The Effect of G. applanatum Crude Polysaccharide Extract on Proinflammatory Cytokines and Proapoptotic Caspases in HeLa Cell Line: An In Vitro Study.

Polysaccharide extracts exhibit promise as potential anticancer agents. Among the fungi rich in polysaccharide content, G. applanatum stands out; however, its anticancer activity necessitates further investigation. This study aims to explore the impact of G. applanatum crude polysaccharide (GACP) extract by assessing its effects on cell viability, levels of proinflammatory cytokines such as TNF-α, IFN-γ, IL-2, and IL-12, and levels of proapoptotic markers including caspase-3 and caspase-9, as well as the percentages of necrosis and apoptosis in the HeLa cell line. Employing the HeLa cell line as a research model, four groups were studied: KN (media and DMSO), K+ (doxorubicin 10 µg/mL), P1 (G. applanatum extract 200 µg/mL), and P2 (G. applanatum extract 400 µg/mL). The G. applanatum extract was obtained via boiling distilled water. Anticancer activity was evaluated through the MTT test (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) conducted over three treatment durations (24, 48, and 72 hours). Cytokine levels and caspase-3 and caspase-9 levels were assessed using the ELISA test. Cell apoptosis was determined using the Annexin V-PI biomarker and analyzed through flow cytometry. The MTT test exhibited optimal results at the 48-hour treatment mark. Cytokine level analysis revealed significant reductions in TNF-α, IFN-γ, IL-2, and IL-12 levels (p < 0.005). Concurrently, caspase-3 and caspase-9 levels exhibited substantial increases (p < 0.005). Flow cytometry highlighted the highest percentage of apoptosis in HeLa cells. In conclusion, G. applanatum's polysaccharide extract demonstrates potential as an anticancer and therapeutic agent for cancer treatment.

Effect of crude Ganoderma applanatum polysaccharides as a renoprotective agent against carbon tetrachloride-induced early kidney fibrosis in mice.

Background and Aim: Interstitial fibrosis is the final stage of chronic kidney injury, which begins with an inflammatory process. Crude Ganoderma applanatum polysaccharides are known to have anti-inflammatory properties. The potential role of crude G. applanatum polysaccharides in renal fibrosis through pro-inflammatory cytokines needs further investigation. This study aimed to determine the renoprotective effect of crude G. applanatum polysaccharide extract in mice with carbon tetrachloride (CCL4)-induced early kidney fibrosis. Materials and Methods: This study was conducted for 4 weeks using 24 male BALB/c mice selected for their metabolic stability. The mice were randomly divided into six groups, including control (CG), model (MG), silymarin group and crude G. applanatum polysaccharide extract groups comprising doses of 25, 50, and 100 mg/kg body weight. After sacrificing the mice, whole blood was analyzed for urea and creatine levels, and kidney tissue was prepared to assess tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), hyaluronic acid (HA), and laminin levels, both using enzyme-linked immunosorbent assay. Kidney histology was determined using hematoxylin and eosin staining, while the extracellular matrix (ECM) components were stained using Masson's trichome staining. The α-smooth muscle actin (α-SMA) concentration was determined using immunohistochemistry. These parameters were measured to determine the effectiveness of the crude G. applanatum polysaccharide extract in preventing interstitial fibrosis. Results: Administration of crude G. applanatum polysaccharides effectively prevented increases in kidney weight and physiological enzymes, pro-inflammatory cytokines, and ECM production compared with those in the MG, as evidenced by the low levels of urea, creatinine, TNF-α, IL-6, HA, and laminin. Histopathological results also showed that crude G. applanatum polysaccharides prevented the occurrence of inflammatory infiltration, desquamated nuclei, cytoplasm debris, rupture at the brush border, dilatation of the glomeruli space and lumen of the proximal tubule, and necrotic cells compared with the MG. Masson's trichrome staining revealed lower collagen levels in the interstitial tubules of kidney tissue than those in the MG. Immunohistochemical analysis revealed low α-SMA expression in the crude G. applanatum polysaccharides treatment groups than that in the MG. Conclusion: The crude polysaccharide extract of G. applanatum has a protective effect that prevents the progression of kidney fibrosis in mice.

