Antimicrobial Activity of Chalcones with a Chlorine Atom and Their Glycosides

Chalcones, secondary plant metabolites, exhibit various biological properties. The introduction of a chlorine and a glucosyl substituent to the chalcone could enhance its bioactivity and bioavailability. Such compounds can be obtained through a combination of chemical and biotechnological methods. Therefore, 4-chloro-2′-hydroxychalcone and 5′-chloro-2′-hydroxychalcone were obtained by synthesis and then glycosylated in two filamentous fungi strains cultures, i.e., Isaria fumosorosea KCH J2 and Beauveria bassiana KCH J1.5. The main site of the glycosylation of both compounds by I. fumosorosea KCH J2 was C-2′ and C-3 when the second strain was utilized. The pharmacokinetics of these compounds were predicted using chemoinformatics tools. Furthermore, antimicrobial activity tests were performed. Compounds significantly inhibited the growth of the bacteria strains Escherichia coli 10536, Staphylococcus aureus DSM 799, and yeast Candida albicans DSM 1386. Nevertheless, the bacterial strain Pseudomonas aeruginosa DSM 939 exhibited significant resistance to their effects. The growth of lactic acid bacteria strain Lactococcus acidophilus KBiMZ 01 bacteria was moderately inhibited, but strains Lactococcus rhamnosus GG and Streptococcus thermophilus KBM-1 were completely inhibited. In summary, chalcones substituted with a chlorine demonstrated greater efficacy in inhibiting the microbial strains under examination compared to 2′-hydroxychalcone, while aglycones and their glycosides exhibited similar effectiveness.

The promising strategy for the glycosylation of chalcones can be the use of microbial enzymes-among others-as well as whole-cell biotransformation using filamentous fungi as biocatalysts [13,14].Xie and coworkers employed genome mining and heterologous expression techniques to discover functional modules of glycosyltransferasemethyltransferase (GT-MT) in these fungi.These modules exhibit substrate promiscuity and regiospecificity, allowing them to methylglucosylate flavonoids, as shown in Figure 1 [15].
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 2 of 22 chlorflavonin, exhibited strong antituberculosis potential with (MIC90 1.56 μM) and was superior to streptomycin treatment [11].Halogenated chalcones, while not naturally occurring, can be synthesized through the Claisen-Schmidt condensation [8,9,12].The promising strategy for the glycosylation of chalcones can be the use of microbial enzymes-among others-as well as whole-cell biotransformation using filamentous fungi as biocatalysts [13,14].Xie and coworkers employed genome mining and heterologous expression techniques to discover functional modules of glycosyltransferase-methyltransferase (GT-MT) in these fungi.These modules exhibit substrate promiscuity and regiospecificity, allowing them to methylglucosylate flavonoids, as shown in Figure 1 [15].[15]).
The lack of in vivo data makes it difficult to generalize the influence of glycosylation on flavonoids bioactivity.Based on some reports, it appears that O-glycosylation may reduce anti-inflammation activity, antioxidant activity, and antimicrobial activity.Simultaneously, some data show that O-glycosylation can enhance specific bioactivity, including anti-HIV activity, tyrosinase inhibition, antirotavirus activity, and antiallergic activity.Furthermore, glycosylation positively affects their bioavailability [16].The impact of glycosylation on flavonoid bioactivity in vitro may not necessarily mirror the effects observed in vivo.Consequently, additional research in this field is essential.Therefore, the purpose of this work was to obtain two chalcones with a chlorine atom, i.e., 4-chloro-2′-hydroxychalcone and 5′-chloro-2′-hydroxychalcone, and afterward glycosylate them in entomopathogenic filamentous fungi I. fumosorosea KCH J2 and B. bassiana KCH J1.5 cultures.As a result, we obtained novel flavonoid derivatives.Subsequently, we evaluated the pharmacokinetics of the received compounds using computer-aided simulations.Furthermore, the antimicrobial activities of the chalcones with chlorine atoms, their main biotransformation products, and 2′-hydroxychalcone used for comparison were assessed to explore how chlorine substitution and glucose attachment influence their effectiveness.
The lack of in vivo data makes it difficult to generalize the influence of glycosylation on flavonoids bioactivity.Based on some reports, it appears that O-glycosylation may reduce anti-inflammation activity, antioxidant activity, and antimicrobial activity.Simultaneously, some data show that O-glycosylation can enhance specific bioactivity, including anti-HIV activity, tyrosinase inhibition, antirotavirus activity, and antiallergic activity.Furthermore, glycosylation positively affects their bioavailability [16].The impact of glycosylation on flavonoid bioactivity in vitro may not necessarily mirror the effects observed in vivo.Consequently, additional research in this field is essential.Therefore, the purpose of this work was to obtain two chalcones with a chlorine atom, i.e., 4-chloro-2 ′ -hydroxychalcone and 5 ′ -chloro-2 ′ -hydroxychalcone, and afterward glycosylate them in entomopathogenic filamentous fungi I. fumosorosea KCH J2 and B. bassiana KCH J1.5 cultures.As a result, we obtained novel flavonoid derivatives.Subsequently, we evaluated the pharmacokinetics of the received compounds using computer-aided simulations.Furthermore, the antimicrobial activities of the chalcones with chlorine atoms, their main biotransformation products, and 2 ′ -hydroxychalcone used for comparison were assessed to explore how chlorine substitution and glucose attachment influence their effectiveness.

