Maximum Non-toxic Dose Research Articles

Anti-dengue viral activity of Glycyrrhiza glabra roots in Vero cells

Despite being a global public health problem, there are no antiviral agents for dengue. Plants are the sources of most approved drugs and many phytochemicals have exhibited in vitro antiviral activity. We explored the antiviral potential of the aqueous extract of Glycyrrhiza glabra roots (GGaq) on dengue viruses. Plaque reduction antiviral assay for half maximal inhibitory concentrations (IC50) was done in Vero cells infected with dengue serotypes 1–4 and exposed to varying concentrations of GGaq. Half-maximal cytotoxic concentration (CC50) of the GGaq was 651.9 µg/mL. The IC50 of the four dengue serotypes (10–50 µg/mL) indicated moderate inhibition by GGaq. 98–100% inhibition of all dengue serotypes was seen with GGaq at the maximum nontoxic dose. Preparative thin layer chromatography of GGaq, isolated sub fractions E and F which had low to moderate antiviral activity (IC50 12.65–85.95 and IC50 13.14–69.27 respectively). They exhibited good therapeutic potential only for dengue serotype-4. Virus adsorption to cells was significantly inhibited by GG (50%) and sub fraction E (24.9%). Chloroquine diphosphate was used as the positive control in all assays. The aqueous extract of GG was non-toxic and had better antiviral activity than sub fractions E and F.

Olive Mill Wastewater Extract: In Vitro Genotoxicity/Antigenotoxicity Assessment on HepaRG Cells.

Olive mill wastewater (OMWW), with its high level of phenolic compounds, simultaneously represents a serious environmental challenge and a great resource with potential nutraceutical activities. To increase the knowledge of OMWW's biological effects, with an aim to developing a food supplement, we performed a chemical characterisation of the extract using the Liquid Chromatography-Quadrupole Time-of-flight spectrometry (LC-QTOF) and an in vitro genotoxicity/antigenotoxicity assessment on HepaRG ™ cells. Chemical analysis revealed that the most abundant phenolic compound was hydroxytyrosol. Biological tests showed that the extract was not cytotoxic at the lowest tested concentrations (from 0.25 to 2.5 mg/mL), unlike the highest concentrations (from 5 to 20 mg/mL). Regarding genotoxic activity, when tested at non-cytotoxic concentrations, the extract did not display any effect. Additionally, the lowest tested OMWW concentrations showed antigenotoxic activity (J-shaped dose-response effect) against a known mutagenic substance, reducing the extent of DNA damage in the co-exposure treatment. The antigenotoxic effect was also obtained in the post-exposure procedure, although only at the extract concentrations of 0.015625 and 0.03125 mg/mL. This behaviour was not confirmed in the pre-exposure protocol. In conclusion, the present study established a maximum non-toxic OMWW extract dose for the HepaRG cell model, smoothing the path for future research.

Antiviral Activity of Physta®, Standardised Water Extract of Eurycoma longifolia against SARS-CoV-2: An in vitro Study

Eurycoma longifolia Jack (Simaroubaceae) root extract is known to exhibit antiinflammatory, antiviral and immunomodulatory activities. Therefore, E. longifolia, through a multimodal approach could potentially be used in COVID-19 management. However, to date there has been no investigation into the antiviral activity of E. longifolia root extract against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The objective of the study is to investigate the antiviral activity of Physta® (a standardised water extract of E. longifolia) against SARS-CoV-2. A mitochondrial metabolic activity assay using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was used to determine the cytotoxicity of the Physta® extract in Vero cells, with concentrations ranging from 1.95 µg/mL to 1,000 µg/mL. Physta® was tested for antiviral activity at six different concentrations, ranging from 3.12 µg/mL to 50 µg/mL. The half maximal cytotoxic concentration (CC50) value of Physta® against Vero cells was estimated at 1,117 µg/mL and the maximum non-toxic dose (MNTD) value was estimated at 60 µg/mL. Physta® inhibited SARS-CoV-2 replication in a dose-dependent manner, and the half maximal inhibition concentration (IC50) was estimated to be 36.3 µg/mL. This study has demonstrated the antiviral activity of Physta® against SARS-CoV-2. Future evaluations in animal and clinical settings should be conducted to determine whether Physta® can be used alone or in combination with other antiviral agents to alleviate COVID-19.

