Identification of valepotriate as a novel antimycobacterial agent against drug-resistant Mycobacterium tuberculosis through screening of a natural terpenoid compound library

Abstract

Background

The continued emergence of multidrug-resistant Mycobacterium tuberculosis (M. tb) has substantially limited treatment options and highlights the need for new antimycobacterial agents. Although terpenoids comprise one of the largest classes of natural products with diverse biological activities, their antimycobacterial potential has not been systematically investigated.

Methods

A library comprising 354 natural terpenoid compounds were screened against M. tb. Lead compounds were subsequently characterized by determining minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), time–kill kinetics, activity against clinical drug-sensitive and drug-resistant isolates, cytotoxicity in THP-1–derived macrophages and intracellular antimycobacterial activity using colony-forming unit assays.

Results

Primary screening identified 10 compounds with strong antimycobacterial activity, among which valepotriate showed as a novel antimycobacterial agent. Valepotriate inhibited extracellular M. tb growth with an MIC of 10 μM and an MBC of 20 μM. Time–kill assays demonstrated rapid concentration-dependent bactericidal activity, reducing bacterial counts by >2 log₁₀ cfu within 24 h at 4× MIC and completely eradicating viable bacteria within 24 h at 8× and 16× MIC. Valepotriate retained activity against 14 clinical drug-sensitive and drug-resistant isolates with MIC values ranging from 5 to 20 μM. Valepotriate significantly reduced the intracellular bacterial burden at bactericidal concentrations that were well tolerated by THP-1–derived macrophages.

Conclusions

Systematic screening identified valepotriate as a rapidly bactericidal antimycobacterial lead compound with activity against both drug-susceptible and drug-resistant M. tb, highlighting its potential as a natural product-derived scaffold for developing new antimycobacterial agents and motivating further optimization of its antibacterial potency.

Introduction

The emergence and dissemination of multidrug-resistant (MDR) Mycobacterium tuberculosis (M. tb) have become a critical obstacle to global tuberculosis (TB) control, greatly increasing the burden of morbidity, mortality and treatment failure worldwide.¹ The increasing prevalence of MDR and extensively drug-resistant TB has severely compromised the effectiveness of current chemotherapy regimens. However, the global development of novel antimycobacterial drugs remains limited, and progress in antimycobacterial drug discovery has been relatively slow.² Although previous studies have identified antimycobacterial candidates through screening of FDA-approved drug libraries, the discovery of novel antimycobacterial compounds from natural product libraries has been less extensively explored.^3,4 Given the rapid expansion of natural compound collections, screening newly identified natural products for antimycobacterial activity represents a promising avenue for the discovery of novel anti-TB agents.

Terpenoids are a large and structurally diverse class of natural compounds that are widely distributed throughout the plant kingdom. Numerous studies have demonstrated that terpenoids possess a broad range of pharmacological activities, including antibacterial, anti-inflammatory, antioxidant, anticancer and neuroprotective effects.^5–7 Several terpenoid-derived compounds have been successfully developed into clinically important drugs, for example, the sesquiterpene artemisinin⁸ and the diterpene paclitaxel.⁹ Our previous studies demonstrated that several naturally derived terpenoid compounds possess inhibitory activity against M. tb.¹⁰ However, whether other terpenoid compounds also exhibit anti-tubercular activity, particularly against drug-resistant M. tb strains, remains largely unknown. In this study, we systematically screened a newly developed terpenoid compound library for antimycobacterial activity and identified valepotriate as a promising lead compound. Valepotriate demonstrated significant inhibitory activity against both drug-susceptible and drug-resistant clinical isolates of M. tb, suggesting its potential for the development of novel therapies against drug-resistant TB.

Materials and methods

Chemicals and reagents

A natural terpenoid compound library containing 354 compounds was purchased from Selleck Chemicals (L1420, Selleck Chemicals, USA). Valepotriate (E2354) and buddlejasaponin IVb (S9074) were obtained from Selleck Chemicals and dissolved in sterile dimethyl sulphoxide (DMSO; Sigma–Aldrich, USA) to prepare concentrated stock solutions. Aliquots were stored at −80°C until use to avoid repeated freeze–thaw cycles. Isoniazid (INH; Sigma–Aldrich, USA) was used as the positive control throughout all extracellular antimycobacterial assays.

