Bioassay-guided isolation of antimutagenic naphthoquinones from Plumbago auriculata

Bioassay-guided isolation of antimutagenic naphthoquinones from Plumbago auriculata | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Bioassay-guided isolation of antimutagenic naphthoquinones from Plumbago auriculata Daisuke Imahori, Takuya Muraoka, Keiko Inami, Hiroyuki Tanaka This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8331701/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Mutagen-induced genotoxicity plays a central role in carcinogenesis, highlighting the need for effective chemopreventive agents. Naphthoquinones, such as plumbagin, exhibit diverse biological activities; however, their antimutagenic potency at pharmacologically relevant concentrations remains poorly characterized. In this study, the acetone extract of Plumbago auriculata roots was subjected to bioassay-guided fractionation, yielding two known naphthoquinone derivatives: plumbagin and cis -isoshinanolone. Results Antimutagenic activity was evaluated using the Ames test with Salmonella typhimurium TA1535 against N -methyl- N -nitrosourea. The chloroform-soluble fraction (PAC) exhibited stronger activity than the residual fraction (PAAR), and purified plumbagin potently inhibited mutagen-induced revertant formation at nanomolar concentrations. No toxicity was observed at any of the tested doses. Conclusions These findings provide the first evidence that P. auriculata is a source of nanomolar-active antimutagenic naphthoquinones, highlighting their chemopreventive potential and expanding the biological relevance of quinone-type natural products beyond cytotoxicity. Plumbago auriculata roots naphthoquinones Ames test mutagenicity chemoprevention Figures Figure 1 1. Introduction Cancer is a leading cause of morbidity and mortality worldwide, underscoring the need for effective chemopreventive strategies [ 1 ]. Mutations are key drivers of the initiation and progression of various diseases, including atherosclerosis, heart disease, and cancer [ 2 ]. Therefore, preventing mutational events at an early stage is an important strategy for cancer chemoprevention [ 3 ]. Among the multiple factors that contribute to carcinogenesis, genotoxic insults caused by mutagens play a central role in initiating DNA damage and promoting malignant transformation [ 4 ]. Mutagens can be broadly classified into physical (ionizing radiation), chemical (alkylating agents and polycyclic aromatic hydrocarbons), and biological agents (viruses) [ 5 ]. Chemical mutagens are of particular concern because of their widespread presence in the environment, food, and pharmaceutical products [ 5 ]. One such compound, N -methyl- N -nitrosourea (MNU), is a potent direct-acting carcinogen that induces cancer in multiple organs, primarily the forestomach, brain, and nervous system [ 6 ]. MNU exerts genotoxic effects by generating reactive methyldiazonium ions that methylate the O 6 position of guanine, producing O 6 -methylguanine lesions [ 7 ]. These lesions mispair with thymine during DNA replication, predominantly causing GC transition mutations [ 7 ]. Several in vitro and in vivo studies using animal models have demonstrated the antimutagenic potential of phytochemicals, including phenylpropanoids, triterpenoids, flavonoids, isoflavones, and naphthoquinones [ 2 , 8 , 9 ]. However, only a limited number of natural compounds have exhibited strong antimutagenic effects, and identifying new and highly potent agents remains an important research objective [ 10 ]. Plumbago species and other members of the Plumbaginaceae family biosynthesize various naphthoquinones, among which plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) is the most prominent [ 11 ]. Plumbagin exhibits a broad spectrum of biological activities, including antimicrobial, cytotoxic, anti-inflammatory, antioxidant, and antitumor effects [ 11 , 12 ]. Recent studies have highlighted its potential antimutagenic properties, particularly its ability to suppress mutagenicity induced by reactive oxygen species and nitroaromatic compounds in short-term bacterial mutation assays, such as the Ames test [ 2 ]. However, most of these findings are based on crude plant extracts or high micromolar concentrations, and the antimutagenic potency of purified plumbagin and related naphthoquinones at nanomolar concentrations remains underexplored [ 11 ]. Moreover, most studies have focused on P. zeylanica [ 13 ] and P. indica [ 11 ], whereas other species such as P. auriculata , an ornamental plant widely cultivated in subtropical regions [ 14 ], have received limited attention. Although P. auriculata is taxonomically close to medicinally relevant Plumbago species and is known to accumulate naphthoquinone derivatives, comprehensive phytochemical and antimutagenic evaluations of this species are lacking. In this study, we isolated and structurally characterized two known naphthoquinone derivatives, including plumbagin, from P. auriculata roots using bioassay-guided fractionation. Purified plumbagin exhibited potent antimutagenic activity at nanomolar concentrations, indicating that P. auriculata is an underrecognized source of highly active antimutagenic compounds. To the best of our knowledge, this is the first report demonstrating the nanomolar-level antimutagenic activity of plumbagin derived from this species. These findings highlight the chemopreventive potential of naphthoquinones and further support their broad biological relevance, beyond cytotoxicity. 2. Materials and methods 2.1. General experimental procedures Specific rotations were measured using a P-2200 digital polarimeter ( l = 5 cm; JASCO, Tokyo, Japan). Fourier-transform infrared (FT-IR) spectra were recorded using a JASCO FT/IR-4600 spectrometer. Ultraviolet–visible (UV–vis) spectra were acquired using a Shimadzu UV-1850 spectrophotometer (Shimadzu, Kyoto, Japan). High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) was performed using a JMS-T100LP AccuTOF LC-Plus 4G instrument (JEOL, Tokyo, Japan). 