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Herbal Extract-Induced DNA Damage, Apoptosis, and Antioxidant Effects in C. elegans: A Comparative Study of M. longifolia, S. orientalis, and E. biebersteinii

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31 May 2025

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02 June 2025

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Abstract
Background: Medicinal herbs are increasingly used as functional foods and therapeutics, yet their biological effects and potential toxicity remain incompletely understood. Using Caenorhabditis elegans, we previously identified several herbal extracts with potent cytotoxic effects. Methods: We focused on three herbal extracts—Mentha longifolia, Scrophularia orientalis, and Echium biebersteinii—to investigate their impact on germline development and fertility. Phenotypic analyses, apoptosis quantification, qRT-PCR, and LC-MS profiling were performed to assess cytotoxicity, meiotic defects, DNA damage responses, and chemical composition. Results: All three extracts significantly reduced worm survival, induced larval arrest, and increased the high incidence of males phenotype, suggesting chromosomal mis-segregation. Germline defects included disorganized nuclei, altered meiotic progression, and reduced bivalent formation. These were accompanied by upregulation of DNA damage checkpoint genes, increased pCHK-1 foci, and elevated apoptosis in the pachytene region. We identified 21 major compounds in three herb extracts, with four shared across all extracts. Among them, thymol and carvyl acetate activated checkpoint responses and apoptosis, while thymol and luteolin-7-O-rutinoside exhibited antioxidant activity. Conclusion: These findings emphasize the value of dissecting complex herbal mixtures to uncover specific bioactive compounds, which is essential for evaluating both the therapeutic potential and safety risks of medicinal plants. Also, this study underscores the need to analyze individual phytochemicals within herbal mixtures to understand their distinct biological effects. Such insight is essential for evaluating both the therapeutic potential and safety risks of medicinal plants used in food and supplements.
Keywords: 
Mentha longifolia
;  
Scrophularia orientalis
;  
and Echium biebersteinii
;  
DNA repair
;  
meiosis
;  
germline development
;  
medicinal plants
Subject: 
Biology and Life Sciences  -   Cell and Developmental Biology

Introduction

While medicinal plants have long been used in traditional remedies, their molecular effects—particularly on genome stability and reproductive health—remain poorly understood. This gap is especially significant given the rising use of herbal formulations with uncharacterized toxicological profiles. To address this gap, we investigated three herbal species native to Armenia—Scrophularia orientalis (S. orientalis), Mentha longifolia (M. longifolia), and Echium biebersteinii (E. biebersteinii). All three are known for their strong bioactivity (Figure 1 and Table 1), but their potential roles in DNA damage repair and apoptosis have not been fully validated. We hypothesized that extracts from these herbs may interfere with DNA repair pathways and germline development due to their bioactive components.

1. Scrophularia Orientalis

General Overview

The genus Scrophularia, belonging to the family Scrophulariaceae, comprises approximately 200–300 species distributed primarily across temperate regions of Asia, Europe (particularly the Mediterranean), and North America [1,2]. These plants are characterized by quadrangular, sometimes winged stems and highly variable, undivided to 3-pinnatisect, generally opposite leaves. The fruit is a septicidal capsule, globose to subconical in shape, containing numerous small seeds [3].

Medicinal Properties and Uses

Various Scrophularia species have played a longstanding role in traditional medicine due to their demonstrated antioxidant, anti-tumor, anti-cancer, anti-protozoal, and anti-inflammatory activities [2,4,5]. In particular, S. orientalis has exhibited notable anticancer properties. A dichloromethane extract of this species significantly reduced the viability of neuroblastoma (NB) cells [6]. Similarly, filtered leaf extracts of S. striata have demonstrated anti-proliferative effects on a human astrocytoma cell line [7]. Moreover, extracts from S. floribunda and S. lucida have been shown to induce both necrosis and apoptosis in cancer cells, with S. lucida notably inhibiting tumor cell intravasation into lymph endothelial cells through suppression of NF-κB activity [8].

2. Echium Biebersteinii

General Overview

Echium, a genus in the Boraginaceae family, comprises approximately 60 species native to North Africa, Europe, and the Macaronesian islands [9,10,11]. This genus is closely related to Lobostemon (including Echiostachys), which is endemic to the southwestern Cape region. Although their pollen structures are highly similar—suggesting possible taxonomic convergence—distinct morphological differences separate the two genera. For example, Echium possesses bilobed styles and a unique annulus inside the corolla tube formed by a small collar or tiny hairy lobules, whereas Lobostemon exhibits undivided styles and hairs or scales at the base of the filaments [12].
Although E. biebersteinii has not been extensively studied, other species in the genus, notably E. amoenum, have received considerable pharmacological attention.

Medicinal Properties and Uses

Species of Echium are known for their sedative, anti-inflammatory, antioxidant, and anxiolytic effects. Consequently, they are widely used in traditional medicine to treat respiratory conditions, ulcers, mental health disorders, and for wound healing [10,13,14,15,16,17]. Among these, E. amoenum is the most extensively studied and has been traditionally used since the third century BC. Its applications include managing respiratory ailments, fevers, colds, and mood disturbances. Preparations such as boiling the plant or combining its leaves with wine were historically believed to enhance mood and relieve fever [10].
Although E. biebersteinii is considered a subspecies of E. italicum and remains underexplored, E. italicum has been traditionally employed in Turkey for wound healing, blister treatment, and relief of rheumatic pain through topical applications and infusions [10,18]. This species has also demonstrated strong anti-inflammatory, antioxidant, and anxiolytic activities [10,13,14,15,16,17]..
Recent studies have highlighted E. amoenum’s potential in cancer therapy. Its active compound, rosmarinic acid, has been shown to inhibit the growth and metastasis of gastric cancer cells through the inactivation of STAT3, AKT, and ERK1/2 signaling pathways [19].

3. Mentha Longifolia

General Overview

The genus Mentha (mint; Lamiaceae) includes between 18 and 30 species [20]. M. longifolia (L.) L. is an aromatic perennial herb widely distributed across Northern Pakistan, Europe, Nepal, India, western China, Germany, the United Kingdom, Egypt, Nigeria, and Turkey [21]. Members of the Mentha genus are herbaceous plants with spreading stolons both above and below ground [22]. Their branched stems are square in cross-section, and their aromatic leaves are oppositely arranged with either serrated or entire margins, typically oblong-elliptical to lanceolate in shape [23].

