Physicochemical Characterization and Antimicrobial Activity against Erwinia amylovora, Erwinia vitivora, and Diplodia seriata of a Light Purple Hibiscus syriacus L. Cultivar

Phytochemicals are essential raw materials for the production of formulations that can be helpful in crop protection. In particular, Hibiscus spp., which are often used in traditional medicine, are rich in potential bioactive molecules. This study presents an analysis of the thermal, vibrational, and phytochemical characteristics of a light purple variety of Hibiscus syriacus, using thermal gravimetric and differential scanning calorimetry, Fourier-transform infrared spectroscopy, and gas chromatography-mass spectroscopy techniques. Further, with a view to its valorization, the antimicrobial activity of its extracts has been investigated in vitro against Erwinia amylovora (the phytopathogen responsible for fire blight in apples, pears, and some other members of the family Rosaceae), Erwinia vitivora (the causal agent of the “maladie d’Oléron” in grapevines), and Diplodia seriata (responsible for “Bot canker”). Higher heating values and thermal features showed similarities with kenaf biomass. The main compounds identified in the hydro-methanolic extracts were: in flowers, 1-heptacosanol, heptacosane, 1-tetracosanol, hexadecenoic acid, 9,12,15-octadecatrienoic acid, and 9,12-octadecadienoic acid; and in leaves, the coumarin derivative 4,4,6,8-tetramethyl-2-chromanone, vitamin E, phytol, and sitosterol. MIC values of 500 and 375 μg·mL−1 were obtained against E. amylovora for flower and leaf extracts, respectively, upon conjugation with chitosan oligomers (to improve solubility and bioavailability). In the case of E. vitivora, MIC values of 250 and 500 μg·mL−1, respectively, were registered. Regarding the antifungal activity, EC90 values of 975.8 and 603.5 μg·mL−1, respectively, were found. These findings suggest that H. syriacus (cv. ‘Mathilde’) may be a promising source of antimicrobials for agriculture.


Introduction
Crop protection is key to global food sustainability and security (in line with Sustainable Development Goal 2 in the 2030 Agenda). Synthetic pesticides have traditionally been used by farmers to control and eradicate pests, but they have detrimental effects on the health of consumers and the environment. To ensure sustainable production patters (SDG Target 12.4) and increase food security, current legislative frameworks promote the use of integrated pest management. In particular, the use of plant extracts as "green agrochemicals" should be intensified. Plants produce a wide range of primary and secondary metabolites (carbohydrates, cyanogenic glycosides, amino acids, lipids, phenols, flavonoids, (BDA), caused by Botryosphaeriaceae fungi. Among the latter, D. seriata is one of the most abundant, and affects a wide range of woody hosts, including not only grapevine [28,29], but also apples, causing "Bot canker", frog-eye leaf spot, and black rot [30][31][32].

Elemental Analysis and Calorific Values Calculation
The C, H, N, and S percentages of Hibiscus syriacus components (wt.% of dry material) were in the 34.4-42.8%, 6.3-6.4%, 2.2-2.8%, and 0.1-0.2% ranges, respectively ( Table 1). The distribution of nitrogen content showed maximum values in the flowers and slightly lower in the leaves and stems. The calculated (from elemental analysis data) higher heating values (HHV) for flowers and leaves were 16.98 and 12.96 kJ·g −1 , respectively, with a mean value of 14.97 kJ·g −1 .

Thermal Characterization
The DSC curve for H. syriacus flowers ( Figure S1) showed exothermal effects at 315, 425, and 443-450-470 • C. The ash content at 550 • C, according to the TG curve, was 6.3%. In turn, the DSC curve of H. syriacus leaves showed exothermal effects at 325 and 445 • C. The ash content at 500 • C was 20.6% ( Figure S2).

Vibrational Characterization
An inspection of the absorption bands, summarized in Table 2, revealed a composition rich in fatty alcohols, fatty acids, and esters. The broad band at around 3300 cm −1 is assigned to the OH stretching vibration, and indicates the presence of primary alcohols. The two intense bands at 2920 and 2850 cm −1 are due to CH 2 asymmetric and symmetric stretching vibrations, respectively. The band at 1734 cm −1 is assigned to the C=O stretching vibration of the carboxylic groups in esters. At 1441 cm −1 , there is a band that can be ascribed to CH 2 bend (scissors) deformation vibration. Several bands also attributed to CH 2 vibrations (wagging and twisting) are observed in the 800-1400 cm −1 range. The band at 719 cm −1 , assigned to the CH 2 rocking mode, is indicative (when it appears together with the other CH 2 vibrations) of the presence of long-chain linear aliphatic molecules. In the spectrum of leaves, the C=C vibration at 1634 cm −1 points to the presence of coumarin derivatives, as discussed below.

