Sea buckthorn (Hippophae rhamnoides L.) fermentation liquid protects against alcoholic liver disease linked to regulation of liver metabolome and the abundance of gut microbiota.

BACKGROUND: Alcoholic liver disease (ALD) refers to liver damage caused by long-term heavy drinking, which causes oxidative stress and changes in gut microbiota. In this paper, we investigated the hepatoprotective effect of sea buckthorn fermentation liquid on ALD in mice and the interaction between ALD and gut microbiota using animal experiments and gut microbiota measurements. RESULTS: We found that the contents of total flavonoids, total triterpenes and related short-chain fatty acids (SCFAs) in sea buckthorn fermentation liquid (SFL) were significantly greater. Liver index, kidney index, spleen index, serum indexes of liver injury - alanine aminotransferase (ALT) and spartate aminotransferase (AST), inflammatory factors in liver tissues - tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), oxidation indexes - malondialdehyde (MDA) and superoxide dismutase (SOD), and lipid metabolism indexes - high-density liptein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), and triglyceride (TG), suggested that SFL significantly ameliorates liver injury caused by alcohol. By measuring gut microbiota in mice feces samples, we found that the high-dose group of SFL reversed the declining trend of the gut microbiota Firmicutes/Bacteroidetes (F/B) ratio caused by alcohol, reducing the number of gram-negative bacteroidetes. Patescibacteria was tightly connected with the indicators of ALD. At the genus level, high-dose SFL significantly downregulated Akkermansia, Turicibacter, Alistipes and Ruminiclostridium, and improved the abundance of beneficial bacteria in Lactobacillus. In addition, Alistipes and Ruminiclostridium was closely connected with the indicators of ALD. CONCLUSION: Sea buckthorn fermentation liquid protected against alcoholic liver disease and modulated the composition of gut microbiota. © 2020 Society of Chemical Industry.

Simultaneous determination of six coptis alkaloids in urine and feces by LC-MS/MS and its application to excretion kinetics and the compatibility mechanism of Jiao-Tai-Wan in insomniac rats.

Jiao-Tai-Wan (JTW) is a well-known traditional Chinese medicine prescription composed of Rhizoma Coptidis (RC) and Cortex Cinnamon (10:1, g/g). It has been used to treat insomnia in China for centuries. This study investigates the excretion properties of coptis alkaloids from RC and JTW in normal and insomniac rats, and it examines the compatibility mechanism for this prescription. A new liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of six alkaloids - berberine, epiberberine, coptisine, jatrorrhizine, palmatine and magnoflorine - in rat urine and feces. The normal and model rats were orally treated with RC and JTW powder at a dosage containing 3.0 g kg-1  day-1 RC once per day for 7 days. Briefly, the results showed that the cumulative amounts of urinary and fecal excretion of the six alkaloids were significantly different in the pathological condition, as well as in compatibility. In normal rats, the urinary and fecal excretion of coptis alkaloids, especially berberine, coptisine and palmatine, increased significantly in the JTW group compared with the RC group, while the urinary and fecal excretion of six alkaloids decreased in insomniac rats. These data suggested that pathological conditions might have a notable influence on the excretion of alkaloids in rats, and demonstrated that the compatibility could promote better therapeutic effects through the accumulation of alkaloids in the body. These results might explain the compatibility of JTW.

Pharmacokinetics, metabolism, and excretion of cycloastragenol, a potent telomerase activator in rats.

1. The objective of this study was to investigate the pharmacokinetics, excretion, and metabolic fate of cycloastragenol (CA) in rats. 2. An LC-MS method was developed and used to quantify CA in biological samples. Rats were orally administrated with CA at 10, 20, and 40 mg/kg or intravenously administrated at 10 mg/kg to determine pharmacokinetic parameters of CA. For excretion experiment, urine, feces, and bile were collected at 24 h after oral administration (40 mg/kg), also at 12 h after intravenous administration (10 mg/kg). An LC-MS/MS method was developed to identify the metabolites of CA. 3. The results showed that the oral bioavailability of CA was about 25.70% at 10 mg/kg. CA was excreted through bile and feces and eliminated predominantly by the kidney in rats. It also might exist an enterohepatic circulation of CA in rats. CA could be metabolized widely in vivo in rat, seven, six, and one phase I metabolites were found in feces, urine, and bile samples respectively, but no phase II metabolite was found. 4. In summary, this study defined pharmacokinetics characteristics of CA, described its excretion, and established its in vivo metabolism in rats.

[In vitro effects of Genkwa Flos chloroform extract on activity of human liver microsomes UGTs and UGT1A1].

