Positive Control Research Articles

Herbs Used for the Management of Hypertension: A Systematic Review

Background: Hypertension is characterized by persistent high blood pressure and has emerged as a critical risk factor for severe cardiovascular diseases. Although several drugs have been designated to reduce blood pressure, these drugs can potentially cause side effects for patients. Therefore, medicinal plants are used to complement conventional drugs to treat various ailments. Methods: This study reviews the anti-hypertensive potential of herbs and plants and their mechanism of action in reducing blood pressure at their evaluated effective doses. Databases such as PubMed, Scopus, Science Direct, and Google Scholar were used to search articles from January 2016 to Sept 2022. The key search terms included “hypertension”, “lowers blood pressure”, “anti-hypertensive”, and “plants”. These generated 807 documents and using the PRISMA guidelines, thirty plants were identified for this review. Research studies with similar plant parts extracted from similar solvents, more than one dose, and animal models with a positive control were considered in this review. Studies that did not show significant blood pressure reduction were excluded. Results: Based on this criterion, the mechanism of actions of these plants was thematically grouped into three categories, namely 1) antioxidant, 2) angiotensin-converting enzyme (ACE) inhibition, and 3) calcium influx in vascular smooth muscle. The effective doses, plant parts used, and traditional medicine usage are presented in this review. Conclusion: Further research is highly recommended to identify the active compounds and to potentially develop them into anti-hypertensive drugs as well as to establish the safe doses and standardization of these plant extracts. This review is partially funded by grants from the Ministry of Higher Education.

Hederagenin Improves Adriamycin-induced Nephropathy by Inhibiting the JAK/STAT Signaling Pathway

ABSTRACT Background: Glomerulonephritis, a common kidney disease and major cause of end-stage renal disease, lacks effective treatment options. Hederagenin (HDG) exerts potent anti-inflammatory and protective effects on the kidneys and exhibits promise for the treatment of glomerulonephritis. This study aimed to investigate the therapeutic effects and mechanism of action of hederagenin in the context of adriamycin-induced nephropathy (ADN). Methods: C57BL/6 mice were randomly divided into 5 groups that included the control, model, low-dose HDG (20 mg/kg), high-dose HDG (40 mg/kg), and positive control (10 mg/kg irbesartan) groups. ADN was established in mice by administering a single injection of 10 mg/kg adriamycin. Renal pathology and fibrosis were assessed using haematoxylin and eosin (H & E) and Masson’s trichrome staining, whereas in vitro studies were conducted using cultured mouse podocytes (MPC5). Immunofluorescence staining and western blotting were performed to detect inflammation and the protein levels of signaling pathways. Results: The results revealed that HDG significantly improved adriamycin-induced abnormal serum creatinine, albumin, and urea nitrogen levels. HDG treatment reduced glomerular injury and fibrosis, particularly at high doses. Additionally, HDG effectively reduced adriamycin-induced activation of Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling and renal fibrosis while suppressing CD4+/CD8+ cell ratios in the kidneys and enhancing the immune response. Interestingly, when the JAK/STAT signaling pathway was activated by an agonist, the ameliorative effects of HDG on ADN were inhibited, thus suggesting that JAK/STAT signaling is a key target of HDG. Conclusion: HDG may represent a promising treatment option for glomerulonephritis by inhibiting JAK/STAT-mediated immune-inflammatory responses.

Probiotic potential of a novel endophytic Streptomyces griseorubens CIBA-NS1 isolated from Salicornia sp. against Vibrio campbellii infection in shrimp

