Cleistocalyx nervosum var. paniala berry extract and cyanidin-3-glucoside inhibit hepatotoxicity and apoptosis.

Excessive oxidative toxicity in liver cells is a significant risk factor that can cause cellular injury, leading to the development of chronic liver disease (CLD). Natural anthocyanins have been shown to prevent the harmful effects of oxidative toxicity in mammalian cells. Ripe Cleistocalyx nervosum var. paniala berry fruits are rich in anthocyanins, which have been reported to possess many health benefits. Therefore, this study examined the protective effect of ethanolic fruit extract of C. nervosum var. paniala (CNPE) against hydrogen peroxide (H2O2)-induced oxidative damage and cell death in human hepatoma HepG2 cells. Results showed that CNPE had strong antioxidant capabilities and high amounts of total phenolics and anthocyanins. HPLC analysis showed that CNPE consists of cyanidin-3-glucoside (C3G). Our investigations found that HepG2 cells pretreated with CNPE or anthocyanin C3G inhibited H2O2-induced cellular damage and apoptosis by increasing the viability of cells, the expression of antiapoptotic Bcl-2 protein, and the activities of cellular antioxidant enzymes, namely SOD, CAT, and GPx. Moreover, both CNPE and C3G significantly suppressed expression of apoptotic proteins (Bax and cytochrome c) and the activities of cleaved caspase-9 and caspase-3 caused by H2O2. Our results indicate that CNPE and C3G can suppress H2O2-induced hepatotoxicity and cell death through stimulation of endogenous antioxidant enzyme activities and inhibition of apoptosis pathway in HepG2 cells. These findings might support development of CNPE as an alternative natural product for preventing CLD.

Neuroprotective mechanisms of luteolin in glutamate-induced oxidative stress and autophagy-mediated neuronal cell death.

Neurodegenerative diseases, characterized by progressive neuronal dysfunction and loss, pose significant health challenges. Glutamate accumulation contributes to neuronal cell death in diseases such as Alzheimer's disease. This study investigates the neuroprotective potential of Albizia lebbeck leaf extract and its major constituent, luteolin, against glutamate-induced hippocampal neuronal cell death. Glutamate-treated HT-22 cells exhibited reduced viability, altered morphology, increased ROS, and apoptosis, which were attenuated by pre-treatment with A. lebbeck extract and luteolin. Luteolin also restored mitochondrial function, decreased mitochondrial superoxide, and preserved mitochondrial morphology. Notably, we first found that luteolin inhibited the excessive process of mitophagy via the inactivation of BNIP3L/NIX and inhibited lysosomal activity. Our study suggests that glutamate-induced autophagy-mediated cell death is attenuated by luteolin via activation of mTORC1. These findings highlight the potential of A. lebbeck as a neuroprotective agent, with luteolin inhibiting glutamate-induced neurotoxicity by regulating autophagy and mitochondrial dynamics.

Neuroprotective Effects of Albizia lebbeck (L.) Benth. Leaf Extract against Glutamate-Induced Endoplasmic Reticulum Stress and Apoptosis in Human Microglial Cells.

Endoplasmic reticulum (ER) stress caused by excessive glutamate in the central nervous system leads to neurodegeneration. Albizia lebbeck (L.) Benth. has been reported to possess neuroprotective properties. We aimed to investigate the effect and mechanism of A. lebbeck leaf extracts on glutamate-induced neurotoxicity and apoptosis linked to ER stress using human microglial HMC3 cells. A. lebbeck leaves were extracted using hexane (AHE), mixed solvents, and ethanol. Each different extract was evaluated for cytotoxic effects on HMC3 cells, and then non-cytotoxic concentrations of the extracts were pretreated with the cells, followed by glutamate. Our results showed that AHE treatment exhibited the highest protective effect and was thus selected for finding the mechanistic approach. AHE inhibited the specific ER stress proteins (calpain1 and caspase-12). AHE also suppressed the apoptotic proteins (Bax, cytochrome c, cleaved caspase-9, and cleaved caspase-3); however, it also increased the antiapoptotic Bcl-2 protein. Remarkably, AHE increased cellular antioxidant activities (SOD, CAT, and GPx). To support the activation of antioxidant defense and inhibition of apoptosis in our HMC3 cell model, the bioactive phytochemicals within AHE were identified by HPLC analysis. We found that AHE had high levels of carotenoids (α-carotene, ß-carotene, and lutein) and flavonoids (quercetin, luteolin, and kaempferol). Our novel findings indicate that AHE can inhibit glutamate-induced neurotoxicity via ER stress and apoptosis signaling pathways by activating cellular antioxidant enzymes in HMC3 cells, suggesting a potential mechanism for neuroprotection. As such, A. lebbeck leaf might potentially represent a promising source and novel alternative approach for preventing neurodegenerative diseases.

