Identification of in vivo metabolites of Citri Sarcodactylis Fructus by UHPLC-Q/Orbitrap HRMS.

INTRODUCTION: Citri Sarcodactylis Fructus has the effects of relieving cough, removing phlegm, and reducing asthma, but little is known about the metabolic and distribution of its chemical constituents in vivo. Therefore, it is necessary to study the metabolism of Citri Sarcodactylis Fructus in vivo. OBJECTIVE: We aimed to (1) analyze the distribution of prototype compounds and metabolites of the chemical constituents of Citri Sarcodactylis Fructus in rat and (2) infer the metabolites and metabolic pathways of the chemical constituents. MATERIALS AND METHODS: A C18 column (3 × 100 mm, 2.6 µm) was used. The mobile phase was water containing 0.1% formic acid (eluent A) and acetonitrile containing 0.1% formic acid (eluent B) at a discharge rate of 0.3 mL/min. Mass spectra of biological samples were collected in electrospray ionization (ESI) positive ion mode in the m/z 100-1500 scan range. The obtained biological samples were then subjected to chemical analysis, including plasma, urine, feces, and heart, liver, spleen, lungs, kidneys, stomach, and small intestine tissues. Prototype compounds and metabolites were identified. RESULTS: In all, 40 prototype compounds and 78 metabolites, including 26 phase I metabolites and 52 phase II metabolites, were identified using UHPLC-Q/Orbitrap HRMS. Eight possible metabolic pathways (reduction, hydrolysis, dehydration, methylation, hydroxylation, sulfation, glucuronidation, and demethylation) were proposed. The prototype compounds were predominantly distributed in lung tissues. The metabolites were mainly distributed in plasma and kidney tissues. CONCLUSION: We systematically investigated the metabolites of Citri Sarcodactylis Fructus in vivo. We suggest metabolic pathways that might be relevant for further metabolic studies and screening of active ingredients of Citrus Sarcodactylis Fructus in vivo.

Intense Pulsed Light Treatment for Itch Associated with Allergic Keratoconjunctivitis: A Retrospective Study of 35 Cases.

Objective: To conduct a preliminary assessment of intense pulsed light (IPL) treatment for allergic keratoconjunctivitis (AKC)-associated ocular itch. Background: Current control measures for AKC rely primarily on drugs. IPL is effective for dry eye disease (DED). Furthermore, phototherapy is effective for managing skin inflammation and pruritus, suggesting that eye itching could decrease in some patients having AKC complicated with DED following IPL treatment to control dry eye symptoms. Methods: Thirty-five patients having DED complicated with mid-to-severe AKC were administered three IPL treatments to the periorbital skin. The eye scores of subjective symptoms and signs of AKC and tear film breakup time (TBUT) were retrospectively assessed before and after each treatment. Results: The scores for AKC-related symptoms and signs were determined four times: on Day 1 (time 0), Day 15 (time 1), Day 45 (time 2), and Day 75 (time 3) before each treatment. The average symptom score significantly decreased with treatments (time 0: 30.97, time 1: 15.03, time 3: 10). The average sign score for both eyes decreased after the first IPL treatment (left eye: 7.97 vs. 11.38; right eye: 8.1 vs. 11.1). There were no further improvements in the signs after the last treatment. The TBUT value in the right eye increased from times 0 to 3 (2.31 vs. 4.66 vs. 7.71 vs. 7.74). The TBUT value in the left eye increased from times 0 to 3 (2.50 vs. 6.97 vs. 7.57 vs. 8.24). Conclusions: Symptoms and signs improved after IPL treatment in patients with AKC. Eye itching was gradually controlled and rarely recurred. IPL may be effective for AKC treatment.

Preliminary Assessment of Intense Pulsed Light Treatment on the Upper Eyelids for Meibomian Gland Dysfunction.

Objective: To conduct a preliminary assessment of the intense pulsed light (IPL) treatment on the upper eyelid. Background: IPL therapy is an effective therapy for meibomian gland dysfunction (MGD). The normal treatment region includes the skin below the lower eyelids and both temporal parts, but not the upper eyelid. Methods: Thirty patients with mid-to-severe dry eye disease caused by MGD were selected and randomly divided into two groups. Fifteen controls in group A received normal IPL treatment. Fifteen patients in Group B underwent additional IPL treatment on the upper eyelid. The change in noninvasive tear breakup time (BUT), Ocular Surface Disease Index (OSDI) questionnaire, patient satisfaction, and adverse events were measured and recorded at each visit, before each treatment. Results: The dry eye symptoms of the patients in both groups improved. The patients in group B had better recovery than those in group A: BUT (right eye: 11.6 ± 3.67 sec vs. 7.73 ± 1.99 sec; left eye: 12.73 ± 4.19 sec vs. 7.73 ± 1.40 sec), OSDI (8.87 ± 4.31 vs. 14.93 ± 3.47); patient satisfaction in group B remained good, but decreased in group A with prolonged treatment duration. No serious skin and eye complications were found in any patient. Conclusions: This study showed the patients who received IPL treatment on the upper eyelid experienced improved symptoms of MGD. A further long-term study should be conducted to increase our understanding of the effect of expanded IPL treatment.

