Novel Strategy for Strengthening Dermatoporotic Skin by Managing Cellular Senescence.

BACKGROUND: Dermatoporosis (DP) is a condition associated with thinning skin layers and resultant fragility. Much of the thinning is related to fibroblast dysfunction, production of destructive inflammatory cytokines, breakdown of the extracellular matrix (ECM), and weakening of the dermo-epidermal junction. A major contributor to this change in the ECM milieu, previously under-considered, is cellular senescence, particularly involving the papillary dermal fibroblasts. METHODS: A series of experiments were undertaken to explore the impact of a combination of known actives on senescent cell status. Human keratinocytes and fibroblasts were cultured, and cytotoxicity tests were performed to determine the ideal concentration to avoid cell toxicity. Microdoses of Centella asiatica (0.005%) and mandelic acid (0.05%) were found to be ideal in avoiding any cytotoxicity. However, the challenge was then to assess the efficacy of these actives in this microdosed form. After exposing the cells to the compounds, RNA was isolated and sequenced. Moreover, a well-described ex vivo model using photodamaged skin was subjected to immunofluorescence to identify senescent cells (via p16INK4a), particularly in the papillary dermis, using the microdose formulation compared to untreated skin. In addition, JAG/NOTCH expression in the epidermal basal cells was evaluated to further understand the cellular senescence signaling mechanism. RESULTS: Microdosing these two well-known agents had surprisingly significant synergistic effects in vitro, decreasing senescence-associated secretory phenotype (SASP) cytokines and the associated inflammation involved in the process. The ex vivo model revealed a significant (P<0.05) decrease in senescent cells in the papillary dermis and a significant increase (P<0.001) of JAG/NOTCH expression in the basal cells of the epidermis. CONCLUSION: Using microdoses of two known agents, a novel approach produced an unexpected effect of reversal of dermal senescent cells and promoting an anti-inflammatory milieu. A gene expression analysis of the individual and combined actives validated these observations, followed by full formulation testing in an ex vivo model. The approach of limiting cellular senescence in dermal fibroblasts for managing DP is novel and provides an exciting new direction to address dermatoporosis. Clinical studies will follow. J Drugs Dermatol. 2024;23(9):748-756. doi:10.36849/JDD.8388.

Review of Malaysian medicinal plants with potential wound healing activity.

Wound is defined as the damage to biological tissues including skin, mucous membranes and organ tissues. The acute wound heals in less than 4 weeks without complications, while a chronic wound takes longer than 6 weeks to heal. Wound healing occurs in 4 phases, namely, coagulation, inflammatory, proliferative and remodeling phases. Triclosan and benzalkonium chloride are commonly used as skin disinfectants in wound healing. However, they cause allergic contact dermatitis and antibiotic resistance. Medicinal plants are widely studied due to the limited availability of wound healing agents. The present review included six commonly available medicinal plants in Malaysia such as Aloe barbadensis Miller, Carica papaya Linn., Centella asiatica Linn., Cymbopogon nardus Linn., Ficus benghalensis Linn. and Hibiscus rosa sinensis Linn. Various search engines and databases were used to obtain the scientific findings, including Google Scholar, ScienceDirect, PubMed Central and Research Gate. The review discussed the possible mechanism of action of medicinal plants and their active constituents in the wound healing process. In addition, their application in nanotechnology and wound dressings was also discussed in detail.

Centella asiatica extract ameliorates deoxygenation-induced neurological dysfunction in zebrafish larvae.

