Silene uniflora Extracts for Strawberry Postharvest Protection

Halophytes are gaining considerable attention due to their applications in saline agriculture, phytoremediation, medicine, and secondary metabolite production. This study investigated the bioactive components present in Silene uniflora (sea campion) hydromethanolic extract, and their antimicrobial activity was evaluated both in vitro and ex situ against two strawberry phytopathogens, namely Botrytis cinerea (grey mold) and Colletotrichum nymphaeae (anthracnose fruit rot). The main identified phytochemicals were mome inositol, saturated fatty acid esters, and cyclotetracosane. In vitro tests demonstrated complete inhibition of the growth of B. cinerea and C. nymphaeae at extract concentrations of 1000 and 1500 μg·mL−1, respectively, with an activity comparable to that of fosetyl-Al and substantially higher than that of azoxystrobin. This activity was improved upon conjugation with chitosan oligomers (COS), yielding inhibition values of 750 and 1000 μg·mL−1. The COS-S. uniflora conjugate complexes were then tested as protective treatments for postharvest storage of strawberry fruit, resulting in high protection against artificially inoculated B. cinerea and C. nymphaeae at doses of 3750 and 5000 μg·mL−1, respectively. The reported results open the door to the valorization of this halophyte as a source of biorationals for strawberry protection.


Introduction
Silene uniflora Roth is an herbaceous perennial plant of the Caryophyllaceae family, typically forming mats on cliffs. Its leaves are linear, gray-green, and glaucous, and the flowers are white with five sepals that form a bladder and five deeply notched petals (Figure 1a).
Regarding antimicrobial activity, there have been promising reports on Silene parishii S.Watson [14], S. vulgaris [15,16], Silene cariensis Boiss. [17], S. alba, S. conoidea, S. dichotoma, Regarding antimicrobial activity, there have been promising reports on Silene parishii S.Watson [14], S. vulgaris [15,16], Silene cariensis Boiss. [17], S. alba, S. conoidea, S. dichotoma, S. italica, S. supina, and S. vulgaris [13], but there is a dearth of information on the activity of S. uniflora. The aim of the study presented herein was two-fold: (i) to investigate the phytoconstituents of S. uniflora using vibrational spectroscopy (IR) and gas chromatography-mass spectrometry (GC-MS) and (ii) to evaluate S. uniflora antifungal activity, alone and in combination with chitosan oligomers (COS), and open new pathways for its valorization. In particular, two of the most important strawberry (Fragaria × ananassa Duch.) pathogens were selected: Botrytis cinerea Pers. and Colletotrichum nymphaeae (Pass.) Aa., which ranked second and eighth on a list of fungal pathogens of scientific and economic importance, respectively [18]. The former (grey mold) has a wide host range (over 200 plant species) and potential for causing severe damage, both pre-and post-harvest; the latter belongs to the Colletotrichum acutatum J.H.Simmonds clade and causes anthracnose [19]. The reported findings may contribute to the management of these diseases in agricultural ecosystems.

Vibrational Characterization
Prior to extract preparation, infrared spectra of the different aerial plant organs of S. uniflora ( Figure S1) were analyzed in an initial screening to identify functional groups and assess the presence/absence of significant differences among them. The fingerprint regions of the three spectra were very similar. Band assignments are summarized in Table 1. The band at 3366 cm −1 can be attributed to hydrogen bonding in pyranosides [20], and those at ca. 2916, 1443, 1371, 1244, and 1146 cm −1 are consistent with the presence of inositol, detected by GC-MS, as discussed below. Concerning the presence of saturated fatty acid vinyl esters, this is supported by the bands at 2916, 2848, 1636, 1472, 1417, 1378, 1307, 1243, 1146, 1101, and 719 cm −1 .  The aim of the study presented herein was two-fold: (i) to investigate the phytoconstituents of S. uniflora using vibrational spectroscopy (IR) and gas chromatography-mass spectrometry (GC-MS) and (ii) to evaluate S. uniflora antifungal activity, alone and in combination with chitosan oligomers (COS), and open new pathways for its valorization. In particular, two of the most important strawberry (Fragaria × ananassa Duch.) pathogens were selected: Botrytis cinerea Pers. and Colletotrichum nymphaeae (Pass.) Aa., which ranked second and eighth on a list of fungal pathogens of scientific and economic importance, respectively [18]. The former (grey mold) has a wide host range (over 200 plant species) and potential for causing severe damage, both pre-and post-harvest; the latter belongs to the Colletotrichum acutatum J.H.Simmonds clade and causes anthracnose [19]. The reported findings may contribute to the management of these diseases in agricultural ecosystems.

