and arid climates.
The tested gloriosaols had significantly stronger activity than the reference compound (quercetin), Yus A-E and RV [14,15].
In fact, the biological activity of spiro-flavostilbenoids is still undiscovered and requires in-depth studies, which is supported by the research results so far. On the other hand, their biosynthetic precursors, namely RV and naringenin, have been well studied in in vitro, ex vivo, in vivo and clinical trials for the prevention and treatment of neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD) [16]. Importantly for the treatment of these disorders, our recent study showed weak and moderate inhibitory activity of Yus and Glos against acetylcholinesterase (AChE) and butyrylocholinesterase (BChE) in in vitro assays, respectively, [7] and YuB and GloA (Figure 1) showed the highest inhibitory capacity against both enzymes. Their IC 50 values against BChE were almost 2-fold (GloA) and 1.5 (YuB) lower than those of galantamine [7].
Molecules 2022, 26, x FOR PEER REVIEW 3 of 19 result, the fraction together with individual spiro-flavostilbenoids and RV showed significant activity, higher than the standard compound BHT (2,6-di-tert-butyl-4-methoxyphenol) at 120 min of assay. THMS was inactive in this model. In contrast, THMS and its spiroderivatives, Glos A-E, exhibited strong radical scavenging activity in the ABTS •+ assay. The tested gloriosaols had significantly stronger activity than the reference compound (quercetin), Yus A-E and RV [14,15]. In fact, the biological activity of spiro-flavostilbenoids is still undiscovered and requires in-depth studies, which is supported by the research results so far. On the other hand, their biosynthetic precursors, namely RV and naringenin, have been well studied in in vitro, ex vivo, in vivo and clinical trials for the prevention and treatment of neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD) [16]. Importantly for the treatment of these disorders, our recent study showed weak and moderate inhibitory activity of Yus and Glos against acetylcholinesterase (AChE) and butyrylocholinesterase (BChE) in in vitro assays, respectively, [7] and YuB and GloA ( Figure  1) showed the highest inhibitory capacity against both enzymes. Their IC50 values against BChE were almost 2-fold (GloA) and 1.5 (YuB) lower than those of galantamine [7]. Our current study was designed to evaluate the neuropharmacological effects of spiro-flavostilbenoids and phenolic fractions derived from YS bark on scopolamine (Sco)induced models of anxiety and amnesia in zebrafish (Danio rerio). In vivo bioassays were associated with in-vitro AChE/BChE and 15-LOX inhibition assays. This study is the first to evaluate the neuroprotective potential of YS phenolics. Either single spiro-flavostilbenoids-YuB and GloA-or phenolic fractions i.e., unpurified (YS unpur), purified (YS pur) and polymeric fractions (YS poly) were tested. Detailed phytochemical analysis of the studied fractions was carried out using different chromatographic techniques and NMR studies.

Chemical Characterization of Y. schidigera Fractions. Quantitative Analyses
Phenolic fractions of YS i.e., YS unpur, YS pur, YS poly and single metabolites YuB and GloA were isolated from powdered yucca bark. The YS pur and YS poly were obtained from the unpurified fraction using the combination of gel filtration and silica gel chromatography. As a result, 10 g of YS unpur yielded 5.95 g of YS pur and 3.03 g of YS poly. In YS bark, the percentages of YS unpur, YS pur and YS poly fractions were 5.15%, 3.09% and 1.55%, respectively.

Chemical Characterization of YS Unpur and YS Pur
Chemical characterization of YS unpur and YS pur phenolic fractions was performed using UHPLC-UV-MS analysis. Phenolic metabolites were identified from retention times Our current study was designed to evaluate the neuropharmacological effects of spiroflavostilbenoids and phenolic fractions derived from YS bark on scopolamine (Sco)-induced models of anxiety and amnesia in zebrafish (Danio rerio). In vivo bioassays were associated with in-vitro AChE/BChE and 15-LOX inhibition assays. This study is the first to evaluate the neuroprotective potential of YS phenolics. Either single spiro-flavostilbenoids-YuB and GloA-or phenolic fractions i.e., unpurified (YS unpur), purified (YS pur) and polymeric fractions (YS poly) were tested. Detailed phytochemical analysis of the studied fractions was carried out using different chromatographic techniques and NMR studies.