Effect of the Ethanol Extract of Red Okra Pods (Abelmoschus esculentus (L.) Moench) to Inhibit Cervical Cancer Cells Growth through Cell Cycle-Associated Oncogenes.

This study aims to evaluate the potency of ethanol extract of red okra pods (EEROP) in inhibiting growth of cervical cancer cells through repression of the cell cycle-associated oncogenes. The EEROP treatment was given to HeLa cells cultured with RPMI medium and incubated at 37°C with 5% CO2. The MTT method was used to measure HeLa cell growth and IC50 values. The mRNA levels of the three cell cycle-associated oncogenes (MYC, TYMS, and MDM2) were evaluated by qRT-PCR to determine the effect of EEROP treatment on the cell cycle. The lowest percentage of viable cells at 24, 48, and 72 hours after EEROP treatment was in the dose of 1000 µg/mL with a growth percentage of 71.60% at 24 hours, 55.61% at 48 hours, and 46.97% at 72 hours. The IC50 values were 2845, 1153, and 776.8 µg/mL for 24, 48, and 72 hours, respectively. The three oncogenes at a dose of 1000 µg/mL significantly decreased the lowest mRNA levels compared to other doses with MYC oncogene that experienced the greatest decrease. The mRNA level of dose 1000 µg/mL EEROP at the MYC oncogene was 0.014-fold changes, at the TYMS oncogene was 0.097-fold changes, and at the MDM2 oncogene was 0.028-fold changes. The EEROP has been shown to decrease the expression of three cell cycle-associated oncogenes. This is also supported by the growth of HeLa cells that did not increase throughout 24, 48, and 72 hours. However, further research is needed on the main active components in red okra that function as anticancer, so that in the future, okra can not only stop cancer cell growth but also induce cancer cell death.

Dual role of immunomodulation by crude polysaccharide from okra against carcinogenic liver injury in mice.

Polysaccharides have long been recognized as the anticancer agent with low toxicity and slight side effects. Okra (Abelmoschus esculentus L.), a flowering plant from the Malvaceae family that is found in tropical, subtropical, and warm temperate regions around the world. Hence, no in vivo studies have addressed the anticancer and immunomodulatory potential of polysaccharides from okra pods grown in Indonesia. This study aims to investigate the effect of okra raw polysaccharide extract (ORPE) to immune cells and cytokines of mice with hepatocarcinogenic conditions induced by diethylnitrosamine (DEN). Thirty-six male mice (BALB/C, 3-4 months old) were divided into six groups: the normal control group (CN), negative control (C-), positive control giving doxorubicin (C+), and three groups of ORPE treatment given the dose of 50 (P1), 100 (P2) and 200 (P3) mg/kg body weight. The administration of ORPE directly suppressed the regulatory T cells accumulations, suppressed macrophages activations, and balanced the number of effector T cells. However, it promoted CD8+ T cell activation at a low dose and increased interleukin-2 levels at all doses. These results suggest that ORPE has unique dual-functions as immunosuppressant and immunostimulant which can be a foundation for the application of the ORPE in the nutraceutical and pharmaceutical industries.

Utilization of bull fertility-associated antigen to improve the quality of frozen bull semen.

