Study on the Evolution Mechanism of Ecosystem Services in Karst Mountainous Areas from the Perspective of Humanities.

Anthropogenic activities have altered ecosystem service functions in the karst mountainous areas. The implementation of ecological restoration projects by the government, the behavior, attitude, and willingness of farmers to participate in their implementation, the application of pesticides and fertilizers, in addition to other socio-economic activities, have had a significant impact on the ecosystem services (ESS) of the region. Taking Guangnan County, a typical karst mountainous area in Yunnan Province, as an example, this study analyzes the evolutionary characteristics of six types of ESS and the driving mechanism of the change in ESS from the anthropogenic macro and micro perspective using questionnaire surveys and the multivariate logistic model. The results showed that (1) ecological restoration projects in the past 20 years have promoted an overall ecological transformation in the typical karst mountainous areas of the Yunnan Province (2) from the macro perspective, and the implementation of such ecological projects is beneficial in increasing soil conservation, carbon sequestration, habitat support, and cultural services. The reduction in agricultural population is beneficial in improving habitat support services, and the increase in the annual average tourism income and the tertiary industry is beneficial in increasing cultural services. Among them, the impact of hydraulic engineering on water production and the tertiary industry on cultural services are the most significant, with the change in the human disturbance index having the most substantial impact on soil conservation, carbon sequestration, and habitat support (3) at the micro level. Increasing pesticide and fertilizer application, willingness and use by farmers has a positive impact on food supply and a negative impact on habitat quality. An increase in the number and willingness of farmers participating in restoring farmland to forests and water conservancy projects was observed. This has a positive impact on soil conservation, water production, and carbon sequestration. Among them, the application of chemical fertilizers and pesticides has the most significant impact on food supply and habitat support, and the willingness to implement the projects on restoring farmlands to forests has the most significant impact on carbon sequestration. The willingness to implement terracing has the greatest impact on water production and soil conservation, and aesthetic value has the greatest impact on cultural services.

Fibroblast growth factor 21 Ameliorates diabetes-induced endothelial dysfunction in mouse aorta via activation of the CaMKK2/AMPKα signaling pathway.

Endothelial dysfunction initiates and exacerbates hypertension, atherosclerosis and other cardiovascular complications in diabetic mellitus. FGF21 is a hormone that mediates a number of beneficial effects relevant to metabolic disorders and their associated complications. Nevertheless, it remains unclear as to whether FGF21 ameliorates endothelial dysfunction. Therefore, we investigated the effect of FGF21 on endothelial function in both type 1 and type 2 diabetes. We found that FGF21 reduced hyperglycemia and ameliorated insulin resistance in type 2 diabetic mice, an effect that was totally lost in type 1 diabetic mice. However, FGF21 activated AMPKα, suppressing oxidative stress and enhancing endothelium-dependent vasorelaxation of aorta in both types, suggesting a mechanism that is independent of its glucose-lowering and insulin-sensitizing effects. In vitro, we identified a direct action of FGF21 on endothelial cells of the aorta, in which it bounds to FGF receptors to alleviate impaired endothelial function challenged with high glucose. Furthermore, the CaMKK2-AMPKα signaling pathway was activated to suppress oxidative stress. Apart from its anti-oxidative capacity, FGF21 activated eNOS to dilate the aorta via CaMKK2/AMPKα activation. Our data suggest expanded potential uses of FGF21 for the treatment of vascular diseases in diabetes.

In Vitro Intestinal Absorption and Metabolism of Magnoflorine and its Potential Interaction in Coptidis Rhizoma Decoction in Rat.

BACKGROUND AND OBJECTIVES: In our previous studies, it was found that there existed pharmacokinetic interactions between magnoflorine and the rest of the ingredients in Coptidis Rhizoma. In this study, the pharmacokinetic interaction mechanism of magnoflorine with the rest of the components in Coptidis Rhizoma was researched based on the intestinal absorption and metabolism characteristics. METHODS: The absorption characteristics of magnoflorine in each rat intestinal segments were evaluated by non-everted intestinal sac model. To identify the metabolites of magnoflorine, the acceptor solutions of each intestinal segment at 120 min were analyzed by HPLC-LTQ-Orbitrap MS. RESULTS: The accumulative absorption (Q), the absorption rate (J) and the apparent permeability coefficient (P app) of magnoflorine were increased in duodenum, jejunum, ileum and colon of the Coptidis Rhizoma group as compared to the magnoflorine group, but there was no statistical difference between the two groups (P > 0.05). Four phase I metabolites of magnoflorine were identified in intestinal acceptor solutions of pure compound, while eight metabolites were detected in that of Coptidis Rhizoma decoction including six phase I metabolites and two phase II metabolic products. CONCLUSIONS: It was shown that the rest of the ingredients in Coptidis Rhizoma accelerated the absorption of magnoflorine weakly and promoted the metabolism of magnoflorine in the gut. The effects of other processes in the pharmacokinetics should be further evaluated.