Chemical Characterization of Y. schidigera Fractions. Quantitative Analyses
Phenolic fractions of YS i.e., YS unpur, YS pur, YS poly and single metabolites YuB and GloA were isolated from powdered yucca bark. The YS pur and YS poly were obtained from the unpurified fraction using the combination of gel filtration and silica gel chromatography. As a result, 10 g of YS unpur yielded 5.95 g of YS pur and 3.03 g of YS poly. In YS bark, the percentages of YS unpur, YS pur and YS poly fractions were 5.15%, 3.09% and 1.55%, respectively.

Chemical Characterization of YS Poly
Understanding the chemical composition of the YS poly fraction, which represents 1 / 3 of the YS unpur, was problematic due to, among other things, the lack of chromatographic peaks during LC-UV-MS analyses and after thiolysis [17], typically used to study the chemical composition of proanthocyanidins by depolymerization (data not shown). The lack of released flavan-3-ol monomers during thiolysis led us to think that YS poly is rather a polymer of THMS and naringenin, like the spiro-flavostilbenoids that predominate the YS unpur and YS pur fractions.
Identification of such a complex mixture of compounds is beyond the scope of this publication, nevertheless we decided to perform 2D 1 H-13 C HSQC and 1 H-13 C HMBC NMR analyses, which gave the information regarding different stilbene (St) and flavanone (FA and FB) units present in the polymeric fraction of YS ( Figure 2). Signals from methoxy (MeO) belonging to THMS stilbenic moiety at δ C /δ H 60.66/3.56-3.86 and parahydroxyphenyl groups from ring B of flavanone moiety (FB) from C 2,6 /H 2,6 at δ C /δ H 127.6-128.9/6.65-7.36 and C 3,5 /H 3,5 at δ C /δ H 115.6/6.30-7.00 are dominant in the HSQC spectra, accompanied by cross peak from flavanone ring A (FA) at δ C /δ H 96.9/5.87-6.00 (Figure 2A). The HMBC spectrum, on the other hand, shows the connection between the methoxy group and the signal of C 1 (St 1 ) at δ C 136.1, and from protons FB 2/6 to carbon FB 4 at δ C 157.3-158.0 ( Figure 2B).
The HMBC spectrum, on the other hand, shows the connection between the methoxy group and the signal of C1 (St1) at δC 136.1, and from protons FB2/6 to carbon FB4 at δC 157.3-158.0 ( Figure 2B).

In Vivo Bioassays
In vivo studies evaluated the memory and anxiety-like behaviors exerted by YS pur and YS poly, and pure YuB and GloA in a model of Sco-induced cognitive deficits and

In Vivo Bioassays
In vivo studies evaluated the memory and anxiety-like behaviors exerted by YS pur and YS poly, and pure YuB and GloA in a model of Sco-induced cognitive deficits and increase in anxiety level in zebrafish (Danio rerio). Y-maze (memory evaluation) and NTT (anxiety assessment) tests were performed.