Mycobacterial strains and culture conditions

The laboratory reference strain M. tb H37Rv and a collection of clinical drug-susceptible and drug-resistant isolates, including MDR strains, were obtained from Beijing Chest Hospital, Capital Medical University. Drug susceptibility of all clinical isolates had been confirmed by routine phenotypic drug susceptibility testing. Before each experiment, frozen bacterial stocks were recovered on Middlebrook 7H10 (BD Biosciences, USA) agar supplemented with 10% OADC enrichment (BD Biosciences, USA). Single colonies were transferred into Middlebrook 7H9 broth supplemented with 10% OADC and 0.05% Tween 80 (Sigma–Aldrich, USA) and incubated at 37°C until reaching the mid-logarithmic growth phase.

Screening of the terpenoid compound library

The antimycobacterial activity of the terpenoid compound library was evaluated using a 96-well microplate screening assay. Each compound was diluted in Middlebrook 7H9 medium to a final concentration of 40 μM. The bacterial culture was grown to an optical density at 600 nm (OD₆₀₀) of 0.6, diluted 20-fold in fresh medium. Subsequently, an equal volume of M. tb suspension was added to each well to obtain a final volume of 200 μL. Following incubation at 37°C for 14 days without shaking, bacterial growth was quantified by measuring OD₆₀₀ using a microplate reader. The wells containing 7H9 medium alone served as blank controls, whereas INH-treated wells were included as positive controls. Compounds exhibiting marked inhibition of bacterial growth relative to the vehicle control were selected for subsequent validation. All compounds were tested in triplicate.

Determination of minimum inhibitory concentration and minimum bactericidal concentration

The extracellular activity of valepotriate and buddlejasaponin IVb against M. tb was determined using the microplate Alamar Blue assay as previously described.¹¹ Briefly, 2-fold serial dilutions of valepotriate were prepared in Middlebrook 7H9 broth to obtain final concentrations ranging from 0.625 to 80 μM. The bacterial culture was adjusted to an OD₆₀₀ of 0.3 with fresh Middlebrook 7H9 broth supplemented with OADC, followed by a 20-fold dilution using the same medium. Then equal volumes of bacterial suspension were added to each well to achieve a final assay volume of 200 μL. After incubation at 37°C for 7 days, Alamar Blue working solution containing 20 μL of AlamarBlue^™ HS Cell Viability Reagent (Bio-Rad, USA) and 50 μL of 0.05% Tween 80 was added to each well, and the plates were further incubated until colour development. The minimum inhibitory concentration (MIC) was defined as the lowest drug concentration completely preventing the colour transition of Alamar Blue from blue to pink. To determine the minimum bacterial concentration (MBC), bacterial suspensions treated with different concentrations of valepotriate were serially diluted and plated onto Middlebrook 7H10 agar. After incubation for approximately 3 weeks, cfu were counted, and the inhibitory activity at each drug concentration was expressed as the percentage reduction in viable bacterial counts relative to the initial inoculum and calculated using the following formula: inhibition rate = [1 − (cfu treatment/cfu original)] × 100%. Bactericidal activity was determined based on the MBC/MIC ratio, with compounds exhibiting an MBC/MIC ratio of ≤4 classified as bactericidal and those with a ratio of > 4 classified as bacteriostatic.¹²

Time–kill kinetic assay

The bactericidal kinetics of valepotriate were evaluated using a time–kill assay. Exponentially growing M. tb H37Rv cultures (∼5×  10⁵ cfu/mL) were exposed to valepotriate at concentrations of 1/2×, 1×, 2×, 4×, 8× and 16 × MIC. At designated timepoints, aliquots were collected, serially diluted and plated on Middlebrook 7H10 agar for colony enumeration after 0, 1, 3, 6 and 10 days of compound treatment. The cfu were determined after incubation, and bacterial killing curves were generated by plotting log₁₀ cfu/mL against time to evaluate concentration- and time-dependent bactericidal activity.

