A combined polyherbal formulation containing tongkat ali (Eurycoma longifolia) and kacip fatimah (Labisia pumila) aqueous extracts was evaluated for its safety aspect. A repeated dose 28-day toxicity study using Wistar rats was conducted where the polyherbal formulation was administered at doses 125, 500 and 2000 mg/kg body weight to male and female treatment groups daily via oral gavage, with rats receiving only water as the control group. In-life parameters measured include monitoring of food and water consumption and clinical and functional observations. On day 29, blood was collected for haematological and biochemical analysis. The rats were necropsied and the organs were collected for histopathological examination. This study showed that the combined formulation did not induce any significant toxicity effect at any dose level in terms of morbidity, mortality, behaviour, functional observation, body weight, food and water consumption, whole blood haematology and serum biochemistry. However, there were some microscopic changes in the histopathological examinations of some organs given 2000 mg/kg body weight, which may suggest an early response to the polyherbal formulation. From this study, the no observed adverse effect level is estimated to be more than 500 mg/kg body weight but not exceeding 2000 mg/kg body weight. The observed effects at the highest dose indicate the need for further study of longer dosing duration.

This protocol paper outlines a robust and reproducible framework for a ¹H nuclear magnetic resonance (NMR) metabolomics analysis of rodent plasma, designed to facilitate preclinical biomarker discovery. The protocol details optimised steps for plasma collection in a preclinical rodent model, sample preparation, and NMR data acquisition using presaturation Carr–Purcell–Meiboom–Gill (PRESAT-CPMG) pulse sequences, ensuring high-quality spectral data and effective suppression of macromolecule signals. Comprehensive spectral processing and metabolite assignment are described, with guidance on multivariate and univariate statistical analyses to identify metabolic changes and potential biomarkers. The framework emphasises methodological rigour and reproducibility, enabling accurate quantification and interpretation of metabolites relevant to disease mechanisms or therapeutic interventions. By providing a standardised approach, this protocol supports longitudinal and translational studies, bridging findings from rodent models to clinical applications and advancing the reliability of metabolomics-based biomarker discovery in preclinical research.

In population health the highly cited Atherosclerosis Risk in Communities study indicated a U-shaped association between carbohydrate intake and mortality, whilst the Prospective Urban Rural Epidemiology study linked higher fat intake to lower mortality risks. The Malaysia Lipid Study reported high-fat and high-carbohydrate dietary patterns were associated with increased cardiometabolic risks, including insulin resistance and small dense LDL particles generation. This animal model study therefore was purposely designed to evaluate metabolic outcomes of carbohydrate–fat permutations in Zucker diabetic fatty (ZDF) and Zucker lean (Z_lean) rats by using ¹H Nuclear Magnetic Resonance (NMR) metabolomics. Twenty-four ZDF rats were randomly divided into four groups (n = 6 per group): control (standard diet), Diet A (54%-energy carbohydrate, 32%-energy fat, 14%-energy protein) mimicking a recommended adult Malaysian diet, Diet B (49%-energy carbohydrate, 37%-energy fat, 14%-energy protein) mimicking a low-carbohydrate, moderate high-fat diet, and metformin treatment (100 mg/kg), which effectively represents a 5%-energy exchange in isocaloric meals. An additional six Z_lean were given Diet A (n = 3) and Diet B (n = 3). The intervention lasted eight weeks. Using log-transformed data, analysis of variance (ANOVA) revealed significant differences between the groups for eleven metabolites (1,6-anhydro-β-D-glucose, 2-hydroxyvalerate, acetate, 3-aminoisobutyrate, 3-hydroxybutyrate, carnitine, choline, citrate, creatine, lactate, and N-methylhydantoin) (all p < 0.05) which remained significant even after false discovery rate (FDR) correction. Majorly elevated metabolites in both ZDF and Z_lean rats were 3-hydroxybutyrate, N-methylhydantoin, 3-aminoisobutyrate, and carnitine, indicating dietary influences independent of diabetes status. Conversely, citrate and 1,6-anhydro-β-D-glucose levels showed distinct patterns across the groups, with Z_lean rats exhibiting lower levels and ZDF rats showing higher levels compared to healthy controls, suggesting potential genetic or physiological influences. Other metabolites such as creatine, acetate, choline and 2-hydroxyvalerate showed varied trends, highlighting metabolic complexities. Compared to the control group, metformin treatment generally resulted in lower levels of metabolites, except for acetate, which was higher, indicating improved insulin sensitivity. The findings indicated moderate high-fat diets may exacerbate metabolic disturbances as seen in both ZDF and Z_lean rats, while metformin treatment generally improved metabolic profiles.