Y-Maze Test
The locomotor tracking pattern in the control group was demonstrated by normal swimming all over the Y-maze tank ( Figure S1). The Sco group exhibited deficits in the response to novelty, which is represented by abnormal tracking pattern, as assessed by less exploration in the novel arm. The locomotor tracking pattern of YS pur-and YS poly-, YuB-and GloA-treated groups at the doses (1, 3 and 5 µg/L) showed attenuation of Sco effect and a swimming pattern nearly similar to control group as shown in Figure S1, Supplementary Materials. Figure 3a shows that YS pur affected memory and locomotor activity in Sco-treated zebrafish in the Y-maze test. Two-way ANOVA showed significant effect of the treatment (F (4, 135) = 4.775; p = 0.0012), time spent on arms (F (2, 135) = 7.644; p = 0.0007 and interactions (F (8, 135) = 4.345; p = 0.0001). Tukey's post hoc test confirmed that Sco decreased the time spent in the novel arm (p < 0.05) in comparison with control group. As Sco-exposed zebrafish explored the novel arm less, this suggests deficits in response to novelty and impaired memory performance. YS pur significantly improved memory deficits as evidenced by increased time spent in the novel arm (1 and 5 µg/L-p < 0.01 and 3 µg/L-p < 0.05) compared with the Sco-treated group. Moreover, a statistically significant effect of treatment was observed in total distance traveled (one-way ANOVA F (4, 45) = 11.21, p < 0.0001) and turn angle (one-way ANOVA F (4, 45) = 9.46, p < 0.0001). Hypolocomotion induced by Sco treatment was observed as a decrease in distance travelled (p < 0.01) and turn angle (p < 0.001). YS pur prevented Sco-induced hypolocomotion by significantly increasing the total distance traveled in the tank (p < 0.001 for 1 µg/L, p < 0.0001 for 3 µg/L and p < 0.01 for 5 µg/L) and turn angle (p < 0.001 for 1 µg/L, p < 0.0001 for 3 µg/L and p < 0.001 for 5 µg/L) compared to the Sco-treated group. Figure 3b shows that YS poly affected memory and locomotor activity in Sco-treated zebrafish in the Y-maze test. Two-way ANOVA showed significant effect of the time spent in arms (F (2, 162) = 9.118, p = 0.0002) and interaction (F (10, 162) = 4.833; p < 0.0001) without treatment (F (5, 162) = 0.5385; p = 0.7469). Tukey's post hoc test confirmed that Sco decreased the time spent in the novel arm (p < 0.05) in comparison with control group. YS poly significantly increased the time spent in the novel arm in a dose-dependent manner (3 µg/L-p < 0.01 and 5 µg/L-p < 0.0001) compared to Sco-treated group. Moreover, the statistically significant effect of treatment was observed in total distance traveled (one-way ANOVA F (4, 45) = 27.14, p < 0.0001) and the turn angle (one-way ANOVAF (4, 45) = 13.42, p < 0.0001). Sco induced decrease in the distance travelled and turn angle (p < 0.0001). YS poly prevented Sco-induced hypolocomotion by significantly increasing total distance traveled in the tank and turn angle (p < 0.01 for 1 µg/L, p < 0.0001 for 3 µg/L and 5 µg/L) as compared with Sco-treated group. Figure 3c shows that YuB affected memory and locomotor activity in Sco-treated zebrafish in the Y-maze test. Two-way ANOVA showed a significant effect of the time spent in arms (F (2, 162) = 31.45; p < 0.0001)) and interaction (F (10, 162) = 5.287; p < 0.0001) without treatment (F (5, 162) = 0.5616; p = 0.7293). Tukey's post hoc test confirmed that Sco decreased the time spent in the novel arm (p < 0.05) in comparison with control group. YuB significantly increased the time spent in the novel arm (3 µg/L-p < 0.01 and 5 µg/Lp < 0.0001) as compared with Sco-treated group. Moreover, the statistically significant effect of treatment was observed in total distance traveled (one-way ANOVA F (4, 45) = 20.17, p < 0.0001) and the turn angle (one-way ANOVA F (4, 45) = 15.86, p < 0.0001). Sco treatmentinduced hypolocomotion was observed as decreased distance travelled (p < 0.001) and turn angle (p < 0.0001). YuB prevented Sco-induced hypolocomotion, significantly increasing the total distance traveled in the tank and turn angle at all concentrations (p < 0.0001) as compared with Sco-treated group.  Figure 3d shows that GloA affected memory and locomotor activity in the Sco-treated group in the Y-maze test. Two-way ANOVA showed a significant effect of the time spent in arms (F (2, 162) = 31.45; p < 0.0001) and interactions (F (10, 162) = 5.287; p < 0.0001) without treatment effect (F (5, 162) = 0.5616; p = 0.7293). Tukey's post hoc test confirmed that Sco decreased the time spent in the novel arm (p < 0.05) in comparison with control group. GloA significantly improved memory deficits induced by Sco observed as an increase in the time spent in the novel arm (3 µg/L-p < 0.01 and 5 µg/L-p < 0.0001) as compared with Sco-treated group. Moreover, the statistically significant effect of treatment was observed in total distance traveled (one-way ANOVA F (4, 45) = 12.25, p < 0.0001) and the turn angle (one-way ANOVA F (4, 45) = 12.44, p < 0.0001). Sco treatment-induced hypolocomotion was observed as decreased distance travelled (p < 0.001) and turn angle (p < 0.0001). GloA prevented Sco-induced hypolocomotion by significantly increasing the total distance traveled in the tank (p < 0.001 for 1 µg/L, p < 0.0001 for 3 µg/L and 5 µg/L) and turn angle (p < 0.01 for 1 µg/L, p < 0.0001 for 3 µg/L and 5 µg/L) as compared with Sco-treated group.
All YS preparations tested exhibited memory-enhancing properties in the Y-maze task. The improvement in cognitive function was accompanied by an increase in locomotor activity.