To predict the mechanism of liver injury induced by Genkwa Flos, we investigated the effect of chloroform extract on UGTs and UGT1A1 activities of the liver microsomes in rat and human. In the present study, 4-nitrophenol(4-NP) and ß-estradiol were elected as substrates to determine activities of UGTs and UGT1A1 by UV and HPLC. The results showed that there were 1.00% of apigenin, 6.40% of hydroxygenkwanin and 18.38% of genkwanin in chloroform extract; and total diterpene mass fraction was 31.40%. Compared with the control group, chloroform extract could significantly inhibit the activity of UGTs in rat liver microsomes(RLM) system, while the inhibitory effect was not obvious in human liver microsomes(HLM) system. UGT1A1 activity was inhibited by chloroform extract in rat liver microsomes and human liver microsomes (based on genkwanin, IC50=8.76, 10.36 µmol•L⁻¹). The inhibition types were non-competitive inhibition(RLM) and uncompetitive inhibition(HLM). In conclusion, the results indicated that chloroform extract showed different inhibitory effects on UGTs and UGT1A1 activity, which may be one of the mechanisms of liver injury induced by Genkwa Flos.

[In vitro and In vivo effects of six Coptidis alkaloids on liver microsomes UGTs and UGT1A1 activities in rats and mice].

In the present study, the effects of six Coptidis alkaloids (berberine, epiberberine, coptisine, jatrorrhizine, palmatine and magnoflorine) on liver microsomes UGTs and UGT1A1 activities in rats and mice were investigated in vitro and in vivo to study the mechanism of metabolic drug-drug interactions of Coptidis Rhizoma with other drugs. In vitro rat and mice liver microsomal incubation systems combined with UDPGA were applied, as well as mice liver microsomes after administration of six Coptidis alkaloids. 4-Nitrophenol and ß-estradiol were selected as substrates to determine activities of UGTs and UGT1A1 by UV and HPLC, respectively. According to the in vitro rat study, berberine, epiberberine, coptisine and jatrorrhizine significantly inhibited rat liver microsome UGTs activity, particularly epiberberine showed the strongest inhibition. UGT1A1 activity was lowly inhibited by jatrorrhizine, with IC50 at about 227 µmol•L⁻¹, whereas coptisine and magnoflorine significantly activated UGT1A1. According to the in vitro mice study, berberine, coptisine, jatrorrhizine and palmatine significantly inhibited mice liver microsome UGTs activity, and the six alkaloids all significantly activated UGT1A1. According to the in vivo mice study, UGTs activity was significantly activated only in berberine group, while UGT1A1 activity was significantly activated only in jatrorrhizine group. In conclusion, the effects of Coptidis alkaloids on UGT activity showed significant differences in species and between in vitro and in vivo. Meanwhile, the changes in structures of Coptidis alkaloids also have a big impact on UGT activity, which may be one of the causes for the drug-drug interactions between Coptidis Rhizoma and other drugs.

[Inhibitory effect of different species of hydroxygenkwanin on UGTs and UGT1A1 activities].

To predit the mechanism of metabolic drug-drug interactions of hydroxygenkwanin with other drugs, we investigated the inhibition inhibitory effect of hydroxygenkwanin on UGTs and UGT1A1 activities of different liver microsomes. In the present study, 4-nitrophenol (4-NP) and ß-estradiol were elected as substrates to determine activities of UGTs and UGT1A1 by UV and HPLC, respectively. The results showed that, hydroxygenkwanin significantly inhibited UGTs activity in rat, mouse and human liver microsomes. UGT1A1 activity was inhibited by hydroxygenkwanin to varying degrees, with IC50 about 190, 10.93, 20.07, 76.31 µmol•L⁻¹ in mouse liver microsome(MLM), rat liver microsome (RLM) and recombinant UGT1A1, and human liver microsome (HLM), respectively. The inhibition types were competitive inhibition (RLM, HLM) and linear mixed-typed linear inhibition (recombinant UGT1A1). The order for the inhibitory intensity was RLM>rUGT1A1>HLM>MLM. In conclusion, hydroxygenkwanin has an inhibitory effect on UGTs and UGT1A1 activities of different liver microsomes, with differences in species, indicating its potential drug interactions based on UGT1A1 enzyme. This study aims to provide a reliable experimental basis for its further research and development of hydroxygenkwanin, and provide theoretical reference for the clinic drug combination research.

Lactobacillus co-fermentation of Cerasus humilis juice alters chemical properties, enhances antioxidant activity, and improves gut microbiota.

