Higher circulating levels of the branched-chain amino acids (BCAAs; i.e., isoleucine, leucine, and valine) are strongly associated with higher type 2 diabetes risk, but it is not known whether this association is causal. We undertook large-scale human genetic analyses to address this question. Genome-wide studies of BCAA levels in 16,596 individuals revealed five genomic regions associated at genome-wide levels of significance (p < 5 × 10⁻⁸). The strongest signal was 21 kb upstream of the PPM1K gene (beta in standard deviations [SDs] of leucine per allele = 0.08, p = 3.9 × 10⁻²⁵), encoding an activator of the mitochondrial branched-chain alpha-ketoacid dehydrogenase (BCKD) responsible for the rate-limiting step in BCAA catabolism. In another analysis, in up to 47,877 cases of type 2 diabetes and 267,694 controls, a genetically predicted difference of 1 SD in amino acid level was associated with an odds ratio for type 2 diabetes of 1.44 (95% CI 1.26–1.65, p = 9.5 × 10⁻⁸) for isoleucine, 1.85 (95% CI 1.41–2.42, p = 7.3 × 10⁻⁶) for leucine, and 1.54 (95% CI 1.28–1.84, p = 4.2 × 10⁻⁶) for valine. Estimates were highly consistent with those from prospective observational studies of the association between BCAA levels and incident type 2 diabetes in a meta-analysis of 1,992 cases and 4,319 non-cases. Metabolome-wide association analyses of BCAA-raising alleles revealed high specificity to the BCAA pathway and an accumulation of metabolites upstream of branched-chain alpha-ketoacid oxidation, consistent with reduced BCKD activity. Limitations of this study are that, while the association of genetic variants appeared highly specific, the possibility of pleiotropic associations cannot be entirely excluded. Similar to other complex phenotypes, genetic scores used in the study captured a limited proportion of the heritability in BCAA levels. Therefore, it is possible that only some of the mechanisms that increase BCAA levels or affect BCAA metabolism are implicated in type 2 diabetes. Evidence from this large-scale human genetic and metabolomic study is consistent with a causal role of BCAA metabolism in the aetiology of type 2 diabetes.

Centella asiatica (CA) is a culinary vegetable and well-known functional food that is widely used as a medicinal herb and dietary supplement. CA is rich in pentacyclic triterpenes (TTs), including asiaticoside (AS), madecassoside (MS) and the related aglycones asiatic acid (AA), madecassic acid (MA). Traditionally, TTs have been associated with the bioactivity and health promoting effect of CA. Recently, mono-caffeoylquinic acids (MonoCQAs) and di-caffeoylquinic acids (DiCQAs) have been found to contribute to the bioactivity of CA as well. This work reports an analytical strategy based on liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM-MS) for the simultaneous rapid and accurate quantification of 12 bioactive compounds in CA, namely AS, MS, AA, MA, 5-CQA, 4-CQA, 3-CQA, 1,3-DiCQA, 3,4-DiCQA, 1,5-DiCQA, 3,5-DiCQA, 4,5-DiCQA. Method selectivity, accuracy, precision, repeatability, robustness, linearity range, limit of detection (LOD), and limit of quantitation (LOQ) were validated. The validated LC-MRM-MS method has been successfully applied to quantify the 12 bioactive compounds in CA aqueous extracts and two related formulations: a standardized CA product (CAP) used in a phase I clinical trial and formulated CA rodent diets used in preclinical studies. The validated method allows us to support the standardization of CA products used for clinical trials and conduct routine LC-MRM-MS analyses of formulated preclinical diets to confirm correct levels of CA phytochemical markers.

Common symptoms of depressive disorders include anhedonia, sleep problems, and reduced physical activity. Drugs used to treat depression mostly aim to increase serotonin signaling but these can have unwanted side effects. Depression has also been treated by traditional medicine using plants like Centella asiatica (CA) and this has been found to be well tolerated. However, very few controlled studies have addressed CA’s protective role in depression, nor have the active compounds or mechanisms that mediate this function been identified. To address this issue, we used Drosophila melanogaster to investigate whether CA can improve depression-associated symptoms like anhedonia and decreased climbing activity. We found that a water extract of CA provides resilience to stress induced phenotypes and that this effect is primarily due to mono-caffeoylquinic acids found in CA. Furthermore, we describe that the protective function of CA is due to a synergy between chlorogenic acid and one of its isomers also present in CA. However, increasing the concentration of chlorogenic acid can overcome the requirement for the second isomer. Lastly, we found that chlorogenic acid acts via calcineurin, a multifunctional phosphatase that can regulate synaptic transmission and plasticity and is also involved in neuronal maintenance.

