Therapeutic Effects of Saponins for the Prevention and Treatment of Cancer by Ameliorating Inflammation and Angiogenesis and Inducing Antioxidant and Apoptotic Effects in Human Cells.

Saponins are natural compounds found in plants and have a diverse range of applications. However, the therapeutic potential of saponins in regulating cytotoxicity, angiogenesis, and inflammation in mammalian cells is yet to be explored. Here, we investigated the therapeutic effects of saponins from green tea by exploring the cytotoxic effects of saponins by inducing apoptosis in the human cancer cell lines hepatocellular carcinoma (HEPG2) and colorectal adenocarcinoma (HT29). The anti-angiogenesis effect of saponins was also investigated in human umbilical vein endothelial cells (HUVEC). We explored the ability of saponins to attenuate inflammation in a dose-dependent manner in normal human cells. It was found that saponins exhibit cytotoxic effects in cancer cells and not in normal cells at the same concentration. Cytotoxicity was measured by inducing apoptosis by enhancing caspase-3 (cas-3) activation and B-cell lymphoma-2 (Bcl-2)-associated X protein (BAX) gene expression and suppressing the antiapoptotic protein, Bcl-2. The inhibition of HUVEC proliferation was due to the suppression of the phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), vascular endothelial growth factor receptor-2 (VEGFR-2), and nuclear factor kappa B (NF-κB). We also observed the antioxidant potential of green tea-derived saponins against free radicals in reactive oxygen species (ROS)-induced cells. Here we observed that the saponins exhibited free radical scavenging activities and activated nuclear factorerythroid 2-related factor 2 (NRF-2) leading to the upregulation of antioxidant-related genes in human embryonic kidney 293 (HEK293) cells. Furthermore, we demonstrated that the anti-inflammatory effects were due to the suppression of pro-inflammatory cytokines interleukin (IL)-1ß, IL-6, tumor necrosis factor-alpha (TNF-α), and inducible nitric oxide synthase (iNOS) in HEK293 cells. The significance of the work is we are the first to report on the anti-cancer effects of saponins based on the anti-inflammatory, antioxidant, anti-angiogenesis, and apoptosis induction properties. In conclusion, green tea-derived saponins could be effective therapeutics for the treatment of cancer.

Neuroprotective Effects of Green Tea Seed Isolated Saponin Due to the Amelioration of Tauopathy and Alleviation of Neuroinflammation: A Therapeutic Approach to Alzheimer's Disease.

Tauopathy is one of the major causes of neurodegenerative disorders and diseases such as Alzheimer's disease (AD). Hyperphosphorylation of tau proteins by various kinases leads to the formation of PHF and NFT and eventually results in tauopathy and AD; similarly, neuroinflammation also exaggerates and accelerates neuropathy and neurodegeneration. Natural products with anti-tauopathy and anti-neuroinflammatory effects are highly recommended as safe and feasible ways of preventing and /or treating neurodegenerative diseases, including AD. In the present study, we isolated theasaponin E1 from ethanol extract of green tea seed and evaluated its therapeutic inhibitory effects on tau hyper-phosphorylation and neuroinflammation in neuroblastoma (SHY-5Y) and glioblastoma (HTB2) cells, respectively, to elucidate the mechanism of the inhibitory effects. The expression of tau-generating and phosphorylation-promoting genes under the effects of theasaponin E1 were determined and assessed by RT- PCR, ELISA, and western blotting. It was found that theasaponin E1 reduced hyperphosphorylation of tau and Aß concentrations significantly, and dose-dependently, by suppressing the expression of GSK3 ß, CDK5, CAMII, MAPK, EPOE4(E4), and PICALM, and enhanced the expression of PP1, PP2A, and TREM2. According to the ELISA and western blotting results, the levels of APP, Aß, and p-tau were reduced by treatment with theasaponin E1. Moreover, theasaponin E1 reduced inflammation by suppressing the Nf-kB pathway and dose-dependently reducing the levels of inflammatory cytokines such as IL-1beta, IL-6, and TNF-alpha etc.