Effects of Cannabidiol on the Functions of Chimeric Antigen Receptor T Cells in Hematologic Malignancies.

Introduction: CD19-chimeric antigen receptor (CAR) T cell therapy is a promising immunotherapy for cancer treatment that has shown remarkable clinical responses, leading to approval by the FDA for relapsed and refractory B cell hematological malignancy treatment. Cannabidiol (CBD) is a nonpsychoactive cannabinoid compound that has been utilized as a palliative treatment in cancer patients due to its immunosuppressive properties. Currently, studies on using CBD during immunotherapy have gained increasing attention. However, the possible interaction between CBD and CAR T cell therapy has not been studied. Therefore, in this study, we aimed to examine the direct effects of CBD on CD19-CAR T cell function against hematologic malignancies. Materials and Methods: The cytotoxic effect of CBD was determined by a cell proliferation reagent water-soluble tatrazolium salt (WST-1) assay. CAR T cells were generated by retroviral transduction and treated with CBD at a nontoxic dose. The effect of CBD on immune characteristics, including transgene expression, T cell subset, and memory phenotype, was analyzed by flow cytometry. Proliferation, apoptosis, and cell cycle distribution were analyzed with standard methods. The effect on cytotoxic function was evaluated using degranulation assays, and antitumor activity was evaluated using flow cytometry. Results: The half-maximum inhibitory concentration (IC50) of CBD on NALM6, Raji, and T cells ranged from 16 to 22 μM. The maximum nontoxic dose of CBD that maintained cell viability at ∼100% was 8 μM. For the generation of CD19-CAR T cells, primary T cells were activated and transduced with a retroviral vector encoding CD19-CAR. CBD did not alter the surface expression or immune characteristics, including the T cell subset and memory phenotype, of CD19-CAR T cells. However, CBD suppressed CD19-CAR T cell proliferation by inducing apoptosis, as evidenced by an increase in the proportion of cells in the Sub-G1 phase in cell cycle arrest. However, the antitumor activity and cytokine secretion of CD19-CAR T cells were not altered by exposure to CBD in this study. Conclusions: In this study, a nontoxic dose of CBD affected CD19-CAR T cell proliferation but not its immune characteristics or cytotoxic function.

A newly-synthesized compound CP-07 alleviates microglia-mediated neuroinflammation and ischemic brain injury via inhibiting STAT3 phosphorylation.

Overactivated glial cells, especially microglia, are core components in the progression of pathologic neuroinflammation, and the application of anti-inflammatory reagents has been regarded as a potential therapy in the management of infarction/reperfusion (I/R) brain injury. This research aims to clarify the anti-inflammatory efect of a novel lipophilic compound N-(2-[4-tert-butylphenyl]-2-[pyrrolidine-1-yl]ethyl)-7-methyl-4-oxo-4H-chromene-2-carboxamide (named CP-07 in this study) in LPS-stimulated BV2 cell line and primary mouse microglia, and its therapeutic effect on I/R brain injury. Cell Counting Kit-8 assay was used to determine the maximal nontoxic dose of CP-07. The mRNA levels of representative proinflammatory cytokines were determined by quantitative real-time polymerase chain reaction both in vitro and in vivo. TTC staining was performed to calculate infarct volumes while behavioral tests were used to assess the neurological deficits at 24 h after middle cerebral artery occlusion (MCAO). Flow cytometry analysis and immunofluorescence staining were performed to calculate the percentage of pro-inflammatory microglia in vivo.A selective JAK2/STAT3 pathway inhibitor, AG490 was used to block STAT3 phosphorylation before the CP-07 anti-inflammation tests in vitro. CP-07 could effectively suppress the mRNA levels of IL-6, IL-1β, iNOS and TNF-α induced by lipopolysaccharide (LPS) in vitro, and markedly block the evaluation of the fluorescence intensity of Iba-1 in primary mouse microglia. In middle cerebral arteryocclusion models, intraperitoneal injection with 1 mg/kg CP-07 significantly reduced cerebral infarct volumes at 24 h after surgery compared with vehicle treatment group, and promoted the recovery of neurological functions in MCAO mice. Further studies validated that CP-07 administration reduced the percentage of CD86 positive microglia after I/R injury, and the expression level of p-STAT3 was also markedly reduced in both microglial cells and the penumbra tissues. Blocking STAT3 phosphorylation with AG490 could completely eliminate the anti-inflammatory effects of CP-07, at least in vitro. We showed that a newly synthesized compound, CP-07, could effectively reduce the inflammatory responses in LPS-stimulated BV2 cells and primary mouse microglia, and overproduction of cytokines in middle cerebral artery occlusion mouse models by inhibiting STAT3 phosphorylation, leading to a neuroprotective effect on I/R brain injury.

