Antibacterial Effects of Flavonoids and Their Structure-Activity Relationship Study: A Comparative Interpretation

According to the latest report released by the World Health Organization, bacterial resistance to well-known and widely available antibacterial drugs has become a significant and severe global health concern and a grim challenge to tackle in order to cure infections associated with multidrug-resistant pathogenic microorganisms efficiently. Consequently, various strategies have been orchestrated to cure the severe complications related to multidrug-resistant bacteria effectively. Some approaches involved the retardation of biofilm formation and multidrug-resistance pumps in bacteria as well as the discovery of new antimicrobial agents demonstrating different mechanisms of action. In this regard, natural products namely alkaloids, terpenoids, steroids, anthraquinone, flavonoids, saponins, tannins, etc., have been suggested to tackle the multidrug-resistant bacterial strains owing to their versatile pharmacological effects. Amongst these, flavonoids, also known as polyphenolic compounds, have been widely evaluated for their antibacterial property due to their tendency to retard the growth of a wide range of pathogenic microorganisms, including multidrug-resistant bacteria. The hydroxylation of C5, C7, C3′, and C4′; and geranylation or prenylation at C6 have been extensively studied to increase bacterial inhibition of flavonoids. On the other hand, methoxylation at C3′ and C5 has been reported to decrease flavonoids’ antibacterial action. Hence, the latest information on the antibacterial activity of flavonoids is summarized in this review, with particular attention to the structure–activity relationship of this broad class of natural compounds to discover safe and potent antibacterial agents as natural products.

Flavonoids, the most important class of phenolic compounds, are secondary metabolites produced by plants and are found in a non-glycosylated form (aglycone) or glycosidic form (attached to a sugar molecule, also known as glycone) [44,46,[51][52][53]. All flavonoids display a standard structure of two phenyl rings (A and B) connected to a heterocyclic ring (C, the ring containing the embedded oxygen). This carbon structure can be abbreviated as C6-C3-C6. These compounds frequently display hydroxylation at positions 5 and 7 at A ring and oxidation at the 3′,4′ or 3′,4′,5′ positions at B ring due to their biosynthesis routes [46,54,55]. Flavonoids are frequently found in nature and have a significant chemical diversity mainly because of the changes in B-and C-ring locations, degree of hydroxylation, oxidation, and saturation of ring C [51,56,57]. Flavonoids are classified according to their chemical structures into chalcones, flavanones, flavanonols, flavones, flavonols, isoflavones, flavan-3-ols (catechins), and anthocyanidins, as shown in Figure 1.
The numbering system of a basic flavonoid structure is also illustrated in Figure 2. The presence of a ketonic functional group at position 4, a double bond between carbon atoms 2 and 3 (C2=C3 double bond), and the presence of a hydroxyl group at position 3  in the ring C are the primary factors that precisely determine flavonoids subdivision.   Flavonoids have been identified as polyphenolic compounds capable o tibacterial activities via various mechanisms of action. According to mu studies, flavonoids can suppress nucleic acid synthesis, cytoplasmic memb and energy metabolism [58][59][60][61][62][63]. Flavonoids have also been found to reduce biofilm formation, porin on the cell membrane, membrane permeability, an ity, all of which are crucial for bacterial growth [58,60,61,63]. Furthermore, noids have been reported to reverse antibiotic resistance and improve the present antibiotics [60,61,[64][65][66][67]. As a result, the development and deploy noid-based medications could be a potential method for antibiotic-resist This review is an attempt to increase our knowledge and understanding antibacterial effects, emphasizing on their structure-activity relationship (S

Literature Sources and Search Strategy
An online literature search utilizing phrases such as 'structure-activit study of flavonoids as antibacterial agent'; 'flavonoids as antibacterial age rial effect of flavonoids'; 'in vitro antibacterial activity or property of flavon flavonoids as antibacterial agent' yielded information about antibacterial pr vonoids and their SAR analysis. The online databases used were Google S Online, Springer Link, Science Direct, PubMed, and the International Islam Malaysia (IIUM) Discovery Service. The online search was tailored between to 2021, giving almost 400 easily accessible studies on the SAR of flavonoid rials that were predominantly published in English. The retrieved data wa amined by looking for descriptions of flavonoids' antibacterial activities a Additional data was gathered by reviewing and analyzing the references c lected articles. As a result, the current review accounts for previous and m formation on flavonoids' antibacterial effects, focusing on their SAR.

Anti-Bacterial Activity of Flavonoids
Flavonoids have been reported to exert varying degrees of antibac Otsuka et al. (2008) evaluated the antibacterial potential of laurel leaves ( and consequently isolated two anti-methicillin resistant Staphylococcus a agents, namely kaempferol 3-O-α-L-(2‴,4‴-di-E-p-coumaroyl)-rhamnosi kaempferol 3-O-α-L-(2″-Z-p-coumaroyl-4‴-E-p-coumaroyl)-rhamnoside (1 Table 1, both compounds outperformed antibacterial agents such as tetracy Flavonoids have been identified as polyphenolic compounds capable of exerting antibacterial activities via various mechanisms of action. According to multiple research studies, flavonoids can suppress nucleic acid synthesis, cytoplasmic membrane function, and energy metabolism [58][59][60][61][62][63]. Flavonoids have also been found to reduce adhesion and biofilm formation, porin on the cell membrane, membrane permeability, and pathogenicity, all of which are crucial for bacterial growth [58,60,61,63]. Furthermore, certain flavonoids have been reported to reverse antibiotic resistance and improve the efficacy of the present antibiotics [60,61,[64][65][66][67]. As a result, the development and deployment of flavonoid-based medications could be a potential method for antibiotic-resistant infections. This review is an attempt to increase our knowledge and understanding of flavonoids' antibacterial effects, emphasizing on their structure-activity relationship (SAR).

Literature Sources and Search Strategy
An online literature search utilizing phrases such as 'structure-activity relationship study of flavonoids as antibacterial agent'; 'flavonoids as antibacterial agent'; 'antibacterial effect of flavonoids'; 'in vitro antibacterial activity or property of flavonoids'; 'SAR of flavonoids as antibacterial agent' yielded information about antibacterial properties of flavonoids and their SAR analysis. The online databases used were Google Scholar, Wiley Online, Springer Link, Science Direct, PubMed, and the International Islamic University Malaysia (IIUM) Discovery Service. The online search was tailored between the years 2000 to 2021, giving almost 400 easily accessible studies on the SAR of flavonoids as antibacterials that were predominantly published in English. The retrieved data was critically examined by looking for descriptions of flavonoids' antibacterial activities and their SAR. Additional data was gathered by reviewing and analyzing the references cited in the selected articles. As a result, the current review accounts for previous and most recent information on flavonoids' antibacterial effects, focusing on their SAR.

Discussion
Anti-Bacterial Activity of Flavonoids  Table 1, both compounds outperformed antibacterial agents such as tetracycline, erythromycin, norfloxacin, ciprofloxacin, and oxacillin in terms of anti-MRSA activity with minimum inhibitory concentration (MIC) of 0.5-2.0 µg/mL. SAR studies for both compounds revealed that 5,7-dihydroxylation of the ring A and 4 -hydroxylation of the ring B are important structural features (pharmacophores) for eliciting an in vitro anti-MRSA activity [68]. Furthermore, substituting 3-O-acyl chains with C8 and C10 in the ring C increased the antistaphylococcal action of flavonoids belonging to the flavan-3-ol class [69]. Both compounds possessed 3-O-acylated rhamnose. Both compounds include a ten-carbons acyl (coumaroyl) group and showed that this portion would likewise contribute to the strong anti-MRSA activity of 1a and 1b. Figure 3 shows the summary of SAR analysis of tested flavonoids [70].   Moreover, Šmejkal et al. (2008) successfully isolated six C6-geranylflavon shown in Table 2 and Figure 4) from Paulownia tomentosa fruits ethanol extract a antibacterial properties were further explored against six different bacterial strain reported that all the isolated compounds showed comparable antibacterial activi compared to commercialized drugs, viz., ciprofloxacin and nystatin. Šmejkal et a lated that the presence of OH at C5 further increased the action of the flavanone The hydroxyl group at C5 can establish an intramolecular hydrogen bond with bonyl group at position C4, resulting in more electron delocalization inside the m However, the introduction of methoxy groups at any R position was found to d the antibacterial potential of the aforementioned isolated compounds by retardin hibition of bacterial growth. In addition, the presence of the geranyl group at C6 in the antibacterial activity by increasing compounds' lipophilicity characteristics. oxy-4′,5′-dihydroxyphenyl groups of ring B of compound 3′-O-meth droxydiplacone (2a) and 3′,5′-dimethoxy-4-hydroxy substitution of compound 3 thyl-5′-O-methyldiplacone (2b) appeared to be crucial for activity due to an increa molecule's planar character [71].  Moreover, Šmejkal et al. (2008) successfully isolated six C6-geranylflavonoids (as shown in Table 2 and Figure 4) from Paulownia tomentosa fruits ethanol extract and their antibacterial properties were further explored against six different bacterial strains. It was reported that all the isolated compounds showed comparable antibacterial activity when compared to commercialized drugs, viz., ciprofloxacin and nystatin. Šmejkal et al. postulated that the presence of OH at C5 further increased the action of the flavanones tested. The hydroxyl group at C5 can establish an intramolecular hydrogen bond with the carbonyl group at position C4, resulting in more electron delocalization inside the molecule. However, the introduction of methoxy groups at any R position was found to diminish the antibacterial potential of the aforementioned isolated compounds by retarding the inhibition of bacterial growth. In addition, the presence of the geranyl group at C6 increased the antibacterial activity by increasing compounds' lipophilicity characteristics. 3 -methoxy-4 ,5 -dihydroxyphenyl groups of ring B of compound 3 -Omethyl-5 -hydroxydiplacone (2a) and 3 ,5 -dimethoxy-4-hydroxy substitution of compound 3 -O-methyl-5 -O-methyldiplacone (2b) appeared to be crucial for activity due to an increase in the molecule's planar character [71].    Similarly,  revealed the important structural aspect in Escherichia col DNA gyrase inhibition exerted by the flavonoids, as shown in Figure 5. DNA gyrase is an essential enzyme for bacterial replication as it can relieve the strain within doublestranded DNA during DNA replication. By looking at the bioactivity results in Table 3 kaempferol with a hydroxyl group at C3′, quercetin with a hydroxyl group at C3′, and galangin without 4′-OH all demonstrated that 3′-OH decreased activity and emphasizing the importance of 4′-OH in the ring B. In addition, myricetin and quercetin, which possess hydroxyl group at C5′, suggested that 5′-OH also decreased the flavonoids' antibacteria activity. Next, baicalein with 6-OH was less efficient as an antibacterial agent, showing that the hydroxyl group at C6 in ring A is not essential and may hinder the compound's biological action. It was also reported that 3-OH in ring C could decrease the DNA gyrase inhibition, as shown in galangin when compared to chrysin. Apart from that, it has been observed that substituting a lipophilic functional group, such as OCH3, at C6 or C8 in creases antibacterial activity of the resultant flavonoids [72]. Tangeritin, for example, dis played more excellent activity than 5,6,7,4′-tetramethoxyflavone, with 8-OCH3 in the ring A being the only structural difference. However, the substitution of 3′-OCH3 in ring B reduces the inhibitory effect on bacteria, as can be seen in nobiletin. Next, the authors reported that hydroxylation at C5 boosted the activity as seen in baicalein, while the meth oxy group at the same position decreased activity as shown by 5,6,7,4-tetramethoxyfla vone. Summary of the structural requirement as shown in Figure 5 [62].  Similarly,  revealed the important structural aspect in Escherichia coli DNA gyrase inhibition exerted by the flavonoids, as shown in Figure 5. DNA gyrase is an essential enzyme for bacterial replication as it can relieve the strain within double-stranded DNA during DNA replication. By looking at the bioactivity results in Table 3, kaempferol with a hydroxyl group at C3 , quercetin with a hydroxyl group at C3 , and galangin without 4 -OH all demonstrated that 3 -OH decreased activity and emphasizing the importance of 4 -OH in the ring B. In addition, myricetin and quercetin, which possess hydroxyl group at C5 , suggested that 5 -OH also decreased the flavonoids' antibacterial activity. Next, baicalein with 6-OH was less efficient as an antibacterial agent, showing that the hydroxyl group at C6 in ring A is not essential and may hinder the compound's biological action. It was also reported that 3-OH in ring C could decrease the DNA gyrase inhibition, as shown in galangin when compared to chrysin. Apart from that, it has been observed that substituting a lipophilic functional group, such as OCH 3 , at C6 or C8 increases antibacterial activity of the resultant flavonoids [72]. Tangeritin, for example, displayed more excellent activity than 5,6,7,4 -tetramethoxyflavone, with 8-OCH 3 in the ring A being the only structural difference. However, the substitution of 3 -OCH 3 in ring B reduces the inhibitory effect on bacteria, as can be seen in nobiletin. Next, the authors reported that hydroxylation at C5 boosted the activity as seen in baicalein, while the methoxy group at the same position decreased activity as shown by 5,6,7,4-tetramethoxyflavone. Summary of the structural requirement as shown in Figure 5 [62].