Pharmacological Approaches to Attenuate Inflammation and Obesity with Natural Products Formulations by Regulating the Associated Promoting Molecular Signaling Pathways.

Obesity is a public health problem characterized by increased body weight due to abnormal adipose tissue expansion. Bioactive compound consumption from the diet or intake of dietary supplements is one of the possible ways to control obesity. Natural products with adipogenesis-regulating potential act as obesity treatments. We evaluated the synergistic antiangiogenesis, antiadipogenic and antilipogenic efficacy of standardized rebaudioside A, sativoside, and theasaponin E1 formulations (RASE1) in vitro in human umbilical vein endothelial cells (HUVECs), 3T3-L1 preadipocytes respectively, and in vivo using a high-fat and carbohydrate diet-induced obesity mouse model. Orlistat was used as a positive control, while untreated cells and animals were normal controls (NCs). Adipose tissue, liver, and blood were analyzed after dissection. Extracted stevia compounds and green tea seed saponin E1 exhibited pronounced antiobesity effects when combined. RASE1 inhibited HUVEC proliferation and tube formation by suppressing VEGFR2, NF-κB, PIK3, and-catenin beta-1 expression levels. RASE1 inhibited 3T3-L1 adipocyte differentiation and lipid accumulation by downregulating adipogenesis- and lipogenesis-promoting genes. RASE1 oral administration reduced mouse body and body fat pad weight and blood cholesterol, TG, ALT, AST, glucose, insulin, and adipokine levels. RASE1 suppressed adipogenic and lipid metabolism gene expression in mouse adipose and liver tissues and enhanced AMP-activated protein kinase levels in liver and adipose tissues and in serum adiponectin. RASE1 suppressed the NF-κB pathway and proinflammatory cytokines IL-10, IL-6, and TNF-α levels in mice which involve inflammation and progression of obesity. The overall results indicate RASE1 is a potential therapeutic formulation and functional food for treating or preventing obesity and inflammation.

Green Tea Seed Isolated Theasaponin E1 Ameliorates AD Promoting Neurotoxic Pathogenesis by Attenuating Aß Peptide Levels in SweAPP N2a Cells.

Alzheimer's disease (AD) is the most frequent type of dementia affecting memory, thinking and behaviour. The major hallmark of the disease is pathological neurodegeneration due to abnormal aggregation of Amyloid beta (Aß) peptides generated by ß- and γ-secretases via amyloidogenic pathway. Purpose of the current study was to evaluate the effects of theasaponin E1 on the inhibition of Aß producing ß-, γ-secretases (BACE1, PS1 and NCT) and acetylcholinesterase and activation of the non-amyloidogenic APP processing α-secretase (ADAM10). Additionally, theasaponin E1 effects on Aß degrading and clearing proteins neprilysin and insulin degrading enzyme (IDE). The effect of theasaponin E1 on these crucial enzymes was investigated by RT-PCR, ELISA, western blotting and fluorometric assays using mouse neuroblastoma cells (SweAPP N2a). theasaponin E1 was extracted and purified from green tea seed extract via HPLC, and N2a cells were treated with different concentrations for 24 h. Gene and protein expression in the cells were measured to determine the effects of activation and/or inhibition of theasaponin E1 on ß- and γ-secretases, neprilysin and IDE. Results demonstrated that theasaponin E1 significantly reduced Aß concentration by activation of the α-secretase and neprilysin. The activities of ß- and γ-secretase were reduced in a dose-dependent manner due to downregulation of BACE1, presenilin, and nicastrin. Similarly, theasaponin E1 significantly reduced the activity of acetylcholinesterase. Overall, from the results it is concluded that green tea seed extracted saponin E1 possess therapeutic significance as a neuroprotective natural product recommended for the treatment of Alzheimer's disease.

