Halomicrobium is a genus of haloarchaea, microorganisms that grow in highly saline environments. The study’s isolate was recovered on medium containing 25% sodium chloride. Haloarchaea can tolerate salt concentrations and other environmental stresses, while producing compounds including carotenoids, exopolysaccharides and antimicrobial substances that have been investigated for biotechnological uses.

The researchers collected 17 brine samples from Bahía de Lobos in April 2022. At the time of collection, the brine measured 27 ± 2 °C, had a pH of 7.6 and had electrical conductivity greater than 20 mS/cm. Cultivation produced 96 haloarchaeal isolates, from which red-pigmented colonies were selected for analysis. The resulting strain AS8 formed circular, slightly mucoid colonies measuring 0.5–2 mm and was described as Gram-negative and able to grow with 25% NaCl.

For molecular identification, the researchers extracted genomic DNA, amplified and sequenced the archaeal 16S rRNA gene, and compared the sequence with records in the NCBI GenBank database. They also performed maximum-likelihood phylogenetic analysis using 14 nucleotide sequences and 1,497 positions. The analysis assigned AS8 to Halomicrobium mukohataei with 100% sequence similarity and an E value of 0.0. The researchers described this as the species’ first report from Mexico and the first study of its pigment activity. They also noted the recognized limitation of 16S rRNA analysis for distinguishing closely related species.

For pigment production, AS8 was grown in liquid medium with 25% NaCl for 10 days with shaking at 150 rpm. The researchers extracted separate biomass pigments and supernatant pigments. The yields were 28 mg per gram of biomass and 140 mg per liter of supernatant, respectively. Thin-layer chromatography showed similar profiles for the two preparations, although the supernatant pigments produced a stronger red-pink signal at an Rf value of 0.45. Fourier-transform infrared spectroscopy showed several shared bands with a β-carotene standard; a band at 1,267 cm−1 attributed to COH vibrations was presented as evidence consistent with bacterioruberin, although the chemical characterization was preliminary.

Both pigment preparations showed concentration-dependent radical-scavenging activity in triplicate DPPH and ABTS assays. In the DPPH assay, the reported scavenging values were 74% for supernatant pigments, 67% for biomass pigments and 38% for the β-carotene control. Corresponding ABTS values were 67%, 52% and 34%. These laboratory assay results indicate greater activity for the tested supernatant preparation than for the biomass preparation and the control under the conditions used; they do not demonstrate effects in people.

In triplicate disk-diffusion tests, both preparations produced inhibition zones against six bacterial species: Bacillus sp., Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Biomass-pigment inhibition zones ranged from 7 to 13 mm, while supernatant-pigment zones ranged from 7 to 17 mm. Bacillus sp. was the most susceptible organism, with mean zones of 13 ± 1 mm for biomass pigments and 17 ± 0.5 mm for supernatant pigments. Neither preparation inhibited the tested yeast, Candida albicans, under the assay conditions.

The researchers also conducted an animal-based preliminary lethality assay using nauplii of the brine shrimp Artemia salina rather than testing the pigments in humans or cancer cell lines. Biomass pigments produced 40% nauplii mortality at both 0.1 and 1 mg/mL. Supernatant pigments produced 45% mortality at 1 mg/mL and 40% at 0.1 mg/mL. The authors classified both preparations as moderately toxic under the cited classification, while describing the test as an initial screening of possible cytotoxic activity.

Overall, the study provides laboratory evidence that pigments produced by the Mexican AS8 isolate can scavenge assay radicals, inhibit several tested bacteria and cause brine-shrimp mortality. Thin-layer chromatography and infrared spectroscopy suggest carotenoid-like components, including a possible bacterioruberin component, in the extracts. The findings establish biological activity in laboratory assays, but the antimicrobial, antioxidant and cytotoxicity results do not establish clinical efficacy or safety, and the reported work did not test the pigments in people.