ANTI-ADIPOGENIC EFFECT OF ELATERIOSPERMUM TAPOS ON 3T3-L1 CELLS

Obesity is defined as an excessive fat accumulation that causes metabolic syndrome such as diabetes, hypertension, and abnormal cholesterol levels. Adipocytes are a major part of the adipose tissue that grows the abnormal lipid droplets which leads to fat accumulation. In modern drugs, treating obesity without any side effects is challenging. Natural products such as plants and herbs are widely used to cure obesity. Natural products are safer because of their reduced toxicity and infrequent negative effects. This study focuses on examining the potential anti-adipogenesis effects of Elateriospermum tapos on 3T3-L1 cells. The extracts from both seed and shell of the fruit were extracted using hot, cold and ethanol extraction. The extracts were tested for the cytotoxicity on 3T3-L1 cells and on zebrafish embryo in vivo. Zebrafish study on heartbeat, heart rate and scoliosis formation, shell extract show less toxic compared to seed extract. This shell extracts showed a positive correlation determination of R2 = 0.96. Therefore, shell extracts were used to indicate the maximum non-toxic dose (MNTD) of each extraction. The lowest MNTD was observed in ethanol extract at 5.2 ± 0.01 µg/mL, hot extract at 7.6 ± 0.25 µg/mL and cold extract at 8.1 ± 0.31 µg/mL. Oil Red O staining to determine the amount of lipid accumulation showed a significant (P < 0.05) decrease of 70% when compared to adipocytes when treated with ethanol shell extract, and a significant (P < 0.05) decrease of 60% by hot shell extract. With the reduction in lipid accumulation, lipolysis was measured by accounting the amount of glycerol produced. The highest production of glycerol was significant when treated with hot shell extract at 42 mg/L, followed by cold extract at 38 mg/L. This is concluded that the hot shell extract has potential to curb obesity by reducing lipid accumulation through lipolysis at a maximum non-toxic dosage.

Anti-Cholinesterase and Anti-α-Amylase Activities and Neuroprotective Effects of Carvacrol and p-Cymene and Their Effects on Hydrogen Peroxide Induced Stress in SH-SY5Y Cells.

Several researchers have demonstrated the health and pharmacological properties of carvacrol and p-cymene, monoterpenes of aromatic plants. This study investigated these compounds' possible anti-cholinesterase, anti-α-amylase, and neuroprotective effects. We evaluated the anti-acetylcholinesterase and anti-α-amylase activities at different concentrations of the compounds. The maximum non-toxic dose of carvacrol and p-cymene against SH-SY5Y neuroblastoma cells was determined using an MTT assay. The neuroprotective effects of the compounds were evaluated on H2O2-induced stress in SH-SY5Y cells, studying the expression of caspase-3 using Western blotting assays. Carvacrol showed inhibitory activities against acetylcholinesterase (IC50 = 3.8 µg/mL) and butyrylcholinesterase (IC50 = 32.7 µg/mL). Instead, the anti-α-amylase activity of carvacrol resulted in an IC50 value of 171.2 μg/mL After a pre-treatment with the maximum non-toxic dose of carvacrol and p-cymene, the expression of caspase-3 was reduced compared to cells treated with H2O2 alone. Carvacrol and p-cymene showed in vitro anti-enzymatic properties, and may act as neuroprotective agents against oxidative stress. Further studies are necessary to elucidate their possible use as coadjutants in preventing and treating AD in diabetic patients.

