The Insight of In Silico and In Vitro evaluation of Beta vulgaris phytochemicals against Alzheimer's disease targeting acetylcholinesterase.

B. vulgaris extracts possess antioxidant, anti-inflammatory along with its role in improving memory disorders. Subsequently, in vitro and in silico studies of its purified phytochemicals may expand complementary and alternative Alzheimer's therapeutic option. Super activation of acetylcholinesterase enzyme is associated explicitly with Alzheimer's disease (AD) ultimately resulting in senile dementia. Hence, acetylcholinesterase enzyme inhibition is employed as a promising approach for AD treatment. Many FDA approved drugs are unable to cure the disease progression completely. The Present study was devised to explore the potential bioactive phytochemicals of B. vulgaris as alternative therapeutic agents against AD by conducting in vitro and in silico studies. To achieve this, chemical structures of phytochemicals were recruited from PubChem. Further, these compounds were analyzed for their binding affinities towards acetylcholinesterase (AChE) enzyme. Pharmacophoric ligand-based models showed major characteristics like, HBA, HBD, hydrophobicity, aromaticity and positively ionizable surface morphology for receptor binding. Virtual screening identified three hit compounds including betanin, myricetin and folic acid with least binding score compared to the reference drug, donepezil (-17 kcal/mol). Further, in vitro studies for anti-acetylcholinesterase activity of betanin and glycine betaine were performed. Dose response analysis showed 1.271 µM and 1.203 µM 50% inhibitory concentration (IC50) values for betanin and glycine betaine compounds respectively. Our findings indicate that phytoconstituents of B. vulgaris can be implicated as an alternative therapeutic drug candidate for cognitive disorders like Alzheimer's disease.

Phytochemical Screening and Protective Effects of Prunus persica Seeds Extract on Carbon Tetrachloride-Induced Hepatic Injury in Rats.

BACKGROUND AND PURPOSE: Carbon tetrachloride (CCl4) is a dynamic environmental toxin released from chemical factories and its concentration in the atmosphere is accelerating at an alarming proportion. The potential presence of CCl4 in the human body causes liver injury via free radical stimulated inflammatory responses. OBJECTIVES: In this study, protective effects of hydromethanolic seeds extract of Prunus persica (PPHM) were evaluated for free radical scavenging potential in CCl4 mediated acute liver toxicity in the murine model. EXPERIMENTAL APPROACH: Followed by acute oral toxicity analysis, liver cells of Sprague-Dawley (SD) rats were treated with CCl4 and subsequently, the chemoprophylactic effect of extract (400 mg/Kg dose) was evaluated using in vivo studies including, silymarin as the positive control. Biochemical parameters, staining (hematoxylin and eosin (H & E) and Masson's Trichome) and quantitative gene expression analysis via real-time PCR were used to evaluate hepatic damage control. RESULTS: The results illustrated that PPHM extract exhibit strong anti-oxidant activity, comparable to the positive control, gallic acid. Research study results also demonstrated that the extract treatment at 400 mg/Kg concentration is highly effective in protecting liver damage due to CCl4 exposure. Mechanistic investigations indicated that the therapeutic action of PPHM was correlated with the increase in Nrf2, NQO-1 and decrease in collagen III mRNA genes expression compared to CCl4 treated group. CONCLUSIONS AND IMPLICATIONS: Accordingly, our research study indicated that PPHM alleviated CCl4-mediated oxidative stress through Nrf2/NQO-1 pathway, thereby protecting liver damage against environmental toxins. Our findings provide supportive evidence to suggest PPHM as a novel nontoxic hepatoprotective agent.

Tectona grandis leaf extract ameliorates hepatic fibrosis: Modulation of TGF- ß /Smad signaling pathway and upregulating MMP3/TIMP1 ratio.

ETHNOBOTANICAL RELEVANCE: Tectona grandis L.f (or syn: Jatus grandis (L.f.) Kuntze Revis), from family Lamiaceae, also known as Teak, is widely recognized in ayurvedic system of medicine and confer curative potential against inflammation, liver disorders, biliousness, diabetes, bronchitis, leprosy and dysentery. Its leaves are rich source of edible food colorant and reported nontoxic for liver and various organs. AIM OF STUDY: Hepatic injury progression to liver cirrhosis and cancer is a serious health issue across the world. Currently, anti-fibrotic therapeutic options are limited and expensive with no FDA approved direct anti-hepato-fibrotic drug validated in clinic. Thus, the aim of this study was to understand ameliorative effect of Tectona grandis L.f, leaves in early liver fibrosis. METHOD AND RESULTS: C57BL/6 mice suffering from CCl4 induced liver injury, were orally administered at three different doses (50, 100 & 200 mg/kg) of Tectona grandis L.f, leaf extract, thrice a week, up to 4 and 8 weeks. Anti-fibrotic effect was evaluated through animal body/liver weight measurements, serological tests (AST, ALT, GSH, MDA and LDH assays), tissue hydroxyproline content, and histochemical analysis (H&E, Masson trichrome, Sirius red and αSMA localization). Moreover, transcriptional and post-transcriptional expression of fibrosis associated biomarkers and TGF-ß/Smad cascade were analyzed. It was observed that 100 mg/kg dose optimally downregulated TGF-ß1/Smad2 with upregulation of Smad7 and regulated αSMA, Col 1, PDGF, TIMP1 and MMP3 expression, post 8 weeks of treatment. In addition, MMP3/TIMP1 ratio was upregulated to 0.7, 2.5 and 1.7 fold at 50 mg/kg, 100 mg/kg & 200 mg/kg treatments respectively, in comparison to untreated liver fibrosis models. The extract contains gallic acid, caffeic acid, sinapinic acid and myricetin when analyzed through high performance liquid chromatography. CONCLUSION: Tectona grandis L.f, leaves have potential to ameliorate liver fibrosis induced by CCl4 in mice via modulation of TGF-ß1/Smad pathway and upregulated MMP3/TIMP1 ratio.