Novel Tank Diving Test (NTT)
In the NTT test, Sco treatment significantly decreased the time spent in the top zone of the tank (p < 0.0001) as compared with the control group, suggesting Sco-induced anxiogenic effect (Figure 4). Sco treatment induced a hypolocomotor effect as compared with the control group observed as a decrease in the distance top/bottom ratio (p < 0.01) and the velocity (p < 0.001).   In YS pur and YS poly and pure compounds the anxiolytic-like effect was noticed by increasing the time spent in the top zone of the tank (p < 0.001) as compared with the Sco-alone treated animals. Moreover, treatment with the tested fractions and compounds (1, 3 and 5 µg/L) prevents the hypolocomotor effect of Sco on the velocity (p < 0.001) as compared with Sco-alone-treated fish. An increase in the distance top/bottom ratio was also observed for YS pur, YS poly, and YuB (p < 0.001) and GloA (p < 0.001 for 1 and 3 µg/L, p < 0.05 for 5 µg/L).
Overall, YS pur and YS poly as well as YuB and GloA showed anxiolytic effects. The tested YS fractions/compounds were safe for zebrafish at the selected doses.

Discussion
Numerous studies show the implication of oxidative stress in the physiopathology of neurodegenerative diseases such as AD and PD. The imbalance between pro-oxidant compounds and antioxidant endogenous systems can be re-established by, for example, administering plant extracts rich in polyphenol carboxylic acids, flavonoids and anthocyanins [18]. In our experiments, we focused on 15-LOX, an enzyme which catalyzes the oxidation of unsaturated fatty acids, leading to the formation of peroxides involved in oxidative stress, inflammation and neurodegeneration [19,20]. YS unpur, YS pur, YS poly, YuB and GloA showed a strong in vitro 15-LOX inhibition capacity, stronger compared to vitamin C. The results obtained are consistent with previous studies [13][14][15]. This can be explained by the presence of compounds with hydrophobic functional groups, which are able to reach the active site of the enzyme, as well as the presence of compounds with hydrophilic groups, which donate protons and electrons and block the redox reaction. The active phenolic metabolites of YS can block the enzyme through various mechanisms: (1) by blocking the reversible redox conversion Fe 2+ → Fe 3+ -compounds that donate hydrogen act as inhibitors in this mechanism; (2) by blocking access of the substrate (linoleic acid) to the enzyme active site-this applies to compounds with significant and large lipophilic groups; (3) by modifying the spatial structure of the enzyme, which indirectly leads to the modification of the enzyme's active site and subsequently to the lack of interaction between enzyme and substrate-attributed to compounds with large lipophilic groups, compounds with positively or negatively ionized functional groups, and compounds capable of forming hydrogen bonds with functional groups present in the enzyme structure [19].
Yucca phenolic fractions showed weak in vitro inhibition against AChE and moderate inhibition against BChE. These results correspond with our previous studies [7] on the inhibitory capacity of pure YS spiro-flavostilbenoids, stilbenoids and flavonoids. In brief, flavonoids (naringenin, dihydrokaempferol) and one of the major stilbenoids, THMS, did not inhibit cholinesterase activity (Table S4). The major spiro-flavostilbenoids, namely: YuC and YuD showed very low activity, a galantamine-like effect was obtained at 74/1.9 times higher doses (for AChE/BChE) of YuC, and 193/5.5 of YuD. YuB and GloA were the most active isolates, their effective concentrations compared to galantamine were 19 and 20 times higher for AChE, respectively. In contrast, YuB and GloA were more active than galantamine in assays with BChE. Cholinesterase inhibitors have significant beneficial effects on attention, information acquisition, memory and language, but do not improve visuospatial abilities. Intense inhibition of these enzymes may worsen symptomatology in patients with cognitive deficits and PD [21].
Since weak cholinesterase inhibition was observed, we decided to search for mechanisms other than cholinergic ones underlying the behavioral effects of the tested fractions and compounds. Accordingly, we were first to investigate the effects of yucca spiroflavostilbenoids on Sco-induced memory deterioration and anxiogenic effects in adult zebrafish. Sco, by blocking cholinergic muscarinic receptors, affects the cholinergic system. The negative effects of Sco (100 µM) i.e., decreased locomotor activity, deficits in response to novelty, increased anxiety, were attenuated in zebrafish that were incubated with YS pur, YS poly, YuB and GloA (at concentrations of 1, 3, 5 µg/L). Yucca preparations were active in both Y-maze and NTT-tests. No visible developmental defects were observed after incubation with the tested substances, suggesting that YS phenolic fractions and pure compounds are not toxic. This is consistent with the GRAS status of the YS products.
