Antibacterial Modes of Herbal Flavonoids Combat Resistant Bacteria

The increasing dissemination of multidrug resistant (MDR) bacterial infections endangers global public health. How to develop effective antibacterial agents against resistant bacteria is becoming one of the most urgent demands to solve the drug resistance crisis. Traditional Chinese medicine (TCM) with multi-target antibacterial actions are emerging as an effective way to combat the antibacterial resistance. Based on the innovative concept of organic wholeness and syndrome differentiation, TCM use in antibacterial therapies is encouraging. Herein, advances on flavonoid compounds of heat-clearing Chinese medicine exhibit their potential for the therapy of resistant bacteria. In this review, we focus on the antibacterial modes of herbal flavonoids. Additionally, we overview the targets of flavonoid compounds and divide them into direct-acting antibacterial compounds (DACs) and host-acting antibacterial compounds (HACs) based on their modes of action. We also discuss the associated functional groups of flavonoid compounds and highlight recent pharmacological activities against diverse resistant bacteria to provide the candidate drugs for the clinical infection.


INTRODUCTION
The worldwide spreading of pathogenic resistant bacteria threatens public health. Currently, the infections caused by Gram-negative (G − ) bacteria occur more frequently than Gram-positive (G + ) bacteria in clinics. A report from the China Antimicrobial Resistance Surveillance System (CARSS) 1 shows that G − bacteria accounted for 71.1% of the 3,249,123 clinical isolated strains, while Gram-positive (G + ) bacteria for 28.9% ( Figure 1A). Among them, the high number of multidrug resistant (MDR) bacterial infections, such as carbapenem-resistant G − bacteria (CRGNB), are life-threating (Bassetti et al., 2021;Palacios-Baena et al., 2021). As shown in Figure 1A, many more MDR pathogens are emerging, including the resistant G − bacteria like carbapenem-resistant E. coli (CREC, 1.6%), carbapenem-resistant K. pneumoniae (CRKPN, 10.9%), carbapenem-resistant P. aeruginosa (CRPsA, 18.3%), carbapenemresistant A. baumannii (CRAB, 53.7%), and the resistant G + bacteria such as methicillin-resistant S. aureus (MRSA, 29.4%) and methicillin-resistant coagulase-negative S. aureus (MRCNS, 74.4%). Worse still, global patient deaths due to antibiotic resistance are approximately 700, 000 and the numbers are expected to increase to 10 million by 2050 if no effective measures are introduced 2 . Therefore, alternative strategies to antibiotics and the discovery of novel antibacterial drugs to combat resistant bacteria are in high demand.
In the resistant era, the major therapy of MDR still rely the efficacy and safety antibacterial agents, while the discovery and development of antibacterial drugs are barrier by the unknown infective route of pathogen Song et al., 2020;Zulauf and Kirby, 2020). Effective antibacterial therapies need to explore more antibacterial compounds and their pharmacological activities and targets to sustainably combat the resistant bacteria (Theuretzbacher et al., 2020). Our previous work has demonstrated that the host defenses are critical for both bacterial infections and antibacterial drug therapy (Liu F. et al., 2020;Liu et al., 2021). Therefore, host factors are vital for the management of MDR bacterial infections. In most situations, bacteria evolve or develop a variety of strategies to infect the human host (Culyba and Van Tyne, 2021). Thus, antibiotics cannot effectively fight against various bacterial mutations, while large doses and frequent usage of antibiotics can cause bacteria to be constantly exposed to drug stress, which will trigger the emergence of drugresistant bacteria.
Unlike the direct stress of antibiotics to bacteria, host-directed therapy (HDT) is a sensible strategy against unknown resistant bacteria, and host-acting antibacterial compounds (HACs) are worthy of developing (Liu et al., 2022). The intrinsic advantages of HACs therapy are mobilizing the host cells to protect themselves from unknown infections with less emergence of resistant bacteria, due to the reduced selective pressure from directly targeting bacteria. Similar to HDT and HACs, the main therapeutic principle of traditional Chinese medicine (TCM) against bacteria is dependent on "reinforcing healthy Qi and expelling pathogenic factors," which also refers to its abilities of both enhancing host defense forces and eliminating pathogenic bacteria ( Figure 1B). In terms of the TCM theory, we divide the antibacterial actions of TCM to direct-acting and host-acting antibacterial modes ( Figure 1B). Upon infections, bacterial diseases belong to heat syndrome (Wang et al., 2018). Heatclearing Chinese medicines symptomatically treat bacteriacaused internal heat syndrome. Therefore, the heat-clearing herbs may serve as potential drug libraries for screening the lead compounds to combat MDR bacterial infection. Flavonoids are abundant in plants, such as in most herbs, and recent research shows that flavonoids have excellent antibacterial activities that are regulated tightly with their functional groups (Song et al., 2021a).
Overall, this review focuses on the flavonoids from heatclearing herbs to reveal the therapeutic strategies of resistant pathogens. In the following sections, we discuss the availabilities and antibacterial pipelines of herbal flavonoids.