Phytochemical analysis of Berberis lyceum methanolic extract and its antiviral activity through the restoration of MAPK signaling pathway modulated by HCV NS5A

Objective: To evaluate the antiviral activity and phytochemicals of selected plant extracts and their effect on the mitogen-activated protein kinase (MAPK) signaling pathway modulated by hepatitis C virus (HCV) nonstructural protein 5A (NS5A). Methods: A total of ten plant extracts were initially screened for their toxicities against HepG2 cells. The non-toxic plants were tested for their inhibitory effect on the expression of HCV NS5A at both mRNA and protein levels using real-time PCR and Western blotting assays, respectively. The differential expression of the genes associated with MAPK pathway in the presence of NS5A gene and plant extract was measured through real-time PCR. Subsequently, the identification of secondary metabolites was carried out by phytochemical and HPLC analysis. Results: The phytochemical profiling of Berberis lyceum revealed the presence of alkaloids, phenols, saponins, tannins, flavonoids, carbohydrates, terpenoids, steroids, and glycosides. Similarly, quercetin, myricetin, gallic acid, caffeic acid, and ferulic acid were identified through HPLC analysis. The methanolic extract of Berberis lyceum strongly inhibited HCV RNA replication with an IC50 of 11.44 μg/mL. RT-PCR and Western blotting assays showed that the extract reduced the expression of HCV NS5A in a dose-dependent manner. Berberis lyceum extract also attenuated NS5A-induced dysregulation of the MAPK signaling pathway. Conclusions: Our findings suggest that Berberis lyceum extract strongly inhibits HCV propagation by reducing HCV NS5A-induced perturbation of MAPK signaling.

Anti-hepatitis C virus activity and synergistic effect of Nymphaea alba extracts and bioactive constituents in liver infected cells.

BACKGROUND: Without an effective vaccine, hepatitis C virus (HCV) remains a global threat, inflicting 170-300 million carriers worldwide at risk of cirrhosis and hepatocellular carcinoma (HCC). Though various direct acting antivirals have been redeemed the hepatitis C treatment, a few restraints persist including possible side effects, viral resistance emergence, excessive cost which restricts its availability to a common person. HYPOTHESIS: There is no preventive HCV vaccine available today so the discovery of potent antiviral natural flora and their bioactive constituents may help to develop preventive cures against HCV infection. STUDY DESIGN: In current study, we aim to clarify anti-HCV activity of methanol and acetone extracts along with the purified fractions of Pakistani local plant, Nymphaea alba L (N. alba) using Huh-7 cell line as transfection model. Synergistic study of purified fractions with interferon was performed using MDBK cell line (expressing interferon receptors) as transfection model. MATERIALS AND METHODS: Recent study by our research group has observed potent anti-HCV NS3 protease activity of methanol and acetone extracts of N. alba. Effect of N. alba extracts, its fractions precisely, the N1 and N8 fractions on HCV replication was demonstrated by analyzing viral gene expression using in vitro transfection model. Considering NS3 protease as a dynamic drug target, fourteen phytochemicals of N. alba were selected as ligands for interaction with NS3 protein using Molecular Operating Environment (MOE) software. Boceprevir, FDA approved NS3 protease inhibitor, was used as standard for comparative study in docking screening. RESULTS: Herein we report 84% and 94% reduction of 3a genotype of HCV NS3/4A gene expression at mRNA level at non-toxic concentration. Specifically, two fractions 'N1' & 'N8' isolated from acetone extract suppressed HCV NS3 gene expression in transfected target cells with an EC50 value of 37 ±â€¯0.03 µg/ml and 20 ±â€¯0.02 µg/ml respectively. Similarly, viral genotype 1a replication is strongly suppressed in target cells by N. alba flower extracts and purified fractions. Moreover, combination of fractions with standard antiviral drug displayed synergistic effects for inhibition of HCV replication. Phytochemicals including Isoquercetin, Hyperoside, Quercetin, Reynoutrin, Apigenin and Isokaempferide displayed minimum binding energies as compared to standard protease inhibitor. CONCLUSION: N. alba and its purified phytochemicals with new scaffolds might significantly serve as valuable and alternative regimen against HCV either alone or in combination with other potential anti-HCV agents.