Biotransformation of 4-Chloro-2′-Hydroxychalcone
Five distinctive proton signals, with δH values between 3.22 and 3.84 ppm, verified the presence of a glucose moiety in biotransformation product 3a in the 1 H-NMR spectrum The products 3a-3b structures were elucidated via NMR spectroscopy (Tables 1 and 2, Figures 5 and 6 below showing key COSY and HMBC correlations) and confirmed using LC-MS (section Materials and Methods and Supplementary Materials: Figures S31 and S52).
Product 6a, analogous to 3a but with a different chlorine positioning, featured 4″-Omethylglucopyranose in a β-configuration at C-2′.This was evidenced by the disappearance of the 2′-OH signal and the presence of characteristic glucose signals in 1 H-NMR (Supplementary Materials: Figures S96, S98 and S101).Additionally, a C-α and C-β double-bond reduction occurred, similarly to 3a, which was indicated by characteristic shifts in the 1 H-NMR and 13 C-NMR spectra (Supplementary Materials: Figures S19, S21, S22, S98 and S101).NMR spectroscopy elucidated the structure of product 6b (Tables 3 and 4, key COSY and HMBC correlations in Figure 12).LC-MS confirmed its molecular mass (Materials and Methods, Supplementary Materials: Figure S115).
The glycosylation of product 6b was confirmed by characteristic 4″-O-methylglucosyl signals in the 1 H-NMR and 13 C-NMR spectra (Supplementary Materials: Figures S119 The structure of product 6a was elucidated via NMR spectroscopy (Tables 3 and 4 in section, key COSY and HMBC correlations in Figure 10) and confirmed using LC-MS (section Materials and Methods and Supplementary Materials: Figure S94).The structure of product 6a was elucidated via NMR spectroscopy (Tables 3 and 4 in section, key COSY and HMBC correlations in Figure 10) and confirmed using LC-MS (section Materials and Methods and Supplementary Materials: Figure S94).
Product 6a, analogous to 3a but with a different chlorine positioning, featured 4″-Omethylglucopyranose in a β-configuration at C-2′.This was evidenced by the disappearance of the 2′-OH signal and the presence of characteristic glucose signals in 1 H-NMR (Supplementary Materials: Figures S96, S98 and S101).Additionally, a C-α and C-β double-bond reduction occurred, similarly to 3a, which was indicated by characteristic shifts in the 1 H-NMR and 13 C-NMR spectra (Supplementary Materials: Figures S19, S21, S22, S98 and S101).NMR spectroscopy elucidated the structure of product 6b (Tables 3 and 4, key COSY and HMBC correlations in Figure 12).LC-MS confirmed its molecular mass (Materials and Methods, Supplementary Materials: Figure S115).
The glycosylation of product 6b was confirmed by characteristic 4″-O-methylglucosyl signals in the 1 H-NMR and 13 C-NMR spectra (Supplementary Materials: Figures S119   The structure of product 6a was elucidated via NMR spectroscopy (Tables 3 and 4 in section, key COSY and HMBC correlations in Figure 10) and confirmed using LC-MS (section Materials and Methods and Supplementary Materials: Figure S94).
Product 6a, analogous to 3a but with a different chlorine positioning, featured 4″-Omethylglucopyranose in a β-configuration at C-2′.This was evidenced by the disappearance of the 2′-OH signal and the presence of characteristic glucose signals in 1 H-NMR (Supplementary Materials: Figures S96, S98 and S101).Additionally, a C-α and C-β double-bond reduction occurred, similarly to 3a, which was indicated by characteristic shifts in the 1 H-NMR and 13 C-NMR spectra (Supplementary Materials: Figures S19, S21, S22, S98 and S101).NMR spectroscopy elucidated the structure of product 6b (Tables 3 and 4, key COSY and HMBC correlations in Figure 12).LC-MS confirmed its molecular mass (Materials and Methods, Supplementary Materials: Figure S115).