Subject of Research: Isolation and laboratory bioactivity testing of pigments from Halomicrobium mukohataei collected in Sonora, Mexico

Article Title: First report of Halomicrobium mukohataei in Mexico and its biological activity

Article References: Cruz-Luna, D., Pina Canseco, S., Hernández Morales, J. L., Aquino Bolaños, T., & García-Sánchez, E. (2025). First report of Halomicrobium mukohataei in Mexico and its biological activity. Neotropical Biology and Conservation, 20(2), 79-92. https://doi.org/10.3897/neotropical.20.e144508

Image Credits: AI Generated

DOI: 10.3897/neotropical.20.e144508

Keywords: Halomicrobium mukohataei, Bahía de Lobos, haloarchaea, microbial pigments, antioxidant assays, antibacterial activity, bacterioruberin, Artemia salina, Mexico, First, report, Halomicrobium

The stakes of the question are considerable. Liver diseases claim more than two million lives every year across the globe, and in regions where access to diagnostic laboratories and hepatology specialists is limited, traditional medicine often fills the gap. Ethiopia is home to a rich pharmacopoeia of native plants used for jaundice, hepatitis-like illnesses, and other hepatic complaints, yet the majority of these botanicals have never been subjected to systematic toxicological evaluation. That gap between widespread use and thin evidence is precisely what motivated the research team, led by Abel Alemayehu and colleagues with corresponding author Seyoum Gizachew, to put three of the most commonly cited Gamo liver remedies through a standardized lethality screen. Their study was supported by Arba Minch University, which also supplied the brine shrimp eggs used in the assay, and received no external funding.

The technical heart of the study is the brine shrimp lethality assay, or BSLA, a bioassay that has earned its place in natural product research because it is cheap, fast, and surprisingly informative. Brine shrimp eggs, typically of the species Artemia salina, are hatched in artificial seawater under light and aeration, producing a swarm of free-swimming nauplii within a day or two. These nauplii, each only a few hundred micrometers long, are then distributed into wells or vials containing serial dilutions of the plant extract under investigation. After a fixed incubation period, in this case twenty-four hours, the researcher simply counts how many animals have died at each concentration. From those counts, a statistical technique called probit analysis converts the dose-response relationship into a single, comparable number: the lethal concentration fifty, or LC50, the dose that kills half of the exposed population.

The convention used in the field is straightforward. Extracts with an LC50 below 100 micrograms per milliliter are considered highly toxic, those between 100 and 500 micrograms per milliliter are regarded as moderately toxic, and values above 500 micrograms per milliliter are generally taken as relatively safe. The thresholds are not a direct prediction of human toxicity, since brine shrimp are invertebrates and mammalian physiology differs in important ways, but decades of use have shown that very low LC50 values flag plants that contain potent bioactive compounds and warrant caution and further investigation. In this sense the assay functions as an early alarm rather than a final verdict, prioritizing which botanicals should move forward to more sophisticated and expensive testing in cell cultures and animal models.

The Arba Minch team prepared their plant materials in two ways that mirror real-world practice. Aqueous extracts, made by decoction, represent the water-based infusions and decoctions that traditional healers actually administer to patients. The 80 percent methanol extracts, produced by maceration, were included because organic solvents pull out a broader and often more concentrated range of plant chemicals, including compounds that are poorly soluble in water. Methanol extraction is a standard approach in phytochemistry for capturing the full chemical repertoire of a plant, and comparing the two extraction methods allowed the researchers to see whether the water-soluble fraction alone carries the toxic burden or whether less polar constituents contribute as well. Across all three plants, the 80 percent methanol method yielded more extract by weight, with yields ranging from 5.4 to 8.1 percent of the starting plant material.

The results were unambiguous and, in places, startling. All three aqueous extracts fell into the highly toxic category, each with an LC50 below 100 micrograms per milliliter. The most potent of the water extracts came from Amaranthus caudatus, the amaranth known in many parts of the world as a food plant, which killed half of the nauplii at a concentration of just 1.91 micrograms per milliliter. Among the methanol extracts, the highest toxicity was recorded for Clutia lanceolata, with an LC50 of 1.28 micrograms per milliliter, a figure that places it among the more lethal plant extracts reported in this type of screening. Every one of the tested 80 percent methanol extracts also crossed the high-toxicity threshold, meaning that neither the traditional water preparations nor the more exhaustive solvent extractions could be described as benign.

