Alginate oligosaccharide assimilation by gut microorganisms and the potential role in gut inflammation alleviation.

Dietary fiber metabolism by gut microorganisms plays important roles in host physiology and health. Alginate, the major dietary fiber of daily diet seaweeds, is drawing more attention because of multiple biological activities. To advance the understanding of alginate assimilation mechanism in the gut, we show the presence of unsaturated alginate oligosaccharides (uAOS)-specific alginate utilization loci (AUL) in human gut microbiome. As a representative example, a working model of the AUL from the gut microorganism Bacteroides clarus was reconstructed from biochemistry and transcriptome data. The fermentation of resulting monosaccharides through Entner-Doudoroff pathway tunes the metabolism of short-chain fatty acids and amino acids. Furthermore, we show that uAOS feeding protects the mice against dextran sulfate sodium-induced acute colitis probably by remodeling gut microbiota and metabolome. IMPORTANCE: Alginate has been included in traditional Chinese medicine and daily diet for centuries. Recently discovered biological activities suggested that alginate-derived alginate oligosaccharides (AOS) might be an active ingredient in traditional Chinese medicine, but how these AOS are metabolized in the gut and how it affects health need more information. The study on the working mechanism of alginate utilization loci (AUL) by the gut microorganism uncovers the role of unsaturated alginate oligosaccharides (uAOS) assimilation in tuning short-chain fatty acids and amino acids metabolism and demonstrates that uAOS metabolism by gut microorganisms results in a variation of cell metabolites, which potentially contributes to the physiology and health of gut.

Antiperspirant effects and mechanism investigation of Mulisan decoction in rats based on plasma metabolomics.

CONTEXT: Mulisan decoction (MLS) is a classic formula of traditional Chinese medicine for treating hyperhidrosis. The mechanism remains unclear. OBJECTIVE: To investigate the antiperspirant effect and underlying mechanisms of MLS. MATERIALS AND METHODS: Fifty rats were divided into control, model, and three doses of MLS intervention groups (n = 10). Rats except for control group were induced diseases features of the applicable scope of MLS via i.p. reserpine (0.5 mg/kg/d) for 10 days. From day 11, MLS groups were administrated orally MLS at 0.6, 3, and 15 g/kg once a day for 14 days, respectively. After the last administration, sweating was induced in all rats via s.c. pilocarpine (25 mg/kg), the right hind foot of rats was stained, and sweat point numbers were observed. Rat serum was collected to detect IL-2, IL-6, IFN-γ, and TNF-α. Rat plasma was collected for endogenous metabolite analysis via UPLC-QE-Focus-MS. RESULTS: Rats treated with MLS presented a significant decrease in sweat point numbers (13.5%), increase in body weight (13.2%), and promotion in the balance of Th1/Th2 cytokine ratio via increasing IL-2 (38.3%), IFN-γ (20.1%), and TNF-α (22.0%) and decreasing IL-6 (24.7%) compared with the model group (p < 0.05). Plasma metabolomics disclosed 15 potential biomarkers related to model rats, of which two could be significantly reversed by MLS (p < 0.05). The involved pathways were pantothenate and CoA biosynthesis, and porphyrin metabolism. CONCLUSIONS: MLS demonstrated a good antiperspirant effect and metabolism improvement. These findings inspire more clinical study validation on immune improvement and antiperspirant effect.

Haimufang decoction, a Chinese medicine formula for lung cancer, arrests cell cycle, stimulates apoptosis in NCI-H1975 cells, and induces M1 polarization in RAW 264.7 macrophage cells.

