Screening of the Active Substances for the Assessment of Walnut Kernel in the Treatment of Scopolamine-Induced AD Animals.

SCOPE: Alzheimer's disease (AD) has been a challenge and hotspot in the field of neuroscience research due to the high morbidity. As we all know, walnut kernel (WK) ingestion has been linked to benefits to brain health and has the function of improving memory. This study follows the AD model induced by scopolamine to reveal the active fractions and substances of walnut in the treatment of AD. METHODS AND RESULTS: The histopathological analysis and brain tissue biochemistry assay are revealed the active fractions of WK, and this result determines that walnut kernel organic acids have significant therapeutic effect on AD. The strategy of studying ingredients pointed at lesions is integrated to ascertain the selected brain-targeted effective substances of WK for blood-brain barrier by ultra-performance liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry, and a total of eight organic acids are figured out definite absorptivity in rat brains. Finally, the binding interaction between the effective substances and target proteins is analyzed by molecular docking, and the main function related active markers are ascertained as glansreginin A, glansreginic acid, ellagic acid, and ellagic acid 4-O-xyloside. CONCLUSIONS: The comprehensive process is helpful to the clinical application of WK as a promising cholinesterase inhibitors for nutritional intervention.

The attenuation effect of licorice on the hepatotoxicity of Euodiae Fructus by inhibiting the formation of protein conjugates and GSH depletion.

ETHNOPHARMACOLOGICAL RELEVANCE: As a traditional Chinese medicine and food, Euodiae Fructus (EF) is widely used in clinics to relieve pain and prevent vomiting and for making tea for more than a thousand years. In recent years, hepatotoxic reactions to EF have been reported. The intermediates produced by evodiamine and rutaecarpine metabolism in vitro were captured by glutathione (GSH), suggesting that the toxicity of EF may be related to metabolic activation. Whether licorice can inhibit the metabolic activation of EF has not been reported, which needed an effective strategy to clarify the correlation between protein conjugates and hepatotoxicity and the attenuation mechanism of licorice processing. AIM OF THE STUDY: This study aimed to explore the toxic components and mechanisms of EF based on metabolic activation and the detoxification of licorice. MATERIALS AND METHODS: The content and toxicity index of protein conjugates in the liver were determined by orally administering mice and rats with EF. The attenuation mechanism of licorice was examined in cell and enzymology experiments. RESULTS: The change in evodiamine-cysteinylglycine (EVO-Cys-Gly) and evodiamine-cysteine (EVO-Cys) levels was consistent with the change in hepatotoxicity. Licorice inhibited the formation of the protein conjugates of EF and increased the content of GSH in L02 cells. CONCLUSION: EF mediated by P450 enzymes produced toxic intermediates, which combined with cysteine residues in animal liver and inactivate them, leading to hepatotoxicity. Interestingly, licorice can alleviate the GSH depletion caused by EF and inhibit the production of protein conjugates by inhibiting P450 enzymes.

Effect of CYP3A inducer/inhibitor and licorice on hepatotoxicity and in vivo metabolism of main alkaloids of Euodiae Fructus based on UPLC-Q-Exactive-MS.

ETHNOPHARMACOLOGICAL RELEVANCE: As a traditional Chinese medicine, Euodiae Fructus (EF) has been used to treat stomachache, belching, and emesis for more than a thousand years. Ancient records and modern research have shown that EF has mild toxicity, which needs to be processed with licorice juice to reduce its toxicity. Research suggested that the toxicity of EF can be caused by in vivo metabolism, but whether its metabolites are related to hepatotoxicity and whether licorice can affect the metabolism of EF have not been reported, which needed an effective strategy to clarify the correlation between metabolites and toxicity and the attenuation mechanism of licorice processing. AIM OF THE STUDY: The poisonous substances and metabolic pathways were clarified by comparing the mechanism in vivo process of the main alkaloids of EF in normal rats and rats treated with dexamethasone (DXMS), ketoconazole (KTC), and EF processed with licorice (EFP). MATERIALS AND METHODS: Rats were given EF and EFP by oral administration, respectively. The EF + DXMS and EF + KTC groups were pretreated with DXMS and KTC, respectively, by i. p. for seven days, and their toxicity differences were compared. The comprehensive strategy based on UPLC-Q-Exactive-MS and Orthogonal Partial Least Squares Discriminant Analysis was developed to compare the types and contents of metabolites and clarify the metabolic pathways of alkaloids among EF, EFP, EF + KTC, and EF + DXMS groups. RESULTS: EF + DXMS group significantly increased the hepatotoxicity, whereas the EF + KTC and EFP groups reduced the hepatotoxicity compared with the EF group. One hundred and thirty-five metabolites were detected, and the metabolic pathways of the main alkaloid components related to toxicity were inferred in the plasma, urine, feces, and bile of rats. KTC and licorice similarly inhibited the production of toxic metabolites, changed metabolism in vivo, and produced many new II and a few phases I metabolites, while the contents of toxic metabolites increased in the DXMS group. CONCLUSION: Licorice and KTC could inhibit the production of metabolites of EF related to toxicity, increase the production of other metabolites and promote the excretion of alkaloids, which may be why licorice and KTC can minimize EF toxicity.