Medicinal Properties and Uses

Traditionally, M. longifolia has been used to treat gastrointestinal ailments, respiratory infections, and inflammatory diseases. Its essential oil, particularly menthol, exhibits antimicrobial properties against bacteria such as Staphylococcus aureus and Streptococcus mutans, as well as antifungal activity against Candida albicans [24]. Additionally, its flavonoid content has been suggested to possess anti-HIV potential [25].
Antioxidant activity has also been reported, with significant DPPH radical-scavenging and inhibition of linoleic acid oxidation observed in various assays [26]. Furthermore, crude extracts of M. longifolia have demonstrated antiproliferative effects in adrenocortical carcinoma SW13 cells, supporting its potential as an anti-cancer agent [27].
Taken together, the Scrophularia, Echium and Mentha species illustrate the remarkable pharmacological diversity found across different botanical families. Despite their phylogenetic divergence, these plants share a spectrum of bioactivities, including antioxidant, anti-inflammatory, cytotoxic, anti-proliferative, pro-apoptotic, and antimicrobial effects. These overlapping properties suggest that common molecular mechanisms may underpin the phenotypes observed in our study.
Despite the documented bioactivities of these herbs, their effects on DNA damage and reproductive function remain largely unexplored. To explore this hypothesis, we investigated the biological activity of the three herbal species using the Caenorhabditis elegans (C. elegans) model system. This model provides a powerful in vivo platform for studying the effects of bioactive compounds on development, reproduction, and genomic stability.
Plant-specific solvents were used to prepare the extracts—E. biebersteinii with butanol, M. longifolia with DMSO, and S. orientalis with water. All extracts were subsequently resuspended in a standardized DMSO–water mixture to ensure consistency in treatment conditions.
Comparative analysis revealed that exposure to each of the three herbal extracts significantly reduced worm survival compared to untreated controls. Treated worms exhibited larval arrest or lethality, suggesting that impaired survival may be linked to disruptions in mitotic cell division during larval development. Notably, all three extracts induced a high incidence of male progeny (HIM phenotype), implying disruption of sex chromosome segregation and potential interference with meiotic processes.
Further analysis revealed a reduced number of DAPI-stained bodies and abnormal meiotic progression in the germline of treated worms, providing additional evidence for impaired meiotic development. Consistently, treatment with any of the three extracts activated the DNA damage checkpoint response via the ATM/ATR and CHK-1 pathways. This response was accompanied by defective germline development, indicating that the extracts interfere with DNA damage repair mechanisms and ultimately lead to fertility defects.
To elucidate the molecular basis of these phenotypes, we performed LC-MS analysis of the herbal extracts. Several shared components—luteolin-7-O-rutinoside, thymol, carvyl acetate, and menthyl acetate—were identified, each has been previously associated with oxidative stress regulation, apoptosis induction, or genotoxic effects. These compounds are likely contributors to the observed disruptions in worm development and reproduction.
Interestingly, S. orientalis and E. biebersteinii shared 79% of their major compounds, indicating a high degree of chemical similarity. In contrast, M. longifolia shared only 42% of its compounds with the other two and possessed seven unique compounds (58%), reflecting a more distinct chemical profile. These differences may underlie the variable biological responses observed in the C. elegans assays and suggest plant-specific mechanisms of action.
This study reveals that extracts from Mentha longifolia, Scrophularia orientalis, and Echium biebersteinii induce reproductive defects in C. elegans by activating DNA damage checkpoints and apoptotic pathways. High-resolution imaging of germline architecture linked structural abnormalities—such as disorganized nuclei, impaired meiotic progression, and reduced bivalent formation—to molecular stress responses. All three extracts significantly decreased survival, caused larval arrest, and increased the high incidence of males (HIM) phenotype, indicating chromosomal mis-segregation.
We identified 21 major compounds, including four shared across the extracts. Among them, thymol and carvyl acetate were associated with pro-apoptotic activity, while thymol and luteolin-7-O-rutinoside exhibited antioxidant effects. These findings highlight both conserved and compound-specific mechanisms of herbal reproductive toxicity and support the use of C. elegans as a model for functional toxicological screening of traditional remedies. Also, this study underscores the need to analyze individual phytochemicals within herbal mixtures to understand their distinct biological effects.

Materials and Methods

Strains and Alleles

C. elegans strains were cultured at 20°C under standard laboratory conditions, following established protocols [28]. The N2 Bristol strain, used as the wild-type control, was obtained from the Caenorhabditis Genetics Center (CGC).

Herb Extraction

Herbal materials were sourced from Armenia and processed as previously reported [29,30]. In summary, plant samples were washed, air-dried, and coarsely ground before undergoing methanol extraction. The resulting methanolic extract was concentrated, reconstituted in 90% aqueous methanol, and partitioned with n-hexane. The hydroalcoholic residue was further fractionated sequentially using dichloromethane and n-butanol, yielding hexane-, butanol-, and water-soluble fractions. Solvent selection was tailored to each plant species: E. biebersteinii was extracted using butanol, M. longifolia with DMSO, and S. orientalis with water. All hexane-based extracts were redissolved in DMSO and standardized to 1 mg/mL before being diluted in M9 buffer to a working concentration of 0.03 µg/mL for most assays, unless otherwise noted.

Survival, Larval Arrest/Lethality, and High Incidence of Males (HIM) Assay

Synchronized L1 larvae were prepared by collecting gravid hermaphrodites from NGM plates, using the method described by Kim and Colaiacovo [31,32]. The larvae were then exposed to 180 µL of herbal extract solution in 96-well plates. Following brief agitation, the plates were incubated at 20°C for 24 hours, with phenotypic observations extending up to 48 hours. Worm survival was determined based on movement after 24 hours of treatment. Brood size was calculated by counting the total number of eggs laid per worm over a 4–5 day period following the L4 stage. Larval arrest or lethality was expressed as the percentage of hatched larvae that failed to reach adulthood. The High Incidence of Males (HIM) phenotype was assessed by calculating the percentage of males among the adult population. Differences among genotypes were analyzed using the two-tailed Mann–Whitney test, applying a 95% confidence interval (C.I.). Each experiment was independently replicated three times to ensure consistency. This procedure was modified from the protocol established by Kim and Colaiacovo [31].