BACKGROUND AND AIM: The implementation of artificial insemination (AI) is one of the strategies to use superior male semen optimally to improve the genetic quality of livestock. One of the factors that influence AI is a fertility-associated antigen (FAA). This research aimed to examine the effects of FAA extracted from the accessory sex glands of a bull from a slaughterhouse that was added in bull semen freezing medium to increase cattle (bull) fertilization. MATERIALS AND METHODS: This research used a randomized complete block design. It consisted of two research phases, namely, explorative and experimental phases. The first phase involved determining the FAA molecular weight using the SDS-PAGE method, and the second phase consisted of laboratory and field testing, including testing the quality of frozen semen supplemented with FAA extracted from the accessory glands of a bull's genital organ from a slaughterhouse with various doses (0, 5, 10, and 15 µg in every 200 million progressively motile spermatozoa). RESULTS: The results showed that the percentages of bull sperm motility between the groups without and with the additional administration of FAA with a dose of 5 µg did not significantly differ. However, there was a difference between the groups without and with the additional administration of FAA with doses of 10 and 15 µg. After further testing, the highest percentage of sperm progressive motility occurred at a dose of 15 µg/200 million progressively motile spermatozoa (P3), which was equal to 2.59±46.88b (%). CONCLUSION: This research found that not all of the accessory glands (seminal vesicles) of bulls taken from the slaughterhouse contain the FAA. An FAA level between the accessory glands (seminal vesicles) of one cattle to another is different. The addition of the FAA protein from the accessory sex glands of a bull's organ in cattle semen can improve fertility by increasing the percentage of viability, motility, intact plasma membrane of spermatozoa, and pregnancy rate of bulls and decreasing the sperm capacitation post-thawing.

Fe/Ni Bimetallic Organic Framework Deposited on TiO2 Nanotube Array for Enhancing Higher and Stable Photoelectrochemical Activity of Oxygen Evaluation Reaction.

Photoelectrochemical (PEC) water splitting is a promising strategy to improve the efficiency of oxygen evolution reactions (OERs). However, the efficient adsorption of visible light as well as long-term stability of light-harvesting electrocatalysis is the crucial issue in PEC cells. Metal-organic framework (MOF)-derived bimetallic electrocatalysis with its superior performance has wide application prospects in OER and PEC applications. Herein, we have fabricated a nickel and iron bimetallic organic framework (FeNi-MOF) deposited on top of anodized TiO2 nanotube arrays (TNTA) for PEC and OER applications. The FeNi-MOF/TNTA was incorporated through the electrochemical deposition of Ni2+ and Fe3+ onto the surface of TNTA and then connected with organic ligands by the hydrothermal transformation. Therefore, FeNi-MOF/TNTA demonstrates abundant photoelectrocatalytic active sites that can enhance the photocurrent up to 1.91 mA/cm2 under 100 mW/cm2 and a negligible loss in activity after 180 min of photoreaction. The FeNi-MOF-doped photoanode shows predominant photoelectrochemical performance due to the boosted excellent light-harvesting ability, rapid photoresponse, and stimulated interfacial energy of charge separation under the UV-visible light irradiation conditions. The results of this study give deep insight into MOF-derived bimetallic nanomaterial synthesis for photoelectrochemical OER and provide guidance on future electrocatalysis design.

Ultrasensitive Detection of Tetracycline Using Boron and Nitrogen Co-Doped Graphene Quantum Dots from Natural Carbon Source as the Paper-Based Nanosensing Probe in Difference Matrices.

Herein, the boron and nitrogen co-doped 0-dimensional graphene quantum dots (B,N-GQDs) with high quantum yield (QY) were synthesized via microwave-assisted hydrothermal method at 170 °C for 20 min using fresh passion fruit juice and boric acid as the starting materials. The 3-6 layers of B,N-GQDs with mean particle size of 9 ± 1 nm were then used for ultra-sensitive and selective detection of tetracycline in aqueous and biological media. The hybridization of boron and nitrogen atoms into the GQD structures increases the intensity of electronegative, resulting in the enhancement of QY to 50 ± 1%. The B,N-GQDs show their excellent analytical performance on tetracycline determination after 2 min of reaction under an optimal condition at pH 5. The linear range of 0.04-70 µM and with limits of detection (LOD) of 1 nM in phosphate buffer saline (PBS), 1.9 nM in urine and 2.2 nM in human serum are obtained. Moreover, the high selectivity of tetracycline by B,N-GQDs over the other 23 interferences is observed. The π-π interaction and electron donor-acceptor principle play pivotal roles in enhancing the ultra-sensitivity and selectivity of B,N-GQDs toward TC detection. Moreover, the B, N-GQD based paper nanosensor exhibits an excellent analytical performance on visual detection of 0.1-30 µM TC in human serum. Results of this study clearly indicate the feasibility of synthesis of B,N-GQDs derived from passion fruit juice for ultrasensitive tetracycline detection, which can open an avenue to use natural products for the preparation of environmentally benign and biocompatible carbon nanomaterials for highly sensitive detection of drugs, antibiotics, organic compounds and biomarkers.

