Antibacterial potential of danthron, embelin, sanguinarine and shikonin against multidrug-resistant Neisseria gonorrhoeae in vitro

Abstract

Background

Neisseria gonorrhoeae (N. gonorrhoeae) poses a public health challenge due to escalating incidence and growing antimicrobial resistance, necessitating urgent exploration of alternative therapies. Presently, clinical management relies on ceftriaxone monotherapy or its combination with azithromycin. However, emergence and spread of strains resistant to previously recommended regimens, particularly the FC428 clone strain, which also exhibits azithromycin resistance, present significant challenges. Therefore, investigation of alternative strategies is paramount.

Methods

55 multidrug-resistant N. gonorrhoeae isolates, including those with high-level resistance to ceftriaxone, high-level resistance to azithromycin, and dual-resistant clones, were collected from Guangdong province between 2017 and 2021. Drug susceptibility of natural products was assessed using broth microdilution methods. Cytotoxicity of the four natural products against renal epithelial (293T) and human vaginal epithelial (VK2/E6E7) cells was evaluated by Cell Counting Kit-8 assay.

Results

Our findings indicated that danthron exhibited good antibacterial activity, followed by embelin, sanguinarine and shikonin, with MIC 50/90 values of 2.4/4.8, 3/6, 3.3/6.6 and 6/12 mg/L, respectively. These results indicated that even low concentrations could effectively inhibit growth of N. gonorrhoeae. Furthermore, all four natural products demonstrated potent antibacterial effects against dangerous strains, including those exhibiting ceftriaxone resistance (MIC ≥ 0.125 mg/L), high-level azithromycin resistance (MIC ≥ 256 mg/L), or dual-resistance characteristics. Additionally, preliminary toxicological assessments on cell lines suggested the following order of selective toxicity: danthron < embelin < sanguinarine < shikonin.

Conclusion

Danthron, embelin, sanguinarine and shikonin demonstrated preliminary effectiveness in eliminating multidrug-resistant N. gonorrhoeae with appropriate selective toxicity. Our study suggests they may represent promising candidates for further investigation and potential therapeutic development.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10123-026-00783-7.

Introduction

Gonorrhea, caused by N. gonorrhoeae, is a sexually transmitted infection that remains global public health concern (Sancta St et al. 2020). It is estimated that there are 82.4 million new cases annually (Landhuis 2024). Clinically, N. gonorrhoeae infects the mucosal epithelia of the human genitourinary system, leading to symptoms like urethritis and cervicitis, as well as increasing the risk of human immunodeficiency virus if left untreated (Susan and Demetre 2018).

Current treatments like penicillin and tetracycline are rapidly becoming ineffective due to resistance, leaving ceftriaxone as the last reliable antibiotic (Unemo et al. 2019). However, the emergence of ceftriaxone-resistant strains, such as the FC428 clone, threatens its efficacy (Lin et al. 2022a, b, c). To address this, dual therapy with extended-spectrum cephalosporin and azithromycin is suggested to curb the spread of ceftriaxone resistance and relieve treatment pressure prior to the introduction of novel antibiotics (Bignell and Unemo 2013). Yet, the rise of azithromycin-resistant strains (St Cyr et al. 2020), especially those with high-level resistance (MIC ≥ 256 mg/L) found in several countries like China (Lin et al. 2022a, b, c), Australia (Stevens et al. 2015), Ireland (Lynagh et al. 2015), Japan (Yasuda et al. 2017), Ontario (Allen et al. 2014), Hawaii (Katz et al. 2017), United Kingdom (Fifer et al. 2016), challenges the effectiveness of dual therapy. Hence, the urgent need for the development of new drugs is underscored by the critical issue of antibiotic resistance.

