A strategy for the rapid discovery and validation of active diterpenoids as quality markers in different habitats of Langdu using ultrahigh-performance liquid chromatography-tandem mass spectrometry with multivariate statistical analysis.

Langdu, known as a traditional Chinese medicine, was identified as the roots of species of Euphorbia ebracteolata Hayata and Euphorbia fischeriana Steud, displaying anti-tuberculosis activity. To clarify the potent quality markers of Langdu, this research first developed a fast and sensitive ultrahigh-performance liquid chromatography-tandem mass spectrometry method for the quantification of 13 diterpenoids in Langdu. The developed method was further applied in the analyses of 12 authentic E. ebracteolata and E. fischeriana samples collected in northern and southeastern China. Then, the anti-tuberculosis evaluation of 12 batches of Langdu samples was performed in vitro. Finally, partial least squares discrimination analysis was used in the discrimination of E. ebracteolata and E. fischeriana from different origins and processing methods. Jolkinolide A (1), jolkinolide E (3), yuexiandajisu D (6), and ebractenone A (11) were identified as key, potent diterpenoids for the quality control of E. ebracteolata Hayata and E. fischeriana Steud. The present study established a qualitative chemical analysis method for Langdu (E. ebracteolata and E. fischeriana) and suggested the key bioactive components that will improve qualitative control methodology for this important medicine.

Application of comprehensive pharmaceutical care program in identifying and addressing drug-related problems in hospitalized patients with osteoporosis.

BACKGROUND: More information about the impacts of comprehensive pharmaceutical care program (CPCP) on the identification and resolution of drug-related problems (DRPs) is needed. This study aimed at researching the characteristics of DRPs in osteoporosis patients and evaluating the effect of CPCP in identifying and addressing DRPs. METHODS: We performed a prospective interventional study in a teaching hospital. CPCP was established and conducted to identify and resolve DRPs by a multidisciplinary team (MDT) based on the Pharmaceutical Care Network Europe (PCNE) classification V9.0. Six pharmacists and one doctor worked directly in the study. All data was obtained from electronic medical records, direct observation and visits. The statistical analyses were performed using the SPSS Statistics software version 26.0. RESULTS: Two hundred nineteen patients with osteoporosis were included in the final analysis. A total of 343 DRPs were identified, with an average of 1.57 DRPs per patient. The most common DRPs identified were "treatment safety P2" (66.8%; 229/343), followed by "other P3" (21.0%; 72/343) and "treatment effectiveness, P1" (12.2%; 42/343). The primary causes of DRPs were "dose selection C3" (35.9%; 211/588), followed by "drug use process C6" (28.9%; 170/588) and "drug selection C1" (12.6%; 74/588). Seven hundred eleven interventions were proposed to address the 343 DRPs, with an average of 2.1 interventions per DRP. The acceptance rate reached 95.9, and 91.0% of these accepted interventions were fully implemented. As a result, only 30 DRPs were unsolved before discharge. Additionally, the number of drugs was found to be associated with the number of DRPs significantly (p = 0.023). CONCLUSION: DRPs frequently occurred in hospitalized osteoporosis patients. CPCP could be an effect option to solve and reduce DRPs for osteoporosis patients and should be implemented widely to increase patient safety.

Aromatic rosane diterpenoids from the roots of Euphorbia ebracteolata and their inhibitory effects against lipase.

The bioactive chemical constituents of Euphorbia ebracteolata have been investigated in the present work using various techniques. On the basis of chromatographic methods, such as silica gel, RP C-18 column chromatography, five novel rosane type diterpenoids with an aromatic A-ring (1-5) have been isolated from the roots of Euphorbia ebracteolata. Their structures were elucidated by widely spectroscopic data, including HRESI-MS, 1D and 2D NMR. Additionally, the inhibitory effects on lipase of these isolated diterpenoids were evaluated in vitro. Compound 1 as a new diterpenoid displayed significant inhibitory effect on lipase (IC50 = 1.0 µM). And, the inhibitory kinetics has been studied fully, which determined a competitive inhibition model for compound 1 on the enzymatic activity of lipase (Ki = 1.8 µM).

Gambogic acid attenuates liver fibrosis by inhibiting the PI3K/AKT and MAPK signaling pathways via inhibiting HSP90.