BACKGROUND: Lung cancer has the highest morbidity and mortality in the world and novel treatment strategies are still needed. Haimufang decoction (HMF) is a patented clinical prescription of traditional Chinese medicine for lung cancer treatment. HMF is composed of four herbs and has been applied clinically in advanced cancer patients. However, its therapeutic mechanisms are still unclear. This study aims to elucidate the possible mechanisms of HMF for the treatment of lung cancer. METHODS: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay was applied for evaluating the proliferative effect of HMF in lung cancer cells and monocyte macrophage RAW264.7 cells. Flow cytometer was used to detect the effects of HMF on cell cycle and apoptosis, and western blotting was employed to explore the potential apoptotic mechanisms of HMF on lung cancer cells. For immunomodulatory effect, co-culture system was used to detect the activation of macrophage RAW264.7 cells when treated with HMF, and neutral red assay was used to measure the effect of HMF on the phagocytosis of the activated macrophages. Enzyme linked immunosorbent assay, flow cytometer, and immunofluorescence staining method were employed for the investigation on the underlying mechanisms of the immunomodulatory effect on RAW264.7 induced by HMF. RESULTS: HMF inhibited the proliferation, induced S phase cell cycle arrest, and stimulated apoptosis in lung cancer NCI-H1975 cells, while had negligible cytotoxicity on macrophage RAW264.7 cells. Moreover, HMF could activate macrophage RAW264.7 cells and promote the inhibition activity of RAW264.7 cells against lung cancer cells. And also, HMF activated macrophages and increased their phagocytic activity in a concentration-dependent manner. HMF increased the expression of macrophage activation marker CD40, the level of nitric oxide, the generation of intracellular reactive oxygen species, as well as M1 macrophages cytokines including tumor necrosis factor-α, interleukin-1ß, interleukin 12 p70, and interleukin 6. Further investigation showed that HMF induced M1 but not M2 phenotype polarization in RAW264.7 cells. CONCLUSIONS: HMF can mainly exert anticancer activity via (1) cytotoxicity to human lung cancer cells by proliferation inhibition, cell cycle arrest, and apoptosis induction; and also via (2) immunomodulation via macrophage cells activation and M1 phenotype polarization induction.

Substitution of one calcium-binding amino acid strengthens substrate binding in a thermophilic alginate lyase.

Ligand binding is sensitive to temperatures since noncovalent bonds between the binding site and ligand could be broken by heat. How metal ion-binding amino acids in alginate lyase evolve to achieve tight substrate binding in a hostile environment remains unknown. An endolytic alginate lyase AlgAT0 specifically cleaved the M-G glycosidic bond and released disaccharides as the main end product. Four conserved calcium-binding sites were predicted and the supplement of Ca2+ led to enhanced substrate binding and protein stability. Among the four conserved calcium-binding sites, one substitution of aspartate for glutamate in AlgAT0 was proved to stimulate Ca2+ affinity. This study suggested that substrate affinity of polysaccharide lyases could be improved by tight binding to Ca2+ via one amino acid substitution.

Comparisons of the chemical profiles, cytotoxicities and anti-inflammatory effects of raw and rice wine-processed Herba Siegesbeckiae.

ETHNOPHARMACOLOGICAL RELEVANCE: Although slightly toxic, the Chinese medicinal herb Herba Siegesbeckiae (HS) has long been used as a remedy for traditional Chinese medicine symptoms that resemble inflammatory joint disorders, because it can eliminate the wind-dampness and soothe painful joints. Proper processing can reduce the toxicity and/or enhance the efficacy of raw herbs. In this study, we aim to examine if processing with rice wine reduces the cytotoxicities and/or enhances the anti-inflammatory effects of HS, and to explore the chemical basis behind the potential changes of medicinal properties caused by the processing. MATERIALS AND METHODS: We used cell models to examine the cytotoxicities and anti-inflammatory effects of HS and rice wine-processed HS (WHS). The chemical profiles of HS and WHS were compared using the ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS) analysis. RESULTS: We found that WHS was less toxic than HS in cultured cells as shown in the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Both HS and WHS had anti-inflammatory effects as demonstrated by their abilities to reduce nitric oxide (NO) production as well as protein and mRNA expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Interestingly, the anti-inflammatory effects of WHS were more potent than that of HS at the concentration of 100 µg/mL. By comparing the chemical profiles, we found that 19 peaks were lower, while 2 other peaks were higher in WHS than in HS. Four compounds including neo-darutoside, darutoside, stigmasterol and 16-O-acetyldarutoside corresponding to 4 individual changed peaks were tentatively identified by matching with empirical molecular formulae and mass fragments. CONCLUSION: Our study showed that processing with rice wine significantly reduced the cytotoxicities and enhanced the anti-inflammatory effects of HS as demonstrated in cell models. We also developed a UPLC/Q-TOF-MS method to clearly differentiate HS from WHS by their different chemical profiles. Further study is warranted to establish the relationship between the alteration of chemical profiles and the changes of medicinal properties caused by processing with rice wine.

