Getting insights into chemical composition and antiherpetic capability of jujube (Ziziphus jujuba mill.) drupes

Food plant diversity in bioactive compounds makes them an exploitable resource in the search for effective natural products to prevent or treat viral infections. Therefore, in the framework aimed at studying the antiviral properties of extractive mixtures from fruits (and their waste) grown in the Campania Region (Italy), jujube drupes (Zizyphus jujuba Mill.) were our focus. The drupes were dissected into their peel, pulp and seed parts, each of which was extracted by ultrasound-assisted maceration and further fractionated, thus obtaining, beyond the sugar fraction, a polyphenolic fraction and a lipid fraction. UHPLC-HR MS/MS tools highlighted that the polyphenolic component of the seed was strongly dissimilar from that of the edible parts, being constituted by swertisin and its derivatives. Moreover, the peel mostly accounted for triglycosylated flavonols, whereas the pulp was rich in volatile aromatic glycosides. Among lipids, p-coumaroyl triterpenes mainly characterized the peel. All fractions were screened for their cytotoxicity, and non-toxic concentrations of each extract were tested against herpes simplex virus type 1 (HSV-1) by plaque assays. Molecular tests and Western blot analyses were also carried out. The jujube mixtures, in detail the peel and pulp polyphenolic fractions, and peel lipophilic fraction (the latter enriched mainly in ursane-type triterpenes), showed a marked inhibitory activity against HSV-1 acting in the early stages of viral infection and preventing attachment of the virus to the host cell. The acquired data suggest jujube active mixtures as promising candidates for the prevention and treatment of herpetic lesions.


Introduction
The incidence of viral infections rose significantly worldwide, as well as the number of viral strains resistant to routinely used drugs.Most herpetic infections pathogenic to humans are due to herpes simplex viruses (HSVs), which consist of two types, i.e., HSV-1 and HSV-2, capable of inducing infections mainly (but not exclusively) in the orofacial and genital areas [1,2].Rarely, HSV-1 induces severe diseases reaching the brain, provoking a diffuse infection resulting in herpes simplex encephalitis (HSE) [3].Another serious disease caused by HSV-1 is neonatal herpes, associated with permanent neurological disabilities in the newborn [4][5][6].Currently, the antiviral arsenal for treating herpetic infections is limited and includes nucleoside analogs, such as acyclovir, interfering with DNA polymerase/thymidine kinase and helicase-primase inhibitors such as amenamevir [7,8].However, especially for severe herpetic infections, early diagnosis and therapy are imperative, and the research and identification of new compounds with different mechanisms of action and able to target a broad spectrum of viruses, should be intensified.Indeed, plant specialized metabolites are of increasing interest due to their broadly reported bioactive properties, including antimicrobial, antioxidant, anti-tumoral and anti-inflammatory [9][10][11][12][13][14].Among these compounds, polyphenols gained a lot of attention, as these compounds, which are commonly involved in defense against pathogens by destroying lipid membranes and thus altering the integrity of cellular components [15], are able also to inhibit or inactivate enzymes, leading to the microbial death.Certainly, the great structural variability of polyphenols strongly affects their bioactivity, and getting insights into the polyphenol identity appears to be mandatory for better targeting biomedical applications.In this context, three Vitis vinifera cvs., namely Fiano, Aglianico and Greco, were diversely extracted to achieve procyanidin or stilbenoid extracts, which showed to be active against Gram-positive bacteria and viruses [12][13][14].Greco cv.extracts excelled for their ability against herpesviruses by targeting the early steps of infection [12].Similarly, a Vitis vinifera leaf extract, mainly accounting for flavonols and flavones, was observed to exert antiviral potential against HSV-1 and the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) [14], while a novel polyphenol-based nutraceutical, Taurisolo [13], showed an anti-herpetic effect, directed on the viral surface.This recent and promising evidence suggests that plant mixtures with high phenolic content need to be investigated and explored as new anti-viral agents.Thus, continuing within this framework, jujube drupes (Ziziphus jujuba Mill.) turn into the focus.
Jujube, also called Chinese date or Chinese jujube, is one of the oldest cultivated fruit plant in the world and is the most important species of the large cosmopolitan Rhamnaceae family, given its economic, ecological, and social importance.This super-fruit [16], which is ethno-pharmacologically used for its diuretic, emollient, and laxative properties, is a rich source, beyond macro and micronutrients, of valuable non-nutritive bioactive compounds, like triterpenic acids, alkaloids, poly-phenols, and other pigments.The search on the Pubmed database showed that 1357 papers concerning plants of the genus Zizyphus, mainly Z. jujuba Mill., were published in 2003-2023.In particular, in the last five years, new research papers were aimed at highlighting the role of the fruit phytochemicals in preventing brain damage [17], counteracting anti-inflammatory diseases [18] or helping as a supplementary strategy against Covid-19 [19,20].However, all these reports did not distinguish among the different parts of the fruits.Although an attempt was made by [21], their attention was mainly devoted to the evaluation of the antioxidant capacity of extracts from seeds, pulp, and peel, without linking the results to the identification of the bioactive chemical constituents.Instead, it is well-known that the phytochemical composition of plant organs or tissues is strongly influenced by biotic and abiotic factors, where the extraction procedure (e.g.technique, solvent polarity, experimental parameters) has a pivotal role in the enrichment of compounds of interest [22], promoting diffusive and/or osmosis processes that otherwise can deplete the plant matrix.In this context, taking into account the high saccharide content of jujube fruit, ultrasound-assisted maceration (UAM) in pure ethanol and further fractionation were performed to minimize saccharide extraction, while enhancing the recovery of specialized metabolites, mainly polyphenols and lipid components, such as triterpenes and fatty acids.Thus, deepening the chemical aspects of jujube produced in the Campania Region (Italy) paved the way to define the profile in specialized metabolites of the three parts of the fruit (peel, pulp, and seed) by an untargeted approach in high-resolution mass spectrometry while originally exploring the antiviral capability of the prepared jujube mixtures.
HRMS analyses were performed by using the AB SCIEX TripleTOF® 4600 spectrometer (AB Sciex, Concord, ON, Canada), equipped with a DuoSpray™ ion source operating in negative electrospray (ESI) ion mode.The APCI probe was used for automated mass calibration in all scan functions using the Calibrant Delivery System (CDS).An untargeted approach was developed, consisting of a full scan TOF survey, and eight information-dependent acquisition (IDA) MS/MS scans.Detailed HR mass spectrometry parameters are reported in supplementary materials (Table S2).The instrument was controlled by Analyst® TF 1.7 software, while data processing was carried out using PeakView® software version 2.2.