The glycosylation of product 6b was confirmed by characteristic 4″-O-methylglucosyl signals in the 1 H-NMR and 13 C-NMR spectra (Supplementary Materials: Figures S119 NMR spectroscopy elucidated the structure of product 6b (Tables 3 and 4, key COSY and HMBC correlations in Figure 12).LC-MS confirmed its molecular mass (Materials and Methods, Supplementary Materials: Figure S115).[18].These results indicate that oxidation and subsequent glycosylation occurred in ring A when ring B was already substituted with a chlorine atom or a methyl group, and this may be related to some steric hindrance of the enzyme action.Conversely, B. bassiana KCH J1.5 attached the 4″-O-methylglucosyl moiety at C-3 in ring B of both biotransformation substrates 3 and 6 in a very similar reaction.It should also be emphasized that the introduction of the sugar unit was most likely preceded by hydroxylation of the C-3 carbon.However, substrate 3 was also hydrogenated to form 3c, while an α,βunsaturated double bond in 6b remained intact.These results show that the enzyme systems of B. bassiana KCH J1.5 and I. fumosrosoea KCH J2 differ, with the former being able to introduce a 4″-O-methylglucopyranose unit, despite the presence of a substituent in the B ring.Previous studies on methylchalcones [18,19] showed less efficient glycosylation of the 2′-hydroxyl group by I. fumosorosea KCH J2, suggesting a positive influence of the chlorine substituent on the glycosylation site.Earlier work on chalcone glycosylation with a C-5′ methyl group in both B. bassiana KCH J1.5 and I. fumosorosea KCH J2 cultures yielded a product similar to 6b but with a methyl group instead of chlorine [20].
To summarize, compounds 4-chloro-2 ′ -hydroxychalcone (3) and 5 ′ -chloro-2 ′ -hydroxychalcone (6) were glycosylated in cultures of both entomopathogenic fungi strains.The resulting compounds revealed different regioselectivity of the glycosyltransferase-methyltransferase functional modules of the two strains.In I. fumosorosea KCH J2, the primary products (3a and 6a) resulted from attaching a 4 ′′ -O-methylglucosyl group to the C-2 ′ hydroxyl moiety in the A ring and reducing the C-α and C-β double bond.For 4chloro-2 ′ -hydroxychalcone (3), glycosylation also occurred at C-5 ′ , yielding 3b.Compound 3b is analogous to the products obtained earlier in the cultures of this fungal strain, i.e., 2-chloro-2 ′ -hydroxydihydrochalcone 5 ′ -O-β-d-(4 ′′ -O-methyl)-glucopyranoside from 2-chloro-2 ′ -hydroxychalcone and 3-chloro-2 ′ -hydroxydihydrochalcone 5 ′ -O-β-d-(4 ′′ -O-methyl)-glucopyranoside from 3-chloro-2 ′ hydroxychalcone [17], and also 2 ′ -hydroxy-4-methyldihydrochalcone 5 ′ -O-β-D-(4 ′′ -O-methyl)-glucopyranoside from 2 ′ -hydroxy-4methylchalcone [18].These results indicate that oxidation and subsequent glycosylation occurred in ring A when ring B was already substituted with a chlorine atom or a methyl group, and this may be related to some steric hindrance of the enzyme action.Conversely, B. bassiana KCH J1.5 attached the 4 ′′ -O-methylglucosyl moiety at C-3 in ring B of both biotransformation substrates 3 and 6 in a very similar reaction.It should also be emphasized that the introduction of the sugar unit was most likely preceded by hydroxylation of the C-3 carbon.However, substrate 3 was also hydrogenated to form 3c, while an α,β-unsaturated double bond in 6b remained intact.These results show that the enzyme systems of B. bassiana KCH J1.5 and I. fumosrosoea KCH J2 differ, with the former being able to introduce a 4 ′′ -O-methylglucopyranose unit, despite the presence of a substituent in the B ring.Previous studies on methylchalcones [18,19] showed less efficient glycosylation of the 2 ′ -hydroxyl group by I. fumosorosea KCH J2, suggesting a positive influence of the chlorine substituent on the glycosylation site.Earlier work on chalcone glycosylation with a C-5 ′ methyl group in both B. bassiana KCH J1.5 and I. fumosorosea KCH J2 cultures yielded a product similar to 6b but with a methyl group instead of chlorine [20].
C. albicans DSM 1386 yeast strain was highly sensitive to the tested compounds.Except for compounds 6a and 7, which allowed slight growth (ΔOD = 0.35 and ΔOD = 0.11, respectively), all others completely inhibited yeast growth.The C. albicans growth patterns under exposure to the tested compounds are presented in Figure 16.C. albicans DSM 1386 yeast strain was highly sensitive to the tested compounds.Except for compounds 6a and 7, which allowed slight growth (∆OD = 0.35 and ∆OD = 0.11, respectively), all others completely inhibited yeast growth.The C. albicans growth patterns under exposure to the tested compounds are presented in Figure 16.
