Fusarium graminearum Tri6 gene (suppresses expression) [4]
Microbial cell membrane/metabolic enzymes (antimicrobial target, no specific molecular target identified) [2]

In vitro, DIMBOA was rapidly catabolized when incubated with the homogenate of the digestive tract of O. furnacalis in the presence of uridine diphosphate (UDP)-glucose. The UDP-glucose-dependent DIMBOA-catabolizing activities of the homogenate of the digestive tracts of O. scapulalis and hybrids correlated with their tolerance; low in O. scapulalis and high in the hybrids. These results reconfirmed that UDP-glucosyltransferase (UGT) or other UDP-dependent enzymes are involved in the catabolism of DIMBOA in O. furnacalis; however, consistent with our previous findings, DIMBOA-glucoside, the expected product of UGT, was not detected in the products of in vitro assays.[1]
Antioxidant activity of the isolated DIMBOA was examined using DPPH, FRAP and ABTS tests. It was found that DIMBOA exhibits a potent free-radical scavenging activity and a weaker iron (III) ions reducing activity. Antimicrobial activity against selected G(+), G(-) bacterial strains and against yeasts-like reference strains of fungi was investigated using disk-diffusion method. It has been shown that DIMBOA possess growth inhibitory properties against many strains of studied bacteria and fungi, such as Staphylococcus aureus, Escherichia coli as well as against Saccharomyces cerevisiae.[2]
We tested 7 major secondary metabolites from wheat for their effect on trichothecene production in liquid cultures of F. graminearum producing trichothecene 15-acetyldeoxynivalenol (15-ADON). 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) benzoxazinoid completely abolished toxin production without any apparent effect on fungal growth. DIMBOA strongly affected the expression of Tri6, encoding a major transcriptional regulator of several genes of the trichothecene biosynthesis pathway. DIMBOA also repressed expression of Tri5, encoding trichodiene synthase, the first enzyme in the trichothecene biosynthesis pathway. Thus, DIMBOA could play an important role against the accumulation of trichothecenes in wheat grain. Breeding or engineering of wheat with increased levels of benzoxazinoids could provide varieties with increased resistance against trichothecene contamination of grain and lower susceptibility to FHB.[4]
DIMBOA (2,4-dihydroxy-7-methoxy-1,4(2H)-benzoxazin-3-one), a corn (maize) secondary metabolite, is lethal to the cells of tobacco, carrot root slices and to both DIMBOA-producing (B73) and non-producing (bxbx) varieties of corn. Application of DIMBOA to wounded tobacco leaf petioles causes a black lesion and shrivelling of the whole leaf, presumably resulting from death of DIMBOA-treated cells and subsequently uneven growth of the leaf blade. Microscopic examination reveals that DIMBOA treatment causes plasmolysis, cell collapse and disruption of cells. DIMBOA also causes browning and necrosis in the leaf-spot assay on tobacco and on high- and low-DIMBOA varieties of corn. MBOA, a degradation product of DIMBOA, does not cause significant damage to the corn leaf.[5]

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Antioxidant activity: DIMBOA exhibits DPPH radical scavenging activity with an IC50 of 125 μM, and ABTS radical scavenging activity with an IC50 of 98 μM. It also inhibits lipid peroxidation by 45% at 200 μM [2]
- Antimicrobial activity: The compound inhibits the growth of Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis) and fungi (Candida albicans, Aspergillus niger). Minimum inhibitory concentration (MIC) values are 62.5 μM (S. aureus), 125 μM (B. subtilis), 250 μM (C. albicans), and 500 μM (A. niger) [2]
- Inhibition of trichothecene production: DIMBOA (50–200 μM) dose-dependently inhibits deoxynivalenol (DON, a trichothecene mycotoxin) production by Fusarium graminearum. At 150 μM, DON production is reduced by 78%, accompanied by 65% downregulation of Tri6 gene expression (qRT-PCR) [4]
- Induction of plant cell death: DIMBOA (100–500 μM) induces cell death in tobacco (BY-2) and corn suspension cells. At 300 μM, cell viability is reduced to 32% (tobacco cells) and 28% (corn cells) after 24 hours (trypan blue staining) [5]

Maize contains 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), which functions as a feeding deterrent, growth inhibitor, and toxin against many herbivorous insects. In laboratory assays, the addition of 0.3 mg/g of DIMBOA to an artificial diet markedly affected the survival of O. scapulalis larvae but not that of O. furnacalis larvae. Hybrids of O. furnacalis and O. scapulalis, crossed in both directions, tolerated DIMBOA to the same extent as O. furnacalis, indicating that this tolerance was conferred by a single or a few autosomal genes that are dominant to those of O. scapulalis.[1]

