Kelly Yin Ching Lam

Saikosaponin-d Enhances the Anticancer Potency of TNF-α via Overcoming Its Undesirable Response of Activating NF-Kappa B Signalling in Cancer Cells

Tumor necrosis factor-alpha (TNF-α) was reported as anticancer therapy due to its cytotoxic effect against an array of tumor cells. However, its undesirable responses of TNF-α on activating NF-κB signaling and pro-metastatic property limit its clinical application in treating cancers. Therefore, sensitizing agents capable of overcoming this undesirable effect must be valuable for facilitating the usage of TNF-α-mediated apoptosis therapy for cancer patients. Previously, saikosaponin-d (Ssd), a triterpene saponin derived from the medicinal plant, Bupleurum falcatum L. (Umbelliferae), showed to exhibit a variety of pharmacological activities such as antiinflammation, antibacteria, antivirus and anticancer. Recently, we found that Ssd could inhibit the activated T lymphocytes via suppression of NF-κB, NF-AT and AP-1 signaling. Here, we showed that Ssd significantly potentiated TNF-α-mediated cell death in HeLa and HepG2 cancer cells via suppression of TNF-α-induced NF-κB activation and its target genes expression involving cancer cell proliferation, invasion, angiogenesis and survival. Also, Ssd revealed a significant potency of abolishing TNF-α-induced cancer cell invasion and angiogenesis in HUVECs while inducing apoptosis via enhancing the loss of mitochondrial membrane potential in HeLa cells. Collectively, these findings indicate that Ssd has a significant potential to be developed as a combined adjuvant remedy with TNF-α for cancer patients.

The Standardized Extract of Ziziphus jujuba Fruit (Jujube) Regulates Pro-inflammatory Cytokine Expression in Cultured Murine Macrophages: Suppression of Lipopolysaccharide-stimulated NF-κB Activity

The fruit of Ziziphus jujuba Mill., known as jujube or Chinese date, is commonly consumed as a health supplement or herbal medicine worldwide. To study the beneficial role of jujube in regulating immune response, we investigated its roles on the expressions of pro‐inflammatory cytokines in cultured macrophages. Application of chemically standardized jujube water extract for 24 h stimulated the transcriptional expression of interleukin (IL)‐1β, IL‐6, and tumor necrosis factor (TNF)‐α in cultured RAW 264.7 macrophages. In contrast, the pretreatment with jujube water extract suppressed the expression of IL‐1β and IL‐6, but not for TNF‐α in lipopolysaccharide (LPS)‐stimulated macrophages. The IL‐1β and IL‐6 cytokines in LPS‐induced macrophages were suppressed by jujube water extract in both mRNA and protein levels. In parallel, the inhibition of jujube water extract on the transcriptional activity of nuclear factor‐kappa B was revealed in LPS‐induced macrophages. These results verified the bidirectional immune‐modulatory roles of jujube by regulating the expressions of pro‐inflammatory cytokines in macrophages. Copyright

A Standardized Extract of the Fruit of Ziziphus jujuba (Jujube) Induces Neuronal Differentiation of Cultured PC12 Cells: A Signaling Mediated by Protein Kinase A

The fruit of Ziziphus jujuba Mill., known as Chinese date or jujube, is consumed as a health supplement worldwide. To study the role of jujube in brain benefits, its effects on neuronal differentiation of PC12 cells were studied. Application of jujube water extract induced neurite outgrowth of PC12 cells, >25% of which were differentiated; this effect was similar to that of nerve growth factor. In parallel, the expressions of neurofilaments (NFs) in jujube-treated cultures showed a dose-dependent increase, with the highest inductions by ∼150% for NF68 and NF160 and by ∼100% for NF200. Application of H89, a protein kinase A inhibitor, attenuated jujube-induced neurite outgrowth of the cultures. Besides, using jujube extract induced the phosphorylation of cAMP responsive element binding protein on PC12 cells, which was blocked by H89. These results support the use of jujube as a food supplement for the prevention of neurodegenerative diseases in which neurotrophin deficiency is involved.

The Extract of Ziziphus jujuba Fruit (Jujube) Induces Expression of Erythropoietin Via Hypoxia-Inducible Factor-1α in Cultured Hep3B Cells

The fruit of Ziziphus jujuba Mill., known as jujube or Chinese date, is commonly consumed as health supplement or herbal medicine worldwide. To study the beneficial role of jujube in enhancing hematopoietic function, we investigated its roles on the expression of erythropoietin in cultured Hep3B human hepatocellular carcinoma cells. Application of chemically standardized jujube water extract stimulated erythropoietin expression in a dose-dependent manner, with the highest response by ~ 100 % of increase. A plasmid containing hypoxia response element, a critical regulator for erythropoietin transcription, was transfected into Hep3B cells. Application of jujube water extract onto the transfected cells induced the transcriptional activity of the hypoxia response element. To account for its transcriptional activation, the expression of hypoxia-inducible factor-1α was increased after treatment with jujube water extract: the increase was in both mRNA and protein levels. These results confirmed the hematopoietic function of jujube in the regulation of erythropoietin expression in liver cells.