HERBAL FLAVONOIDS IN HEAT-CLEARING MEDICINES
TCM is identified by organic wholeness and treatment based on syndrome differentiation. The syndrome differentiation of infectious diseases belongs to internal heat syndrome, which can be treated with heat-clearing medicines. Internal heat The occurrence of resistant pathogens in China from 10/2019 to 12/2020. Reported data are collected from China Antimicrobial Resistance Surveillance System (CARSS) 1 . The isolated resistant strains from the report that account for Gram-negative (G − ) bacteria and Gram-positive (G + ) bacteria are 71.1% and 28.9%, respectively. (B) Scheme of the infectious therapy of traditional Chinese medicine (TCM). Resistant bacterial infections lead to the treatment failures of antibiotics. While therapeutic principles of TCM focus on the organic wholeness referring to "reinforcing healthy Qi and expelling pathogenic factors," which displays that both suppressing bacteria and enhancing host defense. The TCM can divide to two types including direct-acting antibacterial mode and host-acting antibacterial mode. It is worth noting that "Qi" in TCM denoted the gas of host healthy, mostly meaning the forces of host defense. syndrome manifests itself in many forms including Qi aspect heat, blood aspect heat, dampness-heat, toxin-heat, and deficiency-heat ( Figure 2A) (Chen, 1965). The heat syndrome occurs in the spatiotemporal axis of infectious diseases' correspondence with acute phase, disorders of coagulation system, chronic infection, fever, pathological changes, and chronic illness with weakness ( Figure 2A). For these heat syndromes, heat-clearing medicines have five of therapy: 1) heat-clearing and fire-purging herbs, 2) heat-clearing and blood-cooling herbs, 3) heat-clearing and damp-eliminating herbs, 4) heat-clearing and toxin-relieving herbs, and 5) asthenic-heat clearing herbs ( Figure 2B).
Antibacterial approaches and the main targets of the flavonoids are important for clinical applications. Within the decoding of active function, the classifications of flavonoids based on the chemical structures are intuitive, simple, and critical for understanding of antibacterial activates of flavonoids.

CHEMICAL STRUCTURE CLASSIFICATION OF FLAVONOIDS
To assess the antibacterial activities of flavonoids, the chemical structures of flavonoid compounds need to be clarified. Thus, the structural classifications of herbal flavonoids are detailed in Figure 3. Generally, flavonoids refer to a series of compounds in which two benzene rings (A and B rings) with phenolic hydroxyl groups are connected to each other through the central three carbon atoms (Wen et al., 2021). As shown in Figure 3A, the nuclear skeleton of flavonoids forms the C 6 -C 3 -C 6 system. This subsequently classifies the flavonoids according to the degree of oxidation of the central three carbon atoms, the linking position of the B ring, and whether the three carbon chains constitutes a ring (C ring) or not (Xie et al., 2015). Major subclasses of flavonoids are detailed in Figure 3B; subclass I includes flavones and flavonols, Subclass II includes flavanones and flavanonols, Subclass III includes chalcones and dihydrochalcones, Subclass IV includes isoflavones and dihydroisoflavones, Subclass V includes flavan-3-ols, flavan-3.4-diols, and anthocyanidins, and Subclass VI includes xathones and mangiferin, Subclass VII includes other flavonoids such as bioflavonoids, homoisoflavonoids, aurones, isoaurones, and so on. The common skeletons are marked by blue and the green R groups denote substitutable groups. Finally, the functional groups of flavonoids including prenyl, methoyl, and methyl (highlighted in red) decide the antibacterial activities ( Figures 3B,C), resulting in various modes of action on resistant bacteria.