Luteolin directly binds to KDM4C and attenuates ovarian cancer stemness via epigenetic suppression of PPP2CA/YAP axis

Long-term use of low-toxic natural products holds the promise for eradicating cancer stem cells. In this study, we report that luteolin, a natural flavonoid, attenuates the stemness of ovarian cancer stem cells (OCSCs) by directly binding to KDM4C and epigenetic suppression of PPP2CA/YAP axis. Ovarian cancer stem like cells (OCSLCs) isolated by suspension culture and CD133 + ALDH+ cell sorting was employed as OCSCs model. The maximal non-toxic dose of luteolin suppressed stemness properties, including sphere-forming capacity, the expression of OCSCs markers, sphere-initiating and tumor-initiating capacities, as well as the percentage of CD133 + ALDH+ cells of OCSLCs. Mechanistic study showed that luteolin directly binds to KDM4C, blocks KDM4C-induced histone demethylation of PPP2CA promoter, inhibits PPP2CA transcription and PPP2CA-mediated YAP dephosphorylation, thereby attenuating YAP activity and the stemness of OCSLCs. Furthermore, luteolin sensitized OCSLCs to traditional chemotherapeutic drugs in vitro and in vivo. In summary, our work revealed the direct target of luteolin and the underlying mechanism of the inhibitory effect of luteolin on the stemness of OCSCs. This finding thus suggests a novel therapeutic strategy for eradicating human OCSCs driven by KDM4C.

In Vitro Dengue Virus Inhibition by Aqueous Extracts of Aegle marmelos, Munronia pinnata and Psidium guajava.

Dengue is an arboviral (insect-transmitted) infection of global concern. Currently, there are still no specific dengue antiviral agents to treat the disease. Plant extracts have been used in traditional medicine for treating various viral infections - thus, in the present study, aqueous extracts of dried flowers of Aegle marmelos (AM), whole plant of Munronia pinnata (MP) and leaves of Psidium guajava (PG) were investigated for their potential capacity to inhibit dengue virus infection of Vero cells. The maximum non-toxic dose (MNTD) and the 50% cytotoxic concentration (CC50) were determined by using the MTT assay. A plaque reduction antiviral assay was carried out with dengue virus types 1 (DV1), 2 (DV2), 3 (DV3) and 4 (DV4), in order to calculate the half-maximum inhibitory concentration (IC50). AM extract inhibited all four virus serotypes tested; MP extract inhibited DV1, DV2 and DV4, but not DV3; PG extract inhibited DV1, DV2 and DV4, but not DV3. Thus, the results suggest that AM is a promising candidate for the pan-serotype inhibition of dengue viral activity.

Extraction of Anthocyanins from Black Grape By-Products and Improving Their Stability Using Cobalt(II) Complexation.

This study was conducted to investigate the effect of cobalt complexation on the spectral properties of anthocyanins (AC) extracted from black grape pomace (Black Magic) and the effect of complexation on the pH stability of AC during storage. Initially, cobalt acetate tetrahydrate aqueous solution was complexed with AC crude extract and diluted separately in buffer solutions with different pH (3.5, 4.5, 5.5, and 6.5). Afterward, spectral changes were determined spectrophotometrically. pH stability was investigated using the same buffer solutions and stored for 7 days in the dark at room temperature, and the absorbance of each solution was measured daily using a spectrophotometer. Results indicated that complexation caused similar hypsochromic and hyperchromic shifts in λmax at all pH values. With regard to pH stability, the degradation of complexed AC followed first-order reaction kinetics causing half-lives to increase up to 80-fold as compared with noncomplexed AC, which was due to the sharp decrease in K (per day), indicating an improved pH stability as compared with noncomplexed AC. Therefore, Co(II) could be used in the stabilization of grape AC for the coloration of a wide range of foods and food products at near-neutral pH environments considering the health benefits of grape AC and the maximum nontoxic dose of Co(II) salt.