Gambogic acid (GA), a major ingredient of Garcinia hanburryi, is known to have diverse biological effects. The present study was designed to evaluate the anti-fibrotic effects of GA on hepatic fibrosis and reveal its underlying mechanism. We investigated the anti-fibrotic effect of GA on dimethylnitrosamine and bile duct ligation induced liver fibrosis in rats in vivo. The rat and human hepatic stellate cell lines (HSCs) lines were chose to evaluate the effect of GA in vitro. Our results indicated that GA could significantly ameliorate liver fibrosis associated with improving serum markers, decrease in extracellular matrix accumulation and HSCs activation in vivo. GA significantly inhibited the proliferation of HSC cells and induced the cell cycle arrest at the G1 phase. Moreover, GA triggered autophagy at early time point and subsequent initiates mitochondrial mediated apoptotic pathway resulting in HSC cell death. The mechanism of GA was related to inhibit heat shock protein 90 (HSP90) and degradation of the client proteins inducing PI3K/AKT and MAPK signaling pathways inhibition. This study demonstrated that GA effectively ameliorated liver fibrosis in vitro and in vivo, which provided new insights into the application of GA for liver fibrosis.

Cytotoxic ent-Abietane-type diterpenoids from the roots of Euphorbia ebracteolata.

Euphoroids A-C (1-3), three new ent-abietane-type diterpenoids, together with ten known analogues (4-13) were obtained from the roots of Euphorbia ebracteolata. The structures of these compounds were determined by extensive spectroscopic data analysis, including UV, HRESIMS, 1D-, and 2D-NMR data. The inhibitory effects of compounds 1-13 on human cancer cells were determined using the MTT assay. The results revealed that new compounds 2 and 3 showed moderate cytotoxic activities against human cancer cell lines. Especially, compound 3 displayed selective cytotoxic effect agains cancer cell lines.

[Chemical Constituents of the Aerial Parts from Plumbago zeylanica].

Objective: To study the anti-oxidative constituents of the aerial parts of Plumbago zeylanica. Methods: The ethanol extract of Plumbago zeylanica was separated and purified by various chromatographic techniques. On the basis of various spectroscopic data, the structures of isolated compounds were elucidated. ABTS+radical scavenging were carried out in antioxidant activity evaluation of the isolated compounds. Results: Eleven compounds were isolated and identified as cis-isoshinanolone-4-O-ß-D-glucopyranoside( 1),tachioside( 2),2,6-dimethoxy-p-hydroquinone-1-O-ß-D-glucopyranoside( 3),3-( ß-D-glucopyranosyloxy)-4-methoxybenzoic acid( 4),3'-O-ß-D-glucopyranosyloxy-plumbagic acid( 5),3'-O-ß-D-glucopyranosyloxy-plumbagic acid methyl ester( 6),plumbagic acid( 7),plumbagine A( 8),plumbagine C( 9),syringate-4-O-ß-D-glucopyranoside( 10) and 2-methyl-5-hydroxychromone( 11). Compounds 2,3,and5 displayed significant scavenging effect on ABTS+. Conclusion: Compounds 1 ~ 4,10,11 are obtained from this plant for the first time. Compounds 2,3,and 5 show significant anti-oxidative effects.

Protostane Triterpenoids from the Rhizome of Alisma orientale Exhibit Inhibitory Effects on Human Carboxylesterase 2.

Twelve new and 10 known protostane triterpenoids were isolated from the rhizome of Alisma orientale. Their structures were elucidated based on physical data analyses, including UV, HRESIMS, NMR experiments ((1)H, (13)C NMR, (1)H-(1)H COSY, HSQC, HMBC, and NOESY), and induced electronic circular dichroism. New compounds 1-12 were classified as protostanes (1-10), 29-norprotostane (11), and 24-norprotostane (12) by structure analyses. Furthermore, the inhibitory effects on human carboxylesterases (hCE-1, hCE-2) of compounds 1-22 were evaluated. Compounds 2, 6, 9, and 11 showed moderate inhibitory activities and were selective toward hCE-2 enzymes, with IC50 values of 8.68, 4.72, 4.58, and 2.02 µM, respectively. The inhibition kinetics of compound 11 toward hCE-2 were established, and the Ki value was determined as 1.76 µM using a mixed inhibition model. The interaction of bioactive compound 11 with hCE-2 was shown using molecular docking.

