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Acori Tatarinowii Rhizoma (ATR), the rhizome of Acorus tatarinowii Schott, is a common traditional Chinese medicine being used clinically for mental disorder. However, other Acorus species herbs are all having the same Chinese name 'Chang Pu', making the confusion in herbal market. Acori Graminei Rhizoma (AGR) and Acori Calami Rhizoma (ACR) are common adulterants of ATR. Here, we aim to provide a comparative analysis between ATR, AGR, and ACR in potentiating neuronal differentiation. Volatile oil, derived from Acorus species, was applied onto cultured PC12 cells, and various parameters were determined: (i) transcriptional activation of neurofilament promoters was determined by the promoter-driven luciferase activity assay; (ii) the neurite outgrowth of PC12 cells was captured and measured; and (iii) the neurofilament expression and its underlying mechanism were analyzed by western blotting. The co-treatment of ATR, AGR, or ACR volatile oil with low concentration of nerve growth factor (NGF) could potentiate the NGF-induced neuronal differentiation in cultured PC12 cells. In addition, application of protein kinase A inhibitor H89 in cultures blocked the induction of neurofilament. Among these three Acorus species, ATR volatile oil showed the highest NGF-induced induction in neurite outgrowth and neurofilament expression, as compared with that of AGR and ACR. Copyright © 2017 John Wiley & Sons, Ltd.

Ethnopharmacological relevance The fruit of Ziziphus jujuba (Mill.), known as J ujuba F ructus (JF) or jujube, is a well-known T raditional C hinese M edicine (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 G ui z hi T ang (GZT), N eibu D angguijianzhong T ang (NDT) and Z a o T ang (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. g lutamate- c ysteine l igase c atalytic subunit (GCLC), g lutamate- c ysteine l igase m odulatory subunit (GCLM), g lutathione S - t ransferase (GST) and N AD(P)H q uinone o xidoreductase (NQO1) via the activation of a nti-oxidant r esponse e lement (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.

Xiao-Er-An-Shen Decoction (XEASD), a Chinese herbal formula, has been used in clinic for treating insomnia and mental excitement in children and adolescents. However, less of scientific data supports its effectiveness in clinic. Here, we aim to study the role of XEASD in regulating neuron differentiation and antioxidant activity. An HPLC-MS was used to chemically standardize herbal extract of XEASD. The standardized herbal extracts of XEASD (0.3–3.0 mg/mL) were applied onto cultured PC12 cells for 48 hours. The treatment with XEASD extract induced neurite outgrowth of PC12 cells in a dose-dependent manner, having the highest response by ~50% of differentiated cells. Application of XEASD extract dose dependently stimulated expressions of NF68, NF160, and NF200 in cultured PC12 cells. Furthermore, XEASD activated the phosphorylation of cAMP responsive element binding protein on PC12 cells, the effect of which was blocked by H89, a protein kinase A inhibitor. Moreover, XEASD showed free radical scavenging activity and stimulated the transcriptional activity of ARE. These results supported the neurobeneficial effects of XEASD in the induction of neurite outgrowth and protection against oxidative stress and could be useful for neurological diseases, in which neurotrophin deficiency and oxidation insult are involved.

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.

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.

Edible bird’s nest (EBN; Yan Wo), or cubilose, is originated from the salivary secretion of Aerodramus fuciphagus. In Asia, EBN is famous for its unproven skin lightening function. Here, we aim to reveal the active ingredients of EBN responsible for skin lightening function. Three major fractions were isolated from EBN water extract by chromatography using LC-MS/MS, bioactivities of these isolated fractions were analyzed by assays of tyrosinase, melanocytes and 3D human skin model, from which, N-acetylneuraminic acid (NANA), the second isolated fraction showed an inhibition effect on tyrosinase activity in a dose-dependent manner. The IC50 of tyrosinase originated from mushroom and human was 16.93 mM and 0.10 mM respectively. Furthermore, only EBN with higher content of NANA (e.g. White and Red EBN), but not EBN with less NANA (e.g. Grass EBN), showed promising skin lightening function. This is the first report to reveal NANA being an active ingredient of EBN on skin lightening function.

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 © 2014 John Wiley & Sons, Ltd.

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 © 2014 John Wiley & Sons, Ltd.

Chemical change during boiling of herbal mixture is a puzzle. By using Danggui Buxue Tang (DBT), a herbal decoction that contains Astragali Radix (AR) and Angelicae Sinensis Radix (ASR), we developed a model in analyzing the hydrolysis of flavonoid glycosides during the boiling of herbal mixture in water. A proper preparation of DBT is of great benefit to the complete extraction of bioactive ingredients. Boiling of DBT in water increased the solubility of AR-derived astragaloside IV, calycosin, formononetin, calycosin-7-O-β-D-glucoside, and ononin in a time- and temperature-dependent manner: the amounts of these chemicals reached a peak at 2 h. The glycosidic resides of AR, calycosin-7-O-β-D-glucoside, and ononin could be hydrolyzed during the moderate boiling process to form calycosin and formononetin, respectively. The hydrolysis efficiency was strongly affected by pH, temperature, and amount of herbs. Interestingly, the preheated herbs were not able to show this hydrolytic activity. The current results supported the rationality of ancient preparation of DBT in boiling water by moderate heat.