A Preliminary Study on the Anti-dengue Viral Activity of Munronia pinnata

Dengue infection is a major health care problem in tropical and subtropical countries. The recently approved dengue vaccine has limitations, and there is no antiviral drug for treatment at present.For centuries plants and plant extracts have been used in traditional medicine for the treatment of various infections. The whole plant of Munronia pinnata, which has been used for treating fever patients in Sri Lankan traditional medicine, was tested for anti-dengue viral activity.The cytotoxicity assay of M. pinnata on Vero cells using 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) revealed Maximum Nontoxic Dose (MNTD) as 125 μg/ml and 50% cytotoxic concentration (CC50) as 428.9 ± 21.55 μg/ml. Plaque reduction antiviral assay performed on dengue-4 virus infected Vero cells demonstrated half-maximal inhibitory concentration (IC50) as 26.12 ± 0.91 μg/ml. The selectivity index (SI) of Dengue infection is a major health care problem in tropical and subtropical countries. The recently approved dengue vaccine has limitations, and there is no antiviral drug for treatment at present.For centuries plants and plant extracts have been used in traditional medicine for the treatment of various infections. The whole plant of Munronia pinnata, which has been used for treating fever patients in Sri Lankan traditional medicine, was tested for anti-dengue viral activity.The cytotoxicity assay of M. pinnata on Vero cells using 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) revealed Maximum Nontoxic Dose (MNTD) as 125 μg/ml and 50% cytotoxic concentration (CC50) as 428.9 ± 21.55 μg/ml. Plaque reduction antiviral assay performed on dengue-4 virus infected Vero cells demonstrated half-maximal inhibitory concentration (IC50) as 26.12 ± 0.91 μg/ml. The selectivity index (SI) of M. pinnata was 16.42.Based on the selectivity index, Munronia pinnata appears to be a viable candidate for identifying biologically active compounds with anti-dengue viral activity.Keywords: Dengue, Munronia pinnata, Antiviral, Plaque reduction Assay, Vero Cells was 16.42.Based on the selectivity index, Munronia pinnata appears to be a viable candidate for identifying biologically active compounds with anti-dengue viral activity.
 Keywords: Dengue, Munronia pinnata, Antiviral, Plaque reduction Assay, Vero Cells

Antiamoebic Properties of Metabolites against Naegleria fowleri and Balamuthia mandrillaris.

Naegleria fowleri and Balamuthia mandrillaris are free-living, opportunistic protists, distributed widely in the environment. They are responsible for primary amoebic meningoencephalitis (PAM) and granulomatous amoebic encephalitis (GAE), the fatal central nervous infections with mortality rates exceeding 90%. With the rise of global warming and water shortages resulting in water storage in tanks (where these amoebae may reside), the risk of infection is increasing. Currently, as a result of a lack of awareness, many cases may be misdiagnosed. Furthermore, the high mortality rate indicates the lack of effective drugs available. In this study, secondary metabolites from the plants Rinorea vaundensis and Salvia triloba were tested for their anti-amoebic properties against N. fowleri and B. mandrillaris. Three of the nine compounds showed potent and significant anti-amoebic activities against both N. fowleri and B. mandrillaris: ursolic acid, betulinic acid, and betulin. Additionally, all compounds depicted limited or minimal toxicity to human cells and were capable of reducing amoeba-mediated host cell death. Moreover, the minimum inhibitory concentration required to inhibit 50% of amoebae growth, the half-maximal effective concentration, and the maximum non-toxic dose against human cells of the compounds were determined. These effective plant-derived compounds should be utilized as potential therapies against infections due to free-living amoebae, but future research is needed to realize these expectations.

Anti-neuroinflammatory of Chloroform Extract of Panax ginseng Root Culture on Lipopolysaccharide-stimulated BV2 Microglia Cells.

It is believed that activation of microglia in the central nervous system upon detection of stimulus like lipopolysaccharides provokes neuroinflammation via the production of pro-inflammatory mediators and cytokines. The cytoprotective and anti-inflammatory properties of various folk medicine has been gaining attention as a strategy to combat various disease. This study aimed to assess the anti-neuroinflammatory properties of chloroform extract of in vitro Panax ginseng root culture based on nitric oxide and cytokines production. The study was initiated with the determination of maximum non-toxic dose (MNTD) of P. ginseng root culture chloroform extract using the MTT assay. The lipopolysaccharides-stimulated BV2 microglia cells were treated with MNTD and ½MNTD of the extract and its anti-neuroinflammatory properties were assessed by measuring the production of nitric oxide (NO) via Griess assay, as well as TNF-α, IL-6 and IL-10 using Quantikine ELISA. It was found that the MNTD and ½MNTD of the extract did not play a significant role in the production of pro-inflammatory cytokines such as NO, TNF-α and IL-6. However, the MNTD and ½MNTD of chloroform extract significantly increased the anti-inflammatory IL-10 compared to the untreated cells. With this, the chloroform extract of P. ginseng root culture potentially exerts anti-neuroinflammatory properties.