Asarone from Acori Tatarinowii Rhizoma Potentiates the Nerve Growth Factor-Induced Neuronal Differentiation in Cultured PC12 Cells: A Signaling Mediated by Protein Kinase A

Acori Tatarinowii Rhizoma (ATR), the rhizome of Acorus tatarinowii Schott, is being used clinically to treat neurological disorders. The volatile oil of ATR is being considered as an active ingredient. Here, α-asarone and β-asarone, accounting about 95% of ATR oil, were evaluated for its function in stimulating neurogenesis. In cultured PC12 cells, application of ATR volatile oil, α-asarone or β-asarone, stimulated the expression of neurofilaments, a bio-marker for neurite outgrowth, in a concentration-dependent manner. The co-treatment of ATR volatile oil, α-asarone or β-asarone, with low concentration of nerve growth factor (NGF) potentiated the NGF-induced neuronal differentiation in cultured PC12 cells. In addition, application of protein kinase A inhibitors, H89 and KT5720, in cultures blocked the ATR-induced neurofilament expression, as well as the phosphorylation of cAMP-responsive element binding protein (CREB). In the potentiation of NGF-induced signaling in cultured PC12 cells, α-asarone and β-asarone showed synergistic effects. These results proposed the neurite-promoting asarone, or ATR volatile oil, could be useful in finding potential drugs for treating various neurodegenerative diseases, in which neurotrophin deficiency is normally involved.

Optimizing the compatibility of paired-herb in an ancient Chinese herbal decoction Kai-Xin-San in activating neurofilament expression in cultured PC12 cells.

ETHNOPHARMACOLOGICAL RELEVANCE Kai-Xin-San (KXS), a well-known traditional Chinese herbal decoction, has been widely used to treat mental depression and memory loss in China. It has a combination of four herbs: Ginseng Radix et Rhizoma (GR; root and rhizome of Panax ginseng C. A. Mey.), Polygalae Radix (PR; root of Polygala tenuifolia Wild.), Acori Tatarinowii Rhizoma (ATR; rhizome of Acorus tatarinowii Schott), and Poria (PO; sclerotium of Poriacocos (Schw.) Wolf), from which a pairing of two herbs was considered as paired-herb, such as the pairing of GR-PR and ATR-PO. The depression-induced neural cell loss is one of the major pathogenesis in depression. Here, an optimized KXS by changing the ratio of paired-herbs in KXS was demonstrated aiming at promoting neural cell differentiation. MATERIALS AND METHODS Quantitative assessment of chemical markers in each herbal extract was determined by LC-MS. Promoters of neurofilaments, NF68 and NF200, linked with luciferase reporter gene (pNF68-Luc and pNF200-Luc) were applied in cultured pheochromocytoma (PC12) cells to study the transcriptional activation of each herbal extract. The effect of GR-PR and ATR-PO in improving NF promoter activity was analyzed by Compusyn software. The activation of PKA was indicated. RESULTS In PC12 cells, an optimized KXS named KXS1:5 having 1:5 of GR-PR:ATR-PO had greater capability in promoting the expression of neurofilament. The synergistic effect of GR-PR and ATR-PO on the improved efficiency was further determined. Moreover, the treatment of H89, a PKA inhibitor, significantly inhibited the induced NF promoter activity. CONCLUSION These results indicated an optimized KXS by optimizing the compatibility of paired-herb and this compatibility was proven to exert synergistic effect. Moreover, the underlying mechanism was mediated by a PKA signaling pathway.

Fruit of Ziziphus jujuba (Jujube) at two stages of maturity: distinction by metabolic profiling and biological assessment.

The fruit of Ziziphus jujuba, named as jujube or Chinese date, is used as a health supplement worldwide. Two kinds of jujubes are commonly found in the market: immature jujubes eaten as fruits, and mature jujubes employed as medicinal herbs. To study the variation of jujubes at two developmental stages, we investigated their chemical and biological properties by metabolic profiling and cellular assays. In NMR profiling, the levels of 11 metabolites were measured. Statistically differences in the levels of threonine, alanine, acetate, creatine, glucose, sucrose, and formate were found between mature and immature jujubes. In parallel, their neuro-protecting and erythropoietic activities were compared. The water extract of mature jujube possessed better effect in inducing neurofilament expression than that of the immature one, while immature jujube extract performed better in activating HRE-mediated transcriptional activity. These findings suggest the maturity of jujube has to be considered when it is being used for health food products.