Cleistocalyx nervosum var. paniala Berry Seed Protects against TNF-α-Stimulated Neuroinflammation by Inducing HO-1 and Suppressing NF-κB Mechanism in BV-2 Microglial Cells.

Sustained inflammatory responses have been implicated in various neurodegenerative diseases (NDDs). Cleistocalyx nervosum var. paniala (CN), an indigenous berry, has been reported to exhibit several health-beneficial properties. However, investigation of CN seeds is still limited. The objective of this study was to evaluate the protective effects of ethanolic seed extract (CNSE) and mechanisms in BV-2 mouse microglial cells using an inflammatory stimulus, TNF-α. Using LC-MS, ferulic acid, aurentiacin, brassitin, ellagic acid, and alpinetin were found in CNSE. Firstly, we examined molecular docking to elucidate its bioactive components on inflammation-related mechanisms. The results revealed that alpinetin, aurentiacin, and ellagic acid inhibited the NF-κB activation and iNOS function, while alpinetin and aurentiacin only suppressed the COX-2 function. Our cell-based investigation exhibited that cells pretreated with CNSE (5, 10, and 25 µg/mL) reduced the number of spindle cells, which was highly observed in TNF-α treatment (10 ng/mL). CNSE also obstructed TNF-α, IL-1ß, and IL-6 mRNA levels and repressed the TNF-α and IL-6 releases in a culture medium of BV-2 cells. Remarkably, CNSE decreased the phosphorylated forms of ERK, p38MAPK, p65, and IκB-α related to the inhibition of NF-κB binding activity. CNSE obviously induced HO-1 protein expression. Our findings suggest that CNSE offers good potential for preventing inflammatory-related NDDs.

Anti-neuroinflammatory effects of Cleistocalyx nervosum var. paniala berry-seed extract in BV-2 microglial cells via inhibition of MAPKs/NF-κB signaling pathway.

Neuroinflammation is an essential contributor to multiple neurodegenerative disorders. Cleistocalyx nervosum var. paniala, an edible berry, has been reported to exhibit a neuroprotective effect. However, only limited research is available on this fruit seed, which is classified as agricultural food waste. We therefore focused on the anti-neuroinflammatory effects and mechanisms of C. nervosum var. paniala seed extract (CNSE) on lipopolysaccharide (LPS)-induced inflammatory response in BV-2 mouse microglial cells. HPLC analysis showed that CNSE consists of resveratrol (RESV). For cell-based studies, BV-2 cells were pre-treated with CNSE or RESV, followed by LPS. We found that CNSE and RESV inhibited LPS-induced inflammation in a dose-dependent manner. CNSE and RESV inhibited gene expression and activity of iNOS, leading to a decrease in nitric oxide production. Both CNSE and RESV suppressed the gene expression and the activities of TNF-α, IL-1ß, and IL-6. Our results revealed that LPS stimulated the protein levels of MAPKs (JNK, ERK1/2, and p38), while pretreatment of cells with CNSE or RESV attenuated these proteins expressions. CNSE also suppressed NF-κB activation. These results suggest that CNSE and RESV can inhibit LPS-induced inflammatory response through MAPKs/NF-κB pathways in BV-2 cells. Taken together, CNSE have potential as a functional anti-neuroinflammatory agent.

Cleistocalyx nervosum var. paniala Berry Promotes Antioxidant Response and Suppresses Glutamate-Induced Cell Death via SIRT1/Nrf2 Survival Pathway in Hippocampal HT22 Neuronal Cells.