Acori Tatarinowii Rhizoma (ATR, the dried rhizome of Acorus tatarinowii Schott.) is a traditional Chinese medicine widely used to treat brain diseases, e.g. depression, forgetfulness, anxiety and epilepsy. Several lines of evidence support that ATR has neuronal beneficial functions in animal models, but its action mechanism in cellular level is unknown. Here, we identified α-asarone and β-asarone could be the major active ingredients of ATR, which, when applied onto cultured rat astrocytes, significantly stimulated the expression and secretion of neurotrophic factors, i.e. nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and glial derived neurotrophic factor (GDNF), in dose-dependent manners. These results suggested that the neuronal action of ATR, triggered by asarone, might be mediated by an increase of expression of neurotrophic factors in astrocytes, which therefore could support the clinical usage of ATR. In addition, application of PKA inhibitor, H89, in cultured astrocytes partially blocked the asarone-induced neurotrophic factor expression, suggesting the involvement of PKA signaling. The results proposed that α-asarone and β-asarone from ATR could serve as potential candidates for drug development in neurodegenerative diseases.

Acori Tatarinowii Rhizoma (ATR; rhizome of Acorus tatarinowii Schott) (Shi Chang Pu) is widely used in Chinese medicine (CM) to resuscitate, calm the mind, resolve shi (dampness) and harmonize the wei (stomach). Seven different species have been identified as belonging to the genus Acorus, all of which can be found in China. However, it can be difficult to distinguish the different species of Acorus because of their morphological similarities. The aim of this study was to authenticate Acorus species using macroscopic and microscopic techniques, chemical analysis and DNA authentication and to compare the resolution power and reliability of these different methods. Four batches of ATR, Acori Graminei Rhizoma (AGR), Acori Calami Rhizoma (ACR) and Anemones Altaicae Rhizoma (AAR) (totaling 16 samples) were collected from Hong Kong and mainland China. The major characteristic features of these Acorus species were identified by macroscopic and microscopic examination. The identified samples were also analyzed by UHPLC analysis. Principal component analysis (PCA) and hierarchal clustering analysis (HCA) on UHPLC results were used to differentiate between the samples. An internal transcribed spacer (ITS) was selected as a molecular probe and a modified DNA extraction method was developed to obtain trace amounts of DNA from the different Acorus species. All extracted DNA sequences were edited by Bioedit and aligned with the ClustalW. And the sequence distances were calculated using the Maximum Parsimony method. Macroscopic and microscopic analyses allowed for AAR to be readily distinguished from ATR, AGR and ACR. However, it was difficult to distinguish between ATR, AGR and ACR because of their similar morphological features. Chemical profiling revealed that α- and β-asarone were only found in the ATR, AGR and ACR samples, but not in the AAR samples. Furthermore, PCA and HCA allowed for the differentiation of these three species based on their asarone contents. Morphological authentication and chemical profiling allowed for the partial differentiation of ATR, AGR ACR and AAR. DNA analysis was the only method capable of accurately differentiating between all four species. DNA authentication exhibited higher resolution power and reliability than conventional morphological identification and UHPLC in differentiating between different Acorus species.

Background HerboChip is an array of different fractions deriving from herbal extracts. This study aimed to identify effective components from Chinese medicine (CM) that interact with nerve growth factor (NGF) as a target using HerboChip. Methods Fifty types of CM that are traditionally used as remedies for emotion imbalance were selected and extracted with 50 % ethanol. Biotinylated-NGF was hybridized with over 300 chips coated with different HPLC-separated fractions from CM extracts and straptavidin-Cy5 was used to identify the NGF-bound fractions. Results Over 300 chips were screened within a week, and 17 positive hits were identified. The interaction of the identified herbal extracts with NGF was confirmed in cultured PC12 cells. Co-application of NGF and herbal extract interfered with NGF-induced expression of neurofilaments, including NF68 and NF200 in cell cultures. Western blot analysis comparing the intensity of phosphorylated cAMP response element-binding protein (CREB) over total CREB showed NGF-induced CREB phosphorylation was modulated by the identified herbal extracts. Five CM herbs showed activating activities on the NGF response and nine CM herbs showed inhibiting activities. Conclusion The current result supported the applicability of HerboChip for screening NGF binding components from herbal extracts. Electronic supplementary material The online version of this article (doi:10.1186/s13020-016-0107-8) contains supplementary material, which is available to authorized users.

