Effects of commercial beverages on the neurobehavioral motility of Caenorhabditis elegans.

To study the effects of different types of commercially available drinks/beverages on neurobehavior using the model organism C. elegans, and critically review their potential health hazards. Eighteen kinds of beverages from the supermarket were randomly selected and grouped into seven categories namely functional beverage, tea beverage, plant protein beverage, fruit juice beverage, dairy beverage, carbonated beverage and coffee beverage. The pH value, specific gravity and osmotic pressure were also examined. The L4 stage N2 worms were exposed to different concentration of tested beverages (0, 62.5, 125, 250 and 500 µL/mL) for 24 h to measure the survival rate and locomotory behavior such as head thrashing, body bending as well as pharyngeal pumping. All the 18 beverages tested did not induce any visible lethal effects in the nematodes. However, exposure to different types of tested beverages exhibited different effects on the behavioral ability of C. elegans: (1) sports functional beverage and herbal tea drink accelerated the head thrashing and body bending of nematodes when compared to the control group (P < 0.05). (2) The vibration frequency of the pharyngeal pump of nematodes was significantly accelerated after treated with three plant protein beverages (almond milk, coconut milk and milk tea) and dairy products A and B (P < 0.05), and decelerated after treatment with other tested beverages. (3) Carbonated beverage significantly inhibits the head thrashing, body bending and pharyngeal pumping vibration (P < 0.05). Our results indicate that 18 kinds of popular beverages in the market have different influence on the neurobehavior in C. elegans, which may be related to their different components or properties. Further research would be required to conduct a systematic analysis of the effect of beverages by appropriate kinds, taking into consideration other endpoints such as reproduction, lifespan and molecular stress response, etc., and to elucidate the mechanism for its potential health hazards.

The indole alkaloids from the roots of Isatidis Radix.

The root of Isatis indigotica is used as a traditional Chinese medicine (termed Isatidis Radix) due to its antiviral effects. We examined compounds isolated from Isatidis Radix and elucidated the structures of three new natural alkaloids, and we examined the possible mechanisms or active targets of indole alkaloids occurring in blood of rats treated by gavage. Three new natural products were isolated from Radix Isatidis for the first time, including 1-methoxy-2-indoleacetonitrile, 1-hydroxy-3-indoleacetonitrile, 8-Methoxy-1, 2-dihydroquinoline, and 4 compounds isolated from this medicinal material for the first time. Their structures were elucidated using nuclear magnetic resonance. The components of Isatidis Radix were analysed using liquid chromatography tandem mass spectrometry, and 33 compounds were detected in plasma of treated rats; 24 of these compounds were indole alkaloids, and they included the newly identified compounds. Molecular docking and in vitro antithrombin activity tests showed HA inhibition activity of indoleacetonitriles.

Indole-3-carboxaldehyde regulates RSV-induced inflammatory response in RAW264.7 cells by moderate inhibition of the TLR7 signaling pathway.

Human respiratory syncytial virus (RSV) is highly contagious and the leading cause of severe respiratory tract illness in infants, elderly, and immunocompromised individuals. Toll-like receptor 7 (TLR7), a pattern recognition receptor recognising the ssRNA of RSV, activates proinflammatory pathways and triggers secretion of interferons (IFNs). On the one hand, the inflammatory responses help clear out virus. On the other hand, they lead to severe lung damage. Banlangen is a traditional Chinese herbal medicine commonly prescribed for respiratory virus infection treatment, but the mechanisms of action and active components remain largely unknown. In the present study, we investigated the effects of the main active components of total alkaloids from banlangen (epigoitrin, indole-3-carboxaldehyde, indole-3-acetonitrile and 4-methoxyindole-3-acetonitrile) on the RSV-induced inflammatory responses in mouse macrophage cells (RAW264.7). Our results demonstrated that RSV-induced IFN-α excessive secretion was moderately inhibited by indole-3-carboxaldehyde through downregulation of mRNA expression in a dose-dependent manner, in comparison, the inhibitory effects of ribavirin were too strong. Furthermore, we revealed that indole-3-carboxaldehyde suppressed transcription of IFN-α by inhibiting RSV-induced TLR7 expression in RAW264.7 cells. Additionally, indole-3-carboxaldehyde inhibited RSV-induced NF-κB signalling activation in a TLR7-MyD88-dependent manner. Together, our findings suggest that indole-3-carboxaldehyde inhibited RSV-induced inflammatory injury by moderate regulation of TLR7 signaling pathway and did not significantly affect the viral clearance competence of the innate immune system.