Background: Oxygen deprivation (OD) is a critical condition that can lead to brain damage and even death. Current hypoxia management approaches are limited in effectiveness. Centella asiatica (CA), known for its neuroprotective properties, offers a potential alternative for OD treatment. Aims: This study aims to investigate the neuroprotective effects of CA on the expression of brain-derived neurotrophic factor (BDNF) and vesicular glutamate transporter 1 (VGLUT1) in zebrafish larvae under oxygen-deficient conditions. Methods: Zebrafish embryos were subjected to low oxygen levels (1.5 mg/l) 0-2 hours post-fertilization (hpf) until 3 days post-fertilization (dpf), simulating the early stages of OD. Subsequent treatment involved varying concentrations of CA (1.25-5 µg/ml) up to 9 days post-fertilization. The expression levels of BDNF and VGLUT1 were measured using PCR methods. Statistical analysis was conducted using a two-way analysis of variance to evaluate the impact of CA on the expression of BDNF and VGLUT1 in zebrafish larvae aged 3 and 9 dpf in oxygen-deprived conditions. Results: CA significantly influenced the expression of BDNF and VGLUT1 under OD (p < 0.001). An increase in BDNF expression (p < 0.001) and a decrease in VGLUT1 (p < 0.01) were observed in zebrafish larvae experiencing OD and treated with CA. There was no significant difference in BDNF and VGLUT1 expression across age variations in zebrafish larvae at 3 dpf and 9 dpf in the treatment groups (p > 0.05). CA concentration of 2.5 µg/ml effectively enhanced BDNF and reduced VGLUT1 in 3-9 dpf zebrafish larvae. Conclusion: CA demonstrates potential as a neuroprotective agent, modulating increased BDNF expression and reduced VGLUT1 under OD conditions. These findings lay a foundation for further research in developing therapies for oxygen deficiency.

Investigating the proliferative inhibition of HepG2 cells by exosome-like nanovesicles derived from Centella asiatica extract through metabolomics.

Nano-particles demonstrating excellent anticancer properties have gradually found application in cancer therapy. However, their widespread use is impeded by their potential toxicity, high cost, and the complexity of the preparation process. In this study, we achieved exosome-like Centella asiatica-derived nanovesicles (ADNVs) through a straightforward juicing and high-speed centrifugation process. We employed transmission electron microscopy and nanoparticle flow cytometry to characterize the morphology, diameter, and stability of the ADNVs. We evaluated the in vitro anticancer effects of ADNVs using Cell Counting Kit-8 and apoptosis assays. Through sequencing and bicinchoninic acid protein analysis, we discovered the abundant presence of proteins and microRNAs in ADNVs. These microRNAs can target various diseases such as cancer and infection. Furthermore, we demonstrated the effective internalization of ADNVs by HepG2 cells, resulting in an increase in reactive oxygen species levels, mitochondrial damage, cell cycle arrest at the G1 phase, and apoptosis. Finally, we analyzed changes in cellular metabolites post-treatment using cell metabolomics techniques. Our findings indicated that ADNVs primarily influence metabolic pathways such as amino acid metabolism and lipid biosynthesis, which are closely associated with HepG2 treatment. Our results demonstrate the potential utility of ADNVs as anticancer agents.

Centella asiatica (L.) Urb. and Hydrocotyle umbellata L. identification and quality assessment: A methodology comparison.

INTRODUCTION: Centella is an important genus in the Apiaceae family. It includes Centella asiatica, which has significant edible and medicinal values. However, this species is easily confused due to its similar morphological traits to Hydrocotyle umbellata, hindering its utilization in the consumer and pharmacological industries. OBJECTIVE: The study aims to differentiate these two closely related plant species using reliable methods of confirming the authenticity of natural herbal medicines. METHODS: Our work mainly focuses on the basic morphological characteristics, chemical markers, genetic fingerprints, and their biological responses. RESULTS: The plants can be clearly differentiated using their leaf shapes, stipules, petioles, inflorescences, and fruit structures. Although the phytochemical compositions of the C. asiatica extract were similar to that of H. umbellata which included flavonoids, tannins, and saponins important to the plant's ability to reduce inflammation and promote healing of wounds, the H. umbellata extract showed significantly higher toxicity than that of C. asiatica. High-performance liquid chromatography analysis was used to identify chemical fingerprints. The result revealed that C. asiatica had major triterpene glycoside constituents including asiaticoside, asiatic acid, madecassoside, and madecassic acid, which have a wide range of medicinal values. In contrast, triterpenoid saponins were not identified in H. umbellata. Furthermore, using SCoT1-6 primers was possible to effectively and sufficiently created a dendrogram which successfully identified the closeness of the plants and confirmed the differences between the two plant species. CONCLUSION: Therefore, differentiation can be achieved through the combination of morphometrics, molecular bioactivity, and chemical analysis.

Effect of pegagan (Centella asiatica) nanoparticle coated with chitosan on the cytokine profile of chronic diabetic mice.