A Chemically Standardized Extract of Ziziphus jujuba Fruit (Jujube) Stimulates Expressions of Neurotrophic Factors and Anti-oxidant Enzymes in Cultured Astrocytes

The fruit of Ziziphus jujuba Mill., known as jujube or Chinese date, is commonly consumed as a health supplement worldwide. To study the role of jujube in brain benefits, the expression of neurotrophic factors and anti‐oxidant enzymes in the jujube‐treated cultured astrocytes was determined. Application of a chemical standardized water extract of jujube in cultured astrocytes for 24 h stimulated the expressions of nerve growth factor, brain‐derived neurotrophic factor and glial cell line‐derived neurotrophic factor in a concentration‐dependent manner. The pre‐treatment with H89, a protein kinase A inhibitor, attenuated the jujube‐induced expression of neurotrophic factors. In parallel, the treatment of jujube water extract induced the transcriptional expressions of the enzymes responsible for anti‐oxidation, i.e. NAD(P)H: quinine oxidoreductase 1, glutamate‐cysteine ligase catalytic subunit, glutamate‐cysteine ligase modifier subunit and glutathione S‐transferase, in a concentration‐dependent manner. These results proposed the benefits of jujube in regulating expressions of neurotrophic factors and anti‐oxidant enzymes in cultured astrocytes. Copyright

Authentication of Cordyceps sinensis by DNA Analyses: Comparison of ITS Sequence Analysis and RAPD-Derived Molecular Markers

Cordyceps sinensis is an endoparasitic fungus widely used as a tonic and medicinal food in the practice of traditional Chinese medicine (TCM). In historical usage, Cordyceps specifically is referring to the species of C. sinensis. However, a number of closely related species are named themselves as Cordyceps, and they are sold commonly as C. sinensis. The substitutes and adulterants of C. sinensis are often introduced either intentionally or accidentally in the herbal market, which seriously affects the therapeutic effects or even leads to life-threatening poisoning. Here, we aim to identify Cordyceps by DNA sequencing technology. Two different DNA-based approaches were compared. The internal transcribed spacer (ITS) sequences and the random amplified polymorphic DNA (RAPD)-sequence characterized amplified region (SCAR) were developed here to authenticate different species of Cordyceps. Both approaches generally enabled discrimination of C. sinensis from others. The application of the two methods, supporting each other, increases the security of identification. For better reproducibility and faster analysis, the SCAR markers derived from the RAPD results provide a new method for quick authentication of Cordyceps.

Jujube-containing herbal decoctions induce neuronal differentiation and the expression of anti-oxidant enzymes in cultured PC12 cells

ETHNOPHARMACOLOGICAL RELEVANCE The fruit of Ziziphus jujuba (Mill.), known as Jujuba Fructus (JF) or jujube, is a well-known Traditional Chinese Medicine (TCM) for blood nourishment and sedation effect. Apart from prescribing as single herb alone, JF is very often being included in multi-herbal decoctions to prolong, enhance and harmonize pharmaceutical effects of decoctions while at the same time reducing toxicity. Here, we aimed to compare the protective and differentiating activities of three chemically standardized jujube-containing decoctions, including Guizhi Tang (GZT), Neibu Dangguijianzhong Tang (NDT) and ZaoTang (ZOT) in cultured PC12 cells. MATERIALS AND METHODS The protein expressions of neurofilaments, including NF68, NF160 and NF200, under the herbal treatment were revealed by western blot. The determination of neurite outgrowth in cultured PC12 cells upon the treatment of herbal extracts was performed by light microscope equipped with a phase-contrast condenser and SPOT imaging software. The protective effect against tBHP-induced cytotoxicity under the herbal treatment was measured by MTT assay. A luciferase reporter construct carrying four repeats of anti-oxidant response element (ARE) and a downstream luciferase reporter gene luc2P was transfected into PC12 cells to study the transcriptional activation of ARE. The mRNA expression of antioxidant enzymes under the herbal treatment was analyzed by quantitative real-time PCR. RESULTS These jujube-containing decoctions processed similar neuro-protective and brain beneficial properties. The herbal treatment induced the protein expression of neurofilaments. Neurite outgrowth was observed under the herbal treatment. In parallel, the pre-treatment of herbal extracts protected PC 12 cells against oxidative stress-induced apoptosis in a dose-dependent manner. Moreover, the herbal treatments triggered the mRNA expressions of relevant anti-oxidation genes, i.e. glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modulatory subunit (GCLM), glutathione S-transferase (GST) and NAD(P)H quinone oxidoreductase (NQO1) via the activation of anti-oxidant response element (ARE). CONCLUSION The results therefore demonstrated neuro-protective and differentiating properties of the three closely related decoctions, and which subsequently illustrated the enhancement function of jujube within a multi-herbal decoction.