Anti-allergic effect of a combination of Citrus unshiu unripe fruits extract and prednisolone on picryl chloride-induced contact dermatitis in mice

Effect of 50% ethanolic extract of unripe fruits of Citrus unshiu (CU-ext) on type IV allergic reaction was examined by inhibitory activity of ear swelling of picryl chloride-induced contact dermatitis (PC-CD) in mice. Oral administration of CU-ext and subcutaneous administration of prednisolone showed inhibition of ear swelling during both induction and effector phases of PC-CD. The inhibitory activities of combinations of CU-ext (p.o.) and prednisolone (s.c.) during induction phase of PC-CD were more potent than those of CU-ext alone and prednisolone alone. Successive oral administration of hesperidin, a major flavanone glycoside of CU-ext, inhibited ear swelling during induction phase of PC-CD. The inhibitory activities of combinations of hesperidin (p.o.) and prednisolone (s.c.) were more potent than those of hesperidin alone and prednisolone alone. These results indicated that the combinations of prednisolone and CU-ext or hesperidin exerted a synergistic effect.     

Diphlorethohydroxycarmalol,Isolated from Ishige okamurae,Increases Prostaglandin E2 through the Expression of Cyclooxygenase-1 and -2 in HaCaT Human Keratinocytes

Prostaglandin (PG) E₂, the most abundant prostaglandin in the human body, is synthesized from arachidonic acid via the actions of cyclooxygenase (COX) enzymes. PGE₂ exerts homeostatic, cytoprotective, inflammatory, and in some cases anti-inflammatory effects. Also, it has been reported that PGE₂ is involved in hair growth. Diphlorethohydroxycarmalol (DPHC) is a phlorotannin compound isolated from the brown algae Ishige okamurae, with various biological activities in vitro and in vivo. In this study, the biological effect and mechanism of action of DPHC on prostaglandin synthesis in HaCaT human keratinocytes was examined. The results showed that, in these cells, DPHC significantly and dose-dependently induced PGE₂ synthesis by increasing the protein and mRNA levels of COX-1 and COX-2. Interestingly, DPHC-induced COX-1 expression preceded that of COX-2. Also, while both rofecoxib and indomethacin inhibited PGE₂ production, the latter was seems to be the more potent. From above results, we can expect that DPHC has some beneficial effects via increasing of PGE₂ production.     

The Role of Intestinal Microflora in Anti-Inflammatory Effect of Baicalin in Mice

Baicalin, a main constituent of the rhizome of Scutellaria baicalensis, is metabolized to baicalein and oroxylin A in the intestine before its absorption. To understand the role of intestinal microflora in the pharmacological activities of baicalin, we investigated its anti-inflammatory effect in mice treated with and without antibiotics. Orally administered baicalin showed the anti-inflammatory effect in mice than intraperitoneally treated one, apart from intraperitoneally administered its metabolites, baicalein and oroxylin A, which potently inhibited LPS-induced inflammation. Of these metabolites, oroxylin A showed more potent anti-inflammatory effect. However, treatment with the mixture of cefadroxil, oxytetracycline and erythromycin (COE) significantly attenuated the anti-inflammatory effect of orally administered baicalin in mice. Treatment with COE also reduced intestinal bacterial fecal β-glucuronidase activity. The metabolic activity of human stools is significantly different between individuals, but neither between ages nor between male and female. Baicalin was metabolized to baicalein and oroxylin A, with metabolic activities of 1.427 ± 0.818 and 1.025 ± 0.603 pmol/min/mg wet weight, respectively. Baicalin and its metabolites also inhibited the expression of pro-inflammatory cytokines, TNF-α and IL-1β, and the activation of NF-κB in LPS-stimulated peritoneal macrophages. Of them, oroxylin A showed the most potent inhibition. Based on these findings, baicalin may be metabolized to baicalein and oroxylin A by intestinal microflora, which enhance its anti-inflammatory effect by inhibiting NF-κB activation.     