Excessive glutamate neurotransmitters result in oxidative neurotoxicity, similar to neurodegeneration. An indigenous berry of Thailand, Cleistocalyx nervosum var. paniala (CNP), has been recognized for its robust antioxidants. We investigated the effects and mechanisms of CNP fruit extracts on antioxidant-related survival pathways against glutamate-induced neurotoxicity. The extract showed strong antioxidant capability and had high total phenolic and flavonoid contents, particularly resveratrol. Next, the protective effects of the CNP extract or resveratrol on the glutamate-induced neurotoxicity were examined in HT22 hippocampal cells. Our investigation showed that the pretreatment of cells with the CNP extract or resveratrol attenuated glutamate-induced neuronal death via suppression of apoptosis cascade by inhibiting the levels of cleaved- and pro-caspase-3 proteins. The CNP extract and resveratrol suppressed the intracellular ROS by increasing the mRNA expression level of antioxidant enzymes (SODs, GPx1, and CAT). We found that this extract and resveratrol significantly increased SIRT1 expression as a survival-related protein. Moreover, they also promoted the activity of the Nrf2 protein translocation into the nucleus and could bind to the promoter containing the antioxidant response element, inducing the expression of the downstream GPx1-antioxidant protein. Our data illustrate that the CNP extract and resveratrol inhibit apoptotic neuronal death via glutamate-induced oxidative neurotoxicity in HT22 cells through the activation of the SIRT1/Nrf2 survival mechanism.

Evidence of Potential Plant-derived Compounds With Anticancer Effects on Lung Cancer: Clinical and Molecular Pharmacology Approaches.

The effects of plant-derived active compounds on cancer cells have been intensively investigated, leading to the possibility of dietary-based cancer prevention regimens and recommendations for patients with cancer. Many studies have revealed that several compounds can attenuate oxidative stress, suppress survival and proliferative signals, and diminish or suppress cancer stem cells (CSCs). These may provide novel lead compounds for drug development and benefit cancer therapy. The important pharmacological shift in anticancer therapy is the transition of drug discovery for cytotoxic drugs toward targeted therapy and more specific therapy like CSC-targeted therapy. Cancer-driven signaling, as well as survival pathways, have become vital targets for targeted therapeutic drug action. Furthermore, in aggressive cancers, such as lung cancer, it was shown that CSCs drive cancer initiation, progression, metastasis, and therapeutic failure. Moreover, plant-derived compounds are found as a component in diet and are considered safe. Here, we review cancer-protective elements found in plants, including phenolic compounds such as curcumin, carotenoids (ß-carotene and lycopene), epigallocatechin-3-gallate, ginsenoside Rg3, resveratrol, and sulforaphane, for their possible anticancer, anti-metastasis, and cancer-preventive actions against lung cancer, especially in clinical and molecular pharmacological approaches. This review comprehensively summarizes the anticancer properties, target proteins, and CSC suppression capabilities of these plant-derived compounds that may potentially benefit the development of novel anticancer drugs or dietary recommendations for patients with lung cancer.

Caffeine Induces G0/G1 Cell Cycle Arrest and Inhibits Migration through Integrin αv, ß3, and FAK/Akt/c-Myc Signaling Pathway.

Lung cancer is recognized as a major cause of mortality worldwide owing to its metastatic activity. Given the lack of solid information regarding the possible effects of caffeine, one of the most consumed natural psychoactive substances, on molecular signaling pathways implicated in the aggressive behavior of lung cancer, our study aimed to evaluate the effect and mechanism of caffeine on metastasis-related mechanisms. The results revealed that caffeine treatment at concentrations of 0-500 µM caused no direct cytotoxic effects on NCI-H23 cells. Treatment of cells with caffeine showed good potential to inhibit cell proliferation at 48 h and induced significant cell cycle arrest at the G0/G1 phase. Concerning metastasis, caffeine was shown to reduce filopodia formation, inhibit migration and invasion capability, and reduce the ability of cancer cells to survive and grow in an anchorage-independent manner. Moreover, caffeine could attenuate the formation of 3D tumor spheroids in cancer stem cell (CSC)-enriched populations. With regard to mechanisms, we found that caffeine significantly altered the integrin pattern of the treated cells and caused the downregulation of metastasis-associated integrins, namely, integrins αv and ß3. Subsequently, the downstream signals, including protein signaling and transcription factors, namely, phosphorylated focal adhesion kinase (p-FAK), phosphorylated protein kinase B (p-Akt), cell division cycle 42 (Cdc42), and c-Myc, were significantly decreased in caffeine-exposed cells. Taken together, our novel data on caffeine-inhibiting mechanism in relation to metastasis in lung cancer could provide insights into the impact of caffeine intake on human diseases and conditions.

