From Medical Herb to Functional Food: Development of a Fermented Milk Containing Silybin and Protein from Milk Thistle.

Milk thistle is a traditional medicinal herb. Silybin is a medicinal component found in the seed coat of milk thistle, which has liver-protective and anti-cancer properties. Conventional studies have focused on the extraction of silybin with organic reagents, which was inapplicable to the food industry. This study aims to develop a fermented milk containing silybin and protein from the milk thistle seeds. A three step procedure was developed, comprising homogenization of milk thistle seeds, NaHCO3 heat treatment, and microbial fermentation. The silybin was characterized by high performance liquid chromatography, and the protein was quantified and electrophorized. It was found that the homogenization step was essential for the preparation of protein, and the NaHCO3 heat treatment was the crucial step in obtaining silybin. The optimal NaHCO3 treatment settings were 1% NaHCO3, 60°C, and 3 h, and the optimal strains for microbial fermentation were L131 (Rummeliibacillus stabekisii) and RS72 (Lactobacillus plantarum). The silybin yield in the fermented milk reached 11.24-12.14 mg/g seeds, accounting for 72.6-78.4% of the total silybin in the milk thistle seeds, and the protein yield reached 121.8-129.6 mg/g seeds. The fermented milk had a slightly sweet yoghurt-like flavor and could be used as a dietary supplement for silybin and protein.

Recent advances on bioactive compounds, biosynthesis mechanism, and physiological functions of Nelumbo nucifera.

Nelumbo nucifera Gaertn, commonly known as lotus, is a genus comprising perennial and rhizomatous aquatic plants, found throughout Asia and Australia. This review aimed to cover the biosynthesis of flavonoids, alkaloids, and lipids in plants and their types in different parts of lotus. This review also examined the physiological functions of bioactive compounds in lotus and the extracts from different organs of the lotus plant. The structures and identities of flavonoids, alkaloids, and lipids in different parts of lotus as well as their biosynthesis were illustrated and updated. In the traditional medicine systems and previous scientific studies, bioactive compounds and the extracts of lotus have been applied for treating inflammation, cancer, liver disease, Alzheimer's disease, etc. We suggest future studies to be focused on standardization of the extract of lotus, and their pharmacological mechanisms as drugs or functional foods. This review is important for the lotus-based food processing and application.

In vitro effects of Pueraria extract on ethanol-exposed microglia and neurons.

Predominant health impacts from alcoholism are chronic neurologic deficits and hepatic dysfunction. Pueraria extract (PE) is a solution obtained from the dried root of Pueraria lobate and can reverse alcohol-induced hepatic damage. The present study aimed to elucidate the effects of PE on ethanol-induced injury in microglia and neurons. To confirm the reliability of the experimental approach, an in vivo demonstration of PE activity was used to verify its impact on hepatic damage in mice exposed to ethanol (ETOH). Subsequently, an in vitro assay was used to verify the effects of PE on ETOH-exposed microglia and neurons.PE reversed fibrosis and hyperplasia, adipocyte infiltration, hepatomegaly, hepatic function, lipid metabolism, indicators of oxidative stress, and morphological changes in hepatic cells, induced by ETOH exposure. The reliability of the experimental approach was thus confirmed. PE also reversed the activation of microglia and inflammatory-related cytokines and proteins induced by ETOH exposure. PE showed protective effects on neurons via inhibition of mitochondrial fission. in vivo and in vitro evidence indicated that PE might be useful in the treatment of both hepatic injury and neurologic deficits commonly observed in chronic alcoholism.

Combination of baicalein and 10-hydroxy camptothecin exerts remarkable synergetic anti-cancer effects.