Chronic Treatment with Metformin Has No Disrupting Effect on the Hepatic Circadian Clock in Mice.

Background and Objectives: The antidiabetic agent metformin is known to activate AMP-activated protein kinase (AMPK) in various tissues. Because AMPK can modulate intracellular circadian clocks through regulating the stability of clock components, a single dose of metformin has been reported to affect circadian clocks in the peripheral tissues. In this study, therefore, we investigated whether chronic treatment with metformin causes the impairment of circadian clocks, especially if given at an inappropriate time. Materials and Methods: Non-diabetic C57BL/6J mice were allowed access to food only during 4 h at the beginning of the dark period, and repeatedly i.p. injected with a nearly maximum non-toxic dose of metformin, once daily either at 4 h after the beginning of the dark period or at the beginning of the light period. Diabetic ob/ob mice were given free access to food and treated with metformin in drinking water. Results: Under the controlled feeding regimen, 8-day treatment with metformin did not alter the mRNA expression rhythms of clock genes in both liver and adipose tissue of C57BL/6J mice, regardless of dosing time. In addition, chronic treatment with metformin for 2 weeks affected hepatic AMPK activation rhythm but did not disrupt the circadian clocks in the liver and adipose tissues of the ob/ob mice. Conclusions: These results mitigate concerns that treatment with metformin impairs peripheral circadian clocks, although confirmation is needed in humans.

Ocimum sanctum: in vitro antiviral potential against animal viruses

Ocimum sanctum, a well and widely used medicinal plant has been reported to be efficient for many ailments quoted in Ayurvedic literature. It has been reported to treat the various ailments due to viral infections in humans like measles, chickenpox, influenza and Smallpox. Besides, it has also shown its potential to give protection in malignancy related disorders. The present study was carried out to screen its potential against animal viruses infecting the dairy animals. Bovine Herpes Virus-type-1 (BHV-1) and Foot and Mouth disease virus (FMDV) affecting cattles and Newcastle Disease Virus (NDV) affecting poultry were screened. Cytopathic inhibition test was used to check the antiviral effect of O. sanctum against BHV-1 and FMDV in MDBK and BHK cell lines, respectively. Chick embryo fibroblasts were cultured to propagate NDV and tested by haemagglutination inhibition test. The maximum non-toxic dose of Ocimum sanctum leaves was determined in MDBK and BHK cell lines as well as in chick fibroblast by MTT assay. Ocimum sanctum nontoxic concentrations, 2.5 mg/mL, 5 mg/mL and 10 mg/mL were found in MDBK cell line, BHK cell line and chick fibroblast culture respectively. Concentrations lower than MNTD were used for studies. 85.3% and 98.4% protection were recorded against BHV-1 and NDV, respectively. However, no significant effect against FMD virus was observed. Thus, it can be effectively utilised in curing these viral diseases in farm animals.

Tannic Acid Exhibits Adjuvant Activity by Enhancing Humoral and Cell-Mediated Immunity Against BSA as a Protein Antigen.