Diabetes is closely related to immune response problems when it occurs chronically. Pegagan (Centella asiatica) is a medicinal plant with active compounds. Madecassoside is beneficial in treating diabetes, and nanoparticle technology is expected to enhance the medicinal potential and availability of pegagan compounds. The aim of this study was to determine the effect of chitosan-coated pegagan nanoparticles on the cytokine profile of chronic diabetic mice, which included CD4+TNF-α+, CD8+TNF-α+, CD4+IFN-γ+, CD8+IFN-γ+ and IL-6+. An experimental study with a randomized complete block design (CRD) consisting of six treatments with seven replicates was conducted. The groups were: healthy mice as negative control; diabetic mice treated with distilled water as positive control and diabetic mice treated with nanoparticle coated with chitosan (NPC) 20 mg/kg, 30 mg/kg, 40 mg/kg, and metformin 130 mg/kgBW. The data were tested using one-way analysis of variance (ANOVA) with a significance level of 5% and continued with the Duncan's multiple range test. The results showed that pegagan NPC could significantly reduce the relative number of CD4+TNF-α+, CD8+TNF-α+, CD4+IFN-γ+ and CD8+IFN-γ+ and IL-6 in the dose of 20 mg/kg, 30 mg/kg and 40 mg/kg (p<0.05). The treatment dose of 20 mg/kg reduced CD4+TNF-α+, CD8+TNF-α+, CD4+IFN-γ+, CD8+IFN-γ+ to the levels of healthy mice and a dose of 30 mg/kg could reduce IL-6 as in healthy mice. These findings suggest that chitosan-coated pegagan nanoparticles are a promising therapy for diabetes, as they have the potential to modulate the immune response associated with chronic diabetes.

Exploring the composition and properties of Centella asiatica metabolites and investigating their impact on BSA glycation, LDL oxidation and α-amylase inhibition.

Centella asiatica (L.) Urb. is a small herbaceous plant belonging to the Apiaceae family that is rich in triterpenes, such as asiaticoside and madecassoside. Centella asiatica finds broad application in promoting wound healing, addressing skin disorders, and boosting both memory and cognitive function. Given its extensive therapeutic potential, this study aimed not only to investigate the Centella asiatica ethanolic extract but also to analyze the biological properties of its organic fractions, such as antioxidant antiglycation capacity, which are little explored. We also identified the main bioactive compounds through spectrometry analysis. The ethanolic extract (EE) was obtained through a static maceration for seven days, while organic fractions (HF: hexane fraction; DF: dichloromethane fraction; EAF: ethyl acetate fraction; BF: n-butanol fraction and HMF: hydromethanolic fraction) were obtained via liquid-liquid fractionation. The concentration of phenolic compounds, flavonoids, and tannins in each sample was quantified. Additionally, the antiglycation (BSA/FRU, BSA/MGO, and ARG/MGO models) and antioxidant (FRAP, ORAC, and DPPH) properties, as well as the ability to inhibit LDL oxidation and hepatic tissue peroxidation were evaluated. The inhibition of enzyme activity was also analyzed (α-amylase, α-glycosidase, acetylcholinesterase, and butyrylcholinesterase). We also evaluated the antimicrobial and cytotoxicity against RAW 264.7 macrophages. The main compounds present in the most bioactive fractions were elucidated through ESI FT-ICR MS and HPLC-ESI-MS/MS analysis. In the assessment of antioxidant capacity (FRAP, ORAC, and DPPH), the EAF and BF fractions exhibited notable results, and as they are the phenolic compounds richest fractions, they also inhibited LDL oxidation, protected the hepatic tissue from peroxidation and inhibited α-amylase activity. Regarding glycation models, the EE, EAF, BF, and HMF fractions demonstrated substantial activity in the BSA/FRU model. However, BF was the only fraction that presented non-cytotoxic activity in RAW 264.7 macrophages at all tested concentrations. In conclusion, this study provides valuable insights into the antioxidant, antiglycation, and enzymatic inhibition capacities of the ethanolic extract and organic fractions of Centella asiatica. The findings suggest that further in vivo studies, particularly focusing on the butanol fraction (BF), may be promising routes for future research and potential therapeutic applications.