Induction of Biologically Active Flavonoids in Cell Cultures of Morus nigra and Testing their Hypoglycemic Efficacy

The antidiabetic activity of both leaves and MJ-treated cell cultures of Morus nigra was evaluated after their oral administration to streptozotocin-induced diabetic rats. The antidiabetic activity of extracts from leaves given to streptozotocin (STZ)-diabetic rats for 10 days increased with increasing doses of leaves extract up to 500 mg/kg/day. The administration of 500 mg/kg/day of leaves extract reduced the concentration of glucose from 370 ± 7.31 mg/dl (control) to 154 ± 6.27 mg/dl, and a significant increase in the insulin level from 11.3 ± 0.31 μU/ml (control) to 14.6 ± 0.43 μU/ml was recorded. Cell suspension cultures were established from the young leaves of Morus nigra cultivated on modified MS medium supplemented with 2.0 mg/l 1-naphthaleneacetic acid (NAA), 0.2 mg/l 6-(furfurylamino)purine (kinetin). The changes in cell weight and flavonoid content were monitored between day zero and 12. The linear increase in fresh weight was found to be parallel to flavonoids production. Cell cultures treated with 100 μM methyl jasmonate for 24 hours showed a noticeable increase in level of flavonoids and significant and more effective hypoglycemic activity than that for extract from leaves. The major flavonoids were isolated by TLC and HPLC and identified as rutin, quercetin, Morusin and cyclomorusin by co-chromatography and mass spectrometry in comparison to samples of authentic reference compounds.     

Ophiophagus hannah Venom: Proteome,Components Bound by Naja kaouthia Antivenin and Neutralization by N. kaouthia Neurotoxin-Specific Human ScFv

Venomous snakebites are an important health problem in tropical and subtropical countries. King cobra (Ophiophagushannah) is the largest venomous snake found in South and Southeast Asia. In this study, the O. hannah venom proteome and the venom components cross-reactive to N. kaouthia monospecific antivenin were studied. O. hannah venom consisted of 14 different protein families, including three finger toxins, phospholipases, cysteine-rich secretory proteins, cobra venom factor, muscarinic toxin, L-amino acid oxidase, hypothetical proteins, low cysteine protein, phosphodiesterase, proteases, vespryn toxin, Kunitz, growth factor activators and others (coagulation factor, endonuclease, 5’-nucleotidase). N. kaouthia antivenin recognized several functionally different O. hannah venom proteins and mediated paratherapeutic efficacy by rescuing the O. hannah envenomed mice from lethality. An engineered human ScFv specific to N. kaouthia long neurotoxin (NkLN-HuScFv) cross-neutralized the O. hannah venom and extricated the O. hannah envenomed mice from death in a dose escalation manner. Homology modeling and molecular docking revealed that NkLN-HuScFv interacted with residues in loops 2 and 3 of the neurotoxins of both snake species, which are important for neuronal acetylcholine receptor binding. The data of this study are useful for snakebite treatment when and where the polyspecific antivenin is not available. Because the supply of horse-derived antivenin is limited and the preparation may cause some adverse effects in recipients, a cocktail of recombinant human ScFvs for various toxic venom components shared by different venomous snakes, exemplified by the in vitro produced NkLN-HuScFv in this study, should contribute to a possible future route for an improved alternative to the antivenins.     

Regional Decoupling of N-acetyl-aspartate and Glutamate in Schizophrenia

Proton magnetic resonance spectroscopy (¹H-MRS) allows the non-invasive measurement of several metabolites, including N-acetyl-aspartate (NAA), an amino acid exclusively synthesized in the mitochondria of neurons, and glutamate, an amino acid involved in excitatory neurotransmission and metabolism. In view of recent postmortem studies in schizophrenia (SZ) revealing mitochondrial abnormalities as well as perturbed expression of the enzymes regulating the glutamate–glutamine cycle, we hypothesized that a disruption in the homeostasis of NAA and glutamate in SZ is present. Fifty subjects with SZ and 48 matched healthy controls (HC) were enrolled in this ¹H-MRS study. Voxels were placed in the anterior cingulate cortex (ACC) and hippocampus; NAA/Cr and glutamate + glutamine (Glx)/Cr ratios were obtained. We did not find any significant differences between the groups in metabolite levels in both the ACC and hippocampus. In the hippocampus we found that NAA/Cr and Glx/Cr ratios were significantly correlated in HC (r=0.40, p<0.01 (corrected p=0.048)) but not in SZ (r=−0.06; p=0.71), a difference that was statistically significant (z=2.22, p=0.02). Although no differences in neurometabolites between SZ and HC were apparent, correlations between NAA/Cr and Glx/Cr in healthy subjects in the hippocampus were found, and this correlation was lost in subjects with SZ. To our knowledge, this is the first study to suggest decoupling of these metabolites, a pathophysiological change that may be unique to SZ. However, these results warrant replication and further exploration before definite conclusions can be drawn.     