Design, synthesis and antifungal activity evaluation of coumarin-3-carboxamide derivatives.

A series of coumarin-3-carboxamides/hydrazides have been designed and synthesized, all the target compounds were evaluated in vitro for their antifungal activity against Botrytis cinerea, Alternaria solani, Gibberella zeae, Rhizoctorzia solani, Cucumber anthrax and Alternaria leaf spot, some of the designed compounds 4a-4g exhibited potential activity in the primary assays, this highlighted by the compounds 4a, 4d, 4e and 4f, EC50 values of which against Rhizoctorzia solani were as low as 1.80 µg/mL, 2.50 µg/mL, 2.25 µg/mL and 2.10 µg/mL, respectively, exhibiting more effective control with that of the positive control than Boscalid. Furthermore, compounds 4a and 4e represented equivalent antifungal activity with Boscalid against Botrytis cinerea.

An efficient synthesis and antifungal evaluation of natural product streptochlorin and its analogues.

Streptochlorin, a small indole alkaloid isolated from marine Streptomyces sp., exhibits a wide range of potent biological activities. An efficient and economic synthetic protocol for streptochlorin has been developed and validated, 4 steps from indole in a total yield of 45%, and further applied for the synthesis of its analogues. Biological testing showed that most of the target compounds exhibited potential antifungal activity in the primary assays, especially compounds 6, 7 and 9c were the most active ones, representing effective activity against the phytopathogenic fungi screened in preliminary test and might be explored for the study of mode of action in the future.

Overexpression of G-protein-coupled receptor 40 enhances the mitogenic response to epoxyeicosatrienoic acids.

The cytochrome P450 epoxygenase-dependent arachidonic acid metabolites, epoxyeicosatrienoic acids (EETs), are potent survival factors and mitogens for renal epithelial cells, but the molecular identity in the cells that initiates the mitogenic signaling of EETs has remained elusive. We screened kidney cell lines for the expression of G-protein-coupled receptor 40 (GPR40) and found that the porcine renal tubular epithelial cell line LLCPKcl4, which has been previously demonstrated to be sensitive to the mitogenic effect of EETs, expresses higher levels of GPR40 mRNA and protein than the human embryonic kidney cell line HEK293. EETs induced only a weak mitogenic EGFR signaling and mild cell proliferation in HEK293 cells. To determine whether GPR40 expression level is what mediates the mitogenic sensitivity of cells to EETs, we created a human GPR40 (hGPR40) cDNA construct and transfected it into HEK293 cells and picked up a number of stable transfectants. We found that GPR40 overexpression in HEK293 cells indeed significantly enhanced EET-induced cell proliferation and markedly augmented EGFR phosphorylation ERK activation, which were inhibited by the EGFR tyrosine kinase inhibitor, AG1478, or the HB-EGF inhibitor, CRM197. EETs significantly enhanced release of soluble HB-EGF, a natural ligand of EGFR, into the culture medium of hGPR40-transfected HEK293 cells, compared to empty vector-transfected cells. In mouse kidneys, markedly higher level of GPR40 protein was found in the cortex and outer stripe of outer medulla compared to the inner stripe of outer medulla and inner medulla. In situ hybridization confirmed that GPR40 mRNA was localized to a subset of renal tubules in the kidney, including the cortical collecting duct. Thus, this study provides the first demonstration that upregulation of GPR40 expression enhances the mitogenic response to EETs and a relatively high expression level of GPR40 is detected in a subset of tubules including cortical collecting ducts in the mammalian kidney.

Meta-analysis of green tea drinking and the prevalence of gynecological tumors in women.

We performed a meta-analysis of the correlation between drinking green tea and the risk of female ovarian tumors. Related literature (2000-2010) was retrieved from PubMed, EMbase, CBMdisc, CNKI, and Wanfang databases. The relationship between the prevalence of ovarian cancer and drinking tea in cohort studies was explored. RevMan5.1.0 software was used for the meta-analysis. A total of 6 case control studies and cohort studies were included. A total of 9113 participants, 3842 cases, and 5271 control cases were included in our analysis. Our analysis indicates that drinking green tea can significantly decrease the risk of ovarian cancer (odds ratio = 0.81; 95% confidence interval = 0.73-0.89; P < .0001). Further research is needed to explore the relationship between drinking green tea and the risk of ovarian tumor in different groups of people and with different tea types and dosages.