BACKGROUND: 10-Hydroxy camptothecin (HCPT), a naturally occurring alkaloid, is a clinical drug for cancer chemotherapy. Baicalein (BA) is a flavonoid extracted from the root of Scutellaria baicalensis. The synergistic anti-cancer effect of BA and HCPT has not been reported. PURPOSE: To explore whether and how BA enhances the anti-cancer effect of HCPT in BGC823 cells. METHODS: Cell viability was measured by MTT assay. Apoptosis and cell cycle were analyzed through flow cytometry and western blotting analysis. DNA damage was determined by a comet assay. The activity of topoisomerase I (Topo I) was detected by the plasmid DNA relaxation assay. The synergistic anti-cancer effect of BA and HCPT in vivo was tested by BGC823 xenografted tumor model. RESULTS: BA at non-toxic doses prominently enhanced the anti-cancer activities of HCPT in BGC823, MCF7 and SMMC7721 cells. Combination treatment of BA and HCPT induced BGC823 cells apoptosis mainly via intrinsic rather than extrinsic pathways, and preferentially arresting cell cycle in G1 and G2 phases with the aid of p21. Of note, p53, the upstream regulator of cell apoptosis and cycle, was increased by 5 folds in combination group. It helped to further trigger DNA damage and inhibit Topo I catalytic activity after combination treatment of BA and HCPT. Moreover, the BGC823 xenografted tumor growth rate in nude mice was repressed in a greater degree (P< 0.01) in the combinational group than the single-drug group. CONCLUSION: HCPT and BA, a new and effective combination therapy, synergistically target Topo I and up-regulate p53 to induce cell apoptosis and cell cycle arrest.

BA-j as a novel CDK1 inhibitor selectively induces apoptosis in cancer cells by regulating ROS.

Cyclin-dependent kinase 1 (CDK1) is the only necessary CDK in cell proliferation and a novel target in the development of anticancer drugs. 8-Hydroxypiperidinemethyl-baicalein (BA-j) is a novel selective CDK1 inhibitor with broad spectrum anti-cancer activity (IC50 12.3 µM) and 2 tumor xenografts. Because of the differential mechanisms controlling redox-states in normal and cancer cells, BA-j can capture oxygen free radicals ((·)O2(-)) and selectively increase the level of H2O2 in cancer cells, thereby specifically oxidize and activate the intrinsic apoptosis pathway bypassing the extrinsic death receptor pathway, thus inducing apoptosis in cancer cells rather than in normal cells. BA-j is different from cytotoxic anticancer drugs which can activate both the intrinsic apoptosis pathway and the extrinsic death receptor pathway, and therefore harm normal cells while killing cancer cells. The molecular and biochemical mechanisms of reactive oxygen species (ROS) regulation suggest that BA-j may be developed into a novel anticancer agent.

Effects of resibufogenin on voltage-gated sodium channels in cultured rat hippocampal neurons.

Voltage-gated sodium channels (VGSCs) play important roles in maintaining the excitability of hippocampal neurons. The present study investigated the effects of resibufogenin (RBG, a main component of bufadienolides) on voltage-gated sodium channel currents (I(Na)) in rat hippocampal neurons using whole-cell patch clamp recording. According to the results, RBG activated I(Na) in a concentration-dependent manner. RBG at 1 µM concentration could alter some channel kinetics of I(Na), such as activation thresholds, steady-state activation and inactivation curves, time constant of recovery, and activity-dependent attenuation of I(Na). RBG influenced peak amplitude, overshoot and half-width of the evoked single action potential, and simultaneously lessened the firing rate of evoked repetitive firing. These findings suggested that I(Na) is probably a target of RBG, which may explain the mechanisms for the pathological effects of RBG on central nervous system.

Apoptosis of BGC823 cell line induced by p-hydroxymethoxybenzobijuglone, a novel compound from Juglans mandshurica.

p-Hydroxymethoxybenzobijuglone (HMBBJ), a new quinone compound isolated from Juglans mandshurica (by bioassay-guided fractionation), showed cytotoxic activity in the gastric carcinoma cell line BGC823. The growth of BGC823 cells was inhibited as demonstrated by MTT assay and several cellular characteristic changes, such as cell shrinkage, chromatin condensation and apoptotic body formation with programmed cell death. Flow cytometry analysis revealed that the BGC823 cell cycle was arrested at G2/M phase by HMBBJ, and the apoptotic rate of BGC823 cells increased with respect to HMBBJ in a dose-dependent manner. HMBBJ also activated caspase-3, decreased the expression of Bcl-2 and caused a decrease in the mitochondrial membrane potential (Delta Psi(m)). These findings suggest that HMBBJ could significantly induce apoptosis in BGC823 cells and should be considered as a potential candidate for a chemotherapeutic drug against cancer.