To our knowledge, there are no literature data on the neuroprotective properties of spiro-flavostilbenoids and yucca phenolics tested in vivo. YS spiro-flavostilbenoids are composed of THMS/RV and naringenin units, as well as polymers-THMS and naringenin. Based on scientific reports [22][23][24], we hypothesized that dimers, trimers, i.e., spiroflavostilbenoids and their polymers are metabolized by the gut microbiota to monomeric units that reach the blood serum and brain [16,25], causing the observed biological effect of YS phenolics. Wang et al. described that the a grape-derived polyphenolic preparation was metabolized to corresponding monomeric forms of catechin and epicatechin conjugates (glucuronides and methylated ones), which exerted direct neuroprotective effects [22]. The monomeric derivatives were accumulated from blood into mouse brain tissue, and the bioavailability of methylated glucuronides was higher. On the other hand, the same authors pointed out the lack of activity of oligo-and polymeric fractions, due to the low bioavailability of catechin and epicatechin conjugates. This indicates different absorption mechanisms of spiro-flavostilbenoids compared to flavan-3-ols.
Our results indicate a positive effect of YS pur and YS poly fractions along with YuB and GloA on Sco-induced memory deterioration and anxiogenic effects. Both pure compounds (YuB and GloA) and fractions (YS pur and YS poly) contain single or mixtures of compounds with similar chemical structures (spiro-flavostilbenoids) which may have been converted into similar metabolites by intestinal metabolism of zebrafish microbiota, and then these metabolites were distributed to fish brains through the blood-brain barrier (BBB). This theory may be supported by the high activity of YS poly, which contains mostly medium-to-high molecular weight compounds and thus is most likely incapable of passively penetrating the BBB [26]. However, to know what compounds actually penetrated the BBB requires further studies to be performed. The obtained in-vitro and in-vivo results highlight the neuroprotective action of yucca phenolics and link it to antioxidant and anti-inflammatory activities, as well as to muscarinic receptor activation.
The mentioned neuroprotective mechanism is also reported for RV and its derivatives [16,18]. RV exhibits potent anti-oxidant activity (scavenges reactive oxygen species, increases glutathione levels, improves endogenous antioxidants), modulates neuroinflammation (attenuates inflammatory mediators NO, TNF-α, IL, MCP-1, reduces NF-kB transcription and microglia activation) and prevents neuronal death by activating SIRT1 [27], and activates cholinergic pathway [16]. Methoxylation of RV units (e.g., pterostilbene) enhances the metabolic stability and bioavailability due to increased lipophilicity, cellular uptake, and oral absorption compared to RV [16]. Pterostilbene improves cognitive deficits in the older rodents (19-month-old male Fischer 344 rats) in the Moris water maze test by increasing muscarinic receptor sensitivity and calcium buffering [28]. An in vivo study showed that dietary supplementation with pterostilbene or RV improved cognitive function in mice with age-related AD (SAMP8) [25]. Pterostilbene was more potent than RV in activating endogenous antioxidant (increased manganese superoxide dismutase (MnSOD) activity) and anti-inflammatory (reduced NFκβ p65 levels) mechanisms by up-regulating peroxisome proliferator-activated receptor (PPAR) alpha activity. Moreover, tau phosphorylation was decreased by pterostilbene, not RV.
The neuroprotective effects of naringenin are well studied in in vitro and in vivo models, and its multi-target mechanism of action is well documented [29]. Zhang et al. [30] found that naringenin promotes microglia polarization towards the M2 anti-inflammatory phenotype (upregulation of mediators such as arginase-1, transforming growth factor-β (TGF-β), IL-4, and IL-10). On the other hand, this flavanone attenuates the release of proinflammatory substances (TNF-α, IL-1β, iNOS) and inhibits LPS-induced microglial activation. Naringenin showed potent anti-oxidant activity at a dose of 50 mg/kg (p.o.) in male Wistar rats co-administrated with 50 mg/kg iron-dextran injection over 4 weeks. Naringenin supplementation reduced iron-induced neurotoxicity and anxiety-like behavioral deficit in rodent [31]. The tested flavanone exhibited neuroprotective effects by attenuation of reactive oxygen species (ROS) formation, increasing endogenous antioxidant capacity (significantly preventing the alterations in the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxides (GPx)), upregulating AChE activity and protecting against oxidative DNA damage mediated by apoptosis in the cerebral cortex. However, naringenin has poor bioavailability and low cerebral accessibility [29].