Galangin
OH OH  H  OH  H  H  H  H  53  Quercetin  OH  OH  H  OH  H  OH  OH  H  36  Baicalein  H  OH  OH  OH  H  H  H  H  71  Luteolin  H  OH  H  OH  H  OH  OH  H  67  Kaempferol  OH  OH  H  OH  H  H  OH  H  25  Myricetin  OH  OH  H  OH  H  OH  OH OH 142 Figure 5. The SAR of flavonoids as antibacterial agents by inhibiting the DNA gyrase activity [62] In another similar research work performed by , the authors evalu ated the antibacterial activity of several flavonoids (viz. chrysin, kaempferol, 5,6,7,4′-tet ramethoxyflavone, luteolin, baicalein, quercetin, tangeritin, daidzein, puerarin, genistin and ononin) on E. coli via membrane interaction effect. The bioactivity results are dis played in Table 4. Results indicated that the hydroxyl group at position 3 in ring C wa found important to manifest the antibacterial properties of flavonoids. For example, quer cetin (a flavonol) showed a higher antibacterial ability than luteolin (a flavone) which lacked the OH group at position 3 compared to quercetin. It was also revealed that th presence of methoxy groups reduced the antibacterial properties of flavonoids, i.e., tan geritin (4′,5,6,7,8-pentamethoxyflavone) and 5,6,7,4′-tetramethoxyflavone showed lowe antibacterial activity than the flavonoids bearing hydroxyl groups. However, the methox group at C8 in ring A appeared to increase the antibacterial activity. Tangeritin showed higher activity than 5,6,7,4′-tetramethoxyflavone despite the similarity in their structure except for a methoxy group at C8 position in ring A. It was also suggested that the anti bacterial activity of isoflavonoids decreased with the glycosylation at C7 and C8 in th ring A, such as daidzein which exhibited the highest inhibitory activity compared t puerarin, genistin, and ononin ( Figure 6) [73].   In another similar research work performed by , the authors evaluated the antibacterial activity of several flavonoids (viz. chrysin, kaempferol, 5,6,7,4tetramethoxyflavone, luteolin, baicalein, quercetin, tangeritin, daidzein, puerarin, genistin, and ononin) on E. coli via membrane interaction effect. The bioactivity results are displayed in Table 4. Results indicated that the hydroxyl group at position 3 in ring C was found important to manifest the antibacterial properties of flavonoids. For example, quercetin (a flavonol) showed a higher antibacterial ability than luteolin (a flavone) which lacked the OH group at position 3 compared to quercetin. It was also revealed that the presence of methoxy groups reduced the antibacterial properties of flavonoids, i.e., tangeritin (4 ,5,6,7,8-pentamethoxyflavone) and 5,6,7,4 -tetramethoxyflavone showed lower antibacterial activity than the flavonoids bearing hydroxyl groups. However, the methoxy group at C8 in ring A appeared to increase the antibacterial activity. Tangeritin showed higher activity than 5,6,7,4 -tetramethoxyflavone despite the similarity in their structures except for a methoxy group at C8 position in ring A. It was also suggested that the antibacterial activity of isoflavonoids decreased with the glycosylation at C7 and C8 in the ring A, such as daidzein which exhibited the highest inhibitory activity compared to puerarin, genistin, and ononin ( Figure 6) [73].   Figure 6. The SAR of flavonoids as antibacterial by inhibiting the growth of E. coli [73]. Figure 6. The SAR of flavonoids as antibacterial by inhibiting the growth of E. coli [73]. Next, Yadav et al. (2013) studied the SAR of 15 selected flavonoids and their antitubercular effect against Mycobacterium tuberculosis. It was reported that luteolin (MIC = 25 µg/mL) had the most strong antibacterial activity among the all flavonoids evaluated. However, quercetin, baicalein, and myricetin exhibited similar activity (MIC = 50 µg/mL) and hispidulin showed the least antibacterial effect (MIC = 100 µg/mL), while the others were found inactive. They discovered that the presence of hydroxyl groups at the positions 5, 6, and 7 in ring A such as baicalein, or the 3 ,4 positions in ring B such as luteolin, quercetin, and myricetin, or the 4 position in 5,7-dihydroxy-6-methoxy arrangement as in hispidulin were indispensable for inhibiting the growth of M. tuberculosis. However, the addition of random hydroxyl substitutions did not influence the antitubercular action of the flavonoids studied in the same research work. Additionally, this SAR study also demonstrated that antitubercular activity requires the presence of two hydroxyl groups in neighboring locations. However, compounds become inactive when the hydroxyl group is methylated or glycosylated, as seen with casticin and hesperidin [74]. The overall requirements for the antitubercular activity of flavonoids are shown in Figure 7. Next, Yadav et al. (2013) studied the SAR of 15 selected flavonoids and their antitubercular effect against Mycobacterium tuberculosis. It was reported that luteolin (MIC = 25 μg/mL) had the most strong antibacterial activity among the all flavonoids evaluated. However, quercetin, baicalein, and myricetin exhibited similar activity (MIC = 50 μg/mL) and hispidulin showed the least antibacterial effect (MIC = 100 μg/mL), while the others were found inactive. They discovered that the presence of hydroxyl groups at the positions 5, 6, and 7 in ring A such as baicalein, or the 3′, 4′ positions in ring B such as luteolin, quercetin, and myricetin, or the 4′ position in 5,7-dihydroxy-6-methoxy arrangement as in hispidulin were indispensable for inhibiting the growth of M. tuberculosis. However, the addition of random hydroxyl substitutions did not influence the antitubercular action of the flavonoids studied in the same research work. Additionally, this SAR study also demonstrated that antitubercular activity requires the presence of two hydroxyl groups in neighboring locations. However, compounds become inactive when the hydroxyl group is methylated or glycosylated, as seen with casticin and hesperidin [74]. The overall requirements for the antitubercular activity of flavonoids are shown in Figure 7. subtilis, S. aureus, Shigella flexneri, Pseudomonas aeruginosa, and Salmonella typhi. The structural-activity analysis revealed that 4-iminoflavones exhibited better activities compared to 4-thioflavones, due to the presence of 2,4-dinitrophenyl hydrazine moiety. Next, it was also found out that the antibacterial activity of all the derivatives of 4-thioflavone and 4- Ullah et al. (2006) synthesized several 4-thioflavones and 4-iminoflavones derivatives, and revealed their antibacterial properties on linear growth inhibition of E. coli, B. subtilis, S. aureus, Shigella flexneri, Pseudomonas aeruginosa, and Salmonella typhi. The structuralactivity analysis revealed that 4-iminoflavones exhibited better activities compared to 4-thioflavones, due to the presence of 2,4-dinitrophenyl hydrazine moiety. Next, it was also found out that the antibacterial activity of all the derivatives of 4-thioflavone and 4-iminoflavone was increased in the presence of methoxy group at 4 -position in ring B. The same pattern of activity was also found when OCH 3 group in ring B was replaced by NO 2 group; however, the bacterial percentage inhibition was decreased slightly (Figure 8). Fluorine at C4 showed remarkable inhibitory properties against E. coli, B. subtilis, S. flexneri, S. typhi, and P. aeruginosa but were inactive against S. aureus. It was discovered that methoxy groups at C3 and C4 of ring B can enhance the inhibition of E. coli, P. aeruginosa and S. typhi growth. On the other hand, NO 2 group at C5 lowered the inhibition properties [75].