Inhibitory Effects of Highly Oxygenated Lanostane Derivatives from the Fungus Ganoderma lucidum on P-Glycoprotein and α-Glucosidase.

Twelve new highly oxygenated lanostane triterpenoids and nine known ganoderic acids were isolated from the fruiting body of Ganoderma lucidum. The new compounds were lanostane nortriterpenoids with 27 carbons (1-5 and 8), lanostane nor-triterpenoids with 25 carbons (6 and 7), and lanostane triterpenoids (9-12) based on multiple spectroscopic data analysis, including HRESIMS, 1D-NMR, 2D-NMR, and CD. Compounds 1-5 were identified as rare nor-lanostanoids that contain a 17ß-pentatomic lactone ring. Compound 13, possessing a lactone ring, had been isolated previously. The P-glycoprotein (P-gp) inhibitory effects of compounds 1-21 were evaluated at a concentration of 20 µM using an adriamycin (ADM)-resistant human breast adenocarcinoma cell line (MCF-7/ADR). Compounds 1, 5, 18, and 20 and verapamil increased the accumulation of ADM in MCF-7/ADR cells approximately 3-fold when compared with the negative control. These data support the significant P-glycoprotein inhibitory activities of compounds 1, 5, 18, and 20. In silico docking analysis suggested these compounds had similar P-gp recognition mechanisms compared with those of verapamil (a classical inhibitor). Furthermore, in an in vitro bioassay, compounds 2, 4, 5, 6, and 18 showed moderate inhibitory effects against α-glucosidase compared with those of the positive control acarbose.

Phenolic constituents from the roots of Phyllodium pulchellum.

Three new phenolic constituents 1-3 were obtained from the 95% ethanol extract of the roots of Phyllodium pulchellum (Leguminosae). Their structures were elucidated on the basis of spectroscopic analyses, such as NMR, UV, IR, HR-ESI-MS, and CD. Furthermore, in an in vitro bioassay, all compounds were tested for inhibitory effects against the proliferation of acetaldehyde-stimulated HSC-T6 cells, and compound 3 exhibited potent inhibitory activity with the IC50 value of 7.6 µM.

Inverse interaction between tropomyosin and phosphorylated myosin in the presence or absence of caldesmon.

In the present study, co-sedimentation assay, intrinsic fluorescence intensity measurement, and Mg²âº-ATPase activity analysis were carried out to investigate the direct effect of tropomyosin (TM) on unphosphorylated myosin (UM) or phosphorylated myosin (PM) in the presence or absence of caldesmon (CaD). Results showed that TM significantly decreased the sedimentation, intrinsic fluorescence intensity, and the Mg²âº-ATPase activity of PM, but not UM. In the presence of CaD, TM also significantly decreased these parameters irrespective of myosin phosphorylation, suggesting that the interaction between TM and CaD abolished the effects of TM on PM or UM and that there was an inverse interaction between TM and PM, characterized by the decreased PM sedimentation and intrinsic fluorescence intensity.

Two new ent-kaurane diterpenoids from Pteris semipinnata.

In this study, two new compounds and six known compounds were isolated from the aerial parts of Pteris semipinnata. The chemical structures of these two new compounds were elucidated as 6ß,11α-dihydroxy-15-oxo-ent-kaur-16-en-19-oic acid (1) and 7α,11α-dihydroxy-15-oxo-ent-kaur-16-en-19-oic acid (2) by the extensive spectral methods including 2D NMR and HR-MS techniques.