The discovery of new drugs typically requires over a decade and incurs costs exceeding US$1 billion. Nevertheless, approximately 95% of drugs ultimately fail to demonstrate efficacy (The 2014). The development of new antimicrobial drugs is progressing at a significantly slower pace than the emergence of antimicrobial resistance. To address this challenge, high-throughput screening techniques have been employed to rapidly identify numerous drugs. Natural products, known for their diverse structures, have historically been vital sources for new drug development and may offer solutions to overcome existing drug resistance (Newman and Cragg 2012; Mishra and Tiwari 2011; Carter 2011; Cragg and Newman 2013; Dias et al. 2012; Álvarez-Martínez et al. 2020, 2021).

To address the limitations of traditional drug development, our research previously used high-throughput screening (data currently being published), identifying four promising antibacterial drugs: danthron, embelin, sanguinarine and shikonin (refer to Supplemental Fig. 1). Subsequent validation confirmed that these four drugs exhibited significant inhibitory efficacy against multidrug-resistant N. gonorrhoeae, particularly strains exhibiting high-level resistance to ceftriaxone or azithromycin, as well as dual-resistant strains (Table 1 and Fig. 1).

Table 1.

AST of four compounds such as danthron, embelin, sanguinarine and Shikonin against 55 multidrug-resistant N. gonorrhoeae

(A) MIC50 and MIC90 analysis of compounds against 55 clinical multidrug-resistant N. gonorrhoeaae
	MIC(mg/L)
	Danthron	Embelin		Sanguinarine		Sanguinarine		Shikonin
MIC50	2.4	3		3.3		3.3		6
MIC90	4.8	6		6.6		6.6		12
Range	0.001-4.8	0.0015-6		0.0016- 13.2		0.0016-13.2		0.0014-12
(B) Comparison of MIC50/MIC90 of four compounds against antibiotics sensitive and resistant strains.
		MIC(mg/L)
		Danthron		Embelin		Sanguinarine		Shikonin
		MIC50	MIC90	MIC50	MIC90	MIC50	MIC90	MIC50	MIC90
Penicillin	S	/	/	/	/	/	/	/	/
	I	1.2	4.8	3	6	3.3	6.6	6	12
	R	2.4	4.8	3	6	3.3	6.6	6	12
Tetracycline	S	0.6	4.8	1.5	6	0.4125	13.2	1.5	12
	R	2.4	4.8	3	6	3.3	6.6	6	12
Ciprofloxacin	S	/	/	/	/	/	/	/	/
	I	/	/	/	/	/	/	/	/
	R	2.4	4.8	3	6	3.3	6.6	6	12
Spectinomycin	S	2.4	4.8	3	6	3.3	6.6	6	12
	R	/	/	/	/	/	/	/	/
Ceftriaxone	S	2.4	4.8	3	6	3.3	6.6	6	12
	DS	2.4	4.8	3	6	3.3	6.6	6	12
Cefixime	S	2.4	4.8	3	6	3.3	6.6	6	12
	DS	2.4	4.8	3	6	6.6	6.6	6	12
Azithromycin	S	2.4	4.8	3	6	3.3	6.6	6	12
	R	2.4	4.8	3	6	3.3	6.6	6	12
(C) MICs of traditional antibiotics and four compounds against FC428-related clone.
	sample 17	sample 26		sample 32		sample 39		sample 42
Penicillin	1	2		0.125		1		≥ 32
Tetracycline	1	2		0.5		2		2
Ciprofloxacin	≥ 32	32		8		16		32
Spectinomycin	8	16		8		8		16
Ceftriaxone	0.25	0.125		0.125		≥ 1		0.125
Cefixime	≥ 1	≥ 1		≥ 1		≥ 1		≥ 1
Azithromycin	0.125	1		0.125		2		0.25
Danthron	2.4	2.4		0.6		4.8		4.8
Embelin	0.375	1.5		1.5		6		3
Sanguinarine	0.825	3.3		0.4125		6.6		3.3
Shikonin	0.0014	3		1.5		6		3
(D) MICs of traditional antibiotics and four compounds against high-level azithromycin resistant strains.
	sample 8	sample 12	sample 15	sample 34		sample 37		sample 45
Penicillin	1	0.5	≥ 32	1		≥ 32		2
Tetracycline	≥ 32	4	≥ 32	4		≥ 32		≥ 32
Ciprofloxacin	16	16	16	16		16		16
Spectinomycin	16	16	16	16		16		16
Ceftriaxone	0.03	0.008	0.015	0.015		0.015		0.03
Cefixime	0.03	0.06	0.06	0.06		0.06		0.03
Azithromycin	≥ 2048	≥ 2048	≥ 2048	≥ 2048		≥ 2048		≥ 2048
Danthron	2.4	0.001	4.8	1.2		4.8		4.8
Embelin	6	0.0015	6	6		6		6
Sanguinarine	3.3	0.0016	6.6	6.6		6.6		3.3
Shikonin	6	0.0014	12	6		12		12
(E) MICs of traditional antibiotics and four compounds against dual-resistant strains.
	sample 6	sample 14	sample 19	sample 26	sample 39	sample 40		sample 54
Penicillin	2	≥ 32	2	2	1	1		1
Tetracycline	2	≥ 32	2	2	2	2		4
Ciprofloxacin	≥ 32	16	≥ 32	32	16	8		≥ 32
Spectinomycin	8	32	8	16	8	16		16
Ceftriaxone	0.125	0.125	0.125	0.125	≥ 1	0.125		0.125
Cefixime	0.25	0.25	0.25	≥ 1	≥ 1	0.25		0.25
Azithromycin	32	1	32	1	2	1		1
Danthron	2.4	1.2	1.2	2.4	4.8	2.4		1.2
Embelin	3	0.75	0.75	1.5	6	6		6
Sanguinarine	3.3	0.0016	3.3	3.3	6.6	6.6		3.3
Shikonin	3	0.0014	1.5	3	6	12		12
(F) IC50 and SI of four compounds against 293T and VK2/E6E7 cell lines.
	IC50 (mg/L) and SI
Cell lines	Danthron			Embelin		Sanguinarine		Shikonin
MIC50	2.4			3		3.3		6
MIC90	4.8			6		6.6		12
293T	20.79			22.80		5.507		6.026
293T SI50 index	8.6625			7.6		1.669		1.004
293T SI 90 index	4.33125			3.8		0.8345		0.502
VK2/E6E7	32.83			23.65		4.871		4.798
VK2/E6E7 SI50 index	13.68			7.883		1.476		0.799
VK2/E6E7 SI90 index	6.84			3.9415		0.738		0.3995
Concentration range (mg/L)	0.6–76.8			0.75-48		0.106–26.4		0.09-48