Antimycobacterial activity against drug-resistant clinical isolates

To investigate whether drug resistance affected the antibacterial efficacy of valepotriate, MIC values were determined against a panel of clinical drug-resistant M. tb isolate strains, including MDR strains, using the same microplate Alamar Blue assay described above. The susceptibility profiles of valepotriate were compared among drug-susceptible and drug-resistant isolates to evaluate its spectrum of antimycobacterial activity.

Cytotoxicity assay

Human THP-1 monocytes were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA). To generate macrophages, cells were treated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma, USA) for 48 h, followed by a 12 h recovery period in PMA-free medium. Cell viability following valepotriate treatment was evaluated using the Cell Counting Kit-8 (CCK-8, TargetMol, China). THP-1–derived macrophages, either infected with M. tb or uninfected, were incubated with different concentrations of valepotriate for 48 h before the addition of CCK-8 reagent. Absorbance at 450 nm was measured using a microplate reader, and cell viability was expressed relative to vehicle-treated controls.

Intracellular antimycobacterial assay

Differentiated THP-1 macrophages were infected with M. tb at a multiplicity of infection of 5. Following bacterial uptake, extracellular bacilli were removed by washing three times with phosphate-buffered saline. Then, the cells were treated with fresh culture medium containing different concentrations of valepotriate (5 and 10 μM) for an additional 72 h, and the infected macrophages were subsequently analysed at the indicated timepoints. Cells were lysed with 0.1% Triton X-100, and intracellular bacterial viability was assessed by determining cfu through plating serial dilutions of cell lysates onto Middlebrook 7H10 agar plates and incubating them for 2–3 weeks at 37°C. Colony-forming units were determined after incubation to assess intracellular bactericidal activity.

Statistical analysis

All experiments were performed independently at least three times. Data were analysed using GraphPad Prism software and expressed as mean ± standard deviation (SD). Statistical significance between groups was determined using Student's t-test or one-way ANOVA, with P < 0.05 considered statistically significant.

Results

Screening of terpenoid compounds against M. tb

To identify natural terpenoid compounds with antimycobacterial activity, we screened a commercially available terpenoid compound library (Selleck Chemicals) containing 354 structurally diverse compounds using an in vitro antimycobacterial assay. The library comprised 38 monoterpenes, 96 sesquiterpenes, 66 diterpenes, 137 triterpenes, 5 tetraterpenes and 12 other terpenoid compounds. The M. tb H37Rv was cultured with each compound at a final concentration of 20 μM for 14 days, and antimycobacterial activity was subsequently evaluated by measuring the optical density at 600 nm (OD₆₀₀), identifying 10 compounds that reduced bacterial growth to levels comparable to those of the negative control [Figure 1; Table S1 (available as Supplementary data at JAC-AMR Online)]. Notably, the antimycobacterial activities of these identified compounds were comparable to that of INH, showing an almost identical bactericidal effect against M. tb. In addition, we identified 99 compounds in the terpenoid library that exhibited moderate antimycobacterial activity, inhibiting M. tb growth by 30%–70% (Figure 1). Importantly, the antimycobacterial activities of several identified compounds, including celastrol,¹³ parthenolide,¹⁴ oridonin,¹⁵ β-thujaplicin, tanshinone IIA,¹⁶ tanshinone, dihydrotanshinone I and cryptotanshinone,¹¹ have been previously reported in independent studies, further supporting the reliability and robustness of our screening platform. Together, based on the primary screening results, valepotriate and buddlejasaponin IVb were selected as the novel candidate compounds for subsequent experimental validation and comparative analyses.

Figure 1.

Primary screening of a terpenoid compound library against M. tb H37Rv. M. tb H37Rv was cultured in Middlebrook 7H9 medium supplemented with OADC and exposed to individual terpenoid compounds at a final concentration of 20 μM for 14 days. Bacterial growth was assessed by OD₆₀₀ measurement, and compounds showing marked growth inhibition were identified as primary hits. Untreated bacteria served as the negative control, whereas INH-treated bacteria served as the positive control. Data represent the mean of three independent experiments.