Cell line and virus
The kidney epithelial cells of the African green monkey, named Vero 76 (ATCC CRL-1587, CVCL-0603), were grown in Dulbecco's Modified Eagle Medium (DMEM; Gibco; Thermo Fisher Scientific, Waltham, MA, USA) with 4.5 g/L glucose, 2 mm L-glutamine, 100 IU/mL penicillin/streptomycin solution and 10 % fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Waltham, MA, USA) in a humidified atmosphere with 5 % CO 2 at 37 • C. We confirmed that Vero 76 cells used in this work have been authenticated within the last three years; additionally, we also tested them using a mycoplasma detection kit (Mycoplasma PCR Detection Kit, Cat.No. G238, abm, New York, United States) and confirmed they were mycoplasma free.HSV-1 (strain SC16) was propagated on Vero 76 cells as previously reported [23].

Antiviral assays
The antiviral activity was investigated by plaque reduction assays as described elsewhere [25,26].Vero 76 (1.4 × 10 5 cells/well) were plated in 24-well plates and incubated at 37 • C and 5 % CO 2 for 24 h.To better understand in which stage of viral infection the jujube extracts were active, four different treatments were conducted.a) Co-treatment: HSV-1 at a multiplicity of infection (MOI) of 0.01 and non-cytotoxic concentrations of jujube extracts were inoculated simultaneously on cell monolayer for 1 h at 37 • C; b) Virus pre-treatment: virus at MOI of 0.1 and jujube extracts were incubated together for 1 h at 37 • C.After that, the mixture (virus/extract) A. Chianese et al. was inoculated on cell monolayer for 1 h; c) Cell pre-treatment: jujube extracts were first placed on cell monolayer for 1 h, and then cells were infected with HSV-1 at MOI of 0.01 for 1 h; d) Post-treatment: cell monolayer was first infected with the virus at MOI of 0.01 for 1 h, and after exposed to jujube extracts for another hour at 37 • C. At the end of each treatment, Vero 76 cells were washed with citrate buffer and incubated with DMEM supplemented with 10 % FBS and 3 % carboxymethylcellulose (CMC), (Sigma-Aldrich, St. Louis, MO, USA) for 48 h.Cells were fixed with 4 % formaldehyde (Sigma-Aldrich) and stained with 0.5 % crystal violet (Sigma-Aldrich).Viral plaques were counted, and the % of viral inhibition was calculated according to the following formula: % of viral inhibition = (1-(plaques counted in treated cells)/(plaques counted in infected cells (CTRL -))) × 100