Antibacterial Activity
The antibacterial activity against E. amylovora and E. vitivora of chitosan oligomers (COS), H. syriacus flower and leaf hydromethanolic extracts, their main constituents (heptacosanol, DHTMC and vitamin E, Figure 1), and their corresponding conjugate complexes with COS are summarized in Table 5. Both the flower and leaf extracts showed an antimicrobial activity higher than (or comparable to, in the case of E. vitivora for the leaf extract) that of chitosan. Moreover, the flower extract resulted in lower MIC values than those attained with the leaf extract against both pathogens (750 vs. 1000 µg·mL −1 against E. amylovora, and 500 vs. 1500 µg·mL −1 against E. vitivora). This is an unexpected result, given that the main constituent of the flower extract (heptacosanol) showed a lower efficacy than the two main compounds present in the leaf extract (DHTMC and vitamin E) in the case of E. amylovora, and comparable to that of vitamin E in the case of E. vitivora. Hence, other constituents of the flower extract must contribute to its activity, as discussed below.
Upon conjugation with COS, a noticeable enhancement in the antibacterial activity was attained for all the assayed products. In particular, MIC values of 500 and 375 µg·mL −1 were obtained against E. amylovora for flower and leaf extracts, respectively. In the case of E. vitivora, MIC values of 250 and 500 µg·mL −1 , respectively, were registered. Concerning the main constituents, the best results against E. amylovora (MIC = 250 µg·mL −1 ) were registered for COS-vitamin E, while COS-heptacosanol and COS-DHTMC led to the lowest MIC values against E. vitivora (187.5 µg·mL −1 ).

Antifungal Activity
The results of the antifungal susceptibility test (mycelial growth inhibition using the agar dilution method) are summarized in Figure 2. For all the assayed products, an increase in the concentration led to a decrease in the radial growth of the mycelium, resulting in statistically significant differences.
The two H. syriacus extracts showed a lower antifungal activity than COS, for which full inhibition was attained at 1500 µg·mL −1 . Nonetheless, the main constituents of the extracts, viz. heptacosanol, DHTMC, and vitamin E, showed a stronger antifungal action (reaching full inhibition at 375, 1000, and 750 µg·mL −1 , respectively).
The formation of conjugate complexes again led to an improvement in terms of antifungal activity: full inhibition was attained at 1000 µg·mL −1 for both COS-flower and COS-leaf extracts conjugates (a value lower than that obtained with COS alone), and at 250, 500, and 500 µg·mL −1 for COS-heptacosanol, COS-DHTMC, and COS-vitamin E, respectively ( Figure S5). This enhancement is clearly observed in the effective concentration values summarized in Table 6.  Calculation of synergy factors, presented in Table 7, confirmed the aforementioned strong synergistic behavior for COS-heptacosanol and COS-vitamin E (with SFs of 2.59 and 3.14 for the EC 90 , respectively). Nonetheless, SFs > 1 were obtained in all cases.

On the Elemental Analysis Results, Calorific Values, and Ash Contents
Regarding the elemental analysis results, upon comparison with those reported for H. rosa-sinensis leaves by Subramanian et al. [34] (C, 40.8%; H, 4.7%; N, 4.9%), significant differences in the C/N ratio could be observed (15.6 in this work vs. 8.4 for H. rosa-sinensis).
In turn, such differences in C and N contents explain the differences in the calorific values: 13 vs. 23.2 kJ·g −1 . As for the ash content in leaves, the reported content (20.6%) is substantially higher than that found in H. rosa-sinensis (12%). Nonetheless, a comparison with H. cannabinus (C, 38.3%; H, 5.8%; N; 1.7%) [35] results in a closer match, with a C/N ratio of 22.5, a calorific value of 16.68 kJ·g −1 and an ash content of 6.1% (the latter two values are very close to the ones reported for flowers in this work: 16.98 kJ·g −1 and 6.3%, respectively). In view of these similarities with H. cannabinus, a possible valorization for biofuel production may be explored [36,37].