Extracellular antimycobacterial activity of valepotriate

To validate the screening results obtained from the compound library and determine the MICs of valepotriate and buddlejasaponin IVb, we further evaluated their antimycobacterial activities using a microplate-based assay with Alamar Blue as the viability indicator. The results showed that valepotriate exhibited potent inhibitory activity against extracellular M. tb growth (Figure 2a), whereas buddlejasaponin IVb showed relatively weaker inhibitory activity (Figure S1). Notably, valepotriate showed the most potent inhibitory activity against M. tb, with an MIC value of 10 μM, whereas buddlejasaponin IVb exhibited relatively weaker activity, with an MIC of at least 40 μM. Then, we next assessed the bactericidal activity of valepotriate by determining its MBC. As shown in Figure 2(b), valepotriate exhibited concentration-dependent bactericidal activity against M. tb, with an MBC of 20 μM (Figure 2b). Consistent with the MIC results, the MBC/MIC ratio was 2, indicating that valepotriate acts as a bactericidal compound under the tested conditions.

Figure 2.

In vitro antimycobacterial activity of valepotriate against M. tb H37Rv, including the determination of MIC and MBC. Serial 2-fold dilutions of valepotriate (0.625–80 μM) were tested against M. tb H37Rv cultured in Middlebrook 7H9 broth supplemented with OADC. MIC values were determined using the microplate Alamar Blue assay, with the MIC defined as the lowest concentration preventing the colour change from blue to pink (a). MBC values were determined by plating bacterial cultures after drug exposure and quantifying viable bacteria by cfu (b). Untreated bacteria served as the drug-free control. Data represent the mean from at least three independent experiments.

Time–kill kinetics of valepotriate against M. tb

To further characterize the bactericidal activity of valepotriate, we performed time–kill kinetic assays. As shown in Figure 3, valepotriate exhibited rapid and concentration-dependent bactericidal activity against M. tb. At 4× MIC, treatment reduced the bacterial burden by more than 2 log₁₀ cfu within 24 h, whereas complete bacterial eradication was achieved within 1 day at 8× MIC and 16× MIC. Notably, the bactericidal activity was both concentration and time dependent. Increasing drug concentrations markedly accelerated bacterial killing, with complete eradication achieved within 1 day at 8× and 16× MIC, whereas treatment at 2× and 4× MIC required more than 10 days to achieve complete bacterial clearance. Collectively, these findings demonstrate that valepotriate is a potent bactericidal compound with rapid and sustained antimycobacterial activity against M. tb.

Figure 3.

Time–kill kinetics of valepotriate against M. tb H37Rv. Bactericidal activity of valepotriate against M. tb H37Rv cultured in Middlebrook 7H9 medium supplemented with OADC for 10 days. Bacteria were treated with valepotriate at concentrations ranging from 1/2×  to 16× MIC, and viability was determined by cfu. Untreated bacteria served as the drug-free control. Data represent the mean ± SD of at least three independent experiments.

Activity against drug-resistant clinical isolates

To further evaluate the therapeutic potential of valepotriate against diverse drug-resistant phenotypes, we assessed its in vitro antimycobacterial activity against 14 clinical isolates of M. tb, including multidrug-resistant tuberculosis, rifampicin-resistant tuberculosis and isoniazid-resistant tuberculosis isolates as previously descripted.¹¹ The MIC values of valepotriate, together with those of standard anti-TB drugs, are summarized in Table 1. Notably, valepotriate exhibited potent and consistent inhibitory activity against all tested clinical isolates, irrespective of their resistance profiles to first-line and second-line anti-TB drugs. Among these isolate strains, valepotriate displayed remarkably low MIC values, ranging from 5 to 20 μM in the majority of strains, indicating strong activity against drug-resistant M. tb. Importantly, the antimycobacterial efficacy of valepotriate was not affected by the resistance patterns of the clinical isolates. Notably, several drug-resistant clinical isolates exhibited susceptibility to valepotriate at concentrations comparable to those required for drug-sensitive strains.