Chemicals and Reagents
Methanol, ethyl acetate, n-hexane as well as tert-BuOH, all of analytical reagent grade, were purchased from Fisher Chemical (Loughborough, UK) and Merck (Darmstadt, Germany), respectively. Acetonitrile and methanol (LC-MS grade) were purchased from Merck (Darmstadt, Germany), while MS-grade formic acid and other chemicals were purchased from Sigma-Aldrich (Steinheim, Germany). Ultrapure water was prepared using a Milli-Q water purification system (Millipore, Milford, MA, USA). Yuccaols A-E, Yuccalide A, gloriosaols A, C-E, and THMS were in-house prepared at the Department of Biochemistry and Crop Quality, IUNG [8]. Trans-resveratrol (Sigma Aldrich, Steinheim, Germany, >99% HPLC based) was used as a group standard for determination of yucca phenolics.
NMR spectra were recorded at 25 • C in the mixture of acetone-d 6 (99.9% D) and deuterated water (D 2 O, 99.96% D)-1 H, HSQC and HMBC spectra or DMSO-d 6 (99.8%D) for the determination of purity of isolated compounds and fractions, and were purchased from Merck (Darmstadt, Germany).

Extraction and Isolation
The plant material-Yucca schidigera Roezl ex Ortgies bark was purchased from a commercial source (Desert King Int., Chula Vista, CA, USA). The extraction of unpurified phenolic fraction and isolation procedures of single stilbenoids were described in detail in our previous work [8]. Briefly, the powdered material was extracted 3 times with a new portion of 100% MeOH in 15-fold excess, using an ultrasonic bath (Polsonic 33, Warsaw, Poland) for 20 min of each series. All the procedures were performed at ambient temperature (20-21 • C) and in the dark. The partially evaporated extract was dissolved in water and defatted with n-hexane, then water-methanolic fraction was evaporated at 35 • C to eliminate the methanol. Subsequently the water fraction was extracted with ethyl acetate in the separatory funnel, evaporated and finally lyophilized. The obtained reddish-brown powder was YS unpur phenolic fraction, which was tested only in in vitro bioassays.
YuB and GloA were isolated from the YS unpur phenolic fraction as documented in [8]. YS pur, YS poly, YuB and GloA were tested either in vitro or in vivo.

NMR Spectroscopy, Purity of the Studied Fractions and Compounds
Multidimensional spectra (2D HSQC, 2D HMBC) were recorded with 12 mg of YS poly fraction dissolved in 0.65 mL of acetone-d 6 :D 2 O (7:3, v/v). Spectra were calibrated to the residual signal of acetone (δ C /δ H 29.8/2.05). The HSQC experiment used Bruker's standard hsqcedetgpsisp2.3 pulse program, the HMBC experiment used Bruker's standard clhmbcetgpl3nd pulse program.

Quantitative Analyses
The UHPLC-UV-MS was carried out on a Waters ACQUITY UPLC system (Waters Corp., Milford, MA, USA), comprising a binary pump system, sample manager, column manager, and PDA detector (Waters Corp., Milford, MA, USA). The acquisition and data processing were performed using Waters MassLynx software v. where: A EFI -is the difference between the absorbance of the enzyme solution without inhibitor after 5 min and the initial absorbance of the same solution (0 s); A ECI -represents the difference between the absorbance of the enzyme solution treated with inhibitor after 5 min and the initial absorbance of the same solution (0 s).
For each sample, the EC 50 value was calculated and expressed as µg/mL final solution. Galantamine was used as a positive control.