A herbal formula consisting of Rosae Multiflorae Fructus and Lonicerae Japonicae Flos inhibits inflammatory mediators in LPS-stimulated RAW 264.7 macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: A herbal formula (RL) consisting of Rosae Multiflorae Fructus (Yingshi) and Lonicerae Japonicae Flos (Jinyinhua) has been traditionally used to treat inflammatory disorders. This study aims to investigate the anti-inflammatory mode and mechanism of action of the ethanol extract of RL so as to provide a pharmacological basis for the use of RL in treating inflammatory diseases. MATERIALS AND METHOD: RL consisting of Yingshi and Jinyinhua (in 5:3 ratio) was extracted using absolute ethanol. We investigated its effects on nitric oxide (NO), interleukin-6 (IL-6), tumour necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NFκB) in mouse RAW 264.7 macrophages activated with lipopolysaccharide (LPS). RESULTS: RL could decrease the secretion of NO, IL-6 and TNF-α into the culture medium and the cellular protein levels of iNOS and COX-2, which were associated with the reduction of the phosphorylation/activation of JNK and p38, and the inhibition of the transcriptional activity of NF-κB. CONCLUSIONS: The present study demonstrated an inhibitory effect of RL on the inflammatory mediators regulated by the NF-κB and MAPK signalling pathways in LPS-stimulated RAW 264.7 macrophages, providing a pharmacological basis for RL in the control of inflammatory disorders.

UHPLC UHD Q-TOF MS/MS analysis of the impact of sulfur fumigation on the chemical profile of Codonopsis Radix (Dangshen).

Over recent decades sulfur fumigation has been becoming abused in processing some freshly harvested Chinese medicinal herbs, although it is questioned whether sulfur fumigation can result in changes in efficacy and safety of the herbs. One of the herbs commonly processed by sulfur fumigation is Codonopsis Radix (Dangshen). A report showed that lobetyolin content in sulfur-fumigated Dangshen was lower than in air-dried Dangshen. Whereas there is no investigation designed to compare the chemical profiles of the sulfur-fumigated Dangshen and the air-dried Dangshen. In the present study, a rapid and versatile ultra-high-performance liquid chromatography coupled with ultra-high resolution quadrupole time-of-flight mass spectrometry (UHPLC UHD Q-TOF MS/MS) method was developed for comprehensive analysis of the chemical profiles of sulfur-fumigated and air-dried Dangshen samples. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) demonstrated that there were significant chemical differences between sulfur-fumigated and air-dried Dangshen samples. Among the changed components, 57 compounds were identified, in which 15 sulfur-containing compounds were detected only in sulfur-fumigated samples. The established methods were successfully applied to discriminate sulfur-fumigated Dangshen among commercial samples. Whether the chemical changes caused by sulfur fumigation affect the clinical efficacy and safety of Dangshen needs to be further investigated.

Absorptive constituents and their metabolites in drug-containing urine samples from Wuzhishan miniature pigs orally administered with Buyang Huanwu decoction.

Buyang Huanwu decoction (BYHWD), a famous traditional Chinese medicine prescription for the treatment of cerebrovascular diseases, is composed of seven commonly used Chinese herbs--Astragali Radix, Angelicae Sinensis Radix, Paeoniae Radix Rubra, Chuanxiong Rhizoma, Carthami Flos, Persicae Semen and Pheretima. To determine the main absorptive constituents and the metabolites of BYHWD in vivo, urine samples from Wuzhishan (WZS) miniature pigs orally administered with BYHWD (13.6 g crude drugs/kg) were collected to investigate the characteristic compounds. By comparing the high-performance liquid chromatography of a drug-containing urine sample with that of a drug-free sample, 17 characteristic compounds were isolated from the methanol extract of a drug-containing urine sample by column chromatography. Their structures, including 11 isoflavanoids, 2 pterocarpanoids and 4 isoflavonoids, were identified by spectroscopic means. Of the 17 compounds, 8 (1-8) were new compounds with the following structures: 3S-7,3',4'-trihydroxyisoflavan-3'-O-ß-D-glucuronide (1), 3S-7,3',4'-trihydroxyisoflavan-4'-O-ß-D-glucuronide (2), 3S-7,2',4'-trihydroxyisoflavan-2'-O-ß-D-glucuronide (3), 3R-7,2'-dihydroxy-3',4'-dimethoxyisoflavan-2'-O-ß-D-glucuronide (4), 3R-7,2'-dihydroxy-3',4'-dimethoxyisoflavan-2'-O-ß-D-glucuronide-6"-methyl ester (5), 3R-7,2'-dihydroxy-3',4'-dimethoxyisoflavan-7-O-ß-D-glucuronide-6"-methyl ester (6), 3R-7,2',3'-trihydroxy-4'-methoxyisoflavan-3'-O-ß-D-glucuronide-6"-methyl ester (7), and 3S-7,4',5'-trihydroxy-2',3'-dimethoxyisoflavan-5'-O-ß-D-glucuronide (8). Based on the possible relationship and metabolic pathways of the 17 compounds in vivo, 3R-7,2'-dihydroxy-3',4'-dimethoxyisoflavan (isomucronulatol, 11), 6aR,11aR-3-hydroxy-9,10-dimethoxypterocarpan (methylnissolin, astrapterocarpan, 13), 7,3'-dihydroxy-4'-methoxyisoflavone (calycosin, 16) and 7-hydroxy-4'-methoxyisoflavone (formononetin, 17) were thought to be the most important absorptive original isoflavonoid constituents of BYHWD in vivo, which underwent reactions of glucuronidation, hydroxylation, demethylation and reduction. The other 13 compounds were deduced to be their metabolites.