Table 1.

In vitro MICs of valepotriate and anti-TB drugs against clinical isolates of M. tb with different drug resistance profiles

Strains	Valepotriate (μM)	First-line drugs (mg/L)				Rifamycins (mg/L)		Fluoroquinolones (mg/L)		Second-line injectable drugs (mg/L)			Other oral drugs (mg/L)
	VAL	INH	RIF	EMB	SM	RFT	RFB	LVX	MXF	AMK	CPM	KAN	PTO	PAS	CLF	PI	PNB	CLR
MDR1	5	1.6	2	S	S	2	2	1	0.25	S	S	S	S	S	S	0.5	S	S
MDR2	10	1.6	2	5	S	2	2	1	0.25	S	S	S	S	S	S	2	S	S
MDR3	10	1.6	2	5	S	2	0.5	4	1	S	S	S	S	S	S	2	S	S
RR1	20	S	2	S	S	2	0.5	S	S	S	S	S	S	S	S	S	S	S
RR2	20	S	2	S	S	2	2	S	S	S	S	S	S	S	S	S	S	S
RR3	20	S	2	S	S	2	2	S	S	S	S	S	S	S	S	S	S	S
HR1	10	1.6	S	S	8	S	S	S	S	S	S	S	S	S	S	0.5	S	S
HR2	20	1.6	S	S	8	S	S	S	S	S	S	S	S	S	S	2	S	S
HR3	10	1.6	S	S	S	S	S	S	S	S	S	S	S	S	S	0.6	S	S
S1	10	S	S	S	S	S	S	S	S	S	S	S	S	S	S	S	S	S
S2	20	S	S	S	S	S	S	S	S	S	S	S	S	S	S	S	S	S
S3	5	S	S	S	S	S	S	S	S	S	S	S	S	S	S	S	S	S
S4	20	S	S	S	S	S	S	S	S	S	S	S	S	S	S	S	S	S
S5	20	S	S	S	S	S	S	S	S	S	S	S	S	S	S	S	S	S

MDR, multidrug-resistant (resistant to isoniazid and rifampicin); RR, rifampicin-resistant; HR, isoniazid-resistant; S, susceptible to all test drugs (the test drugs can inhibit H37Rv based on proportion method); INH, isoniazid; RIF, rifampicin; EMB, ethambutol; SM, streptomycin; RFT, rifapentine; RFB, rifabutin; LVX, levofloxacin; MXF, moxifloxacin; AMK, amikacin; CPM, capreomycin; KAN, kanamycin; PTO, protionamide; PAS, para-aminosalicylic acid; CLF, clofazimine; PI, isoniazid para-aminosalicylate; PNB, p-nitrobenzoic acid; CLR, clarithromycin.

Cytotoxicity and intracellular killing activity of valepotriate in THP-1–derived macrophages

To further characterize the intracellular anti-TB activity of valepotriate, we first evaluated its cytotoxicity in both uninfected and M. tb-infected THP-1–derived macrophages. Cells were treated with valepotriate at a 2-fold serial concentration range of 1.25–40 μM for 48 h, covering concentrations relevant to its in vitro antimycobacterial activity. As shown in Figure 4(a and b), valepotriate exhibited minimal cytotoxicity in both uninfected and M. tb-infected THP-1 macrophages. The cytotoxicity profiles were comparable between infected and uninfected cells, suggesting that M. tb infection did not significantly alter the cellular response with the treatment of valepotriate. Specifically, valepotriate at concentrations up to 40 μM did not significantly reduce cell viability in either uninfected or infected macrophages compared with the DMSO control. These results indicate that Valepotriate has a favourable safety profile at concentrations exhibiting antimycobacterial activity.

Figure 4.

Cytotoxicity and intracellular bactericidal activities of valepotriate against M. tb in THP-1 macrophages. (a) Viability of uninfected- and M. tb-infected THP-1–derived macrophages after treatment with valepotriate (1.25–40 μM) for 48 h. (b) THP-1–derived macrophages infected with M. tb H37Rv were treated with valepotriate (5 and 10 μM) for 48 and 72 h, and intracellular bacterial burden was quantified by cfu assay after cell lysis (n = 3). Significant differences compared to the untreated control group at each timepoint are indicated: ***P < 0.001 (one-way ANOVA).