Fermentation with Lactobacillus has been shown to improve the nutritional value of juice. In this study, Cerasus humilis juice was fermented using two commercial probiotics, namely, Lactobacillus acidophilus and Lactobacillus plantarum. The total antioxidant capacity (TAOC), viable count, chemical properties, antioxidant activity after in vitro digestion, and alterations in the gut microbiota composition of the fermented juice were investigated. After fermentation, the TAOC increased from 107.66 U mL-1 to 126.72 U mL-1; viable count increased from 5.85 lg (CFU mL-1) to 8.17 lg (CFU mL-1); and the contents of total phenols, total flavonoids, proanthocyanins, four organic acids, and 29 amino acids had changed. Overall, 47 compounds were identified in the juice, 20 of which were enriched after fermentation. Furthermore, Lactobacillus co-fermentation improved the antioxidant properties of the juice after in vitro digestion and increased the abundance of probiotics to regulate the gut microbiota. These findings illustrate the potential use of Lactobacillus acidophilus and Lactobacillus plantarum in the co-fermentation of C. humilis juice to enhance its nutritional and functional properties.

Preventive effect of Chinese dwarf cherry [Cerasus humilis (Bge.) Sok.] fermentation juice on dextran sulfate sodium-induced ulcerative colitis rats through the regulation of IgA and the intestinal immune barrier.

Ulcerative colitis (UC) is a modern, refractory disease, and studies have shown that UC is closely associated with the gut microbiota and intestinal immune barrier. This study evaluated the protective effects and regulatory mechanism of Chinese dwarf cherry [Cerasus humilis (Bge.) Sok.] fermentation juice (CFJ) on UC induced by dextran sulfate sodium (DSS). The results indicated that CFJ could significantly modulate the oxidative stress index in the serum and colon, observably reduce MPO and NO activity, and increase the SOD level. CFJ significantly downregulated the levels of TNF-α, IL-1ß and IL-6 and reduced inflammation caused by DSS. SIgA and short-chain fatty acids (SCFAs) levels were effectively improved in the CFJ group, especially the acetic acid and butyric acid levels. Intestinal flora analysis showed that DSS could enrich harmful bacteria such as Alistipes and Oribacterium and that CFJ could increase the abundance of beneficial bacteria (Parasutterella, Bacteroides, Roseburia and Blautia). SIgA in the colon was positively correlated with Lachnoclostridium, Blautia, Lachnospiraceae_UCG-004, Prevotellaceae_NK3B31_group and other beneficial bacteria. The results showed that DSS group rats had immunity and signalling pathway disorders and that CFJ could regulate immune disorders, mainly by regulating the expression of IgA pathway components. Taken together, our results demonstrated that CFJ could regulate changes in the gut microbiota, improve the expression of immune protein-related genes, further regulate intestinal mucosal immune function and maintain intestinal mucosal barrier homeostasis.

Jiaotai Pill () Alleviates Insomnia through Regulating Monoamine and Organic Cation Transporters in Rats.

OBJECTIVE: To reveal the effect and mechanism of Jiaotai Pill (, JTP) on insomniac rats. METHODS: The insomniac model was established by intraperitoneal injection of p-chlorophenylalanine (PCPA). In behavioral experiments, rats were divided into control, insomniac model, JTP [3.3 g/(kg•d)], and diazepam [4 mg/(kg•d)] groups. The treatment effect of JTP was evaluated by weight measurement (increasement of body weight), open field test (number of crossings) and forced swimming test (immobility time). A high performance liquid chromatography-electrochemical detection (HPLC-ECD) method was built to determine the concentration of monoamine transmitters in hypothalamus and peripheral organs from normal, model, JTP, citalopram [30 mg/(kg•d)], maprotiline [40 mg/(kg•d)] and bupropion [40 mg/(kg•d)] groups. Expressions of serotonin transporter (SERT), dopamine transporter (DAT), and norepinephrine transporter (NET) were analyzed by quantitative polymerase chain reaction (qPCR) and Western blot in normal, model and JTP groups. A high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS/MS) method was established to determine the pharmacokinetics, urine cumulative excretion of metformin in vivo, and tissue slice uptake in vitro, which were applied to assess the activity of organic cation transporters (OCTs) in hypothalamus and peripheral organs. RESULTS: Compared with the insomniac model group, the body weight and spontaneous locomotor were increased, and the immobility time was decreased after treatment with JTP (P<0.01). Both serotonin and dopamine contents in hypothalamus and peripheral organs were increased (P<0.01). The norepinephrine content was increased in peripheral organs and decreased in hypothalamus (P<0.05 or P<0.01). At the same time, SERT, DAT, OCT1, OCT2, and OCT3 were down-regulated in hypothalamus and peripheral organs (P<0.05). NET was down-regulated in peripheral organs and up-regulated in hypothalamus (P<0.05 or P<0.01). Moreover, the activity of OCTs in hypothalamus and peripheral organs was inhibited (P<0.05). CONCLUSION: JTP alleviates insomnia through regulation of monoaminergic system and OCTs in hypothalamus and peripheral organs.

Assessing Volatiles in Three Chinese Dwarf Cherry Cultivars during Veraison and Maturation Using Headspace-Solid Phase Microextraction with Gas Chromatography-Mass Spectrometry.