Real-Time PCR and Western-blot analyses
The antiviral activity of jujube extracts was also investigated by Real-Time PCR and Western-blot [23].Virus pre-treatment was performed as described above.Thirty hours after infection, RNA and protein extract were collected using TRIzol reagent (Thermo Fisher, Waltham, MA, USA) and lysis buffer, respectively.After that, RNA was retro-transcribed in cDNA by BlasTaq TM 2X qPCR MasterMix (Applied Biological Materials, Richmond, Canada) and Real-time PCR was performed to analyze expression levels of the gene UL27.The relative target threshold cycle (Ct) was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as a housekeeping gene.In Table 1, the primer sequences are reported.

Statistical analysis
All experiments were carried out in triplicate and expressed as mean ± Standard Deviation (SD) calculated by GraphPad Prism (version 8.0.1;Software for 2D graphing and statistics; GraphPad Software Inc.: San Diego, CA, US, 2018; www.graphpad.com[27]).One-way ANOVA followed Dunnett's multiple comparisons test was performed; a value of p ≤ 0.0001 was considered significant.

HRMS-based characterization of polyphenolic fractions (Pe 3/2 , Pu 3/2 and S 3/2 )
Flavonoid glycosides are the representative compounds in all parts of the fruit investigated, although their relative content in the peel fraction (Pe 3/2 ) appeared 3.74-fold higher than in the pulp sample (Pu 3/2 ).The extract from the seeds (S 3/2 ) showed a total amount relatively comparable to the peel extract, although the identified metabolites differed.The TOF-MS data of the identified compounds in these fractions are listed in Table 2.
Aromatic glycosides are also part of the three fractions, representing 12.7 %, 29.5 %, and 10.1 % of Pe 3/2 , Pu 3/2 , and S 3/2 , respectively.Benzyl glycosides, differing in the saccharide moiety, were tentatively identified.Compound 5, with the deprotonated molecular ion at m/z 401.1440 was likely benzyl primeveroside.Supporting this hypothesis, following the loss of a pentosyl unit, the ion at m/z 269.1036 was formed [28], while the ion at m/z 161.0379 consisted in the dehydrated hexose (Fig. S1).The two isomers 1 and 3, with deprotonated molecular ions at m/z 431.1565, are benzyldisiloxane derivatives.In particular, the benzyl sophoroside (1), namely zizybeoside I, was previously reported as jujube constituent [29].Compound 3 was likely a gentiobiosyl derivative of benzyl alcohol.The TOF MS/MS spectra of both compounds yielded fragment ions [M-H-162] -at m/z 269.09 and [M-H-162-108] -at m/z 161.04 (Fig. S1).The other identified compounds, as described above, are flavonoid O-and C-glycosides.Compound 6, which is most abundant in S 3/2 and Pu 3/2 fractions, was putatively identified as the dihydrokaempferol 7-O-glucoside.In fact, in the TOF-MS/MS spectrum the aglycone ion at m/z 287.06 was detected, together with the ions at m/z 259.06 and m/z 269.05, generated from the latter after the loss of CO and water, respectively (Fig. S2).Dihydrokaempferol was previously isolated from the root of Zizyphus jujuba Mill.var.spinosa [30].

