Non-alcoholic steatohepatitis patients exhibit reduced CD47, and increased sphingosine, cholesterol and MCP1 levels in the erythrocyte membranes

Non-alcoholic steatohepatitis (NASH) constitutes a significant cause of deaths, liver transplantations and economic costs worldwide. Despite extended research, investigations on the role of erythrocytes are scarce. Red blood cells from experimental animals and human patients with NASH, present phosphatidylserine exposure which is then recognized by Kupffer cells. This event leads to erythrophagocytosis, and amplification of inflammation through iron disposition. In addition, it has been shown that erythrocytes from NASH patients release the chemokine MCP1, leading to increased TNF-α release from macrophages RAW 264.7. However, erythrophagocytosis can also be caused by reduced CD47 levels. In addition, increased MCP1 release could be either signal-induced, or caused by higher MCP1 levels on the erythrocyte membrane. Finally, erythrocyte efferocytosis could provide additional inflammatory metabolites. In this study, we measured the erythrocyte membrane levels of CD47 and MCP1 by ELISA, and cholesterol and sphingosine with thin-layer chromatography. 18 patients (8 men, 10 women aged 56.7+/-11.5 years) and 14 healthy controls (7 men, 7 women aged 39.3+/-15.5 years) participated in our study. The erythrocyte CD47 levels were decreased in the erythrocyte membranes of NASH patients (844+/-409 pg/ml) compared to healthy controls (2969+/-1936 pg/ml) with P(Healthy>NAFLD)=99.1%, while the levels of MCP1 were increased in NASH patients (389+/-255 pg/ml), compared to healthy controls (230+/-117 pg/ml) with P(Healthy<NAFLD)=88.9%. Moreover, in erythrocyte membranes there was a statistically significant accumulation of sphingosine and cholesterol in NASH patients, compared to healthy controls. Our results imply that erythrocytes release chemotactic (find me signals) MCP1, while containing reduced (do not eat me signals) CD47. These molecules can lead to erythrophagocytosis. Next, increased (goodbye signals) sphingosine and cholesterol could augment inflammation by metabolic reprogramming.

Optimization of Gamma Aminobutyric Acid Production Using High Pressure Processing (HPP) by Lactobacillus brevis PML1

In the present research, the production potential of gamma aminobutyric acid (GABA) using Lactobacillus brevis PML1 was investigated. In addition, the microorganism viability was examined in MAN, ROGOSA, and SHARPE (MRS) after undergoing high hydrostatic pressure at 100, 200, and 300 MPa for 5, 10, and 15 min. Response surface methodology (RSM) was applied to optimize the production conditions of GABA as well as the bacteria viability. Analysis of variance (ANOVA) indicated that both the independent variables (pressure and time) significantly influenced the dependent ones (GABA and bacteria viability) ( P < 0.05 ). The optimum extraction conditions to maximize the production of GABA included the pressure of 300 MPa and the time of 15 min. The amount of the compound was quantified using thin-layer chromatography (TLC) and spectrophotometry. For the process optimization, a central composite design (CCD) was created using Design Expert with 5 replications at the center point, whereby the highest content of GABA was obtained to be 397.73 ppm which was confirmed by high performance liquid chromatography (HPLC). Moreover, scanning electron microscopy (SEM) was utilized to observe the morphological changes in the microorganism. The results revealed that not only did have Lactobacillus brevis PML1 the potential for the production of GABA under conventional conditions (control sample) but also the content of this bioactive compound could be elevated by optimizing the production parameters.

Simultaneous Determination of Caffeine and Paracetamol in Commercial Formulations Using Greener Normal-Phase and Reversed-Phase HPTLC Methods: A Contrast of Validation Parameters