Electrophysiological and Histologic Evaluation of the Time Course of Retinal Degeneration in the rd10 Mouse Model of Retinitis Pigmentosa

Among several animal models of retinitis pigmentosa (RP), the more recently developed rd10 mouse with later onset and slower rate of retinal degeneration than rd1 mouse is a more suitable model for testing therapeutic modalities. We therefore investigated the time course of retinal degeneration in rd10 mice before adopting this model in our interventional studies. Electroretinogram (ERG) recordings were carried out in postnatal weeks (PW) 3~5 rd10 (n=23) and wild-type (wt) mice (n=26). We compared the amplitude and implicit time of the b-wave of ERG records from wt and rd10 mice. Our results showed that b-wave amplitudes in rd10 mice were significantly lower and the implicit time of b-waves in rd10 mice were also significantly slower than that in wt mice (20~160 µV vs. 350~480 µV; 55~75 ms vs. 100~150 ms: p<0.001) through PW3 to PW5. The most drastic changes in ERG amplitudes and latencies were observed during PW3 to PW4. In multichannel recording of rd10 retina in PW2 to PW4.5, we found no significant difference in mean spike frequency, but the frequency of power spectral peak of local field potential at PW3 and PW3.5 is significantly different among other age groups (p<0.05). Histologic examination of rd10 retinae showed significant decrease in thickness of the outer nuclear layer at PW3. TUNEL positive cells were most frequently observed at PW3. From these data, we confirm that in the rd10 mouse, the most precipitous retinal degeneration occurs between PW3~PW4 and that photoreceptor degeneration is complete by PW5.     

Mass Spectrometry-Based Method of Detecting and Distinguishing Type 1 and Type 2 Shiga-Like Toxins in Human Serum

Shiga-like toxins (verotoxins) are responsible for the virulence associated with a variety of foodborne bacterial pathogens. Direct detection of toxins requires a specific and sensitive technique. In this study, we describe a mass spectrometry-based method of analyzing the tryptic decapeptides derived from the non-toxic B subunits. A gene encoding a single protein that yields a set of relevant peptides upon digestion with trypsin was designed. The ¹⁵N-labeled protein was prepared by growing the expressing bacteria in minimal medium supplemented with ¹⁵NH₄Cl. Trypsin digestion of the ¹⁵N-labeled protein yields a set of ¹⁵N-labeled peptides for use as internal standards to identify and quantify Shiga or Shiga-like toxins. We determined that this approach can be used to detect, quantify and distinguish among the known Shiga toxins (Stx) and Shiga-like toxins (Stx1 and Stx2) in the low attomole range (per injection) in complex media, including human serum. Furthermore, Stx1a could be detected and distinguished from the newly identified Stx1e in complex media. As new Shiga-like toxins are identified, this approach can be readily modified to detect them. Since intact toxins are digested with trypsin prior to analysis, the handling of intact Shiga toxins is minimized. The analysis can be accomplished within 5 h.     

The effect of cassia fistula emulsion on pediatric functional constipation in comparison with mineral oil: a randomized,clinical trial

Background

The prevalence of Pediatric Functional Constipation (FC) has been reported between 0.7% to 29.6%. This study was conducted to compare the laxative effect of cassia fistula emulsion (CFE) with mineral oil (MO) on FC. Cassia fistula is named in Traditional Iranian Medicine (TIM) as “Folus”.

Materials and methods

A randomized clinical trial was carried on 81 children (age range: 4–13 years) with FC, according to Rome III criteria in Amirkola Children’s Hospital, Babol, Iran. They received CFE or MO randomly for three weeks. CFE was produced according to the order of TIM references. Children were counted as improved when they exited from Rome III criteria of FC. Frequency of defecation, fecal incontinence, retentive posturing, severity of pain, consistency of stool and anal leakage of oily material were compared between the two groups and with baselines. An intent-to-treat analysis was used. Safety of drugs was assessed with the evaluation of clinical adverse effects.