A novel endophytic Streptomyces griseorubens CIBA-NS1 was isolated from a salt marsh plant Salicornia sp. The antagonistic effect of S. griseorubens against Vibrio campbellii, was studied both in vitro and in vivo. The strain was validated for its endophytic nature and characterized through scanning electron microscopy, morphological and biochemical studies and 16SrDNA sequencing. The salinity tolerance experiment has shown that highest antibacterial activity was at 40‰ (16 ± 1.4 mm) and lowest was at 10 ‰ salinity (6.94 ± 0.51 mm). In vivo exclusion of Vibrio by S. griseorubens CIBA-NS1 was studied in Penaeus indicus post larvae and evaluated for its ability to improve growth and survival of P. indicus. After 20 days administration of S. griseorubens CIBA-NS1, shrimps were challenged with V. campbellii. The S. griseorubens CIBA-NS1 reduced Vibrio population in test group when compared to control, improved survival (60.5 ± 6.4%) and growth, as indicated by weight gain (1.8 ± 0.05g). In control group survival and growth were 48.4 ± 3.5% and 1.4 ± 0.03 g respectively. On challenge with V. campbellii, the S. griseorubens CIBA-NS1 administered group showed better survival (85.6 ± 10%) than positive control (64.3 ± 10%). The results suggested that S. griseorubens CIBA-NS1 is antagonistic to V. campbellii, reduce Vibrio population in the culture system and improve growth and survival. This is the first report on antagonistic activity of S. griseorubens isolated from salt marsh plant Salicornia sp, as a probiotic candidate to prevent V. campbellii infection in shrimps.

Isolation and characterization of secondary metabolites from Bryophylum pinnatum (Lam.) Oken and assessment of wound healing efficacy using animal model

Wound healing is a complex and intrinsic process that implicates various stages in skin tissue regeneration, may further improvise using secondary metabolites. During a screening of plants traditionally utilized for wound healing, especially by tribals, we encountered Bryophyllum pinnatum (Lam.) Oken Plant. Further, we considered the leaf extract of Bryophyllum pinnatum (Lam.) Oken in various solvents for In vivo characterization of wound healing. The leaf extracts were prepared in petroleum ether, ethyl acetate, and ethanol and further identified the phytochemicals via advanced analytical techniques. The solvent extracts in the ointment base (1 % and 2 % w/w) were prepared and tested against the acute and burn wound models along with control and positive control (Cipladine 1 %). The ethanolic extract had higher phytochemical constituents was tested against the rat model, was found that 2 % w/w ethanolic ointment base stimulated the wound healing area, hydroxyproline content, and wound index and simultaneously reduced the epithelization period compared to the control. The present study concluded that the Ethanolic extract of Bryophyllum pinnatum leaves loaded ointment further promote the % of wound contraction rate, epithelization, and promotion of angiogenesis, exhibited the potential to be utilized to treat wounds.

Two Lactones Non-toxic to Humans Are Effective Repellents for the German Cockroach.

Lactones are cyclic esters of hydroxy carboxylic acids, present in several fruits and animals consumed by humans. There is evidence that some lactones modify insect behavior. The aim of the present study was to evaluate the repellent effect of four lactones (γ- and δ-nonalactone, and γ- and δ-dodecalactone) in first instar nymphs of the German cockroach, Blattella germanica (Linnaeus). To assess repellency, a nymph was placed on a circle of filter paper, half of which had been treated with lactone dissolved in acetone and the other half with acetone alone. The behavior of the nymph was recorded and the time the nymph spent in each half of the paper was quantified using Ethovision XT 10.1 software. Values of Distribution Coefficient (DC) were calculated: DC = (Tt - TA) / Tt, where Tt is the experimental time and TA is the time the nymph spent in the area treated with the repellent agent. DC can vary between 0 and 1. Values significantly higher than 0.5 indicate repellency. N,N-diethyl-meta-toluamide (DEET) was used as a positive control. DEET, δ- and γ-nonalactone caused repellency as from 77.9µg/cm2, whereas γ- and δ-dodecalactone had a repellent effect starting at 779.0µg/cm2. The values of DC for these concentrations were 0.89 (DEET), 0.86 (γ-nonalactone), 0.87 (δ-nonalactone), 0.83 (γ-dodecalactone), and 0.72 (δ-dodecalactone). To our knowledge, this is the first report of repellency produced by lactones in the German cockroach. This work allowed to identify two lactones that have a repellent effect similar to DEET.

