Developing a Rational, Optimized Product of Centella asiatica for Examination in Clinical Trials: Real World Challenges

Botanical products are frequently sold as dietary supplements and their use by the public is increasing in popularity. However, scientific evaluation of their medicinal benefits presents unique challenges due to their chemical complexity, inherent variability, and the involvement of multiple active components and biological targets. Translation away from preclinical models, and developing an optimized, reproducible botanical product for use in clinical trials, presents particular challenges for phytotherapeutic agents compared to single chemical entities. Common deficiencies noted in clinical trials of botanical products include limited characterization of the product tested, inadequate placebo control, and lack of rationale for the type of product tested, dose used, outcome measures or even the study population. Our group has focused on the botanical Centella asiatica due to its reputation for enhancing cognition in Eastern traditional medicine systems. Our preclinical studies on a Centella asiatica water extract (CAW) and its bioactive components strongly support its potential as a phytotherapeutic agent for cognitive decline in aging and Alzheimer's disease through influences on antioxidant response, mitochondrial activity, and synaptic density. Here we describe our robust, scientific approach toward developing a rational phytotherapeutic product based on Centella asiatica for human investigation, addressing multiple factors to optimize its valid clinical evaluation. Specific aspects covered include approaches to identifying an optimal dose range for clinical assessment, design and composition of a dosage form and matching placebo, sourcing appropriate botanical raw material for product manufacture (including the evaluation of active compounds and contaminants), and up-scaling of laboratory extraction methods to available current Good Manufacturing Practice (cGMP) certified industrial facilities. We also address the process of obtaining regulatory approvals to proceed with clinical trials. Our study highlights the complexity of translational research on botanicals and the importance of identifying active compounds and developing sound analytical and bioanalytical methods for their determination in botanical materials and biological samples. Recent Phase I pharmacokinetic studies of our Centella asiatica product in humans (NCT03929250, NCT03937908) have highlighted additional challenges associated with designing botanical bioavailability studies, including specific dietary considerations that need to be considered.

Development of Hairy Root Cultures for Biomass and Triterpenoid Production in Centella asiatica

Centella asiatica (Apiaceae) is a tropical/subtropical medicinal plant, which contains a variety of triterpenoids, including madecassoside, asiaticoside, madecassic acid, and asiatic acid. In this study, we tested the efficiency of hairy root (HR) induction in C. asiatica from leaf and petiole explants. Leaves and petioles collected from C. asiatica plants were suspended in agro-stock for 30 min and co-cultured with Agrobacterium rhizogenes for 3 days to induce HR formation. The transformation efficiency of leaf and petiole explants was approximately 27% and 12%, respectively. A total of 36 HR lines were identified by PCR-based amplification of rol genes, and eight of these lines were selected for further analysis. Among all eight HR lines, the petiole-derived lines HP4 and HP2 displayed the highest growth index (37.8) and the highest triterpenoids concentration (46.57 mg∙g−1), respectively. Although triterpenoid concentration was >2-fold higher in leaves than in petioles of C. asiatica plants, the accumulation of triterpenoids in petiole-derived HR cultures was 1.4-fold higher than that in leaf-derived HR cultures. Additionally, in both leaf- and petiole-derived HR cultures, terpenoid production was higher in HRs than in adventitious roots. These results demonstrate that the triterpenoid content in the explant does not affect the triterpenoid content in the resultant HRs. The HR culture of C. asiatica could be scaled up to enable the mass production of triterpenoids in bioreactors for the pharmaceutical and cosmetic industries.

Withania somnifera and Centella asiatica Extracts Ameliorate Behavioral Deficits in an In Vivo Drosophila melanogaster Model of Oxidative Stress

Due to an increase in the aging population, age-related diseases and age-related changes, such as diminished cognition and sleep disturbances, are an increasing health threat. It has been suggested that an increase in oxidative stress underlies many of these changes. Current treatments for these diseases and changes either have low efficacy or have deleterious side effects preventing long-time use. Therefore, alternative treatments that promote healthy aging and provide resilience against these health threats are needed. The herbs Withania somnifera and Centella asiatica may be two such alternatives because both have been connected with reducing oxidative stress and could therefore ameliorate age-related impairments. To test the effects of these herbs on behavioral phenotypes induced by oxidative stress, we used the Drosophila melanogaster sniffer mutant which has high levels of oxidative stress due to reduced carbonyl reductase activity. Effects on cognition and mobility were assessed using phototaxis assays and both, W. somnifera and C. asiatica water extracts improved phototaxis in sniffer mutants. In addition, W. somnifera improved nighttime sleep in male and female sniffer flies and promoted a less fragmented sleep pattern in male sniffer flies. This suggests that W. somnifera and C. asiatica can ameliorate oxidative stress-related changes in behavior and that by doing so they might promote healthy aging in humans.

Potential remediators in the rice production area of Zambales, Philippines contaminated with mine tailing

High concentration of cadmium and lead are hazardous to environment. The study isolated and identified potential fungal, bacterial and hyperaccumulating plants as bioremediators in contaminated rice ecosystem. Fungi were identified morphologically and with the use of internal transcribed spacer (ITS) region sequencing. Bacteria were identified using 16S ribosomal RNA sequences. Plants were analyzed for Cadmium and Lead accumulation in root and shoot tissues using atomic absorption spectrophotometer (AAS). Fungal species including Penicillium janthinellum, Trichoderma hamatum, Trichoderma harzianum, and Curvularia lunata along with bacterial species such as Bacillus cereus, Bacillus thuringiensis, Pseudomonas gessardii, Lysinibacillus xylanilyticus, Lysinibacillus sphaericus, and two species of unidentified bacteria were identified. Plants predominant in the area includes Cyperus difformis, Scirpus juncoides, Fimbristylis miliacea, Centella asiatica, Sphagneticola trilobata, and Monochoria vaginalis. Cadmium was detected in the shoots of S. trilobata (3.2 mg kg−1) and roots of C. asiatica (3.6 mg kg−1). Lead was found in the shoots of C. asiatica (2.8 mg kg−1) and roots of both S. juncoides (15.00 mg kg−1) and F. miliacea (15.00 mg kg−1). Phytoremediation potential of S. juncoides, F. miliacea, C. asiatica and S. trilobata was observed. Heavy metal resistant microbes can be harnessed as a very useful biological tool for in-situ bioremediation.