Immunization or vaccination is the process of inducing artificial immunity against an antigen taking advantage of the mechanisms of immunological memory. Current vaccines include substances known as adjuvants, which tend to improve the immunogenicity of the antigen, reduce the antigen quantity employed, and boost the immune response in weak responders. Unfortunately, only a few vaccine adjuvants are approved for human use. Thus, the objective of this study was to investigate the effect of Tannic acid on humoral and cell-mediated immunity against bovine serum albumin (BSA) as a protein antigen in Wistar rats. In order to establish the Tannic acid concentration to test it as an adjuvant, the lethal dose 50 and maximum non-toxic dose were calculated through cytotoxicity and hemolytic assays with J774 A.1 cell line and rat erythrocytes by resazurin reduction method and UV/vis spectrophotometry. Thirty Wistar rats were divided into 5 groups that included two controls without antigen and three treatment groups of adjuvants plus BSA as a protein antigen. The rats were immunized in a 30-day scheme. Blood samples were collected for humoral immunity analysis by means of immunoglobulin quantification, isotyping and antigen-antibody precipitation inhibition analysis. Rat peritoneal macrophages and splenocytes were isolated for cell-mediated immunity analysis by means of nitric oxide quantification from adjuvant stimulated peritoneal macrophages and lymphocytes proliferation assay. Tannic acid was capable of increasing the immunogenicity of the antigen; besides, it was able to stimulate cell-mediated immunity by means of increased lymphocyte proliferation. Moreover, Tannic acid improved the humoral response by means of increased specific antibodies titers. These activities may be attributed to pattern recognition receptors stimulation. Tannic acid was considered biocompatible when tested in vivo because the concentration tested did not show cytotoxicity or hemolytic effect, and there was no detrimental effect observed on the animals' health. These results show Tannic acid as a promising candidate for vaccine adjuvant.

Experimental evaluation of the cytoprotective effect of probiotic metabolites of Bacillus subtilis B-9909 strain on the culture of isolated hepatocytes

The aim of the study was to study cytoprotective effect of BAS (metabolites produced by probiotic microorganisms of the genus Bacillus) of a sterile puffer culture of probiotic microorganisms of the Bacillus subtilis VCPM B-9909 on the culture of isolated hepatocytes when modeling their toxic damage. The preparation of a sterile puffer culture of Bacillus subtilis B-9909 was carried out by sterilizing filtration of the culture fluid of this strain. The work was carried out using a transferable L-41 cell culture line, which allows assessing the toxicity of various substrates on cell cultures and withstanding toxic effects in different concentrations. At the first stage of the research, the maximum non-toxic dose of sterile fugate and the minimum toxic dose of CCl₄ in relation to the cells of the L-41 culture were determined to solve the tasks set. At the next stage of research, cytoprotective effect of BAS, which are part fugate strain Bacillus subtilis B-9909, was studied and proved in relation to the cells of the L-41 line. cytoprotective and regenerative effects of BAS in the composition fugate strain Bacillus subtilis B-9909 were demonstrated on the model of toxic damage to the culture of isolated hepatocytes. In preclinical studies to assess the toxicity and safety of an experimental sample biohepatoprotector on experimental animals, it was found that the complex of biologically active substances (metabolites) administered intragastrically intraperitoneally is non-toxic and safe for laboratory animals and does not cause any pathological changes in their internal organs and tissues. The basis biohepatoprotector, which has a multifunctional mechanism of action that allows to effectively restore depressed liver functions, while simultaneously normalizing immunological parameters, is its active biocomponent — metabolites of probiotic spore-forming bacteria, which, when introduced into the body, produce a complex of biologically active metabolites (antibiotics, proteolytic, amylolytic, etc.enzymes, immunoglobulins, as well as interleukins, vitamins, proteins, amino acids, and others bioactive substances). The experimentally established cytoprotective effect of the complex of BAS, which are part fugate culture of Bacillus subtilis strain B-9909, will allow us to develop new promising medical immunobiological drugs that have a protective effect on human organs and tissues in the future. Thus, as a result of the conducted studies, it was found that the BAS in the composition fugate of the Bacillus subtilis B-9909 strain not only have a pronounced cytoprotective effect, but also have a positive effect on the regenerative abilities of liver cells, which is a significant factor for the further use of this strain biocomponent immunotropic biohepatoprotector.