Isolation, molecular characterization, immunological and anticoagulatant activities of polysaccharides from frankincense and its vinegar processed product.

Frankincense (FRA), the oily resin consisting of essential oils, boswellic acids (BAs) and polysaccharides, has been used to improve the blood circulation and relieve pain against carbuncles. According to the theory of traditional Chinese medicine, vinegar processed frankincense (VPF) can increase the effects of promoting blood circulation and relieving pain. Existing studies have carried out much on BAs and essential oils. However, the comparative analysis of polysaccharides from FRA and VPF has not been reported. In this paper, two polysaccharides were isolated and purified from FRA and the other two were from VPF, and their structures and physicochemical properties were analyzed. The immunological and anticoagulatant activities of the four polysaccharides were tested in RAW 264.7 cell and Sprague-Dawley rats, respectively. The polysaccharides purified from VPF showed better immunological and anticoagulatant activities than those in FRA. Therefore, polysaccharides may be one of the active substances for the synergistic effect of VPF.

Systematic characterization of the metabolites of defatted walnut powder extract in vivo and screening of the mechanisms against NAFLD by UPLC-Q-Exactive Orbitrap MS combined with network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Walnut kernel, a well-known TCM, is often used after being defatted in tradition. And defatted walnut powder extract (DWPE) has the actions of tonifying the liver and kidney, dissipating stagnation and removing blood stasis, which has the effect on non-alcoholic fatty liver disease (NAFLD). However, the effective components of DWPE in vivo were unclear and the multiple mechanisms of DWPE against NAFLD have not been explored. AIM OF THE STUDY: The studies were performed to screen the effective substances in vivo by identification of the metabolites of DWPE in rats and to seek the potential mechanisms of DWPE on NAFLD by construction of the network pharmacology based on metabolites and verification of the highly correlated pathway. MATERIALS AND METHODS: To explore the effective substances in vivo, the metabolites of DWPE were identified in SD rats' bio-samples through UPLC-Q-Exactive Orbitrap MS. To analyze the mechanisms of DWPE on NAFLD, a Metabolite-Target-Disease network was established and the potential mechanisms were predicted. Then, highly correlated pathway was verified in animal and cells studies. RESULTS: A total of 52 metabolites of DWPE were identified in vivo, which were derived from gallic acid, ellagic acid (EA) and glansreginin A (Gla A). The possible metabolic pathways were phase Ⅰ (hydroxylation, hydrolyzation, etc) and phase Ⅱ metabolic reactions (methylation, sulfation and glucuronidation). Furthermore, in the network pharmacology, 54 core targets were enriched into pathways in cancer, nitrogen metabolism and other 9 pathways, which were essential pathways of DWPE against NAFLD. And the mechanism of nitrogen metabolism was verified in both of animal and cells studies. The results showed that DWPE could decline the concentration of ammonia and increase the expressions of carbonic anhydrase 2 (CA2) and carbamoylphosphate synthetase (CPS1) in nitrogen metabolism. CONCLUSION: Taken together, the study revealed the absorption components and their metabolic pathways and demonstrated the mechanism of nitrogen metabolism of DWPE on anti-NAFLD.

