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.

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.

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.

Development and validation of a Lao Juan questionnaire.

OBJECTIVES: Lao Juan (LJ, ) is a syndrome described in Chinese medicine (CM) that manifests with fatigue, fever, spontaneous sweating, indigestion, work-induced pain, weakness of the limbs, and shortness of breath. The present study was conducted to examine the reliability and validity of a Lao Juan Questionnaire (LJQ). METHODS: A total of 151 outpatients and 73 normal subjects were asked to complete the LJQ. Seventy-three normal subjects were additionally asked to complete the Chalder Fatigue Scale (CFS). Twelve clinicians determined whether the 151 outpatients exhibited LJ or not. The internal consistency and construct validity for the LJQ were estimated using data from the outpatient subjects. The CFS data were used to examine the concurrent validity of the LJQ. Total LJQ scores and the clinicians᾿diagnoses of the outpatients were used to perform receiver operating characteristics (ROC) curve analyses and to define an optimum cut-off score for the LJQ. RESULTS: The 19-item LJQ had satisfactory internal consistency (α=0.828) and concurrent validity, with significant correlations between the LJQ and the CFS subscales. In the test of construct validity using principal component analysis, a total of six factors were extracted, and the overall variance explained by all factors was 59.5%. In ROC curve analyses, the sensitivity, specificity, and area under the curve were 76.0%, 59.2%, and 0.709, respectively. The optimum cut-off score was defined as six points. CONCLUSIONS: Our results suggest that the LJQ is a reliable and valid instrument for evaluating LJ.

Immunomodulatory effect of tea saponin in immune T-cells and T-lymphoma cells via regulation of Th1, Th2 immune response and MAPK/ERK2 signaling pathway.

The anti-cancer activity of saponins and phenolic compounds present in green tea was previously reported. However, the immunomodulatory and adjuvanticity activity of tea saponin has never been studied. In this study, we investigated the immunomodulatory effect of tea saponin in T-lymphocytes and EL4 cells via regulation of cytokine response and mitogen-activated protein kinases (MAPK) signaling pathway. Quantitative analysis of mRNA expression level of cytokines were performed by reverse transcription polymerase chain reaction following stimulation with tea saponin, ovalbumin (OVA) alone or tea saponin in combination with OVA. Tea saponin inhibited the proliferation of EL4 cells measured in a dose-dependent manner. No cytotoxicity effect of tea saponin was detected in T-lymphocytes; rather, tea saponin enhanced the proliferation of T-lymphocytes. Tea saponin with OVA increased the expression of interleukin (IL)-1, IL-2, IL-12, interferon-γ and tumor necrosis factor (TNF)-α and decreased the expression level of IL-10 and IL-8 in T-lymphocytes. Furthermore, tea saponin, in the presence of OVA, downregulated the MAPK signaling pathway via inhibition of IL-4, IL-8 and nuclear factor kappaB (NF-κB) in EL4 cells. Th1 cytokines enhancer and Th2 cytokines and NF-κB inhibitor, tea saponin can markedly inhibit the proliferation and invasiveness of T-lymphoma (EL4) cells, possibly due to TNF-α- and NF-κB-mediated regulation of MAPK signaling pathway.

Development of a valid and reliable phlegm pattern questionnaire.

OBJECTIVES: The present study was conducted to examine the reliability and validity of a Phlegm Pattern Questionnaire (PPQ) that was developed within the context of Traditional Chinese Medicine. METHODS: A total of 292 normal subjects and 61 inpatients were asked to complete the PPQ. Among the 292 normal subjects, 73 college students were additionally asked to complete the Profile of Mood States (POMS) and the Chalder Fatigue Scale (CFS). Three (3) clinicians determined whether the 61 inpatients exhibited the Phlegm Pattern (PP). The authors estimated the internal consistency and construct validity for the PPQ using data from the normal and inpatient subjects. The POMS and CFS data were used to evaluate the concurrent validity of the PPQ. PPQ data and the clinicians' diagnoses of the inpatient subjects were used to estimate the sensitivity and specificity and to define the optimum cut-off point of the PPQ. RESULTS: The PPQ had satisfactory internal consistency (α=0.902 in normal subjects, α=0.919 in inpatient subjects) and concurrent validity, with significant correlations between the total score of the PPQ and all of the POMS and CFS subscales. In a test of construct validity using principal component analysis, a total of six factors were extracted, and the overall variance explained by all factors was 58.9%. In the test of discriminant validity using receiver operating characteristic curve analysis, the sensitivity, specificity, and area under the curve were 83.78%, 83.33%, and 0.860, respectively. The optimum cut-off level was defined as five points. CONCLUSIONS: These results suggest that the PPQ is a reliable and valid self-rating instrument for estimating the severity of PP.