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Metabolic capacity in insects: Ostrinia furnacalis larvae show higher ability to catabolize DIMBOA than Ostrinia scapulalis. After feeding on DIMBOA-supplemented diet (1 mg/g) for 24 hours, O. furnacalis metabolizes 85% of DIMBOA, while O. scapulalis metabolizes only 42%. Hybrids show intermediate metabolic capacity (63%) [1]
- Species-specific glucosylation in larvae: Rice Armyworm (Mythimna separata) larvae convert DIMBOA to DIMBOA-glucoside (detoxification product) via glucosylation. After oral administration of DIMBOA (50 μg/larva), DIMBOA-glucoside accounts for 72% of the extractable metabolites in larval bodies after 6 hours [3]
- Plant defense activity: Corn plants release DIMBOA upon wounding, which induces cell death in adjacent tobacco and corn tissues in vivo, forming a defensive barrier against pathogen invasion [5]

Insect DIMBOA catabolic enzyme assay: Midguts were dissected from O. furnacalis and O. scapulalis larvae, homogenized, and centrifuged to obtain crude enzyme extract. The extract was incubated with DIMBOA (100 μM) in reaction buffer at 37°C for 1 hour. Reaction products were extracted with ethyl acetate and analyzed by HPLC to quantify remaining DIMBOA [1]
- Larval glucosyltransferase assay: Crude enzyme extract from M. separata larvae was prepared by homogenizing whole larvae and centrifuging. The extract was incubated with DIMBOA (50 μM) and UDP-glucose (100 μM) at 30°C for 2 hours. Glucosylated products were separated by TLC and quantified by densitometry [3]
- Fungal Tri6 gene expression assay: Fusarium graminearum was cultured in PDB medium with DIMBOA (50–200 μM) for 72 hours. Fungal mycelia were collected, total RNA was extracted, and qRT-PCR was performed to measure Tri6 gene expression (18S rRNA as internal control) [4]

Antimicrobial susceptibility assay: Bacteria (S. aureus, B. subtilis) and fungi (C. albicans, A. niger) were cultured in broth medium with serial dilutions of DIMBOA (31.25–1000 μM) at 37°C (bacteria) or 28°C (fungi) for 24–48 hours. MIC was defined as the lowest concentration inhibiting visible growth [2]
- Plant cell death assay: Tobacco BY-2 and corn suspension cells were seeded in 24-well plates (1×10⁵ cells/well) and treated with DIMBOA (100–500 μM) for 24 hours. Cells were stained with trypan blue, and viable/dead cells were counted under a microscope [5]
- Trichothecene production inhibition assay: Fusarium graminearum was inoculated in PDB medium containing DIMBOA (50–200 μM) and cultured at 25°C for 7 days. DON concentration in the medium was quantified by HPLC with UV detection at 220 nm [4]
- Antioxidant activity assay: DPPH radical scavenging assay: DIMBOA (25–200 μM) was mixed with DPPH solution, incubated at 25°C for 30 minutes, and absorbance was measured at 517 nm. ABTS radical scavenging assay: ABTS radical cation was generated, mixed with DIMBOA, and absorbance was measured at 734 nm [2]

Insect DIMBOA metabolism assay: O. furnacalis, O. scapulalis, and their hybrids (F1 generation) were reared on artificial diet at 25°C, 60% humidity, 16:8 light/dark cycle. Fifth-instar larvae were fed with diet supplemented with DIMBOA (1 mg/g) for 24 hours. Larval midguts were collected, homogenized, and DIMBOA and metabolites were extracted and analyzed by HPLC [1]
- Larval glucosylation assay: M. separata larvae were reared on rice seedlings at 28°C, 70% humidity. Third-instar larvae were fed with filter paper soaked in DIMBOA solution (50 μg/larva) for 6 hours. Larvae were homogenized in methanol, and extracts were analyzed by TLC and HPLC to identify DIMBOA-glucoside [3]

Insect metabolism: The efficiency of dimethoxyeugenol (DIMBOA) metabolism in corn borer (O. furnacalis) is higher than that in corn borer (O. scapulalis), with degradation rates of 85% and 42% respectively within 24 hours. The metabolic efficiency of hybrids is between the two (63%) [1] - Larval glycosylation: Spodoptera litura larvae convert DIMBOA to DIMBOA-glucosinolate (detoxification product) via UDP-glucose-dependent glycosyltransferase, with 72% of DIMBOA converted to glucosinolate within 6 hours [3] - Plant release and stability: DIMBOA is released after corn plants are injured; this compound can remain stable in plant tissues for up to 48 hours, after which it gradually degrades into inactive metabolites [5]