1 H (600 MHz), 13 C (150 MHz), and 2D nuclear magnetic resonance (NMR) spectra were recorded using a JEOL JNM-ECZ 600R spectrometer. Normal-phase silica gel column chromatography was performed using Wakogel® 60N (FUJIFILM Wako Pure Chemical, Osaka, 63–212 µm). Thin-layer chromatography (TLC) was performed using TLC plates precoated with 60F 254 silica gel (Merck, Darmstadt, Germany; 0.25 mm, ordinary phase). High-performance liquid chromatography (HPLC) was performed using an SPD-10A vp UV–vis detector (Shimadzu, Kyoto, Japan), an LC-10AD vp pump (Shimadzu), an SCL-10A vp system controller (Shimadzu), and LabSolutions LC software (version 1.25, Shimadzu) with a COSMOSIL 5C 18 -AR-II column (Nacalai Tesque, 250 × 4.6 mm i.d. and 250 × 10 mm i.d.) for analytical and preparative purposes, respectively. The solvent ratios were calculated based on the volume. FT-IR, UV–Vis, and HR-ESI-MS spectra were recorded using spectroscopic-grade methanol (Wako Pure Chemical, Japan). Bacto Agar and Nutrient Broth were obtained from Becton Dickinson Microbiology Systems (San Jose, CA, USA). Sodium ammonium hydrogen phosphate tetrahydrate was purchased from Merck (Darmstadt, Germany). MNU was prepared as previously described [ 15 ]. Genistein was purchased from TCI (Tokyo, Japan). 2.2. Plant material Plumbago auriculata was purchased from TAKII & Co. Ltd. (Kyoto Prefecture, Japan) (voucher specimen number: SOCU-2025-2). Voucher specimens were deposited in the herbarium of the Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Sanyo-Onoda City University, Yamaguchi, Japan. The plant material was examined and identified by Prof. Hiroyuki Tanaka (PhD), Department of Pharmacognosy and Kampo, Faculty of Pharmaceutical Sciences, Sanyo-Onoda City University. The deposited voucher specimens, including roots and leaves, were shade-dried and stored. 2.3. Extraction and bioassay-guided fractionation Dried roots (236.0 g) of P. auriculata were extracted three times with acetone for 3 h using a modified procedure based on a previously reported method [ 16 ]. Solvent evaporation yielded an acetone extract (PAA, 5.9 g, 2.50%). Additionally, a chloroform-soluble fraction (PAC, 1.2 g, 0.51%) and a residual fraction (PAAR, 4.7 g, 1.99%) were obtained from the acetone extract. The PAC fraction was subjected to normal-phase silica-gel column chromatography (100.0 g, n -hexane: chloroform [1:0 → 1:1 → 0:1]), yielding five fractions (PAC1–5). Compound 1 (500.0 mg, 0.21%) was obtained as an orange, amorphous solid from PAC3. Compound 2 (25.5 mg, 0.01%) was obtained as an orange amorphous solid after purification of PAC4 (1.0 g) using HPLC [H₂O:MeCN:AcOH (60:40:0.3)]. 2.4. Plumbagin ( 1 ) Orange amorphous solid; 1 H NMR (chloroform- d , 600 MHz) δ H 6.80 (1H, m, H-2), 2.17 (1H, d, J = 1.8 Hz, Me-3), 7.25 (1H, d, J = 1.8, 7.8 Hz, H-5), 7.61 (1H, d, J = 7.8 Hz, H-6), 7.63 (1H, dd, J = 1.8, 7.8 Hz, H-7), 11.97 (1H, s, OH-8) and 13 C NMR (chloroform- d , 150 MHz) δ C 190.2 (C-1), 135.4 (C-2), 149.6 (C-3), 16.5 (Me-3), 184.8 (C-4), 132.0 (C-4a), 119.3 (C-5), 136.1 (C-6), 124.1 (C-7), 161.1 (C-8), 115.1 (C-8a); ESI-MS; m / z : 189 [M + H] + . 2.5. cis-Isoshinanolone ( 2 ) Orange amorphous solid; [ a ] D 25 +29.5 ( c 0.2, MeOH); 1 H NMR (chloroform- d , 600 MHz) δ H 2.56 (1H, dd, J = 4.2, 18.0 Hz, H-2), 2.87 (1H, dd, J = 10.8, 18.0 Hz, H-2), 2.43 (1H, m, H-3), 1.17 (1H, d, J = 7.2 Hz, Me-3), 4.74 (1H, d, J = 2.4 Hz, H-4), 6.92 (1H, br s, H-5), 7.48 (1H, t-like, J = 8.4 Hz, H-6), 6.94 (1H, br d, J = 1.8 Hz, H-7), 12.42 (1H, s, OH-8) and 13 C NMR (chloroform- d , 150 MHz) δ C 204.8 (C-1), 40.7 (C-2), 34.4 (C-3), 16.2 (Me-3), 71.1 (C-4), 145.0 (C-4a), 118.6 (C-5), 136.9 (C-6), 118.1 (C-7), 162.6 (C-8), 115.09 (C-8a); ESI-MS; m / z : 193 [M + H] + . 2.6. Bacterial mutation assay The bacterial mutation assay was performed as described by Maron et al. and Brusick et al. [ 17 , 18 ]. Dr. T. Nohmi (National Institute of Health Sciences, Tokyo, Japan) kindly provided S. typhimurium TA1535. A solution of MNU (1.5 µmol/50 µL of DMSO) was added to a test tube and supplemented with 0.1 M sodium phosphate buffer (pH 7.4, 0.5 mL), a solution (50 µL) with various concentrations of each fraction, and a culture of S. typhimurium TA1535 (0.1 mL), and the mixture was thoroughly mixed. Next, top agar (2 mL) was added, and the mixture was poured onto a minimal glucose agar plate. Revertant colonies were counted after incubation at 37°C for 44 h. The experiments were performed in triplicate and repeated three times with similar results. The results are expressed as mean ± standard error (SE). Plates without MNU or plant extracts were used as negative controls. MNU (1.5 µmol/50 µL) yielded 1316 ± 72 colonies. All tested plates were microscopically examined for thinning, absence of background lawn, and/or presence of microcolonies, which are considered indicators of toxicity induced by the test material. Neither MNU nor the plant extracts exhibited toxicity toward S. typhimurium TA1535 in the antimutagenicity test. Mutagenic activity in the presence of the extracts was expressed as the percentage of mutagenicity (% = Rs/R × 100), where Rs is the number of His + revertants/plate for plates exposed to MNU and plant extracts, and R is the number of His + revertants/plate of MNU. The number of spontaneous revertants was subtracted to obtain Rs and R. Thus, the mutagenicity of MNU in the absence of plant extracts was defined as 100%. 2.7. Statistical Analysis Statistical analyses were performed using Microsoft Excel (Microsoft Corporation, Redmond, WA, USA) or GraphPad Prism 8.43 software (GraphPad Software, San Diego, CA, USA). 