The research team, led by Setiasih Setiasih and colleagues at Indonesia’s National Research and Innovation Agency (BRIN) and partner institutions, published their findings in Results in Chemistry. Their strategy centered on a single deceptively simple question: what exactly is inside rose myrtle leaves, and how does the answer change depending on how you extract it? To find out, they harvested leaves from an experimental garden in Riau Province, dried them at 40 degrees Celsius for three days, and ground them into a flour. That flour was then subjected to maceration with three solvents spanning the polarity spectrum: 96 percent ethanol as a polar solvent, ethyl acetate as a semipolar solvent, and n-hexane as a nonpolar solvent. Each extract was subsequently analyzed by liquid chromatography coupled to high-resolution mass spectrometry, or LC-HRMS, at BRIN’s Advanced Characterization Laboratories.

The choice of solvent turned out to matter enormously. LC-HRMS profiling tentatively identified 191 distinct compounds in the ethanol extract, 226 in the ethyl acetate extract, and 206 in the n-hexane extract. Ethanol preferentially pulled out polar molecules, yielding a profile dominated by flavonoid glycosides, phenolic acids and related derivatives, including myricitrin, myricetin, quinic acid derivatives and multiple dihydroxybenzoic acids. These hydroxyl-rich compounds dissolve readily in polar solvents through hydrogen bonding and are classic free-radical scavengers, which helps explain the high antioxidant activity long associated with the plant. The ethyl acetate extract displayed the greatest metabolite diversity of all, dominated by pentacyclic triterpenoids such as ursolic acid, asiatic acid, oleanolic acid, arjungenin and glycyrrhetinic acid derivatives, alongside phenolics including ellagic acid, gallic acid, myricetin and taxifolin. The n-hexane extract, by contrast, was rich in lipophilic sesquiterpenes such as caryophyllene oxide, alpha-farnesene and curcumene, together with assorted triterpenoid derivatives.

The analytical power of the method lies in its ability to go beyond a simple mass readout. Using higher-energy collisional dissociation fragmentation, the researchers could watch molecules break apart in characteristic ways, dramatically raising confidence in their identifications. Myricitrin, for example, produced a precursor ion at mass-to-charge ratio 463.0881 that fragmented to give a product ion at 316.0224, corresponding to the loss of its rhamnose sugar and formation of the myricetin aglycone. Oleanolic acid showed a precursor at 455.3534 with a diagnostic fragment at 411.3472 reflecting loss of carbon dioxide, while caryophyllene oxide in the n-hexane extract yielded sequential ring-cleavage fragments at 161.0962 and 109.1014. The authors are careful to stress that this was qualitative screening rather than quantitative metabolomics, conducted without biological replicates, so peak areas reflect relative signal intensity within a run rather than absolute concentrations. Even so, the picture that emerges is of a plant whose chemistry shifts meaningfully with every solvent a chemist chooses.

With the chemical inventory in hand, the team turned to biological testing. Using the agar disc diffusion method, they challenged four bacterial strains with each extract at concentrations of 25, 50 and 80 percent: Escherichia coli ATCC 25922 and Salmonella typhimurium ATCC 14028 as Gram-negative representatives, and Staphylococcus aureus ATCC 25923 and Listeria monocytogenes ATCC 19115 as Gram-positive representatives. The result was a clean split. Every extract, regardless of solvent, inhibited both Gram-positive species and failed entirely to inhibit either Gram-negative one. Chloramphenicol, the reference antibiotic, produced a 14.82 millimeter zone against S. aureus, larger than the 7.11 to 7.70 millimeter zones produced by the extracts, but the n-hexane extract at 80 percent actually outperformed chloramphenicol against Listeria, generating a 7.17 millimeter zone compared with the antibiotic’s 5.23 millimeters.

The selectivity is rooted in bacterial architecture. Gram-negative bacteria wrap themselves in an outer membrane studded with lipopolysaccharides, an exceptionally effective permeability barrier that excludes hydrophobic molecules and is reinforced by selective porins and efflux pumps that expel antimicrobials before they reach lethal intracellular concentrations. Gram-positive bacteria, lacking that outer membrane, rely on a thick but porous peptidoglycan layer some 20 to 80 nanometers deep, through which small plant metabolites can diffuse relatively freely toward the cytoplasmic membrane. The researchers also observed that solvent type shaped potency: ethanol extracts, rich in phenolics and flavonoids, were most effective against S. aureus, while n-hexane extracts, loaded with membrane-disrupting terpenoids, were the champions against Listeria, a reminder that even within the Gram-positive category, membrane composition and defensive machinery differ enough to matter. The findings align with earlier reports that rose myrtle extracts and the pure compound rhodomyrtone, an antibiotic candidate with activity comparable to vancomycin, are most effective against Gram-positive organisms.