Network analysis identifies a gene biomarker panel for sepsis-induced acute respiratory distress syndrome.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is characterized by non-cardiogenic pulmonary edema caused by inflammation, which can lead to serious respiratory complications. Due to the high mortality of ARDS caused by sepsis, biological markers that enable early diagnosis are urgently needed for clinical treatment. METHODS: In the present study, we used the public microarray data of whole blood from patients with sepsis-induced ARDS, patients with sepsis-alone and healthy controls to perform an integrated analysis based on differential expressed genes (DEGs) and co-expression network to identify the key genes and pathways related to the development of sepsis into ARDS that may be key targets for diagnosis and treatment. RESULTS: Compared with controls, we identified 180 DEGs in the sepsis-alone group and 152 DEGs in the sepsis-induced ARDS group. About 70% of these genes were unique to the two groups. Functional analysis of DEGs showed that neutrophil-mediated inflammation and mitochondrial dysfunction are the main features of ARDS induced by sepsis. Gene network analysis identified key modules and screened out key regulatory genes related to ARDS. The key genes and their upstream regulators comprised a gene panel, including EOMES, LTF, CSF1R, HLA-DRA, IRF8 and MPEG1. Compared with the healthy controls, the panel had an area under the curve (AUC) of 0.900 and 0.914 for sepsis-alone group and sepsis-induced ARDS group, respectively. The AUC was 0.746 between the sepsis-alone group and sepsis-induced ARDS group. Moreover, the panel of another independent blood transcriptional expression profile dataset showed the AUC was 0.769 in diagnosing sepsis-alone group and sepsis-induced ARDS group. CONCLUSIONS: Taken together, our method contributes to the diagnosis of sepsis and sepsis-induced ARDS. The biological pathway involved in this gene biomarker panel may also be a critical target in combating ARDS caused by sepsis.

ROS-dependent catalytic mechanism of melatonin metabolism and its application in the measurement of reactive oxygen.

Melatonin (Mel) is an endogenous active molecule whose metabolism progress significantly influences its bioactivity. However, the detailed metabolic pathway of Mel in the pathological state has not yet been fully illustrated. In this study, 16 metabolites of Mel in cancer cells and human liver microsomes were identified, of which seven novel metabolites were newly discovered. Among them, 2-hydroxymelatonin (2-O-Mel), as the major metabolite in cancer cells, was revealed for the first time, which was different from the metabolite found in the human liver. Furthermore, CYP1A1/1A2- and reactive oxygen species (ROS)-mediated 2-hydroxylation reactions of Mel were verified to be the two metabolic pathways in the liver and cancer cells, respectively. ROS-dependent formation of 2-O-Mel was the major pathway in cancer cells. Furthermore, the underlying catalytic mechanism of Mel to 2-O-Mel in the presence of ROS was fully elucidated using computational chemistry analysis. Therefore, the generation of 2-O-Mel from Mel could serve as another index for the endogenous reactive oxygen level. Finally, based on the ROS-dependent production of 2-O-Mel, Mel was successfully used for detecting the oxygen-carrying capacity of hemoglobin in human blood. Our investigation further enriched the metabolic pathway of Mel, especially for the ROS-dependent formation of 2-O-Mel that serves as a diagnostic and therapeutic target for the rational use of Mel in clinics.

Inhibition effect of 1-acetoxy-6α-(2-methylbutyryl)eriolanolide toward soluble epoxide hydrolase: Multispectral analysis, molecular dynamics simulation, biochemical, and in vitro cell-based studies.

Soluble epoxide hydrolase (sEH) serves as a potential target in inflammation-related diseases. Based on the bioactivity-guided separation, a new sesquiterpenoid inulajaponoid A (1) was isolated from Inula japonica with a sEH inhibitory effect, together with five known compounds, such as 1-O-acetyl-6-O-isobutyrylbritannilactone (2), 6ß-hydroxytomentosin (3), 1ß,8ß-dihydroxyeudesma-4(15),11(13)-dien-12,6α-olide (4), (4S,6S,7S,8R)-1-O-acetyl-6-O-(3-methylvaleryloxy)-britannilactone (5), and 1-acetoxy-6α-(2-methylbutyryl)eriolanolide (6). Among them, compounds 1 and 6 were assigned as mixed and uncompetitive inhibitors, respectively. The result of immunoprecipitation (IP)-MS demonstrated the specific binding of compound 6 to sEH in the complex system, which was further confirmed by the fluorescence-based binding assay showing its equilibrium dissociation constant (Kd = 2.43 µM). The detail molecular stimulation revealed the mechanism of action of compound 6 with sEH through the hydrogen bond of amino acid residue Gln384. Furthermore, this natural sEH inhibitor (6) could suppress the MAPK/NF-κB activation to regulate inflammatory mediators, such as NO, TNF-α, and IL-6, which confirmed the anti-inflammatory effect of inhibition of sEH by 6. These findings provided a useful insight to develop sEH inhibitors upon the sesquiterpenoids.