On the Total Phenol and Flavonoid Contents
The TPC results obtained for H. syriacus flower and leaf extracts (800 and 425 mg GAE/100 mg) are within the ranges reported by Wong et al. [38] for the methanolic extracts of other Hibiscus species: 264-2420 mg GAE/100 mg for flowers and 301-2080 mg GAE/100 mg for leaves, respectively, being close to those found for H. rosa-sinensis. The TFC results (315 and 280 mg CE/100 mg for flower and leaf extracts, respectively) were similar to those found after pulsed ultrasonic assisted extraction in methanol of H. cannabinus leaves (290 mg CE/100 mg) [39].

On the Composition of H. syriacus Extracts
To date, the only analyses available on H. syriacus flower or leaf extracts are those reported by Kim et al. [16] (methanolic formic acid extract of petals, analyzed by 1 H-NMR and fast atom bombardment mass spectroscopy, FABMS); by Wei et al. [17] (leaf extract, analyzed by 1 H-NMR and 13 C-NMR); and by Zhang et al. [18] (ethanolic flower extract, analyzed by 1 H-NMR and 13 C-NMR). However, to the best of the authors' knowledge, no studies based on GC-MS or HPLC are available for H. syriacus, so comparisons with other Hibiscus spp. extracts are provided instead.
Regarding the main identified flower extract phytoconstituents, 1-heptacosanol has also reported in the essential oil of H. sabdariffa flowers by Inikpi et al. [3]. The presence of hexadecanoic and 9,12-octadecadienoic acids and their esters has also been referred in the essential oil of H. sabdariffa by Inikpi et al. [3] and in the flowers of H. tiliaceus by Melecchi et al. [4]. Concerning the presence of 9,12-octadecadienoic acid (in a 4.4%), it is worth noting that it has also been found by Dingjian et al. [5] in the essential oil of H. syriacus. Regarding DDMP-4-one, a principal reducing Maillard compound [40], it has been identified in H. tiliaceus [6] and in H. rosa-sinensis flowers [7]. In relation to nonanoic acid, albeit present in small amounts (0.82%), it had been previously found in the root of H. syriacus [8].
With reference to the phytochemicals found in the leaf extract, coumarin derivatives have been reported in H. rosa-sinensis leaf ethanol and water extracts [9]. α-tocopherol (vitamin E) has been identified in the ethanolic leaf extract of H. sabdariffa by Subhaswaraj et al. [10]. Phytol has been reported in the essential oil of kenaf (H. cannabinus) by Kobaisy et al. [11], in the ethanolic leaf extract of H. sabdariffa [10], and in the aqueous methanol fraction of H. asper leaves by Olivia et al. [12]. In the latter two works, 9,12,15-octadecatrienoic, 9,12-octadecadienoic, and hexadecanoic acids were also found (as in the GC-MS analyses reported herein). β-sitosterol has been identified in H. sabdariffa and H. mutabilis leaves [13,14].