Table 1
Forward and reverse sequences of primers used in Real-time PCR. the disaccharide derivatives 23 and 25, sharing the neutral loss of 308 Da to achieve the aglycone ion, were in accordance with rutin and its isomer, likely quercetin 3-O-robinobioside (quercetin 3-rhamnosyl-(1 → 6)-galactoside) [31].Rutin was the first flavonol found in jujube leaves [32] and is considered a bitterness indicator in jujube fruit [33].Based on literature data, compounds 36 and 37, whose deprotonated molecular ions lost 278 Da according to a dehydrated pentosyl-deoxyhexose, were tentatively identified as quercetin 3-O-arabinorhamnoside and quercetin 3-O-xylorhamnoside, respectively [34]; Fig. 2. Compounds 8, 9, 15, 16 were isomers, whose saccharidic part accounted for two hexose residues and one deoxyhexose.The first two, eluting at shorter retention times, were differently abundant in the three fruit parts investigated, while the last two were present only in the peel.Finally, the saccharidic moiety of compounds 13 and 19 consisted in a deoxyhexose, a hexose and a pentose, so much so that in the TOF-MS/MS spectrum of compound 19 a loss of 440.16 Da was observed.This latter neutral loss was also observed for compound 28, which was one of the four kaempferol trisaccharides identified in jujube extracts, together with compounds 10, 12 and 33, which differed from the previous one as their sugar moiety was due two dehydrated hexoses and one deoxyhexose (Fig. S3).The kaempferol disaccharides 32 and 35 were distinguishable based on the relative abundance of the aglycone ion, suggesting two isomers in which the glycosylation site is at C-3 aglycone carbon (32) or at C-7 (35).Several C-glycosides were detected, and identified thanks to characteristic neutral losses due to internal cleavages within the sugar rings (Fig. S4).Among these, compound 7, which constitutes 17 % and 16 % of the Pe 3/2 and Pu 3/2 fractions, respectively, was likely naringenin 6,8-di-C-glucoside.Compounds 11, 17, and 18 were other di-C-glycosyl compounds, while compound 14 has been tentatively identified as saponarin, an apigenin O,C-diglucoside [21].Compound 20 was a C-(O-glucosyl) glucoside of apigenin, which also was the aglycone of compounds 11 and 17.These three compounds shared a common deprotonated molecular ion, but differed significantly in their TOF-MS/MS spectra.In particular, the presence of the ion at m/z 431.0996 in the spectrum of compound 14, also known as 7-O-(β-D-glucosyl)isovitexin, highlighted the loss of a dehydrated hexose as per an O-glycosidic bond.For compound 20, the loss of 180 Da suggested the loss of the sugar residue from the hydroxymethyl function of the C-linked glucose.Compound 17 has been tentatively identified as a C-pentoside-C-hexoside derivative of apigenin.Confirming this hypothesis, literature data highlight the isolation of apigenin 6-C-α-L-arabinopyranoside-8-C-β-D-glucopyranoside [35].Compound 18 was the di-C-glucosyl derivative of chrysoeriol (i.e.3′-methoxy luteolin).The dihydrochalcone phloretin 3,5-di-C-glucoside (29) has been identified in all parts of the fruit, although it appears to be more abundant in the peel.
All the other identified compounds were detected only in the S 3/2 fraction, and are derivatives of swertisin (Fig. 3).This latter was also identified, hereinafter labeled as compound 31, based on its deprotonated molecular ion at m/z 445.1160.Swertisin, a C-glucosylflavone, is known for its antidiabetic, anti-inflammatory, and antioxidant effects, and also for its being an antagonist of the adenosine A1 receptor [36].Among its derivatives, the isomers isospinosin (21) and spinosin (26) were identified.Spinosin, previously isolated from the seeds of Ziziphus jujuba var.spinosa, has been characterized as 2″-β-O-glucopyranosyl swertisin.Spinosin may aid the sleep mechanism and interact with 5-hydroxytryptophan and 5-hydroxytryptamine 1A receptors [37].It has also been reported that subchronic low dose spinosin application for 2 weeks significantly increases latency time in the passive avoidance task in healthy adult rodents.Spinosin reverses cognitive impairment, improves memory and learning, and reduces Aβ1-42 oligomers in the brain of AD-induced mice.Compounds 22, 24, 30, 34, 38-40 are acylated derivatives of spinosin.Compound 22, with a deprotonated molecular ion at m/z 919.2470, has been tentatively identified as (hexosyl)vanilloylspinosin (Fig. 3) [38].
Compound 24, with a deprotonated molecular ion at m/z 889.2379, corresponded to 6‴-(4‴'-O-β-D-glucopyranosyl)-benzoylspinosin, previously reported from the seeds of Ziziphus mauritiana (Rhamnaceae) [39].Other acylated C-glycosyl flavonoids, 6‴-(− )-phaseoylspinosin (40), 6‴-sinapoylspinosin (38), and 6‴-feruloylspinosin (39), were also previously isolated from the seeds of Z. mauritiana [40].Furthermore, the glucosylated derivative of feruloylspinosin, never reported before in the literature, was also putatively identified.Finally, based on the TOF-MS/MS spectrum, compound 34 was putatively acetylspinosin.In fact, the deprotonated molecular ion lost 42 Da (acetyl moiety) to provide the ion at m/z 607.17, attributable to spinosin.Lastly, in S 3/2 fraction two deprotonated molecular ions at m/z 577.14 (2 and 4) were detected, whose TOF MS/MS spectra were consistent with two procyanidins B-type (Fig. S5).Both fragmented giving rise to characteristic product ions through the quinone methide (QM) cleavage pathway of the interflavanoid bond, as well as by heterocyclic ring fission (HRF) and retro-Diels-Alder (RDA) cleavage within the heterocyclic ring of one of the two subunits [41].The relative quantification, obtained by considering the areas under each peak in their respective XICs (Extracted Ion Chromatograms), evidenced that jujube polyphenols were differently distributed within the three jujube drupe parts (Fig. S6).When the data were plotted considering the percentage of each compound in its relative fraction, it was highlighted that quercetin triglycosides were mainly abundant in the peel fraction, volatile glycosylated compounds were more representative of the pulp component, while swertisin-derivated compounds made the seed fraction unique.