C. albicans DSM 1386 yeast strain was highly sensitive to the tested compounds.Except for compounds 6a and 7, which allowed slight growth (ΔOD = 0.35 and ΔOD = 0.11, respectively), all others completely inhibited yeast growth.The C. albicans growth patterns under exposure to the tested compounds are presented in Figure 16.The effect of the tested compounds on the lactic acid bacteria varied by species.L. rhamnosus GG and S. thermophilus KBM-1 growth were totally inhibited by all compounds.However, complete growth inhibition of the L. acidophilus KBiMZ 01 bacteria occurred because of the action of compounds 3 and 7, and significant inhibition occurred when molecule 6 was used (∆OD = 0.12).The glycosylated flavonoids with a chlorine atom (3a, 6a, and 6b) prolonged the microbial lag phase and limited this lactic bacteria growth.The lowest level of inhibition was observed in the case of compound 6b.detail the growth of these bacteria in response to the compounds used.The effect of the tested compounds on the lactic acid bacteria varied by species.L. rhamnosus GG and S. thermophilus KBM-1 growth were totally inhibited by all compounds.However, complete growth inhibition of the L. acidophilus KBiMZ 01 bacteria occurred because of the action of compounds 3 and 7, and significant inhibition occurred when molecule 6 was used (ΔOD = 0.12).The glycosylated flavonoids with a chlorine atom (3a, 6a, and 6b) prolonged the microbial lag phase and limited this lactic bacteria growth.The lowest level of inhibition was observed in the case of compound 6b.detail the growth of these bacteria in response to the compounds used.The effect of the tested compounds on the lactic acid bacteria varied by species.L. rhamnosus GG and S. thermophilus KBM-1 growth were totally inhibited by all compounds.However, complete growth inhibition of the L. acidophilus KBiMZ 01 bacteria occurred because of the action of compounds 3 and 7, and significant inhibition occurred when molecule 6 was used (ΔOD = 0.12).The glycosylated flavonoids with a chlorine atom (3a, 6a, and 6b) prolonged the microbial lag phase and limited this lactic bacteria growth.The lowest level of inhibition was observed in the case of compound 6b.detail the growth of these bacteria in response to the compounds used.Antimicrobial activity tests of the obtained compounds 3, 6, 3a, 6a, 6b, and 7 showed that the substitution of a chlorine atom in the chalcone structure had a positive effect on their activity against the tested microorganisms: E. coli 10536, S. aureus DSM 799, P. aeruginosa DSM 939, and C. albicans DSM 1386.Prasad and coworkers also showed a positive effect of pharmacophores like chloro-, dichloro-, and fluoro-moiety on antibacterial activity against the used strain of E. coli bacteria [8].On the other hand, antimicrobial activity tests against the S. aureus AM-176 strain performed by Alcaraz and coworkers showed that 4-chlorochalcone inhibited its growth less effectively than 2′-hydroxychalcone and chalcone (PID (Percent Inhibition Degree) = 34.7,98.3, and 38.3 respectively) [27].These results indicate that the hydroxyl group within the chalcone structure strongly influences its antimicrobial activity.However, researchers have not investigated how the combined effects of a 2′-hydroxyl moiety and a chlorine atom impact the antimicrobial properties of the resulting compound.In the presented work, chlorinated 2′-hydroxychalcones showed significantly stronger antibacterial activity against S. aureus DSM 799.Furthermore, their derivatives with the blocked 2′-hydroxyl group by the attached glucosyl moiety were not less effective.Konečná and coworkers also proved that introducing a bromine or chlorine atom into pyrazine-based chalcones yielded receiving compounds with strong antistaphylococcal and antienterococcal activity [10].The impact of glucosyl groups on chalcones' antimicrobial properties remains poorly understood.However, in the antimicrobial activity tests of another group of flavonoids with this moiety, i.e., flavonol 3-O-glycosides, researchers