Lipoxygenase (15-LOX) Inhibition Activity (Modified Malterud Method)
Briefly, 0.9 mL borate buffer solution 0.1 M (pH 9) is mixed with 0.05 mL lipoxygenase solution in borate buffer pH 9 and with 0.05 mL samples i.e., YS unpur, YS pur, YS poly, YuB and GloA solution (of various concentrations, in DMSO). The mixture is left to stand for 10 min at room temperature, after which 2 mL linoleic acid solution 0.16 mM in borate buffer pH 9 is added. For each sample, a blank is prepared by substituting the enzyme solution with 0.05 mL borate buffer pH 9 solution. The absorbance of the solution was measured at 234 nm, in the 0-120 s interval [34]. The fish were divided by 10 in a group and stored in 24 L thermostatted (26 ± 1 • C) tanks, kept under water filtration and aeration (7.20 mg O 2 /L) using Tetratec ® air pumps (Tetra, Melle, Germany). The animals were maintained on 14/10 h light/dark cycle and were fed twice a day with Norwin Norvitall flake (Norwin, Gadstrup, Denmark).

Behavioral Assay
In behavioral studies, acclimatized zebrafish were randomly assigned to the controluntreated group; the Sco (100 µM) treated group; groups treated with Sco 100 µM and the studied samples i.e., YS pur, YS poly, YuB and GloA (at doses 1, 3, and 5 µg/L). The doses of the tested yucca preparations as well as Sco were selected according to our previous studies [35]. The tested doses (1, 3, and 5 µg/L) were diluted with 1% DMSO solution and administered to zebrafish by immersion for 1 h, once daily for 8 days, while Sco (100 µM) was administered for 30 min before each behavioral test. The control group was immersed only in unchlorinated water with a 1% DMSO solution. The animals were then transferred individually to a Y-maze glass tank or a trapezoid-shaped test tank filled with home tank water. Their swimming behavior was recorded with a Logitech C922 Pro HD Stream webcam (Logitech, Lausanne, Switzerland), and the recordings were analyzed using ANY-maze software v6.3 (Stoelting Co., Wood Dale, IL, USA).
All experiments were carried out following scrutiny by the Ethics Committee on Animal Research of the Alexandru Ioan Cuza University of Iasi, Faculty of Biology (Iasi, Romania) under license no. 02/30.06.2020 and fully complied with the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the safety of animals. The health status and the well-being of all animals involved in the research have been tested regularly during the behavioral tests. No procedures have caused serious pain or long-lasting damage to the zebrafish, and no experimental subject has died during the experimental procedures (fish housing and behavioral tests).

Y-Maze Test
Spatial memory and the response to novelty in zebrafish was assessed using the Ymaze task [36]. The location in the Y-maze task was considered to be a memory index [37]. The apparatus ( Figure 5) consisted of a Y-maze glass tank with three identical arms (25 cm long, 8 cm wide, and 15 cm high), filled with 3 L of the home aquarium water. The water height in the Y-maze was 13 cm. Explicit geometric shapes (squares, circles, and triangles) were placed on the outer walls and were visible from the inside. The Y-maze test consisted of two trials separated by a 1 h interval. During the first trial, the fish could freely swim in the start arm and the other arm for 5 min while the novel arm was blocked by a dividing wall. In the second trial, the wall was removed, and the fish could explore for 5 min all three arms including the novel environment constituted by the novel arm. Fish were placed in different arms as starting points and the maze was rotated in each experiment to randomize the maze cues. The water was changed between groups and trials. Assessing time spent in each arm (percent of the total time), total distance traveled (m), and turn angle ( • ) were determined. In the NTT, the zebrafish exhibits robust behavioral responses to novelty-provoked anxiety. The NTT protocol applied in this study was described before by [38,39]. The testing apparatus (Figure 6) consisted of a trapezoidal shaped glass tank filled with 1.5 L of home tank water and having the following dimensions: 23.9 cm along the bottom × 28.9 cm at the top × 15.1 cm high with 15.9 cm along the diagonal side, 7.4 cm wide at the top and 6.1 cm wide at the bottom. The fish were individually placed in the testing tank and their behavior was recorded for 6 min with a webcam placed at 40 cm in the front of the tank. The tank was virtually divided into the top zone and bottom zone, respectively. To measure anxiety-like behavior and the locomotor activity of the zebrafish, we used the behavioral endpoints described previously by Cachat et al. [38].