Synthesis of New Glucose-containing 5-Arylisoxazoles and their Enzyme Inhibitory Activity

Abstract: Carbohydrates are an important group of biomolecules that have received special attention due to their significant role in the design and synthesis of new bioactive compounds. In this study, a new class of 5-arylisoxazole-glucose hybrids was designed and synthesized for evaluation of their inhibitory effects on α-glucosidase, α-amylase, and tyrosinase. The target compounds depicted selective α-glucosidase inhibitory activity over α-amylase, which is an important factor in reducing probable gastrointestinal problems in the treatment of type 2 diabetes. In this respect, compound 9a, possessing the phenylisoxazole group, was found to be the most potent α-glucosidase inhibitor (IC50 = 292.2 ± 0.1 μM) compared to acarbose (IC50 = 750.2 ± 0.1 μM) as the positive control. All compounds were also evaluated for their anti-tyrosinase effect, and among them, compound 9j, containing a fluoroaryl moiety, showed potent activity (IC50 = 50.1 ± 6.3 μM) in comparison to kojic acid (IC50 = 23.6 ± 2.6 μM). Also, docking studies were performed to investigate the probable mode of action, which indicated the construction of important H-bonding interactions between the sugar moiety and the enzyme’s active sites. According to the results, hybrids containing heterocycles attached to glucose can be used to inhibit α-glucosidase.

Pterostilbene-Loaded Polydopamine Nanoparticles Down-Regulate Tumor Necrosis Factor-α and Improve Myocardial Function in Mice with Acute Myocardial Infarction

TNF-α expression is related to myocardial function damage and recovery in patients with acute myocardial infarction, but its mechanism is not clear. 50 SD mice, 10 in each group, were in this study divided into TNF-α group, Notch1/eIF3a agonist group, model group, positive control group, and control group. The cardiac function score, myocardial infarction volume, myocardial cell apoptosis index, TNF-α expression, and Notch1/eIF3a pathway factor expression were observed. The size of polydopamine nanoparticles carrying pterostilbene was about 15.5 nm, and cardiac function score, myocardial infarction volume, myocardial cell apoptosis index, and myocardial cell apoptosis number in the model group and Notch1/eIF3a agonist group were higher than model group and Notch1/eIF3a agonist group (P < 0.05). Compared with model group, the Notch1/eIF3a agonist group, TNF-α group, and positive control group showed no differences (P > 0.05). The model group and Notch1/eIF3a agonist group had highest inflammatory response and lowest oxidative stress, which were significantly different from other groups (P < 0.05). The expression of TNF-α in Notch1/eIF3a agonist group, model group, and positive control group all decreased, which was significantly different from other groups (P < 0.05). The expressions of p-IL-6 and p-eIF3a in model group, Notch1/eIF3a agonist group and positive control group were all highest (P < 0.05). Carrying Pterostilbene-loaded polydopamine nanoparticles (PPNs) therefore inhibits apoptosis of cardiomyocytes, Notch1/eIF3a signaling pathway and inflammatory response and oxidative stress of myocardial system, and protects cardiomyocytes of model mice.

Sesquiterpene coumarins with anti-vitiligo and cytotoxic activities from Ferula samarkandica

Two undescribed sesquiterpene coumarins (1–2) and eight known analogues (3–10) were isolated from the roots of Ferula samarkandica Korovin, growing in Uzbekistan. Their structures were established by comprehensive spectroscopic analysis including NMR, HRESIMS, UV and IR data. The absolute configurations were determined based on ECD calculations and confirmed by single-crystal X-ray diffraction analysis. Six compounds carrying 7′-carbonyl, 3′-carbonyl and 3′-acetoxy (4, 5, 6, 7, 9, 10) demonstrated anti-vitiligo potentials, in addition to five (4, 7, 8, 9, 10) with cytotoxic effects. Evaluation of anti-vitiligo activity in B16 cells revealed that compounds 4–7 and 9 exhibited promotion effects on melanin synthesis in B16 cells more potent than the positive control 8-methoxypsoralen (8-MOP), while 5, 7, 9 and 10 could also stimulate the tyrosinase activity superior to 8-MOP. Besides, compounds 4 and 7–10 also presented certain cytotoxic activities against HeLa, HT-29 and A549 cell lines, wherein 8 displayed a significant cytotoxicity against HT-29 cells with an IC50 value of 8.33 ± 0.34 μM.