S Susceptible, DS Decreased Susceptibility, I Intermediate susceptibility, R Resistant.

As reported, (I) danthron, an anthraquinone compound isolated from a Marine Fungus, exerts antimicrobial activity, particularly against Staphylococcus, by disrupting the cell wall and membrane, leading to cell damage (Anisha et al. 2018; Wei et al. 2015); (II) embelin, found in Embelia schimperi, demonstrated potent excellent antibacterial activity. An ointment containing embelin exhibited inhibitory effects on two gram-positive bacteria (Staphylococcus aureus and Streptococcus epidermidis) as well as gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) (Rosalyn et al. 2016; Mahendran 2018); (III) sanguinarine, an alkaloid found in corydalis ophiocarpa, exhibited potent activity against Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus agalactiae, Bacillus subtilis, Methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumanii (Razan et al. 2014, 2015), comparable to that of antibiotics, through the mechanism involving interference with cell wall and membrane permeability, as well as induction of reactive oxygen species production in bacteria (Yeqing et al. 2023); (IV) shikonin, naturally occurring hydroxynaphthoquinones, has been shown to hinder the growth of MRSA and Candida through multiple pathways, as evidenced by its ability to induce nucleotide protein leakage, bacteriolysis and inhibition of biofilm formation (Qianqian et al. 2022; Kirandeep et al. 2023). Additionally, shikonin has been found to inhibit Adenosine triphosphate (ATP) synthase and impede cell growth in Escherichia coli (Xiaoru et al. 2017).