Further pharmacological evidence of the neuroprotective effect of catalpol from Rehmannia glutinosa.

We have previously evaluated the neuroprotective effect of catalpol on aging mice induced by d-galactose, in which catalpol treatment ameliorated cognition deficits and attenuated oxidative damage in mice brain. To thoroughly elucidate the anti-aging effects of catalpol, the liver and spleen antioxidative systems and energy metabolism in senescent mice induced by d-galactose have been studied. Except control group, mice were subcutaneously injected with d-galactose (150mgkg(-1)body weight) for 6 weeks. Meanwhile, drug group mice were treated with catalpol (2.5, 5, 10mgkg(-1)body weight) and piracetam (300mgkg(-1)body weight) for the last 2 weeks. The activities of endogenous antioxidants and the level of glutathione (GSH) and lipid peroxide in the liver and spleen were assayed. Compared to control group, model group mice had significantly lower spleen index (spleen weight/body weight), lower level of GSH, lower activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), higher level of malondialdehyde (MDA) in the liver and spleen. However, catalpol administration markedly reversed these effects of senescence induced by d-galactose. Simultaneously, catalpol noticeably elevated the decreased activities of lactate dehydrogenase (LDH), glutamine synthetase (GS), Na(+)-K(+)-ATPase, Ca(2+)-Mg(2+)-ATPase and decreased the elevated activity of creatine kinase (CK) in mice liver or spleen. These results implied that the anti-aging effects of catalpol were achieved at least partly by promoting endogenous antioxidant enzyme activities and normalizing energy disturbance. Catalpol may be a potential anti-aging agent and worth testing for further preclinical study aimed for senescence or neurodegenerative diseases such as Alzheimer's and Parkinson's diseases.

Benzobijuglone, a novel cytotoxic compound from Juglans mandshurica, induced apoptosis in HeLa cervical cancer cells.

A new quinone compound, p-hydroxymethoxybenzobijuglone (HMBBJ), isolated from Juglans mandshurica by bioassay-guided fractionation, showed cytotoxic activity against HeLa cell line. Its chemical structure was determined by NMR and HREIMS spectra. In this paper, its ability to induce apoptosis in HeLa cells was studied for the first time. After treated with HMBBJ, the growth of HeLa cells was inhibited and cells displayed typical morphological apoptotic characteristics. Data from flow cytometry analysis showed that the HeLa cell cycle was arrested in the G2/M phase by HMBBJ, and the apoptotic rate of HeLa cells increased in a dose-dependent manner. Meanwhile, HMBBJ increased the expression of caspase-8, -3 and Bax, decreased the expression of Bcl-2, and lowered the DeltaPsi(m). These findings reveal that HMBBJ could efficiently induce HeLa cells apoptosis through mitochondria dependent pathway and activation of the caspase cascade, and it may be a potential chemotherapeutic candidate for the treatment of cancer.

Catalpol increases hippocampal neuroplasticity and up-regulates PKC and BDNF in the aged rats.