Sulforaphane decreases oxidative stress and inhibits NLRP3 inflammasome activation in a mouse model of ulcerative colitis

Excessive oxidative stress and NLRP3 inflammasome activation are considered the main drivers of inflammatory bowel disease (IBD), and inhibition of inflammasomes ameliorates clinical symptoms and morphological manifestations of IBD. Herein, we examined the roles of NLRP3 activation in IBD and modulation of NLRP3 by sulforaphane (SFN), a compound with multiple pharmacological activities that is extracted from cruciferous plants. To simulate human IBD, we established a mouse colitis model by administering dextran sodium sulfate in the drinking water. SFN (25, 50 mg·kg−1·d−1, ig) or the positive control sulfasalazine (500 mg/kg, ig) was administered to colitis-affected mice for 7 days. Model mice displayed pathological alterations in colon tissue as well as classic symptoms of colitis beyond substantial tissue inflammation. Expression of NLRP3, ASC, and caspase-1 was significantly elevated in the colonic epithelium. The expression of NLRP3 inflammasomes led to activation of downstream proteins and increases in the cytokines IL-18 and IL-1β. SFN administration either fully or partially reversed these changes, thus restoring IL-18 and IL-1β, substantially inhibiting NLRP3 activation, and decreasing inflammation. SFN alleviated the inflammation induced by LPS and NLRP3 agonists in RAW264.7 cells by decreasing the levels of reactive oxygen species. In summary, our results revealed the pathological roles of oxidative stress and NLRP3 in colitis, and indicated that SFN might serve as a natural NLRP3 inhibitor, thereby providing a new strategy for alternative colitis treatment.

The effect of garlic formulation on some hematological parameters and organ weight of male and female Wister albino rats

The study was conducted to investigate the impact of garlic formulation on certain haematological parameters and organ weights in both male and female rats. Haematological data, including the count of red blood corpuscles (RBC), the volume of packed cells (PCV), the count of white blood corpuscles (WBC), and the count of platelets, were evaluated using a fully automated haematology analyser. The liver, right kidney, left kidney, heart, and spleen of the rats were weighed to determine their respective organ weights. A total of thirty (30) albino Wister rats, consisting of both males and females, were randomly assigned to five groups, with six animals in each group. Group 1 acted as the positive control and was given 100% standard feed. Group II rats were given a mixture of 10% garlic and 90% feed. Group III animals were provided with a diet consisting of 20% garlic and 80% feed. Group IV received a diet containing 40% garlic and 60% feed. The final group, Group V, was fed a diet consisting of 80% garlic and 20% feed daily for a period of 21 days. The feed formulation was assessed at concentrations of 10, 20, 40, and 80 g/dl, based on the weight of the animal. The findings indicated a noteworthy (P<0.05) reduction in white blood cell count (WBC) and lymphocyte count (LYM). There were no statistically significant changes (P>0.05) observed in the levels of haemoglobin (HGB), haematocrit (HCT), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), and mean corpuscular haemoglobin (MCH) when compared to the control group. Furthermore, there was a noteworthy (P<0.05) reduction in HGB, MCV, and MCH when the rats reached a body weight of 80 g/100dl. The results indicated a statistically significant (P<0.05) reduction in the weight of the left kidney, heart, and spleen, as well as in the liver and right kidney weights at 20, 40, and 80 g/dl body weight. There were no statistically significant differences (P>0.05) in the body weight of the liver and right kidney, which were both 10g/dl. Ultimately, the use of garlic formulation in appropriate dosages can impact certain haematological markers and the weights of organs.