LC–MS/MS Analysis

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) was carried out according to established protocols [29,30]. Briefly, the analysis was conducted using a Shimadzu LC-30A system equipped with a C18 column, with all procedures performed by YanBo Times (Beijing, China). Compound identification was verified through comparison with a standardized reference database. All detected compounds were authenticated through this stringent methodology. The English names in the LC–MS output were translated from the original Chinese names supplied by YanBo Times.

Immunofluorescence Assay

Whole-mount gonads were stained for immunofluorescence following previously described methods [32,33,34]. The primary antibody used was rabbit anti-phospho-CHK-1 (Ser345) at a 1:250 dilution (Cell Signaling Technology), followed by Cy3-conjugated anti-rabbit secondary antibody at a 1:300 dilution (Jackson). Fluorescent images were captured using a Nikon Eclipse Ti2-E inverted microscope paired with a DS-Qi2 camera. Imaging was conducted at 0.2 μm Z-steps using a 60x objective lens with an additional 1.5x magnification. Image processing and deconvolution were performed using Nikon NIS Elements software. Figures display either full or partial nuclear projections.

pCHK-1 Foci Quantification

The number of pCHK-1 foci was quantified following established protocols ([32,34]). For each condition, five to ten germlines were examined. Statistical analysis was performed using either a two-tailed Mann–Whitney U test or a standard T-test, applying a 95% confidence interval.

Assessment of Germline Apoptosis

Germline apoptosis was evaluated via acridine orange staining in synchronized animals, 20 hours after reaching the L4 stage, as previously described [35]. Between 20 and 30 gonads were scored per condition using a Nikon Ti2-E fluorescence microscope. Statistical comparisons were conducted using the two-tailed Mann–Whitney test, with significance set at a 95% confidence level.

qRT-PCR

Total RNA was isolated from young adult hermaphrodites and reverse-transcribed into cDNA using the ABscript II First Strand Synthesis Kit (ABclonal, RK20400). qRT-PCR was carried out using ABclonal 2X SYBR Green Fast Mix (RK21200) on the LineGene 4800 system (BIOER, FQD48A). Thermal cycling conditions included an initial denaturation at 95°C for 2 minutes, followed by 40 amplification cycles at 95°C for 15 seconds and 60°C for 20 seconds, with extension. A melting curve analysis (60°C–95°C) was performed to confirm product specificity. The tba-1 gene, which encodes tubulin, was used as an internal reference, based on previously published C. elegans microarray data. All PCR reactions were repeated at least twice to ensure reproducibility.