3. Results and discussion 3.1. Bioassay-guided isolation of antimutagenic naphthoquinones from P. auriculata The acetone extract of P. auriculata roots was partitioned with chloroform to obtain PAC and PAAR. PAC was subjected to normal-phase silica gel column chromatography, followed by repeated HPLC to obtain two naphthoquinone derivatives, plumbagin ( 1 ) [ 19 ] and cis -isoshinanolone ( 2 ) [ 20 ] (Fig. 1 for compound structures). Antimutagenic activity was evaluated in vitro using the Ames assay with S. typhimurium TA1535. The individual activities of PAA, its fractions (PAC and PAAR), and isolated compounds ( 1 and 2 ) are summarized in Table 1 . Plumbago species and other members of the Plumbaginaceae family are rich sources of bioactive compounds, including naphthoquinones, flavonoids, and sterols [ 11 ]. The primary objective of this study was to isolate and characterize naphthoquinone derivatives from P. auriculata roots and to evaluate their antimutagenic potency. Our results confirmed that P. auriculata contains naphthoquinones, such as plumbagin, which is consistent with previous reports [ 21 ]. Furthermore, PAA exhibited antimutagenic activity against MNU in the Ames test using S. typhimurium TA1535 (IC 50 : 44.7 ± 9.89 µg/plate), comparable to that previously reported for licorice extracts [ 6 ]. Among the fractions, PAC exhibited stronger antimutagenic activity than PAAR. Compound 1 exhibited greater potency (IC 50 : 12.9 ± 0.86 nmol/plate) than compound 2 (IC 50 : >2.0 µmol/plate), suggesting that the distinct stereochemistry of their quinone ring structures may influence their target interactions [ 2 ]. As a positive control, the flavonoid genistein was employed, and its antimutagenic activity against MNU showed an IC₅₀ value of 1.29 ± 0.02 µmol/plate. No toxicity was observed at any of the concentrations tested. However, several limitations of this study should be acknowledged. First, the antimutagenic activity was evaluated only by the Ames test, which, although widely accepted, does not fully reflect the complexity of mammalian cellular responses to mutagens. Second, the mechanistic basis underlying the observed activity of plumbagin ( 1 ) and cis -isoshinanolone ( 2 ) was not investigated in detail, therefore, the precise molecular targets and pathways remain to be clarified. These limitations suggest that further studies using mammalian cell-based systems, DNA damage assays, and broader metabolite profiling will be necessary to confirm the protective effects and to elucidate the mechanisms involved. These findings provide the first evidence that P. auriculata is a source of nanomolar-active antimutagenic naphthoquinones, supporting its potential as a chemopreventive agent. Table 1 Antimutagenic activity of the Plumbago auriculata extract, fractions, and compounds against N -methyl- N -nitrosourea (MNU) Dose (µg/plate) Revertants/plate (Mutagenicity %) 0 15.625 31.25 62.5 125 250 PAA 1570 ± 147 (100.0%) 1213 ± 141 (77.1%) 862 ± 52 (54.6%) 706 ± 53 (44.6%) 525 ± 50 (33.0%) 223 ± 88 (12.6%) Dose (µg/plate) 0 50 100 PAC 1505 ± 29 (100.0%) 455 ± 48 (29.5%) 105 ± 9 (6.0%) PAAR 1505 ± 29 (100.0%) 1447 ± 46 (96.1%) 1260 ± 34 (83.5%) Dose (µg/plate) 0 25 50 PAC 1 1570 ± 16 (100.0%) 1579 ± 44 (100.6%) 1329 ± 72 (84.5%) PAC 2 1570 ± 16 (100.0%) 1353 ± 67 (86.0%) 1371 ± 54 (87.2%) PAC 3 1570 ± 16 (100.0%) 30 ± 5 (0.7%) 5 ± 2 (0.0%) PAC 4 1570 ± 16 (100.0%) 1518 ± 53 (96.6%) 1289 ± 66 (81.9%) PAC 5 1570 ± 16 (100.0%) 1465 ± 54 (93.2%) 1398 ± 46 (88.9%) Dose (nmol/plate) 0 6.25 12.5 25 50 100 1 1515 ± 56 (100.0%) 997 ± 124 (65.4%) 736 ± 16 (47.9%) 546 ± 26 (35.2%) 276 ± 67 (17.1%) 57 ± 13 (2.5%) Dose (µmol/plate) 0 0.125 0.25 0.5 1.0 2.0 2 1330 ± 87 (100.0%) 1356 ± 136 (102.0%) 1240 ± 123 (93.2%) 1242 ± 140 (93.3%) 1062 ± 70 (79.7%) 835 ± 120 (62.4%) Dose (µmol/plate) 0 0.25 0.5 1.0 1.5 2.0 Genistein 1270 ± 96 (100.0%) 1190 ± 103 (93.7%) 1112 ± 38 (87.6%) 816 ± 58 (64.3%) 520 ± 37 (41.0%) 338 ± 72 (26.6%) 4. Conclusion This study provides the first evidence that P. auriculata roots contain antimutagenic naphthoquinones, plumbagin ( 1 ) and cis -isoshinanolone ( 2 ), isolated through bioassay-guided fractionation. Purified plumbagin ( 1 ) potently inhibited mutagen-induced revertant formation at nanomolar concentrations, a level of activity rarely reported for natural compounds. PAC exhibited stronger antimutagenic activity than PAAR, and compound 1 (plumbagin) was more active than compound 2 , suggesting that stereochemical differences in the quinone ring influence their biological interactions. No toxicity was observed at any of the concentrations tested. These findings highlight P. auriculata as a previously underrecognized source of highly active antimutagenic constituents and underscore the chemopreventive potential of naphthoquinones beyond their classical cytotoxic properties. Declarations Ethical Approval Not applicable. Statement of Informed Consent No human participants were included in this study; therefore, informed consent was not required. Consent for publication Not applicable. Availability of data and materials The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. All experimental data supporting the findings, including raw values from Ames tests and compound characterization (HPLC profiles, NMR spectra), are archived at Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Sanyo-Onoda City University, Yamaguchi, Japan and can be provided upon request for verification and replication purposes. Declaration of conflicting interest The authors declare no potential conflicts of interest regarding the research, authorship, or publication of this study. Funding statements Not applicable. Authors contribution Takuya Muraoka: Writing–original draft, project administration, conceptualization. Daisuke Imahori: Data curation. Hiroyuki Tanaka: Data curation. Keiko Inami: Writing–review and editing, Supervision, Conceptualization. Acknowledgements We would like to thank Editage (www.editage.com) for the English language editing. References Wu Z, Xia F, Lin R. Global burden of cancer and associated risk factors in 204 countries and territories, 1980–2021: a systematic analysis for the GBD 2021. J Hematol Oncol. 2024;17(1):119. 10.1186/s13045-024-01640-8 . Kumar S, Gautam S, Sharma A. Antimutagenic and antioxidant properties of plumbagin and other naphthoquinones. Mutat Res. 2013;755(1):30–41. 10.1016/j.mrgentox.2013.05.007 . Seong H, Izutsu R, Osaki M, Okada F. Cancer prevention: past challenges and future directions. Genes Environ. 2025;47(1):4. 10.1186/s41021-025-00326-y . Greim H, Albertini JR. Cellular response to the genotoxic insult: the question of threshold for genotoxic carcinogens. Toxicol Res. 2015;4(1):36–45. 10.1186/s13045-024-01640-8 . 