Equally telling was the pattern of mortality over time and dose. The researchers observed that death rates climbed both with increasing extract concentration and with longer exposure, a classic dose- and time-dependent relationship that strengthens confidence in the finding. At the maximum concentration tested, 1000 micrograms per milliliter, all six extracts achieved complete mortality, wiping out every nauplius within twenty-four hours. By contrast, the control group, treated only with distilled water, showed minimal deaths, confirming that the lethality came from the plant chemistry rather than from the test conditions themselves. This internal control matters because brine shrimp nauplii are delicate organisms, and a poorly run assay can produce false positives through simple handling stress or poor water quality. The clean control results indicate the assay behaved as expected.

What might be responsible for such potency? The study did not identify the specific molecules involved, and the authors are careful not to speculate beyond their data. But each of the three plants carries chemical families known to produce strong biological effects. Solanum incanum, a spiny relative of the tomato and eggplant, is notorious for steroidal glycoalkaloids such as solasonine and solamargine, compounds with documented cytotoxic and membrane-disrupting activity. Clutia lanceolata belongs to a genus whose members contain diterpenes and other irritant principles. Amaranthus species contain saponins and other secondary metabolites whose safety profile depends heavily on dose and preparation. Any or all of these could account for the low LC50 values, and teasing out the responsible compounds will require phytochemical fractionation followed by testing of isolated molecules, a natural next step for the team and for other groups working on Ethiopian medicinal flora.

The authors’ conclusion is measured but firm: the aqueous and 80 percent methanol extracts of all three plants are highly toxic in this assay, and their use therefore demands caution. It is worth being precise about what that means and what it does not. A low LC50 in brine shrimp does not establish that a traditional dose harms a human liver, and some celebrated drugs, including several chemotherapy agents, would also fail this screen spectacularly. Toxicity and therapeutic value can coexist, separated only by dose. But when a plant is taken repeatedly, self-administered, and unstandardized, as these Gamo remedies typically are, the margin between a helpful amount and a harmful one becomes a matter of luck rather than measurement. For patients already suffering from liver disease, whose organs may have diminished capacity to handle chemical stress, that uncertainty is especially troubling.

The broader lesson of the study reaches well beyond southern Ethiopia. Around the world, the appeal of natural remedies rests heavily on the intuition that plants, having coexisted with humanity for millennia, must be safe. Toxicology tells a different story: plants are, in effect, untitrated chemical cocktails, and many of the most dangerous poisons known to science come from leaves, roots, and seeds. What the Arba Minch researchers have shown is that a screening assay costing pennies per sample, requiring no specialized animal facility, and deliverable in a university laboratory within days, can flag potentially hazardous botanicals before they cause harm. Their work offers a template that other communities and research groups can replicate cheaply, pairing the preservation of traditional knowledge with the scientific rigor needed to decide which remedies deserve trust and which deserve a warning label.

Subject of Research: Toxicity screening of Ethiopian medicinal plants used for liver diseases using the brine shrimp lethality assay

Article Title: Evaluation of the toxicity of aqueous and 80% methanol extracts of selected medicinal plants used for liver diseases in Gamo Community, South Ethiopia, using the brine shrimp lethality assay

Article References: Alemayehu, A., Gashaye, A., Getachew, F., Dilbar, R., Adane, T., Tufer, S., & Gizachew, S. (2026). Evaluation of the toxicity of aqueous and 80% methanol extracts of selected medicinal plants used for liver diseases in Gamo Community, South Ethiopia, using the brine shrimp lethality assay. BMC Complementary Medicine and Therapies. https://doi.org/10.1186/s12906-026-05575-0

Image Credits: AI Generated

DOI: 10.1186/s12906-026-05575-0

Keywords: brine shrimp lethality assay, Clutia lanceolata, Amaranthus caudatus, Solanum incanum, medicinal plants, liver diseases, toxicity, LC50, Ethiopia, Gamo community, traditional medicine, Artemia salina