cules 2022, 27, x 9 of iminoflavone was increased in the presence of methoxy group at 4′-position in ring B. T same pattern of activity was also found when OCH3 group in ring B was replaced by N group; however, the bacterial percentage inhibition was decreased slightly (Figure 8). F orine at C4′ showed remarkable inhibitory properties against E. coli, B. subtilis, S. flexn S. typhi, and P. aeruginosa but were inactive against S. aureus. It was discovered that me oxy groups at C3′ and C4′ of ring B can enhance the inhibition of E. coli, P. aeruginosa a S. typhi growth. On the other hand, NO2 group at C5′ lowered the inhibition propert [75].  Table  It can be seen that flavones were more active against Gram-positive bacterial inhibiti compared to flavanones. Therefore, it can be concluded that C2=C3 double bond may essential for inhibiting bacterial growth. In flavanones, the Gram-positive bacterial in bition became weaker when the number of OH groups was greater as seen in naringen and 7-O-methyl eriodictyol with three OH groups. In comparison, pinocembrin with t hydroxyl groups at ring A showed the lowest MIC. In addition, the authors also point out that the number of OH groups was not important but the position of two hydro groups in the flavanones was found to be essential and important in exerting antibacter effects. Therefore, it was stressed that the presence of two OH groups in ring A and t absence of any hydroxylation in ring B influenced good antibacterial activity, as seen the most active flavanone, pinocembrin. In flavones, with galangin and 3-O-meth galangin as the most active compound, it can be deduced that the OH groups are i portant for antibacterial activity. It is because galangin has three OH groups (two in ri A), followed by 3-O-methylgalangin with two OH in ring A in their structures. Similar flavanones, the lack of substitutions in ring B seemed to be essential for the activity. Th the authors also mentioned that the methylation of OH groups at ring A would decrea the antibacterial action. It was proved as 3-O-methylgalangin with the methylation of o OH group in ring A had weaker antibacterial activity than the galangin. Furthermore, methylation of both OH groups at ring A (as seen in 3,7-O-dimethylgalangin) led to in tivity in antibacterial inhibition. Figure 9 summarizes all the above-mentioned criti  Table 5. It can be seen that flavones were more active against Gram-positive bacterial inhibition compared to flavanones. Therefore, it can be concluded that C2=C3 double bond may be essential for inhibiting bacterial growth. In flavanones, the Gram-positive bacterial inhibition became weaker when the number of OH groups was greater as seen in naringenin and 7-O-methyl eriodictyol with three OH groups. In comparison, pinocembrin with two hydroxyl groups at ring A showed the lowest MIC. In addition, the authors also pointed out that the number of OH groups was not important but the position of two hydroxyl groups in the flavanones was found to be essential and important in exerting antibacterial effects. Therefore, it was stressed that the presence of two OH groups in ring A and the absence of any hydroxylation in ring B influenced good antibacterial activity, as seen in the most active flavanone, pinocembrin. In flavones, with galangin and 3-Omethylgalangin as the most active compound, it can be deduced that the OH groups are important for antibacterial activity. It is because galangin has three OH groups (two in ring A), followed by 3-O-methylgalangin with two OH in ring A in their structures. Similar to flavanones, the lack of substitutions in ring B seemed to be essential for the activity. Then, the authors also mentioned that the methylation of OH groups at ring A would decrease the antibacterial action. It was proved as 3-O-methylgalangin with the methylation of one OH group in ring A had weaker antibacterial activity than the galangin. Furthermore, the methylation of both OH groups at ring A (as seen in 3,7-O-dimethylgalangin) led to inactivity in antibacterial inhibition. Figure 9 summarizes all the above-mentioned critical structural features of the flavonoids for antibacterial activity [76].   Figure 9. Chemical structure of several tested flavonoids and the SAR of flavonoids studied for antibacterial properties against four Gram-positive and four Gram-negative bacteria [76]. Ávila et al. (2008) studied the SAR of chalcones (described as minor flavonoids), as aromatic ketones with two phenyl rings that are also intermediates in the synthesis of many biological compounds, including flavonoids and isoflovonoids. The closure of hydroxychalcones causes the formation of the flavonoid structure in antibacterial activity by testing 31 chalcones compounds. The bacteria used in this study were S. aureus ATCC 25923, P. aeruginosa ATCC 27853, E. coli ATCC 25922, and B. cereus ATCC 11778. The results showed that 4-hydroxyderricin (MIC ≥ 3.9 µg/mL) had the highest antibacterial activity, followed by 2,4,4 -trihydroxy-3-prenyl-3 -geranylchalcone (MIC ≥ 15.6 µg/mL) and 2 ,4,4 -trihydroxy-3 -geranylchalcone (MIC ≥ 15.6 µg/mL). After comparing all 31 chalcones, several necessary structural requirements for antibacterial action were confirmed, as shown in Figure 10. Firstly, hydroxylation at C2 seemed to be an important feature as acetylation or methylation at those positions weakened the bioactivity. Secondly, hydroxyl group at C4 position of ring B was also one of the important characteristics as the chalcone containing the hydroxyl group at the same position became inactive once it underwent methylation. Moreover, the absence of double bonds was also found to decrease the chalcones' activity. Additionally, the hydrophobic group such as prenyl and geranyl groups at C3 position of ring A was also found important to improve the chalcones lipophilicity and facilitate its entry into the bacteria cell membrane [77]. , as aromatic ketones with two phenyl rings that are also intermediates in the synthesis of many biological compounds, including flavonoids and isoflovonoids. The closure of hydroxychalcones causes the formation of the flavonoid structure in antibacterial activity by testing 31 chalcones compounds. The bacteria used in this study were S. aureus ATCC 25923, P. aeruginosa ATCC 27853, E. coli ATCC 25922, and B. cereus ATCC 11778. The results showed that 4-hydroxyderricin (MIC ≥ 3.9 μg/mL) had the highest antibacterial activity, followed by 2,4,4′-trihydroxy-3-prenyl-3′-geranylchalcone (MIC ≥ 15.6 μg/mL) and 2′,4,4′-trihydroxy-3′-geranylchalcone (MIC ≥ 15.6 μg/mL). After comparing all 31 chalcones, several necessary structural requirements for antibacterial action were confirmed, as shown in Figure 10. Firstly, hydroxylation at C2′ seemed to be an important feature as acetylation or methylation at those positions weakened the bioactivity. Secondly, hydroxyl group at C4′ position of ring B was also one of the important characteristics as the chalcone containing the hydroxyl group at the same position became inactive once it underwent methylation. Moreover, the absence of double bonds was also found to decrease the chalcones' activity. Additionally, the hydrophobic group such as prenyl and geranyl groups at C3 position of ring A was also found important to improve the chalconesʹ lipophilicity and facilitate its entry into the bacteria cell membrane [77].  Table 6, it was reported that myricetin, datiscetin, kaempferol, quercetin, and luteolin were active against MRSA. Meanwhile, only myricetin was found active on multidrug-resistant B. cepacia and VRE. Several structural features requirements have been discussed, as illustrated in Figure 11. Firstly, presence of polyhydroxylation may lead to higher bioactivity as seen as myricetin, datiscetin, quercetin, luteolin, and kaempferol. The authors stressed that trihydroxylation at C3′, C4′, and C5′ of ring B was important, especially against multidrug-resistant B.  Table 6, it was reported that myricetin, datiscetin, kaempferol, quercetin, and luteolin were active against MRSA. Meanwhile, only myricetin was found active on multidrug-resistant B. cepacia and VRE. Several structural features requirements have been discussed, as illustrated in Figure 11. Firstly, presence of polyhydroxylation may lead to higher bioactivity as seen as myricetin, datiscetin, quercetin, luteolin, and kaempferol. The authors stressed that trihydroxylation at C3 , C4 , and C5 of ring B was important, especially against multidrug-resistant B. cepacian and VRE. It was also confirmed that hydroxylation at ring B was important as the absence of the hydroxyl groups led to inactivities, which was seen in pinocembrin, chrysin, galangin, kaempferide, and tamarixetin. Hydroxylation at the C3 position of ring C was also found essential for a good activity. It was also observed that the glycosylation at C3 position instead of hydroxylation in myricitrin and rutin caused inactivity. The methoxylation at C3 position also led to inactivities such as in ombuin and rhamnetin. Moreover, when quercetin and inactive catechin were compared, it was suggested that C2=C3 double bond might also be important for the manifestation of antibacterial activity [64]. cepacian and VRE. It was also confirmed that hydroxylation at ring B was important as the absence of the hydroxyl groups led to inactivities, which was seen in pinocembrin, chrysin, galangin, kaempferide, and tamarixetin. Hydroxylation at the C3 position of ring C was also found essential for a good activity. It was also observed that the glycosylation at C3 position instead of hydroxylation in myricitrin and rutin caused inactivity. The methoxylation at C3 position also led to inactivities such as in ombuin and rhamnetin. Moreover, when quercetin and inactive catechin were compared, it was suggested that C2=C3 double bond might also be important for the manifestation of antibacterial activity [64].  