Effect of ethanolic stem bark extract of Annona muricata (soursop) on reproductive hormones and ovary in female Wistar rats

The effect of ethanol extract of stem bark of Annona muricata (soursop) on reproductive hormones and ovaries of Wistar rats was studied. The phytochemical analysis revealed the presence of alkaloids, flavonoids, flavones, flavonols, xanthones, flavanonols, flavonones, and condensed tannins. Twenty (20) experimental animals were randomly grouped into five groups of 4 rats per group and were allowed to acclimatize for 2 weeks. Rats in group A served as the control and were fed with rat feed and water, rats in group B severed as positive control and received low dose clomiphene citrate (20 mg/kg) daily for 21 days, rat feed and water, rats in groups C to E were fed with normal rat feed, water and received different doses of fresh stem bark of Annona muricata extract at 250, 500 and 1000 mg/kg respectively for 21 days. The animals were sacrificed under anesthesia after the experiment and the ovaries were surgically extracted, weighed and sent for histology. Blood samples were collected for analysis (hormonal assay of luteinizing hormone (LH), estrogen, follicle stimulating hormone (FSH), progesterone and prolactin. This was carried out using enzyme linked immunosorbent assay (ELISA) technique. The results showed that there were significant changes in the hormone level of the test groups when compared with the control rats. Significant increase was recorded in progesterone, estrogen, prolactin, follicle stimulating hormone and luteinizing hormone p< 0.05. Hence Annona muricata improves fertility in female Wistar rats. There were no histological changes in the ovaries and hence, it was concluded that Annona Muricata does not cause organ damage with increasing dose.

Therapeutic efficacy of candidate antischistosomal drugs in a murine model of schistosomiasis mansoni.

Schistosomiasis is a neglected tropical disease associated with considerable morbidity. Praziquantel (PZQ) is effective against adult schistosomes, yet, it has little effect on juvenile stages, and PZQ resistance is emerging. Adopting the drug repurposing strategy as well as assuming enhancing the efficacy and lessening the doses and side effects, the present study aimed to investigate the in vivo therapeutic efficacy of the widely used antiarrhythmic, amiodarone, and diuretic, spironolactone, and combinations of them compared to PZQ. Mice were infected by Schistosoma mansoni "S. mansoni" cercariae (Egyptian strain), then they were divided into two major groups: Early- [3weeks post-infection (wpi)] and late- [6 wpi] treated. Each group was subdivided into seven subgroups: positive control, PZQ, amiodarone, spironolactone, PZQ combined with amiodarone, PZQ combined with spironolactone, and amiodarone combined with spironolactone-treated groups. Among the early-treated groups, spironolactone had the best therapeutic impact indicated by a 69.4% reduction of total worm burden (TWB), 38.6% and 48.4% reduction of liver and intestine egg load, and a significant reduction of liver granuloma number by 49%. Whereas, among the late-treated groups, amiodarone combined with PZQ was superior to PZQ alone evidenced by 96.1% reduction of TWB with the total disappearance of female and copula in the liver and intestine, 53.1% and 84.9% reduction of liver and intestine egg load, and a significant reduction of liver granuloma number by 67.6%. Comparatively, spironolactone was superior to PZQ and amiodarone in the early treatment phase targeting immature stages, while amiodarone had a more potent effect when combined with PZQ in the late treatment phase targeting mature schistosomes.

Evaluation of anthelmintic efficacy of ethanolic leaf extract of Juglans regia L. on Ascaridia galli: a comprehensive in vitro and in vivo study.

Anthelmintic resistance in livestock animals has been spreading across the world in prevalence and severity. As a result, researchers are exploring alternative strategies to combat this issue, and one promising avenue is the utilization of medicinal plants. This study aims to investigate the anthelmintic efficacy of the crude ethanolic extract (CEE) derived from the leaves of Juglans regia against one of the most detrimental nematode parasites affecting poultry, namely Ascaridia galli (A. galli). For the in vitro studies, adult A. galli worms were collected from the naturally infected chickens and the efficacy of CEE was measured at the concentration of 25, 50, and 100mg/ml using adult worm motility inhibition (WMI) assay. In addition, levamisole (0.55mg/ml) was used as the positive control. Likewise, Phosphate buffered saline (PBS) was used as the negative control. For the in vivo studies, CEE of J.regia at the doses of 500, 1000, and 2000mg/kg were evaluated in chickens experimentally infected with A. galli. The anthelmintic efficacy was monitored using faecal egg count reduction (FECR) and worm count reduction (WCR) assays. In vitro studies revealed significant (P < 0.001) anthelmintic effects of CEE of J.regia on the motility of A. galli worms at different hours post-exposure. At the concentration of 100mg/ml, CEE resulted in 96.5% inhibition of worm motility at 24h post-exposure. While the synthetic anthelmintic drug, levamisole caused the highest inhibition of worm motility (100%) at the same time period. The in vivo anthelmintic activity of CEE of J. regia demonstrated a maximum effect on day 14 post-treatment by inducing 67.28% FECR and 65.03% WCR. We observed no significant difference (P > 0.05) in worm counts between the negative control group and the chickens treated with CEE at the dosage of 500mg/kg. Together, the results of the present study suggest that CEE of J. regia leaves possess anthelmintic properties and could be a potential source of novel anthelmintic compounds for controlling helminth parasites.