Results

41 children were assigned randomly to receive CFE and 40 children received MO. After three weeks of medication, 84% of children in CFE group and 50% in MO group (p = 0.002) exited from the criteria of FC, so called improved. All measurable criteria improved in both groups. The frequency of defecation in CFE group improved from 1.7 per week (before the study) to 10.6 per week (at the third week) while this parameter differed in MO group from 2 to 6.1 (p < 0.001). The severity of pain during defecation and consistency of stool improved significantly better in CFE group than MO group (p < 0.05), but there were not any significant differences between the two groups in fecal incontinence and retentive posturing. Anal leakage of oily material occurred as an important complication in MO group while the children in CFE group did not complaint it. Drug’s compliances were not significantly different in the two groups. CFE and MO did not cause clinically significant side effects.

Conclusions

CFE was most effective than MO in the 3-week treatment of children with FC.     

Long-Term Memory Deficits are Associated with Elevated Synaptic ERK1/2 Activation and Reversed by mGluR5 Antagonism in an Animal Model of Autism

A significant proportion of patients with autism exhibit some degree of intellectual disability. The BTBR T⁺ Itpr3^tf/J mouse strain exhibits behaviors that align with the major diagnostic criteria of autism. To further evaluate the BTBR strain''s cognitive impairments, we quantified hippocampus-dependent object location memory (OLM) and found that one-third of the BTBR mice exhibited robust memory, whereas the remainder did not. Fluorescence deconvolution tomography was used to test whether synaptic levels of activated extracellular signal-regulated kinase 1/2 (ERK1/2), a protein that contributes importantly to plasticity, correlate with OLM scores in individual mice. In hippocampal field CA1, the BTBRs had fewer post-synaptic densities associated with high levels of phosphorylated (p-) ERK1/2 as compared with C57BL/6 mice. Although counts of p-ERK1/2 immunoreactive synapses did not correlate with OLM performance, the intensity of synaptic p-ERK1/2 immunolabeling was negatively correlated with OLM scores across BTBRs. Metabotropic glutamate receptor (mGluR) 5 signaling activates ERK1/2. Therefore, we tested whether treatment with the mGluR5 antagonist MPEP normalizes synaptic and learning measures in BTBR mice: MPEP facilitated OLM and decreased synaptic p-ERK1/2 immunolabeling intensity without affecting numbers of p-ERK1/2+ synapses. In contrast, semi-chronic ampakine treatment, which facilitates memory in other models of cognitive impairment, had no effect on OLM in BTBRs. These results suggest that intellectual disabilities associated with different neurodevelopmental disorders on the autism spectrum require distinct therapeutic strategies based on underlying synaptic pathology.     

Pachymic Acid Enhances Pentobarbital-Induced Sleeping Behaviors via GABAA-ergic Systems in Mice

This study was investigated to know whether pachymic acid (PA), one of the predominant triterpenoids in Poria cocos (Hoelen) has the sedative-hypnotic effects, and underlying mechanisms are mediated via γ-aminobutyric acid (GABA)-ergic systems. Oral administration of PA markedly suppressed locomotion activity in mice. This compound also prolonged sleeping time, and reduced sleep latency showing synergic effects with muscimol (0.2 mg/kg) in shortening sleep onset and enhancing sleep time induced by pentobarbital, both at the hypnotic (40 mg/kg) and sub-hypnotic (28 mg/kg) doses. Additionally, PA elevated intracellular chloride levels in hypothalamic primary cultured neuronal cells of rats. Moreover, Western blotting quantitative results showed that PA increased the amount of protein level expression of GAD_65/67 over a broader range of doses. PA increased α- and β-subunits protein levels, but decreased γ-subunit protein levels in GABA_A receptors. The present experiment provides evidence for the hypnotic effects as PA enhanced pentobarbital-induced sleeping behaviors via GABA_A-ergic mechanisms in rodents. Taken together, it is proposed that PA may be useful for the treatment of sleep disturbed subjects with insomnia.     

Effect of an isolated active compound (Cg-1) of Cassia glauca leaf on blood glucose, lipid profile, and atherogenic index in diabetic rats

Objectives:

The objective of present study was to evaluate the effect of active principle (Cg-1) from Cassia glauca leaf on serum glucose and lipid profile in normal and diabetic rats.