observed a potent suppression of Gram-positive bacteria and a weaker inhibition of Gram-negative bacteria [16].In our studies, E. coli 10536 were exceptionally susceptible to the actions of chlorinated aglycones and the glycosides of 2′-hydroxychalcone.However, P. aeruginosa DSM 939 was quite opposing.In our previous studies with 2chloro-2′-hydroxychalcone, 3-chloro-2′hydroxychalcones, and their glycosides, we also observed that chlorinated chalcones were more active as inhibitors of the tested microbial strains' growth compared to their unchlorinated counterparts.However, aglycones showed slightly greater efficacy than their glycoside forms [17].By comparing all the obtained results, one can observe differences in the inhibition of microbial growth depending on the chlorine atom substitution position in the tested chalcones.Comparing all of the results obtained, differences in microbial growth inhibition can be observed depending on a chlorine atom substitution position in chalcones tested.Interestingly, 5′-  Antimicrobial activity tests of the obtained compounds 3, 6, 3a, 6a, 6b, and 7 showed that the substitution of a chlorine atom in the chalcone structure had a positive effect on their activity against the tested microorganisms: E. coli 10536, S. aureus DSM 799, P. aeruginosa DSM 939, and C. albicans DSM 1386.Prasad and coworkers also showed a positive effect of pharmacophores like chloro-, dichloro-, and fluoro-moiety on antibacterial activity against the used strain of E. coli bacteria [8].On the other hand, antimicrobial activity tests against the S. aureus AM-176 strain performed by Alcaraz and coworkers showed that 4-chlorochalcone inhibited its growth less effectively than 2 ′ -hydroxychalcone and chalcone (PID (Percent Inhibition Degree) = 34.7,98.3, and 38.3 respectively) [27].These results indicate that the hydroxyl group within the chalcone structure strongly influences its antimicrobial activity.However, researchers have not investigated how the combined effects of a 2 ′ -hydroxyl moiety and a chlorine atom impact the antimicrobial properties of the resulting compound.In the presented work, chlorinated 2 ′ -hydroxychalcones showed significantly stronger antibacterial activity against S. aureus DSM 799.Furthermore, their derivatives with the blocked 2 ′ -hydroxyl group by the attached glucosyl moiety were not less effective.Konečná and coworkers also proved that introducing a bromine or chlorine atom into pyrazine-based chalcones yielded receiving compounds with strong antistaphylococcal and antienterococcal activity [10].The impact of glucosyl groups on chalcones' antimicrobial properties remains poorly understood.However, in the antimicrobial activity tests of another group of flavonoids with this moiety, i.e., flavonol 3-O-glycosides, researchers observed a potent suppression of Gram-positive bacteria and a weaker inhibition of Gram-negative bacteria [16].In our studies, E. coli 10536 were exceptionally susceptible to the actions of chlorinated aglycones and the glycosides of 2 ′ -hydroxychalcone.However, P. aeruginosa DSM 939 was quite opposing.In our previous studies with 2-chloro-2 ′ -hydroxychalcone, 3-chloro-2 ′ hydroxychalcones, and their glycosides, we also observed that chlorinated chalcones were more active as inhibitors of the tested microbial strains' growth compared to their unchlorinated counterparts.However, aglycones showed slightly greater efficacy than their glycoside forms [17].By comparing all the obtained results, one can observe differences in the inhibition of microbial growth depending on the chlorine atom substitution position in the tested chalcones.Comparing all of the results obtained, differences in microbial growth inhibition can be observed depending on a chlorine atom substitution position in chalcones tested.Interestingly, 5 ′ -chlorodihydrochalcone 2 ′ -O-β-D-(4 ′′′ -O-methyl)-glucopyranoside (6a) showed significant activity against all tested microbial strains but was the least effective against the C. albicans DSM 1386 strain.
The physical data of compounds 3 and 6 (color, form, molecular ion mass, molecular formula, melting point (