There has been no assessment of the greenness of the described analytical techniques for the simultaneous determination (SMD) of caffeine and paracetamol. As a result, in comparison to the greener normal-phase high-performance thin-layer chromatography (HPTLC) technique, this research was conducted to develop a rapid, sensitive, and greener reversed-phase HPTLC approach for the SMD of caffeine and paracetamol in commercial formulations. The greenness of both techniques was calculated using the AGREE method. For the SMD of caffeine and paracetamol, the greener normal-phase and reversed-phase HPTLC methods were linear in the 50–500 ng/band and 25–800 ng/band ranges, respectively. For the SMD of caffeine and paracetamol, the greener reversed-phase HPTLC approach was more sensitive, accurate, precise, and robust than the greener normal-phase HPTLC technique. For the SMD of caffeine paracetamol in commercial PANEXT and SAFEXT tablets, the greener reversed-phase HPTLC technique was superior to the greener normal-phase HPTLC approach. The AGREE scores for the greener normal-phase and reversed-phase HPTLC approaches were estimated as 0.81 and 0.83, respectively, indicated excellent greenness profiles for both analytical approaches. The greener reversed-phase HPTLC approach is judged superior to the greener normal-phase HPTLC approach based on numerous validation parameters and pharmaceutical assays.

Phytochemical characterization of the ethanolic extract of Kaempferia galanga rhizome for anti-oxidant activities by HPTLC and GCMS

Background The rhizome of Kaempferia galanga (K. galanga) was collected from Meghalaya, India, and its ethanolic extract was obtained by freeze-drying or lyophilization process, which was then assessed for its in vitro anti-oxidant activity and phytochemical characterization using high-performance thin-layer chromatography (HPTLC) and gas chromatography-mass spectroscopy (GCMS). Results In vitro anti-oxidant activity analysis shows an inhibitory concentration (IC50) value of 1.824 mg/mL and 0.307 mg/mL for, α, α-diphenyl-ρ-picrylhydrazyl (DPPH) and 2, 2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays, respectively. Total polyphenol content (TPC) of 23.55 ± 0.5 mg gallic acid equivalent (GAE)/g dry weight of extract and total flavonoid content (TFC) of 100 ± 1.414 mg rutin equivalents (RE)/g dry weight of extract were found. High-performance thin-layer chromatography (HPTLC) analysis shows the best separation of bands at different retention factor (Rf) values, when employing the solvent system 2-butanol/1-propanol/water in the ratio of 3:1:1 (v/v/v). Gas chromatography-mass spectroscopy (GCMS) analysis confirms the presence and identification of various phytocompounds, with ethyl p-methoxycinnamate identified as the major active compound. Conclusion Freeze-dried ethanolic extract of K. galanga (rhizome) possesses anti-oxidant activity. Ethyl p-methoxycinnamate is present as the major bioactive component (about 94.87% of the total area composition), and since it has very important and diverse medicinal properties, a freeze-drying process (lyophilization) can be utilized for its isolation and extraction.

Feeding Brassica vegetables to rats leads to the formation of characteristic DNA adducts (from 1-methoxy-3-indolylmethyl glucosinolate) in many tissues

Juices of Brassica vegetables are mutagenic and form characteristic DNA adducts in bacteria and mammalian cells. In this study, we examined whether such adducts are also formed in vivo in animal models. Rats fed raw broccoli ad libitum in addition to normal laboratory chow for 5 weeks showed one major adduct spot and sometimes an additional minor adduct spot in liver, kidney, lung, blood and the gastrointestinal tract, as determined by 32P-postlabelling/thin-layer chromatography. Adducts with the same chromatographic properties were formed when herring sperm DNA (or dG-3’-phosphate) was incubated with 1-methoxy-3-indolylmethyl glucosinolate (phytochemical present in Brassica plants), in the presence of myrosinase (plant enzyme that hydrolyses glucosinolates to bioactive breakdown products). UPLC–MS/MS analysis corroborated this finding: 1-Methoxy-3-indolylmethyl-substituted purine nucleosides were detected in the hepatic DNA of broccoli-fed animals, but not in control animals. Feeding raw cauliflower led to the formation of the same adducts. When steamed rather than raw broccoli was used, the adduct levels were essentially unchanged in liver and jejunum, but elevated in large intestine. Due to inactivation of myrosinase by the steaming, higher levels of the glucosinolates may have reached the large bowl to be activated by glucosidases from intestinal bacteria. In conclusion, the consumption of common Brassica vegetables can lead to the formation of substantial levels of DNA adducts in animal models. The adducts can be attributed to a specific phytochemical, neoglucobrassicin (1-methoxy-3-indolylmethyl glucosinolate).