Materials and Methods:

Diabetes was induced by streptozotocin in neonates. Oral administration of petroleum ether, chloroform, acetone, and methanol of C. glauca leaf (100 mg/kg, p.o.) for 21 days caused a decrease in fasting blood glucose (FBG) in diabetic rats. Among all the extracts, acetone extract was found to lower the FBG level significantly in diabetic rats. Glibenclamide was used as standard antidiabetic drug (5 mg/kg, p.o). Acetone extract was subjected to column chromatography that led to isolation of an active principle, which was given trivial name Cg-1. Cg-1 (50 mg/kg, p.o.) was studied for its hypoglycemic and hypolipidemic potential. The unpaired t-test and analysis of variance (ANOVA) followed by post hoc test was used for statistical analysis.

Results:

Cg-1 caused a significant reduction in FBG level. It also caused reduction in cholesterol, triglycerides, and LDL levels and improvement in the atherogenic index and HDL level in diabetic rats.

Conclusion:

Improvement in the FBG and the atherogenic index by Cg-1 indicates that Cg-1 has cardioprotective potential along with antidiabetic activity and provides a scientific rationale for the use as an antidiabetic agent.     

Presence of Potential Toxin-Producing Cyanobacteria in an Oligo-Mesotrophic Lake in Baltic Lake District,Germany: An Ecological,Genetic and Toxicological Survey

Massive developments of potentially toxic cyanobacteria in Lake Stechlin, an oligo-mesotrophic lake in the Baltic Lake District of Germany raised concerns about toxic contamination of these important ecosystems. Field samples in the phase of mass developments of cyanobacteria were used for genetic and toxicological analyses. Microcystins and microcystin genes were detected in field samples of the lake for the first time. However, the toxins were not produced by the dominant taxa (Dolichospermum circinale and Aphanizomenon flos-aquae) but by taxa, which were present only in low biomass in the samples (Microcystis cf. aeruginosa and Planktothrix rubescens). The phytoplankton successions during the study period revealed an increase of cyanobacterial populations. The findings contribute to the changes that have been investigated in Lake Stechlin since the mid-1990s. The possible reasons behind these developments may be climate change, special weather conditions and an increased nutrient pool.     

Formulation and in vivo evaluation for anti-aging effects of an emulsion containing basil extract using non- invasive biophysical techniques

Background and the purpose of study

Skin aging is a complex process induced by constant exposure to ultraviolet (UV) irradiation and damages human skin. UV generates reactive oxygen species leading to collagen deficiency and eventually skin wrinkling. Basil contains a number of phenolics and favonoids which possess antioxidant properties. The aim of this study was to formulate and investigate the antiaging potential of a cream containing Basil extract.

Methods

A single blinded study was conducted using non-invasive methods. Formulation containing 3% of the concentrated extract of Basil was developed by entrapping in the inner aqueous phase of w/o emulsion and base contained no extract. Both creams were stored at different storage conditions of 8°C, 25°C, 40°C and 40°C+ 75% relative humidity to predict their stabilities. The formulation and base were evaluated for their effects on various skin parameters i.e., moisture and trans epidermal water loss (TEWL), volume, energy and surface evaluation of the living skin (SELS).

Results

Significant effects (p≤0.05) were observed for both creams in the case of TEWL. The base showed insignificant (p≤0.05) while formulation showed significant effects on skin moisture. Volume, SELS SEr (skin roughness), SEsc (skin scaliness), SEsm (skin smoothness), SEw (skin wrinkles) parameter showed significant decline while texture parameter of ‘Energy’ showed significant increase.

Conclusion

The results statistically indicated that the active formulation containg extract of Basil exert antiaging effects when applied topically.     