Microorganisms
Microbial glycosylation of chlorochalcones 3 and 6, obtained by chemical synthesis, was achieved in cultures of I. fumosorosea KCH J2 and B. bassiana KCH J1.5 filamentous fungi belonging to the collection of the Faculty of Biotechnology and Food Microbiology of the Wrocław University of Environmental and Life Sciences in Poland.Our previous studies detailed the genetic identification, collection methods, and reproduction of these fungi [13,33].

Analysis
Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) were used to monitor biotransformation progress, specifically substrate transformation [18].All compounds were 95%-98% pure according to HPLC analysis.The separation of biotransformation products on a semi-preparative scale was achieved using preparative silica gel TLC plates with thicknesses of 500 µm and 1000 µm (Supelco, Darmstadt, Germany) and mixture of chloroform and methanol (9:1 volume ratio) [17,19].

Screening Procedure
A biotransformation screening procedure was conducted to determine the time required for the complete conversion of substrates 3 and 6 in preparation for subsequent experiments at a semi-preparative scale.Entomopathogenic fungi were cultivated using a modified Sabouraud medium.Fungal strains were initially grown for 72 h and then transferred to fresh medium.Substrates 3 or 6 (10 mg) were added to flasks containing either Isaria fumosorosea KCH J2 or Beauveria bassiana KCH J1.5, with a final concentration of 0.39 mM.Samples were collected after 3, 6, and 8 days.Biotransformation products were extracted with ethyl acetate, which were then dried and concentrated.The experi-ments were concluded after 8 days or when complete substrate conversion was confirmed.Controls included substrate stability and cultivation without substrates [17,19].