Thunbergia laurifolia Leaf Extract Inhibits Glutamate-Induced Neurotoxicity and Cell Death through Mitophagy Signaling.

Oxidative stress plays a crucial role in neurodegeneration. Therefore, reducing oxidative stress in the brain is an important strategy to prevent neurodegenerative disorders. Thunbergia laurifolia (Rang-jued) is well known as an herbal tea in Thailand. Here, we aimed to determine the protective effects of T. laurifolia leaf extract (TLE) on glutamate-induced oxidative stress toxicity and mitophagy-mediated cell death in mouse hippocampal cells (HT-22). Our results reveal that TLE possesses a high level of bioactive antioxidants by LC-MS technique. We found that the pre-treatment of cells with TLE prevented glutamate-induced neuronal death in a concentration-dependent manner. TLE reduced the intracellular ROS and maintained the mitochondrial membrane potential caused by glutamate. Moreover, TLE upregulated the gene expression of antioxidant enzymes (SOD1, SOD2, CAT, and GPx). Interestingly, glutamate also induced the activation of the mitophagy process. However, TLE could reverse this activity by inhibiting autophagic protein (LC3B-II/LC3B-I) activation and increasing a specific mitochondrial protein (TOM20). Our results suggest that excessive glutamate can cause neuronal death through mitophagy-mediated cell death signaling in HT-22 cells. Our findings indicate that TLE protects cells from neuronal death by stimulating the endogenous antioxidant enzymes and inhibiting glutamate-induced oxidative toxicity via the mitophagy-autophagy pathway. TLE might have potential as an alternative or therapeutic approach in neurodegenerative diseases.

Melatonin and its derivative disrupt cancer stem-like phenotypes of lung cancer cells via AKT downregulation.

Cancer stem cells (CSCs), a small subpopulation of tumour cells, have properties of self-renewal and multipotency, which drive cancer progression and resistance to current treatments. Compounds potentially targeting CSCs have been recently developed. This study shows how melatonin, an endogenous hormone synthesised by the pineal gland, and its derivative suppress CSC-like phenotypes of human non-small cell lung cancer (NSCLC) cell lines, H460, H23, and A549. The effects of MLT and its derivative, acetyl melatonin (ACT), on CSC-like phenotypes were investigated using assays for anchorage-independent growth, three-dimensional spheroid formation, scratch wound healing ability, and CSC marker and upstream protein signalling expression. Enriched CSC spheroids were used to confirm the effect of both compounds on lung cancer cells. MLT and ACT inhibited CSC-like behaviours by suppression of colony and spheroid formation in NSCLC cell lines. Their effects on spheroid formation were confirmed in CSC-enriched H460 cells. CSC markers, CD133 and ALDH1A1, were depleted by both compounds. The behaviour and factors associated to epithelial-mesenchymal transition, as indicated by cell migration and the protein vimentin, were also decreased by MLT and ACT. Mechanistically, MLT and ACT decreased the expression of stemness proteins Oct-4, Nanog, and ß-catenin by reducing active AKT (phosphorylated AKT). Suppression of the AKT pathway was not mediated through melatonin receptors. This study demonstrates a novel role, and its underlying mechanism, for MLT and its derivative ACT in suppression of CSC-like phenotypes in NSCLC cells, indicating that they are potential candidates for lung cancer treatment.

Cyanidin-3-glucoside activates Nrf2-antioxidant response element and protects against glutamate-induced oxidative and endoplasmic reticulum stress in HT22 hippocampal neuronal cells.