Given that M. tb is an intracellular pathogen, we next evaluated the ability of valepotriate to inhibit intracellular bacterial survival in M. tb-infected THP-1 macrophages. Following valepotriate treatment, intracellular bacterial loads were determined by cfu enumeration at both 48 and 72 h post-treatment (Figure 4c). Valepotriate treatment resulted in a significant reduction of intracellular M. tb burden compared with the negative control at 48 and 72 h post-treatment. Notably, valepotriate treatment at 5 μM exhibited a sustained inhibitory effect on intracellular M. tb survival, with comparable cfu reductions observed at both 48h and 72 h. In contrast to the progressive bacterial growth observed in the DMSO-treated control group, valepotriate-treated macrophages maintained intracellular M. tb burden at a consistently lower level throughout the treatment period, suggesting a durable intracellular antimycobacterial activity. Collectively, these findings demonstrate that valepotriate effectively restricts intracellular M. tb survival in macrophages without host-cell toxicity, supporting its potential as a promising anti-TB candidate.

Discussion

In this study, we systematically screened a structurally diverse terpenoid library containing 354 natural terpenoid compounds and identified valepotriate as a promising antimycobacterial candidate, while the broad range of growth inhibition observed across the library further highlights terpenoids as a valuable source of anti-TB lead compounds. Subsequent characterization demonstrated that valepotriate exhibited potent extracellular bactericidal activity with rapid concentration-dependent killing kinetics and retained strong activity against clinically drug-resistant M. tb isolates, including MDR-, RR- and INH-resistant strains. Furthermore, valepotriate effectively restricted intracellular M. tb survival in macrophages with minimal host-cell toxicity. Collectively, these findings identify valepotriate as a promising lead compound for the development of new anti-TB therapeutics.

Terpenoids represent one of the largest and most chemically diverse groups of bioactive natural products. Several terpenoid compounds have previously demonstrated antimycobacterial activity or inhibitory effects against M. tb, highlighting the potential of this chemical class for TB drug discovery.^11–15 Consistent with previous reports, our screening platform successfully identified more than 100 terpenoid compounds with reported antimycobacterial activity, further validating the reliability of our discovery strategy. Among the identified candidates, valepotriate, an iridoid-type monoterpene ester derived from Valeriana species, exhibited the strongest antimycobacterial activity. Valeriana officinalis contains two major groups of bioactive constituents, including volatile sesquiterpene oils and valepotriate.¹⁷ Previous studies have shown that valerenic acid, a representative sesquiterpene constituent of Valeriana species, exhibits antimicrobial activity against various microorganisms, including Helicobacter pylori, Staphylococcus aureus and Candida albicans.^18,19 However, to our knowledge, this is the first study demonstrating that valepotriate possesses direct bactericidal activity against M. tb. Importantly, valepotriate exhibited comparable inhibitory and bactericidal effects against both drug-sensitive and clinically resistant M. tb isolates, indicating that its activity is largely unaffected by existing resistance mechanisms associated with current anti-TB drugs. The time–kill assays in this study were conducted under static drug exposure conditions, in which valepotriate concentrations remained constant over time, unlike the fluctuating drug concentrations encountered in vivo.²⁰ Importantly, despite this limitation, valepotriate exhibited rapid concentration-dependent bactericidal activity, with transient exposure to high drug concentrations achieving complete eradication of M. tb. Notably, these bactericidal concentrations were well tolerated by THP-1–derived macrophages, indicating a favourable therapeutic window. Together, these findings suggest that maximizing peak drug exposure may be an effective strategy for enhancing the antimycobacterial efficacy of valepotriate while maintaining acceptable host-cell safety. Future PK/PD studies are warranted to determine whether this exposure profile can be translated into effective dosing regimens in vivo. Although its current effective concentrations remain higher than those of established anti-TB drugs, the relatively simple iridoid scaffold provides opportunities for further medicinal chemistry optimization to improve antibacterial potency, metabolic stability and pharmacokinetic properties.