HRMS-based characterization of lipid fractions (Pe 2/2 , Pu 2/2 and S 2/2 )
The organic fractions (Pe 2/2 , Pu 2/2 and S 2/2 ) resulting from liquid-liquid extraction were characterized by the presence of lipidic components with a wide structural variety, and differentially abundant in the three parts of the jujube fruit.
Triterpenes represent one of the most representative classes in the Ziziphus genus, attracting significant attention in recent scientific literature [42][43][44].Specifically, for Z. jujuba, more than 120 triterpenoids have been identified to date, in the form of aglycones, esters, ethers, and glycosides.The aglycone triterpenes, despite having a pentacyclic skeleton composed of 30 carbon atoms, can be distinguished into different subclasses related to lupane, oleanane, and ursane [45].Table 3A summarizes the UHPLC-TOF/MS data related to the tentatively identified triterpenes.The limited fragmentation in tandem mass spectrometry experiments and the unavailability of commercial standards for individual metabolites belonging to this class for comparing retention times in UHPLC have made it challenging to confirm the identity.Therefore, identification is based almost exclusively on the molecular formula and the elution order in reverse-phase chromatography of isomeric compounds.The compound with the formula C 30 H 48 O 3 (m/z 455.3552;T14) can be attributed to ursolic acid.Compounds T15 and T16 likely correspond to oleanonic and ursonic acids, in which the OH substituent at C-3 is replaced by a ketonic group [39].The presence of the ketonic function in ursonic acid, identified as one of the main compounds in ripe fruits, seems responsible for various enhanced biological properties compared to those exhibited by ursolic acid (e. g. antihyperglycemic, anti-inflammatory, antiviral, antitumor) [20].
The deprotonated molecular ion at m/z 471.349(8) was in accordance with the presence of alphitolic acid (T2), maslinic acid (T3), and pomolic acid (T13), previously described as components of the leaves and fruits of Z. jujuba [46,47].The esterification at C-3 of these metabolites with a p-coumaric acid residue led to the formation of compounds T11 and T12 with a deprotonated molecular ion at m/z 617.3877 (6).Although the fragmentation was limited also in this case, the TOF-MS/MS spectrum showed a fragment ion at m/z 145.03, corresponding to the dehydrated hydroxycinnamic acid portion, confirming its presence (Fig. S7(A)).Similarly, esterification can occur with ceanothic or epiceanothic acid (T1 and T8; m/z 485.3274), leading to the formation of metabolites T4 and T9 at m/z 631.366(5) (C 39 H 52 O 7 , RDB = 14) (Fig. S7(B)).These ones, along with the hypothetical hydroxycinnamoyl derivative of tormentic acid (T5; m/z 633.3816) (Fig. S7(C)), were detected only in the epicarp of the fruit.Finally, the deprotonated molecular ion at m/z 469.334 for triterpenes with Rt = 5.979 and 6.310 min, consistent with the molecular formula C 30 H 46 O 4 , were attributed to two isomers of ziziberanalic acid (T6 and T10).Ziziberanalic acid differs from (epi)ceanothic acid for a formyl group instead of a carboxyl group.This hypothesis is supported by the absence of decarboxylation, characteristic of the TOF-MS/MS spectrum of ceanothic acid (Fig. S7).The composition of free fatty acids in the investigated organic fractions includes molecules with a carbon skeleton of 16 or 18 carbon atoms, exhibiting different unsaturation and hydroxylation degrees.In Table 3B UHPLC-TOF/MS data related to the tentatively identified fatty acids and derivatives are reported.The composition and content of fatty acids in jujube fruits can be influenced by both their cultivars and the production areas due to differences in climate, soil, water, and sunlight, as well as the fruit's maturation state and harvesting time [32,48].Beyond palmitic acid (F24; m/z 255.2334), the presence of the polyunsaturated fatty acids linoleic (F23; m/z 279.2336) and oleic (F25; m/z 281.2488) was detected, along with their oxygenated derivatives, concentrated mainly in the fruit mesocarp.
In the case of the compound F26 with the empirical formula C 25 H 49 O 12 P, palmitic acid was esterified to glycerol, which in turn was linked to a phosphate group and an inositol residue.A similar structure, with the only difference being the presence of stearic acid (18:0) instead of palmitic acid, was hypothesized for the compound F30.In both cases, a neutral loss of inositol (180 Da) from the ).The rationalization of the fragmentation patterns led to the putative identification of these molecules as PG (16:0) and LPA (16:0), respectively [49].TOF-MS/MS spectra and proposed structures are reported in Fig. S8.Lastly, palmitic acid is also part of the compound F27 with a deprotonated molecular ion at m/z 452.2776, tentatively identified as LPE (16:0).In this case, the glycerophosphate moiety binds an ethanolamine residue, highlighted by the diagnostic fragment at m/z 196.0385 (C 5 H 11 NO 5 P − , exact mass m/z 196.0380) [50], and its structure is depicted in Fig. S8.
The tissue-specific accumulation was highlighted also for lipid constituents (Figura S8).In particular, triterpenes were mainly found in Pe 2/2 fraction, whose content in keto derivatives of oleanolic and ursolic acid (T15 and T16) was estimated to be equal to 39.5 %.Ursolic acid (T14) appeared to be relatively more abundant in seed fraction than in edible components, together with oleic and linoleic acids.All other lipid representatives were primarily concentrated in Pu 2/2 .Cluster analysis highlighted the diversity in triterpenes of peel fraction, which segregated from the other two components, while highlighting that seed extract was in a separate group when fatty acids and their derivatives were considered (Fig. S9).