MODES OF ACTION OF FLAVONOIDS ON RESISTANT BACTERIA
Antibacterial modes of flavonoids depend on the structures, that is the substitutions on the aromatic rings. As more antibacterial The nuclear skeleton of flavonoids contains a 2-phenyl-chromone core with the C 6 -C 3 -C 6 system. The A and B benzene rings connect to each other through the central three carbon atoms, which can or cannot form the C ring. (B) The major subclasses of flavonoids. Subclass I, flavones and flavonols; Subclass II, flavanones and flavanonols; Subclass III, chalcones and dihydrochalcones; Subclass IV, isoflavones and dihydroisoflavones; Subclass V, flavan-3-ols, flavan-3.4-diols and anthocyanidins; Subclass VI, xathones and mangiferin; Subclass VII, other flavonoids such as bioflavonoids, homoisoflavonoids, aurones, isoaurones, and so on. The common skeletons are marked by blue and the green R groups denote substitutable groups (C) The main functional groups of flavonoids include phenolic hydroxy, prenyl, methoxyl, and methyl (highlighted in red).
For a clearer understanding of the two modes, the related three subcategories are attributed to these two modes. According to the antibacterial activities of inhibition of bacterial membrane, bacterial biofilm, efflux pump, and virulence factor, DAC divides to DAC IM , DAC IB , DAC IE , and DAC IV , respectively. HAC is divided into HAC AO , HAC AI , HAC MI , and HAC RP based on the effects on the antioxidation, anti-inflammatory, modulation of immune cells, and regulating pathway of host ( Figure 4A and Table 1). Furthermore, the specific modes of action on herbal flavonoids that combat resistant bacteria are illustrated in the following.