Rehmannia, a traditional Chinese medical herb, has a long history in age-related disease therapy. Previous work has indicated that catalpol is a main active ingredient performing neuroprotective effect in rehmannia, while the mechanism underlying the effect remains poorly understood. In this study, we attempt to investigate the effect of catalpol on presynaptic proteins and explore a potential mechanism. The hippocampal levels of GAP-43 and synaptophysin in 3 groups of 4 months (young group), 22-24 months (aged group) and catalpol-treated 22-24 months (catalpol-treated group) rats were evaluated by western blotting. Results clearly showed a significant decrease in synaptophysin (46.6%) and GAP-43 (61.4%) levels in the aged group against the young animals and an increase (45.0% and 31.8% respectively) in the catalpol-treated aged rats in comparison with the untreated aged group. In particular, synaptophysin immunoreactivity (OD) in the dentate granule layer of the hippocampus was increased 0.0251 in the catalpol-treated group as compared with the aged group. The study also revealed a catalpol-associated increase of PKC and BDNF in the hippocampus of the catalpol-treated group in comparison with the aged rats and highly correlated with synaptophysin and GAP-43. Such positive correlations between presynaptic proteins and signaling molecules also existed in the young group. These results suggested that catalpol could increase presynaptic proteins and up-regulate relative signaling molecules in the hippocampus of the aged rats. Consequently, it seemed to indicate that catalpol might ameliorate age-related neuroplasticity loss by "normalizing" presynaptic proteins and their relative signaling pathways in the aged rats.

Catalpol modulates the expressions of Bcl-2 and Bax and attenuates apoptosis in gerbils after ischemic injury.

Our previous study described the neuroprotective effects of catalpol in gerbil ischemic model, in which catalpol was shown to prevent hippocampal neurons from death and ameliorate the cognitive ability of the animals. In the study, we focused on investigating the neuroprotective mechanism of catalpol. Animals were randomly assigned three groups as sham-operated, ischemia-treated with saline and ischemia-treated with catalpol. Transient global ischemia was produced by a 5 min occlusion of the bilateral common carotid arteries. Catalpol was intraperitoneally injected at the dose of 5 mg/kg immediately after reperfusion and repeatedly at 12, 24, 48 and 72 h. Histology as well as immunohistochemistry and TUNEL (the terminal deoxynucleotidyl transferase-mediated UTP nick end label) analysis were performed on serial slices through the dorsal hippocampus after gerbils were sacrificed. The results showed that 5 min transient global ischemia followed by 4 days reperfusion caused significant increases in TUNEL-positive and Bax-positive cells in hippocampal CA1 subfield. Catalpol not only significantly reduced TUNEL-positive and Bax-positive cells but also significantly increased Bcl-2-positive cells. All these suggested that catalpol could effectively inhibit apoptosis by modulating the expressions of Bcl-2 and Bax genes.

Screening and analysis of an antineoplastic compound in Rhizoma Chuanxiong by means of in vitro metabolism and HPLC-MS.

A new screening and analysis method that combines in vitro metabolism with high-performance liquid chromatography-mass spectrometry (HPLC-MS) was developed for the screening and analysis of an antineoplastic compound, coniferyl ferulate, which is present in the rhizome of Rhizoma Chuanxiong. Infrared (IR), ultraviolet visible spectroscopy (UV-Vis), nuclear magnetic resonance (NMR) and element analysis were used to identify the molecular structure of coniferyl ferulate. The quantitative analysis of coniferyl ferulate in different extracts of Rhizoma Chuanxiong was carried out, and the metabolism of coniferyl ferulate was investigated by in vitro incubation with rat liver homogenate. The metabolite of coniferyl ferulate, ferulic acid ethyl ester, was identified by HPLC-MS, UV-Vis and IR. In addition, antineoplastic activities of coniferyl ferulate and ferulic acid ethyl ester were detected by the MTT assay. The observed inhibition rate of coniferyl ferulate on the activity of HeLa cells was over 80% at 5.4 ng microl(-1). However, its metabolite, ferulic acid ethyl ester, showed no antineoplastic activity in vitro.

Alpinia protocatechuic acid protects against oxidative damage in vitro and reduces oxidative stress in vivo.