Empirical Study of Monthly Economic Losses Assessments for "Standard Unit Lockdown" Due to COVID-19.

Background: The pandemic of COVID-19 has been shaping economic developments of the world. From the standpoint of government measures to prevent and control the epidemic, the lockdown was widely used. It is essential to access the economic losses in a lockdown environment which will provide government administration with a necessary reference for decision making in controlling the epidemic. Methods: We introduce the concept of "standard unit incident" and an economic losses assessment methodology for both the standard and the assessed area. We build a "standard unit lockdown" economic losses assessment system and indicators to estimate the economic losses for the monthly lockdown. Using the comprehensive assessment system, the loss infected coefficient of monthly economic losses during lockdown in the 40 countries has been calculated to assess the economic losses by the entropy weighting method (EWM) with data from the CSMAR database and CDC website. Results: We observe that countries in North America suffered the most significant economic losses due to the epidemic, followed by South America and Europe, Asia and Africa, and Oceania and Antarctica suffered relatively minor economic losses. The top 10 countries for monthly economic losses during lockdown were the United States, India, Brazil, France, Turkey, Russia, the United Kingdom, Italy, Spain, and Germany. The United States suffered the greatest monthly economic losses under lockdown ($65.3 billion), roughly 1.5 times that of China, while Germany suffered the least ($56.4 billion), roughly 1.3 times that of China. Conclusion: Lockdown as a control and mitigation strategy has great impact on the economic development and causes huge economic losses. The economic impact due to the pandemic has varied widely among the 40 countries. It will be important to conduct further studies to compare and understand the differences and the reasons behind.

Microbial transformation of capsaicin by several human intestinal fungi and their inhibitory effects against lysine-specific demethylase 1.

Capsaicin widely exists in the Capsicum genus (e.g., hot peppers) and is commonly used as a food additive or medicinal material. In this work, microbial transformation of capsaicin was performed based on the three cultivated human intestinal fungi. Fourteen metabolites were obtained, and their chemical structures were elucidated by spectroscopic data analysis, including 13 compounds with undescribed structures. Hydroxylation, lactylation, succinylation, citric acylation, and acetylation were observed for these microbial metabolites derived from capsaicin, which indicated diverse catalytic characteristics of human intestinal fungi. In an in vitro bioassay, four metabolites and capsaicin inhibited the activity of lysine-specific demethylase 1 (LSD1) with a more than 70% inhibitory rate at 10 µM. In particular, 9,5'-dihydroxycapsaicin displayed the strongest inhibitory effect with an IC50 of 1.52 µM. Therefore, capsaicin analogs displayed potential application as LSD1 inhibitors against the invasion and migration of cancer cells.

Metabolites isolated from the human intestinal fungus Penicillium oxalicum SL2 and their agonistic effects on PXR and FXR.

Intestinal commensal fungi are vital to human health, and their metabolites play a key role in the reciprocal relationship. In the present work, eighteen alkaloids and seven monoterpenoids were isolated from the fermentation of the human intestinal fungus Penicillium oxalicum SL2, including seven undescribed alkaloids (penicilloxalines A-G), three undescribed monoterpenoids (penicilloxalines H-J), and fifteen reported compounds. The structures of the isolated compounds were identified by HRESIMS, 1D and 2D NMR, electronic circular dichroism spectra and quantum chemical calculations. Some metabolites displayed moderate agonistic effects against the pregnane X receptor (PXR), whereas (6R)3,7-dimethyl-6,7-dihydroxy-2(Z)-octenoic acid displayed a significant agonistic effect against the farnesoid X receptor (FXR) with an EC50 value of 0.43 µM, which was verified by investigating FXR downstream target genes and proteins, such as small heterodimer partner 1 (SHP1), fibroblast growth factor (FGF), and bile salt export pump (BSEP).

Potent Inhibition of Human Cytochrome P450 3A4 by Biflavone Components from Ginkgo Biloba and Selaginella Tamariscina.