Capsella bursa-pastoris (L.) Medic. extract alleviate cataract development by regulating the mitochondrial apoptotic pathway of the lens epithelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Capsella bursa-pastoris (L.) Medic. (CBP) is a cruciferous plant valuable in reducing fever, improving eyesight and calming the liver. This herb was recorded in the Compendium of Materia Medica for cataract treatment. AIM OF THE STUDY: To determine the effects and mechanism of CBP on cataract prevention and treatment using a selenite cataract model. MATERIALS AND METHODS: The main compounds in CBP extract were analyzed by UPLC, 1H-NMR and 13C-NMR spectroscopic techniques. Flavonoids formed a significant proportion of its compounds, thus necessitating an evaluation of their inhibitory effects on the development of cataract using a selenite cataract model. The protective effects of CBP flavonoids (CBPF) against oxidative damage and the modulation of mitochondrial apoptotic pathway were subsequently verified on H2O2-treated SRA01/04 lens epithelial cells. RESULTS: CBPF significantly alleviated the development of cataract by decreasing the MDA level and increasing the GSH-Px and SOD levels in the lens. It also inhibited H2O2-induced apoptosis in SRA01/04 cells, increased the expression of Bcl-2 protein and decreased the expressions of Caspase-3 and Bax proteins. CONCLUSION: CBPF exerts a significant preventive effect on cataract development by regulating the mitochondrial apoptotic pathway of the lens epithelial cells. It is thus a potent traditional Chinese medicine (TCM) whose application should be further developed for the clinical treatment of cataract.

Cytotoxicity evaluation and metabolism of hepatotoxicity components of Euodiae Fructus in L02 cells.

Euodiae Fructus (EF), the dried unripe scented fruit of Euodia rutaecarpa (Juss.) Benth., was reported to show anti-hypertensive, antitumor, and anti-obesity effects. The main alkaloids of EF were reported as the reason for toxicity of EF by metabolic activation majority through CYP3A. Up till the present moment, the cytotoxicity mechanisms of EF have not yet to be fully clarified. For the purposes of this article, the influence of CYP3A inducer and inhibitor on cytotoxicity of EF and metabolism in L02 cells of five alkaloids related to toxicity of EF were evaluated. The results indicated that CYP3A inducer aggravated the toxicity and CYP3A inhibitor alleviated the toxicity. UPLC-Q-Exactive-MS was used for the identification of five alkaloids of EF in L02 cells. A total of 13 metabolites were detected in L02 cells. In general, five alkaloids were widely metabolized in L02 cells such as oxygenation, demethylation, dehydrogenation, and etc. In addition, oxygenation was the main metabolic pathway. It was inferred that the toxicity of EF was closely related to the CYP3A and the metabolic intermediate might be one of the reasons for the toxicity of EF. Hence, the choice of optimal dose might be critical to avoid the adverse reactions owing to combination of EF and CYP3A inducer.

The hepatoprotective efficacy and biological mechanisms of three phenylethanoid glycosides from cistanches herba and their metabolites based on intestinal bacteria and network pharmacology.

Echinacoside (ECH), acteoside (ACT), and isoacteoside (ISAT), the typical phenylethanoid glycosides (PhGs) in cistanches herba, have various pharmacological activities. However, the ECH, ACT and ISAT have extremely low oral bioavailability, which is related to their metabolism under the intestinal flora. Previous studies showed that intestinal metabolites were the hepatoprotective substances in vivo, but the research on whether PhGs has effects without intestinal bacteria has not been studied. In this paper, ECH, ACT and ISAT were incubated with human or rat intestinal bacteria for 36 h. After incubating with human bacteria for 36 h, three prototype compounds were not detected and were mainly biotransformed to 3-HPP and HT. In the network pharmacology, a total of 6 common targets were obtained by analysing the prototypes, the metabolites and the liver injury. It was found that the combinations of three metabolites and common targets were more stable than those of the prototypes and common targets by molecular docking. Meanwhile, hepatocellular apoptosis, proliferation, inflammation and oxidative responses might play important roles in the mechanisms of the metabolites exerting hepatoprotective activities. Then normal and pseudo-sterile mice experiments were adopted to further compare the hepatoprotective activities of prototypes and metabolites. Animal experiment results showed that the prototypes and the metabolites in the normal mice had significantly hepatoprotective activity. Interestingly, in the pseudo-germfree mice, the metabolites showed significant hepatoprotective effect, but the prototypes had not effect. It indicated that the prototype cannot exert liver protective activity without the effect of intestinal bacteria.

UPLC-Q-Exactive-MS analysis for hepatotoxicity components of Evodiae Fructus based on spectrum-toxicity relationship.