Results

All three plant extracts exhibited potent nematocidal activity after 48 hours of treatment at 20 °C, with survival rates ranging from 24% to 38%, compared to 89.4% in the DMSO-treated control group (Figure 2A). In addition to reduced survivability, extract-treated worms exhibited a larval arrest or lethality (93% vs. 38% for DMSO and M.l., p = 0.0002; 93% vs. 29% for S.o., p < 0.0001; 93% vs. 40% for E.b., p < 0.0001; two-tailed t-test), suggesting that decreased viability is likely linked to mitotic growth defects.
Also, all three extracts also significantly increased the incidence of the High Incidence of Males (HIM) phenotype, indicative of potential sex chromosome mis-segregation and aberrant meiotic development (0.29% vs. 3.12% for DMSO and M.l., p = 0.002; 3.47% for S.o., p = 0.0015; 9.82% for E.b., p = 0.0044; two-tailed t-test; [36]).
Since C. elegans feed on E. coli, we tested whether the observed nematocidal effects might result from indirect toxicity due to impaired bacterial growth. However, bacterial growth curves showed no significant changes following treatment with any of the three extracts at 0.03 μg/mL—the same concentration that induced phenotypes in C. elegans—indicating minimal impact on bacterial proliferation (Figure 2B). After 24 hours of incubation, the OD600 values were comparable across groups: 0.12 for DMSO + E. coli, 0.13 with M.l., 0.13 with S.o., and 0.14 with E.b.
In C. elegans, germline nuclei are organized in a well-defined spatial and temporal pattern during germline development. Actively dividing mitotic nuclei are located at the distal end within the premeiotic tip (PMT), and as cells move proximally, they enter meiotic prophase, beginning at the transition zone (TZ), where nuclei display a characteristic crescent-shaped morphology [36]. To assess effects on germline architecture, adult hermaphrodites were dissected, DAPI-stained, and analyzed. In controls, germline nuclei maintained orderly progression from the premeiotic tip (PMT) through the transition zone (TZ) to the pachytene region (Figure 3A, 3B). However, S.o. and E.b. treatments caused increased nuclear gaps, especially in the pachytene region, while S.o. additionally affected the PMT. In contrast, M.l. had no visible impact on nuclear organization.
Crescent-shaped nuclei, normally restricted to the transition zone (TZ) in controls, appeared ectopically in both the pre-meiotic tip (PMT) and pachytene regions of extract-treated worms (Figure 3C). While control animals showed proper localization of these nuclei to the TZ, all three herbal extracts induced their mislocalization into adjacent germline regions. This mislocalization increased significantly: M.l. (1.2 vs. 1.9, 1.58-fold, p = 0.0103), S.o. (1.2 vs. 2.6, 2.17-fold, p = 0.0649), and E.b. (1.2 vs. 2.0, 1.67-fold, p = 0.0088). These findings suggest premature entry into meiosis and disrupted developmental timing.
At the diakinesis stage, control worms showed the expected six DAPI-stained bivalents, whereas S.o.-treated animals showed five bivalents in 3.8% of cases, indicating potential homologous recombination or synapsis defects (Figure 3D; [37]). No abnormal bivalent numbers were detected in M.l. or E.b. groups.
Proper spatial organization of germline nuclei reflects normal developmental progression, and its disruption is often associated with reduced germline size. A significant decrease in germline length was observed only in worms treated with S. orientalis extract. The TZ and pachytene region lengths decreased from 45 μm to 29 μm (Figure 3E, p = 0.0006) and from 280 μm to 247 μm (p = 0.0175), respectively. No significant changes were observed in the PMT length (60 μm vs. 42 μm, p = 0.0519).
These developmental defects correlated with reduced fertility, as evidenced by a decrease in brood size over four days. The most notable reduction occurred on day 3. S.o.-treated worms showed a 3.08-fold decline in brood size (Figure 3F, 148 to 48, p = 0.0022), while M.l. and E.b. led to 1.59-fold (to 93, p = 0.0022) and 1.44-fold (to 103, p = 0.0050) reductions, respectively. These results suggest that impaired germline development ultimately leads to reduced fertility.
We hypothesized that impaired germline progression would activate the DNA damage checkpoint and initiate DNA repair mechanisms. To determine whether germline disruption was associated with activation of the DNA damage response, we assessed expression of DNA damage checkpoint genes. All three extracts significantly upregulated atm-1 and atl-1 mRNA, two key DNA damage checkpoint kinases: M.l. (Figure 4A, 1.52- and 1.51-fold), S.o. (2.26- and 2.24-fold), and E.b. (1.93- and 1.41-fold); p = 0.0007 for all (Mann–Whitney test).
Consistent with the upregulation of key DNA damage checkpoint genes, an increase in pCHK-1 foci was observed in the pachytene region following treatment with M.l. (1.6 vs. 4.9, p = 0.0049), S.o. (2.6, p = 0.0076), and E.b. (5.9, p = 0.0049) (Figure 4B). Additional pCHK-1 foci appeared in the PMT for M.l. (1.7 vs. 2.3, p = 0.0263) and E.b. (1.7 vs. 6.5, p < 0.0001), but not significantly for S.o. (1.7 vs. 2.0, p = 0.0981).
Activation of DNA damage checkpoint along with meiotic defects would lead to DNA damage mediated Cell death in pachytene stage of germline in C. elegans. In line with this idea, Apoptosis in the pachytene region increased significantly in S.o. (Figure 4C, 1 vs. 2.3, p = 0.0008) and E.b. (1 vs. 2.1, p = 0.0024) treated groups. M.l. induced a mild, non-significant increase (1 vs. 1.7, p = 0.0776). This apoptotic response was especially pronounced in worms treated with Stachys orientalis and Euphorbia biebersteinii, underscoring their stronger detrimental effects on germline integrity.
Among the three, S.o. induced the most pronounced phenotypes—altered nuclear organization, reduced bivalents, shortened germline regions, decreased brood size, and elevated expression of DNA damage markers—prompting further analysis of DNA repair. To further investigate this, we analyzed RAD-51 foci, which mark sites of double-strand break (DSB) repair [32,34]. RAD-51 foci were significantly increased in S.o.-treated worms at both the PMT (Figure 4D, 0.04 vs. 0.12, p = 0.023) and late pachytene stages (0.71 vs. 2.14, p = 0.0028), suggesting impaired double-strand break (DSB) repair (Figure 4D). Although RAD-51 foci levels were mildly increased in the transition zone, early pachytene, mid pachytene, and diplotene stages (0.12 vs. 0.11 in TZ, P = 0.7430; 1.36 vs. 1.64 in early pachytene, P = 0.1443; 4.14 vs. 4.54 in mid pachytene, P = 0.0752; 0.05 vs. 0.04 in diplotene, P = 0.7317), these differences were not statistically significant.
To explore the molecular basis of the distinct phenotypic effects observed in C. elegans, we conducted LC-MS analysis on each of the three herbal extracts, as detailed in our previous report [29,30,38]. This analysis identified 21 major compounds across the extracts (Figure 5), with four—luteolin-7-O-rutinoside, thymol, carvyl acetate, and menthyl acetate—common to all. Mentha longifolia contained the highest number of unique compounds, including caryophyllene, genistein, and ursolic acid, totaling seven unique constituents. Scrophularia orientalis featured one exclusive compound, resveratrol, while Echium biebersteinii uniquely contained vitexin-4'-rhamnoside. These findings highlight both common and unique chemical profiles that may explain the distinct biological activities of the extracts (Table 2).
Since all three herbs produced common phenotypes—upregulation of DNA damage checkpoint regulators and elevated germline apoptosis—we next investigated whether the four shared compounds could contribute to these effects. Specifically, we examined the expression levels of key DNA damage checkpoint genes following treatment with each compound.
Thymol and carvyl acetate significantly upregulated atm-1 and atl-1 (Figure 6A, thymol: 2.0- and 1.7-fold; carvyl acetate: 1.58- and 1.8-fold; P = 0.0005 for all), whereas luteolin-7-O-rutinoside and menthyl acetate had no significant effect (luteolin-7-O-rutinoside: P = 0.5396 for atm-1, P = 0.1870 for atl-1; menthyl acetate: P = 0.6029 for atm-1, P = 0.1459 for atl-1).
To determine whether these compounds also influence germline apoptosis, we quantified DNA damage-induced apoptosis. Consistent with their effects on checkpoint gene expression, thymol and carvyl acetate promoted germline apoptosis (Figure 6B, thymol: 1.33 to 2.47, p < 0.0001; carvyl acetate: 1.3 to 1.7, p = 0.0359). In contrast, luteolin-7-O-rutinoside induced only a marginal, non-significant change (1.05-fold, P = 0.7281), and menthyl acetate showed no effect (P = 0.9797). Thus, thymol and carvyl acetate may mediate the pro-apoptotic effects of the extracts. These findings suggest that among the common constituents, thymol and carvyl acetate may play an active role in DNA damage signaling and apoptosis, thereby contributing to the biological activities of the herb extracts.
Given antioxidant properties associated with these herbs (Figure 1, Figure 5, Supplemental Table 1 and 2), we next assessed their antioxidant capacity using the DPPH radical scavenging assay. All three herb extracts exhibited dose-dependent antioxidant activity, with M. longifolia showing the strongest effect, followed by S. orientalis and E. biebersteinii (Figure 6C). At 3 µg/ml, inhibition percentages were 39.15% for E.b. (P < 0.0001), 30.95% for S.o. (P < 0.0001), and 23.05% for M.l. (P < 0.0001).
To further dissect the contribution of individual compounds, we assessed the antioxidant activity of the four common constituents. Among them, luteolin-7-O-rutinoside and thymol displayed measurable radical-scavenging activity. Luteolin-7-O-rutinoside produced a modest but significant dose-dependent inhibition (Figure 6D, 2.15% at 20 µM, P < 0.0001; 3.17% at 30 µM, P < 0.0001). In contrast, thymol exhibited a much stronger antioxidant effect, reaching 18.05% inhibition at 2 mM and 18.08% at 3 mM (P < 0.0001 for both). Meanwhile, carvyl acetate and menthyl acetate did not show significant antioxidant activity at tested concentrations (carvyl acetate: max 0.37%, P > 0.87; menthyl acetate: max 0.55%, P > 0.13), indicating they are unlikely to contribute to the antioxidant effects of the extracts.