Direct-Acting Antibacterial Modes
Direct-acting antibacterial modes of herbal flavonoids means that the antibacterial agents directly target the bacterial themselves, such as the bacterial membrane functions, biofilm formation, efflux pumps, and virulence factors ( Figure 4A). Of the activities of DAC flavonoids, the first category is the biophysical barrier of the bacterial inner membrane that directly increases bacteria survival . Similar to most antibacterial drugs, the main therapeutic treatment option of flavonoids is damaging bacterial membrane functions. A recent report shows that the antibacterial mechanisms of flavonoids rely on distinctive modes of action to bind the phospholipids of bacterial membrane, which result in the disruption of proton motive force and metabolic disturbance (Song et al., 2021a). DAC flavonoids such as isobavachalcone (AMG) and α-mangostin (IBC) target the phosphatidylglycerol (PG) of bacterial membrane   Figure 4B). These two DAC flavonoids have 3′-prenyl and 2, 8prenyl, respectively ( Figure 3B). Active prenyl flavonoids endow the antibacterial activities that combat most bacteria, even MRSA ( Table 1). In addition, the prenyl flavonoid compounds are abundantly distributed in herbal flavonoids (Wen et al., 2021). For instance, as typical heat-clearing and damp eliminating herbs, Scutellariae Radix has the main flavonoes of baicalein and baicalin using the direct-acting antibacterial activity to fight against MRSA, S. aureus, and Streptococcus suis infections via inhibition of bacterial membrane functions (Rajkumari et al., 2017;Chen et al., 2018;Zhang et al., 2020;Lu et al., 2021) (Table 1). In addition, other flavonoid compounds in heat-clearing herbs also have the antibacterial abilities of damaging the bacterial membrane, such as quercetin from Gardeniae Fructus (iii, heat-clearing and damp eliminating herb) (Mohamed et al., 2020;Yang et al., 2020), kaempferol origin from Moutan Coetrx (ii, heat-clearing and blood-eliminating herb) (Lin et al., 2020;He et al., 2021), luteolin and lonicerin from Lonicerae Japonica (iv, heat-clearing and toxinrelieving herb) (Zhang et al., 2018;Singh et al., 2021), hyperin and hyperoside origin from Lonicerae Japonica (iv, heat-clearing and toxin-relieving herb) (Rodrigo Cavalcante de Araújo et al., 2019), rutin from Forshiae Fructus (iii, heat-clearing and damp eliminating herb) (Deepika et al., 2019;Di Lodovico et al., 2020;Motallebi et al., 2020), and mangiferin from Anemarrhenae Rhizome (i, heat-clearing and blood-cooling herbs) (Tchinda et al., 2019). These DAC IM flavonoids that treat bacterial strains are detailed in Table 1.
In order to gain more survival options from the antibiotic stress, resistant bacteria evolved various approaches to survive, such as biofilm, efflux pumps, and virulence factors (Blair et al., 2015;Narendran et al., 2016;Ercoli et al., 2018;Yelin and Kishony, 2018). Flavonoids limited the spread of resistant bacteria by serving as the inhibitors of bacterial efflux pumps (Solnier et al., 2020) and bacterial virulence factors . We summarized the effect of flavonoids such as DAC IB , DAC IE, and DAC IV in Table 1. For the DAC IB mode, flavonoids directly target the bacterial biofilm formation as therapeutic strategies. For instance, the baicalein-fabricated gold nanoparticles have antibiofilm activity against Pseudomonas aeruginosa PAO1 (Rajkumari et al., 2017). The flavone luteolin and the flavonols myricetin, morin, and quercetin strongly reduce the extracellular matrix to interrupt the Escherichia coli macrocolony biofilms by directly inhibiting the assembly of amyloid curli fibers by driving CsgA subunits into an off-pathway leading to SDS-insoluble oligomers (Pruteanu et al., (Brown et al., 2015;Tang et al., 2017;Wang et al., 2019;Ciumarnean et al., 2020). Additionally, wogonin can suppress the EtBr efflux pumps of S. aureus, Mycobacterium aurum, and Mycobacterium smegmatis (Solnier et al., 2020). Luteolin inhibits MsrA efflux pump in Trueperella pyogenes (Guo et al., 2022), phloridzin inhibits the msrA and norA efflux proteins of S. aureus (Lopes et al., 2017;Kamdi et al., 2021), isobavachalcone inhibits AcrAB, TolC efflux pumps of G − bacteria (Kuete et al., 2010), and daidzein inhibits AcrB efflux pump of E. coli (Aparna et al., 2014). It is worth noting that quercetin that belongs to flavanols have the inhibitory potential to effectively act as the efflux pump to treat CRNB due to its polyphenol hydroxyl group structure (Pal and Tripathi, 2020).
On the other side, flavonoid compounds have the direct-acting antibacterial effect of inhibiting bacterial virulence factors, such as α-hemolysin (Hla) of S. aureus He et al., 2021). The Hla can cause bacterial entry of host cells and also lead to bacterial coinfection, while flavonoids can target Hla to control bacterial infection . Altogether, the flavonoid compounds that inhibit virulence factors are denoted as DAC IV flavonoids and summarized in Table 1.