CD62-L down-regulation after L18-MDP stimulation as a complementary flow cytometry functional assay for the diagnosis of XIAP deficiency.

X-linked inhibitor of apoptosis (XIAP) deficiency is an infrequent inborn error of immunity caused by mutations in XIAP gene. Most cases present with absence of XIAP protein which can be detected by flow cytometry (FC), representing a rapid diagnostic method. However, since some genetic defects may not preclude protein expression, it is important to include a complementary functional test in the laboratory workup of these patients. L-selectin (CD62-L) is a molecule that is cleaved from the surface membrane of leukocytes upon stimulation of different receptors such as toll like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs), including NOD2. Considering that XIAP deficiency impairs NOD2 signaling, we decided to assess CD62-L down-regulation by FC post-stimulation of neutrophils and monocytes with L18-muramyl Di-Peptide (L18-MDP), a NOD2 specific agonist, in order to develop a novel assay for the functional evaluation of patients with suspicion of XIAP defects. Whole blood samples from 20 healthy controls (HC) and four patients with confirmed molecular diagnosis of XIAP deficiency were stimulated with 200 ng/mL of L18-MDP for 2 h. Stimulation with 100 ng/mL of lipopolysaccharide (LPS) was carried out in parallel as a positive control of CD62-L shedding. CD62-L expression was evaluated by FC using an anti CD62-L- antibody and down-regulation was assessed by calculating the difference in CD62-L expression before and after stimulation, both in terms of percentage of CD62-L expressing cells (Δ%CD62-L) and median fluorescence intensity (ΔMFI%). Neutrophils and monocytes from XIAP deficient patients displayed a significantly diminished response to L18-MDP stimulation compared with HC (p < 0.0001), indicating a severely altered mechanism of CD62-L down-regulation following activation of NOD2-XIAP axis. On the other hand, the response to LPS stimulation was comparable between patients and heathy controls, suggesting preserved CD62-L shedding with a different stimulus. FC detection of CD62-L down-regulation in monocytes and neutrophils after whole blood stimulation with L18-MDP results in an effective and rapid functional test for the identification of XIAP deficient patients.

Microhardness and elemental analysis of ion-releasing restoration/ dentin interface following enzymatic chemomechanical caries excavation

BackgroundThis study was conducted to compare chemical, elemental and surface properties of sound and carious dentin after application of two restorative materials resin-modified glassionomer claimed to be bioactive and glass hybrid restorative material after enzymatic chemomechanical caries removal (CMCR) agent.MethodsForty carious and twenty non-carious human permanent molars were used. Molars were randomly distributed into three main groups: Group 1 (negative control) - sound molars, Group 2 (positive control) - molars were left without caries removal and Group 3 (Test Group) caries excavated with enzymatic based CMCR agent. After caries excavation and restoration application, all specimens were prepared Vickers microhardness test (VHN), for elemental analysis using Energy Dispersive Xray (EDX) mapping and finally chemical analysis using Micro-Raman microscopy.ResultsVickers microhardness values of dentin with the claimed bioactive GIC specimens was statistically higher than with glass hybrid GIC specimens. EDX analysis at the junction estimated: Calcium and Phosphorus of the glass hybrid GIC showed insignificantly higher mean valued than that of the bioactive GIC. Silica and Aluminum mean values at the junction were significantly higher with bioactive GIC specimens than glass hybrid GIC specimen. Micro-raman spectroscopy revealed that bioactive GIC specimens showed higher frequencies of v 1 PO 4, which indicated high level of remineralization.ConclusionsIt was concluded that ion-releasing bioactive resin-based restorative material had increased the microhardness and remineralization rate of carries affected and sound dentin. In addition, enzymatic caries excavation with papain-based CMCR agent has no adverse effect on dentin substrate.

