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%), carbapenem-resistant 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 (Liu et al., 2019; 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 drug-resistant 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). Heat-clearing Chinese medicines symptomatically treat bacteria-caused 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 heat-clearing herbs to reveal the therapeutic strategies of resistant pathogens. In the following sections, we discuss the availabilities and antibacterial pipelines of herbal flavonoids.

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 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).

Flavonoids are widely distributed heat-clearing medicines and exhibit multiple biological activities such as antibacterial, anti-inflammatory, and antioxidation effects (Lan et al., 2021; Waditzer and Bucar, 2021). The chemical structures of more than 8000 flavanoids have been determined (Wen et al., 2021) and most existing flavonoids have been reported to defend against MDR bacterial infections (Song et al., 2021a). As shown in Figure 2B, the typical heat-clearing and fire-purging herbs include Anemarrhenae Rhizome, Gardeniae Fructus, and Mangosteen peel. Mangiferin and isomangiferin are the main compounds from Anemarrhenae Rhizome (Piwowar et al., 2020), quercetin and isoquercitrin can be extracted from fruits of Gardeniae Fructus (Kim et al., 2006), and mangostin exsits in the Mangosteen peel (Weerayuth et al., 2014). Heat-clearing and blood-cooling herbs contain Moutan Coetrx, Rehmanniae Radix, and Scrophulariae Radix; among these, only Moutan Coetrx is reported to contain flavonoid compounds of catechins, picetin, and kaempferol (Zhang et al., 2017), while the others have no detected flavonoids. Three heat-clearing and damp-eliminating herbs with large flavonoids are apigenin, luteolin, andquercetin in Cancrinia discoidea (Su et al., 2011), baicalin, baicalein, wogonin, and wogonoside in Scutellariae Radix (Guowei et al., 2018), and kurarinone and norkurarinone in Sophora flavescens (Dong et al., 2021). Heat-clearing and toxin-relieving herbs such as Lonicerae Japonica and Forsythiae Fructus have luteolin, lonicerin, hyperin, and rutin (Huang et al., 2020; Wei et al., 2020). Finally, asthenic-heat clearing herbs include Artemisia annua herba and Stellariae Radix and these exist in tamarixetin and quercetin, correspondingly (Hao et al., 2020).

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.

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 C₆-C₃-C₆ 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.

Antibacterial modes of flavonoids depend on the structures, that is the substitutions on the aromatic rings. As more antibacterial activities of natural flavonoids have been found, numerous flavonoids have been confirmed to have existing antibacterial activity-structure relationships. For instance, the flavonoid compounds that have prenyl groups with hydrophobic substituents inhibit bacterial membrane function and biofilm formation (Rashid et al., 2007; Prawat et al., 2013; Wenzel, 2013; Narendran et al., 2016). The flavonoid compounds that have phenolic hydroxy groups with scavenging oxygen free radicals can modulate antioxidation and anti-inflammatory activities (Wenzel, 2013; Guowei et al., 2018; Farhadi et al., 2019; Jordan et al., 2020).

According to different modes of action, flavonoid compounds can be grouped into two types, one with a direct-acting antibacterial mode (DAC) regulated by prenyl (Dong et al., 2017; Kariu et al., 2017; Jordan et al., 2020; Song et al., 2021a), the other with host-acting antibacterial mode (HAC) regulated by phenolic hydroxy (Xie et al., 2015; Echeverria et al., 2017; Farhadi et al., 2019; Liu et al., 2022).

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.

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.