Host-Acting Antibacterial Modest
To establish persistent infection in the host, resistant bacteria toned to interrupt the host defense and trigger inflammation (Ercoli et al., 2018;Mathur et al., 2019;Pham et al., 2020;Rowe et al., 2020). The host is the key point to drug therapy and HDT may be a novel approach for controlling resistant bacteria (Kaufmann et al., 2018;Ahmed et al., 2020). Many natural plant compounds exhibited the antibacterial activity of HDT, such as the bacterial infection therapy obtained from bedaquiline from Perucian bark that activates host innate immunity (Giraud-Gatineau et al., 2020). Additionally, the host-acting antibacterial compounds were systematically summarized in our previous works (Liu et al., 2022). Based on these foundations, as shown in Figure 4C, we divide HAC flavonoids into four groups: antioxidation (HAC AO ), anti-inflammatory (HAC AI ), modulation of immune cells (HAC MI ), and regulating cellular pathways (HAC RP ). HAC AO flavonoids are able to combat bacterial infection with their antioxidation function, by reducing the free radicals and lipid peroxidation to inhibit oxygen-derived radicals or nitrogen-derived radicals to avoid oxidative damage (Kamdi et al., 2021;Palierse et al., 2021) In addition, this antibacterial activity mainly relies on the phenolic hydroxy group of flavonoid compounds, such as quercetin and hyperoside ( Figures 3C, 4A) (Xie et al., 2015). For the HAC AI flavonoids, the major anti-inflammatory modes of flavonoid compounds are inhibition of protein kinases (COX, cyclooxygenase, LOX, lipoxygenase and PLA2, phospholipase A2), inflammatory factors (IL-1, IL-4, IL-10, and IL-13) and related transcription factors (NF-κB, GATA-3, and STAT-6) (Park et al., 2018;Ren et al., 2019;Kamdi et al., 2021;Miao et al., 2021). The anti-inflammatory flavonoid compounds include apigenin, baicalein, baicalin, luteolin, lonicerin, tamarixetin, rutin, phloridzin, isoliguirtigentin, puerarin, and catechin ( Table 1). The HAC MI flavonoids are marked as modulation of immune cells, which is usually accompanied by anti-inflammatory effects, like luteolin and kurarinol A (Singh et al., 2021;Xu et al., 2021). These functions of activating immune cells to downregulate inflammation contains decreasing CD80/CD86 of dendritic cells and histamine, prostaglandin, pro-inflammatory, and cytokines of mast cells to suppress excessive inflammation. In addition, it also increases immune cells (T cell, macrophage, PMNs and Th2 cell) to activate innate immunity ( Figure 4C). The HAC RP flavonoids are the kinds of compounds that target cellular signaling pathways to combat bacteria. As shown in Table 1 and Figure 4C, the flavonoids of Sophora flavescens are potential agents in Tuberculosis (TB) infection therapy by enhancing macrophage autophagy to promote cellular survival and then reducing inflammation. (Kan et al., 2020). The Baicalein-7-O-β-D-glucuronide of Scutellariae Radi and Luteolin-7-O-β-D-glucuronide of Impatiens balsamina target MAPKs or Wnt/β-catenin pathway to inhibit the inflammation from bacterial LPS (Li et al., 2009;Kawai, 2018;Liu F. et al., 2020;Pereira et al., 2020).

CONCLUSION
The antibacterial resistance crisis has led to a prolonged period of infection control in clinics. High-efficiency and novel antimicrobial drugs remain the most effective strategies for the treatment of multidrug-resistant and unknown pathogen infections (Brown and Wright, 2016;Theuretzbacher et al., 2020). It is clear that the main measures for the prevention and control of drug-resistant bacteria are to vigorously develop green and effective new antibacterial drugs and restore existing antibacterial drugs safely and stably. However, unclear mechanisms of antibacterial action is the main reason that hinders the development of drugs. Therefore, target identification of herbal products is necessary. In this review, we summarized both the direct-acting and host-acting antibacterial flavonoids derived from heat-clearing herbs and focused on the antibacterial modes. The current reports show the antibacterial effects of flavonoid compounds on MDR bacteria by both the direct-acting antibacterial mode and host-acting antibacterial mode ( Table 1). It also proves that herbal flavonoids should be the better source for alternative antibiotics ( Figure 1B and Figure 4). However, the focus on the discovery of antibacterial targets of flavonoids are the main topic in this review. In addition, the screening principles of lead flavonoids based on the antibacterial targets need more illustration. Hence, further studies ought to devote more attention to the multi-targets of flavonoids. Thus, we believe the crisis in antibiotic discovery will rapidly be solved though exploring more herbs in future. Altogether, the basis of this review aims to find the flavonoid lead compounds to guide the future drug modifications based on the structure and active antibacterial mechanism based on functional groups.

AUTHOR CONTRIBUTIONS
XL conceived the projects. XL, LS, and XH prepared the figures and table. JL and XR performed data collection. XL, LS, and XH wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

FUNDING
This work was funded by "Beijing Natural Science Foundation, grant number 6224060", "2021 youth project of high-end technology innovation think tank, grant number 2021ZZZLFZB1207120", "2022 Research and Innovation ability improvement plan for young teachers of Beijing University of Agriculture, grant number QJKC2022028" and "Beijing University of Agriculture science and Technology innovation Sparkling support plan, grant number, BUA-HHXD2022007".