BACKGROUND: Cyanidin-3-glucoside (C3G), a major anthocyanin present in berries, exhibits a strong antioxidant and has been shown to possess a neuroprotection. Prolonged exposure to glutamate will lead to oxidative damage and endoplasmic reticulum stress which could play a key detrimental role in the development of neurodegenerative disorders (NDs). In the present study, we investigated the neuroprotective effect and underlying mechanisms of C3G on the reduction of oxidative/ER stress-induced apoptosis by glutamate in HT22 mouse hippocampal neuronal cells. METHOD: Cells were pre-treated with C3G in various concentrations, followed by glutamate. Cell viability and toxicity were examined using MTT and LDH assays. The apoptotic and necrotic cell death were carried out by Annexin V-FITC/propidium iodide co-staining assays. Generation of intracellular reactive oxygen species (ROS) in cells was measured by flow cytometry using DCFH-DA probe. Expression of antioxidant genes was evaluated by Real-time polymerase chain reaction analysis. The possible signaling pathways and proteins involved were subsequently demonstrated by Western blot analysis. RESULT: The pretreatment of the HT22 cells with C3G protected cell death from oxidative toxicity induced by glutamate. We demonstrated that treatment cells with glutamate caused several radical forms of ROS formation, and they were abolished by specific ROS inhibitors. Interestingly, C3G directly scavenged radical activity and inhibited intracellular ROS generation in our cell-based system. In addition, C3G pretreatment suppressed the up-regulation of specific ER proteins namely calpain, caspase-12 and C/EBP homologous proteins (CHOP) induced by glutamate-mediated oxidative and ER stress signal by up-regulating the expressions of survival proteins, including extracellular regulated protein kinase (ERK) and nuclear factor E2-related factor 2 (Nrf2). Furthermore, dramatically activated gene expression of endogenous antioxidant enzymes (i.e. superoxide dismutases (SODs), catalase (CAT) and glutathione peroxidase (GPx)), and phase II enzymes (glutathione-S-transferases (GSTs)) was found in C3G-treated with cells. CONCLUSIONS: Our finding suggest that C3G could be a promising neuroprotectant via inhibition of glutamate-induced oxidative and ER stress signal and activation of ERK/Nrf2 antioxidant mechanism pathways.

Antiaging, Stress Resistance, and Neuroprotective Efficacies of Cleistocalyx nervosum var. paniala Fruit Extracts Using Caenorhabditis elegans Model.

Plant parts and their bioactive compounds are widely used by mankind for their health benefits. Cleistocalyx nervosum var. paniala is one berry fruit, native to Thailand, known to exhibit various health benefits in vitro. The present study was focused on analyzing the antiaging, stress resistance, and neuroprotective effects of C. nervosum in model system Caenorhabditis elegans using physiological assays, fluorescent imaging, and qPCR analysis. The results suggest that the fruit extract was able to significantly extend the median and maximum lifespan of the nematode. It could also extend the healthspan by reducing the accumulation of the "age pigment" lipofuscin, inside the nematode along with regulating the expression of col-19, egl-8, egl-30, dgk-1, and goa-1 genes. Further, the extracts upregulated the expression of daf-16 while downregulating the expression of daf-2 and age-1 in wild-type nematodes. Interestingly, it could extend the lifespan in DAF-16 mutants suggesting that the extension of lifespan and healthspan was dependent and independent of DAF-16-mediated pathway. The fruit extract was also observed to reduce the level of Reactive Oxygen Species (ROS) inside the nematode during oxidative stress. The qPCR analysis suggests the involvement of skn-1 and sir-2.1 in initiating stress resistance by activating the antioxidant mechanism. Additionally, the fruit could also elicit neuroprotection as it could extend the median and maximum lifespan of transgenic strain integrated with Aß. SKN-1 could play a pivotal role in establishing the antiaging, stress resistance, and neuroprotective effect of C. nervosum. Overall, C. nervosum can be used as a nutraceutical in the food industry which could offer potential health benefits.

Eggplant fruits protect against DNA damage and mutations.