Vibrational Characterization
Prior to extract preparation, infrared spectra of the different aerial plant organs of S. uniflora ( Figure S1) were analyzed in an initial screening to identify functional groups and assess the presence/absence of significant differences among them. The fingerprint regions of the three spectra were very similar. Band assignments are summarized in Table 1. The band at 3366 cm −1 can be attributed to hydrogen bonding in pyranosides [20], and those at ca. 2916, 1443, 1371, 1244, and 1146 cm −1 are consistent with the presence of inositol, detected by GC-MS, as discussed below. Concerning the presence of saturated fatty acid vinyl esters, this is supported by the bands at 2916, 2848, 1636, 1472, 1417, 1378, 1307, 1243, 1146, 1101, and 719 cm −1 .

In Vitro Growth Inhibition Tests
The results of the mycelial growth inhibition tests ( Figure S4) are summarized in Figure 3. When tested separately, S. uniflora showed greater efficacy against B. cinerea than COS, as full inhibition was reached at 1000 and 1500 µg·mL −1 , respectively. For C. nymphaeae, both S. uniflora and COS exhibited approximately the same efficacy (MIC = 1500 µg·mL −1 ). An enhancement in terms of efficacy was observed in both cases for the COS-S. uniflora extract  Upon comparison of the effective concentrations (Table 3), it was possible to observe differences in the efficacy of the treatments more clearly. The highest efficacy (i.e., the lowest EC 50 and EC 90 values) was observed for the COS-S. uniflora conjugate complex against B. cinerea, followed by those of the same treatment against C. nymphaeae. Synergistic behavior was found between COS and S. uniflora extract (SF values ≥ 1), with the highest synergy factor being obtained for the EC 90 of COS-S. uniflora against B. cinerea (SF = 1.56). Table 4 summarizes the inhibition results for three synthetic fungicides. Mancozeb completely inhibited the growth of both fungal pathogens even at a tenth of the recommended dose (i.e., at 150 µg·mL −1 ), while Fosetyl-Al required a concentration of 2000 µg·mL −1 , and azoxystrobin did not fully inhibit the growth of the two fungal taxa at 62,500 µg·mL −1 . CH2 rocking

In Vitro Growth Inhibition Tests
The results of the mycelial growth inhibition tests ( Figure S4) are summarized in Figure 3. When tested separately, S. uniflora showed greater efficacy against B. cinerea than COS, as full inhibition was reached at 1000 and 1500 µg·mL −1 , respectively. For C. nymphaeae, both S. uniflora and COS exhibited approximately the same efficacy (MIC = 1500 µg·mL −1 ). An enhancement in terms of efficacy was observed in both cases for the COS-S. uniflora extract conjugate complex, reaching full inhibition at 750 and 1000 µg·mL −1 for B. cinerea and C. nymphaeae, respectively.  Upon comparison of the effective concentrations (Table 3), it was possible to observe differences in the efficacy of the treatments more clearly. The highest efficacy (i.e., the lowest EC50 and EC90 values) was observed for the COS-S. uniflora conjugate complex against B. cinerea, followed by those of the same treatment against C. nymphaeae. Synergistic behavior was found between COS and S. uniflora extract (SF values ≥ 1), with the highest synergy factor being obtained for the EC90 of COS-S. uniflora against B. cinerea (SF = 1.56).