HRMS-based characterization of sugar fractions (Pe 3/1 , Pu 3/1 and S 3/1 )
The aqueous fractions resulting from GPC on Amberlite XAD-4 consisted of two hexose monosaccharides (fructose and glucose) and the disaccharide sucrose [51].The identification was carried out by comparison with commercial standards analyzed under the same experimental conditions as the samples under study (Fig. S10a).The abundance of drupe saccharides required fractionation procedures that better unmasked the bioactive components, mainly flavonoid glycosides, triterpenes and fatty acid derivatives.Although particularly abundant in the pulp, the relative abundance of sucrose in the sugar fraction of the three parts highlights its equal distribution, where fructose and glucose decrease moving from the peel to the seed (Fig. S10b).

Cytotoxicity of jujube fraction towards Vero 76 cells
Before exploring the antiviral potential of jujube extracts, their cytotoxicity was analyzed via MTT assay.This test was employed to measure cell viability as it assesses the reduction of the tetrazolium salt MTT to formazan by the mitochondrial dehydrogenases occurring only in viable cells.Different concentrations of jujube fractions were tested within the range from 200 to 3.1 μg/mL.Cell viability was quantified in respect to untreated cells (CTRL+).No toxicity was observed at all the tested concentrations for all the fractions of peel, seed and pulp (Fig. 4).Our results are in agreement with the data reported by Rajaei et al.They evaluated the cytotoxic effect of Z. jujuba on several cellular systems confirming no toxicity also at the highest concentrations tested [52].