These reported broad-spectrum activities and multi-target mechanisms prompted us to investigate their potential against the priority pathogen N. gonorrhoeae, for which specific activity has not been previously documented. This exploration is positioned within a critical therapeutic landscape. While the development of new anti-gonococcal agents remains urgent, most clinical-stage candidates—such as the topoisomerase inhibitor zoliflodacin (Taylor et al. 2018), the novel triazaacenaphthylene gepotidacin (Scangarella-Oman et al. 2018), and the ribosome-targeting lefamulin (Jacobsson et al. 2017) —are single-target, synthetic or semi-synthetic molecules whose mechanisms (inhibiting DNA replication or protein synthesis) may remain vulnerable to resistance evolution. In contrast, the natural products in this study are characterized by a multi-target, membrane-centric mechanistic profile involving direct membrane/cell wall disruption, induction of oxidative stress, and biofilm inhibition (Wei et al. 2015; Rosalyn et al. 2016; Mahendran 2018; Razan et al. 2014, 2015; Yeqing et al. 2023; Qianqian et al. 2022; Kirandeep et al. 2023; Xiaoru et al. 2017). We hypothesized that this distinct mode of action could potentially help overcome resistance mechanisms that compromise current and pipeline antibiotics. Therefore, this study aimed to evaluate the in vitro antibacterial efficacy and cytotoxicity of these four compounds against a contemporary collection of multidrug-resistant N. gonorrhoeae clinical isolates, thereby exploring their potential as novel scaffolds for future therapeutic development.

Materials and Methods

Strain collection, cultivation, preservation and antibiotics breakpoints

A total of 55 multidrug-resistant N. gonorrhoeae isolates were collected in Guangdong, China, from 2017 to 2021. These isolates exhibited resistance or decreased susceptibility to at least two classes of antibiotics. Clinical isolates were identified by gram staining and oxidase, catalase and sugar fermentation tests recommended by the World Health Organization (WHO). Minimal inhibitory concentrations (MICs) of antibiotics were determined using agar dilution method and ensured with ATCC49226, WHO-L and WHO-P quality control strains. Antimicrobial susceptibility interpretations followed contemporary guidelines from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2025 and the Western Pacific NG Monitoring Program. For EUCAST criteria, resistance (R) thresholds were defined as: azithromycin MIC ≥ 1 mg/L, ciprofloxacin MIC > 0.06 mg/L, penicillin MIC > 1 mg/L, spectinomycin MIC > 64 mg/L and tetracycline MIC > 0.5 mg/L. Intermediate susceptibility (I) applied to penicillin (0.12 mg/L ≤ MIC≤1 mg/L) and ciprofloxacin (MIC = 0.06 mg/L), while susceptibility (S) was assigned to azithromycin (MIC < 1 mg/L), penicillin (MIC ≤ 0.06 mg/L), tetracycline (MIC ≤ 0.5 mg/L), ciprofloxacin (MIC ≤ 0.03 mg/L), and spectinomycin (MIC ≤ 64 mg/L). For cephalosporins, WHO criteria defined decreased susceptibility (DS) as ceftriaxone MIC ≥ 0.125 mg/L and cefixime MIC ≥ 0.25 mg/L, with susceptibility (S) thresholds at < 0.125 mg/L and < 0.25 mg/L, respectively. High-level AZM (AZM ^HL−R) was defined as an MIC of ≥ 256 mg/L.