CYP3A4-mediated Phase I biotransformation is the rate-limiting step of elimination for many commonly used clinically agents. The modulatory effects of herbal medicines on CYP3A4 activity are one of the risk factors affecting the safe use of drug and herbal medicine. In the present study, the inhibitory effects of nearly hundred kinds of herbal medicines against CYP3A4 were evaluated based on a visual high-throughput screening method. Furthermore, biflavone components including bilobetin (7-demethylginkgetin, DGK), ginkgetin (GK), isoginkgetin (IGK), and amentoflavone (AMF) were identified as the main inhibitory components of Ginkgo biloba L. (GB) and Selaginella tamariscina (P. Beauv.) Spring (ST), which displayed very strong inhibitory effects toward CYP3A4. The inhibitory effects of these biflavones on clinical drugs that mainly undergo CYP3A4-dependent metabolism were evaluated. The IC 50 of GK toward tamoxifen, gefitinib and ticagrelor were found to be of 0.478 ± 0.003, 0.869 ± 0.001, and 1.61 ± 0.039 µM, respectively. These results suggest the potential pharmacokinetic interactions between the identified biflavones and clinical drugs undergoing CYP3A4-mediated biotransformation. The obtained information is important for guiding the rational use of herbal medicine in combination with synthetic pharmaceuticals.

Myosin light chain kinase/actin interaction in phorbol dibutyrate-stimulated smooth muscle cells.

Previous work has suggested that in addition to its kinase activity, myosin light chain kinase (MLCK) exhibits non-kinase properties within its N-terminus that could influence cytoskeletal organization of smooth muscle cells (A. Nakamura et al. Biochem Biophys Res Commun. 2008;369:135-143). Myosin ATPase activity measurements indicate that the 26-41 peptide of MLCK significantly decreases ATPase activity as the concentration of this peptide increases. Sliding velocity of actin-filaments on myosin and stress responses in skinned smooth muscle tissue are also inhibited. Peptide-mediated uptake and the microinjection technique in cells indicate that the peptide was necessary for actin-filament stabilization. Fluorescence resonance energy transfer analysis indicated that in the presence of MLCK, α-actin but not ß-actin remodeled during phorbol 12,13-dibutyrate (PDBu)-induced contractions. PDBu also induced podosomes in the cell. When MLCK expression was down-regulated by introduction of RNAi for MLCK by lentivirus vector into the cells, we failed to observe the podosome induction upon PDBu stimulation. Rescue experiments indicate that the non-kinase activity of MLCK plays an important role in maintaining actin stress fibers and in the PDBu-induced reorganization of actin-filaments in smooth muscle cells.

Tannin extracts from immature fruits of Terminalia chebula Fructus Retz. promote cutaneous wound healing in rats.

BACKGROUND: Tannins extracted from immature fruits of Terminalia chebula Fructus Retz. are considered as effective components promoting the process of wound healing. The objective of this study is to explore the optimal extraction and purification technology (OEPT) of tannins, while studying the use of this drug in the treatment of a cutaneous wound of rat as well as its antibacterial effects. METHODS: The content of tannin extracts was measured by the casein method, and antibacterial ability was studied by the micro-dilution method in vitro. In wound healing experiment, animals in group Ⅰ, Ⅱ and Ⅲ were treated with vaseline ointment, tannin extracts (tannin content: 81%) and erythromycin ointment, respectively (5 mg of ointment were applied on each wound). To evaluate the process of wound healing, selected pharmacological and biochemical parameters were applied. RESULTS: After optimal extraction and purification, content of tannin extracts was increased to 81%. Tannin extracts showed the inhibition of Staphylococcus aureus and Klebsiella Pneumonia in vitro. After excision of wounds, on days 7 and 10, the percent of wound contraction of group Ⅱ was higher than that of group Ⅰ. After being hurt with wounds, on days 3, 7, and 10, the wound healing quality of group Ⅱ was found to be better than that of group Ⅰ in terms of granulation formation and collagen organization. After wound creation, on day 3, the vascular endothelial growth factor expression of group Ⅱ was higher than that of group Ⅰ. CONCLUSION: The results suggest that tannin extracts from dried immature fruits of Terminalia chebula Fructus Retz. can promote cutaneous wound healing in rats, probably resulting from a powerful anti-bacterial and angiogenic activity of the extracts.