Different types of effective fractions from Radix Isatidis revealed a multiple-target synergy effect against respiratory syncytial virus through RIG-I and MDA5 signaling pathways, a pilot study to testify the theory of superposition of traditional Chinese Medicine efficacy.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Isatidis, a commonly used traditional Chinese medicine, is also documented in "Dictionary of Chinese Ethnic Medicine" being as an ethnic herb clinically utilized by different nations in China such as Mongol, Uygur, and Dong et al. It has been reported to have a very strong efficacy on respiratory viruses, but to date the mechanism remains unknown. Similarly, it is unclear how different types of effective fractions of Radix Isatidis interact to exert antiviral effects. AIM OF STUDY: To reveal the underlying mechanisms for the inhibitory effects of three active fractions from Radix Isatidis, i.e. total alkaloids, lignans and organic acids, on respiratory syncytial virus when used alone or in combination. In addition, we investigated whether these three parts worked synergistically in vivo and in vitro. MATERIALS AND METHODS: A mouse model of RSV infection was constructed by intranasal infection, and the pathological changes of lung tissues in different parts were observed. The level changes of IFNß and inflammatory cytokines in the mouse alveolar lavage fluid were detected by enzyme-linked immunosorbent assay (ELISA). The anti-RSV effects of different effective fractions were evaluated by the plaque reduction test. The mRNA and protein expressions of RIG-I, MDA-5, MAVS and IRF3 in RAW264.7 cells were detected by RT-PCR and Western blot respectively. RESULTS: HE staining showed that Radix Isatidis extracts alone or in combination relieved virus-induced mouse lung lesions. Compared with individual drugs, the lung lesions were alleviated more significantly after treatment with the three fractions in combination. ELISA demonstrated that the expression levels of IFNß and inflammatory cytokines were maintained balanced between antiviral and proinflammatory effects. The plaque reduction test indicated that the antiviral effect of combination treatment was much stronger than those of individual drugs. RT-qPCR and Western blot suggested that the mRNA and protein expression levels of key signaling molecules in the RIG-I and MDA5 pathways in mouse macrophages were down-regulated by different effective parts alone or in combination. CONCLUSIONS: The three effective fractions of Radix Isatidis have remarkable synergistic anti-RSV effects in vitro and in vivo, and total alkaloids and lignans show multi-target synergistic effects via the RIG-I and MDA5 signaling pathways.

4(3H)-Quinazolone regulates innate immune signaling upon respiratory syncytial virus infection by moderately inhibiting the RIG-1 pathway in RAW264.7 cell.

The root of Isatis indigotica, also known as Ban-Lan-Gen (BLG), is commonly used for prevention and early treatment of respiratory virus infection, but the underlying mechanisms of action remain unclear. In the early stage of infection, the innate immune system is activated by virus, and related immune cells such as macrophages secrete large amounts of cytokines including IFNs and ILs. On the one hand, these cytokines can remove virus. On the other hand, they dominantly mediate the inflammatory injury caused by viral infection. This study evaluated the effects of the main active plasma components of BLG (tryptanthrin B, 4(3H)-Quinazolone and epigoitrin) on the innate immune response of respiratory syncytial virus (RSV)-activated mouse macrophages. ELISA, real-time PCR and reporter gene assay all showed that 4(3H)-Quinazolone inhibited RSV-induced IFN-ß secretion in mouse macrophages in a dose-dependent manner within a concentration range (0.3125-1.25µM) having no effects on cell viability, but the inhibitory effects were inferior to those of ribavirin. Western blot analyses further revealed that 4(3H)-Quinazolone inhibited RSV-induced expression of RIG-I (Retinoic acid-Inducible Gene-I) in mouse macrophages dose-dependently, thereby suppressing the transcription of IFN-ß, with lower effects than those of ribavirin. This may be one of the important mechanisms by which BLG inhibited inflammatory injury without affecting the immune system to eliminate virus. The results inspire future studies to elucidate the antiviral mechanisms of traditional Chinese medicine drugs.