Mouse (Mus Musculus) Embryonic Cerebral Cortex Cell Death Caused by Carbofuran Insecticide Exposure.

INTRODUCTION: The aim of the study was to describe the process of neuron death in the cerebral cortex caused by embryonic carbofuran exposure. MATERIAL AND METHODS: 81 mouse foetuses from 27 breeding mice were used in the study. Carbofuran was administered by gavage from the 6th to the 15th day of gestation to two groups: one at 0.0208 and the other at 0.0417 mg/kg b.w. On the 17th day, the mice were sacrificed and the foetuses were taken to measure the ROS (malondialdehyde/MDA and superoxide dismutase/SOD) activity in brain tissue, the number of apoptotic embryonic cerebral cortex neurons using a TUNEL assay, and necrotic cells using HE staining. Examination of p53 and caspase 3 expression was done by immunohistochemistry. Data were analysed using analysis of variance (ANOVA) and Duncan's test. RESULTS: Increased activity of cerebral ROS characterised by significant elevation of the MDA level (P < 0.05), decreased SOD (P < 0.01), increased p53 and caspase 3 expression, and cerebral cortical neuron death either by necrosis or apoptosis (P < 0.05) were found. At the low dose carbofuran increased expression of p53, caspase 3, and apoptosis. At the high dose it increased levels of MDA and necrosis. CONCLUSION: Increased expression of p53 and caspase 3 and apoptosis indicated that carbofuran may cause apoptosis through the intrinsic pathway. The increased apoptosis grants an opportunity to prevent and treat the effect of ROS due to gestational carbofuran exposure.

Anticancer Activity of Methanol Extract of Ficus carica Leaves and Fruits Against Proliferation, Apoptosis, and Necrosis in Huh7it Cells.

The polyphenol plant extracts have previously been demonstrated to act as chemopreventive and anticancer agents. Ficus carica is a rich source of polyphenols, yet its antioxidant and anticancer activities remain poorly characterized. This study aimed to determine the anticancer activity of F carica leaf and fruit extracts by investigating their impact on proliferation, apoptosis, and Huh7it cell necrosis. Leaves and fruits were extracted using methanol, and the phytochemical contents were analyzed using Fourier-transform infrared spectroscopy. The antioxidant activity was measured using the 2,2-diphenyl-1-picrylhydrazyl method. Anticancer activities were examined through MTT (3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay on Huh7it liver cancer cells. The apoptosis and necrosis conditions were examined using Annexin biomarkers V-PI and later analyzed in flow cytometry. F carica leaves and fruit examined were found to have strong antioxidant activities with IC50 values of 7.9875 µg/mL and 13.402 µg/mL, respectively. MTT assay results indicated F carica leaves and fruit had IC50 values >653 µg/mL and >2000 µg/mL, respectively. The flow cytometry analysis indicated a higher percentage of Huh7it apoptosis and necrosis in leaf extracts compared with fruit extracts. The difference in anticancer activity was attributed to differing compounds present in each extract.

Hepatoprotective effect of crude polysaccharides extracted from Ganoderma lucidum against carbon tetrachloride-induced liver injury in mice.