Figure 11. Chemical structures of tested flavonoids that were active and the structural feature requirements for the manifestation of flavonoids' antibacterial activity against antibiotic resistant bacteria [64]. The isolated flavonoids were initially tested for their antibacterial potential against two Gram-positive bacteria, namely S. aureus and S. epidermidis. Nine flavonoids, namely corylifol B, neobavaisoflavone, isobavachalcone, 7,8-dihydro-8-(4-hydroxyphenyl)-2,2-dimethyl-2H,6H-benzo[1,2-b:5,4-b ]dipyran-6-one, isoneobavaisoflavone, bavachalcone, bavachin, bavachinin, and one novel compound (erythrinin A) exhibited significant antibacterial activity against both bacteria with an average MIC of 0.009-0.073 mM (Table 7). Isobavachalcone, bavachinin, and erythrinin A showed more potent activity with MIC values of two times lower than two well-known antibacterial agents, namely bakuchiol and magnolol. From the bioactivity, the SAR study of these flavonoids as antibacterial agents revealed that for the chalcone derivatives, prenyl group at ring A led to more active antibacterial activity as exhibited by corylifol B, isobavachalcone, and bavachalcone. It was further proved by observing 1-[2,4-dihydroxy-3-(2-hydroxy-3-methyl-3-butenyl)phenyl]-3-(4-hydroxyphenyl)-2-propen-1-one, bakuchalcone, and brosimacutin G, in which the prenyl group of ring A was either oxygenated or cyclized, the compounds became inactive. As corylifols B with an extra hydroxyl group at C3 and C4 had higher activity compared to bavachalcone with single C4 -OH group, it was seen that the additional hydroxylation led to the stronger antibacterial action. Next, for prenylflavone derivatives, presence of three hydroxyl groups produced more antibacterial active compounds, as shown in 7, dipyran-6-one, bavachinin, and bavachin. Furthermore, the methoxylation at C7, as shown in bavachinin, had stronger activity compared to hydroxylation at C7 which can be seen in bavachin ( Figure 12) [78]. examined the 18 flavonoids comprising chalcones, flavanones, and flavones, and studied the SAR concerning antibacterial activity against MRSA strain using quantitative analysis. Based on the quantitative analysis, several characteristics were pointed out. Firstly, the authors mentioned that chalcones portrayed higher antibacterial activity compared to flavanones and flavones. It can be said that chalcone with the presence of broken ring C may enhance the anti-MRSA action. It was reported that carbonylic group is a crucial structural feature for anti-MRSA. It was also observed that the hydroxylation in unsubstituted flavonoids increased the activity. Hence, the hydroxylation at C2 of chalcones and C5 of flavanones and flavones led to an increase of antibacterial activity. Meanwhile, the -OCH 3 group at the above-mentioned positions reduced the antibacterial action. Lastly, halogen introduction in unsubstituted flavonoids failed to modify the antibacterial activity ( Figure 13) [79]. Alcaraz et al. (2000) examined the 18 flavonoids comprising chalcones, flavanones, and flavones, and studied the SAR concerning antibacterial activity against MRSA strain using quantitative analysis. Based on the quantitative analysis, several characteristics were pointed out. Firstly, the authors mentioned that chalcones portrayed higher antibacterial activity compared to flavanones and flavones. It can be said that chalcone with the presence of broken ring C may enhance the anti-MRSA action. It was reported that carbonylic group is a crucial structural feature for anti-MRSA. It was also observed that the hydroxylation in unsubstituted flavonoids increased the activity. Hence, the hydroxylation at C2′ of chalcones and C5 of flavanones and flavones led to an increase of antibacterial activity. Meanwhile, the -OCH3 group at the above-mentioned positions reduced the antibacterial action. Lastly, halogen introduction in unsubstituted flavonoids failed to modify the antibacterial activity ( Figure 13) [79]. Xie et al. (2017) evaluated antibacterial effects of some selected and structurally alike flavonoids, particularly possessing pyrogallol functionality in different rings and performed a SAR study to discover a potent antibacterial agent. They reported that balcalein and myricetin exhibited the most substantial antibacterial effect among all the flavonoids, with MIC values ranging between 0.5-2.5 mmol/L. The structure of baicalein and myricetin were taken into account to understand their antibacterial potential from SAR perspective. It was observed that both the compounds possessed pyrogallol structure in which three hydroxyl groups were present at the adjacent carbons of the benzene ring namely, balcalein in which trihydroxylation (benzenetriols) existed in ring A at C5, C6, and C7 positions, respectively, however in the case of myricetin, positions C3′, C4′, and C5′ had hydroxyl groups in ring B. Therefore, it was deduced that pyrogallol structure is an important structural feature for the manifestation of good antibacterial activity in the case of flavonoids. Meanwhile, for flavonols and flavanones, hydroxylation at C5 and C7 of ring A also seemed important as shown in quercetin, rutin, naringenin, and hesperitin. It was also observed that flavanones (naringenin) had stronger antibacterial activity than corresponding flavones (apigenin). Hence, the saturation of C2=C3 double bond, which can be seen in flavanones, was also considered an important structural feature for antibacterial activity. The structures of all mentioned flavonoids and their related SAR for antibacterial activity can be viewed in Figure 14 [80]. Hummelova et al. (2014) studied the antibacterial activity of 15 naturally occurring isoflavones against nine Gram-positive and Gram-negative bacteria using the broth microdilution method and the antibacterial effect was expressed as MIC. It was reported that only five isoflavones showed significant antibacterial activity as depicted in Table 8. 6,7,4′trihydroxyisoflavone (3f), 5,7-dihydroxy-4′-methoxyisoflavone (3i), 5,7,4′-trihydroxyisoflavone (3n), 7,3′,4′-trihydroxyisoflavone (3g), and 7,8,4′-trihydroxyisoflavone (3j) showed remarkable antibacterial activity with MIC of ≥16, ≥32, ≥64, ≥128, and ≥128 μg/mL, respectively. Results indicated that 3f was the most active isoflavone among the tested isoflavones. The SAR study revealed that the hydroxylation at C7 and C5 seemed to be important for these isoflavones. It was also mentioned that C4′-OH group could improve the antibacterial activity of isoflavones. In addition, the most active compound, namely 3f had hydroxylation at C7, C4′, and C6 positions. Therefore, hydroxylation at the C6 position was also found to influence the antibacterial activity. Moreover, the presence of methoxy groups in ring A or at C4ʹ led to inactivity, which was seen in several methylated isoflavones, namely 6,4′-dimethoxy-7-hydroxyisoflavone (3a), 6,7,4′-trimethoxyisoflavone (3b), 7,4′-dimethoxyisoflavone (3c), 5,7,4′-trimethoxyisoflavone (3d), 7-hydroxy- Xie et al. (2017) evaluated antibacterial effects of some selected and structurally alike flavonoids, particularly possessing pyrogallol functionality in different rings and performed a SAR study to discover a potent antibacterial agent. They reported that balcalein and myricetin exhibited the most substantial antibacterial effect among all the flavonoids, with MIC values ranging between 0.5-2.5 mmol/L. The structure of baicalein and myricetin were taken into account to understand their antibacterial potential from SAR perspective. It was observed that both the compounds possessed pyrogallol structure in which three hydroxyl groups were present at the adjacent carbons of the benzene ring namely, balcalein in which trihydroxylation (benzenetriols) existed in ring A at C5, C6, and C7 positions, respectively, however in the case of myricetin, positions C3 , C4 , and C5 had hydroxyl groups in ring B. Therefore, it was deduced that pyrogallol structure is an important structural feature for the manifestation of good antibacterial activity in the case of flavonoids. Meanwhile, for flavonols and flavanones, hydroxylation at C5 and C7 of ring A also seemed important as shown in quercetin, rutin, naringenin, and hesperitin. It was also observed that flavanones (naringenin) had stronger antibacterial activity than corresponding flavones (apigenin). Hence, the saturation of C2=C3 double bond, which can be seen in flavanones, was also considered an important structural feature for antibacterial activity. The structures of all mentioned flavonoids and their related SAR for antibacterial activity can be viewed in Figure 14 [80]. Hummelova et al. (2014) studied the antibacterial activity of 15 naturally occurring isoflavones against nine Gram-positive and Gram-negative bacteria using the broth microdilution method and the antibacterial effect was expressed as MIC. It was reported that only five isoflavones showed significant antibacterial activity as depicted in Table 8. 6,7,4 -trihydroxyisoflavone (3f), 5,7-dihydroxy-4 -methoxyisoflavone (3i), 5,7,4 -trihydroxyisoflavone (3n), 7,3 ,4 -trihydroxyisoflavone (3g), and 7,8,4 -trihydroxyisoflavone (3j) showed remarkable antibacterial activity with MIC of ≥16, ≥32, ≥64, ≥128, and ≥128 µg/mL, respectively. Results indicated that 3f was the most active isoflavone among the tested isoflavones. The SAR study revealed that the hydroxylation at C7 and C5 seemed to be important for these isoflavones. It was also mentioned that C4 -OH group could improve the antibacterial activity of isoflavones. In addition, the most active compound, namely 3f had hydroxylation at C7, C4 , and C6 positions. Therefore, hydroxylation at the C6 position was also found to influence the antibacterial activity. Moreover, the presence of methoxy groups in ring A or at C4 led to inactivity, which was seen in several methylated isoflavones, namely 6,4 -dimethoxy-7-hydroxyisoflavone (3a), 6,7,4 -trimethoxyisoflavone (3b), 7,4 -dimethoxyisoflavone (3c), 5,7,4 -trimethoxyisoflavone (3d), 7-hydroxy-6methoxyisoflavone (3e), 7,4 -dimethoxy-5-hydroxyisoflavone (3h), 5,4dihydroxy-7-methoxyisoflavone (3k), 7,4 -dihydroxy-6-methoxyisoflavone (3l), and 7hydroxy-4 -methoxyisoflavone (3o) ( Figure 15) [81]. 6methoxyisoflavone (3e), 7,4′-dimethoxy-5-hydroxyisoflavone (3h), 5,4′-dihydroxy-7methoxyisoflavone (3k), 7,4′-dihydroxy-6-methoxyisoflavone (3l), and 7-hydroxy-4′methoxyisoflavone (3o) ( Figure 15) [81]. OMe OMe   OMe  OH  H  H  H  ------* 3f  H  OH  OH  H  H  OH  32  -128  16  32  64  * 3g  H  H  OH  H  OH  OH  -128 128   2014) conducted the SAR studies while evaluating the antibacterial potential of chalcone (precursor of flavonoids) derivatives. It was reported that the chalcone derivatives exhibited potent antibacterial action against Gram-positive bacteria compared to other cyclized derivatives. It can be observed from Table 9 that (E)-3-(2-(Allyloxy)phenyl)-1(2,4-dihydroxyphenyl)prop-2-en-1-one (4d) with C7, C9-dihydroxyl groups and alloxy group at C2 showed the best activity with MIC of 0.39−6.25 μg/mL. 4d displayed greater activity than (E)-3-(2-(Allyloxy)phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (4i), with hydroxy group at C9 of ring A being the only structural difference, indicating that C9 hydroxylation is important for Gram-positive bacterial inhibition. Next, hydroxylation at C5 of ring A had been reported to reduce Gram-positive bacterial inhibition, as can be seen in (E)-3-(6,6-Dimethylbicyclo Lastly, the presence of one hydroxyl group at C5 or C9 was significant. It is Figure 15. SAR of isoflavones as antibacterial agent [81]. Feng et al. (2014) conducted the SAR studies while evaluating the antibacterial potential of chalcone (precursor of flavonoids) derivatives. It was reported that the chalcone derivatives exhibited potent antibacterial action against Gram-positive bacteria compared to other cyclized derivatives. It can be observed from Table 9 that (E)-3-(2-(Allyloxy)phenyl)-1(2,4-dihydroxyphenyl)prop-2-en-1-one (4d) with C7, C9-dihydroxyl groups and alloxy group at C2 showed the best activity with MIC of 0.39−6.25 µg/mL. 4d displayed greater activity than (E)-3-(2-(Allyloxy)phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (4i), with hydroxy group at C9 of ring A being the only structural difference, indicating that C9 hydroxylation is important for Gram-positive bacterial inhibition. Next, hydroxylation at C5 of ring A had been reported to reduce Gram-positive bacterial inhibition, as can be seen in (E)-3-(6,6-Dimethylbicyclo Lastly, the presence of one hydroxyl group at C5 or C9 was significant. It is because of the presence or absence of hydroxyl group at both these positions led to a weaker antibacterial activity. The summa-rization of the important structural characteristics for antibacterial chalcone derivatives is shown in Figure 16 [82]. because of the presence or absence of hydroxyl group at both these positions led to a weaker antibacterial activity. The summarization of the important structural characteristics for antibacterial chalcone derivatives is shown in Figure 16 [82].   