Crude microcystins extracted from Microcystis aeruginosa exert anti-obesity effects by downregulating angiogenesis and adipogenesis related signaling molecules in HUVEC and 3 T3-L1 cells.

BACKGROUND: Obesity is a risk factor for many diseases including diabetes, cancer, arthritis, and cardiovascular diseases. Angiogenesis nourishes adipose tissues and contributes to obesity; it can be prevented by suppressing the expression of associated signaling molecules. Natural products have garnered attention owing to their safety and efficacy in treating several diseases, including obesity. METHODS: Crude Microcystins were extracted from the blooming Microcystis aeruginosa under stress conditions, by ultrasonication following by solvent extraction. The microcystin extract was evaluated for its potential of inhibiting angiogenesis and adipogenesis. The antiangiogenic activity of the microcystins extract was investigated using human umbilical vein endothelial cells (HUVECs), and its anti-obesity activity was determined in vitro by quantification of the accumulated lipids in mouse 3 T3-L1 cells via Oil Red O staining method. RESULTS: The microcystin extract suppressed HUVECs proliferation and tubes formation in Matrigel in a dose-dependent manner. RT-PCR analysis revealed the downregulation of the mRNA expression of angiogenesis-related signaling molecules, such as PI3K, ß-catenin, vascular endothelial growth factor receptor-2 (VEGFR-2), vascular endothelial-cadherin, Akt1, and NF-κB. Additionally, it inhibited the differentiation of premature 3 T3 cells and lipid accumulation in a dose-dependent manner. It suppressed adipogenesis and lipogenesis by reducing the expression level of peroxisome proliferator-activated receptor γ, CCAAT/enhancer binding protein α, and sterol regulatory element-binding protein. CONCLUSIONS: Crude microcystin exerts anti-angiogenic and anti-obesity effects due to the inhibitory effects on the genes expression of associated signaling molecules and transcriptional factors.

Anthocyanins from Cornus kousa ethanolic extract attenuate obesity in association with anti-angiogenic activities in 3T3-L1 cells by down-regulating adipogeneses and lipogenesis.

Cornus kousa the Korean dogwood has been traditionally used in East Asia as therapeutic traditional medicine however biological activities of Cornus kousa have not been investigated previously. The aim of the present study was to evaluate anti-obesity activities coupled with anti-angiogenic activities of anthocyanins rich fraction of ethanolic leaf extract of Cornus kousa (ELECk) in HUVECs and 3T3- L1 cells. Dried plants leaves were extracted with 70% ethanol and anthocyanin fraction (AnT Fr) was obtained by eluting the ethanolic extract through non-polar macroporous resin and further purification by HPLC. Antiangiogenic activities were determined by antiproliferative effect of AnT Fr on HUVECs. In the presence of various concentrations of AnT Fr, 3T3-L1 preadipocytes were induced to differentiate. Lipid accumulation in differentiated adipocytes were quantified by Oil-Red O staining. AnT Fr significantly suppressed angiogenesis by inhibiting proliferation and tube formation of HUVECs via downregulating VEGRF 2, PI3K, ß-catenin, NF-kB, and Akt1 in a dose dependent manner. AnT Fr inhibited lipid accumulation by down-regulating adipogenesis and lipogenesis promoting signaling proteins, PPARγ, CCAAT, C/EBPα, aP2, FAS, and LPL, however enhanced AMPK activation to p-AMPK in 3T3 cells quantified and expressed by western blotting. AnT Fr inhibit lipid accumulation by regulating adipogenesis and lipogenesis related genes and signaling proteins. The anti-obesity activities exerted by Cornus kousa are associated with antiangiogenic activities of anthocyanins rich fraction of Cornus kousa. Hence the presence of bioactive anthocyanins, Cornus kosa, is a good candidate for nutraceutical and pharmaceutical formulation for treating or controlling obesity.