Total synthesis and anti-viral activities of an extract of Radix isatidis.

Radix isatidis (Banlangen), a famous traditional Chinese medicine, has been used for thousands of years in China due to its anti-viral activity. Through our research, we inferred that the anti-viral activity of Radix isatidis depended on the water-soluble part. Among the components of this extract, the isoquinoline derivative 1 was isolated for the first time and has shown better anti-viral activity than other constituents. In this study, to solve the problem of sourcing sufficient quantities of compound 1, a total synthesis route is described, and several analogues are also evaluated for their anti-viral activities. Among them, compound 8 shown potent anti-viral activity with an IC50 value of 15.3 µg/mL. The results suggested that isoquinoline derivatives possessed potent anti-viral activity and are worthy further development.

[Structures and bioactivity of polysaccharides from isatidis radix].

OBJECTIVE: To investigated the chemical structures and bioactivity of polysaccharides from Isatidis Radix. METHOD: Polysaccharides were extracted and purified by column chromatograph and their chemical structures were identified by UV, IR, NMR, periodic acid oxadation and Smith degradation method and their stimulation effects to macrophage were evaluated by using MTT method. RESULT: Five polysaccharides, polysaccharide A , B, C, D and E were gotten and their molecular weights were 2 000, 1 757.1, 1 34 2.7, 955.6, 11.7 kDa, respectively. Polysaccharide A was composed of arabinose, polysaccharide E was composed of arabinose and galactose, polysaccharides B, C, D were composed of glucose and 1 --> 2, 1 --> 3, 1 --> 4, 1 --> 6 linkages existed in polysaccharides A-E, of A, B, C, D, E were alpha-configurations. Polysaccharides B, C and D showed better bioactivity than polysaccharides A and E with stimulation index (SI) of 5.31, 4.76, 5.17. CONCLUSION: Five polysaccharides are seperated firstly from Isatidis Radix.

Preclinical qualification of a new multi-antigen candidate vaccine for metastatic melanoma.

New therapies are urgently required for the treatment of patients with melanoma. Here we describe the generation and preclinical evaluation of 3 new recombinant ALVAC(2) poxviruses vCP2264, vCP2291, and vCP2292 for their ability to induce the desired cellular immune responses against the encoded melanoma-associated antigens. This was done either in HLA-A2/K transgenic mice or using in vitro antigen-presentation studies. These studies demonstrated that the vaccine was able to induce HLA-A*0201-restricted T-cell responses against gp100 and NY-ESO-1, detectable directly ex vivo, in HLA-A2/K-transgenic mice. The in vitro antigen presentation studies, in the absence of appropriate animal models, demonstrated that target cells infected with the vaccine construct were lysed by MAGE-1, MAGE-3 or MART-1 peptide-specific T cells. These data indicate that ALVAC(2)-encoded melanoma-associated antigens can be properly processed and presented to induce antigen-specific cytotoxic T-cell responses. To enhance the immunogenicity of the melanoma antigens, a TRIad of COstimulatory Molecules (TRICOM) were also cloned into all 3 vectors. Increased in vitro proliferation and IFN-γ production was observed with all ALVAC(2) poxviruses encoding TRICOM, confirming the immune-enhancing effect of the ALVAC-encoded TRICOM. These studies demonstrated that all components of the vaccine were functionally active and provide a rationale for moving this candidate vaccine to the clinic.

[Calcium channel mechanism by which betaine promotes proliferation of lymphocytes in mice].