In vitro potential modulation of baicalin and baicalein on P-glycoprotein activity and expression in Caco-2 cells and rat gut sacs.

Context Previous studies have shown that Scutellariae Radix, the dried root of Scutellaria baicalensis Georgi (Labiatae), has a certain inhibitory effect on P-glycoprotein (P-gp), but the effects of its main active constituents on P-gp are still ambiguous. Objectives In vitro studies were performed to investigate the effects of its main active constituents (baicalin and its aglycone, baicalein) on the activity and expression of P-gp in intestine using Caco-2 cells and rat gut sacs. Materials and methods In Caco-2 cell experiments, the effects of baicalin and baicalein on P-gp activity were investigated using a P-gp substrate, rhodamine 123 and non-substrate fluorescein Na, by determining their intracellular fluorescence accumulation, and their effects on P-gp expression were determined using flow cytometry. In addition, rat gut sac model was selected to investigate the effects of baicalin and baicalein on the transport of verapamil, a classical P-gp substrate. The gut sacs of male Sprague-Dawley rats were filled with 0.4 mL the test solution contained verapamil (0.2575 mg/mL) and the drugs [baicalin and baicalein, at concentrations of 1/8 IC50 (59.875, 41.5 µg/mL), 1/4 IC50 (119.75, 83 µg/mL) and 1/2 IC50 (239.5, 166 µg/mL)], and then incubated in Tyrode's solution for a period of time. After termination of the incubation, the incubated solution was processed for the subsequent detection. Results According to the results of MTT assay, the IC50 values of verapamil, baicalin and baicalein were 104, 479, 332 µg/mL, respectively. The obtained results from the two models were confirmed mutually. As a result, baicalin exhibited no obvious effect on intracellular accumulation of Rh-123, and almost had no effect on P-gp expression and verapamil transportation, while baicalein significantly increased intracellular accumulation of Rh-123 (p < 0.01), down-regulated P-gp expression (p < 0.01) and increased the transport of verapamil (p < 0.05). Discussion and conclusion The results indicated that baicalein may be a P-gp inhibitor, which presented obvious inhibitory effects on P-gp activity and expression level. A comparison of the structures of baicalin and baicalein indicates that the existence of glucosyl plays a decisive role in influencing the activity and expression of P-gp.

[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.

Pharmacokinetics and Brain Distribution and Metabolite Identification of Coptisine, a Protoberberine Alkaloid with Therapeutic Potential for CNS Disorders, in Rats.

Coptisine (COP), a protoberberine alkaloid (PBA) from Chinese medicinal plants (such as family Berberidaceae), may be useful for improving central nervous system disorders. However, its pharmacokinetics, disposition and metabolism are not well defined. In the present study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established for the analysis of COP in biological samples. To better understand its in vivo pharmacological activities, COP concentrations in rat plasma were determined after oral (50 mg/kg) and intravenous administration (10 mg/kg). For the brain distribution study, the concentration of COP in five different regions was examined after intravenous administration at 10 mg/kg. Pharmacokinetic parameters from the COP concentration-time profiles in plasma and brain, and the brain-to-plasma coefficient (Kp, brain) were calculated by non-compartmental analysis. The metabolites of COP in rats in vivo and in vitro (urine, bile, liver microsomes and intestinal bacteria incubation) were also identified. Seventeen metabolites, including 11 unconjugated metabolites formed by hydroxylation, hydrogenation, demethylation, dehydrogenation, demethylation, and 6 glucuronide and sulfate conjugates were identified for the first time. The results suggested that COP had low oral bioavailability of 8.9% and a short (plasma) half-life (T1/2=0.71 h) in rats. After intravenous administration, it quickly crossed the blood-brain barrier, accumulating at higher concentrations and then was slowly eliminated from different brain regions. Moreover, COP was transformed into metabolites through multiple metabolic pathways in vivo and in vitro. These results should help to promote further research on COP and contribute to clarifying the metabolic pathways of PBAs.