The second half of the study addresses climate. Ruminant livestock account for roughly a quarter of anthropogenic greenhouse gas emissions measured by methane’s contribution, with enteric fermentation in the digestive tract responsible for 80 to 89.5 percent of that methane and manure contributing the rest. Methanogenic archaea in the rumen generate the gas, and feed additives that suppress them are among the most actively pursued mitigation strategies. Because flavonoids, tannins and saponins have documented anti-methanogenic effects, the team tested the ethyl acetate extract, the richest in bioactive triterpenoids and phenolics, in an in vitro rumen fermentation system using Brachiaria ruziziensis grass as the basal feed, at inclusion levels of 1 to 4 percent, with methane measured by gas chromatography over 48 hours.

The outcome was striking in one particular way. Total gas production was unaffected across all treatment levels, meaning fermentation efficiency was preserved, but methane output fell steadily as the extract dose increased. The methane percentage of total gas dropped from 7.72 percent in the untreated control to 3.77 percent at the 4 percent inclusion level, a statistically significant reduction, and methane volume declined as well with a P value of 0.056, right at the threshold of significance. The chemical fingerprint offers a plausible mechanism in several complementary parts. Triterpenoids such as ursolic acid are membrane-active against rumen protozoa, which maintain physical symbioses with methanogenic archaea and supply them with hydrogen; suppressing protozoa often drags methane down with it. Phenolic compounds can selectively inhibit methanogens while redirecting metabolic hydrogen toward propionate synthesis, an energetically favorable route for the animal. Terpenoids such as caryophyllene oxide may interfere with membrane integrity and ion gradients in anaerobic microbes. The authors also cite prior molecular docking work showing that rose myrtle compounds bind strongly to methyl coenzyme M reductase, the enzyme at the heart of methanogenesis, with rhodomyrtial A achieving a predicted binding affinity of minus 10.4 kilocalories per mole.

The researchers are appropriately measured about what these results do and do not prove. The antibacterial assay was a preliminary screen, and they recommend follow-up with minimum inhibitory and bactericidal concentration determinations, plus in vivo testing, to establish therapeutic potential; earlier work suggests possible veterinary applications including mastitis treatment in dairy cows. Similarly, they emphasize that the methane findings provide a chemical rationale rather than a demonstrated field benefit, and that in vivo feeding trials will be required to confirm efficacy and safety. Nevertheless, the study is the first LC-HRMS-based phytochemical characterization of rose myrtle in the context of ruminant nutrition, and it reveals an intertwined network of triterpenoids, phenolics, flavonoids and lipophilic terpenoids whose combined effects may prove more stable than any single isolated compound. In an era when agriculture is under intense pressure to cut emissions without compromising animal productivity, a shrub that grows wild across tropical Asia, fights Gram-positive pathogens in the laboratory, and measurably dampens methane in simulated rumen fluid is exactly the kind of natural lead worth chasing.

Subject of Research: Phytochemical characterization of Rhodomyrtus tomentosa leaves and their antibacterial and methane-mitigating bioactivities

Article Title: LC–HRMS-based phytochemical characterization and biological activities of Rhodomyrtus tomentosa leaves extracts: Antibacterial and methane mitigation potentials

Article References: Setiasih, S., Anggraeny, Y. N., Puspito, S., Widodo, S., Wardi, W., Prihandini, P. W., Antonius, A., Shiddieqy, M. I., Sasongko, W. T., Istiqomah, N., Haryanto, B., Kurniawati, A., Prihartini, I., Wulansari, W. I., & Ernawati, A. (2026). LC–HRMS-based phytochemical characterization and biological activities of Rhodomyrtus tomentosa leaves extracts: Antibacterial and methane mitigation potentials. Results in Chemistry, 30, Article 103812. https://doi.org/10.1016/j.rechem.2026.103812

Image Credits: AI Generated

DOI: 10.1016/j.rechem.2026.103812

Keywords: Rhodomyrtus tomentosa, rose myrtle, LC-HRMS, phytochemical profiling, antibacterial activity, triterpenoids, flavonoids, enteric methane, ruminant nutrition, feed additives, methanogens, Results in Chemistry

The stakes are considerable for the poultry industry. Modern laying hens are biological machines optimized for extraordinary output, producing an egg nearly every day, and that productivity depends on an ovary in constant, high-turnover motion. Follicles are recruited, grown, and ovulated in rapid succession, a process that demands enormous energy, a rich blood supply, and tight hormonal orchestration. It is also a process exquisitely vulnerable to reactive oxygen species. When oxidative stress tips the balance, follicles die off in a process called atresia, hormone levels fall, and laying performance plummets. Because consumers and regulators increasingly demand reductions in synthetic additives, the search for natural, sustainable interventions has become urgent within the One Health framework that links animal, human, and environmental well-being.