[Experimental study of extract from Prunella vulgaris inducing B, T lymphoma cell apoptosis].

OBJECTIVE: To investigate the effects of extract from Prunella vulgaris on proliferation of human B lymphoma cell line Raji cells anf T lymphoma cell line Jurkat cells and discuss the mechanism. METHODS: Used different concentrations of extract from Prunella vulgaris to treat Raji and Jurkat cells and collected cells after 48 h respectively, the proliferation inhibition rate, the DNA Ladder and the apoptosis rate of Raji and Jurkat cells were examined by MTT assay, agarose gel electrophoresis and flow cytometry respectively; Western blot was used to detect the change of BCL-2, BAX protein. RESULTS: Different concentrations of the extract from Prunella vulgaris could inhibit the proliferation of both Raji and Jurkat cells remarkably (P < 0.01), the IC50 of Raji cells, 18.01 +/- 0.92 microg/mL, was lower than that of Jurkat, the difference was significant statistically (P < 0.05); Apoptosis related DNA Ladder appeared after treated Raji and Jurkat cells with the extract from Prunella vulgaris; Compared with the control group, with the increase of the concentration of the extract from Prunella vulgaris, the early cell apoptosis rate of Raji and Jurkat were all increased,the early cell apoptosis rate of the extract from Prunella vulgaris of 15, 20 and 25 microg/mL treated Raji and Jurkat cells were (9.46 +/- 0.25)%, (21.68 +/- 0.46)%, (35.03 +/- 0.35)% and (4.06 +/- 0.14)%, (13.59 +/- 0.23)%, (22.92 +/- 0.20)% respectively. With the same concentration, the early apoptosis rate of Raji cells was higher than that of Jurkat cells significantly (P < 0.01); Compared with the control group, with the in- crease of the concentration of the extract from Prunella vulgaris, the expression of BCL-2 protein was down-regulated and BAX up-regulated, With the same concentration, the decline degree of BCL-2 protein expression and the increase degree of BAX protein expression in Raji cells was more remarkable than that in Jurkat cells, the difference was significant (P < 0.05). CONCLUSION: The extract from Prunella vulgaris can inhibit the proliferation of lymphoma cells and the inhibition is realized by inducing apoptosis, the mechanism of inducing cell apoptosis with extract from Prunella vulgaris is probably related with the BAX and BCL-2 protein expression, the inhibition effect on Raji cells is greater than that of Jurkat cells.

[Progress of chronic ocular hypertensive glaucoma in rodents].

Glaucoma is characterized by progressive loss of retinal ganglion cell (RGCs), optic nerve atrophy, cupping of the optic disc, and specific visual field deficits. Elevated intraocular pressure (IOP) is considered to be a major risk factor associated with the development of this neuropathy, therefore, elevated IOP has been induced in animals to simulate key aspects of human glaucoma, in order to unlock the mechanism of this disease and seek out new therapeutic strategies. In the following review: (1) models used for the study of hypertensive glaucomatous neuropathy in rodents, (2) assessment of these animal models and (3) their usage will be described.

[Study on proteomics of Jurkat cells treated with the extracts from Prunella vulgaris].

OBJECTIVE: To analyse the differential proteins of Jurkat cells after treated with the extracts from Prunella vulgaris using two-dimensional electrophoresis and mass spectrum. METHODS: Jurkat cell growth inhibitive effect of the extracts from Prunella vulgaris was analyzed by MTT assay. The total proteins of the cells were extracted after treated with the extracts in a dose of 20 microg/mL. Then 2D and MALDI-TOF-MS were used to assess the differential proteins. RESULTS: The extracts from Prunella vulgaris could depress the proliferation of Jurkat cells in a dose-dependent manner. After 2-DE and MALDI-TOF-MS,11 proteins were identified successfully, including glyceraldehyde-3-phosphate dehydrogenase, coagulation factor VII, Heterogeneous nuclear ribonucleoprotein L, heat shock 70 kDa protein 8 isoform 2, immunoglobulin heavy chain variable region, heterogeneous nuclear ribonucleoprotein A2/B1, zinc finger protein 43, chaperonin containing TCP1, subunit 6A (zeta 1), isoform CRA_b, etc. CONCLUSION: The extracts from Prunella vulgaris could inhibit the growth of Jurkat cells significantly, and lead the proteomics change of Jurkat cells, which may be related to the anti-tumor effect of the extracts from Prunella vulgaris.

[The experimental research about the effect of Prunella vulgaris L. on Raji cells growth and expression of apoptosis related protein].