Statistical Analyses
All results are expressed as mean ± standard error of the mean (S.E.M) and were analyzed by GraphPad Prism 9.0 software (GraphPad Software, Inc., San Diego, CA, USA). The normality of data distribution was evaluated using Shapiro-Wilk-Test. Datasets with multiple comparisons were evaluated using one-way or two-way ANOVA followed by Tukey's post hoc test. p < 0.05 was considered to indicate a statistically significant difference.

Conclusions
All tested phenolic fractions and pure compounds (YuB and GloA) derived from the bark of Yucca schidigera showed significant in vivo locomotor improvement, memory enhancement and anxiolytic effects on the Sco-induced amnesia and anxiety level zebrafish models. The detailed phytochemical analysis of YS unpur, YS pur and YS poly revealed their chemical similarity, which is characterized by the presence of single or mixtures of spiro-flavostilbenoids. The promising neuroprotective effects of yucca phenolic preparations require deeper investigation. In the NTT, the zebrafish exhibits robust behavioral responses to novelty-provoked anxiety. The NTT protocol applied in this study was described before by [38,39]. The testing apparatus ( Figure 6) consisted of a trapezoidal shaped glass tank filled with 1.5 L of home tank water and having the following dimensions: 23.9 cm along the bottom × 28.9 cm at the top × 15.1 cm high with 15.9 cm along the diagonal side, 7.4 cm wide at the top and 6.1 cm wide at the bottom. The fish were individually placed in the testing tank and their behavior was recorded for 6 min with a webcam placed at 40 cm in the front of the tank. The tank was virtually divided into the top zone and bottom zone, respectively. To measure anxiety-like behavior and the locomotor activity of the zebrafish, we used the behavioral endpoints described previously by Cachat et al. [38]. Novel Tank Diving Test (NTT) In the NTT, the zebrafish exhibits robust behavioral responses to novelty-provoked anxiety. The NTT protocol applied in this study was described before by [38,39]. The testing apparatus (Figure 6) consisted of a trapezoidal shaped glass tank filled with 1.5 L of home tank water and having the following dimensions: 23.9 cm along the bottom × 28.9 cm at the top × 15.1 cm high with 15.9 cm along the diagonal side, 7.4 cm wide at the top and 6.1 cm wide at the bottom. The fish were individually placed in the testing tank and their behavior was recorded for 6 min with a webcam placed at 40 cm in the front of the tank. The tank was virtually divided into the top zone and bottom zone, respectively. To measure anxiety-like behavior and the locomotor activity of the zebrafish, we used the behavioral endpoints described previously by Cachat et al. [38].

Statistical Analyses
All results are expressed as mean ± standard error of the mean (S.E.M) and were analyzed by GraphPad Prism 9.0 software (GraphPad Software, Inc., San Diego, CA, USA). The normality of data distribution was evaluated using Shapiro-Wilk-Test. Datasets with multiple comparisons were evaluated using one-way or two-way ANOVA followed by Tukey's post hoc test. p < 0.05 was considered to indicate a statistically significant difference.

Conclusions
All tested phenolic fractions and pure compounds (YuB and GloA) derived from the bark of Yucca schidigera showed significant in vivo locomotor improvement, memory enhancement and anxiolytic effects on the Sco-induced amnesia and anxiety level zebrafish models. The detailed phytochemical analysis of YS unpur, YS pur and YS poly revealed their chemical similarity, which is characterized by the presence of single or mixtures of spiro-flavostilbenoids. The promising neuroprotective effects of yucca phenolic preparations require deeper investigation.

Statistical Analyses
All results are expressed as mean ± standard error of the mean (S.E.M) and were analyzed by GraphPad Prism 9.0 software (GraphPad Software, Inc., San Diego, CA, USA). The normality of data distribution was evaluated using Shapiro-Wilk-Test. Datasets with multiple comparisons were evaluated using one-way or two-way ANOVA followed by Tukey's post hoc test. p < 0.05 was considered to indicate a statistically significant difference.

Conclusions
All tested phenolic fractions and pure compounds (YuB and GloA) derived from the bark of Yucca schidigera showed significant in vivo locomotor improvement, memory enhancement and anxiolytic effects on the Sco-induced amnesia and anxiety level zebrafish models. The detailed phytochemical analysis of YS unpur, YS pur and YS poly revealed their chemical similarity, which is characterized by the presence of single or mixtures of spiroflavostilbenoids. The promising neuroprotective effects of yucca phenolic preparations require deeper investigation.