Discussion

Herbal Extracts Induce Germline-Specific DNA Damage Checkpoint Activation and Meiotic Defects in C. elegans

All three herbal extracts—Mentha longifolia (M.l.), Stachys orientalis (S.o.), and Euphorbia biebersteinii (E.b.)—exhibited strong nematocidal activity, reducing viability and inducing developmental arrest in C. elegans. These phenotypes were accompanied by a significant increase in the High Incidence of Males (HIM) phenotype, indicative of X chromosome nondisjunction and activation of DNA damage checkpoint and defective DNA repair (Figure 2, Figure 3 and Figure 4). These effects were not attributable to indirect E. coli-mediated toxicity, as bacterial growth remained unaffected by extract treatment.
Our multi-layered analysis—linking organism-level phenotypes to cellular, genetic, and molecular markers—demonstrates that these herbal extracts induce germline-specific defects through activation of conserved DNA damage checkpoint pathways. This systems-level approach offers a comprehensive view of the reproductive toxicity caused by botanical mixtures.

Herbal Extracts Lead to Defective Mitotic and Meiotic Progression, Impaired DNA Repair, and DNA Damage Checkpoint Activation, Resulting in Germline Apoptosis

DAPI staining of dissected gonads revealed that S.o. and E.b. disrupted the spatial organization of germline nuclei. The presence of crescent-shaped nuclei beyond the transition zone, as well as increased nuclear gaps, suggest premature meiotic entry and impaired control of the mitosis-to-meiosis switch. S.o. treatment additionally disrupted the premeiotic tip (PMT), pointing to broader developmental dysregulation. These morphological disruptions correlate with reduced germline length and decreased fertility.
All three extracts induced transcriptional upregulation of key DNA damage checkpoint regulators—atm-1 and atl-1—with accompanying increases in pCHK-1 foci and germline apoptosis. These effects were particularly pronounced in S.o. and E.b.-treated animals. This suggests that the extracts induce genotoxic stress or replication challenges sufficient to activate the DNA damage response, leading to checkpoint-mediated apoptotic removal of compromised germ cells.
Although M.l. showed milder phenotypes, it still significantly elevated checkpoint gene expression and pCHK-1 foci, indicating that even low-grade germline stress is sufficient to engage surveillance pathways.
Among the three extracts, S.o. produced the most severe phenotypes, including a reduction in diakinesis-stage bivalents, indicative of defective homolog pairing or recombination. Furthermore, RAD-51 foci were significantly elevated in the PMT and pachytene stages following S.o. treatment, suggesting impaired double-strand break (DSB) repair or persistent recombination intermediates. These disruptions likely compound DNA damage signaling, culminating in heightened apoptosis.

Phytochemical Composition Underlies the Biological Activities of Herbal Extracts: Four Common Compounds Identified—Thymol, Carvyl Acetate, Luteolin-7-O-Rutinoside, and Menthyl Acetate.

LC-MS profiling revealed both shared and species-specific compounds across the three extracts. Notably, four compounds—thymol, carvyl acetate, luteolin-7-O-rutinoside, and menthyl acetate—were common to all extractS. of these, thymol and carvyl acetate significantly upregulated atm-1 and atl-1 and increased germline apoptosis, effectively recapitulating the effects of the full extracts. In contrast, luteolin-7-O-rutinoside and menthyl acetate showed no such activity, underscoring the functional specificity of individual phytochemicals. This finding suggests that a subset of shared compounds may mediate the core genotoxic effects observed across all extracts, while species-specific compounds and the combination of compounds may modulate their severity.

Phytochemical Overlap Explains Parallel DNA Damage Responses Induced by S. orientalis and E. biebersteinii Extracts

We next asked whether the phytochemical similarities between extracts could explain their shared phenotypic profiles. Interestingly, S.o. and E.b. exhibited the most phenotypic similarity among the three extracts—manifesting nearly indistinguishable effects on germline disorganization, apoptosis, and checkpoint activation. This similarity is supported by their phytochemical profiles: 12 out of 13 major compounds in S.o. were also found in E.b., suggesting a shared chemical basis for their biological effects.
In addition to the shared compounds, S.o. and E.b. both contain misoprostol and aucubin, which have been linked to modulation of DNA damage and repair pathways. Misoprostol has demonstrated radioprotective effects in mammalian models by mitigating DNA damage-induced apoptosis [39], while aucubin has been implicated in topoisomerase-mediated DNA repair regulation and has shown therapeutic relevance in cancer settings [40]. These compounds may enhance or synergize with the shared DDR-active constituents to produce stronger germline toxicity.
Moreover, resveratrol, uniquely present in S.o., is a well-known polyphenol with multiple pharmacologic activities including promotion of IR-mediated apoptosis [41,42]. Resveratrol has been shown to sensitize tumor cells to radiation and enhance DNA damage-induced apoptosis, and may contribute to the severity of phenotypes seen in S.o.-treated animals.

Uncoupling Antioxidant Activity from Germline Toxicity in Herbal Extracts

While genotoxicity emerged as a major effect of the extracts, we also considered whether antioxidant properties might modulate or counterbalance these effects. All three extracts showed dose-dependent antioxidant activity in DPPH assays, with M.l. being the most potent. However, the genotoxic and apoptotic effects did not correlate with antioxidant capacity. For example, M.l. had the highest antioxidant activity but the mildest phenotypes, while S.o. and E.b. exhibited stronger toxicity despite moderate antioxidant profiles. Among shared compounds, thymol contributed to both antioxidant and pro-apoptotic activity, whereas carvyl acetate induced apoptosis without radical-scavenging effects.
These findings indicate that the biological effects of the extracts cannot be explained solely by oxidative stress modulation. Instead, distinct compounds within each extract exert functionally divergent effects—some activating protective antioxidant pathways, others engaging pro-apoptotic DNA damage signaling.
Our findings reveal that the germline phenotypes and fertility defects observed in C. elegans upon treatment with Mentha longifolia, Stachys orientalis, and Euphorbia biebersteinii extracts are the result of both shared and species-specific phytochemicals. Among the four compounds common to all three extracts, thymol and carvyl acetate specifically induced DNA damage checkpoint activation and pachytene-stage apoptosis, while thymol and luteolin-7-O-rutinoside contributed to antioxidant activity. The identification of carvyl acetate as a potent apoptosis inducer without antioxidant activity highlights its distinct and potentially toxic function. Meanwhile, species-specific constituents—such as ursolic acid and caryophyllene in M. longifolia, or resveratrol in S. orientalis—may contribute additional, non-overlapping biological effects.
Importantly, this study illustrates how the interaction between shared and unique compounds drives the complex and divergent biological outcomes of each herbal extract. By establishing a clear correlation between LC-MS-derived chemical profiles and in vivo physiological effects, we provide a mechanistic framework for understanding how multi-component herbal formulations act in biological systems. Moreover, our results experimentally demonstrate the potential of these herbs to induce reproductive toxicity and DNA damage responses, emphasizing the need for careful evaluation of herbal products, especially those consumed as food or supplements.