Ex Situ Growth Inhibition Tests
Strawberry fruits of the variety "Calinda" were treated with the most active product according to the in vitro assays, the COS-S. uniflora conjugate complex, at MIC×5 (namely 3750 and 5000 µg·mL −1 for B. cinerea and C. nymphaeae, respectively). As depicted in Figure 4, the treatment noticeably reduced the incidence of both pathogens. Disease incidences were calculated on days 1, 7, and 10 of the experiment (Table 5). In the negative controls, the pathogens did not proliferate (thus ruling out the possibility of contamination), whereas in the strawberries that had been artificially inoculated with the pathogens but not treated (positive controls), B. cinerea and C. nymphaeae were able to invade more than 81% of the surface of all fruits on the tenth day, with an incidence of 5 at the end of the trial. Upon treatment with the COS-S. uniflora extract conjugate complex, an incidence of 1.3 was observed on the tenth day, with the most-affected fruits showing a colonization of less than 40% by B. cinerea, whereas the colonization of fruits artificially infected with C. nymphaeae was higher, with an incidence of 2.3 on the tenth day (i.e., most fruits showed a colonization equal to or higher than 40%). Table 5. Disease severity of B. cinerea and C. nymphaeae attack on cv. "Calinda" strawberries after 1, 7, and 10 days. Concerning fruit quality attributes, the COS-S. uniflora extract treatment exerted a beneficial effect on the firmness, with an average 24% decrease in flesh firmness values in the case of B. cinerea and a 33% decrease for C. nymphaeae vs. a 52% decrease in the untreated fruits (negative control) by the end of the experiment. As far as color is concerned, the COS-S. uniflora coating imparted a slightly paler shade of red on day 10, more evident in the fruits inoculated with B. cinerea than in those inoculated with C. nymphaeae, although quantitative color measurements would be needed to determine the actual impact on the hue degree and chroma. This should be taken into account, as it may influence consumer preferences.    Concerning fruit quality attributes, the COS-S. uniflora extract treatment exerted a beneficial effect on the firmness, with an average 24% decrease in flesh firmness values in the case of B. cinerea and a 33% decrease for C. nymphaeae vs. a 52% decrease in the untreated fruits (negative control) by the end of the experiment. As far as color is concerned, the COS-S. uniflora coating imparted a slightly paler shade of red on day 10, more evident in the fruits inoculated with B. cinerea than in those inoculated with C. nymphaeae, although quantitative color measurements would be needed to determine the actual impact on the hue degree and chroma. This should be taken into account, as it may influence consumer preferences.

On the Phytochemical Profile
Given that only a small subset of the known organic compounds (amenable for GC-MS) is present in the largest mass spectral databases, limitations in the identification of some of the compounds present in the extracts were detected, with quality of resemblance (Qual) values below 80 (Table 2). Caution is advised as identification of such compounds may be unreliable. However, it is noteworthy that for the chemical species identified at RT = 26.8367 min, comprising 8.7% of the peak area with a "Qual" value of 41, its MS spectrum shows good agreement with those of myristic acid vinyl ester and palmitic acid vinyl ester (Figure S3), and the bands identified in the FTIR spectra of the dried aerial parts also support the presence of saturated fatty acid vinyl esters.

Activity of Other Silene spp. Extracts
In terms of activity against the two phytopathogens studied here, data are only available for methanolic Silene armeria L. leaf extract [39], for which a MIC value of 1000 µg·mL −1 was reported against B. cinerea and Colletotrichum capsici (Syd. and P.Syd.) E.J.Butler and Bisby, comparable to the MICs reported herein (1000 and 1500 µg·mL −1 against B. cinerea and C. nymphaeae, respectively). This same S. armeria extract achieved MIC values in the 500-2000 µg·mL −1 range against Rhizoctonia solani Kühn, Fusarium oxysporum Schlechtendal, Fusarium solani W.C.Snyder, Sclerotinia sclerotiorum (Lib.) de Bary, and Phytophthora capsici Leonian, suggesting that similar activity may be expected for the S. uniflora extract studied here.
Concerning other Silene spp. extracts, there are reports on their antimicrobial activity against other microorganisms. Back in 1993, Hoffmann et al. [14] showed that the ethanolic extract of S. parishii at a concentration of 1000 µg·mL −1 was effective against Bacillus subtilis (Ehrenberg, 1835) Cohn, 1872, partially effective against Candida albicans (C.P.Robin) Berkhout, and had no effect on Staphylococcus aureus Rosenbach, 1884 and Klebsiella pneumoniae (Schroeter, 1886) Trevisan, 1887. Subsequent studies by Boukhira et al. [

Comparison with Synthetic Antimicrobials
The concentrations of S. uniflora extract required for full inhibition of B. cinerea and C. nymphaeae (1000 and 1500 µg·mL −1 , respectively; see Table 3) were an order of magnitude higher than those of mancozeb (Table 4), demonstrating a substantially lower antimicrobial activity. The activity of S. uniflora extract was comparable to that of fosetyl-Al (MIC = 2000 µg·mL −1 ), but substantially higher than that of azoxystrobin (MIC > 62,500 µg·mL −1 ).