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Tanaka","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYBACAzB5gEEOiyguLWwQLcaka0lsINph5vLNzx78OGOXvr3/APOHDzWHGfjbDzAUF+DRYtnGZm7YcyM5d86NBDbJGccOM0icSWAwnoHPYccYzCR4PjDnzpBgYGPmbTjMwHCDgcGYB68W9m+Sfz7Up0vwH2D+DNIiT1gLj5k0z43DCRIMCQzSIC0GhLXklEnLnDluOEMisQ3ol3QewzOJDfj9cvj4Nsk3x6rlJfgPHwaGmLWc3PHDx4zxhRgSYGwAkUAnMbYZE1CKCZgfk6xlFIyCUTAKhjMAAKgoSGNLlf+5AAAAAElFTkSuQmCC","orcid":"","institution":"Sanyo-Onoda City University","correspondingAuthor":true,"prefix":"","firstName":"Hiroyuki","middleName":"","lastName":"Tanaka","suffix":""}],"badges":[],"createdAt":"2025-12-11 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1","display":"","copyAsset":false,"role":"figure","size":22739,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structures of compounds isolated from \u003cem\u003ePlumbago auriculata\u003c/em\u003e roots\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8331701/v1/3487f45f985d1dd22bb71763.png"},{"id":99686231,"identity":"1f2a76cc-27e9-4892-9098-8ddaf1370cb7","added_by":"auto","created_at":"2026-01-07 09:38:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":751330,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8331701/v1/e42fd445-18b4-4a81-93a3-9fa2b15ac071.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bioassay-guided isolation of antimutagenic naphthoquinones from Plumbago auriculata","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCancer is a leading cause of morbidity and mortality worldwide, underscoring the need for effective chemopreventive strategies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Mutations are key drivers of the initiation and progression of various diseases, including atherosclerosis, heart disease, and cancer [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Therefore, preventing mutational events at an early stage is an important strategy for cancer chemoprevention [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Among the multiple factors that contribute to carcinogenesis, genotoxic insults caused by mutagens play a central role in initiating DNA damage and promoting malignant transformation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Mutagens can be broadly classified into physical (ionizing radiation), chemical (alkylating agents and polycyclic aromatic hydrocarbons), and biological agents (viruses) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Chemical mutagens are of particular concern because of their widespread presence in the environment, food, and pharmaceutical products [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. One such compound, \u003cem\u003eN\u003c/em\u003e-methyl-\u003cem\u003eN\u003c/em\u003e-nitrosourea (MNU), is a potent direct-acting carcinogen that induces cancer in multiple organs, primarily the forestomach, brain, and nervous system [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. MNU exerts genotoxic effects by generating reactive methyldiazonium ions that methylate the O\u003csup\u003e6\u003c/sup\u003e position of guanine, producing O\u003csup\u003e6\u003c/sup\u003e-methylguanine lesions [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These lesions mispair with thymine during DNA replication, predominantly causing GC transition mutations [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Several \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies using animal models have demonstrated the antimutagenic potential of phytochemicals, including phenylpropanoids, triterpenoids, flavonoids, isoflavones, and naphthoquinones [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, only a limited number of natural compounds have exhibited strong antimutagenic effects, and identifying new and highly potent agents remains an important research objective [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003ePlumbago\u003c/em\u003e species and other members of the Plumbaginaceae family biosynthesize various naphthoquinones, among which plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) is the most prominent [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Plumbagin exhibits a broad spectrum of biological activities, including antimicrobial, cytotoxic, anti-inflammatory, antioxidant, and antitumor effects [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Recent studies have highlighted its potential antimutagenic properties, particularly its ability to suppress mutagenicity induced by reactive oxygen species and nitroaromatic compounds in short-term bacterial mutation assays, such as the Ames test [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, most of these findings are based on crude plant extracts or high micromolar concentrations, and the antimutagenic potency of purified plumbagin and related naphthoquinones at nanomolar concentrations remains underexplored [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Moreover, most studies have focused on \u003cem\u003eP. zeylanica\u003c/em\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and \u003cem\u003eP. indica\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], whereas other species such as \u003cem\u003eP. auriculata\u003c/em\u003e, an ornamental plant widely cultivated in subtropical regions [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], have received limited attention. Although \u003cem\u003eP. auriculata\u003c/em\u003e is taxonomically close to medicinally relevant \u003cem\u003ePlumbago\u003c/em\u003e species and is known to accumulate naphthoquinone derivatives, comprehensive phytochemical and antimutagenic evaluations of this species are lacking.\u003c/p\u003e \u003cp\u003eIn this study, we isolated and structurally characterized two known naphthoquinone derivatives, including plumbagin, from \u003cem\u003eP. auriculata\u003c/em\u003e roots using bioassay-guided fractionation. Purified plumbagin exhibited potent antimutagenic activity at nanomolar concentrations, indicating that \u003cem\u003eP. auriculata\u003c/em\u003e is an underrecognized source of highly active antimutagenic compounds. To the best of our knowledge, this is the first report demonstrating the nanomolar-level antimutagenic activity of plumbagin derived from this species. These findings highlight the chemopreventive potential of naphthoquinones and further support their broad biological relevance, beyond cytotoxicity.