Supplementary Materials

The following supporting information can be downloaded at the website of this paper posted on Preprints.org.

Author Contributions

Methodology: Q.M. and H-M.K.; Validation: A.H. and Q.M.; Investigation: A.H., Q.M. and H-M.K.; Reference collection and verification: A.H.; Resources: R.P.B. and H-M.K.; Writing—original draft: H-M.K. and A.H.; Writing—review and editing: R.P.B. and H-M.K.; Proofreading: A.H., Q.M., R.P.B., and H-M.K.; Supervision: H-M.K.; Project administration: H-M.K.; Funding acquisition: H-M.K. All authors have reviewed and approved the final version of the manuscript.

Funding

This work was funded by the Kunshan Shuangchuang grant award (KSSC202202060).

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and supplementary materials.

Acknowledgments

We thank members of the Kim laboratory for discussions and proofreading, especially Zifei Liu.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Venn diagram summarizing the reported biological activities of the genera Scrophularia, Mentha, and Echium based on published literature. This diagram emphasizes the shared biological properties among the three genera from which the herb extracts were derived. Notably, all three have been consistently reported to exhibit antioxidant, pro-apoptotic, anti-inflammatory, cytotoxic, anti-proliferative, and antimicrobial activities. Non-overlapping regions represent additional, genus-specific effects reported in the literature. Detailed information can be found in Table 1, Supplementary Table 1, and Supplementary Table 2.
Figure 1. Venn diagram summarizing the reported biological activities of the genera Scrophularia, Mentha, and Echium based on published literature. This diagram emphasizes the shared biological properties among the three genera from which the herb extracts were derived. Notably, all three have been consistently reported to exhibit antioxidant, pro-apoptotic, anti-inflammatory, cytotoxic, anti-proliferative, and antimicrobial activities. Non-overlapping regions represent additional, genus-specific effects reported in the literature. Detailed information can be found in Table 1, Supplementary Table 1, and Supplementary Table 2.
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Figure 2. Extracts obtained from Mentha longifolia (M.l), Scrophularia orientalis (S.o), and Echium biebersteinii (E.b) exhibit marked nematocidal, larval arrest/lethality and HIM phenotype of C. elegans, without exerting discernible impact on bacterial growth. A) M.l, S.o, and E.b extracts significantly reduced the survival and larval development and high incidence of male (HIM) of C. elegans. The effect of herb extracts were evaluated by treating worms with different extracts of M.l, S.o, and E.b (indicated by brown, orange, and gray colors) and monitoring their survival, adult formation, and male (HIM) phenotype over a 48 h period. Statistical significance was assessed using a two-tailed t-test, with ** p < 0.01; *** p < 0.001; and **** p < 0.0001, comparing the control (+DMSO) with the treated samples. Nocodazole is a positive control. (B) Assessment of bacterial growth in the presence of herbal extracts. E. coli OP50 was incubated with 0.03 μg/mL of M.l, S.o, and E.b extracts—the same concentration used in C. elegans assays—for 24 hours. No significant inhibition of bacterial growth was observed at Absorbance (600nm), indicating that the extracts' nematocidal effects are unlikely to result from compromised bacterial food source (P=0.100 for all three herb at 24 hour of incubation).
Figure 2. Extracts obtained from Mentha longifolia (M.l), Scrophularia orientalis (S.o), and Echium biebersteinii (E.b) exhibit marked nematocidal, larval arrest/lethality and HIM phenotype of C. elegans, without exerting discernible impact on bacterial growth. A) M.l, S.o, and E.b extracts significantly reduced the survival and larval development and high incidence of male (HIM) of C. elegans. The effect of herb extracts were evaluated by treating worms with different extracts of M.l, S.o, and E.b (indicated by brown, orange, and gray colors) and monitoring their survival, adult formation, and male (HIM) phenotype over a 48 h period. Statistical significance was assessed using a two-tailed t-test, with ** p < 0.01; *** p < 0.001; and **** p < 0.0001, comparing the control (+DMSO) with the treated samples. Nocodazole is a positive control. (B) Assessment of bacterial growth in the presence of herbal extracts. E. coli OP50 was incubated with 0.03 μg/mL of M.l, S.o, and E.b extracts—the same concentration used in C. elegans assays—for 24 hours. No significant inhibition of bacterial growth was observed at Absorbance (600nm), indicating that the extracts' nematocidal effects are unlikely to result from compromised bacterial food source (P=0.100 for all three herb at 24 hour of incubation).
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Figure 3. Effects of herbal extracts on nuclear organization, germline development, and fertility defective outcomes in C. elegans. S.o and E.b herb extracts induced increased spacing between nuclei within the pachytene region. In contrast, the M.l extract did not produce any discernible changes in nuclear spacing or organization. (A) DAPI-stained nuclei during germline development of 24 hours post L4 hermaphrodite with or without treatment of three herb extracts. Yellow Arrows indicate crescent shape nuclei positioned at pachytene. White arrow indicates chromatin bridge. (B) Quantification of the increased nuclear spacing in the PMT and pachytene stages shown in the panel. (C) Quantification of crescent -shaped nuclei in per gonad arm is indicated. Asterisks indicate statistically significant differences compared to the control group. (D) Quantification of DAPI-stained bivalents in the germline. The percentage of bivalent at -1 position oocyte is indicated. Numbers in the bracket indicate the number of bivalents. (E) Germline length was measured in three regions: the premeiotic tip (PMT), transition zone (TZ), and pachytene. S.o. extract shortens specific TZ and pachytene stage. (F) Brood size of herb-exposed C. elegans hermaphrodites. The number of offspring produced by individual hermaphrodite worms was monitored daily over a four-day reproductive period following treatment with herbal extracts. Data are presented as mean ± SEM. Statistical significance was assessed using a two-tailed T-test. Asterisks indicate statistically significant differences compared to the control group.