On the Antimicrobial Activity of H. syriacus Extracts
The antibacterial activity of H. syriacus extracts has been studied by Punasiya et al. [23] against B. cereus, S. aureus, and K. pneumonia; by Mak et al. [41] against S. typhimurium and S. aureus; and by Seyyednejad et al. [42] against B. anthracis, B. cereus, S. aureus, S. epidermidis, L. monocytogenes, S. pyogenes, E. coli, S. typhy, K. pneumonia, and P. aeruginosa, but no assays against E. amylovora and E. vitivora pathogens have been carried out. Regarding the antifungal activity, it has been assayed against C. albicans and S. cerevisiae by Liu et al. [43], and against T. mentagrophytes [8], but no data on Diplodia spp. (or other Botryosphaeriaceae) is available. Hence, a tentative explanation for the observed activity on the basis of the phytoconstituents identified in the extracts is presented.
With respect to the flower extract, 1-heptacosanol has been reported to have antimicrobial and antioxidant activity [44], putative antibacterial activity [45], and significant antifungal activity against all Candida spp. [46]. Nonetheless, its efficacy against the phytopathogens referred herein was variable: moderate against E. amylovora, and high against E. vitivora and D. seriata. As regards other constituents that were not assayed in vitro, hexadecanoic acid and its esters are considered antifungals and antioxidants [47]. The same applies to 1-tetracosanol [48,49], and to the unsaturated linolenic and linoleic fatty acids [50,51]. Moreover, according toČechovská et al. [40], part of the antioxidant activity of H. syriacus flowers can be ascribed to 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one or DDMP-4-one, and such antioxidant activity is generally associated with antibacterial, antifungal, and antimycotoxigenic biological activities [52].
As for the leaf extract, the neoflavonoid 4,4,6,8-tetramethyl-2-chromanone (or 3,4dihydro-4,4,6,8-tetramethyl-coumarin), although not included among the coumarins screened by Souza et al. [53] against B. cereus, E. coli, P. aeruginosa, and S. aureus, nor among those tested by Montagner et al. [54] against C. albicans, A. fumigatus, and F. solani, has shown comparable MIC values to those reported in those works. The lowest efficacy (MIC = 1000 µg·mL −1 ), observed against E. amylovora, may still be regarded as moderate, and would support the observations of Halbwirth et al. [55] on the antimicrobial activity of flavonoids in pome fruit trees for fire blight control (contrary to the opinion of Flachowsky et al. [56], who considered that the accumulation of flavanones did not appear to reduce fire blight susceptibility in apple). As a potential explanation behind such activity, the efficacy of phytoalexins and flavonoids may be connected to their capability to elude the outer membrane protein TolC and the AcrAB transport system in E. amylovora [57,58].
Regarding other leaf extract constituents, vitamin E is also known to have antimicrobial activity [59,60]. In the present study, vitamin E showed a higher efficacy against E. vitivora (MIC = 500 µg·mL −1 ) than against E. amylovora and D. seriata (MIC = 750 µg·mL −1 ). It should also be taken into consideration that the third main compound, phytol, although not assayed in vitro, may also contribute to the observed antimicrobial activity [61].
In relation to the improved antimicrobial activity of the constituents of H. syriacus extracts observed upon conjugation with COS, it may be ascribed to solubility and bioavailability enhancement, as result of an enhanced linkage to negatively charged site-specific binding receptors on the bacterial/fungal membranes. Nevertheless, further research is needed on this specific point, given that no convincing mechanism to explain the synergistic action of above (and other previously reported [62,63]) COS-phytochemical conjugates has been reported to date.

Limitations of the Study
With regard to the evolution of this work, it should be taken into consideration that-even though the in vitro results are promising-in vivo tests are required in order to evaluate the actual field applicability. While no restrictions apply to ex situ and in vivo tests involving Botryosphaeriaceae fungi (which may be conducted on autoclaved grapevine wood or on grafted grapevine plants artificially inoculated with the fungal pathogen), bioassays with highly virulent Erwinia spp. (for which the best MIC values have been attained and which would be most interesting, given that effective and sustainable control measures are lacking) can only be conducted on suitable host materials under carefully controlled laboratory conditions, given that field studies require authorization, especially in protected zones (according to EU Commission Directive 2003/116/EC of 4 December 2003). Further, even if assays were conducted on artificially inoculated seedlings, it is known that there are sensitivity differences depending on whether it is a natural infection or an artificial inoculation, and also depending on the affected organ (flowers, shoots, unripe fruits, etc.). In addition, comparisons with currently allowed chemical and biological treatment products (viz. Fosetyl-aluminium, laminarin, prohexadione calcium and copperderivatives; and Aureobasidium pullulans and B. subtilis) would be needed for the costeffectiveness analysis.

Studied Species
Hibiscus syriacus, colloquially known as 'Rose of Sharon' (or 'Korean Rose'), is one of the 300 species of the genus Hibiscus. Although it was first identified in Syria (as indicated by its name), it is mainly found in south-central and southeast China, India, and much of east Asia. This deciduous shrub grows up to 3 m tall and has flowers with attractive white, pink, purple, lavender, or blue color over a long blooming period, though individual flowers last only a day. The leaves are glabrous, triangular-ovate to rhombic, often 3-lobed ( Figure 3, top-center). About 40 different H. syriacus cultivars, with varying flower color and shape, are commonly cultivated, and many more genotypes exist in different collections [66]. Among the light purple/purplish white cultivars, 'Mathilde' (or Blush Satin ® ), from nursery M. Verweij & Zonen (Boskoop, The Netherlands), released in 1995, is one of the most popular, together with 'Marina' cultivar (or Blue Satin ® ), which looks similar to 'Oiseau Bleu', but is said to have a stronger growth (Figure 3, bottom). The purple color has been referred to anthocyanin pigments [67].
A pharmacognostic and pharmacological overview of H. syriacus is provided in the review paper by Punasiya et al. [68].