The Semi-Preparative Biotransformation
Semi-preparative biotransformation was conducted in 2 L flasks with 500 mL of modified Sabouraud medium to produce sufficient product for NMR analyses, structural determination, and antimicrobial testing.The process began with transferring 1 mL of a preincubated culture of I. fumosorosea KCH J2 or B. bassiana KCH J1.5 to the flasks, followed by a 72-h incubation.Then, 50 mg of substrate 3 or 6 (dissolved in 2.0 mL of dimethyl sulfoxide) was added, maintaining a final concentration of 0.39 mM.The flasks were incubated on a rotary shaker for 8 days.Post-reaction mixtures were extracted with ethyl acetate, which were then dried, filtered, and evaporated.The biotransformation products were separated and purified using preparative TLC plates, which were then visualized under UV light and extracted with ethyl acetate.The chemical structures were analyzed via spectroscopic methods, and yields were determined based on the mass of the isolated products [17,19].

Fungal Biotransformation Products
The physical data of compounds 3a-3c and 6a-6b (color, form, molecular ion mass, molecular formula, melting point ( • C), retention time t R (min), retardation factor Rf, and NMR spectral data) are presented below, in Tables 1-4 in the Results and Discussion section, and in the Supplementary Materials.
Assays were conducted using 100-well microtiter plates with a working volume of 300 µL per well.Each well contained 280 µL of culture medium, 10 µL of microorganism suspension (final density 1 × 10 6 cells/mL), and 10 µL of flavonoids dissolved in dimethyl sulfoxide (final flavonoid concentration 0.1% (m/v)).The dimethyl sulfoxide final concentration in each well was 3.3% (v/v).The plates were incubated at 30 • C with optical density measurements at 560 nm taken every 60 min for 72 h for all microorganisms except lactic acid bacteria, for which the temperature was 37 • C, and measurements were taken every 30 min for 70 h.Each test was performed in triplicate with continuous shaking.Oxytetracycline (10 mg/mL) and cycloheximide (0.1% (m/v) purchased from Sigma-Aldrich (Saint Louis, MO, USA) were used as positive controls.Data were analyzed using Microsoft Excel, and growth curves were created based on the mean absorbance values over time.Antimicrobial activity was assessed by comparing the increase in optical density (∆OD) of treated cultures to controls with only dimethyl sulfoxide [17].

Figure 5 .
Figure 5. Key COSY (on the left) and HMBC (on the right) correlations of product 3a.

Figure 6 .
Figure 6.Key COSY (on the left) and HMBC (on the right) correlations of product 3b.

Figure 5 .
Figure 5. Key COSY (on the left) and HMBC (on the right) correlations of product 3a.

Figure 5 .
Figure 5. Key COSY (on the left) and HMBC (on the right) correlations of product 3a.

Figure 6 .
Figure 6.Key COSY (on the left) and HMBC (on the right) correlations of product 3b.

Figure 6 .
Figure 6.Key COSY (on the left) and HMBC (on the right) correlations of product 3b.

Figure 8 .
Figure 8. Key COSY (on the left) and HMBC (on the right) correlations of product 3c.

Figure 10 .
Figure 10.Key COSY (on the left) and HMBC (on the right) correlations of product 6a.Product 6a, analogous to 3a but with a different chlorine positioning, featured 4 ′′ -O-methylglucopyranose in a β-configuration at C-2 ′ .This was evidenced by the disappearance of the 2 ′ -OH signal and the presence of characteristic glucose signals in1 H-NMR (Supplementary Materials: FiguresS96, S98 and S101).Additionally, a Cα and C-β double-bond reduction occurred, similarly to 3a, which was indicated by characteristic shifts in the 1 H-NMR and 13 C-NMR spectra (Supplementary Materials: Figures S19, S21, S22, S98 and S101).

Figure 12 .
Figure 12.Key COSY (on the left) and HMBC (on the right) of product 6b.

Figure 14 .
Figure 14.The impact of compounds 3, 6, 3a, 6a, 6b, 7 on the growth of P. aeruginosa DSM 939.On the other hand, the bacteria growth of S. aureus DSM 799 was completely inhibited by compound 6a.Other flavonoids with a chlorine atom also prolonged the microbial

Table 1 .
The

Table 1 .
The

Table 1 .
The

Table 4 .
The

Table 5 .
SwissADME online tool analysis of pharmacokinetic properties and drug-likeness for compounds 3

Table 6 .
Antimicrobial activity of compounds 3