Biomarker triterpenoids of Centella asiatica as potential antidepressant agents: Combining in vivo and in silico studies.

Although there are various treatments available for depression, some patients may experience resistance to treatment or encounter adverse effects. Centella asiatica (C. asiatica) is an ancient medicinal herb used in Ayurvedic medicine for its rejuvenating, neuroprotective and psychoactive properties. This study aims to explore the antidepressant-like effects of the major constituents found in C. asiatica, i.e., asiatic acid, asiaticoside, madecassic acid, and madecassoside at three doses (1.25, 2.5, and 5 mg/kg, i.p), on the behavioural and cortisol level of unpredictable chronic stress (UCS) zebrafish model. Based on the findings from the behavioural study, the cortisol levels in the zebrafish body after treatment with the two most effective compounds were measured using enzyme-linked immunosorbent assay (ELISA). Furthermore, a molecular docking study was conducted to predict the inhibitory impact of the triterpenoid compounds on serotonin reuptake. The in vivo results indicate that madecassoside (1.25, 2.5, and 5 mg/kg), asiaticoside and asiatic acid (5 mg/kg) activated locomotor behaviour. Madecassoside at all tested doses and asiaticoside at 2.5 and 5 mg/kg significantly decreased cortisol levels compared to the stressed group, indicating the potential regulation effect of madecassoside and asiaticoside on the hypothalamic-pituitary-adrenal axis overactivity. This study highlights the potential benefits of madecassoside and asiaticoside in alleviating depressive symptoms through their positive effects on behaviour and the hypothalamic-pituitary-adrenal (HPA)- axis in a chronic unpredictable stress zebrafish model. Furthermore, the in silico study provided additional evidence to support these findings. These promising results suggest that C. asiatica may be a valuable and cost-effective therapeutic option for depression, and further research should be conducted to explore its potential benefits.

Centella asiatica mitigates the detrimental effects of Bisphenol-A (BPA) on pancreatic islets.

Bisphenol-A (BPA) is widely used in food packaging and household products, leading to daily human exposure and potential health risks including metabolic diseases like type 2 diabetes mellitus (T2DM). Understanding BPA's mechanisms and developing intervention strategies is urgent. Centella asiatica, a traditional herbal medicine containing pentacyclic triterpenoids, shows promise due to its antioxidant and anti-inflammatory properties, utilized for centuries in Ayurvedic therapy. We investigated the effect of Centella asiatica (CA) ethanol extract on BPA-induced pancreatic islet toxicity in male Swiss albino mice. BPA administration (10 and 100 µg/kg body weight, twice daily) for 21 days caused glucose homeostasis disturbances, insulin resistance, and islet dysfunction, which were partially mitigated by CA supplementation (200 and 400 mg/kg body weight). Additionally, heightened oxidative stress, elevated levels of proinflammatory cytokines, loss of mitochondrial membrane potential (MMP), abnormal cell cycle, and increased apoptosis were implicated in the detrimental impact of BPA on the endocrine pancreas which were effectively counteracted by CA supplementation. In summary, CA demonstrated a significant ability to mitigate BPA-induced apoptosis, modulate redox homeostasis, alleviate inflammation, preserve MMP, and regulate the cell cycle. As a result, CA emerged as a potent agent in neutralizing the diabetogenic effects of BPA to a considerable extent.

Asiatic acid and madecassic acid cause cardiotoxicity via inflammation and production of excessive reactive oxygen species in zebrafish.

Centella asiatica (L.) Urban is a famous Chinese traditional medicine, which is widely used for treating various chronic inflammatory diseases. Although there are reports that Centella total glycosides exhibit heart-protective properties, our previous experiment showed that it has cardiac toxic effects in zebrafish. The components of Centella total glycosides are complex, so we recommend further research to determine their key components and mechanisms. In this study, sample quantification was done using liquid chromatography-tandem mass spectrometry. The cardiotoxicity of Centella total glycosides, asiaticoside, madecassoside, asiatic acid, and madecassic acid was evaluated using zebrafish and cell models. The zebrafish oxidative stress model and myocarditis model were used to explore further the mechanisms through which cardiotoxicity is achieved. Asiatic acid and madecassic acid caused zebrafish cardiotoxicity and H9C2 cell death. However, no toxicity effects were observed for asiaticoside and madecassoside in zebrafish, until the solution was saturated. The results from the cell model study showed that asiatic acid and madecassic acid changed the expression of apoptosis-related genes in myocardial cells. In the zebrafish model, high concentrations of these components raised the levels of induced systemic inflammation, neutrophils gathered in the heart, and oxidative stress injury. Asiatic acid and madecassic acid are the main components causing cardiotoxicity in zebrafish. This may be due to enhanced inflammation and reactive oxygen species injury, which causes myocardial cell apoptosis, which further leads to cardiac toxicity.