OBJECTIVE: To study how the way in which betaine promotes the proliferation of mouse spleen lymphocytes is related to calcium channels. METHOD: BALB/c mice were used for this experiment. Mouse spleen lymphocytes were obtained through in vitro cultivation after they had been separated, and were divided into a negative control group, a Con A group, and 0.04, 0.4, 4, and 20 mmol x L(-1) betaine groups. MTT was used to observe the effect of betaine on the proliferation of mouse spleen lymphocytes; flow cytometry was used to measure the changes in the cell cycle of mouse spleen lymphocytes; and laser confocal scanning microscopy was used to observe the changes in the intracellular [Ca2+]i of mouse spleen lymphocytes after betaine or different calcium channel blockers were applied. RESULT: Betaine was found to promote the proliferation of mouse spleen lymphocytes 12 h after it had been applied in vitro in concentrations of 4 and 20 mmol x L(-1). It was also found to promote the proliferation of mouse spleen lymphocytes 24 h and 48 h after it had been applied in vitro in concentrations of 0.04, 0.4, 4, and 20 mmol x L(-1), with the effect being most marked for the 4 mmol x L(-1) group 24 h after its application. It was found to facilitate the entry of mouse spleen lymphocytes from the G0/G1 to the S phase 4, 6, 18, and 24 h after it had been applied to mouse spleen lymphocytes in a concentration of 4 mmol x L(-1), with the effect being most marked at 18 h after its application. Intracellular [Ca2+]i in mouse spleen lymphocytes increased significantly (P < 0.01) 6, 12, 18 h after 4 mmol x L(-1) betaine had acted on the lymphocytes, with the effect being most marked at 6 h. The calcium channel blockers nifidipine, diltiazem, mibefradil, and genistein had no effect on the increase of the intracellular [Ca2+]i in mouse spleen lymphocytes due to the application of betaine, while verapamil, mycifradin, heparin, and procaine could block such increase. CONCLUSION: Betaine facilitates the entry of mouse spleen lymphocytes from the G0/G1 into the S phase by raising the intracellular [Ca2+]i in these cells, thus promoting their proliferation. Intracellular [Ca2+]i increases mainly in two ways: (1) By affecting the alpha1 subunit of the L-type voltage-gated calcium channel with mediation by G proteins and thus leading to an efflux of intracellular calcium: (2) By affecting the IP3R and RyR calcium channels of the intracellular calcium stores and thus leading to the release of intracellular calcium.

New nonadjacent bis-THF ring acetogenins from the seeds of Annona squamosa.

Two new annonaceous acetogenins named as squamostanin-C and squamostanin-D were isolated from 95% EtOH extract of the seeds of Annona squamosa. Their structures were determined by spectroscopic method, and their cytotoxicities were evaluated by using MTT method.

New sphingolipids from the root of Isatis indigotica and their cytotoxic activity.

Two new sphingolipids were isolated from the 95% EtOH extract of traditional Chinese medicinal plant Isatis indigotica. Their structures were elucidated as (2S,3R)-3-hydroxymethyl-N-(2'-hydroxynonacosanoyl)-trideca-9E-sphingenine(1) and 1-O-beta-D-glucopyranosyl-(2S,3R)-3-hydroxymethyl-N-(2'-hydroxynonacosanoyl)-trideca-9E-sphingenine(2) on the basis of spectroscopic data. Their cytotoxic effects were evaluated by using MTT method.

[Chemical constituents from water extract of Radix isatidis].

AIM: To study the chemical constituents from water extract of Radix isatidis. (Isatis indigotica Fort. ). METHODS: The water extract was underwent absorption by D101 macroporous resin, the portion eluted by ethanol of different concentrations was isolated and purified on silica gel column repeatedly. The obtained compounds were identified and structurally elucidated by their physico-chemical properties and spectral analysis. RESULTS: Five compounds were isolated from water extract of Radix isatidis, and were partly identified separately: 3-[2'-(5'-hydroxymethyl) furyl] -1 (2H) -isoquinolinone-7-O-beta-D-glucoside (I), lariciresinol-4,4'-di-O-beta-D-glucopyranoside (II), lariciresinol-4-O-beta-D-glucopyranoside (III), 2-hydroxy-1, 4-benzenedicarboxylic acid (IV), mannitol (V). CONCLUSION: Compound I is a new compound and compounds IV and V were isolated from the plant for the first time.