Plant Material and Extraction Procedure
Hibiscus syriacus cv. 'Mathilde' samples (PP12660, Satin ® series) were collected in the full flowering stage, in September 2020, in Llanes (Asturias, Spain). A voucher specimen, identified and authenticated by Prof. J. Ascaso, has been deposited at the herbarium of the Escuela Politécnica Superior de Huesca, Universidad de Zaragoza. Aerial parts from different specimens (n = 20) were thoroughly mixed to obtain (separate) flowers and leaves composite samples. The composite samples were shade-dried, pulverized to fine powder in a mechanical grinder, homogenized, and sieved (1 mm mesh).
The flower samples were mixed (1:20 w/v) with a methanol/water solution (1:1 v/v) and heated in a water bath at 50 • C for 30 min, followed by sonication for 5 min in pulse mode with a 1 min stop for each 2.5 min, using a probe-type ultrasonicator (model UIP1000 hdT; 1000 W, 20 kHz; Hielscher Ultrasonics, Teltow, Germany). The solution was then centrifuged at 9000 rpm for 15 min and the supernatant was filtered through Whatman No. 1 paper. Aliquots were lyophilized for CHNS and FTIR analyses. The extraction procedure for leaf samples was identical.

Bacterial and Fungal Isolates
The E. amylovora and E. vitivora bacterial isolates were supplied by CECT (Valencia, Spain), with NCPPB 595 and CCUG 21,976 strain designations, respectively. The former was isolated from pear (Pyrus communis L.) in the UK, and the latter from Vitis vinifera var. 'Sultana' in Greece. D. seriata (code ITACYL_F098, isolate Y-084-01-01a) was isolated from 'Tempranillo' diseased grapevine plants from protected designation of origin (PDO) Toro (Spain) and supplied as lyophilized vials (later reconstituted and refreshed as PDA subcultures) by ITACYL (Valladolid, Spain) [69].
The calculation of calorific values from elemental analysis data was carried out according to the following equation [70]: HHV = (0.341 × %C) + (1.322 × %H) − 0.12(%O + %N), where HHV is the heating value for the dry material, expressed in kJ·g −1 ; and %C, %H, %O, and %N are the mass fractions, expressed in wt.% of dry material.
The infrared vibrational spectra were collected using a Thermo Scientific (Waltham, MA, USA) Nicolet iS50 Fourier-transform infrared spectrometer, equipped with an in-built diamond attenuated total reflection (ATR) system. A spectral resolution of 1 cm −1 over the 400-4000 cm −1 range was used, taking the interferograms that resulted from co-adding 64 scans.
The hydromethanolic plant extracts were studied by gas chromatography-mass spectrometry (GC-MS) at the Research Support Services (STI) at Universidad de Alicante (Alicante, Spain), using a gas chromatograph model 7890A coupled to a quadrupole mass spectrometer model 5975C (both from Agilent Technologies, Santa Clara, CA, USA). The chromatographic conditions were: 3 injections/vial, injection volume = 1 µL; injector temperature = 280 • C, in splitless mode; and initial oven temperature = 60 • C, 2 min, followed by ramp of 10 • C/min up to a final temperature of 300 • C, 15 min. The chromatographic column used for the separation of the compounds was an Agilent Technologies HP-5MS UI of 30 m length, 0.250 mm diameter, and 0.25 µm film. The mass spectrometer conditions were: temperature of the electron impact source of the mass spectrometer = 230 • C and of the quadrupole = 150 • C; and ionization energy = 70 eV. Test mixture 2 for apolar capillary columns according to Grob (Supelco 86501, Sigma Aldrich Química, Madrid, Spain) and PFTBA tuning standards were used for equipment calibration. NIST11 library was used for compound identification.
Total phenolic content, expressed in gallic acid equivalents (GAE), was determined by using the Folin-Ciocalteau method as described by Dudonné et al. [71], and the total flavonoid content, expressed in catechin equivalents (CE), was evaluated according to Mak et al. [42] through the use of the aluminum chloride method. An Agilent UV-Vis Cary 100 spectrometer was used for the colorimetric quantification.