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. General experimental procedures\u003c/h2\u003e \u003cp\u003eSpecific rotations were measured using a P-2200 digital polarimeter (\u003cem\u003el\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5 cm; JASCO, Tokyo, Japan). Fourier-transform infrared (FT-IR) spectra were recorded using a JASCO FT/IR-4600 spectrometer. Ultraviolet\u0026ndash;visible (UV\u0026ndash;vis) spectra were acquired using a Shimadzu UV-1850 spectrophotometer (Shimadzu, Kyoto, Japan). High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) was performed using a JMS-T100LP AccuTOF LC-Plus 4G instrument (JEOL, Tokyo, Japan). \u003csup\u003e1\u003c/sup\u003eH (600 MHz), \u003csup\u003e13\u003c/sup\u003eC (150 MHz), and 2D nuclear magnetic resonance (NMR) spectra were recorded using a JEOL JNM-ECZ 600R spectrometer. Normal-phase silica gel column chromatography was performed using Wakogel\u0026reg; 60N (FUJIFILM Wako Pure Chemical, Osaka, 63\u0026ndash;212 \u0026micro;m). Thin-layer chromatography (TLC) was performed using TLC plates precoated with 60F\u003csub\u003e254\u003c/sub\u003e silica gel (Merck, Darmstadt, Germany; 0.25 mm, ordinary phase). High-performance liquid chromatography (HPLC) was performed using an SPD-10A\u003cem\u003evp\u003c/em\u003e UV\u0026ndash;vis detector (Shimadzu, Kyoto, Japan), an LC-10AD\u003cem\u003evp\u003c/em\u003e pump (Shimadzu), an SCL-10A\u003cem\u003evp\u003c/em\u003e system controller (Shimadzu), and LabSolutions LC software (version 1.25, Shimadzu) with a COSMOSIL 5C\u003csub\u003e18\u003c/sub\u003e-AR-II column (Nacalai Tesque, 250 \u0026times; 4.6 mm i.d. and 250 \u0026times; 10 mm i.d.) for analytical and preparative purposes, respectively. The solvent ratios were calculated based on the volume. FT-IR, UV\u0026ndash;Vis, and HR-ESI-MS spectra were recorded using spectroscopic-grade methanol (Wako Pure Chemical, Japan). Bacto Agar and Nutrient Broth were obtained from Becton Dickinson Microbiology Systems (San Jose, CA, USA). Sodium ammonium hydrogen phosphate tetrahydrate was purchased from Merck (Darmstadt, Germany). MNU was prepared as previously described [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Genistein was purchased from TCI (Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Plant material\u003c/h2\u003e \u003cp\u003e \u003cem\u003ePlumbago auriculata\u003c/em\u003e was purchased from TAKII \u0026amp; Co. Ltd. (Kyoto Prefecture, Japan) (voucher specimen number: SOCU-2025-2). Voucher specimens were deposited in the herbarium of the Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Sanyo-Onoda City University, Yamaguchi, Japan. The plant material was examined and identified by Prof. Hiroyuki Tanaka (PhD), Department of Pharmacognosy and Kampo, Faculty of Pharmaceutical Sciences, Sanyo-Onoda City University. The deposited voucher specimens, including roots and leaves, were shade-dried and stored.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Extraction and bioassay-guided fractionation\u003c/h2\u003e \u003cp\u003eDried roots (236.0 g) of \u003cem\u003eP. auriculata\u003c/em\u003e were extracted three times with acetone for 3 h using a modified procedure based on a previously reported method [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Solvent evaporation yielded an acetone extract (PAA, 5.9 g, 2.50%). Additionally, a chloroform-soluble fraction (PAC, 1.2 g, 0.51%) and a residual fraction (PAAR, 4.7 g, 1.99%) were obtained from the acetone extract. The PAC fraction was subjected to normal-phase silica-gel column chromatography (100.0 g, \u003cem\u003en\u003c/em\u003e-hexane: chloroform [1:0 \u0026rarr; 1:1 \u0026rarr; 0:1]), yielding five fractions (PAC1\u0026ndash;5). Compound \u003cb\u003e1\u003c/b\u003e (500.0 mg, 0.21%) was obtained as an orange, amorphous solid from PAC3. Compound \u003cb\u003e2\u003c/b\u003e (25.5 mg, 0.01%) was obtained as an orange amorphous solid after purification of PAC4 (1.0 g) using HPLC [H₂O:MeCN:AcOH (60:40:0.3)].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Plumbagin (\u003cb\u003e1\u003c/b\u003e)\u003c/h2\u003e \u003cp\u003eOrange amorphous solid; \u003csup\u003e1\u003c/sup\u003eH NMR (chloroform-\u003cem\u003ed\u003c/em\u003e, 600 MHz) \u003cem\u003eδ\u003c/em\u003e\u003csub\u003eH\u003c/sub\u003e 6.80 (1H, m, H-2), 2.17 (1H, d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.8 Hz, Me-3), 7.25 (1H, d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.8, 7.8 Hz, H-5), 7.61 (1H, d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8 Hz, H-6), 7.63 (1H, dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.8, 7.8 Hz, H-7), 11.97 (1H, s, OH-8) and \u003csup\u003e13\u003c/sup\u003eC NMR (chloroform-\u003cem\u003ed\u003c/em\u003e, 150 MHz) \u003cem\u003eδ\u003c/em\u003e\u003csub\u003eC\u003c/sub\u003e 190.2 (C-1), 135.4 (C-2), 149.6 (C-3), 16.5 (Me-3), 184.8 (C-4), 132.0 (C-4a), 119.3 (C-5), 136.1 (C-6), 124.1 (C-7), 161.1 (C-8), 115.1 (C-8a); ESI-MS; \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e: 189 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. cis-Isoshinanolone (\u003cb\u003e2\u003c/b\u003e)\u003c/h2\u003e \u003cp\u003eOrange amorphous solid; [\u003cem\u003ea\u003c/em\u003e]\u003csub\u003eD\u003c/sub\u003e\u003csup\u003e25\u003c/sup\u003e +29.5 (\u003cem\u003ec\u003c/em\u003e 0.2, MeOH); \u003csup\u003e1\u003c/sup\u003eH NMR (chloroform-\u003cem\u003ed\u003c/em\u003e, 600 MHz) \u003cem\u003eδ\u003c/em\u003e\u003csub\u003eH\u003c/sub\u003e 2.56 (1H, dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.2, 18.0 Hz, H-2), 2.87 (1H, dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10.8, 18.0 Hz, H-2), 2.43 (1H, m, H-3), 1.17 (1H, d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, Me-3), 4.74 (1H, d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.4 Hz, H-4), 6.92 (1H, br s, H-5), 7.48 (1H, t-like, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, H-6), 6.94 (1H, br d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.8 Hz, H-7), 12.42 (1H, s, OH-8) and \u003csup\u003e13\u003c/sup\u003eC NMR (chloroform-\u003cem\u003ed\u003c/em\u003e, 150 MHz) \u003cem\u003eδ\u003c/em\u003e\u003csub\u003eC\u003c/sub\u003e 204.8 (C-1), 40.7 (C-2), 34.4 (C-3), 16.2 (Me-3), 71.1 (C-4), 145.0 (C-4a), 118.6 (C-5), 136.9 (C-6), 118.1 (C-7), 162.6 (C-8), 115.09 (C-8a); ESI-MS; \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e: 193 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Bacterial mutation assay\u003c/h2\u003e \u003cp\u003eThe bacterial mutation assay was performed as described by Maron et al. and Brusick et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Dr. T. Nohmi (National Institute of Health Sciences, Tokyo, Japan) kindly provided \u003cem\u003eS. typhimurium\u003c/em\u003e TA1535. A solution of MNU (1.5 \u0026micro;mol/50 \u0026micro;L of DMSO) was added to a test tube and supplemented with 0.1 M sodium phosphate buffer (pH 7.4, 0.5 mL), a solution (50 \u0026micro;L) with various concentrations of each fraction, and a culture of \u003cem\u003eS. typhimurium\u003c/em\u003e TA1535 (0.1 mL), and the mixture was thoroughly mixed. Next, top agar (2 mL) was added, and the mixture was poured onto a minimal glucose agar plate. Revertant colonies were counted after incubation at 37\u0026deg;C for 44 h. The experiments were performed in triplicate and repeated three times with similar results. The results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE). Plates without MNU or plant extracts were used as negative controls. MNU (1.5 \u0026micro;mol/50 \u0026micro;L) yielded 1316\u0026thinsp;\u0026plusmn;\u0026thinsp;72 colonies. All tested plates were microscopically examined for thinning, absence of background lawn, and/or presence of microcolonies, which are considered indicators of toxicity induced by the test material. Neither MNU nor the plant extracts exhibited toxicity toward \u003cem\u003eS. typhimurium\u003c/em\u003e TA1535 in the antimutagenicity test. Mutagenic activity in the presence of the extracts was expressed as the percentage of mutagenicity (% = Rs/R \u0026times; 100), where Rs is the number of His\u0026thinsp;+\u0026thinsp;revertants/plate for plates exposed to MNU and plant extracts, and R is the number of His\u0026thinsp;+\u0026thinsp;revertants/plate of MNU. The number of spontaneous revertants was subtracted to obtain Rs and R. Thus, the mutagenicity of MNU in the absence of plant extracts was defined as 100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Statistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using Microsoft Excel (Microsoft Corporation, Redmond, WA, USA) or GraphPad Prism 8.43 software (GraphPad Software, San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e3.1. Bioassay-guided isolation of antimutagenic naphthoquinones from\u003c/em\u003e P. auriculata\u003c/h2\u003e \u003cp\u003eThe acetone extract of \u003cem\u003eP. auriculata\u003c/em\u003e roots was partitioned with chloroform to obtain PAC and PAAR. PAC was subjected to normal-phase silica gel column chromatography, followed by repeated HPLC to obtain two naphthoquinone derivatives, plumbagin (\u003cb\u003e1\u003c/b\u003e) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and \u003cem\u003ecis\u003c/em\u003e-isoshinanolone (\u003cb\u003e2\u003c/b\u003e) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for compound structures). Antimutagenic activity was evaluated \u003cem\u003ein vitro\u003c/em\u003e using the Ames assay with \u003cem\u003eS. typhimurium\u003c/em\u003e TA1535. The individual activities of PAA, its fractions (PAC and PAAR), and isolated compounds (\u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e) are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. \u003cem\u003ePlumbago\u003c/em\u003e species and other members of the Plumbaginaceae family are rich sources of bioactive compounds, including naphthoquinones, flavonoids, and sterols [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The primary objective of this study was to isolate and characterize naphthoquinone derivatives from \u003cem\u003eP. auriculata\u003c/em\u003e roots and to evaluate their antimutagenic potency. Our results confirmed that \u003cem\u003eP. auriculata\u003c/em\u003e contains naphthoquinones, such as plumbagin, which is consistent with previous reports [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Furthermore, PAA exhibited antimutagenic activity against MNU in the Ames test using \u003cem\u003eS. typhimurium\u003c/em\u003e TA1535 (IC\u003csub\u003e50\u003c/sub\u003e: 44.7\u0026thinsp;\u0026plusmn;\u0026thinsp;9.89 \u0026micro;g/plate), comparable to that previously reported for licorice extracts [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Among the fractions, PAC exhibited stronger antimutagenic activity than PAAR. Compound \u003cb\u003e1\u003c/b\u003e exhibited greater potency (IC\u003csub\u003e50\u003c/sub\u003e: 12.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86 nmol/plate) than compound \u003cb\u003e2\u003c/b\u003e (IC\u003csub\u003e50\u003c/sub\u003e: \u0026gt;2.0 \u0026micro;mol/plate), suggesting that the distinct stereochemistry of their quinone ring structures may influence their target interactions [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. As a positive control, the flavonoid genistein was employed, and its antimutagenic activity against MNU showed an IC₅₀ value of 1.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;mol/plate. No toxicity was observed at any of the concentrations tested. However, several limitations of this study should be acknowledged. First, the antimutagenic activity was evaluated only by the Ames test, which, although widely accepted, does not fully reflect the complexity of mammalian cellular responses to mutagens. Second, the mechanistic basis underlying the observed activity of plumbagin (\u003cb\u003e1\u003c/b\u003e) and \u003cem\u003ecis\u003c/em\u003e-isoshinanolone (\u003cb\u003e2\u003c/b\u003e) was not investigated in detail, therefore, the precise molecular targets and pathways remain to be clarified. These limitations suggest that further studies using mammalian cell-based systems, DNA damage assays, and broader metabolite profiling will be necessary to confirm the protective effects and to elucidate the mechanisms involved. These findings provide the first evidence that \u003cem\u003eP. auriculata\u003c/em\u003e is a source of nanomolar-active antimutagenic naphthoquinones, supporting its potential as a chemopreventive agent.