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.

In vitro and in vivo identification of metabolites of magnoflorine by LC LTQ-Orbitrap MS and its potential pharmacokinetic interaction in Coptidis Rhizoma decoction in rat.

Magnoflorine, an important aporphine alkaloid in Coptidis Rhizoma, is increasingly attracting research attention because of its pharmacological activities. The in vivo and in vitro metabolism of magnoflorine was investigated by LC LTQ-Orbitrap MS. In vivo samples including rat urine, feces, plasma and bile were collected separately after both oral (50 mg kg(-1) ) and intravenous administration (10 mg kg(-1) ) of magnoflorine, along with in vitro samples prepared by incubating magnoflorine with rat intestinal flora and liver microsome. As a result, 12 metabolites were found in biological samples. Phase I metabolites were identified in all biological samples, while phase II metabolites were mainly detected in urine, plasma and bile. In a pharmacokinetic study, rats were not only dosed with magnoflorine via oral (15, 30 and 60 mg kg(-1) ) and intravenous administration (10 mg kg(-1) ) but also dosed with Coptidis Rhizoma decoction (equivalent to 30 mg kg(-1) of magnoflorine) by intragastric administration to investigate the interaction of magnoflorine with the rest of compounds in Coptidis Rhizoma. Studies showed that magnoflorine possessed lower bioavailability and faster absorption and elimination. However, pharmacokinetic parameters altered significantly (p < 0.05) when magnoflorine was administered in Coptidis Rhizoma decoction. Oral gavage of Coptidis Rhizoma decoction decreased the absorption and elimination rates of magnoflorine, which revealed that there existed pharmacokinetic interactions between magnoflorine and the rest of ingredients in Coptidis Rhizoma.

[Stability study in biological samples and metabolites analysis of astragaloside IV in rat intestinal bacteria in vitro].

To figure out the stability and intestinal bacteria metabolites of rats in vitro of astragaloside IV ( AST), this research was done to explore the stability of AST in the artificial gastric juice. artificial intestinal juice and rat liver homogenate and the metabolism in rat intestinal in vitro. HPLC was used to calculate the remaining rate of AST in biological samples by measuring the content of AST, while metabolites were determined by combining the methods of TLC, HPLC and LC-MS/MS. It turned out that AST was difficult to metabolize in the artificial gastric juice, artificial intestinal juice and rat liver. Also, the metabolic pathway of AST was stepped by deglycosylation. Firstly, AST was converted to its secondary etabolites (6-O-ß-D-glucopyranosyl- cycloastragenol, CMG) by removal of xylose moiety at C-3, then transformed into cycloastragenol (CAG) after hydrolytic removal of the glucose moiety at C-6. All the results suggested that the metabolism of AST in vivo occurs mainly in the intestinal by hydrolysis of glycosyl. In conclusion, hydrolysis of intestinal flora is the main reason that AST metabolizes.

[Identification of metabolites of epiberberine in rat liver microsomes and its inhibiting effects on CYP2D6].

Epiberberine, one of the most important isoquinoline alkaloid in Coptidis Rhizoma, possesses extensive pharmacological activities. In this paper, the liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to study phase I and phase II metabolites. A Thermo HPLC system (including Surveyor AS, Surveyor LC Pump, Surveyor PDA. USA) was used. The cocktail probe drugs method was imposed to determine the content change of metoprolol, dapsone, phenacetin, chlorzoxazone and tolbutamide simultaneously for evaluating the activity of CYP2D6, CYP3A4, CYP1A2, CYP2E1 and CYP2C9 under different concentrations of epiberberine in rat liver microsomes. The result showed that epiberberine may have phase I and phase II metabolism in the rat liver and two metabolites in phase I and three metabolites in phase II are identified in the temperature incubation system of in vitro liver microsomes. Epiberberine showed significant inhibition on CYP2D6 with IC50 value of 35.22 µmol L(-1), but had no obvious inhibiting effect on the activities of CYP3A4, CYP1A2, CYP2E1 and CYP2C9. The results indicated that epiberberine may be caused drug interactions based on CYP2D6 enzyme. This study aims to provide a reliable experimental basis for its further research and development of epiberberine.