Clonal integration of stress signal induces morphological and physiological response of root within clonal network.

Clonal integration of defense or stress signal induced systemic resistance in leaf of interconnected ramets. However, similar effects of stress signal in root are poorly understood within clonal network. Clonal fragments of Centella asiaticas with first-young, second-mature, third-old and fourth-oldest ramets were used to investigate transportation or sharing of stress signal among interconnected ramets suffering from low water availability. Compared with control, oxidative stress in root of the first-young, second-mature and third-old ramets was significantly alleviated by exogenous ABA application to the fourth-oldest ramets as well as enhancement of antioxidant enzyme (SOD, POD, CAT and APX) activities and osmoregulation ability. Surface area and volume in root of the first-young ramets were significantly increased and total length in root of the third-old ramets was significantly decreased. POD activity in root of the fourth-oldest and third-old ramets was significantly enhanced by exogenous ABA application to the first-young ramets. Meanwhile, total length and surface area in root of the fourth-oldest and third-old ramets were significantly decreased. Ratio of belowground to aboveground biomass in the whole clonal fragments was significantly increased by exogenous ABA application to the fourth-oldest or first-young ramets. It is suggested that transportation or sharing of stress signal may induce systemic resistance in root of interconnected ramets. Specially, transportation or sharing of stress signal against phloem flow was observed in the experiment. Possible explanation is that rapid recovery of foliar photosynthesis in first-young ramets subjected to exogenous ABA application can partially reverse phloem flow within clonal network. Thus, our experiment provides insight into ecological implication on clonal integration of stress signal.

Pantoea ananatis, a plant growth stimulating bacterium, and its metabolites isolated from Hydrocotyle umbellata (dollarweed).

A bacterium growing on infected leaves of Hydrocotyle umbellata, commonly known as dollarweed, was isolated and identified as Pantoea ananatis. An ethyl acetate extract of tryptic soy broth (TSB) liquid culture filtrate of the bacterium was subjected to silica gel chromatography to isolate bioactive molecules. Indole was isolated as the major compound that gave a distinct, foul odor to the extract, together with phenethyl alcohol, phenol, tryptophol, N-acyl-homoserine lactone, 3-(methylthio)-1-propanol, cyclo(L-pro-L-tyr), and cyclo(dehydroAla-L-Leu). This is the first report of the isolation of cyclo(dehydroAla-L-Leu) from a Pantoea species. Even though tryptophol is an intermediate in the indoleacetic acid (IAA) pathway, we were unable to detect or isolate IAA. We investigated the effect of P. ananatis inoculum on the growth of plants. Treatment of Lemna paucicostata Hegelm plants with 4 × 109 colony forming units of P. ananatis stimulated their growth by ca. five-fold after 13 days. After 13 days of treatment, some control plants were browning, but treated plants were greener and no plants were browning. The growth of both Cucumis sativus (cucumber) and Sorghum bicolor (sorghum) plants was increased by ca. 20 to 40%, depending on the growth parameter and species, when the rhizosphere was treated with the bacterium after germination at the same concentration. Plant growth promotion by Pantoea ananatis could be due to the provision of the IAA precursor indole.

Intermittent fasting along with hydroalcoholic extract of Centella-asiatica ameliorates sub-acute hypoxia-induced ischemic stroke in adult zebrafish.

