Herbal Extract-Induced DNA Damage, Apoptosis, and Antioxidant Effects of C. elegans: A Comparative Study of Mentha longifolia, Scrophularia orientalis, and Echium biebersteinii

Abstract

Background: Herbal medicine represents a rich yet complex source of bioactive compounds, offering both therapeutic potential and toxicological risks. Methods: In this study, we systematically evaluated the biological effects of three traditional herbal extracts-Mentha longifolia, Scrophularia orientalis, and Echium biebersteinii-using Caenorhabditis elegans as an in vivo model. Results: All three extracts significantly reduced worm survival, induced larval arrest, and triggered a high incidence of males (HIM) phenotypes, indicative of mitotic failure and meiotic chromosome missegregation. Detailed analysis of germline architecture revealed extract-specific abnormalities, including nuclear disorganization, ectopic crescent-shaped nuclei, altered meiotic progression, and reduced bivalent formation. These defects were accompanied by activation of the DNA damage response, as evidenced by upregulation of checkpoint genes (atm-1, atl-1), increased pCHK-1 foci, and elevated germline apoptosis. LC-MS profiling identified 21 major compounds across the extracts, with four compounds-thymol, carvyl acetate, luteolin-7-O-rutinoside, and menthyl acetate-shared by all three herbs. Among them, thymol and carvyl acetate significantly upregulated DNA damage checkpoint genes and promoted apoptosis, whereas thymol and luteolin-7-O-rutinoside contributed to antioxidant activity. Notably, S. orientalis and E. biebersteinii shared 11 of 14 major constituents (79%), correlating with their similar phenotypic outcomes, while M. longifolia exhibited a more distinct chemical profile, possessing seven unique compounds. Conclusions: These findings highlight the complex biological effects of traditional herbal extracts, demonstrating that both beneficial and harmful outcomes can arise from specific phytochemicals within a mixture. By deconstructing these extracts into their active components, such as thymol, carvyl acetate, and luteolin-7-O-rutinoside, we gain critical insight into the mechanisms driving reproductive toxicity and antioxidant activity. This approach underscores the importance of component-level analysis for accurately assessing the therapeutic value and safety profile of medicinal plants, particularly those used in foods and dietary supplements.

Keywords: DNA repair; Echium biebersteinii; Mentha longifolia; Scrophularia orientalis; germline development; medicinal plants; meiosis.

Figure 1

Venn diagram summarizing the reported biological activities of the genera Scrophularia, Mentha, and Echium based on the 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 Data S1 and S2.

Figure 2

Extracts obtained from M. longifolia (M.l.), S. orientalis (S.o.), and E. 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 diminished survival and larval development while augmenting the high incidence of males (HIM) phenotype in C. elegans. The effect of herb extracts was evaluated by treating worms with different extracts of M.l., S.o., and E.b. 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 < 0.05; ** 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 h. 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 herbs at 24 h of incubation).

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 h post-L4 hermaphrodite with or without treatment of three herb extracts. Yellow Arrows indicate crescent-shaped nuclei positioned at pachytene. White arrow indicates chromatin bridge. Worms exposed to the herbal extracts often exhibited a reduced number of DAPI-stained bivalent bodies during diakinesis, with a count of 5 indicating five bivalents and a count of 6 indicating six bivalents. Bar = 2 µm. (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 bivalents at −1 position of the oocyte is indicated. Numbers in the brackets in panel A indicate the number of bivalents. (E) Germline length was measured in three regions: the PMT, TZ, and pachytene. S.o. extract shortens specific TZ and pachytene stage. (F) Brood size of herb-exposed 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 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. Bar = 2 µm. (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 5

Comparative LC-MS analysis of major compounds in three herbal extracts. Venn diagram and heat map summarizing the 21 major compounds identified across M. longifolia, S. orientalis, and E. 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. 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 [33,34,35]).

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 (18 µg/mL), thymol (450 µg/mL), 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 arm. 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 inhibition (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 (6–18 µg/mL), thymol (150–450 µg/mL), 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%). 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. Asterisks indicate statistically significant differences compared to the control group.