Green Tea Seed Isolated Saponins Exerts Antibacterial Effects against Various Strains of Gram Positive and Gram Negative Bacteria, a Comprehensive Study In Vitro and In Vivo.

Bacteria are one of the major causes of severe infections and diseases of plants and animals. Salmonella are crucially important due to infection in poultry leading to huge economical loses. Due to high cost and microbial resistance to the currently available chemical antibiotics, demand of screening natural products with antibiotics effects is increased. Plants are rich sources of natural bioactive compounds with antibiotic effects. Saponins are natural compounds of plant sources having a diverse range of applications. In present study we investigated the in vitro and in vivo antibacterial activities of green tea seed extracted saponins. Green tea seeds crude extract was prepared in 70% ethanol by continuous reflux in heating mantel for 5 hours. Crude saponins were extracted from the crude ethanolic extract of green tea seed by column chromatography using macroporous resin (D101). Saponin mixture in fraction 1 (Fr1) was obtained from crude saponins extract via column chromatography. Fr2 and Fr3 were isolated from saponins mixture by preparative HPLC. Antibacterial activities of the isolated saponins fractions were investigated against Escherichia coli (ATCC 25922), Streptococcus aureus (ATCC 12600), and six serovars of Salmonella. In vitro antibacterial activities were determined by disc-diffusion method and growth inhibition in liquid culture using 96-well plate. Results showed that the green tea isolated saponins fractions possess antibacterial effects in the following order Fr1>Fr2>Fr3. Antibacterial mechanism of saponins was elucidated by cell wall and membrane damaging potential of saponins determined by measuring AKP and soluble proteins levels. Fr1 was further used for in vivo antibacterial activities. Five-week grown chickens were selected for in vivo work, divided into three groups as control, infected, and treatment groups. Infected and treatment groups chickens were infected with bacteria and only treatment group chickens were treated with saponins. The qRT- PCR analysis of the blood and feces samples of the different groups' animals shows the presence of bacteria only in infected group while reduced expression levels of the bacterial pathogens were found in the samples of treatment group. Our results demonstrated that the green tea seed saponins used in this study possess strong antibacterial activities.

Taraxacum officinale Weber extracts inhibit LPS-induced oxidative stress and nitric oxide production via the NF-κB modulation in RAW 264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The common dandelion (Taraxacum officinale G.H. Weber ex Wiggers, Asteraceae) has been widely used in folklore medicine to treat dyspepsia, heartburn, and spleen and liver disorders. AIM OF THE STUDY: To compare the antioxidative and anti-inflammatory activities of Taraxacum officinale methanol extract (TOME) and water extract (TOWE) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and assess their constitutional differences, including luteolin, chicoric acid, and total phenol content. MATERIALS AND METHODS: Antioxidative enzyme activities, nitric oxide (NO) production, and inducible NO synthase (iNOS) and nuclear factor (NF)-κB expression were estimated by biochemical analysis, the Griess reaction, reverse transcription-polymerase chain reaction, western hybridization, and electrophoretic mobility shift assay. High-performance liquid chromatography and the Folin-Ciocalteau method were used to analyze functional phytochemicals and total phenol content. RESULTS: TOME and TOWE significantly reduced NO production with an IC(50) of 79.9 and 157.5 µg/mL, respectively, without cytotoxicity. Depleted glutathione (GSH) and antioxidative enzyme activities, including superoxide dismutase, catalase, GSH-peroxidase, and GSH-reductase, were restored by dandelion extracts. Both extracts inhibited LPS-stimulated iNOS gene expression and that of its transcription factor, NF-κB, in parallel with nitrite reduction. TOME showed more potent antioxidative and anti-inflammatory capacities than TOWE, which was attributable to its high total phenol, luteolin, and chicoric acid content. CONCLUSIONS: These results indicate that TOME and TOWE inhibit oxidative stress and inflammatory responses through elevated de novo synthesis of antioxidative enzymes and suppression of iNOS expression by NF-κB inactivation.