Evodiae Fructus (EF) is generally divided into three categories: small flower EF (SEF), medium flower EF (MEF) and big flower EF (BEF) in commodity circulation according to the size of the fruit. It is a well-known and frequently used herbal medicine for treating gastrointestinal disorder-related stomachache and headache, which has aroused wide attention for its hepatotoxicity. However, reports about hepatotoxicity is controversial and hepatotoxic components are inconclusive. The study aimed to explain the controversial hepatotoxicity of EF and screen the components associated with hepatotoxicity of EF based on the spectrum-toxicity relationship. UPLC fingerprints of 39 batches of EF collected from different regions were established. Combined with the results of L02 cell viability assays, the spectrum-toxicity relationship was investigated on the basic of orthogonal partial least squares (OPLS). The results of the research demonstrated that the toxicity of EF was obviously various among the different categories, in particularly, SEF was with less toxicity, MEF except for adulterants and BEF had mild toxicity and adulterants of MEF (A-MEF) produced more damage to L02 cell and no regions specificity in hepatotoxicity of EF. Thereinto, samples, the contents of which do not meet the requirements of Chinese Pharmacopoeia, were adulterants. It was worth noting that P11, P17, P20 and P25 were closely related to hepatotoxicity of EF and they were respectively identified as limonin (LIM), evodiamine (EVO), 1-methyl-2-nonyl-4(1H)-quinolone (MNQ), and 1-methyl-2-undecyl-4(1H)-quinolone (MUQ) by UPLC-Q-Exactive-MS. The hepatoprotection of P11 and hepatotoxicity of P17 were consistent with the results of spectrum-toxicity relationship. In summary, A-MEF was more toxic than other categories and SEF was less toxic than the others. It was noteworthy that EVO was the main hepatotoxic component of EF and LIM was the main hepatoprotective component of EF. The results provided worthy evidence for better utilization and development of EF.

Anti-NAFLD effect of defatted walnut powder extract in high fat diet-induced C57BL/6 mice by modulating the gut microbiota.

ETHNOPHARMACOLOGICAL RELEVANCE: Walnut kernel has the actions of removing meteorism, dissipating stagnation and removing blood stasis and is used after being defatted in TCM. Defatted walnut powder extract (DWPE) has the abilities of anti-oxidation and lowering lipid levels in vivo. However, the effects and the potential mechanisms of DWPE on NAFLD have not been explored. AIM OF THE STUDY: The study were to investigate the anti-NAFLD effect of DWPE in high fat diet-induced C57BL/6 mice and demonstrate that whether DWPE developed the effect on anti-NAFLD by remodeling the compositions and abundances of gut microbiota. MATERIALS AND METHODS: The inhibitory effect of DWPE on the development of NAFLD was conducted on C57BL/6 mice with a high fat diet and the regulation effect of DWPE on gut microbiota was verified on pseudo-sterile mice with treatment of broad spectrum antibiotics. RESULTS: The results showed that the oral administration of DWPE remarkably alleviated hepatic lipid accumulation by decreasing the levels of TG, TC, LDL, MDA and increasing HDL. Meanwhile, the expressions of NF-κB and MAPKs family proteins were reduced by DWPE compared with HFD group. Otherwise, the efficacy of anti-NAFLD of DWPE was significantly decreased after treatment of antibiotics, which indicated the key role of gut microbiota in the therapeutic process. Furthermore, sequencing of 16S rRNA gene revealed that DWPE could revert the decreased relative abundance of gut microbiota caused by the long term of a high fat diet. And the disordered microflora was remodeled by DWPE including the reduction of Erysipelotrichia, Firmicutes and Actinobacteria as well as the increment of Bacteroidetes, Clostridiales, Bacteroidales S24-7, Prevotellaceae and Bacteroides. CONCLUSION: Taken together, DWPE had a preventing effect on NAFLD, which might be associated with the regulation of gut microbiota.

Quality assessment of Typhae Pollen Carbonisata based on chromaticity analysis combined with UPLC fingerprinting and thrombin activity.

INTRODUCTION: Typhae pollen (TP) has been used as an anticoagulant in traditional Chinese medicine and throughout Asia. Typhae Pollen Carbonisata (TPC), a processed product of TP, has hemostatic properties. TPC is produced by frying TP, and the degree of processing can be judged by the colour change from yellow to brown. OBJECTIVE: To establish a novel method for quality assessment of TPC and discriminate TPC from underdone products and overdone products. METHODOLOGY: The Commission Internationale de l'Eclairage (CIE) L* a* b* colour space values of TP and TPC were measured to establish the colour model of TPC. Ultra-performance liquid chromatography was developed for fingerprinting. Thrombin activity promoting rates, clotting time, and bleeding time illustrated the difference in the hemostatic effect of the processed products. Chemometric approaches were performed to reveal the correlation between components and colour values or thrombin activity. RESULTS: Reference ranges of colour values and mathematical functions of TPC were established. The developed method was found to be fast, economic, sensitive, and stable. Fingerprints and thrombin activity in conjunction with partial least squares (PLS) demonstrated that peaks 2, 4, 7, 30, and 36 (isorhamnetin) were the main contributors for colour values and hemostatic activity of TPC. CONCLUSIONS: TPC and its unqualified products can be effectively distinguished based on chromaticity analysis, which provides a powerful tool for the comprehensive evaluation of the quality of Chinese herbal medicines.