Antiviral activity of the extracts against HSV-1
To explore the antiviral activity of the constituted jujube fractions and their potential mechanism of action, plaque reduction assays against HSV-1 was carried out, using four distinct treatment schemes: co-treatment, virus pre-treatment, cell pre-treatment and posttreatment.All fractions were tested at not cytotoxic concentrations, i.e., in the range 200-3.1 μg/mL for all of them.
As shown in Fig. 5A, when the extracts and virus were inoculated on cell monolayer simultaneously (co-treatment), the sugar fraction, mainly accounting of sucrose, fructose and glucose, was the only one not active in inhibiting HSV-1 infection regardless of the nature of extracts.A very strong inhibitory potential was detected for both lipid and polyphenolic fractions, as also evidenced by the half-maximal inhibitory concentration (IC 50 ) (Fig. 5A).In particular, it was observed that lipid fraction was the most active compared to the polyphenolic one for both peel and pulp, meanwhile S 3/2 , which is the only one, among the three polyphenolic fractions, constituted by swertisin and its derivatives, exhibited a higher antiviral activity respect to S 2/2 .
When virus was pre-treated with extracts (virus pre-treatment), an improvement of the antiviral effect was present (Fig. 5B).The trend was very similar to that observed in co-treatment assay, therefore lipid and polyphenolic fractions were the most active.Among all and similarly to co-treatment results, Pe 2/2 fraction showed the best activity with an IC 50 at 3.6 μg/mL, followed by Pu 2/2 (IC 50 = 5.2 μg/mL) and S 3/2 (IC 50 = 6.2 μg/mL).These data suggested that the active fractions interact directly with the viral particles blocking the early stages of viral infection.
Similar results have also been observed with several extracts, such as Vitis vinifera leaf extract, elderberry extract, green tea extract, and others, confirming that extracts rich in polyphenols reduce the viral infection targeting the viral surface [14,53,54].On the other side, no reduction in infection was observed in cell pre-treatment and post-treatment assays, indicating that the extracts could neither interact with the cell surface nor interfere with intracellular targets or infection stages (Fig. S11).This indicates that the mechanism of action of these compounds may be specifically due to interactions that occur during the initial stages of viral infection or when the virus is directly exposed to jujube fractions.Altogether our findings demonstrate that Z. jujuba extract is endowed of an antiviral potential acting in the extracellular phase of infection, at the moment in which the virus attaches to the cellular membrane.No precise mechanism has been elucidated, but, probably, the extract may damage the viral particle interfering with the subsequent step of penetration inside the host cell.

Evaluation of viral gene expression
Considering the higher activity of extract Pe 2/2 , Pu 2/2 and S 3/2 (in descending order), further investigations were carried out to confirm their antiviral effect.Real-Time PCR analysis was performed to assess the extracts impact on the expression of a critical gene associated with HSV-1 viral entry, namely UL27.This gene is a late gene playing a crucial role during the first stages of the viral life cycle: UL27 gene encodes the envelope glycoprotein B (gB), a class III fusion protein crucial for viral membrane and host cell membrane fusion [55].
As previously reported, the extract Pe 2/2 was the most active: as indicated in Fig. 6, the expression level of UL27 was very low up to 6.2 μg/mL.Very similarly, Pu 2/2 significantly reduced the gene expression in the range of concentrations 25-6.2 μg/mL.On the contrary, S 3/2 showed a lower ability in inhibiting the infection: UL27 was detectable already at 12.5 μg/mL, according to data obtained by plaque assays (Fig. 5, panels A and B).