In this study, the neuroprotective effects of Alpinia protocatechuic acid (PCA), a phenolic compound isolated from the dried fruits of Alpinia Oxyphylla Miq. was found. The protective effect of Alpinia PCA against H2O2-induced oxidative damage on PC12 cells was investigated by measuring cell viability via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Rats were injected intraperitoneally with Alpinia PCA at a dose of 5mg/kg per day for 7 days, behavioral testing was performed in Y-maze. In order to make clear the neuroprotective mechanism of Alpinia PCA, the activities of endogenous antioxidants and the content of lipid peroxide in brain were assayed. The results proved that Alpinia PCA significantly prevented the H2O2-induced reduction in cell survival, improved the cognition of aged rats, reduced the content of lipid peroxide, increased the activity of glutathione peroxidase and superoxide dismutase. All these suggested that Alpinia PCA was a potential neuroprotective agent and its neuroprotective effects were achieved at least partly by promoting endogenous antioxidant enzymatic activities and inhibiting free radical generation.

Protective effect of protocatechuic acid from Alpinia oxyphylla on hydrogen peroxide-induced oxidative PC12 cell death.

The neuroprotective effects of protocatechuic acid (PCA), a phenolic compound isolated from the kernels of Alpinia oxyphylla, on hydrogen peroxide (H(2)O(2))-induced apoptosis and oxidative stress in cultured PC12 cells were investigated. Exposure of PC12 cells to 0.4 mM H(2)O(2) induced a leakage of lactate dehydrogenase (LDH) and decreased cell viability denoted by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PCA increased PC12 cellular viability and markedly attenuated H(2)O(2)-induced apoptotic cell death in a dose-dependent manner. By flow cytometric analysis, PCA showed its significant effect on protecting PC12 cells against H(2)O(2)-induced apoptosis. In these cells, the levels of glutathione (GSH) and activity of catalase were augmented, while glutathione peroxidase activity remained unchanged. In addition, PCA also protected against cell damage induced by H(2)O(2) and Fe(2+), which generated hydroxyl radicals (OH) by the Fenton reaction. These results suggest that PCA may be a candidate chemical for the treatment of oxidative stress-induced neurodegenerative disease.

[The protect effect of flavonoids from Cuscuta chinensis in PC12 cells from damage induced by H2O2].

OBJECTIVE: To study the protective effects of flavonoids from Cuscuta chinensis (CF) on oxidative stress in cultured PC12 cells and investigate the mechanism of the effects. METHODS: The cell viability was analyzed by MTT method and the radical scavenging activity of CF was examined by DPPH (1, 1-diphenyl-2-picrylhydrazyl). The morphological changes were observed by Hoechst 33258 staining assay, and the apoptosis rate of PC12 cells was detected by propidium iodide stain flow cytometry (FCM). RESULTS: Application with 0.3-0.5 mM H2O2 induced a dose and time dependent viability loss in PC12 cells; Treatment with 0.5 mM H2O2 for 24 h was shown to cause nearly 50% viabliity loss and apoptosis in PC12 cells; Pretreatment with different concentrations of CF for 0.5 h increased the survival rate of PC12 cells, inhibited apoptosis induced by H2O2; CF had the activity of scavenging free radicals generated by DPPH in a dose-dependent manner. CONCLUSION: CF can protect PC12 cells against oxidative stress. The mechanism of it may be the ability of scavenging ROS and increasing the activity of antioxidant enzyme.

[Screening of antineoplastic components in Radix et Rhizoma Rhei using chromatographic fingerprints].

A new strategy for screening of antineoplastic components in the traditional Chinese medicine of Radix et Rhizoma Rhei has been developed using chromatographic fingerprints before and after metabolism by high performance liquid chromatography-mass spectrometry. The metabolizing method was established based on the in vitro metabolism by Sprague-Dawley (SD) rat liver homogenate. By means of the metabolism methods in vitro, the antineoplastic activity of the extracts, metabolites and components of Radix et Rhizoma Rhei were determined by microculture tetrazolium (MTT) assays in vitro. It was observed that the inhibition rate of the crude extract for HeLa cell was increased from 26.7% to 36.2% after 60 min of metabolism. The changes of activities resulted from the changes of components' structures and the bioactive components were discovered simultaneously in view of metabolism by inhibiting rate assay for the components in Radix et Rhizoma Rhei. It is concluded that the antineoplastic activity of the crude extract from Radix et Rhizoma Rhei was increased after in vitro metabolism because the antineoplastic activity of aloe-emodin, the metabolite of chrysophanol, is higher than its parent compounds.