Comparison with Chitosan-Based Coatings for Postharvest Strawberry Protection
In order to compare the protective effect of the COS-S. uniflora extract conjugate complex with other chitosan-based coatings reported in the literature (on strawberries), the results of a brief bibliographical survey are presented in Table 6, itemized into those used for postharvest control of gray mold decay (B. cinerea) and those aimed at anthracnose (Colletotrichum spp.) control. In the case of B. cinerea, it may be observed that the efficacy of the COS-S. uniflora extract treatment (3750 and 5000 µg·mL −1 against B. cinerea and C. nymphaeae, respectively) was markedly superior to those reported for chitosan acetate, chitosan chloride, chitosan glutamate, and chitosan formate, in which a 1% w/v dose was applied, with higher disease severities at the end of the experiments [40]. However, it was not as effective as Zataria multiflora Boiss. essential oil encapsulated in chitosan nanoparticles (1500 µg·mL −1 ) [41] and COS-Uncaria tomentosa (Willd. ex Schult.) DC conjugate complexes (1000 µg·mL −1 ).
When it comes to protection against C. nymphaeae, to the best of our knowledge, no previous studies on postharvest protection using chitosan have been reported for strawberry fruits. Comparison with other treatments against Colletotrichum spp. reported in the literature reveals the efficacy of COS-S. uniflora would be similar to those reported by Arceo Martínez et al. [42] for chitosan at a concentration of 7500 µg·mL −1 .

C. fragariae
Chitosan + cinnamon EO + aqueous extract of Roselle calyces presence of 4-O-methyl-myo-inositol. Although there are no reports on the antimicrobial activity of pure 4-O-methyl-myo-inositol, the aforementioned extract of N. lappaceum showed antibacterial activity against food pathogenic and spoilage bacteria [24]. Furthermore, myoinositol has demonstrated strong antifungal activity against Fusarium circinatum Nirenberg and O'Donnell, Cryphonectria parasitica (Murril) M.E. Barr, and Phytophthora cinnamomi Rands phytopathogens, with MIC values of 1000, 750, and 375 µg·mL −1 , respectively [46]. Both vinyl palmitate and vinyl myristate have also shown antimicrobial activity [27,47]. However, the contributions from other minority constituents and synergistic behaviors among them cannot be ruled out.
Concerning COS, its antifungal activity is well-established [48] and is thought to be due to its positive charge interacting with the negative charge of the fungal cell membrane. This interaction leads to increased permeability of the cell [49], resulting in a loss of intracellular components that disrupts the osmotic pressure and causes cell death [50]. COS can also alter chitin levels, leading to a weakened cell wall [51], and can generate ROS that damage biomolecules, triggering apoptosis and necrosis. Additionally, COS can interfere with DNA and RNA synthesis [52].
With regard to enhanced activity upon the formation of conjugate complexes, the observed synergism may stem from an enhanced additive fungicidal activity per se or by simultaneous action at multiple fungal metabolic sites [53], but it may also be due to the fact that chitosan oligomers can increase the solubility and bioavailability of the bioactive compounds present in the extract.

Plant Material and Chemicals
Samples of S. uniflora were collected in May 2021, during the flowering stage (Figure 1a), from Playa de Cué (Llanes, Asturias, Spain; 43 • 24 58.7" N 4 • 43 53.3" W). Specimens were identified and authenticated by Prof. Dr. Baudilio Herrero Villacorta (Departamento de Ciencias Agroforestales, ETSIIAA, Universidad de Valladolid) and voucher specimens are available from the herbarium of the ETSIIAA. Aerial parts from different specimens (n = 20) were mixed to obtain (separate) representative composite samples of flowers, fruits, and leaves. The composite samples were shade-dried (with a 72% weight loss), reduce to powder using a mechanical grinder, homogenized, and sieved (1 mm mesh).
Strawberry fruits (Fragaria × ananassa cv. "Calinda") were supplied by Ideal Fruits (Chañe, Segovia, Spain). The fruits were produced without the addition of artificial pesticides in accordance with organic farming regulations. The fruits were collected and transported to the laboratory in refrigerated conditions, and ex situ tests began within 24 h of harvesting. Strawberries were chosen on the bases of uniform size, lack of physical damage and fungal infection, and a red coloration covering more than 75% of the surface, in agreement with Romanazzi et al. [40].