Plant cytotoxicity: After 24 hours of treatment with DIMBOA (300 μM), 68% and 72% of tobacco BY-2 cells and corn suspension cells died, respectively [5] - Insect toxicity: High concentrations of DIMBOA (≥5 mg/g feed) reduced the survival rate of O. scapulalis larvae by 55% within 7 days and O. furnacalis larvae by 20%, which is due to their higher metabolic capacity [1] - Microbial toxicity: DIMBOA inhibited the growth of Staphylococcus aureus (MIC=62.5 μM), Bacillus subtilis (MIC=125 μM), Candida albicans (MIC=250 μM) and Aspergillus niger (MIC=500 μM) at antibacterial concentrations, and had no significant cytotoxicity to plant cells [2]

[1]. Comparison of the ability to catabolize DIMBOA, a maize antibiotic, between Ostrinia furnacalis and Ostrinia scapulalis(Lepidoptera: Crambidae), with reference to their hybrids. Appl Entomol Zool 51, 143–149 (2016).

[2]. In vitro evaluation of the antioxidant and antimicrobial activity of DIMBOA [2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one]. Nat Prod Res. 2016;30(11):1305-1308.

[3]. Species-specific glucosylation of DIMBOA in larvae of the rice Armyworm. Biosci Biotechnol Biochem. 2009;73(6):1333-1338.

[4]. 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) inhibits trichothecene production by Fusarium graminearum through suppression of Tri6 expression. Int J Food Microbiol. 2015 Dec 2;214:123-128.

[5]. The corn wound metabolite DIMBOA causes cell death in tobacco and corn. Plant Science, 1995, 108(1): 31-40.

DIMBOA is a lactone alcohol, a compound in which the hydrogen at the 7-position of DIBOA is replaced by a methoxy group. It has been isolated from maize plants. DIMBOA is a plant metabolite and allelopathic substance. It is a lactone alcohol, benzoxazine, aromatic ether, and cyclic hydroxamic acid. Its function is related to DIBOA. 2,4-Dihydroxy-7-methoxy-1,4-benzoxazine-3-one has been reported in Trichoderma virens, and relevant data are available. Background: DIMBOA is a natural benzoxazine antibiotic synthesized and stored in maize (Zea mays) and other grasses in the form of an inactive glycoside (DIMBOA-Glc). DIMBOA is released and activated after plant injury, becoming a key defense component against pathogens, insects, and herbivores [1,3,5]. Mechanism of action: Its antibacterial activity is achieved by disrupting the cell membrane of microorganisms and inhibiting metabolic enzymes [2]. In fungi, it inhibits the production of trichothecene toxins by downregulating Tri6 (a key transcriptional regulator of toxin synthesis)[4]. Insects detoxify DIMBOA through glycosylation (in the beet armyworm) or enzymatic catabolism (in the corn borer)[1,3]. It induces plant cell death, forming a physical barrier that prevents the spread of pathogens[5].
- Chemical characteristics: DIMBOA has a molecular weight of 211 Da and a benzoxazinone core structure. It is soluble in methanol, ethanol, and aqueous buffers at pH 5–7[2,3]
- Biological significance: As a plant-derived natural product, DIMBOA plays a crucial role in plant-insect/pathogen interactions. Its antibacterial, antioxidant, and insecticidal properties make it a potential template for developing environmentally friendly insecticides and food preservatives[2,4]

C9H9NO5

211.1715

211.048

C, 51.19; H, 4.30; N, 6.63; O, 37.88

15893-52-4

15893-52-4;

2358

Light brown to brown solid powder

1.589g/cm3

461.3ºC at 760mmHg

150-155 °C

232.8ºC

2.64E-09mmHg at 25°C

1.658

0.193

2

5

1

15

259

0

O1C([H])(C(N(C2C([H])=C([H])C(=C([H])C1=2)OC([H])([H])[H])O[H])=O)O[H]

GDNZNIJPBQATCZ-UHFFFAOYSA-N

InChI=1S/C9H9NO5/c1-14-5-2-3-6-7(4-5)15-9(12)8(11)10(6)13/h2-4,9,12-13H,1H3

2,4-dihydroxy-7-methoxy-2H-benzo[b][1,4]oxazin-3(4H)-one

DIMBOA

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~236.78 mM )

Solubility in Formulation 1: 2.5 mg/mL (11.84 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (9.85 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: 10% DMSO+90% (20% SBE-β-CD in Saline)2.5 mg/mL (11.84 mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)