Figure 3. Effects of herbal extracts on nuclear organization, germline development, and fertility defective outcomes in C. elegans. S.o and E.b herb extracts induced increased spacing between nuclei within the pachytene region. In contrast, the M.l extract did not produce any discernible changes in nuclear spacing or organization. (A) DAPI-stained nuclei during germline development of 24 hours post L4 hermaphrodite with or without treatment of three herb extracts. Yellow Arrows indicate crescent shape nuclei positioned at pachytene. White arrow indicates chromatin bridge. (B) Quantification of the increased nuclear spacing in the PMT and pachytene stages shown in the panel. (C) Quantification of crescent -shaped nuclei in per gonad arm is indicated. Asterisks indicate statistically significant differences compared to the control group. (D) Quantification of DAPI-stained bivalents in the germline. The percentage of bivalent at -1 position oocyte is indicated. Numbers in the bracket indicate the number of bivalents. (E) Germline length was measured in three regions: the premeiotic tip (PMT), transition zone (TZ), and pachytene. S.o. extract shortens specific TZ and pachytene stage. (F) Brood size of herb-exposed C. elegans hermaphrodites. The number of offspring produced by individual hermaphrodite worms was monitored daily over a four-day reproductive period following treatment with herbal extracts. Data are presented as mean ± SEM. Statistical significance was assessed using a two-tailed T-test. Asterisks indicate statistically significant differences compared to the control group.
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Figure 4. The three herb extract exposure activates the DNA damage checkpoint pathway and apoptosis. S.o. extract leads to defective DSB repair in the germline. (A) Quantitative PCR analysis of DNA damage checkpoint gene expression in whole worms treated with herbal extracts. Transcript levels of atm-1 and atl-1 were normalized to tba-1 (tubulin) and compared to untreated controls. (B) Quantification of pCHK-1 foci, a downstream marker of ATM/ATR checkpoint activation, in the premeiotic tip (PMT) and pachytene region. All three herb treatments significantly increased pCHK-1 foci in the pachytene stage. Arrows indicate pCHK-1 foci adjacent to chromatin. (C) Quantification of germline apoptosis using acridine orange staining. Apoptotic nuclei were significantly elevated in the pachytene region following S.o. and E.b. treatments, while M.l. treatment caused a mild, non-significant increase compared to the control. (D) RAD-51 foci quantification to assess double-strand break (DSB) repair. S.o. treatment led to significantly increased RAD-51 foci in both the PMT and late pachytene, indicating impaired DSB repair during both mitotic and meiotic stages. All statistical analyses were performed using two-tailed Mann–Whitney tests. Data are presented as mean ± SEM from biological replicates. Asterisks indicate statistically significant differences compared to the control group.
Figure 4. The three herb extract exposure activates the DNA damage checkpoint pathway and apoptosis. S.o. extract leads to defective DSB repair in the germline. (A) Quantitative PCR analysis of DNA damage checkpoint gene expression in whole worms treated with herbal extracts. Transcript levels of atm-1 and atl-1 were normalized to tba-1 (tubulin) and compared to untreated controls. (B) Quantification of pCHK-1 foci, a downstream marker of ATM/ATR checkpoint activation, in the premeiotic tip (PMT) and pachytene region. All three herb treatments significantly increased pCHK-1 foci in the pachytene stage. Arrows indicate pCHK-1 foci adjacent to chromatin. (C) Quantification of germline apoptosis using acridine orange staining. Apoptotic nuclei were significantly elevated in the pachytene region following S.o. and E.b. treatments, while M.l. treatment caused a mild, non-significant increase compared to the control. (D) RAD-51 foci quantification to assess double-strand break (DSB) repair. S.o. treatment led to significantly increased RAD-51 foci in both the PMT and late pachytene, indicating impaired DSB repair during both mitotic and meiotic stages. All statistical analyses were performed using two-tailed Mann–Whitney tests. Data are presented as mean ± SEM from biological replicates. Asterisks indicate statistically significant differences compared to the control group.
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Figure 5. Comparative LC-MS analysis of major compounds in three herbal extracts. Venn diagram and heat map summarizing the 21 major compounds identified across Mentha longifolia, Scrophularia orientalis, and Echium biebersteinii. Four compounds—luteolin-7-O-rutinoside, thymol, carvyl acetate, and menthyl acetate—were common to all three extracts. M. longifolia contained seven unique major compounds, including caryophyllene, genistein, and ursolic acid. S. orientalis had one unique compound, resveratrol, while E. biebersteinii uniquely possessed vitexin-4'-rhamnoside. Compound identification was performed based on methods previously described (see Materials and Methods; [29,30,38]).
Figure 5. Comparative LC-MS analysis of major compounds in three herbal extracts. Venn diagram and heat map summarizing the 21 major compounds identified across Mentha longifolia, Scrophularia orientalis, and Echium biebersteinii. Four compounds—luteolin-7-O-rutinoside, thymol, carvyl acetate, and menthyl acetate—were common to all three extracts. M. longifolia contained seven unique major compounds, including caryophyllene, genistein, and ursolic acid. S. orientalis had one unique compound, resveratrol, while E. biebersteinii uniquely possessed vitexin-4'-rhamnoside. Compound identification was performed based on methods previously described (see Materials and Methods; [29,30,38]).