Radioprotective Effect of Achillea millefolium L Against Genotoxicity Induced by Ionizing Radiation in Human Normal Lymphocytes

The radioprotective effect of Achillea millefolium L (ACM) extract was investigated against genotoxicity induced by ionizing radiation (IR) in human lymphocytes. Peripheral blood samples were collected from human volunteers and incubated with the methanolic extract of ACM at different concentrations (10, 50, 100, and 200 μg/mL) for 2 hours. At each dose point, the whole blood was exposed in vitro to 2.5 Gy of X-ray and then the lymphocytes were cultured with mitogenic stimulation to determine the micronuclei in cytokinesis-blocked binucleated cell. Antioxidant capacity of the extract was determined using free radical-scavenging method. The treatment of lymphocytes with the extract showed a significant decrease in the incidence of micronuclei binucleated cells, as compared with similarly irradiated lymphocytes without any extract treatment. The maximum protection and decrease in frequency of micronuclei were observed at 200 μg/mL of ACM extract which completely protected genotoxicity induced by IR in human lymphocytes. Achillea millefolium extract exhibited concentration-dependent radical-scavenging activity on 1,1-diphenyl-2-picryl hydrazyl free radicals. These data suggest that the methanolic extract of ACM may play an important role in the protection of normal tissues against genetic damage induced by IR.     

Scientific Insights in the Preparation and Characterisation of a Lead-based Naga Bhasma

Naga bhasma is one of the herbo-metallic preparations used in Ayurveda, a traditional Indian System of Medicine. The preparation of Naga bhasma involves thermal treatment of ‘Naga’ (metallic lead) in a series of quenching liquids, followed by reaction with realgar and herbal constituents, before calcination to prepare a fine product. We have analysed the intermediates obtained during different stages of preparation to understand the relevance and importance of different steps involved in the preparation. Our results show that ‘Sodhana’ (purification process) removes heavy metals other than lead, apart from making it soft and amenable for trituration. The use of powders of tamarind bark and peepal bark maintains the oxidation state of lead in Jarita Naga (lead oxide) as Pb²⁺. The repeated calcination steps result in the formation of nano-crystalline lead sulphide, the main chemical species present in Naga bhasma.     

Cross-regulation between protein L-isoaspartyl O-methyltransferase and ERK in epithelial mesenchymal transition of MDA-MB-231 cells

Aim:

Protein L-isoaspartyl O-methyltransferase (PIMT) regulates cell adhesion in various cancer cell lines through activation of integrin αv and the PI3K pathway. The epithelial mesenchymal transition (EMT) enables epithelial cells to acquire the characteristics of mesenchymal cells, and to allow them to migrate for metastasis. Here, we examined the relationship between PIMT and EMT with attached or detached MDA-MB 231 cells.

Methods:

Human breast cancer cell line MDA-MB-231 cells were maintained in a suspension on poly-HEMA in the presence or absence of PIMT siRNA or ERK inhibitor PD98059. The mRNAs and proteins were analyzed using RT-PCR and immunoblotting, respectively.

Results:

During cellular incubation under detached conditions, PIMT, integrin αv and EMT proteins, such as Snail, Slug and matrix metalloproteinase 2 (MMP-2), were significantly increased in correlation with the phosphorylation of ERK1/2. The ERK inhibitor PD98059 (25 μmol/L) strongly suppressed the expression of the proteins and PIMT. Interestingly, PIMT siRNA blocked the phosphorylation of ERK and the expression of the EMT proteins. Additionally, PIMT and ERK phosphorylation were both co-activated by treatment with TGF-β (10 ng/mL) and TNF-α (10 ng/mL).

Conclusion:

A tight cross-regulation exists between ERK and PIMT in regards to their activation and expression during the EMT.     

Cytotoxicity and glycan-binding properties of an 18 kDa lectin isolated from the marine sponge Halichondria okadai

A divalent cation-independent lectin-HOL-18, with cytotoxic activity against leukemia cells, was purified from a demosponge, Halichondria okadai. HOL-18 is a 72 kDa tetrameric lectin that consists of four non-covalently bonded 18 kDa subunits. Hemagglutination activity of the lectin was strongly inhibited by chitotriose (GlcNAcβ1-4GlcNAcβ1-4GlcNAc), fetuin and mucins from porcine stomach and bovine submaxillary gland. Lectin activity was stable at pH 4-12 and temperatures lower than 60 °C. Frontal affinity chromatography with 16 types of pyridylaminated oligosaccharides indicated that the lectin had an affinity for N-linked complex-type and sphingolipid-type oligosaccharides with N-acetylated hexosamines and neuramic acid at the non-reducing termini. The lectin killed Jurkat leukemia T cells and K562 erythroleukemia cells in a dose- and carbohydrate-dependent manner.