BACKGROUND AND AIM: Natural products are currently widely used as alternative treatments for liver disease. The study aimed to determine the hepatoprotective effect of crude polysaccharides extracted from Ganoderma lucidum against liver injury induced by carbon tetrachloride (CCl4). MATERIALS AND METHODS: Twenty-four male BALB/C mice were randomly divided into six groups. Serum and liver samples were taken on day 10 after G. lucidum administration. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were measured using enzyme-linked immunosorbent assays, and the histology of the liver was evaluated using light microscopy. RESULTS: G. lucidum extract significantly decreased the levels of ALT, AST, and MDA and significantly increased the levels of SOD and CAT. In the histological evaluation, the liver tissue of CCl4-treated mice exhibited hydropic degeneration, necrosis, and sinusoidal dilatation. G. lucidum extract administration improved this liver tissue histopathology. CONCLUSION: Crude polysaccharides extracted from G. lucidum showed a hepatoprotective effect, regenerating damaged liver tissue.

Crude Polysaccharides from Okra Pods (Abelmoschus esculentus) Grown in Indonesia Enhance the Immune Response due to Bacterial Infection.

Okra pods were widely consumed by Indonesians to maintain health. The aim of this study was at investigating the potential of crude polysaccharides from okra pods on immune response in mice infected with Staphylococcus aureus. Thirty male Balb/C mice were divided into six groups: normal control, negative control, and treatment groups (administration of crude polysaccharides at doses of 25, 50, 75, and 100 mg/kg). Crude polysaccharides were administrated for fourteen days. Furthermore, mice were exposed to S. aureus at the fifteenth day. Two weeks after the end of treatment, the parameters were measured. This study showed that crude polysaccharides at a dose of 75 and 100 mg/kg improved phagocytic activity, spleen index, and splenocytes proliferation. Rising of TNF-α levels was shown in groups treated with crude polysaccharides at doses of 25, 50, and 100 mg/kg. All treatment groups showed a decreasing level of IL-17. Crude okra polysaccharides also showed a slight increase in NK cells activity and IFN-γ level. Thus, crude okra polysaccharides could act as an effective material to enhance immune response including phagocytic activity, spleen index, splenocytes proliferation, and control immune responses through cytokine production.

Western Blot Analysis and Immunostaining for Prediction of Embryotoxicity in Mus musculus.

A broad range of research must be answered in order to gain a complete understanding of the histological and histochemical profile of teratological exposure in Mus musculus. Continued research is needed to track patterns of teratogen effects on the DNA expression of the embryonic brain and its variation impact. An important technique used in cell and molecular biology is Western blotting. By using a Western blot analysis and immunostaining, researchers are able to predict embryotoxicity in Mus musculus. The method uses three elements to accomplish this task: (1) Nonspecific antibody binding to a nitrocellulose membrane, (2) an incubation using a primary antibody, and (3) the antigen-antibody reaction using a secondary antibody. The proteins are further stained with a substrate/chromogen. In this chapter, the electrophoresis-based protein detection following mice embryonic exposure to a teratogen, 2-methoxyethanol, is described.

Quantitative Structure-Cytotoxic Activity Relationship 1-(Benzoyloxy)urea and Its Derivative.

Drug development is originally carried out on a trial and error basis and it is cost-prohibitive. To minimize the trial and error risks, drug design is needed. One of the compound development processes to get a new drug is by designing a structure modification of the mother compound whose activities are recognized. A substitution of the mother compounds alters the physicochemical properties: lipophilic, electronic and steric properties. In Indonesia, one of medical treatments to cure cancer is through chemotherapy and hydroxyurea. Some derivatives, phenylthiourea, phenylurea, benzoylurea, thiourea and benzoylphenylurea, have been found to be anticancer drug candidates. To predict the activity of the drug compound before it is synthesized, the in-silico test is required. From the test, Rerank Score which is the energy of interaction between the receptor and the ligand molecule is then obtained. Hydroxyurea derivatives were synthesized by modifying Schotten-Baumann's method by the addition of benzoyl group and its homologs resulted in the increase of lipophilic, electronic and steric properties, and cytotoxic activity. Synthesized compounds were 1-(benzoyloxy)urea and its derivatives. Structure characterization was obtained by the spectrum of UV, IR, H NMR, C NMR and Mass Spectrometer. Anticancer activity was carried out using MTT method on HeLa cells. The Quantitative Structure-Cytotoxic Activity Relationships of 1-(benzoyloxy)urea compound and its derivatives was calculated using SPSS. The chemical structure was described, namely: ClogP, π, σ, RS, CMR and Es; while, the cytotoxic activity was indicated by log (1 / IC50). The results show that the best equation of Quantitative Structure-Cytotoxic Activity Relationships (QSAR) of 1- (benzoyloxy)urea compound and its derivatives is Log 1/IC50 = - 0.205 (+ 0.068) σ - 0.051 (+ 0.022) Es - 1.911 (+ 0.020).