Reduced blood flow (hypoxia) to the brain is thought to be the main cause of strokes because it deprives the brain of oxygen and nutrients. An increasing amount of evidence indicates that the Centella-Asiatica (HA-CA) hydroalcoholic extract has a variety of pharmacological benefits, such as antioxidant activity, neuroprotection, anti-inflammatory qualities, and angiogenesis promotion. Intermittent fasting (IF) has neurological benefits such as anti-inflammatory properties, neuroprotective effects, and the ability to enhance neuroplasticity. The current study evaluates the combined effect of IF (for 1, 6, and 12 days) along with HA-CA (daily up to 12 days) in adult zebrafish subjected to hypoxia every 5 min for 12 days followed by behavioral (novel tank and open-field tank test), biochemical (SOD, GSH-Px, and LPO), inflammatory (IL-10, IL-1ß, and TNF-α), mitochondrial enzyme activities (Complex-I, II, and IV), signaling molecules (AMPK, MAPK, GSK-3ß, Nrf2), and imaging/staining (H&E, TTC, and TEM) analysis. Results show that sub-acute hypoxia promotes the behavioral alterations, and production of radical species and alters the oxidative stress status in brain tissues of zebrafish, along with mitochondrial dysfunction, neuroinflammation, and alteration of signaling molecules. Nevertheless, HA-CA along with IF significantly ameliorates these defects in adult zebrafish as compared to their effects alone. Further, imaging analysis significantly provided evidence of infarct damage along with neuronal and mitochondrial damage which was significantly ameliorated by IF and HA-CA. The use of IF and HA-CA has been proven to enhance the physiological effects of hypoxia in all dimensions.

Integrating High-Resolution Mass Spectral Data, Bioassays and Computational Models to Annotate Bioactives in Botanical Extracts: Case Study Analysis of C. asiatica Extract Associates Dicaffeoylquinic Acids with Protection against Amyloid-ß Toxicity.

Rapid screening of botanical extracts for the discovery of bioactive natural products was performed using a fractionation approach in conjunction with flow-injection high-resolution mass spectrometry for obtaining chemical fingerprints of each fraction, enabling the correlation of the relative abundance of molecular features (representing individual phytochemicals) with the read-outs of bioassays. We applied this strategy for discovering and identifying constituents of Centella asiatica (C. asiatica) that protect against Aß cytotoxicity in vitro. C. asiatica has been associated with improving mental health and cognitive function, with potential use in Alzheimer's disease. Human neuroblastoma MC65 cells were exposed to subfractions of an aqueous extract of C. asiatica to evaluate the protective benefit derived from these subfractions against amyloid ß-cytotoxicity. The % viability score of the cells exposed to each subfraction was used in conjunction with the intensity of the molecular features in two computational models, namely Elastic Net and selectivity ratio, to determine the relationship of the peak intensity of molecular features with % viability. Finally, the correlation of mass spectral features with MC65 protection and their abundance in different sub-fractions were visualized using GNPS molecular networking. Both computational methods unequivocally identified dicaffeoylquinic acids as providing strong protection against Aß-toxicity in MC65 cells, in agreement with the protective effects observed for these compounds in previous preclinical model studies.

Sub-chronic oral toxicity of a water-soluble extract of Centella asiatica (Centell-S) in Wistar rats.

BACKGROUND: Centell-S, a water-soluble extract from Centella asiatica, is predominantly composed of madecassoside and asiaticoside, exceeding 80% w/w. Pursuing its development as an herbal medicinal product, Centell-S underwent sub-chronic toxicity assessment adhering to OECD GLP 408 standards. METHODS: In a study involving 100 Wistar rats, varying doses of Centell-S (50, 200, or 800 mg/kg/day) or a vehicle control were administered orally over 90 days. To evaluate Centell-S's safety profile, assessments included clinical observations, health examinations, clinical biochemistry analyses, and detailed anatomical pathology evaluations were conducted. RESULTS: Over the 90 days of treatment, the administration of Centell-S did not lead to any fatalities in the test animals. Clinical observations did not reveal any signs indicative of toxic effects. Notably, an increase in total white blood cell and lymphocyte counts was observed in both sexes, yet these levels returned to normal following a two-week discontinuation period post-treatment. CONCLUSIONS: Under the specific conditions of the OECD GLP 408, Repeated Dose 90-day Oral Toxicity Study in Rodents, the no observed adverse effect level (NOAEL) of Centell-S was 800 mg/kg/day. These findings are promising for the continued development of Centell-S as a phytopharmaceutical for clinical applications.