Drugs susceptibility testing

GC Broth microdilution (Wu et al. 2018) was used to determine drug susceptibility of danthron, embelin, sanguinarine and shikonin. Quality control (QC) was ensured with ATCC49226 strains. GC Broth Base plus medium supplemented with 5% beef extract (No. LP0029B), 1% brain heart infusion (No. CM1135), and 1.5% tryptone (No. LP0042B), 0.5% agar (No. CM0367), and 1% vitox, was purchased from Zhuhai Jinbiao Biotechnology Co., Ltd. This medium has been demonstrated to effectively support the good growth of N. gonorrhoeae. Briefly, the sub-cultured strains were cultured on Thayer-Martin medium and gonococcal agar supplemented with 10% defibrinated sheep blood for 18 h in a 37 incubator at 5% CO2. Afterwards, a bacterial suspension equivalent to 0.5 McFarland standard was prepared and diluted in broth medium. Test agents were added in the first row of the 96-well plates and serially diluted along the plates. Plates were then incubated at 37℃ in the presence of 5% CO₂ for 24 h. In the course of evaluating the antimicrobial efficacy of four natural products against clinical strains, we utilized the ATCC49226 QC strain to perform both agar dilution and GC Broth microdilution tests. These two tests were employed to determine the MICs of antibiotics—specifically, penicillin, tetracycline, ciprofloxacin, spectinomycin, ceftriaxone, cefixime and azithromycin—as well as four natural products: danthron, embelin, sanguinarine and shikonin. The adoption of natural products MICs data in clinical samples only occurred when the MIC values for the seven antibiotics identified by both agar dilution and GC Broth microdilution methods simultaneously fell within the correct range as defined by the WHO in ATCC49226 QC strain (Supplemental Table 1). The susceptibility assay utilized a range of danthron concentrations from 0.001 to 9.6 mg/L, embelin concentrations from 0.0015 to 12 mg/L, sanguinarine concentrations from 0.0016 to 13.2 mg/L, and shikonin concentrations from 0.0014 to 12 mg/L. These four natural compounds—danthron (T0800), embelin (T6485), sanguinarine (T2781) and shikonin (T1125)—were procured from TargetMol Chemicals Inc. These compounds were dissolved in DMSO in accordance with their respective physicochemical properties and the manufacturer’s guidelines.

Cytotoxicity assays

Drug cytotoxicity was assessed by 293T and VK2/E6E7. For the experiment, cells were passaged and counted. Then, 293T cells were seeded at a density of 2 × 10⁴ cells per well, and VK2/E6E7 cells at 1.5 × 10⁴ cells per well, with three replicates for each concentration. After 16 h of incubation to allow cell attachment, the culture medium was removed. Fresh DMEM (CAS: C11995500BT, Gibco) medium containing the test drugs was prepared and added to each well at a volume of 100 µL. Following 24 h of drug treatment, 10 µL/well CCK-8 (CAS: BB-4202-4, Bestbio) was added to each well according to the manufacturer’s instructions. The plates were then incubated for an additional 4 h in the dark. The absorbance at 450 nm (OD₄₅₀) was measured. The half maximal inhibitory concentration (IC₅₀) was calculated using GraphPad Prism 9 software. SI (selectivity index)₅₀ = IC₅₀ / MIC₅₀. SI₉₀= IC₅₀ / MIC₉₀. All experiments were repeated three times independently to ensure reproducibility of the results.

Results

Natural products such as danthron, embelin, sanguinarine and shikonin exhibited strong antibacterial activities

The data in Supplemental Table 2 indicate that 56.36%, 96.36% and 100% of the strains exhibited resistance to penicillin, tetracycline, and ciprofloxacin, with a MIC₅₀ of 8, 4, 16 mg/L respectively and MIC₉₀ above 32 mg/L, underscoring the unsuitability of these antibiotics for ongoing clinical management of gonorrhea. A notable percentage of clinically strains displayed decreased susceptibility to ceftriaxone (54.44%) and cefixime (36.36%), with MIC₅₀ values of 0.125 and 0.25 mg/L and MIC₉₀ values of 0.125 mg/L and ≥ 1 mg/L, respectively. Additionally, 50.91% of the clinical strains demonstrated resistance to azithromycin, with an MIC₅₀ of 4 mg/L and MIC₉₀ values of ≥ 2048 mg/L. These resistance patterns underscore the urgent need for novel antimicrobial agents.