In Vitro Antibacterial Activity Assessment
The antibacterial activity was assessed according to CLSI standard M07-11 [72], using the agar dilution method to determine the minimum inhibitory concentration (MIC). An isolated colony of E. amylovora in TSB liquid medium was incubated at 30 • C for 18 h. Serial dilutions were then conducted, starting from a 10 8 CFU·mL −1 concentration, to obtain a final inoculum of~10 4 CFU·mL −1 . Bacterial suspensions were then delivered to the surface of TSA plates, to which the bioactive products had previously been added at concentrations in the 62.5-1500 µg·mL −1 range. Plates were incubated at 30 • C for 24 h. In the case of E. vitivora, the same procedure was followed, albeit at 26 • C. Readings were taken after 24 h. MICs were visually determined in the agar dilutions as the lowest concentrations of the bioactive products at which no bacterial growth was visible. All experiments were run in triplicate, with each replicate consisting of 3 plates per treatment/concentration.

In Vitro Antifungal Activity Assessment
The antifungal activity of the different treatments was determined using the agar dilution method according to EUCAST standard antifungal susceptibility testing procedures [73], by incorporating aliquots of stock solutions onto the PDA medium to obtain concentrations ranging from 62.5 to 1500 µg·mL −1 range. Mycelial plugs ( = 5 mm), from the margin of 1-week-old PDA cultures of D. seriata, were transferred to plates incorporating the above-mentioned concentrations for each treatment (3 plates per treatment/concentration, with 2 replicates). Plates were incubated at 25 • C in the dark for a week. PDA medium without any amendment was used as the control. Mycelial growth inhibition was estimated according to the formula: ((d c − d t )/d c ) × 100, where d c and d t represent the average diameters of the fungal colony of the control and of the treated fungal colony, respectively. Effective concentrations (EC 50 and EC 90 ) were estimated using PROBIT analysis in IBM SPSS Statistics v.25 (IBM; Armonk, NY, USA) software.
The level of interaction was determined according to Wadley's method [74], which is based on the assumption that one component of the mixture can substitute at a constant proportion for the other. The expected effectiveness of the mixture is then directly predictable from the effectiveness of the constituents if the relative proportions are known (as it is in this case). The synergy factor (SF) is estimated as: where a and b are the proportions of the products A and B in the mixture, respectively, and a + b = 1; ED A and ED B are their equally effective doses; ED(exp) is the expected equally effective dose; and ED(obs) is the equally effective dose observed in the experiment. If SF = 1, the hypothesis of similar joint action (i.e., additivity) can be accepted; if SF > 1, there is synergistic action; and if SF < 1, there is antagonistic action between the two fungicide products.

Statistical Analysis
Given that the homogeneity and homoscedasticity requirements were satisfied (according to Shapiro-Wilk and Levene tests, respectively), the mycelial growth inhibition results for D. seriata were statistically analyzed in IBM SPSS Statistics (IBM, New York, NY, USA) v.25 software using one-way analysis of variance (ANOVA), followed by post hoc comparison of means through Tukey's test at p < 0.05.

Conclusions
Elemental and thermal analysis data of H. syriacus biomass showed similarities with kenaf, a suitable lignocellulosic feedstock for bioenergy production. The GC-MS analysis of H. syriacus extracts revealed that, apart from fatty alcohols and fatty acids, 4,4,6,8tetramethyl-3H-chromen-2-one, vitamin E (and its precursor phytol), phytosterols, selinenes, 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one, Z-12-pentacosene, and 5-HMF were also present. The antimicrobial activity of H. syriacus extracts was then assayed in vitro. Upon conjugation with COS, flower and leaf extracts led to MIC values of 500 and 375 µg·mL −1 , respectively, against E. amylovora; to MIC values of 250 and 500 µg·mL −1 , respectively, against E. vitivora; and to EC 90 values of 976 and 604 µg·mL −1 , respectively, against D. seriata. The strong synergistic behavior observed upon conjugation with COS may be ascribed to solubility and bioavailability enhancement. In view of the observed activity, an alternative valorization approach as a source of bioactive products may be envisaged, although in vivo assays are required to determine the actual operational efficacy.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to their relevance to be part of an ongoing PhD Thesis.