because of the presence or absence of hydroxyl group at both these positions led to a weaker antibacterial activity. The summarization of the important structural characteristics for antibacterial chalcone derivatives is shown in Figure 16 [82].  because of the presence or absence of hydroxyl group at both these positions led to a weaker antibacterial activity. The summarization of the important structural characteristics for antibacterial chalcone derivatives is shown in Figure 16 [82].   because of the presence or absence of hydroxyl group at both these positions led to a weaker antibacterial activity. The summarization of the important structural characteristics for antibacterial chalcone derivatives is shown in Figure 16 [82].   because of the presence or absence of hydroxyl group at both these positions led to a weaker antibacterial activity. The summarization of the important structural characteristics for antibacterial chalcone derivatives is shown in Figure 16 [82].   because of the presence or absence of hydroxyl group at both these positions led to a weaker antibacterial activity. The summarization of the important structural characteristics for antibacterial chalcone derivatives is shown in Figure 16 [82].   because of the presence or absence of hydroxyl group at both these positions led to a weaker antibacterial activity. The summarization of the important structural characteristics for antibacterial chalcone derivatives is shown in Figure 16 [82].  because of the presence or absence of hydroxyl group at both these positions led to a weaker antibacterial activity. The summarization of the important structural characteristics for antibacterial chalcone derivatives is shown in Figure 16 [82].  because of the presence or absence of hydroxyl group at both these positions led to a weaker antibacterial activity. The summarization of the important structural characteristics for antibacterial chalcone derivatives is shown in Figure 16 [82].  because of the presence or absence of hydroxyl group at both these positions led to a weaker antibacterial activity. The summarization of the important structural characteristics for antibacterial chalcone derivatives is shown in Figure 16 [82].   because of the presence or absence of hydroxyl group at both these positions led to a weaker antibacterial activity. The summarization of the important structural characteristics for antibacterial chalcone derivatives is shown in Figure 16 [82].    The isolated flavonoids known as balsacones (also known as derivatives of dihydrocinnamoyl flavans) were tested against ten MRSA strains obtained from Chicoutimi hospital in Saguenay, Province Québec, Canada by using liquid microdilution method. The results showed that balsacone R was the most active compound with a MIC value < 10 μM against all MRSA strains. On the other hand, balsacone Q was the second most active compound as it exhibited MIC value < 10 μM on half of the MRSA strains. Figure 17 shows the crucial structural characteristics of balsacones on their anti-MRSA activity. Firstly, the presence of cinnamoyl or dihydrocinnamoyl chain at C8 (R1) led to stronger anti-MRSA action. Next, p-hydroxycinnamyl group at the C3 position (R2) was said to be supplemental but not essential for anti-MRSA activity. Next, the hydroxylation at C3 (R2) weakened the antibacterial activity compared to the unsubstitution or presence of cinnamyl group at C3. Then, it was found that the 3,4dihydropyran ring fused with ring A at C5 and C6 (R3 and R4) without p-hydroxycinnamyl group at C8′ did not cause any reduction in anti-MRSA activity, but the presence of phydroxycinnamyl group at C8′ completely inactivated the compound. Next, the unsubstitution at C6 (R4) with the presence of p-hydroxycinnamyl was found to be tolerated [83]. Omosa et al. (2016) also evaluated the anti-MRSA activity of various flavonoid derivatives isolated from Kenyan plants. Three chalcone derivatives, namely 32,032,5′-dihydroxy-1′-methoxychalcone (5a), 1′,3′-dihydroxy-2′,5′-dimethoxychalcone (5b), and 1,5-diacetate-3′-methoxychalcone (5c), were found (as shown in Table 10) to be active against four MRSA strains tested, with MIC values ranging from 16 to 128 μg/mL. It can be seen that 5a and 5b were the most active compounds. However, by observing both compounds, it was revealed that hydroxylation at C6 (R2) was responsible for chalcone's anti-MRSA activity. The meta orientation of the methoxy and hydroxyl groups in ring A was also accountable for chalcone's MRSA inhibition. It can be seen that 5b had better inhibitory action than 5a, implying that additional oxygenation at the C1 position can enhance anti-MRSA action. Furthermore, the authors investigated the antibacterial activity of different flavanones and polymethoxylated flavones against MRSA strains. However, due to their high lipophilicity character, all aforementioned compounds demonstrated no activity against tested bacteria. Hence, hydrophilicity was proved to be a critical factor in anti-MRSA action. Then, it was substantiated by the fact that the most lipophilic flavone, namely 5,7-diacetate-3,4′-trimethoxyflavone, displayed minimal activity against certain MRSA bacteria with MIC values ranging from 128 to 256 g/mL. Figure 18 summarizes the The isolated flavonoids known as balsacones (also known as derivatives of dihydrocinnamoyl flavans) were tested against ten MRSA strains obtained from Chicoutimi hospital in Saguenay, Province Québec, Canada by using liquid microdilution method. The results showed that balsacone R was the most active compound with a MIC value < 10 µM against all MRSA strains. On the other hand, balsacone Q was the second most active compound as it exhibited MIC value < 10 µM on half of the MRSA strains. Figure 17 shows the crucial structural characteristics of balsacones on their anti-MRSA activity. Firstly, the presence of cinnamoyl or dihydrocinnamoyl chain at C8 (R 1 ) led to stronger anti-MRSA action. Next, p-hydroxycinnamyl group at the C3 position (R 2 ) was said to be supplemental but not essential for anti-MRSA activity. Next, the hydroxylation at C3 (R 2 ) weakened the antibacterial activity compared to the unsubstitution or presence of cinnamyl group at C3. Then, it was found that the 3,4-dihydropyran ring fused with ring A at C5 and C6 (R 3 and R 4 ) without p-hydroxycinnamyl group at C8 did not cause any reduction in anti-MRSA activity, but the presence of p-hydroxycinnamyl group at C8 completely inactivated the compound. Next, the unsubstitution at C6 (R 4 ) with the presence of p-hydroxycinnamyl was found to be tolerated [83]. Omosa et al. (2016) also evaluated the anti-MRSA activity of various flavonoid derivatives isolated from Kenyan plants. Three chalcone derivatives, namely 32,032,5 -dihydroxy-1 -methoxychalcone (5a), 1 ,3 -dihydroxy-2 ,5 -dimethoxychalcone (5b), and 1,5-diacetate-3 -methoxychalcone (5c), were found (as shown in Table 10) to be active against four MRSA strains tested, with MIC values ranging from 16 to 128 µg/mL. It can be seen that 5a and 5b were the most active compounds. However, by observing both compounds, it was revealed that hydroxylation at C6 (R 2 ) was responsible for chalcone's anti-MRSA activity. The meta orientation of the methoxy and hydroxyl groups in ring A was also accountable for chalcone's MRSA inhibition. It can be seen that 5b had better inhibitory action than 5a, implying that additional oxygenation at the C1 position can enhance anti-MRSA action. Furthermore, the authors investigated the antibacterial activity of different flavanones and polymethoxylated flavones against MRSA strains. However, due to their high lipophilicity character, all aforementioned compounds demonstrated no activity against tested bacteria. Hence, hydrophilicity was proved to be a critical factor in anti-MRSA action. Then, it was substantiated by the fact that the most lipophilic flavone, namely 5,7-diacetate-3,4trimethoxyflavone, displayed minimal activity against certain MRSA bacteria with MIC values ranging from 128 to 256 g/mL. Figure 18 summarizes the structural requirements for the tested chalcones against MRSA strains [84].  OCOMe OMe H OCOMe -128 --* 3′,5′-dihydroxy-1′-methoxychalcone (5a), 1′,3′-dihydroxy-2′,5′-dimethoxychalcone (5b), 1,5-diacetate-3′-methoxychalcone (5c).   Linden et al. (2020) performed a structural investigation on the antibacterial activity of biflavonoids derived from the fruits of the Brazilian Peppertree (Schinus terebinthifolius Raddi). Agathisflavone, amentoflavone (AMF), and tetrahydroamentoflavone were among the biflavonoids examined. The structure of biflavonoids studied can be seen in Figure 19. Tetrahydroamentoflavone was shown to have the best antibacterial activity against B. subtilis and Staphylococcus carnosus, with MIC ≥ 0.063 mg/mL. It implied that reduced Cring influenced tetrahydroamentoflavone's strong antibacterial activity. On the other hand, amentoflavone with an oxidized ring C did not impede the growth of the tested bacteria at concentrations ranging from 0.016 mg/mL to 1 mg/mL. However, at a concentration of 1 mg/mL, agathisflavone, the constitutional isomer of AMF, inhibited bacterial growth of S. carnosus by one log unit. As a result, dimerization and the saturated state of the ring C were shown to affect the antibacterial activity of the biflavonoids studied [85]. Additional oxygenation boosted activity  Linden et al. (2020) performed a structural investigation on the antibacterial activ of biflavonoids derived from the fruits of the Brazilian Peppertree (Schinus terebinthifol Raddi). Agathisflavone, amentoflavone (AMF), and tetrahydroamentoflavone w among the biflavonoids examined. The structure of biflavonoids studied can be seen Figure 19. Tetrahydroamentoflavone was shown to have the best antibacterial activ against B. subtilis and Staphylococcus carnosus, with MIC ≥ 0.063 mg/mL. It implied th reduced C-ring influenced tetrahydroamentoflavone's strong antibacterial activity. the other hand, amentoflavone with an oxidized ring C did not impede the growth of tested bacteria at concentrations ranging from 0.016 mg/mL to 1 mg/mL. However, a concentration of 1 mg/mL, agathisflavone, the constitutional isomer of AMF, inhibit bacterial growth of S. carnosus by one log unit. As a result, dimerization and the saturat state of the ring C were shown to affect the antibacterial activity of the biflavonoids stu ied [85].   Figure 20. According to the authors, the antibacterial impact of liquiritin was roughly two times stronger than the antibacterial effect of liquiritigenin. The only structural variation between these two compounds was at position C4 of ring B, where liquiritigenin included a hydroxyl group; however, liquiritin contained a sugar moiety. It was hypothesized that substitution at C4 of ring B is vital for imparting antibacterial action of these tested flavonoids. Furthermore, the authors speculated that the presence of four free hydroxyl groups in the sugar moiety of liquiritin might be one of the most important reasons contributing to the compound's enhanced antibacterial action [86].