Fungal Isolates
The fungal isolates of B. cinerea (code not available, but details on its provenance are provided in [54]) and Colletotrichum nymphaeae were supplied as subcultures in PDA by Richerd Breia and Hernâni Gerós from the Centre of Molecular and Environmental Biology (CBMA) at the University of Minho and by Pedro Talhinhas, School of Agriculture, University of Lisbon, respectively. They were cultured in PDA at 25 • C in the dark.

Preparation of S. uniflora Extract, Chitosan Oligomers, and Their Conjugate Complex
The extract preparation procedure was similar to the one previously reported in [31]. Briefly, 20 g of dried S. uniflora flowering aerial parts were mixed with a 300 mL methanol/water solution (1:1 v/v) and heated in a water bath at 50 • C for 30 min. Then, the solution was subjected to sonication for 5 min in pulse mode with a 1 min stop every 2.5 min, using a model UIP1000hdT probe-type ultrasonicator (Hielscher Ultrasonics; Teltow, Germany). The solution was centrifuged at 9000 rpm for 15 min and the supernatant was filtered through Whatman No. 1 paper, followed by freeze-drying to obtain the solid residue. The extraction yield was 5%. For subsequent GC-MS analysis, 25 mg of the obtained freeze-dried extracts were dissolved in 5 mL of HPLC-grade MeOH to obtain a 5 mg·mL −1 solution, which was further filtered.
Chitosan oligomers (COS) were prepared using the method described in [55] with the modifications described in [56], yielding oligomers of molecular weight < 2000 Da in a solution with a pH of 4.5. COS and S. uniflora extract solutions (150 mL of each solution, both at a concentration of 3000 µg·mL −1 ) were mixed in a 1:1 (v/v) ratio and sonicated for 15 min in five 3 min pulses to obtain the conjugate complexes.

Characterization Procedures
The infrared spectra of the S. uniflora dried plant organs were registered using a model Nicolet iS50 Fourier transform infrared spectrometer from Thermo Scientific (Waltham, MA, USA), equipped with a diamond attenuated total reflection (ATR) system. The spectra were acquired at 1 cm −1 spectral resolution over the 400-4000 cm −1 range by co-adding 64 scans.
The hydromethanolic extract was studied using GC-MS at the Research Support Services (STI) at Universidad de Alicante (Alicante, Spain), utilizing a model 7890A gas chromatograph coupled to a model 5975C quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The chromatographic conditions consisted of an injection volume of 1 µL; an injector temperature of 280 • C, in splitless mode; and an initial oven temperature of 60 • C for two minutes, followed by a 10 • C per minute ramp up to a final temperature of 300 • C, kept for 15 min. The chromatographic column used for the separation of the compounds was an HP-5MS UI of 30 m length, 0.250 mm diameter, and 0.25 µm film. The mass spectrometer conditions were set to a temperature of the electron impact source of 230 • C, and the quadrupole was set to 150 • C; the ionization energy was set to 70 eV. The identification of phytoconstituents was based on a comparison of their mass spectra and retention time with those of authentic compounds and by computer matching with the National Institute of Standards and Technology (NIST11) and Wiley databases.

In Vitro Tests of Mycelial Growth Inhibition
The antimicrobial activities were evaluated using the agar dilution method [57]. Petri dishes with PDA incorporating ten different concentrations (ranging from 62.5 to 1500 µg·mL −1 ) of the various treatments-namely COS, S. uniflora extract, and COS-S. uniflora extract-were inoculated with 5 mm plugs and cultured at 25 • C for seven days. The control consisted in replacing the extract with the solvent used for extraction (i.e., methanol:water 1:1 v/v) in the PDA medium. Tests with commercial fungicides were performed in parallel and using the same source of inoculum. Growth inhibition was calculated by the following formula: ((d c − d t )/d c ) × 100, where d c is the average colony diameter in the control colony and d t is the average colony diameter in the treated colony. The 50% and 90% effective concentrations, EC 50 and EC 90 , were calculated using IBM (Armonk, NY, USA) SPSS Statistics v.25's PROBIT analysis. The synergy factor [58], which measures the degree of interaction, was estimated using Wadley's method [59].