Expression of vimentin and gfap protein of cerebral cortex and its impact on corticogenesis disorder as a result of 2-methoxyethanol / Expresión de vimentina y proteína gfap de la corteza cerebral y su impacto en el trastorno de corticogenesis como producto del 2-metoxietanol

One of the plastic base material, widely used in the plastics industry in various countries, is a ester phthalate. These compounds will be oxidizedin the body to 2-methoxyethanol (2-ME). Effect of 2-ME on human health and the environment depends on the number, duration and frequency of exposure. 2-ME and its metabolites in the body can damage cells and tissues. The body can be exposed by 2-ME through the air, water and soil. Western blot results showed that the protein Vimentin was detectable in the control group at GD-11 to 17, meanwhile GFAP protein was detachable in the control group atGD- 12 to GD-18. After administration 2-ME, the expression of Vimentinprotein were changed, and started at GD- 12 up to GD-18. whereas the expression of GFAP protein began at GD-11 up to GD-17. The Changes on timetable protein expression of Vimentin and GFAP affect corticogenesis disorder. The disorder caused by the existence of these proteins as a result of 2-Methoxyethanol. Disorder of corticogenesis process were sub-plate and cortical plate of the cerebral cortex of fetus brains of mice at GD-18. Generally, it can be concluded that changes inprotein expression of Vimentin and GFAP causedby 2-ME. The Vimentin more important during the period of fetal brain development. GFAP and Vimentin is a protein involved in response to damage caused by a teratogenic agent, so that cells in the cerebral cortex, has dedifferentiation.

Uno de los materiales a base de plástico, ampliamente utilizado en la industria en varios países, es un éster de ftalato. Estos compuestos se oxidan en el cuerpo a 2-metoxietanol (2-ME). El efecto del 2-ME en la salud humana y el medio ambiente depende de la cantidad, duración y frecuencia de exposición. El 2-ME y sus metabolitos en el cuerpo puede dañar las células y tejidos. El cuerpo puede ser expuesto al 2-ME a través del aire, agua y suelo. Los resultados de Western blot mostraron que la proteína vimentina fue detectable en el grupo de control en GD-11 a 17, por su parte proteína GFAP fue detectable en el grupo de control en GD-12 a GD-18. Después de la administración de 2-ME, la expresión de la proteína vimentina cambió, y comenzó a detectarse en GD-12 hasta GD-18, mientras que la expresión de la proteína GFAP se inició en GD-11 hasta GD-17. Los cambios en el momento de expresión de las proteínas vimentina y GFAP afectan produciendo trastornos de la corticogénesis. El trastorno causado por la existencia de estas proteínas como resultado de 2-metoxietanol a nivel del proceso corticogénesis fue en la subplaca y la placa cortical de la corteza cerebral del cerebro de fetos de ratones en GD-18. En general, se puede concluir que existen cambios en la expresión de las proteínas vimentina y GFAP causados por el 2-ME. La vimentina es muy importante durante el período de desarrollo del cerebro fetal. GFAP y vimentina son proteínas implicadas en la respuesta a los daños causados por un agente teratogénico, de modo que las células en la corteza cerebral presentan desdiferenciación.