To model that stress in a controlled way, the team injected hens with tert-butyl hydroperoxide, or tBHP, a chemical oxidant widely used to induce reproducible oxidative damage in animal studies. Thirty healthy Hy-Line Brown hens were randomly divided into three groups of ten. A control group received a basal diet and saline injections. A stressed group received the basal diet plus tBHP injections every four days for 28 days. The third group received the same tBHP challenge but ate a diet supplemented with 0.6 percent Acer truncatum leaf extract, or ATLE, a preparation exceptionally rich in polyphenols and flavonoids. Over the four-week trial, the researchers tracked egg production, feed efficiency, ovarian anatomy, blood hormones, antioxidant markers, angiogenic factors, and, crucially, the full transcriptomic landscape of the ovary.

The results were striking. Oxidative stress alone drove laying rates down, pushed the feed conversion ratio up, and reduced average daily feed intake, exactly the pattern seen when hens divert resources from reproduction toward survival. It also shrank the pool of hierarchical follicles, the large preovulatory follicles destined for ovulation, and lowered the ovarian stroma index, a measure of the functional tissue supporting follicle development. Histological sections revealed more atretic follicles and necrotic inflammatory foci in the stressed ovaries. Serum concentrations of luteinizing hormone and growth hormone fell, disrupting the hypothalamic-pituitary-gonadal axis that governs follicle recruitment. In hens fed the maple leaf extract, however, nearly all of these parameters rebounded. Laying rate and feed efficiency recovered, hierarchical follicle numbers and stroma index were preserved, and LH and GH concentrations rose significantly compared with the stressed, unsupplemented birds.

The molecular engine behind this rescue appears to be the Nrf2 pathway, the master switch of cellular antioxidant defense. Under normal conditions, the transcription factor Nrf2 is held inactive by its suppressor Keap1; when oxidative pressure rises, Nrf2 escapes, migrates to the nucleus, and switches on a battery of cytoprotective genes. In the stressed hens, the expression of Nrf2 and its downstream targets, including SOD3, GPX3, PRDX4, GSR, and CAT, was significantly suppressed. ATLE supplementation reversed this shutdown, and the biochemical consequences were measurable in both blood and ovarian tissue: superoxide dismutase and catalase activities climbed, while malondialdehyde, a lipid peroxidation product that serves as a fingerprint of oxidative damage, dropped. The authors propose that the extract’s flavonoids and polyphenols act either by directly scavenging reactive oxygen species or by modulating the Keap1-Nrf2 interaction, consistent with prior work showing that resveratrol, theabrownin, and other plant compounds preserve reproductive function through the same axis.

Perhaps the most novel finding concerns blood vessels. Follicle growth is angiogenesis-dependent: each developing follicle must be wrapped in a network of new capillaries that deliver nutrients, oxygen, and hormones. Oxidative stress damages vascular endothelial cells and disrupts hypoxia signaling, suppressing key angiogenic factors such as vascular endothelial growth factor and angiopoietin 1. In the stressed hens, ovarian protein levels of VEGF, ANGPT1, and HIF-1α all fell, along with mRNA expression of VEGFA, ANGPT1, ANGPT2, ITGA5, and MMP9. ATLE supplementation restored these factors, suggesting the extract did more than passively mop up free radicals; it appeared to actively support the reconstruction of a functional vascular scaffold within the ovary. This aligns with known actions of related phytochemicals, such as quercetin promoting angiogenesis through PI3K/Akt signaling and resveratrol stimulating endothelial migration via SIRT1/HIF-1α pathways.

The transcriptomic analysis tied these threads together into a systems-level picture. RNA sequencing of ovarian tissue identified 4,191 differentially expressed genes under oxidative stress and 1,373 genes shifted by ATLE supplementation. The overlap was striking: 91.9 percent of the genes altered by the extract were also among those damaged by oxidative stress, and more than 88 percent of the enriched Gene Ontology terms overlapped between the two comparisons. In other words, the extract appeared to reverse the damage almost point for point. Pathway analysis showed that both injury and repair converged on extracellular matrix organization, focal adhesion, and ECM-receptor interaction, the structural infrastructure that allows endothelial cells to migrate and vascular networks to form. Genes encoding collagens, laminin, fibronectin, and the VEGF receptor KDR were all suppressed by stress and restored by the extract, indicating a coordinated remodeling of the ovarian microenvironment back toward homeostasis.