OBJECTIVE: To investigate the anti-lymphoma effect of Prunella vulgaris L. in order to offer exprimental data for the treatment of lymphoma with Prunella vulgaris L. in clinic. METHODS: Effect of Prunella vulgaris L. injection on inhibition ratio of cell growth of Raji cells and IC50 were tested by MTT assay. The growth curve line of Raji cells was drawn also by MTT assay. The cellular morphology was observed by invert microscope, Giemas staining and MTT assay. The expression of apoptosis related protein bcl-2, bax was measured by immunocytochemistry and the quantitative analysis was made with figure analysis system. RESULTS: 1. Prunella vulgaris L. could obviously suppress the cell proliferation of Raji cells in a concentration-dependent manner (r = 0.97). The IC50 was 0.118 mg/ml. 2. After Raji cells was reacted with injection of Prunella vulgaris L. (50 mg/ml) , the morphlogical of apoptosis were observed by invert microscope, Giemsa staining and MTT assay. RESULTS: The results of immunocytochemistry showed that after Raji cells were treated by the injection of Prunella vulgaris L. (50 mg/ml) for 48 hours, the expression of bcl-1 was up-regulated, and the expression of bax was down-regulated. The differences between process group and control group were significant (P < 0.01). CONCLUSION: Prunella vulgaris L. can suppress the proliferation of Raji cells and may be a new anti-lymphoma drug. Inducing the apoptosis of Raji cells maybe one of anti-lymphoma mechanisms.

Lithium treatment inhibits renal GSK-3 activity and promotes cyclooxygenase 2-dependent polyuria.

The use of LiCl in clinical psychiatry is routinely complicated by overt nephrogenic diabetes insipidus (NDI), the mechanism of which is incompletely understood. In vitro studies indicate that lithium can induce renal medullary interstitial cell cyclooxygenase 2 (COX2) protein expression via inhibition of glycogen synthase kinase-3beta (GSK-3beta). Both COX1 and COX2 are expressed in the kidney. Renal prostaglandins have been suggested to play an important role in lithium-induced polyuria. The present studies examined whether induction of the COX2 isoform contributes to LiCl-induced polyuria. Four days after initiation of lithium treatment in C57 BL/6J mice, urine volume increased in LiCl-treated mice by fourfold compared with controls (P < 0.0001) and was accompanied by decreased urine osmolality. This was temporally associated with increased renal COX2 protein expression and increased urinary PGE(2) excretion, whereas COX1 levels remained unchanged. COX2 inhibition significantly blunted lithium-induced polyuria (P < 0.0001) and reduced urinary PGE(2) levels. Lithium-associated polyuria was also seen in COX1-/- mice and was associated with increased urinary PGE(2). COX2 inhibition completely prevented polyuria and PGE(2) excretion in COX1-/- mice, suggesting that COX2, but not COX1, plays a critical role in lithium-induced polyuria. Lithium also induced renal medullary COX2 protein expression in congenitally polyuric antidiuretic hormone (AHD)-deficient rats, demonstrating that lithium-induced COX2 protein expression is not secondary to altered ADH levels or polyuria. Lithium also decreased renal medullary GSK-3beta activity, and this was temporally related to increased COX2 expression in the kidney from lithium-treated mice, consistent with a tonic in vivo suppression of COX2 expression by GSK-3 activity. In conclusion, these findings temporally link decreased GSK-3 activity to enhanced renal COX2 expression and COX2-derived urine PGE(2) excretion. Suppression of COX2-derived PGE(2) blunts lithium-associated polyuria.

Regulation of renal cortical cyclooxygenase-2 in young rats.

Cyclooxygenase-2 (COX-2) is involved in kidney morphogenesis and is transiently elevated in the immature kidney. In adult rats, renal cortical COX-2 expression is tonically suppressed by mineralocorticoids (MC) and glucocorticoids (GC) and induced by chronic salt restriction. Young rats have low levels of GC and are in a state of relative volume depletion. The present study was designed to investigate the mechanisms underlying elevated cortical COX-2 expression in the immature kidney. Supplementation of GC or MC suppressed cortical COX-2 expression in suckling rats. GC suppression was significantly, but not completely, prevented by either an MC receptor antagonist or a GC receptor antagonist. MC suppression was completely prevented by a mineralocorticoid receptor antagonist. Salt supplementation suppressed cortical COX-2 expression in a dose- and time-dependent pattern in the suckling rats. Cortical COX-2 expression in the weanling rats was upregulated by a low-salt diet and downregulated by a high-salt diet. These results suggest that relative volume depletion and reduced GC levels are involved in elevated cortical COX-2 expression in the immature rodent kidney.