Regulation of carbohydrate metabolism and insulin secretion in diabetic rats following treatment with Hypericum perforatum L. hairy root aqueous methanol extract

Hairy root (HR) cultures of Hypericum perforatum L. have shown promise in controlling hyperglycemia, regulating blood lipid and enzyme profiles, and improving metabolic function in vivo. These benefits are mainly attributed to the phenolic acids, flavonoids, and xanthones present in HR extracts. However, the specific mechanism underlying these effects remains unclear. This study was performed to elucidate the biochemical and molecular mechanisms driving HR the antihyperglycemic and antidiabetic effects of HR extracts. HR extract (200 mg/kg body weight) was administered daily for 14 days to healthy rats and rats with streptozotocin-induced diabetes, with glibenclamide serving as a positive control. The phenolic composition of the HR extracts was confirmed through high-performance liquid chromatography/diode array detection/electrospray ionization mass spectrometry (HPLC/DAD/ESI-MSn ) analysis. The results showed that HR extract treatment increased the plasma insulin level and pancreatic poly (adenosine diphosphate-ribose) polymerase (PARP) activity in diabetic rats, thus normalizing blood glucose levels. Additionally, it reduced the activity of gluconeogenic enzymes, increased the activity of glycolytic enzymes, and normalized the glycogen content in the liver. HR extract-treated rats also exhibited increased hepatic adenosine monophosphate-activated protein kinase (AMPK) mRNA expression and a decreased protein kinase Cε (PKCε) concentration. In conclusion, HR extract demonstrated insulinotropic effects and effectively regulated hepatic carbohydrate metabolism in diabetic rats by modulating AMPK expression and the PKCε concentration. These findings suggest the potential use of HR extract as an herbal medicine for diabetes treatment and a source of antidiabetic drug development.

Identification of DNA Markers from Ant’s Stomach in Zoological Findings

DNA can be extracted from ants and utilised in forensic analyses. Ants can be potential carriers of ambient DNA important to forensic research, as they are common in a variety of locations. In order to assure the validity of results, the study emphasises the significance of positive and negative controls as it examines the difficulties and techniques involved in extracting DNA from ant guts. Overall, the work broadens the scope of ecological and forensic research by highlighting the potential of ant gut DNA as a useful resource in forensic science.

P35 Adaptation of a phenotypic screening assay to incorporate 384-well plates: opening the door to high-throughput screening

Abstract Introduction and aims High-throughput screening has significantly impacted drug discovery by increasing speed of experiments and reducing reagent and sample costs. We have previously developed several screening assays to measure myofibroblast inhibition via quantification of α-smooth muscle actin (α-SMA) expression, in a 96-well format. Currently, there are no drugs for the treatment of hypertrophic scarring, a major side-effect of burn injuries that occurs by myofibroblast transformation, leaving a huge gap in this sector. The aim for this project was to adapt our current assay for use in 384-well microplates. Methods Human primary dermal fibroblasts were transformed to myofibroblasts using 10 ng mL−1 transforming growth factor (TGF)-β1. α-SMA expression was measured using the In-Cell enzyme-linked immunosorbent assay method, with cell viability measured using DRAQ5 nuclear staining. Assay accuracy and reliability was determined by calculating the robust z-factor, with guidelines set to an average score over 0.5, with a minimum score per plate above 0.4. To further validate the assay, concentration-response curves (CRCs) for the drug vehicle [dimethyl sulfoxide (DMSO)] and our tool compound (SB-505124) were constructed at varying concentrations, in coincubation with TGF-β1. Results Optimization of well metrics was performed, producing an average robust z-factor of 0.52, confirming the assay Results are reliable and reproducible. The DMSO CRC produced no significant drop in cell viability at concentrations that are expected to be used in the screening of compounds. Our tool compound (SB-505124) produced a concentration-dependant curve for the inhibition of myofibroblast transformation. Conclusions A 384-well assay has been designed, tested and validated for the determination of myofibroblast transformation in human primary dermal fibroblasts. The assay has been shown to deliver an average robust z-factor of above 0.5, it contains a positive control and is compatible with compounds stored in up to 100% DMSO.