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Figure 6. Functional characterization of shared herbal compounds reveals their roles in DNA damage response, apoptosis, and antioxidant activity. (A) Expression levels of DNA damage checkpoint genes (atm-1 and atl-1) in response to treatment with four common herbal compounds. Young adult hermaphrodites were treated with luteolin-7-O-rutinoside (30 µM), thymol (3 mM), carvyl acetate (8 µg/ml), or menthyl acetate (10 µg/ml), and qRT-PCR was performed to assess expression of atm-1 and atl-1. Thymol and carvyl acetate significantly upregulated both genes, whereas luteolin-7-O-rutinoside and menthyl acetate showed no significant effect. Data are presented as fold change relative to control (mean ± SEM, n ≥ 20 animals per group). (B) Quantification of germline apoptosis in the pachytene region following compound treatment. Germline apoptosis was measured in wild-type C. elegans treated with the four shared compounds. Thymol and carvyl acetate significantly increased apoptotic cell counts, consistent with their induction of atm-1 and atl-1 checkpoint gene expression. Luteolin-7-O-rutinoside and menthyl acetate showed no significant effects. Data are presented as mean apoptotic nuclei per gonad. Mean ± SEM, n ≥ 20 animals per group. (C) DPPH radical scavenging activity of M.l, S.o, and E.b herb extracts at increasing concentrations. All three extracts exhibited dose-dependent antioxidant activity, with E.b showing the strongest effect (39.15% inhibition at 3 µg/ml, P < 0.0001), followed by S.o (30.95%, P < 0.0001) and M.l (23.05%, P < 0.0001). (D) Antioxidant activity of four common constituents found in the herb extracts at three different doses: luteolin-7-O-rutinoside (10–30 µM), thymol (1–3 mM), carvyl acetate (1–8 µg/ml), and menthyl acetate (1–10 µg/ml). Luteolin-7-O-rutinoside and thymol showed dose-dependent radical scavenging activity, with thymol demonstrating stronger inhibition (up to 18.08% at 3 mM). In contrast, carvyl acetate and menthyl acetate showed negligible activity at all tested concentrations. Data are presented as mean ± SEM. Statistical significance was calculated using two-tailed Mann-Whitney test.
Figure 6. Functional characterization of shared herbal compounds reveals their roles in DNA damage response, apoptosis, and antioxidant activity. (A) Expression levels of DNA damage checkpoint genes (atm-1 and atl-1) in response to treatment with four common herbal compounds. Young adult hermaphrodites were treated with luteolin-7-O-rutinoside (30 µM), thymol (3 mM), carvyl acetate (8 µg/ml), or menthyl acetate (10 µg/ml), and qRT-PCR was performed to assess expression of atm-1 and atl-1. Thymol and carvyl acetate significantly upregulated both genes, whereas luteolin-7-O-rutinoside and menthyl acetate showed no significant effect. Data are presented as fold change relative to control (mean ± SEM, n ≥ 20 animals per group). (B) Quantification of germline apoptosis in the pachytene region following compound treatment. Germline apoptosis was measured in wild-type C. elegans treated with the four shared compounds. Thymol and carvyl acetate significantly increased apoptotic cell counts, consistent with their induction of atm-1 and atl-1 checkpoint gene expression. Luteolin-7-O-rutinoside and menthyl acetate showed no significant effects. Data are presented as mean apoptotic nuclei per gonad. Mean ± SEM, n ≥ 20 animals per group. (C) DPPH radical scavenging activity of M.l, S.o, and E.b herb extracts at increasing concentrations. All three extracts exhibited dose-dependent antioxidant activity, with E.b showing the strongest effect (39.15% inhibition at 3 µg/ml, P < 0.0001), followed by S.o (30.95%, P < 0.0001) and M.l (23.05%, P < 0.0001). (D) Antioxidant activity of four common constituents found in the herb extracts at three different doses: luteolin-7-O-rutinoside (10–30 µM), thymol (1–3 mM), carvyl acetate (1–8 µg/ml), and menthyl acetate (1–10 µg/ml). Luteolin-7-O-rutinoside and thymol showed dose-dependent radical scavenging activity, with thymol demonstrating stronger inhibition (up to 18.08% at 3 mM). In contrast, carvyl acetate and menthyl acetate showed negligible activity at all tested concentrations. Data are presented as mean ± SEM. Statistical significance was calculated using two-tailed Mann-Whitney test.
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Table 1. Taxonomy, Characteristics, and Distribution of Three Medicinal Herbs.
Table 1. Taxonomy, Characteristics, and Distribution of Three Medicinal Herbs.
Genus Taxonomy Characteristics Distribution Sample Used in this study
Mentha Defined as 18–30 species across five sections: Mentha, Preslia, Audibertia, Eriodontes, Pulegium. Includes M. spicata, M. aquatica, M. arvensis, M. longifolia [20,21,96,97] Aromatic, herbaceous perennials with extensive stolons [97] Widely distributed: Northern Pakistan, Europe, Nepal, India, Western China, Germany, UK, Egypt, Nigeria, Turkey [21] Mentha longifolia
Scrophularia Genus Scrophularia (Scrophulariaceae); ~300 species [2] Mostly herbaceous perennials; also subshrubs, biennials, or annuals [3] Temperate Asia, Mediterranean Europe, North America [1] Scrophularia orientalis
Echium Genus Echium (Boraginaceae); ~60 species, 30 in Canary Islands, 24 endemic [9] Annual, biennial, or perennial flowering plants [9,10] Native to North Africa, Europe, Macaronesia (Azores, Madeira, Canaries, Cape Verde) [9,10] Echium biebersteinii
Table 2. Reported biological functions of major compounds in three herbal extracts. Each compound—such as antioxidant, DNA damage response/repair, anti-tumor, and anti-inflammatory functions—are based on previous reports. However, many compounds remain insufficiently characterized and require further investigation.
Table 2. Reported biological functions of major compounds in three herbal extracts. Each compound—such as antioxidant, DNA damage response/repair, anti-tumor, and anti-inflammatory functions—are based on previous reports. However, many compounds remain insufficiently characterized and require further investigation.
No Compounds Antioxidant DNA damage response/repair Anti-tumor Anti-inflammatory
1 Luteolin-7-O-Rucoside [43] [44] [45] [46]
2 Thymol [47] [48] [49] [50]
3 Carvyl acetate
4 Menthyl Acetate [51]
5 Luteolin [52] [53] [54] [55]
6 Caryophyllene [56] [57] [58] [59]
7 Geranium lignin/Diosmetin [60] [61] [62] [63]
8 Genistein [64] [65] [66] [67]
9 Isoquercetin [68] [69] [70]
10 Naringin [71] [72] [73]
11 Ursolic acid [74] [75] [76] [77]
12 Phlogistic acid
13 Dihydrocarvone
14 Resveratrol [78] [79] [80] [81]
15 Aucubin [82] [83] [84]
16 Lycopene [85] [86] [87] [88]
17 Linoleic acid [215] [90] [91]
18 Misoprostol [92]
19 Homoplantain [93]
20 Vitexin-4-O-glucoside [94]
21 Vitexin-4-rhamnoside [95]
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