THE EFFECTS OF ORIENTIN ON THP-1 CELLS INFECTED BY DENV2

Orientin, a flavonoid known for its significant antioxidative properties, has also shown to possess antiviral properties against some viruses. Nevertheless, up to now, there was no report on its ability against dengue virus. This study investigated the effects of orientin against DENV2 infecting human monocyte, THP-1 cells. THP-1 cells infected with DENV2 (3.16×102 TCID50/mL) were treated with a maximum non-toxic dose (MNTD) of orientin (0.053 ± 0.006 µM) for two days. Cytopathic effect (CPE) formation was observed after two days, followed by measurement of the percentage of cell viability using 3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The treatment was compared to cells infected with DENV2 only, cells treated with orientin at MNTD only, and control cells. The MNTD of orientin on THP-1 cells were determined to be 0.053 ± 0.006 µM. Treatment of DENV2 infected THP-1 cells with the MNTD of orientin showed CPE formation similar to the THP-1 cells infected with DENV2 only. The MTT assay showed that orientin did not exhibit antiviral activity against DENV2 infecting THP-1 cells. Percentage of cell viability of the orientin treated cells and DENV2-infected cells were not significantly different. Orientin at MNTD does not exhibit antiviral effects on THP-1 cells infected with DENV2.

In vitro and in silico Anti-dengue activity of Supercritical extract of medicinal plants against Dengue serotype-2

Dengue is transmitted by female Aedes mosquitoes. It has been reported that about 2.5 billion peoples are at the risk of dengue virus. Millions of cases of dengue virus occur worldwide each year. There is no antiviral drug available still. Hence, the researchers are in the search of new anti-dengue drugs from natural products. The present study is aimed to determine the anti-dengue activity of supercritical extracts of Andrographis paniculata, Berberis vulgaris, Carica papaya, Euphorbia hirta, Phyllanthus niruri and Tinospora cordifolia in vitro and in silico. The cell viability was evaluated in C6/36 cells line by using MTT assay using a microplate reader at 595 nm. The maximum non-toxic dose of C. papaya and B. vulgaris extracts were reported as 46.87µg/ml, 31.25µg/ml for A. paniculata, P. niruri and E. hirta and 23.43 µg/ml, for T. cordifolia. Further, the anti-dengue activity of plants extract was analyzed by real-time RT-PCR. 100 copies/ml of DENV-2 virus was used for the antiviral assay. A. paniculata supercritical extract showed complete inhibition against the dengue-2 virus at 40ºC temperature and 15Mpa pressure. The other plant extracts showed the inhibition in order of T. cordifolia (83.44%) > C. papaya (34.71%) > E. hirta (28.70%) whereas P. niruri and B. vulgaris failed to inhibit dengue virus. Andrographolide a major compound present in A. paniculata has been reported to have antiviral activity against hepatitis B, C virus, herpes simplex virus, influenza virus, chikungunya virus, dengue virus 2 and 4. Results of molecular docking showed that the interaction between andrographolide and NS1protein shows the maximum binding energy -7.30 Kcal/mol. The docking study was conducted to validate the result against the anti-dengue activity using dengue NS1 protein with andrographolide. It was concluded that A. paniculata could be a source for isolation of therapeutic compounds against the dengue-2 virus.

Ecofriendly preparation of silver nanoparticles-based nanocomposite stabilized by polysaccharides with antibacterial, antifungal and antiviral activities.

In the present work, sustainable and green method was used to prepare silver nanoparticles (Ag-NPs), followed with incorporation into tertiary nanocomposite consisted of starch, oxidized cellulose and ethyl cellulose. The prepared tertiary silver-nanocomposite (Ag-NC) was fully characterized via instrumental analysis (UV-vis, FT-IR, XRD, SEM, EDX and TEM) and evaluated for antibacterial, antifungal, and antiviral activities. Ag-NC significantly suppressed growth of tested bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis) as compared with controls. Antifungal activity revealed that the prepared tertiary Ag-NC has a promising antifungal activity towards unicellular (Candida albicans) and multicellular fungi ( Aspergillus niger, A. terreus, A. flavus and A. fumigatus). In same line, both Ag-NC and free Ag-NPs have shown a dose-dependent reduction in Vero cell line with maximum non-toxic dose at 6.25 and 12.5 μg/mL, respectively. Both Ag-NPs and Ag-NC exhibited antiviral effects against Herpes simplex virus, Adenovirus and Coxsackie B virus in a dose-dependent manner. Combined treatment of Ag-NPs incorporated into tertiary nanocomposite based on starch, oxidized cellulose and ethyl cellulose opens new possibilities to be more efficient nanomaterials for preventing microbial growth. In conclusion, the prepared tertiary Ag-NC has a promising antibacterial, antifungal as well as antiviral activities.