Prior to conducting this study, we employed high-throughput screening techniques to identify four potential antibacterial compounds. Currently, drug sensitivity testing was conducted on the 55 drug-resistant strains mentioned above (Supplemental Table 3). As illustrated in Fig.  1A and Table 1A, danthron demonstrated antibacterial efficacy against all multi-drug N. gonorrhoeae at concentrations less than or equal to 4.8 mg/L, with MIC₅₀ and MIC₉₀ values of 2.4 mg/L and 4.8 mg/L, respectively. In the case of embelin, it exhibited antibacterial activity against 45.45% of the tested isolates at a concentration of 6 mg/L, followed by 23.64% at 3 mg/L, 21.81% at 1.5 mg/L, and 3.64% at both 0.375 mg/L and 0.75 mg/L, with a further 1.82% at 0.0015 mg/L (Fig. 1B). The MIC range for embelin spans from 0.0015 to 6 mg/L, with MIC₅₀ and MIC₉₀ values of 3 mg/L and 6 mg/L, respectively (Table 1A). For sanguinarine, the MIC values were predominantly distributed at 3.3 mg/L and 6.6 mg/L, accounting for 47.27% and 38.18% of the strains, respectively (Fig. 1C). The MIC range for sanguinarine was 0.0016 to 13.2 mg/L, with MIC₅₀ and MIC₉₀ values of 3.3 mg/L and 6.6 mg/L, respectively (Table 1A). Lastly, shikonin exhibited antibacterial activity at higher concentrations, achieving complete inhibition of all clinical isolates at 12 mg/L (Fig.  1D). The MIC range for shikonin was 0.0014 to 12 mg/L, with MIC₅₀ and MIC₉₀ values of 6 mg/L and 12 mg/L, respectively (Table 1A).

Fig. 1.

MIC distribution of four compounds against N. gonorrhoeae isolates. Proportion of 55 N. gonorrhoeae isolates with different MICs for danthron (A), embelin (B), sanguinarine (C) and shikonin (D) (n, proportion%)

Furthermore, regardless of the antibiotic susceptibility profiles of the clinical strains, these four natural products exhibited considerable antibacterial activity (Table 1B). Notably, for strains exhibiting high-level resistance to cephalosporins (Table 1C), high-level resistance to azithromycin (Table 1D), and dual-resistance (Table 1E), the four compounds preliminary demonstrated potent and effective antibacterial activity against multidrug-resistant bacteria, with danthron showing the most pronounced effect.

Last, the cytotoxicity of the four natural products against 293T and VK2/E6E7 cell lines was assessed, with results summarized in Table 1F. The SI calculated as IC₅₀/MIC, was evaluated using both MIC₅₀ (SI₅₀) and the more conservative MIC₉₀ (SI₉₀) to provide a comprehensive view of the therapeutic window. In 293T cells, danthron and embelin exhibited favorable SI₅₀ values of 8.6625 and 7.6, and SI₉₀ values of 4.33125 and 3.8, respectively. Sanguinarine showed a narrower margin (SI₅₀=1.669, SI₉₀=0.8345), while shikonin’s SI values were near or below the threshold of selectivity (SI₅₀=1.004, SI₉₀=0.502). A similar trend was observed in the more physiologically relevant VK2/E6E7 cells: danthron (SI₅₀=13.68, SI₉₀=6.84) and embelin (SI₅₀=7.883, SI₉₀=3.9415) maintained robust selectivity indices. Sanguinarine again presented a constrained window (SI₅₀=1.476, SI₉₀=0.738), and shikonin demonstrated SI₉₀ values below 0.4, indicating that its effective antibacterial concentration (MIC₉₀) exceeds its cytotoxic threshold. These data suggest a distinct gradient of in vitro selectivity: danthron > embelin > sanguinarine > shikonin. It is important to note that SI₉₀ values approaching or falling below 1.0 signify a minimal or non-existent selectivity window, indicating that the concentration required to inhibit 90% of clinical isolates likely exceeds the threshold for significant cytotoxicity.