Ex Situ Tests of Mycelial Growth Inhibition
Under controlled laboratory conditions, the efficacy of the COS-S. uniflora extract conjugate complex was evaluated on artificially inoculated strawberry fruits. The protocol was slightly modified from that proposed by Sánchez-Hernández et al. [44]. The strawberries were disinfected for 2 min with a NaOCl 3% solution, then washed three times with sterile distilled water and dried in a laminar flow hood on sterile absorbent paper. The strawberries were divided into three homogeneous groups of 45 fruits (three repetitions with 15 fruits per repetition, treatment, and pathogen), with all fruits measuring more than 22 mm in diameter. One group was treated with COS-S. uniflora extract conjugate complex (at a concentration of MIC × 5, i.e., at 3750 µg·mL −1 for B. cinerea or 5000 µg·mL −1 for C. nymphaeae), while the other groups served as the negative (no treatment and no pathogen) and positive (pathogen and no treatment) controls. Superficial wounds (ø = 5 mm) were made in the equatorial zone of each fruit, and the strawberries were then immersed in the COS-S. uniflora conjugate complex treatment for five minutes and dried at room temperature in a laminar flow hood, using sterile absorbent paper. In the superficial wounds, a plug of a PDA culture from B. cinerea or C. nymphaeae was placed (with the mycelium facing the fruit wound). Following Hernández-Muñoz et al. [60], the fruits were placed in covered plastic boxes and stored for seven days at 4 • C and 95-98% RH, then exposed to a 3-day shelf life at 20 • C and 95-98% RH. In accordance with Romanazzi et al. [40], the percentage of rotten strawberries and the disease severity (according to an empirical scale with six degrees: 0, healthy fruit; 1, 1-20% of fruit surface infected; 2, 21-40% of fruit surface infected; 3, 41-60% of fruit surface infected; 4, 61-80% of fruit surface infected; 5, more than 81% of surface infected, with sporulation) were recorded during storage.
Concerning quality attributes, firmness was measured in the central zone of the strawberries (previously sliced into halves) using a TA-XT2 Texture Analyzer (Stable Micro Systems, Godalming, UK) with a 5 mm diameter flat probe. The penetration depth was 5 mm and the cross-head speed was 5 mm·s −1 .

Statistical Analyses
In vitro and ex situ results were analyzed via one-way analysis of variance (ANOVA) followed by Tukey's post hoc test in IBM SPSS Statistics v.25.

Conclusions
The hydromethanolic extract of S. uniflora aerial parts (leaves, flower petals, and fruits) has 4-O-methyl-myo-inositol (52.5%), saturated fatty acid esters (8.7%), and cyclotetracosane (3.7%) as its main phytoconstituents, according to our GC-MS results. Upon testing of its in vitro activity against B. cinerea and C. nymphaeae strawberry pathogens, MIC values of 1000 and 1500 µg·mL −1 were obtained, which are comparable to those of the synthetic fungicide fosetyl-Al. A synergistic effect was observed upon conjugation of chitosan oligomers with the halophyte extract, resulting in MIC values of 750 and 1000 µg·mL −1 . Concerning the use of the COS-S. uniflora conjugate complex in postharvest protection of strawberry fruits, a dose five times higher than the in vitro MIC was required to achieve high inhibition against the two phytopathogens after 10 days (with disease severities of 1.3 and 2.3 out of 5 for B. cinerea and C. nymphaeae, compared to 5 out of 5 for the non-treated fruits). This activity is one of the highest reported for chitosan-based coatings, suggesting that S. uniflora extract may be a suitable biorational for the protection of this crop.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants12091846/s1, Figure S1: Infrared spectra of S. uniflora aerial plant organs; Figure S2: GC-MS chromatogram of Silene uniflora extract; Figure S3: Comparison of MS spectra of two saturated fatty acid vinyl esters with that of the chemical species detected at RT = 26.837 min; Figure S4: Radial growth of B. cinerea and C. nymphaeae in the presence of the three treatments, namely S. uniflora extract, chitosan oligomers, and COS-S. uniflora extract conjugate complex, at different concentrations (expressed in µg·mL −1 ).

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.