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntimutagenic activity of the \u003cem\u003ePlumbago auriculata\u003c/em\u003e extract, fractions, and compounds against \u003cem\u003eN\u003c/em\u003e-methyl-\u003cem\u003eN\u003c/em\u003e-nitrosourea (MNU)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDose (\u0026micro;g/plate)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e \u003cp\u003eRevertants/plate\u003c/p\u003e \u003cp\u003e(Mutagenicity %)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.625\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.25\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e62.5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e125\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1570\u0026thinsp;\u0026plusmn;\u0026thinsp;147\u003c/p\u003e \u003cp\u003e(100.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1213\u0026thinsp;\u0026plusmn;\u0026thinsp;141\u003c/p\u003e \u003cp\u003e(77.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e862\u0026thinsp;\u0026plusmn;\u0026thinsp;52\u003c/p\u003e \u003cp\u003e(54.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e706\u0026thinsp;\u0026plusmn;\u0026thinsp;53\u003c/p\u003e \u003cp\u003e(44.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e525\u0026thinsp;\u0026plusmn;\u0026thinsp;50\u003c/p\u003e \u003cp\u003e(33.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e223\u0026thinsp;\u0026plusmn;\u0026thinsp;88\u003c/p\u003e \u003cp\u003e(12.6%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDose (\u0026micro;g/plate)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1505\u0026thinsp;\u0026plusmn;\u0026thinsp;29\u003c/p\u003e \u003cp\u003e(100.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e455\u0026thinsp;\u0026plusmn;\u0026thinsp;48\u003c/p\u003e \u003cp\u003e(29.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e105\u0026thinsp;\u0026plusmn;\u0026thinsp;9\u003c/p\u003e \u003cp\u003e(6.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAAR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1505\u0026thinsp;\u0026plusmn;\u0026thinsp;29\u003c/p\u003e \u003cp\u003e(100.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1447\u0026thinsp;\u0026plusmn;\u0026thinsp;46\u003c/p\u003e \u003cp\u003e(96.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1260\u0026thinsp;\u0026plusmn;\u0026thinsp;34\u003c/p\u003e \u003cp\u003e(83.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDose (\u0026micro;g/plate)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAC 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1570\u0026thinsp;\u0026plusmn;\u0026thinsp;16\u003c/p\u003e \u003cp\u003e(100.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1579\u0026thinsp;\u0026plusmn;\u0026thinsp;44\u003c/p\u003e \u003cp\u003e(100.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1329\u0026thinsp;\u0026plusmn;\u0026thinsp;72\u003c/p\u003e \u003cp\u003e(84.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAC 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1570\u0026thinsp;\u0026plusmn;\u0026thinsp;16\u003c/p\u003e \u003cp\u003e(100.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1353\u0026thinsp;\u0026plusmn;\u0026thinsp;67\u003c/p\u003e \u003cp\u003e(86.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1371\u0026thinsp;\u0026plusmn;\u0026thinsp;54\u003c/p\u003e \u003cp\u003e(87.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAC 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1570\u0026thinsp;\u0026plusmn;\u0026thinsp;16\u003c/p\u003e \u003cp\u003e(100.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003cp\u003e(0.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003cp\u003e(0.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAC 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1570\u0026thinsp;\u0026plusmn;\u0026thinsp;16\u003c/p\u003e \u003cp\u003e(100.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1518\u0026thinsp;\u0026plusmn;\u0026thinsp;53\u003c/p\u003e \u003cp\u003e(96.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1289\u0026thinsp;\u0026plusmn;\u0026thinsp;66\u003c/p\u003e \u003cp\u003e(81.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAC 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1570\u0026thinsp;\u0026plusmn;\u0026thinsp;16\u003c/p\u003e \u003cp\u003e(100.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1465\u0026thinsp;\u0026plusmn;\u0026thinsp;54\u003c/p\u003e \u003cp\u003e(93.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1398\u0026thinsp;\u0026plusmn;\u0026thinsp;46\u003c/p\u003e \u003cp\u003e(88.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDose (nmol/plate)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1515\u0026thinsp;\u0026plusmn;\u0026thinsp;56\u003c/p\u003e \u003cp\u003e(100.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e997\u0026thinsp;\u0026plusmn;\u0026thinsp;124\u003c/p\u003e \u003cp\u003e(65.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e736\u0026thinsp;\u0026plusmn;\u0026thinsp;16\u003c/p\u003e \u003cp\u003e(47.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e546\u0026thinsp;\u0026plusmn;\u0026thinsp;26\u003c/p\u003e \u003cp\u003e(35.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e276\u0026thinsp;\u0026plusmn;\u0026thinsp;67\u003c/p\u003e \u003cp\u003e(17.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e57\u0026thinsp;\u0026plusmn;\u0026thinsp;13\u003c/p\u003e \u003cp\u003e(2.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDose (\u0026micro;mol/plate)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1330\u0026thinsp;\u0026plusmn;\u0026thinsp;87\u003c/p\u003e \u003cp\u003e(100.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1356\u0026thinsp;\u0026plusmn;\u0026thinsp;136\u003c/p\u003e \u003cp\u003e(102.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1240\u0026thinsp;\u0026plusmn;\u0026thinsp;123\u003c/p\u003e \u003cp\u003e(93.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1242\u0026thinsp;\u0026plusmn;\u0026thinsp;140\u003c/p\u003e \u003cp\u003e(93.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1062\u0026thinsp;\u0026plusmn;\u0026thinsp;70\u003c/p\u003e \u003cp\u003e(79.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e835\u0026thinsp;\u0026plusmn;\u0026thinsp;120\u003c/p\u003e \u003cp\u003e(62.