Eggplant belongs to the Solanaceae family, and it has an important antioxidant capability that has been shown to counteract oxidation, which is harmful to health and many diseases. In this present study, we evaluated the antigenotoxic effects of six eggplants ((Solanum aculeatissimum Jacq. 'Ma-khuea-lueang'; ML), (Solanum aculeatissimum Jacq. 'Ma-khuea-pro'; MP), (Solanum aculeatissimum Jacq. 'Ma-khuea-sawoei'; MS), (Solanum melongena Linn. 'Ma-khuea-khai-tao'; MKT), (Solanum melongena Linn. 'Ma-khuea-muang klom'; MM) and (Solanum torvum Sw. 'Ma-khuea-phuang'; MPH)) against urethane-induced somatic mutation and recombination test (SMART) in Drosophila melanogaster and hydrogen peroxide-induced oxidative DNA damage in human lymphocytes. First, we determined all of the eggplant extracts of their antioxidant properties including radical scavenging activities, reducing antioxidant power and total phenolic contents, surprisingly ML extract showed the highest level of activity. In SMART, larvae were fed with each lyophilized eggplant. The results revealed that no sample was mutagenic. Interestingly, we found that all six eggplants had a potent inhibitory effect against urethane-induced mutagenicity. Moreover, the protective effect of each eggplant extract against oxidative DNA damage in human lymphocytes was investigated using the single-cell gel electrophoresis (comet) assay. The treatment cells with six eggplant extracts prevented DNA human lymphocytes in response to hydrogen peroxide, especially ML extract exhibited higher an inhibition percentage than other samples. This study demonstrated that these eggplants seem to be safe for consumption and their extracts could protect against DNA damage. Thus, these eggplants have the potential to provide health benefits associated with prevention or reduced risk of developing chronic diseases, such as cancer.

Cleistocalyx nervosum var. paniala berry fruit protects neurotoxicity against endoplasmic reticulum stress-induced apoptosis.

Oxidative and endoplasmic reticulum (ER) stresses cause neuronal damage leading to neurodegenerative disorders. Cleistocalyx nervosum var. paniala (CNP) berry fruit has been shown to possess powerful antioxidant properties. Here, we investigated the neuroprotective effect of CNP extract against glutamate-mediated oxidative/ER stress-induced cell death in mouse hippocampal neuronal HT22 cells. CNP extract was clarified for its radical scavenging activities, total phenolic and anthocyanin contents. The key anthocyanin cyanidin-3-glucoside was used as a marker to standardize the extract used in the study. We found that pretreated cells with CNP extract (0.05-1 µg/ml) prevented neuronal cell death in response to 5 mM glutamate evaluated by cell viability MTT, LDH and apoptosis/necrosis Annexin V/propidium iodide co-staining assays. For mechanistic approach, glutamate-induced cell death through reactive oxygen species (ROS)-mediated ER stress pathways, indicating the increase of ROS and ER stress signature molecules including calpain, caspases-12 and C/EBP homologous proteins (CHOP). CNP extract inhibited ROS production. Moreover, the extract also suppressed the specific-ER stress apoptotic proteins level in glutamate-induced cells by upregulating the gene expression of cellular antioxidant enzymes (SODs, CAT, GPx and GSTs). Taken together, our results provide information about and the molecular mechanism of CNP extract as a promising neuroprotectant and antioxidant.

Nutrients and bioactive compounds of Thai indigenous fruits.

This study determined the nutritional potential of Thai indigenous fruits in terms of nutrients, bioactive compounds, and antioxidant activities. Three indigenous fruits were collected at two conservation areas in Kanchanaburi province, Thailand. The results showed that Phyllanthus emblica L. exhibited the highest levels of vitamin C (575±452mg/100g), total phenolics (TP) (3703±1244mGAE/100g), and antioxidant activities, as measured by DPPH, FRAP and ORAC assays. Compared to the other two fruits, Antidesma velutinosum Blume contained higher levels of most nutrients and dietary fibre (15.6±5.9g/100g), as well as carotenoids (335±98µg/100g) and phytosterols (22.1±3.9mg/100g). Spondias pinnata (L.f.) Kurz was high in total phenolics (3178±887mGAE/100g) and antioxidant activity. Moreover, high correlations were found between TP and antioxidant activities (r>0.9). These Thai indigenous fruits are potentially good sources of nutrients, bioactive compounds, and antioxidant activities. Conservation and utilisation should be promoted for food security and consumption as part of a healthy diet.