Beyond its extracellular antibacterial activity, the ability of valepotriate to restrict intracellular M. tb survival provides additional support for its therapeutic potential. Macrophages represent the primary intracellular niche of M. tb, and compounds capable of efficiently limiting intracellular bacterial replication are highly desirable for TB treatment.²¹ Notably, valepotriate maintained significant intracellular antibacterial activity at one-half of its extracellular MIC concentration, resulting in sustained suppression of intracellular bacterial burden without detectable host-cell toxicity. This observation suggests that mechanisms beyond direct extracellular bacterial killing may contribute to its intracellular efficacy. Increasing evidence indicates that natural products can influence host antimicrobial defences by inducing inflammatory signalling,²² oxidative stress responses,²³ autophagy²⁴ and lysosomal function.^25,26 In this context, previous studies have shown that valepotriate-rich extracts suppress inflammatory leucocyte migration in a concentration-dependent manner, exhibiting anti-inflammatory and antinociceptive effects comparable to diclofenac sodium.²⁷ Furthermore, valepotriate derivatives can modulate intracellular reactive oxygen species dynamics.²⁸ Although these findings were obtained using valepotriate-containing extracts rather than purified valepotriate, they raise the possibility that valepotriate may possess additional host-associated immunomodulatory properties beyond its direct antimycobacterial activity. Further investigation will be required to determine whether purified valepotriate contributes to host-directed protective mechanisms during M. tb infection.

Nevertheless, several limitations of this study should be considered. Firstly, although valepotriate showed potent antimycobacterial activity in vitro, its clinical potential remains to be confirmed. Future studies should evaluate its therapeutic efficacy and safety in animal models and test a larger, more geographically diverse panel of clinical isolates. Secondly, the precise molecular target and antibacterial mechanisms of valepotriate remain unclear, requiring further investigation. Although the molecular target responsible for valepotriate-mediated antimycobacterial activity remains unknown, previous studies have suggested that valepotriate-related compounds can influence cytochrome P450 (CYP450)-dependent metabolic pathways in mice by inhibiting methacetin metabolism, indicating their potential interaction with CYP450-associated enzymes.²⁹ CYP450 enzymes in M. tb, which participate in essential biological processes, have been proposed as potential targets for anti-TB drug development.³⁰ Although there is currently no direct evidence that valepotriate inhibits mycobacterial CYP450 enzymes, these observations provide a potential mechanistic hypothesis that warrants further investigation. Thirdly, comprehensive pharmacokinetic and toxicity studies are required to assess the translational potential of valepotriate, particularly given its potential chemical instability and rapid enzymatic hydrolysis, which may limit its suitability for oral administration. Furthermore, as a small-molecule natural product, valepotriate may be suitable for combination formulations with existing anti-TB drugs to achieve synergistic antibacterial effects, reduce treatment duration and minimize the emergence of resistance. Furthermore, incorporation into advanced drug-delivery platforms, such as nanoparticle-based formulations, may improve its solubility, stability, intracellular accumulation and targeted delivery to infected tissues. Collectively, these findings establish valepotriate as a natural product-derived anti-TB lead compound with both direct antimycobacterial activity and potential host-modulatory properties.

Transparency declarations

All authors declare no competing interests.

Author contributions

C. Z. and L. P. were responsible for the study design. X. W., X. Z. and C. Z. contributed to data collection, data analysis and visualization. H. J., L. Z. and Z. Z. contributed to data analysis. The draft of the manuscript was written by C. Z. and revised by L. P. and X. W. All authors have reviewed and approved the final manuscript. The authors declare that all data were generated inhouse and that no paper mill was used.

Funding

This study was supported by Beijing Natural Science Foundation (7242025), the National Science and Technology Major Project of China (2025ZD01900903, 2025ZD01903603, 2025ZD01903600), National Natural Science Foundation of China (32394013), and the Beijing High-level Public Health Project (G2024-2–007).

Supplementary data

Figure S1 and Table S1 are available as Supplementary data at JAC-AMR Online.