Centella asiatica (L.) Urban (also known as “gotu kola”) is a perennial plant, used in traditional medicine for promoting resilience to central nervous system (CNS) disorders. C. asiatica is a tropical medicinal herb from the Apiaceae family and is native to Southeast Asian countries. The chemical composition and contaminant profile of commercial C. asiatica is variable. The goal of this study was to guide the future cultivation of organically grown C. asiatica for obtaining optimized plant materials for pre-clinical studies and clinical trials. Optimized plant materials in this case are defined as producing similar amounts of biologically active components as previously studied material. In this study, C. asiatica cultivars were grown in Central Oregon and their phytochemical compositions were examined. Four different cultivars were grown in climate-controlled greenhouses over three different vegetative propagation periods. Aerial parts of the plant were collected at four different harvest times: 8, 10, 12, and 14 weeks from growth initiation. The phytochemical composition of each cultivar was analyzed by liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). Global metabolomic profiles allowed cultivar-specific compositional differences to be distinguished and production trends of phytochemical constituents to be analyzed using multinomial Bayesian hierarchical clustering and Self-Organizing Maps. Production trends of known bioactive phytoconstituents are reported here and will inform cultivation and harvest strategies to obtain C. asiatica materials of desired composition for preclinical and clinical studies. The computational methods for analyzing cultivar-specific and time-course dependent metabolomic profiles can be applied to other medicinal plant cultivation efforts to optimize cultivation and harvest practices.

Background: A water extract of the Ayurvedic plant Centella asiatica (L.) Urban, family Apiaceae (CAW), improves cognitive function in mouse models of aging and Alzheimer’s disease and affects dendritic arborization, mitochondrial activity, and oxidative stress in mouse primary neurons. Triterpenes (TT) and caffeoylquinic acids (CQA) are constituents associated with these bioactivities of CAW, although little is known about how interactions between these compounds contribute to the plant’s therapeutic benefit. Methods: Mouse primary cortical neurons were treated with CAW or equivalent concentrations of four TT combined, eight CQA combined, or these twelve compounds combined (TTCQA). Treatment effects on the cell transcriptome (18,491 genes) and metabolome (192 metabolites) relative to vehicle control were evaluated using RNAseq and metabolomic analyses, respectively. Results: Extensive differentially expressed genes (DEGs) were seen with all treatments, as well as evidence of interactions between compounds. Notably, many DEGs seen with TT treatment were not observed in the TTCQA condition, possibly suggesting CQA reduced the effects of TT. Moreover, additional gene activity seen with CAW as compared to TTCQA indicates the presence of additional compounds in CAW that further modulate TTCQA interactions. Weighted Gene Correlation Network Analysis (WGCNA) identified 4 gene co-expression modules altered by treatments that were associated with extracellular matrix organization, fatty acid metabolism, cellular response to stress and stimuli, and immune function. Compound interaction patterns were seen at the eigengene level in these modules. Interestingly, in metabolomics analysis, the TTCQA treatment saw the highest number of changes in individual metabolites (20), followed by CQA (15), then TT (8), and finally CAW (3). WGCNA analysis found two metabolomics modules with significant eigenmetabolite differences for TT and CQA and possible compound interactions at this level. Conclusions: Four gene expression modules and two metabolite modules were altered by the four treatment types applied. This methodology demonstrated the existence of both negative and positive interactions between TT, CQA, and additional compounds found in CAW on the transcriptome and metabolome of mouse primary cortical neurons.

Background/objectives: A water extract of the plant Centella asiatica (CAW) has been shown to improve cognitive deficits in aged mice when administered for 5 weeks in drinking water. However, the contribution of the constituent compounds within CAW to the beneficial effects of the extract remains unelucidated. This study evaluated the effects of triterpene (TT) and caffeoylquinic acids (CQA) found within CAW, on learning, cognitive flexibility, memory, and anxiety-like behaviors in aged C57BL6 mice. Methods: Eighteen-month-old male and female C57BL6 mice were administered either TT, CQA, or the combination (TT+CQA) in their drinking water for a total of 5 weeks, at concentrations corresponding to their presence in CAW. During the final two weeks of treatment learning, executive function, memory, and anxiety were assessed. Results: Aged mice of both sexes showed significant improvement in learning when treated with TT and CQA separately and in combination. Treatment with TT also improved cognitive flexibility in aged mice of both sexes, but CQA and the combination of TT+CQA did not alter cognitive flexibility in aged male mice. There was no effect on recognition memory or anxiety in any of the treatment groups (TT, CQA, TT+CQA) relative to mice treated with the vehicle control although there was a trend towards improved recognition memory with TT treatment. Conclusions: These results suggest that the TT and CQA present in CAW likely contribute to its previously reported amelioration of age-related cognitive changes, especially in learning and cognitive flexibility, while other constituents may be responsible for CAW’s anxiolytic effects.