Profiling and characterization of volatile components from non-fumigated and sulfur-fumigated Flos Lonicerae Japonicae using comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry coupled with chemical group separation.

Flos Lonicerae Japonicae (FLJ) is a popular herb used for many centuries in Traditional Chinese Medicine as a treatment of fever and inflammation. Non-fumigated processing of FLJ has been the traditional approach used in post-harvest preparation of the commodity for commercial use. However, in recent years, natural drying processing of FLJ has been replaced by sulfur-fumigation for efficiency and pest control. Sulfur-fumigation can induce changes in the volatile compounds of the herb, altering its medicinal properties. A comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOF/MS) method was established for the resolution and determination of volatile components in non-fumigated and sulfur-fumigated FLJ. In this paper, analysis of the volatile oils in non-fumigated and sulfur-fumigated (including lab-prepared sulfur-fumigated and industrial sulfur-fumigated) FLJ was performed using GC×GC-TOF/MS. Seventy-three representative volatile components were identified, including furans, alkalies, acids, aldehydes, ketones, alcohols, terpenes, esters, and others, as the main components of FLJ volatile oils. The proposed method was successfully applied for rapid and accurate quality evaluation of FLJ and its related medicinal materials and preparations.

The antidepressant effect of secoisolariciresinol, a lignan-type phytoestrogen constituent of flaxseed, on ovariectomized mice.

Secoisolariciresinol (SECO) is a natural lignan-type phytoestrogen constituent mainly found in flaxseed. It can be metabolized in vivo to mammalian lignans of enterodiol and enterolactone, which have been proven to be effective in relieving menopausal syndrome. Depression is one of the most common symptoms of menopausal syndrome, and is currently treated with estrogen replacement and antidepressant therapy. However, due to the serious side-effects of such agents, there are urgent needs for safer and more tolerable treatments. In this paper, using two classical depression models, the forced swimming test and the tail suspension test, we report the antidepressant effect of SECO on ovariectomized (OVX) mice by intragastric administration for 14 consecutive days at doses of 5, 10 and 20 mg/kg. The results showed that SECO (10 mg/kg) treatment could significantly reduce the duration of immobility of OVX mice in these two models compared with the control group (OVX mice + vehicle), which was similar to the positive control imipramine. In addition, SECO treatment could substantially increase brain monoamine (norepinephrine and dopamine) levels in OVX mice. The present studies showed that SECO can reverse depressive-like behavior and exhibit monoamine-enhancing effects.

[Study on HPLC fingerprints of Chrysanthemi Flos before and after sulfur-fumigation].

OBJECTIVE: To evaluate the quality of Chrysanthemi Flos before and after sulfur-fumigation by analysis of HPLC fingerprints. METHODS: RP-HPLC method was applied to establish the chromatographic fingerprint. The separation was performed on a Waters C, (250 mm x 4.6 mm, 5 microm) column with a gradient elution composed of acetonitrile-aqueous solution containing 0.5% phosphoric acid. The column temperature was 35 degrees C, the detective wavelength was set at 268 nm. RESULTS: Eleven batches of Chrysanthemi Flos samples composed of both sun-dried and sulfur-fumigated were analyzed and evaluated respectively. The method of clustering analysis was used, and the chromatographic fingerprint common pattern was finally established. CONCLUSION: The HPLC fingerprint method is reliable and suitable for quality evaluation of Chrysanthemi Flos before and after sulfur-fumigation.