Morning glory seed keeps laxative effect while retains less subchronic toxicity after being fried.

ETHNOPHARMACOLOGICAL RELEVANCE: Morning glory seed (MGS), has been widely used in treating constipation especially towards children. Clinically, people usually take fried MGS (MGSF) in formulas to reduce its side effect. However, the safety of MGSF other than MGS has yet to be explored. OBJECTIVE: The study aimed to reveal the potential mechanisms of using MGSF instead of MGS basing on chemistry, pharmacodynamics and toxicology. METHODS: The chemical compositions of the extracts of MGS and MGSF were compared using UPLC-Q-TOF/MS method. Simultaneously, to prove the availability and safety of MGSF, we investigated the laxative effect and subchronic toxicity of MGS and MGSF and addressed the mechanism of laxative effect of them. RESULTS: In this study, less phenolic acids and more fatty acids were detected in MGSF compared with the compounds in MGS. Moreover, we found that MGS group had stronger laxative effect than MGSF group via downregulating the expression of AQP3 protein. As for subchronic toxicity test, the body weights of MGS group were lower than MGSF group. In serum biochemistry and histopathological examinations, MGS group could cause more serious toxicity in liver, kidney and colon than MGSF group with higher values of BUN, Cr, AST and ALP. CONCLUSION: Based on the findings in this study, MGSF with varied compounds contents could still keep the laxative effect while retain less subchronic toxicity, which emphasized the necessity of processing and provided an insight into the rational use of MGSF in clinical practice.

Metabolites of Dietary Acteoside: Profiles, Isolation, Identification, and Hepatoprotective Capacities.

In recent years, cistanche tea has been increasingly used as a major herbal supplement in functional drinks, and it has attracted a growing number of consumers because of its excellent tonic effects and medicinal properties. Acteoside (ACT), which is the principal bioactive component of Chinese cistanche tea, possesses various pharmacological effects. This study profiled, isolated, identified, and investigated the hepatoprotective capacities of metabolites in rat urine after the administration of ACT. Eleven metabolites, including one new compound (M8), were obtained and identified by nuclear magnetic resonance (NMR) spectroscopy for the first time. Compared with native ACT, ACT metabolites such as hydroxytyrosol (HT), 3-hydroxyphenylpropionic acid (3-HPP), and caffeic acid (CA) exhibited higher hepatoprotective activities by regulating oxidative stress, lipid peroxidation, and inflammatory responses in a GalN/LPS-induced-acute-hepatic-injury mouse model. The HT treatment markedly reduced the levels of TNF-α to 280 ± 14.3 ng/L compared with the model group (429 ± 9.20 ng/L, p < 0.01). The results obtained indicated that cistanche tea could be developed as a functional drink for the prevention of hepatic injuries and that ACT metabolites could be responsible for the potent hepatoprotective activity as well as the other therapeutic effects.

Pharmacokinetic comparison of two phenolic acids after oral administration of Typhae pollen to normal rats and rats with acute cold blood stasis.

Typhae Pollen, dried pollen of Typha angustifolia L., Typha orientalis Presl or other plants of the same genus (Typhaeceae), has the effect of activating the circulation to cure blood stasis in traditional Chinese Medicine. The purpose of this study was to set up an ultra-high performance liquid chromatography method that could determine p-hydroxybenzoic acid and vanillic acid simultaneously in rat plasma, and to compare their pharmacokinetics in normal rats and rats with acute cold blood stasis, and further to investigate the influence of different dosages of oral administration. The pharmacokinetic parameters obtained showed that both of the phenolic acids had a higher bioavailability in rats with cold blood stasis than that in normal rats with a higher area under the concentration-time curve and longer mean residence time, and the high dose oral administration group had a higher capacity in blood stasis rats than in normal rats. These results reminded us that changes in health condition could alter the absorption and elimination of both phenolic acids in vivo, and the pharmacokinetic study under pathological conditions provides important information for more rational drug use in clinical situations.