The study is not without caveats, which the authors acknowledge candidly. The trial lasted only four weeks with ten hens per group, a design well suited to illuminating mechanisms under acute chemical stress but insufficient to predict performance across full commercial laying cycles. ATLE is a complex mixture, and the specific compounds responsible for the effects were not isolated. Most importantly, it remains unresolved whether the extract stimulates angiogenesis directly or whether vascular recovery is simply a secondary benefit of reduced oxidative damage; the interplay between Nrf2 and VEGF signaling has been noted in other contexts, including preeclampsia research. The authors call for endothelial cell models to disentangle cause from effect and for longer trials in production settings.

Even with those limitations, the findings carry real weight for animal agriculture and beyond. They demonstrate that a nutritional intervention can act on multiple signaling axes at once, fortifying antioxidant defenses through Nrf2 while simultaneously preserving the vascular and extracellular matrix architecture that follicles require. They also reinforce a growing theme in reproductive biology: ovarian vascular health, long overlooked, is a central determinant of fertility, and its degradation may underlie reproductive aging in species far beyond the hen. For poultry producers, ATLE now has a scientific rationale as a natural feed additive that combines antioxidant action with microenvironment regulation. For researchers, the study provides a template for evaluating how phytochemical cocktails reshape tissue microenvironments, not merely how they quench radicals. And for anyone following the broader movement toward sustainable, plant-based interventions in animal health, the purpleblow maple has just earned a prominent place on the list of plants worth watching.

The choice of tBHP as the oxidative challenge deserves note. Unlike chronic stressors such as heat or high stocking density, this organic peroxide generates intracellular free radicals directly and predictably, allowing researchers to isolate the biochemical cascade from the many confounders of real-world husbandry. That precision explains why the model is a workhorse in poultry reproductive studies, even though the damage it inflicts is more acute than what a commercial flock typically experiences.

The dual emphasis on Nrf2 and VEGF also reflects an emerging understanding that these pathways are not independent. Reactive oxygen species at moderate levels normally participate in hypoxia-inducible signaling, and when oxidative stress overwhelms that system, both antioxidant gene transcription and angiogenic factor production collapse together. A supplement that restores one axis while sparing the other would leave follicles metabolically protected but still starved of blood supply. The coordinated recovery of SOD and CAT activities alongside VEGF, ANGPT1, and HIF-1α in the supplemented hens suggests the polyphenol mixture acted upstream of both branches, a property single-molecule antioxidants often lack.

The extracellular matrix findings extend this picture beyond the vasculature. Focal adhesion and ECM-receptor interaction pathways provide the physical substrate on which granulosa cells and endothelial cells migrate during follicle growth, and their disruption by stress, followed by restoration of fibronectin, collagen, and the VEGF receptor KDR, indicates repair of tissue architecture rather than mere biochemical correction. For feed manufacturers, the practical implication is that a standardized leaf extract could be formulated into layer diets during vulnerable periods, such as the peak-to-post-peak transition, when oxidative burdens on the ovary are greatest. For comparative biologists, the study adds to evidence that ovarian microvascular integrity is a conserved determinant of fertility, making the laying hen a useful model for nutritional approaches to reproductive resilience in mammals as well.

Subject of Research: Use of Acer truncatum leaf extract as a dietary feed additive to protect laying hen ovarian function from oxidative stress via Nrf2-mediated antioxidant defense and VEGF-mediated angiogenesis.

Article Title: Dietary supplementation of Acer truncatum leaf extract alleviates oxidative-stress induced impairment of ovarian function in laying hens via Nrf2-mediated antioxidant defense and VEGF-mediated angiogenesis

Article References: Qin, K., Ma, J., Gao, M., Liu, Y., & Yang, X. (2026). Dietary supplementation of Acer truncatum leaf extract alleviates oxidative-stress induced impairment of ovarian function in laying hens via Nrf2-mediated antioxidant defense and VEGF-mediated angiogenesis. Stress Biology, 6(1), Article 60. https://doi.org/10.1007/s44154-026-00329-x

Image Credits: AI Generated

DOI: 10.1007/s44154-026-00329-x

Keywords: Acer truncatum leaf extract, laying hens, oxidative stress, Nrf2 pathway, angiogenesis, VEGF, ovarian function, poultry nutrition, reproductive hormones, extracellular matrix remodeling, feed additives, transcriptomics