Chinese dwarf cherry is a native shrub in northwest China with a rich and unique fruit aroma. This study aims to determine the changes in volatile profiles during the maturation period, which provides a theoretical basis for the optimal harvest times and the breeding of aroma-rich varieties. The variation in the production of 164 volatile compounds from three Chinese dwarf cherry cultivars, namely, "Jing'ou 1", "Jing'ou 2", and "Jing'ou 3", were investigated by headspace-solid phase microextraction (HS-SPME)-GC-MS. These volatiles mainly constituted alcohols, carbonyls, esters, terpenoids, and hydrocarbons. Their maturation process could be divided into three stages, namely prophase, metaphase, and anaphase. Prophase contained an abundance of hydrocarbons and carbonyls, primarily benzaldehyde being dominant among all volatiles. During metaphase, volatiles remained at a low level of abundance and diversity. Anaphase coincided with full maturation and was associated with esters and terpenoids; in particular, "Jing'ou 3" presented more compound diversity and a high level of acetate esters. The periods including the week prior to veraison and the week during maturation were particularly critical in volatile formation in Chinese dwarf cherries. This study reveals that the low level or lack of hexanal might be one of the distinctive characteristics separating Chinese dwarf cherries from other Cerasus or Rosaceae fruits.

The oral bioavailability, excretion and cytochrome P450 inhibition properties of epiberberine: an in vivo and in vitro evaluation.

Epiberberine (EPI) is a novel and potentially effective therapeutic and preventive agent for diabetes and cardiovascular disease. To evaluate its potential value for drug development, a specific, sensitive and robust high-performance liquid chromatography-tandem mass spectrometry assay for the determination of EPI in rat biological samples was established. This assay was used to study the pharmacokinetics, bioavailability and excretion of EPI in rats after oral administration. In addition, a cocktail method was used to compare the inhibition characteristics of EPI on cytochrome P450 (CYP450) isoforms in human liver microsomes (HLMs) and rat liver microsomes (RLMs). The results demonstrated that EPI was rapidly absorbed and metabolized after oral administration (10, 54 or 81 mg/kg) in rats, with Tmax of 0.37-0.42 h and T1/2 of 0.49-2.73 h. The Cmax and area under the curve values for EPI increased proportionally with the dose, and the oral absolute bioavailability was 14.46%. EPI was excreted mainly in bile and feces, and after its oral administration to rats, EPI was eliminated predominantly by the kidneys. A comparison of the current half-maximal inhibitory concentration and Ki values revealed that EPI demonstrated an obvious inhibitory effect on CYP2C9 and CYP2D6. Furthermore, its effect was stronger in HLM than in RLM, more likely to be a result of noncompetitive inhibition.

Proteomics approach to analyze protein profiling related with ADME/Tox in rat treated with Scutellariae radix and Coptidis rhizoma as well as their compatibility.

ETHNOPHARMACOLOGICAL RELEVANCE: Scutellariae radix (Scutellaria baicalensis Georgi) and Coptidis rhizoma (Coptis chinensis Franch), known as traditional Chinese medicine (TCM), have been widely used with the effects of suppressing fever, dispelling dampness, purging fire and removing toxicosis. Owing to their unimaginable complexity, it is difficult to understand their pharmacokinetic properties in detail. The aim of this study was to develop an optimal proteomics approach to analyze the protein profiling related with ADME/Tox in rat liver treated with S. radix and C. rhizoma as well as their compatibility. MATERIALS AND METHODS: Male rats were respectively administered the extracts of S. radix, C. rhizoma and their mixture for 7 days, and their liver tissue samples were prepared for the comparative proteomic analysis. The significantly differentially expressed proteins between the experimental groups and the control group were found and identified by 2-DE and MALDI-TOF-MS analyses. To validate the proteomic analysis results, glutathion peroxidase, catalase and betaine homocysteine methyl transferase were selected and confirmed by western blotting. RESULTS: Seventy eight significantly differentially expressed proteins between the experimental groups and the control group were found and identified. By querying the relational databases, the identified differentially expressed proteins were summarized and classified into three groups, phase I drug metabolic enzymes, phase II drug metabolic enzymes and the rest proteins which mainly involve in energy metabolism, signal transduction and cytoskeleton. These proteins involved in ADME/Tox may be the targets for metabolic studies or markers for toxicity. CONCLUSIONS: Our findings indicated S. radix and C. rhizoma as well as their compatibility can assuredly influence the expression of the proteins in rat liver. After administration, the majority of these expressions presented a downward trend, which may be closely related to the pharmacological properties of the medicine. The method in this study may open up a new road for the complementary tests for ADME/Tox properties of S. radix and C. rhizoma as well as their compatibility.