Molecules 2022, 27, x sugar moiety of liquiritin might be one of the most important reasons contributin compound's enhanced antibacterial action [86].  Table  bavachalcone exhibited superior bacterial inhibition compared to the commercial able drug, vancomycin. The authors then demonstrated that prenylation might be sible for the activity because another compound from the xanthone class, know mangostin, which contains a prenyl group, also exhibited remarkable inhibitory against bacteria. The structure of isobavachalcone is depicted in Figure 21 [65].    Table 11, isobavachalcone exhibited superior bacterial inhibition compared to the commercially available drug, vancomycin. The authors then demonstrated that prenylation might be responsible for the activity because another compound from the xanthone class, known as α-mangostin, which contains a prenyl group, also exhibited remarkable inhibitory activity against bacteria. The structure of isobavachalcone is depicted in Figure 21 [65]. sugar moiety of liquiritin might be one of the most important reasons contributing to the compound's enhanced antibacterial action [86].  Table 11, isobavachalcone exhibited superior bacterial inhibition compared to the commercially available drug, vancomycin. The authors then demonstrated that prenylation might be responsible for the activity because another compound from the xanthone class, known as αmangostin, which contains a prenyl group, also exhibited remarkable inhibitory activity against bacteria. The structure of isobavachalcone is depicted in Figure 21 [65].   Table 12 demonstrates that 8-bromo-4-(4-chlorophenyl)-N,N-diethyl-4H- [1,3]dithiolo [4,5- [1,3]dithiolo [4,5-c]chromen-2-amine (6e) exhibited good antibacterial activity. The authors reported that 6a-e exerted better inhibition compared to their equivalent flavanones. Therefore, it can be concluded that the third fused cycle as highlighted in Figure 22 was responsible for the antibacterial action. The authors mentioned that the fused cycle's electrophilic atom could interact with nucleophilic moieties of bacterial wall or membrane constituents, particularly in Gram-positive bacteria, which have more nucleophilic sites than Gram-negative bacteria [87]. The statement was backed up by the fact that 6a-e were more sensitive to S. aureus than E. coli. Next, it can be seen that 6b had lower E. coli inhibition compared to 6a. It could be ascribed to the substitution of a chlorine atom to methoxy group at C4 of ring B. Besides, changing the hydrogen atom (6a) at C8 of ring A to iodine or bromine (6c,d) substituent found to decrease the antibacterial activity in both tested strains [88].  be concluded that the third fused cycle as highlighted in Figure 22 was responsible for the antibacterial action. The authors mentioned that the fused cycle's electrophilic atom could interact with nucleophilic moieties of bacterial wall or membrane constituents, particularly in Gram-positive bacteria, which have more nucleophilic sites than Gram-negative bacteria [87]. The statement was backed up by the fact that 6a-e were more sensitive to S. aureus than E. coli. Next, it can be seen that 6b had lower E. coli inhibition compared to 6a. It could be ascribed to the substitution of a chlorine atom to methoxy group at C4′ of ring B. Besides, changing the hydrogen atom (6a) at C8 of ring A to iodine or bromine (6c,d) substituent found to decrease the antibacterial activity in both tested strains [88].    . It can be seen that 7e-l that contained NEt 2 at R 2 exhibited better bacterial inhibition compared to commercialized antibiotics, kanamycin, and ampicillin. Next, halogen substitution at C6 of ring A and C4 of ring B seemed to be important for activity since hydrogen atom substitution at those positions reduced the antibacterial activity, as can be seen in compound 7m [89].     Table 14 shows that all three compounds exhibited good antibacterial properties, especially 4-(€-3-(4-Fluorophenyl)-3-oxoprop-1-enyl)benzoic acid (8b). It can be seen that 8b exerted the maximum zone inhibition and the lowest MIC value compared to other synthesized compounds. 8b was also outperformed the commercially available antibiotic ampicillin in terms of zone inhibition. The summary of SAR analysis is illustrated in Figure 24. The authors discussed that good antibacterial activity exerted by these compounds was due to the presence of COOH moiety at C2 of ring B [90]. Then, as previously stated, 8b demonstrated the highest activity, attributed to the fluorine substituent at C6 of ring A [91]. The authors discussed that higher electronegative substitution improved the antibacterial action [92].     Table 14 shows that all three compounds exhibited good antibacterial properties, e cially 4-(€-3-(4-Fluorophenyl)-3-oxoprop-1-enyl)benzoic acid (8b). It can be seen tha exerted the maximum zone inhibition and the lowest MIC value compared to other thesized compounds. 8b was also outperformed the commercially available antibiotic picillin in terms of zone inhibition. The summary of SAR analysis is illustrated in Fi 24. The authors discussed that good antibacterial activity exerted by these compou was due to the presence of COOH moiety at C2′ of ring B [90]. Then, as previously sta 8b demonstrated the highest activity, attributed to the fluorine substituent at C6 of rin [91]. The authors discussed that higher electronegative substitution improved the anti terial action [92].   Table 15. The authors analyzed several significant characteristics of synthesized chalc for their antibacterial action as illustrated in Figure 25. To begin, substitution at C6 of A, such as NO2, NH2 and OH appeared to play an essential role in chalcones' antibact action [93]. Other than that, electron donating groups such as hydroxyl and meth groups at ring B helped to boost the antibacterial activity against B. subtilis and P. a ginosa, as observed in compounds (E)-3-   Table 15. The authors analyzed several significant characteristics of synthesized chalcones for their antibacterial action as illustrated in Figure 25. To begin, substitution at C6 of ring A, such as NO 2 , NH 2 and OH appeared to play an essential role in chalcones' antibacterial action [93]. Other than that, electron donating groups such as hydroxyl and methoxy groups at ring B helped to boost the antibacterial activity against B. subtilis and P. aeruginosa, as observed in compounds (E)-3-(4-Hydroxyphenyl)-1-(4-Nitrophenyl)prop-2-en-1-one (9a), (E)-3-(3,4,5-Trimethoxyphenyl)-1-(4-Nitrophenyl)Prop-2-en-1-one (9c), and (E)-3-(4-Hydroxy-3-methoxyphenyl)-1-(4-Nitrophenyl)prop-2-en-1-one (9f) (MIC ≥ 1.16 µg/mL). Next, presence of bromine moiety at C2 improved the antibacterial activity against E. coli, B. sub-tilis and P. aeruginosa, as depicted in compound (E)-3-(4-Bromophenyl)-1-(4-Nitrophenyl)prop-2-en-1-one (9h) (MIC ≥ 0.94 µg/mL) [94]. Table 15. Antibacterial activity of the substituted chalcones contaning nitor, amino, hydroxyl, methoxy, and chloro groups (9a-n).  Sadgrove et al. (2020) isolated several isoflavones and derivatives from the bark of Erythrina lysistemon. The compounds were identified as erybraedin A, phaseollidin, abyssinone V-4′ methyl ether, eryzerin C, alpumisoflavone, cristacarpin, and lysisteisoflavone. These compounds' antibacterial activity was also tested, and the findings are reported in Table 16. The authors addressed how prenylation is vital for activity because all molecules except alpumisoflavone have a prenyl group. Following that, hydroxylation at the same aromatic ring bearing the prenyl group can improve antibacterial activity. As demonstrated in abyssinone V-4′ methyl ether and cristacarpin, methoxylation of those hydroxyl groups decreased activity ( Figure 26) [95]. Sadgrove et al. (2020) isolated several isoflavones and derivatives from the bark of Erythrina lysistemon. The compounds were identified as erybraedin A, phaseollidin, abyssinone V-4 methyl ether, eryzerin C, alpumisoflavone, cristacarpin, and lysisteisoflavone. These compounds' antibacterial activity was also tested, and the findings are reported in Table 16. The authors addressed how prenylation is vital for activity because all molecules except alpumisoflavone have a prenyl group. Following that, hydroxylation at the same aromatic ring bearing the prenyl group can improve antibacterial activity. As demonstrated in abyssinone V-4 methyl ether and cristacarpin, methoxylation of those hydroxyl groups decreased activity (Figure 26) [95].  1  2  2  2  20  Phaseollidin  10  10  5  20  20  Abyssinone V-4 methyl ether  26  59  117  260  260  Eryzerin C  10  5  2  5  5  Alpumisoflavone  31  31  125  125  20  Cristacarpin  156  156  412  625  78  Lysisteisoflavone  2  62  26  6  31 Molecules  Phaseollidin  10  10  5  20  20  Abyssinone V-4′ methyl ether  26  59  117  260  260  Eryzerin C  10  5  2  5  5  Alpumisoflavone  31  31  125  125  20  Cristacarpin  156  156  412  625  78  Lysisteisoflavone  2  62 Table 17 clearly shows that dihydromorin inhibited bacteria better than norartocarpetin against all strains tested. Therefore, based on Figure 27, it can be seen that hydroxylation of C3 of ring C and saturated C2-C3 lead to better bacterial inhibitory properties [96]. norartocarpetin against all strains tested. Therefore, based on Figure 27, it can be seen that hydroxylation of C3 of ring C and saturated C2-C3 lead to better bacterial inhibitory properties [96].   