Centella asiatica improves memory and executive function in middle-aged rats by controlling oxidative stress and cholinergic transmission.

ETHNOPHARMACOLOGICAL RELEVANCE: Centella asiatica (L.) Urban, is a medicinal herb with rich history of traditional use in Indian subcontinent. This herb has been valued for its diverse range of medicinal properties including memory booster, and also as a folk treatment for skin diseases, wound healing and mild diuretic. AIM OF STUDY: Aging is a gradual and continuous process of natural decay in the biological systems, including the brain. This work aims to evaluate the effectiveness of ethanolic extract of Centella asiatica (CAE) on age-associated cognitive impairments in rats, as well as the underlying mechanism. MATERIAL AND METHODS: Rats were allocated into five distinct groups of 5 animals each: Young rats (3 months old rats), middle-aged (m-aged) rats (13-14 months old), and the remaining three groups were comprised of m-aged rats treated with different concentrations of CAE, viz., 150, 300, and 450 mg/kg b. w., orally for 42 days. Y-maze, open field, novel object recognition, and elevated plus maze tests were used to assess animal behavior. The malondialdehyde (MDA), superoxide dismutase (SOD), and acetylcholinesterase (AChE) assays; and H&E staining were done in the rat brain to assess the biochemical and structural changes. CAE was also subjected to HPLC analysis, in vitro antioxidant and anti-cholinergic activity. The active compounds of CAE were docked with AChE and BuChE in molecular docking study. RESULTS: The results showed that CAE treatment improves behavioral performance; attenuates the age-associated increase in MDA content, SOD, and AChE activity; and reduces neuronal loss. In vitro study showed that CAE has concentration-dependent antioxidant and anti-AChE activity. Furthermore, the presence of Asiatic acid and Madecassic acid in CAE and their good binding with cholinergic enzymes (in silico) also suggest the anticholinergic effect of CAE. CONCLUSION: The findings of the current study show that the anticholinergic and antioxidant effects of CAE are attributable to the presence of Asiatic acid and Madecassic acid, which not only provide neuroprotection against age-associated cognitive decline but also reverse it.

Exogenous glycine betaine alleviates water-deficit stress in Indian pennywort (Centella asiatica) under greenhouse conditions.

Centella asiatica (Indian pennywort) is a green leafy vegetable containing centelloside' (triterpenoid), a key phytochemical component in traditional medicine. Being a glycophytic species, they exhibit decline in growth performance and yield traits when subjected to water-deficit (WD) conditions. Glycine betaine (GB) is a low molecular-weight organic metabolite that plays a crucial role in abiotic stress conditions in higher plants. The objective of this study was to investigate the potential of GB in alleviating water-deficit stress (in terms of morphological and physiological responses) in two different genotypes of Indian pennywort, "Nakhon Pathom" (NP; high centelloside-yielding genotype) and "Pathum Thani" (PT; low centelloside-yielding genotype). The genotypes of Indian pennywort were propagated by stolon cutting and transplanted into plastic bags containing 2 kg of garden soil. At the flower-initiation stage (30 days after transplantation), uniform plant material was treated exogenously with 0 (control), 25, and 50 mM GB at 100 mL per plant (one-time foliar spray) and then divided into two groups, 1) well watered (WW; irrigated daily with 400 mL fresh water; 98% field capacity) and 2) water deficit (WD; withheld water for 14 days; 72% field capacity). Foliar application of GB (25 mM) significantly improved leaf osmotic potential in NP under WD conditions via osmotic adjustment by free proline and fructose. Differences in leaf temperature (Tleaf) between WD and WW in NP were maximized (+ 1.93 °C) and the gap of Tleaf was reduced in the case of 25-50 mM GB application. Similarly, crop water stress index (CWSI) in NP and PT plants under WD condition was significantly increased by 1.95- and 1.86-fold over the control, respectively; however, it was significantly decreased by exogenous GB application. Increasing Tleaf and CWSI in drought-stressed plants was closely related to stomatal closure, leading to reduced gas exchange parameters, i.e., stomatal conductance (gs), transpiration rate (E), net photosynthetic rate (Pn), and intercellular CO2 concentration (Ci), and consequently decreased plant biomass and total centelloside yield. Overall physiological, morphological, and secondary metabolite traits were enhanced in NP under WD conditions using 25 mM GB exogenous application compared with the control. The study highlights the significance of GB in Indian pennywort production under limited water irrigation (water deficit) with higher vegetable yield and phytochemical stabilization.