Studies of chemical constituents and their antioxidant activities from Astragalus mongholicus Bunge.

OBJECTIVE: To evaluate the antioxidant activities of different chemical constituents from Astragalus mongholicus Bunge and their protection against xanthine (XA)/xanthine oxidase (XO)-induced toxicity in PC12 cells. METHODS: The compounds of Astragalus mongholicus Bunge were isolated by chromatography and the structures were elucidated on the basis of spectral data interpretation. Their antioxidant activities were detected by 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities in a cell-free system. Meanwhile, the effects against XA/XO-induced toxicity were assessed using MTT assay in PC12 cells. RESULTS: Ten principal constituents were isolated and identified as formononetin (I), ononin (II), calycosin (III), calycosin-7-O-beta-D-glucoside (IV), 9,10-dimethoxypterocarpan-3-O-beta-D-glucoside (V), adenosine (VI), pinitol (VII), daucosterol (VIII), beta-sitoster (IX) and saccharose (X) from Astragalus mongholicus Bunge. The compounds I, III, and IV scavenged DPPH free radicals in vitro. Formononetin and calycosin were found to inhibit XA/XO-induced cell injury significantly, with an estimated EC50 of 50 ng/mL. CONCLUSION: Compound II, VI, and VII are first reported in this plant. Calycosin exhibits the most potent antioxidant activity both in the cell-free system and in the cell system.

Isoflavonoids from Astragalus mongholicus protect PC12 cells from toxicity induced by L-glutamate.

lsoflavonoids, formononetin, 9,10-dimethoxypterocarpan 3-O-beta-D-glucoside, ononin, calycosin 7-O-glc and calycosin, were isolated from the roots of Astragalus mongholicus Bunge (Leguminosae). The neuroprotective roles and direct antioxidant effects of these isoflavonoids were investigated by using PC12 cell model and DPPH (1,1-diphenyl-2-picrylhydrazyl) assay. Formononetin, ononin and calycosin were found inhibiting glutamate-induced cell injury, with an estimated 50% effective concentration (EC50) of 0.027 microg/ml, 0.047 microg/ml and 0.031 microg/ml, respectively. Pretreatment with them increased the activities of antioxidant enzymes, including superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and prevented the release of lactate dehydrogenase (LDH) in glutamate-injured PC12 cells. On the other hand, calycosin 7-O-glc and calycosin showed more scavenging activity to DPPH radicals than formononetin in the cell-free system. The inconsistency between the neuroprotective capabilities of isoflavonoids and their directly scavenging activity to DPPH radicals indicated that formononetin, ononin and calycosin probably depended on increasing endogenous antioxidant and stabilizing the cells' membrane structures to inhibit the cell damage induced by glutamate.

Codonopsis pilosula (Franch) Nannf total alkaloids potentiate neurite outgrowth induced by nerve growth factor in PC12 cells.

AIM: To explore the effect of Codonopsis pilosula (Franch) Nannf total alkaloids (DSA) on differentiation induced by nerve growth factor (NGF) in PC12 cells. METHODS: After culturing PC12 cells with DSA in the presence or absence of NGF, neurite outgrowth in PC12 cells and correlated protein kinases were assayed. RESULTS: DSA alone did not exhibit neuritogenic activity, but caused a significant enhancement of NGF (2 microg/L)-induced neurite outgrowth in PC12 cells, and increased the phosphorylation of mitogen-activated protein kinase (MAPK). Furthermore, this enhancing effect was completely blocked by a specific MAPK kinase inhibitor, PD98059. CONCLUSION: DSA enhanced the NGF-induced neurite outgrowth in PC12 cells by amplifying an up-stream step of the MAPK-dependent signaling pathway.