Western blot analyses
The antiviral activity of Pe 2/2 , S 3/2 and Pu 2/2 was further investigated via another molecular analysis.Western blot was employed to assess the extracts ability to reduce the expression of gB at translation level (Fig. 7).A strong reduction of gB protein level was observed, confirming both data obtained through plaque assays and Real-time analysis.We demonstrated that Pe2/2 was the extract with the best antiviral activity and was able to reduce by double gB protein production at 6.2 μg/mL; on the other side, Pu2/2 and S3/2 resulted active until 25 μg/mL in decreasing protein level.Thus, lipid fractions from the different parts of Ziziphus jujuba Mill., i.e., peel, seed and pulp were demonstrated for the first time as antiherpetic promising agents.To date, several studies have been conducted and only report the antiviral effect of compounds isolated from jujube fruit.Hong et al. [56] described that betulinic acid blocked influenza A/PR/8 virus infection at 50 μM, while in another study, authors showed that three compounds derived from jujube roots were endowed with antiviral potential against the porcine epidemic diarrhea virus (PEDV) [57].Indeed, among the triterpenes identified in peel lipid fraction, ursolic acid (T14), which accounted for 13 % of the prepared fraction, when isolated from Mallotus peltatus and tested at 9.0 μg/mL exhibited nearly 100 % inhibition against HSV-1, also inhibiting plaque formation at 80 % level [58].The antiviral activity of ursolic acid was broadly tested towards different viruses: it was observed to counteract human immunodeficiency virus (HIV-1) protease, to be efficacious against chronic hepatitis C (HCV), to be able to interfere with rotavirus replication cycle, and to acts as selective inhibitor of papilloma virus [59].Analogously, although it was less investigated than ursolic acid, its keto derivative, ursonic acid (T16), was found to suppress HSV-1 and HSV-2 cytopathic effects, and its inhibitory effect was ten times higher than that exhibited by ursolic acid.This finding is in line with our data on peel lipid fraction, in which ursonic acid is the most abundant compound followed by oleanonic acid (T15).While searching on jujube antiviral polyphenols, interesting data were found in relation to two purified methoxylated flavones were active against tobacco mosaic virus replication [60].Quercetin and some different its derivatives were also widely screened [61].In particular, quercetin showed to inhibit the expressions of HSV proteins such as glycoprotein D (gD) and Infected Cell Protein 0 (ICP0), and also to suppress TLR-3 dependent inflammatory responses in infected cells [62].
This evidence suggests that constituted jujube fractions could be favorably employed, also considering the established traditional prescription, named Yakammaoto, which consists of 9 ingredients, including jujube fruit, widely used in China for the management of airway symptoms due to its high effect in inhibiting picornavirus infection [63,64].

Conclusions
Recently, a great deal of interest in the medical field has been aroused by natural extracts [65].As reported in several studies, polyphenols particularly flavonoids, are known for their anti-inflammatory, antimicrobial, anticancer, antioxidant and antiviral properties [66].In this study, we investigated the inhibitory potential of jujube drupe extracts against HSV-1, identifying the jujube fruit as a promising candidate for further pharmaceutical applications.
Considering the high sugar content of jujube fruit, with the aim to deplete sugar constituents in favor of the specialized metabolites, ultrasound-accelerated maceration in pure ethanol was carried out [67].This led to the acquisition of alcoholic extracts from each part of the fruit (seed, pulp and peel), further fractionated to create a polyphenolic extract and a lipid extract.The extracts thus obtained were chemically profiled through ultra-high performance liquid chromatography coupled with high resolution mass spectrometry.Finally, fractions were tested for their antiviral effect.Lipid and polyphenol fractions showed a strong antiherpetic activity, which could be due to their ability to target the viral membrane damaging it and hindering the adsorption of virus on the host cell.We found out that the peel lipid fraction, whose triterpene component accounted for 13 % in oleanonic acid and 26.4 % of ursonic acid, was the most active.On the contrary, as expected, the aqueous fractions, containing only sucrose, glucose and fructose, exhibited no antiviral effect.

Funding
Funding for this research was provided by Enforcing the THERapeutic Arsenal againSt Emerging and Reemerging RNA Viruses Prin 2022_Fondi Prin_2022W97H54, CUP: B53D23003630006.

Fig. 3 .
Fig. 3. TOF-MS/MS spectra (a) and hypothesized fragmentation patterns (b) of swertisin (31) and its derivatives from jujube seeds.The theoretical m/z values are reported below each structure.

Fig. 7 .
Fig. 7.Western blot analyses.The assay was performed to evaluate the effect of a. peel fraction (Pe); b. pulp fraction (Pu), and c. seed extracts (S) on gB protein expression.Non-treated cells were used as negative control (− ), while infected cells were used as positive control (+).The amount of gB was compared to that of tubulin.Dunnet's multiple comparison test: ****p < 0.0001; ***p = 0.0003; **p = 0.0017; ns: non-significant.

Table 3
TOF-MS data of A) triterpenes, and B) fatty acids and their derivatives tentatively identified in lipid fractions (Pe 2/2 , Pu 2/2 and S 2/2 ).RDB = Ring Double Bonds.molecularion was observed, along with two diagnostic fragments at m/z 152.9962(1) and 315.049, whose structures are depicted in Fig.S8.The fragment ion at m/z 152.99 was also present in the TOF-MS/MS spectra of metabolites F28 (C 22 H 45 O 9 P; m/z 483.2719) and F29 (C 19 H 39 O 7 P; m/z 409.2356 deprotonated