Pyrrolizidine alkaloids (PAs) are commonly found in many plants including those used in medical therapeutics. The hepatotoxicities of PAs have been demonstrated both in vivo and in vitro; however, the neurotoxicities of PAs are rarely mentioned.In this study, we aimed to investigate in vitro neurotoxicities of clivorine, one of the PAs found in various Ligularia species, in cultured PC12 cells.PC12 cell line was employed to first elucidate the neurotoxicity and the underlying mechanism of clivorine, including cell viability and morphology change, neuronal differentiation marker and signaling pathway.PC12 cells were challenged with series concentrations of clivorine and/or nerve growth factor (NGF). The cell lysates were collected for MTT assay, trypan blue staining, immunocytofluorescent staining, qRT-PCR and western blotting.Clivorine inhibited cell proliferation and neuronal differentiation evidenced by MTT assay and dose-dependently reducing neurite outgrowth, respectively. In addition, clivorine decreased the level of mRNAs encoding for neuronal differentiation markers, e.g. neurofilaments and TrkA (NGF receptor). Furthermore, clivorine reduced the NGF-induced the phosphorylations of TrkA, protein kinase B and cAMP response element-binding protein in cultured PC12 cells.Taken together, our results suggest that clivorine might possess neurotoxicities in PC12 cells via down-regulating the NGF/TrkA/Akt signaling pathway. PAs not only damage the liver, but also possess neurotoxicities, which could possibly result in brain disorders, such as depression.

Jujubae Fructus, known as jujube or Chinese date, is the fruit of Ziziphus jujuba (Mill.), which not only serves as daily food, but acts as tonic medicine and health supplement for blood nourishment and sedation. According to Chinese medicine, jujube is commonly included in herbal mixtures, as to prolong, enhance and harmonize the efficiency of herbal decoction, as well as to minimize the toxicity. Here, we aim to compare the chemical and pharmacological properties of three commonly used jujube-containing decoctions, including Guizhi Tang (GZT), Neibu Dangguijianzhong Tang (NDT) and Zao Tang (ZOT). These decoctions share common herbs, i.e. Glycyrrhizae Radix et Rhizoma Praeparata cum Melle, Zingiberis Rhizoma Recens and Jujube, and they have the same proposed hematopoietic functions. The amount of twelve marker biomolecules deriving from different herbs in the decoctions were determined by LC-MS, and which served as parameters for chemical standardization. In general, three decoctions showed common chemical profiles but with variations in solubilities of known active ingredients. The chemical standardized decoctions were tested in cultured Hep3B cells. The herbal treatment stimulated the amount of mRNA and protein expressions of erythropoietin (EPO) via the activation of hypoxia response elements: the three herbal decoctions showed different activation. The results therefore demonstrated the hematopoietic function of decoctions and explained the enhancement of jujube function within a herbal mixture.

Acori Tatarinowii Rhizome (ATR; the dried rhizome of Acori tatarinowii Schott) is a well-known herb being used for mental disorder in China and Asia. Volatile oil is considered as the active ingredient of ATR, and asarones account for more than 90% of total volatile oil. Here, the protective effects of ATR oil and asarones, both α-asarone and β-asarone, were probed in cultured rat astrocytes. The cyto-protective effect of ATR oil and asarones against tBHP-induced astrocyte injury was revealed, and additionally ATR oil and asarones reduced the tBHP-induced intracellular reactive oxygen species (ROS) accumulation. In parallel, the activity of anti-oxidant response element (ARE) promoter construct (pARE-Luc), being transfected in cultured astrocytes, was markedly induced by application of ATR oil and asarones. The mRNAs encoding anti-oxidant enzymes, e.g. glutathione S-transferase (GST), glutamate-cysteine ligase modulatory subunit (GCLM), glutamate-cysteine ligase catalytic subunit (GCLC) and NAD(P)H quinone oxidoreductase (NQO1) were induced by ATR oil and asarones in a dose-dependent manner. The ATR oil/asarone-induced gene expression could be mediated by Akt phosphorylation; because the applied LY294002, a phosphoinositide 3-kinase inhibitor, fully abolished the induction. These results demonstrated that α-asarone and β-asarone could account, at least partly, the function of ATR being a Chinese medicinal herb.

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.

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.

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.

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.

Linderae Radix, the dry roots of Lindera aggregata (Sims) Kosterm, has long been used as traditional Chinese medicine for treatment of inflammatory diseases. The total alkaloids are believed to be the active components responsible for anti-inflammation of Linderae Radix. Here, the total alkaloids of Linderae Radix were extracted and isolated, including 12 isoquinoline alkaloids and 1 furan sesquiterpene. Within the alkaloids, norisobolidine, boldine, linderaline, isoboldine, reticuline, N-methyllaurotetanine, norjuziphine were found to be the major ingredients. In lipopolysaccharide-treated macrophage RAW 264.7 cells, application of Linderae Radix extract, or total alkaloids, suppressed the transcription of pro inflammatory cytokines, interleukin-1β and interleukin-6. Out of the 12 alkaloids, norisoboldine, boldine, and isoboldine were tested in lipopolysaccharide-treated macrophages, and norisoboldine was the strongest alkaloid in suppressing the cytokine expressions. The current studies suggested that the identification of alkaloids from Linderae Radix could provide a plausible explanation for herbal therapeutic functions.