Evaluating acetate as a renoprotective agent following kidney ischemia in a porcine model.

OBJECTIVE Renoprotection from reperfusion injury appears to be conferred by HIF-2A activation, which can be stimulated by exogenous acetate administration. The study objective was to assess whether administration of acetate in a porcine model can mitigate kidney injury related to ischemia-reperfusion following renal hilar occlusion. METHODS A porcine single-kidney model was created by performing a laparoscopic nephrectomy followed by animal recovery. After seven days, the animals underwent laparoscopic hilar dissection. Block randomization was used to assign pigs into one of four experimental groups. One treatment block of pigs received 150 mEq of sodium acetate intravenously during 90 minutes of en bloc occlusion of the renal hilum (herein noted as 'cross clamping'). Another block received 0.75 g/kg of oral sodium acetate for three days prior to cross clamping. A third block received no acetate and underwent hilar dissection without cross clamping (negative control). The final block received no acetate and underwent cross clamping (positive control). Serum creatinine was used to estimate renal function post-nephrectomy. RESULTS A total of 16 animals (4 pigs in each group), completed the study protocol. Median pig weight was 34.6 kg. One pig receiving IV acetate was excluded from the final analysis due to unrecoverable renal failure following cross-clamping. There was a significantly lower mean serum creatinine for the IV acetate group compared to the positive control group 72 hours after cross clamp (p=0.012). The same effect was not observed for the pigs receiving oral acetate. By day 7, renal function recovered without significant difference in all groups. CONCLUSIONS We observed that the administration of intravenous acetate conferred a significant renoprotective benefit in our single kidney ischemia-reperfusion porcine model 72 hours following hilar occlusion. This work is hypothesis-generating and further work in human subjects undergoing renal hilar occlusion during partial nephrectomy is warrante.

Fetal cardiac troponin I levels decline towards birth in sheep.

Elevated cardiac troponin I (cTnI), a myocardial damage biomarker, has been reported in cord blood of neonates delivered vaginally or by cesarean section. While the neonatal peak likely reflects the physiological adjustment to extrauterine life, a better understanding of serial prepartum changes is required to determine physiological causes of fetal cTnI release. METHODS We longitudinally sampled eight healthy lambs (20 days before spontaneous birth to 5 days postnatal), and from three fetuses receiving intravenous IGF-1. Samples were collected into heparin, and the plasma was stored at -80ºC for later determination of high-sensitivity (hs) cTnI levels (BeckmanCoulter UniCel DxI Access IA; log transformed detection limit=0.30, quantification limit=0.78, 99th%ile=1.78). Positive and negative control samples were drawn from an adult ewe during a terminal experiment (myocardial ischemia) and similarly assessed. hs-cTnI data were log transformed from ng/L. RESULTS Log(hs-cTnI) was 1.47±0.30 (mean±SD) at 20 days before birth and declined to 1.02±0.65 in fetuses 12±4 hour before birth (P<0.0001, R2=0.7869). Birth stimulated a delayed, transient peak in hs-cTnI (P=0.0058). Newborn (43±19 min postnatal) levels were 1.39±0.40 (P=0.0650 vs. fetus on day of birth) and 2.14±0.63 the day after birth (P=0.0331 vs. newborn). The second day after birth, levels declined to 1.65±0.48 (P=0.0238 vs. day 1). IGF-1 infusion increased hs-cTnI levels 25-50% over baseline (P=0.0252, R2=0.9938). Baseline adult ewe log(hs-cTnI) was below the limit of detection; three hours following coronary artery ligation, levels were 3.21. CONCLUSIONS We newly report that fetal hs-cTnI levels decline concomitant with reduced proliferation of cardiomyocytes towards term.