Table 18. Some important structural features of carboxylic chalcones as antibacterial agents have been discussed. Firstly, substitution in ring B at meta position [4′-Carboxy-3-trifluoromethyl-chalcone (10h), 4′-Carboxy-3-bromo-chalcone (10i), 4′-Carboxy-3-chloro-chalcone (10j), 4′-Carboxy-3-nitro-chalcone (10k), 4′-Carboxy-3-hydroxychalcone (10l), and 4′-Carboxy-3-phenoxy-chalcone (10m); (MIC ≥ 40 μM) led to a more potent inhibition compared to para substitution [4′-Carboxy-4-trifluoromethyl-chalcone (10b), 4′-Carboxy-4-chloro-chalcone (10c), 4′-Carboxy-4-methyl-chalcone (10d), 4′-Carboxy-4-methoxy-chalcone (10e), 4′-Carboxy-4-hydroxy-chalcone (10f), and 4′-Carboxy-4phenoxy-chalcone (10g); (MIC ≥ 150 μM). Next, the presence of lipophilic substituents such as bromine, chlorine and CF3 as can be seen in 10b,c, 10h-j, 4′-Carboxy-2-trifluoromethyl-chalcone (10n), 4′-Carboxy-2-bromo-chalcone (10o), 4′-Carboxy-2-chloro-chalcone (10p), 4′-Carboxy-3,5-bis-trifluoromethyl-chalcone (10s), 4′-Carboxy-3,5-dibromo-chalcone (10t), and 4′-Carboxy-3,5-dichloro-chalcone (10u) (MIC ≥ 2 μM) showed better antibacterial action than those compounds possessing polar hydroxy and methoxy groups (10e,f,l), 4′-Carboxy-2-hydroxy-chalcone (10q), and 4′-Carboxy-3,5-dimethoxy-chalcone (10x); )MIC > 300 μM) ( Figure 28). The importance of lipophilic substitution was further supported by the fact that the most active compounds (10s,t; MIC ≥ 2 μM) contained two lipophilic substituents with potencies equivalent to two different antibiotics, namely ciprofloxacin and linezolid [97].   Table 18. Some important structural features of carboxylic chalcones as antibacterial agents have been discussed. Firstly, substitution in ring B at meta position [4 -Carboxy-3-trifluoromethyl-chalcone (10h), 4 -Carboxy-3-bromo-chalcone (10i), 4 -Carboxy-3-chloro-chalcone (10j), 4 -Carboxy-3-nitro-chalcone (10k), 4 -Carboxy-3-hydroxy-chalcone (10l), and 4 -Carboxy-3-phenoxy-chalcone (10m); (MIC ≥ 40 µM) led to a more potent inhibition compared to para substitution [4 -Carboxy-4-trifluoromethyl-chalcone (10b), 4 -Carboxy-4-chloro-chalcone (10c), 4 -Carboxy-4-methyl-chalcone (10d), 4 -Carboxy-4methoxy-chalcone (10e), 4 -Carboxy-4-hydroxy-chalcone (10f), and 4 -Carboxy-4-phenoxychalcone (10g); (MIC ≥ 150 µM). Next, the presence of lipophilic substituents such as bromine, chlorine and CF 3 as can be seen in 10b,c, 10h-j, 4 -Carboxy-2-trifluoromethylchalcone (10n), 4 -Carboxy-2-bromo-chalcone (10o), 4 -Carboxy-2-chloro-chalcone (10p), 4 -Carboxy-3,5-bis-trifluoromethyl-chalcone (10s), 4 -Carboxy-3,5-dibromo-chalcone (10t), and 4 -Carboxy-3,5-dichloro-chalcone (10u) (MIC ≥ 2 µM) showed better antibacterial action than those compounds possessing polar hydroxy and methoxy groups (10e,f,l), 4 -Carboxy-2-hydroxy-chalcone (10q), and 4 -Carboxy-3,5-dimethoxy-chalcone (10x); (MIC > 300 µM) ( Figure 28). The importance of lipophilic substitution was further supported by the fact that the most active compounds (10s,t; MIC ≥ 2 µM) contained two lipophilic substituents with potencies equivalent to two different antibiotics, namely ciprofloxacin and linezolid [97].  4′-Carboxy-3-bromo-chalcone (10i), 4′-Carboxy-3-chloro-chalcone (10j), 4′-Carboxy-3-nitro-chal cone (10k), 4′-Carboxy-3-hydroxy-chalcone (10l), 4′-Carboxy-3-phenoxy-chalcone (10m), 4′-Car boxy-2-trifluoromethyl-chalcone (10n), 4′-Carboxy-2-bromo-chalcone (10o), 4′-Carboxy-2-chloro chalcone (10p), 4′-Carboxy-2-hydroxy-chalcone (10q), 4′-Carboxy-2-butoxy-chalcone (10r), 4′-Car boxy-3,5-bis-trifluoromethyl-chalcone (10s), 4′-Carboxy-3,5-dibromo-chalcone (10t), 4′-Carboxy-3,5 dichloro-chalcone (10u), 4′-Carboxy-3,5-dimethyl-chalcone (10v), 4′-Carboxy-3,5-difluoro-chalcone (10w), and 4′-Carboxy-3,5-dimethoxy-chalcone (10x).  Table 19. Results of the study were found similar to the previous find ings reported by Nielsen et al. (2004) Table 19. Results of the study were found similar to the previous findings reported by Nielsen et al. (2004), lipophilic substitution at the different positions in ring B brought to a better chalcones' antibacterial activity ( Figure 29). It can be seen that chalcone derivatives with lipophilic substituents such as 1-    The bacterial action of the tested compounds can be viewed To start, it was discussed that hydroxylation at C5 and C7 of ring A is import bacterial activity because all compounds contained it [68]. Next, it can be seen with 2′,4′-OH substitution exhibited better activity than the quercetin with 3′ stitution, which proved the importance of hydroxylation at C2′ of ring B in ba bition [59,99]. Next, further investigation revealed that the presence of a sul C5' was only found to enhance bacterial activity against S. aureus and not ag the other tested strains. The summary of SAR for flavonoids and their sulfonic as antibacterial agents is presented in Figure 30 [100]. Oliveira et al. (2021) investigated the effects of fatty acid chain length di anthocyanins, particularly cyanidin-3-glucoside, on antibacterial activity. Th with a fatty acid chain was inserted at C3 of cyanidin's ring A, and the length acid chain ranged from C4 to C12. Their bacterial inhibition can be observed Compared to other compounds with fatty acid chains, cyanidin-3-O-glucosid no fatty acid chain was found inactive. Following that, it was discovered tha length comprising 12 carbons in the sugar moiety [cyanidin-3-O-glucoside-C1 found too long to exert antibacterial action, whereas the chain length containin [cyanidin-3-O-glucoside-C4 (12b)] was too short. It can be concluded that the o for antibacterial activity was six carbons to ten carbons, where cyanidin-3-O C10 (12e) with a fatty acid chain comprising ten carbons was found out to b potent compound. The important structural features of cyanidin as antibacteri be viewed in Figure 31 [101]. The bacterial action of the tested compounds can be viewed in Table 20. To start, it was discussed that hydroxylation at C5 and C7 of ring A is important for antibacterial activity because all compounds contained it [68]. Next, it can be seen that morin with 2 ,4 -OH substitution exhibited better activity than the quercetin with 3 ,4 -OH substitution, which proved the importance of hydroxylation at C2 of ring B in bacterial inhibition [59,99]. Next, further investigation revealed that the presence of a sulfo group at C5' was only found to enhance bacterial activity against S. aureus and not against any of the other tested strains. The summary of SAR for flavonoids and their sulfonic derivatives as antibacterial agents is presented in Figure 30 [100].     Oliveira et al. (2021) investigated the effects of fatty acid chain length differences in anthocyanins, particularly cyanidin-3-glucoside, on antibacterial activity. The glucoside with a fatty acid chain was inserted at C3 of cyanidin's ring A, and the length of the fatty acid chain ranged from C4 to C12. Their bacterial inhibition can be observed in Table 21. Compared to other compounds with fatty acid chains, cyanidin-3-O-glucoside (12a) with no fatty acid chain was found inactive. Following that, it was discovered that the chain length comprising 12 carbons in the sugar moiety [cyanidin-3-O-glucoside-C12 (12f)] was found too long to exert antibacterial action, whereas the chain length containing 4 carbons [cyanidin-3-O-glucoside-C4 (12b)] was too short. It can be concluded that the optimal size for antibacterial activity was six carbons to ten carbons, where cyanidin-3-O-glucoside-C10 (12e) with a fatty acid chain comprising ten carbons was found out to be the most potent compound. The important structural features of cyanidin as antibacterial agent can be viewed in Figure 31 [101].               Table 22, 6-bromo-2-(4-(trifluoromethyl)phenyl)-4H-chromen-4-one (13c) was the mos active compound against all of the tested strains. Based on the MIC value of 13c, it can be concluded that 13c was antibacterially equipotent to ciprofloxacin, particularly against S aureus and P. aeruginosa. As a result, based on the efficacy of 13c, it can be concluded tha halogenation at C6 of ring A enhanced the antibacterial action. The authors then discussed how the trifluoromethyl group at C4′ of ring B appeared to be important for bacterial in hibition because all tested compounds contained this moiety and exerted antibacteria properties. The SAR analysis of tested flavones is illustrated in Figure 32 [102].   Table 22, 6-bromo-2-(4-(trifluoromethyl)phenyl)-4H-chromen-4-one (13c) was the most active compound against all of the tested strains. Based on the MIC value of 13c, it can be concluded that 13c was antibacterially equipotent to ciprofloxacin, particularly against S. aureus and P. aeruginosa. As a result, based on the efficacy of 13c, it can be concluded that halogenation at C6 of ring A enhanced the antibacterial action. The authors then discussed how the trifluoromethyl group at C4 of ring B appeared to be important for bacterial inhibition because all tested compounds contained this moiety and exerted antibacterial properties. The SAR analysis of tested flavones is illustrated in Figure 32 Sato et al. (2006) tested the antibacterial activity of some isoflavones, namely albigenin, erythrinin B, lupiwighteone, and laburnetin against eleven strains of The potency of these compounds was found to be in the given order, isolupa (MIC50 = 3.13 μg/mL) > erythrinin B (MIC50 = 6.25 μg/mL) > lupiwighteone = lab (MIC50 > 25 μg/mL). Results revealed that the prenyl group at C3′ of ring B as p in isolupalbigenin led to increased MRSA inhibition. Similarly, as seen in ery prenylation at C6 of the ring improved anti-MRSA activity. Following that, all of th compounds had hydroxyl groups at C5 of ring A, indicating that hydroxylation Figure 32. The structural-activity analysis of tested flavone derivatives containing halogens [102]. Sato et al. (2006) tested the antibacterial activity of some isoflavones, namely isolupalbigenin, erythrinin B, lupiwighteone, and laburnetin against eleven strains of MRSA. The potency of these compounds was found to be in the given order, isolupalbigenin (MIC 50 = 3.13 µg/mL) > erythrinin B (MIC 50 = 6.25 µg/mL) > lupiwighteone = laburnetin (MIC 50 > 25 µg/mL). Results revealed that the prenyl group at C3 of ring B as presented in isolupalbigenin led to increased MRSA inhibition. Similarly, as seen in erythrin B, prenylation at C6 of the ring improved anti-MRSA activity. Following that, all of the tested compounds had hydroxyl groups at C5 of ring A, indicating that hydroxylation at C5 is critical for antibacterial activity against MRSA. The summarization of the SAR can be viewed in Figure 33 [103]. Fang et al. (2016) used 3D-QSAR and docking studies to investigate the SAR of flavonoids as potential E. coli inhibitors. The study was conducted against DNA gyrase B (GyrB) of E. coli. The findings from both 3D-QSAR and docking investigations revealed that hydroxylation at the C3, C5, and C7 positions of ring A was critical in inhibiting E. coli. It was also discovered that the carbonyl group at position C4 plays a vital function in E. coli inhibition. Following that, 3D-QSAR demonstrated that hydroxylation at C3 of ring B, as well as the presence of C2=C3, were both required for GyrB inhibition. On the other hand, C6 hydroxylation and C8, C3 methoxylation can all result in a decrease in E. coli inhibition. The summary of SAR discussion is depicted in Figure 34 [104].   (6)); evaluated their antimicrobial and antibiofilm properties against resistant bacteria, isolated from organic foods (12 Gram-positive and Gram-negative bacteria). This research work was carried out to find the structural features responsible for their antimicrobial and antibiofilm activities. Among all the synthesized compounds, 2-(3′,4′-sDihydroxyphenyl)-6-nitrochromane-(4 → 4″,2 → O-5″)-phloroglucinol, which had a NO2 group on ring A, exhibited the highest B, as well as the presence of C2=C3, were both required for GyrB inhibition. On the oth hand, C6 hydroxylation and C8, C3′ methoxylation can all result in a decrease in E. c inhibition. The summary of SAR discussion is depicted in Figure 34 [104].    (6)); evaluated their antimicrobial and antibiofilm properties against resistant bacteria, isolated from organic foods (12 Gram-positive and Gram-negative bacteria). This research work was carried out to find the structural features responsible for their antimicrobial and antibiofilm activities. Among all the synthesized compounds, 2-(3 ,4 -sDihydroxyphenyl)-6-nitrochromane-(4 → 4 -,2 → O-5 -)-phloroglucinol, which had a NO 2 group on ring A, exhibited the highest antibacterial activity (MIC of 10 µg/mL). The same compound also displayed the best effect at preventing biofilm formation (up to 40% decreases at 100 µg/mL) and disrupting preformed biofilms (up to 40% reductions at 0.1 µg/mL). As depicted in Figure 35, structure-activity relationships findings showed that electron withdrawing group attached to ring A was found to be responsible to manifest strong antibacterial action [105].
The extensive discussion of flavonoids possessing antibacterial properties and thei structural requirements for antibacterial activity led us to conclude numerous structura requirements frequently noted in the reviewed literature. Table 23 summarizes the major ity of the SAR discussed before for several classes of flavonoids, including chalcones, fla vanones, flavones, isoflavones, flavan-3-ols, and anthocyanidins. Significant structura traits can only be compared and concluded for chalcones, flavanones, flavones, and iso flavones, as flavan-3-ols and anthocyanidins lack sufficient comparison. Moreover, another similar study conducted by Ortega-Vidal et al. (2022) in which nine phenolic compounds (viz. 2 flavan-3-ols (catechin and epicatechin); 1 phenolic glucoside (annphenone); 6 dimeric A-type proanthocyanidins) were isolated from the pruning wood residue of European plum (Prunus domestica L.). All the isolated compounds were further subjected to determine their antibacterial and antibiofilm effects against a selection of multi-resistant strains including foodborne microorganisms. The most potent antibacterial activity was observed on the strain Enterobacter sp. UJA37p (obtained from organic tomato) and with high tolerance to different biocides. All phenolic compounds exhibited MIC values of 100 µg/mL against this resistant bacterium. In contrast, when bacteria were previously allowed to attach and form biofilm before treatments, the interesting results were observed in which disruption of preformed biofilms was noticed with (+)-epiafzelechin-(2β → O→7,4β → 8)-epicatechin, followed by (−)-ent-epiafzelechin-(2α → O → 7,4α → 8)catechin and i.e., annphenone at a concentration of 0.1 µg/mL further confirming the potent antibacterial action of these phenolic compounds. The chemical structures of mentioned flavonoids can be viewed in Figure 36 [106].
The extensive discussion of flavonoids possessing antibacterial properties and their structural requirements for antibacterial activity led us to conclude numerous structural requirements frequently noted in the reviewed literature. Table 23 summarizes the majority of the SAR discussed before for several classes of flavonoids, including chalcones, flavanones, flavones, isoflavones, flavan-3-ols, and anthocyanidins. Significant structural traits can only be compared and concluded for chalcones, flavanones, flavones, and isoflavones, as flavan-3-ols and anthocyanidins lack sufficient comparison. antibacterial activity, while methylation or glycosylation of the hydroxyls can reduce their antibacterial activity. Next, hydroxylation at C5 can enhance flavones' antibacterial activity, whilst methoxylation at C5 can decrease it. C7-OH, C4′-OH, C3-OH, and C2=C3 double bond have all been observed to enhance the antibacterial activity of flavones. In the case of isoflavones, hydroxylation at rings A and B, particularly at C5 and C4′, as well as prenylation at ring A, particularly at C6, might enhance antibacterial action. On the other hand, methoxylation and glycosylation in ring A and B seemed to reduce the antibacterial activity. Then, it can be seen that there is some inconsistency in the findings, specifically whether hydroxylation and methylation of the hydroxyl groups boosted or lowered the antibacterial activity of flavonoids, specifically at the C6 and C3′ positions of flavones. This inconsistency can be explained by the significance of antibacterial agents' amphiphilic balance to enter bacterial strains, where both hydrophilic and hydrophobic characteristics must be present in balance [76].
According to the summary above, modifications to each flavonoids class, particularly in synthetic or semi-synthetic approaches, may be made to enhance the antibacterial activity. However, when looking at the broader picture without regard to the flavonoids class, it was shown that hydroxylation of C5, C7, C3′, and C4′ as well as geranylation or prenylation at C6 are important structural requirements for antibacterial activity. On the other hand, it has been shown that methoxylation at C3′ and C5 reduces the antibacterial activity of flavonoids. Yet, it should be highlighted that flavonoids' local balance of amphiphilicity will still be required for bacterial penetration to exert potent antibacterial effects. Moreover, after thorough review of all the antibacterial research studies of Figure 36. The chemical structures of mentioned flavonoids [106]. Table 23 shows that hydroxylation at C5 and C7, as well as lipophilic substitution at ring B improves chalcones' antibacterial activity. Following that, several authors have observed that C5 hydroxyl group and C2-C3 double bond saturation can enhance the antibacterial activity of flavanones. Hydroxylation of flavanones at ring B can enhance the antibacterial activity, while methylation or glycosylation of the hydroxyls can reduce their antibacterial activity. Next, hydroxylation at C5 can enhance flavones' antibacterial activity, whilst methoxylation at C5 can decrease it. C7-OH, C4 -OH, C3-OH, and C2=C3 double bond have all been observed to enhance the antibacterial activity of flavones. In the case of isoflavones, hydroxylation at rings A and B, particularly at C5 and C4 , as well as prenylation at ring A, particularly at C6, might enhance antibacterial action. On the other hand, methoxylation and glycosylation in ring A and B seemed to reduce the antibacterial activity.
Then, it can be seen that there is some inconsistency in the findings, specifically whether hydroxylation and methylation of the hydroxyl groups boosted or lowered the antibacterial activity of flavonoids, specifically at the C6 and C3 positions of flavones. This inconsistency can be explained by the significance of antibacterial agents' amphiphilic balance to enter bacterial strains, where both hydrophilic and hydrophobic characteristics must be present in balance [76].
According to the summary above, modifications to each flavonoids class, particularly in synthetic or semi-synthetic approaches, may be made to enhance the antibacterial activity. However, when looking at the broader picture without regard to the flavonoids class, it was shown that hydroxylation of C5, C7, C3 , and C4 as well as geranylation or prenylation at C6 are important structural requirements for antibacterial activity. On the other hand, it has been shown that methoxylation at C3 and C5 reduces the antibacterial activity of flavonoids. Yet, it should be highlighted that flavonoids' local balance of amphiphilicity will still be required for bacterial penetration to exert potent antibacterial effects. Moreover, after thorough review of all the antibacterial research studies of flavonoids, it is believed that the above-mentioned key structural features may play a critical role to orchestrate strategies in the modern chemical synthesis to discover more potent flavonoids' based antibacterial drugs.

Conclusions
Flavonoid-protein interactions (enzymes, receptors, transporters, and transcription factors) are basic phenomena that govern the beneficial effects of flavonoids. As has been mentioned in the review, one of the most studied pharmacological effects of flavonoids is their antibacterial property which has been thoroughly evaluated through various SAR studies to discover more potent antibacterial agents as safe natural products. It can be concluded that several flavonoids' structural features may be important for imparting antibacterial effects, including C5, C7, C3 , and C4 hydroxylation as well as geranylation or prenylation at C6. However, the most important aspect of flavonoids is that they must retain their amphiphilic characteristics to penetrate bacteria to exert their potent antibacterial action. Hence, these important structural features of antibacterial flavonoids, if taken into account while orchestrating new synthetic strategies, might play an important role to synthesize better antibacterial drugs to overcome stiff challenges associated with the resistant bacteria.