Discussion

The ongoing evolution of antimicrobial resistance in N. gonorrhoeae poses a significant challenge for healthcare providers tasked with managing the treatment of this widespread global sexually transmitted infection (Lin et al. 2022a, b, c). Currently, the single dose therapy of ceftriaxone endorsed by the WHO, as well as the dual-therapy of cephalosporin and azithromycin, remain efficacious in the treatment of gonorrhea (Susan et al. 2022; Julia 2021). However, the persistently decreased susceptible rate of ceftriaxone is exhibiting a trend of escalation (Lin et al. 2022a, b, c; Kirkcaldy et al. 2015). The efficacy of azithromycin therapy is further jeopardized by instances of unsuccessful treatment with the combination regimen and the escalating resistance to azithromycin observed in certain geographical areas (St Cyr et al. 2020). Hence, the WHO classifies N. gonorrhoeae as a priority pathogen requiring immediate attention for the development of novel pharmaceutical interventions.

In our study, danthron emerged as the most effective agent, with MIC₅₀ and MIC₉₀ values of 2.4 mg/L and 4.8 mg/L, respectively, against all tested strains. This potency is particularly significant given the high prevalence of ceftriaxone resistance (54.44% of isolates) and azithromycin high-level resistance (MIC ≥ 256 mg/L) in our cohort. Embelin, sanguinarine and shikonin also demonstrated antibacterial activity, albeit at slightly higher MIC₅₀ range from 3 mg/L to 6 mg/L. The broad-spectrum efficacy of these compounds against strains with diverse resistance profiles—including the globally disseminated FC428 clone—suggests they may hold potential as standalone or adjunct therapies, especially in regions where resistance to cephalosporins and macrolides is rampant.

The in vitro efficacy of danthron, embelin, sanguinarine and shikonin against multidrug-resistant N. gonorrhoeae, including strains with high-level resistance to ceftriaxone or azithromycin, underscores their potential as novel anti-gonococcal scaffolds. A potential implication of our findings lies in the mechanistic divergence between these natural products and both current therapies and clinical-stage candidates (e.g., zoliflodacin, gepotidacin and lefamulin) (Taylor et al. 2018; Scangarella-Oman et al. 2018; Jacobsson et al. 2017). While the latter are typically single-target enzyme inhibitors, our compounds appear to employ a multi-faceted, membrane-centric strategy. The antimicrobial mechanisms of the four natural products could provide a rationale for their efficacy against multidrug-resistant N. gonorrhoeae. Danthron, an anthraquinone, exerts its effect by disrupting bacterial cell wall and membrane integrity, ultimately leading to cellular damage (Wei et al. 2015). Embelin demonstrates broad-spectrum activity against both Gram-positive and Gram-negative bacteria, though its precise molecular targets are under investigation (Rosalyn et al. 2016; Mahendran 2018). Sanguinarine, a benzophenanthridine alkaloid, compromises cell wall and membrane permeability while also inducing the production of reactive oxygen species within bacterial cells (Razan et al. 2014, 2015; Yeqing et al. 2023). Shikonin, a hydroxynaphthoquinone, employs multiple strategies including the inhibition of biofilm formation and the suppression of ATP synthase activity (Qianqian et al. 2022; Kirandeep et al. 2023; Xiaoru et al. 2017). This polypharmacological approach—simultaneously targeting membrane integrity, elevating oxidative stress, and hindering biofilm formation—could potentially confer a higher barrier to resistance and could explain their maintained activity against strains resistant to conventional antibiotics.