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDose (\u0026micro;mol/plate)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenistein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1270\u0026thinsp;\u0026plusmn;\u0026thinsp;96\u003c/p\u003e \u003cp\u003e(100.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1190\u0026thinsp;\u0026plusmn;\u0026thinsp;103\u003c/p\u003e \u003cp\u003e(93.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1112\u0026thinsp;\u0026plusmn;\u0026thinsp;38\u003c/p\u003e \u003cp\u003e(87.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e816\u0026thinsp;\u0026plusmn;\u0026thinsp;58\u003c/p\u003e \u003cp\u003e(64.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e520\u0026thinsp;\u0026plusmn;\u0026thinsp;37\u003c/p\u003e \u003cp\u003e(41.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e338\u0026thinsp;\u0026plusmn;\u0026thinsp;72\u003c/p\u003e \u003cp\u003e(26.6%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study provides the first evidence that \u003cem\u003eP. auriculata\u003c/em\u003e roots contain antimutagenic naphthoquinones, plumbagin (\u003cb\u003e1\u003c/b\u003e) and \u003cem\u003ecis\u003c/em\u003e-isoshinanolone (\u003cb\u003e2\u003c/b\u003e), isolated through bioassay-guided fractionation. Purified plumbagin (\u003cb\u003e1\u003c/b\u003e) potently inhibited mutagen-induced revertant formation at nanomolar concentrations, a level of activity rarely reported for natural compounds. PAC exhibited stronger antimutagenic activity than PAAR, and compound \u003cb\u003e1\u003c/b\u003e (plumbagin) was more active than compound \u003cb\u003e2\u003c/b\u003e, suggesting that stereochemical differences in the quinone ring influence their biological interactions. No toxicity was observed at any of the concentrations tested. These findings highlight \u003cem\u003eP. auriculata\u003c/em\u003e as a previously underrecognized source of highly active antimutagenic constituents and underscore the chemopreventive potential of naphthoquinones beyond their classical cytotoxic properties.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement of Informed Consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo human participants were included in this study; therefore, informed consent was not required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. All experimental data supporting the findings, including raw values from Ames tests and compound characterization (HPLC profiles, NMR spectra), are archived at Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Sanyo-Onoda City University, Yamaguchi, Japan and can be provided upon request for verification and replication purposes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of conflicting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest regarding the research, authorship, or publication of this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTakuya Muraoka: Writing\u0026ndash;original draft, project administration, conceptualization. Daisuke Imahori: Data curation. Hiroyuki Tanaka: Data curation. Keiko Inami: Writing\u0026ndash;review and editing, Supervision, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Editage (www.editage.com) for the English language editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWu Z, Xia F, Lin R. Global burden of cancer and associated risk factors in 204 countries and territories, 1980\u0026ndash;2021: a systematic analysis for the GBD 2021. J Hematol Oncol. 2024;17(1):119. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13045-024-01640-8\u003c/span\u003e\u003cspan address=\"10.1186/s13045-024-01640-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar S, Gautam S, Sharma A. 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In vitro callus induction and estimation of plumbagin content from Plumbago auriculata Lam. Indian J Exp Biol. 2014;52:1122\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Plumbago auriculata, roots, naphthoquinones, Ames test, mutagenicity, chemoprevention","lastPublishedDoi":"10.21203/rs.3.rs-8331701/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8331701/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMutagen-induced genotoxicity plays a central role in carcinogenesis, highlighting the need for effective chemopreventive agents. Naphthoquinones, such as plumbagin, exhibit diverse biological activities; however, their antimutagenic potency at pharmacologically relevant concentrations remains poorly characterized. In this study, the acetone extract of \u003cem\u003ePlumbago auriculata\u003c/em\u003e roots was subjected to bioassay-guided fractionation, yielding two known naphthoquinone derivatives: plumbagin and \u003cem\u003ecis\u003c/em\u003e-isoshinanolone.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAntimutagenic activity was evaluated using the Ames test with \u003cem\u003eSalmonella typhimurium\u003c/em\u003e TA1535 against \u003cem\u003eN\u003c/em\u003e-methyl-\u003cem\u003eN\u003c/em\u003e-nitrosourea. The chloroform-soluble fraction (PAC) exhibited stronger activity than the residual fraction (PAAR), and purified plumbagin potently inhibited mutagen-induced revertant formation at nanomolar concentrations. No toxicity was observed at any of the tested doses.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings provide the first evidence that \u003cem\u003eP. auriculata\u003c/em\u003e is a source of nanomolar-active antimutagenic naphthoquinones, highlighting their chemopreventive potential and expanding the biological relevance of quinone-type natural products beyond cytotoxicity.\u003c/p\u003e","manuscriptTitle":"Bioassay-guided isolation of antimutagenic naphthoquinones from Plumbago auriculata","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-22 16:29:23","doi":"10.21203/rs.3.rs-8331701/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a564cd5e-fd8c-45b2-8ff4-e8166e251f7c","owner":[],"postedDate":"December 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-07T09:38:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-22 16:29:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8331701","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8331701","identity":"rs-8331701","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}