Standardized Centella asiatica extract ECa 233 alleviates pain hypersensitivity by modulating P2X3 in trigeminal neuropathic pain.

OBJECTIVE: During oral surgery and temporomandibular joint repositioning, pain hypersensitivity often occurs due to irritation or inflammation of the nerve endings in the orofacial region. This study aimed to investigate the effects of ECa 233, a Centella asiatica-standardized extract, on the development of mechanical hyperalgesia and allodynia induced by chronic constriction injury of the infraorbital nerve in mice. METHODOLOGY: The right infraorbital nerves of the mice were ligated. Oral carbamazepine (20 mg/kg) or ECa 233 (30, 100, or 300 mg/kg) was administered daily for 21 days. Von Frey and air-puff tests were performed on both sides of the whisker pad on days 0, 7, 14, and 21. Thereafter, the expression of purinergic receptor subtype 3 (P2X3) and voltage-gated sodium channel 1.7 (NaV1.7), a transmembrane protein, in the trigeminal ganglion and c-fos immunoreactivity-positive neurons in the trigeminal nucleus caudalis was assessed. RESULTS: After 21 days of infraorbital nerve ligation, the mice showed allodynia- and hyperalgesia-like behavior, P2X3 and NaV1.7 were upregulated in the trigeminal ganglion, and nociceptive activity increased in the trigeminal nucleus caudalis. However, the oral administration of carbamazepine (20 mg/kg), ECa 233 (100 mg/kg), or ECa 233 (300 mg/kg) mitigated these effects. Nevertheless, ECa 233 failed to affect NaV1.7 protein expression. CONCLUSION: Carbamazepine and ECa 233 can prevent pain hypersensitivity in mice. Considering the side effects of the long-term use of carbamazepine, ECa 233 monotherapy or combined ECa 233 and carbamazepine therapy can be used as an alternative for regulating the development of hypersensitivity in trigeminal pain. However, further detailed clinical studies should be conducted to provide comprehensive information on the use of ECa 233.

The Standardized Extract of Centella asiatica and Its Fractions Exert Antioxidative and Anti-Neuroinflammatory Effects on Microglial Cells and Regulate the Nrf2/HO-1 Signaling Pathway.

Background: Neuroinflammation and oxidative stress can aggravate the progression of Alzheimer's disease (AD). Centella asiatica has been traditionally consumed for memory and cognition. The triterpenes (asiaticoside, madecassoside, asiatic acid, madecassic acid) have been standardized in the ethanolic extract of Centella asiatica (SECA). The bioactivity of the triterpenes in different solvent polarities of SECA is still unknown. Objective: In this study, the antioxidative and anti-neuroinflammatory effects of SECA and its fractions were explored on lipopolysaccharides (LPS)-induced microglial cells. Methods: HPLC measured the four triterpenes in SECA and its fractions. SECA and its fractions were tested for cytotoxicity on microglial cells using MTT assay. NO, pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß), ROS, and MDA (lipid peroxidation) produced by LPS-induced microglial cells were measured by colorimetric assays and ELISA. Nrf2 and HO-1 protein expressions were measured using western blotting. Results: The SECA and its fractions were non-toxic to BV2 microglial cells at tested concentrations. The levels of NO, TNF-α, IL-6, ROS, and lipid peroxidation in LPS-induced BV2 microglial cells were significantly reduced (p < 0.001) by SECA and its fractions. SECA and some of its fractions can activate the Nrf2/HO-1 signaling pathway by significantly enhancing (p < 0.05) the Nrf2 and HO-1 protein expressions. Conclusions: This study suggests that the inhibitory activity of SECA and its fractions on pro-inflammatory and oxidative stress events may be the result of the activation of antioxidant defense systems. The potential of SECA and its fractions in reducing neuroinflammation and oxidative stress can be further studied as a potential therapeutic strategy for AD.