Our cytotoxicity data reveal distinct selectivity profiles among the compounds and lend preliminary support to their therapeutic potential. While danthron and embelin showed favorable SI₅₀ and SI₉₀ values (> 3) in VK2/E6E7 cells, sanguinarine presented a narrower margin (SI=0.738–1.669). Notably, shikonin exhibited SI₉₀ values below 1 in both cell lines, indicating that its effective antibacterial concentration (MIC₉₀) approaches or exceeds its cytotoxic threshold. This suggests a limited safety margin for shikonin and tempers its potential for direct therapeutic development without substantial chemical optimization to improve selectivity. Consequently, this work identifies promising hit compounds and suggests a complementary mechanistic avenue for anti-gonococcal drug discovery.

Several limitations of this study should be considered. First, the in vitro findings reported here require further validation in in vivo models to confirm therapeutic efficacy and systemic safety. Future studies should include time-kill assays to characterize the bactericidal/bacteriostatic profile, evaluate potential synergism with existing antibiotics (e.g., ceftriaxone), and assess resistance emergence risk using appropriate animal models of infection. Second, although our mechanistic discussion is based on reported modes of action, the precise molecular targets of these compounds in N. gonorrhoeae remain to be elucidated. Third, the cytotoxicity assessment was limited to two cell lines. While VK2/E6E7 represents a relevant vaginal epithelial model, the kidney-derived 293T line may not fully reflect tissue-specific toxicity in the urogenital tract. More comprehensive toxicological profiling in primary human cells or advanced tissue models relevant to infection sites is warranted. Finally, the clinical isolates used were from a single geographical region. Expanding the sample size and including strains from diverse geographical origins would strengthen the generalizability of these findings. Addressing these limitations in future work will be essential to advance these natural products toward preclinical development.

Our study also underscores the importance of high-throughput drug screening in identifying novel antibacterial agents. The rapid identification and validation of these four natural products demonstrate the efficiency of this approach in accelerating drug discovery. Given the urgent need for new treatments for gonorrhea, high-throughput screening offers a cost-effective and time-saving strategy to identify potential therapeutic candidates.

Conclusion

In conclusion, our study provides preliminary evidence that natural products such as danthron, embelin, sanguinarine, and shikonin may hold promise as antibacterial agents against multidrug-resistant N. gonorrhoeae. These findings suggest a potential pathway toward new therapeutic options to combat the growing threat of antimicrobial resistance in gonorrhea. Further research remains necessary to explore the clinical applicability of these compounds and evaluate their role in future treatment strategies.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

AMR

antibiotic resistance

MIC

minimum inhibitory concentration

WHO

World Health Organization

QC

quality control

EUCAST

European Committee on Antimicrobial Susceptibility Testing

ATP

Adenosine triphosphate

Author contributions

All authors made a significant contribution to the work reported. Xiaomian Lin designed the research, wrote the manuscript, and provided research fundings. Yuqiu Qi, Ran Zhang performed most of the experiments. Yingxian Tang help provide the clinical samples. Hui Peng, Chenlong Tao, Qiankun Ke, Songwei Lu, Shangzhu Shen, and Zhuo Wang provided assistance in MIC calculations. Peng Hui and Chenlong Tao jointly completed the cytotoxicity experiment during the article revision period. All authors participated in the drafting, revision, and critical review of the manuscript; provided final approval for the version to be published; consented to the choice of journal for submission; and accepted responsibility for all aspects of the work.

Funding

This study was supported by the National Natural Science Foundation of China (No. 82302578) and Shanghai Municipal Health Commission Health Industry Clinical Research Project (20244Y0232).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

A total of 55 multidrug-resistant N. gonorrhoeae isolates were collected in Guangdong, China, from 2017 to 2021. The clinic strains were part of the routine hospital laboratory procedure.

Consent for publication

All authors read and revise the article. Agree to publish.

Competing interests

The authors declare no competing interests.