Phytochemical profiling and antiviral activity of Ajuga bracteosa, Ajuga parviflora, Berberis lycium and Citrus lemon against Hepatitis C Virus.

Hepatitis C is a serious health issue and cause liver disorders in millions of people. Available therapeutic agents require long term administration with numerous side effects. Therefore, there is a dire need to find alternative treatment options for this disease. Since ancient times, medicinal plants are widely used to cure various diseases with no or less harmful effects. Therefore, this study was designed to find out phytochemicals and investigate antiviral activity of methanol extract of Ajuga bracteosa, Ajuga parviflora, Berberis lycium and Citrus lemon against Hepatitis C Virus (HCV infection). Phytochemical analysis of the plant extract was performed using various chemical tests. Toxicity of the plant extract was determined against using trypan blue exclusion method. Antiviral activity of the selected plant extract was find out against HCV infected HepG2 cells. For this purpose, HepG2 cells were seeded with HCV positive and negative serum and nontoxic doses of plant extract for 24 and 48 h. After this RNA was extracted and viral load was determined using Real-time PCR. Phytochemical analysis showed the presence of flavonoids and phenols in all plant extracts while amino acids, alkaloids and tannins were present in B. lycium and saponins were detected in C. lemon. Toxicity assay showed that all plant extracts were nontoxic at maximum concentration of 200 µg/ml except B. lycium, which showed mild toxicity at 40 µg/ml and were extremely toxic at 60 µg/ml and above doses. Real-time PCR quantitation result revealed that after 24 h treatments A. parviflora showed highest antiviral activity, followed by A. bracteosa, while B. lycium extract had low (35%) and C. lemon has no antiviral effects. The 48 h treatments showed an increase antiviral activity by A. bracteosa followed by A. parviflora and B. lycium while C. lemon showed negative effect. Our results depicted that mentioned plants might be used as an alternative therapeutic regime or in combination with existing treatments against HCV.

Comparison of Anti-HCV Activity of Multiple Punica granatum Extracts and Fractions in Virus-infected Human Hepatocytes.

BACKGROUND: Plants extracts and their bioactive constituents can provide an alternative approach for new treatment. Pakistani flora reveals a huge, largely untapped source of potential antiviral constituents. OBJECTIVE: High-cost concerns of direct-acting anti-HCV drugs limit their employment specifically in developing countries like Pakistan. Therefore, discovery of inexpensive and nontoxic agents is needed for HCV treatment. METHODOLOGY: In this study, we used plasmid constructs of pCR3.1/FLAGtag/HCV NS3/4A (genotype 1a & 3a) and Punica granatum extracts (PK AV 003) and semi-purified fractions (P1-P11) were evaluated for their anti-HCV activity. Acetone extract along with two fractions (P4 & P11) revealed a useful therapeutic index. RESULTS: The fractions P4 (IC50=28.5±0.02 µg/ml; IC25=16±0.02 µg/ml) and P11 (IC50=72±0.02 µg/ml; IC25=41±0.03 µg/ml) dramatically suppressed HCV replication as measured by quantitative PCR (qPCR) and HCV NS3 protein expression level in transient transfection model. Consistent with suppression in genome replication, inhibition of HCV infectious particles by PK AV 003 extract was also judged in an infectious model system. This data is the first description of Pakistani indigenous cultivated fruit-producing plant, Punica granatum, possessing anti-HCV activity. Further analyses are being performed for investigating the mechanistic studies and structural characterization of purified fractions of PK AV 003. CONCLUSION: Our findings suggest that PK AV 003 fruit extract might be useful as an add-on therapeutic candidate for treating HCV infection.

Therapeutic potential of Taraxacum officinale against HCV NS5B polymerase: In-vitro and In silico study.

Discovery of alternative and complementary regimens for HCV infection treatment is a need of time from clinical as well as economical point of views. Low cost of bioactive natural compounds production, high biochemical diversity and inexistent/milder side effects contribute to new therapies. Aim of this study is to clarify anti-HCV role of Taraxacum officinale, a natural habitat plant rich of flavonoids. In this study, methanol extract of T. officinale leaves was initially analyzed for its cytotoxic activity in human hepatoma (Huh-7) and CHO cell lines. Hepatoma cells were transfected with pCR3.1/Flagtag/HCV NS5B gene cloned vector (genotype 1a) along with T. officinale extract. Considering NS5B polymerase as potential therapeutic drug target, twelve phytochemicals of T. officinale were selected as ligands for molecular interaction with NS5B protein using Molecular Operating Environment (MOE) software. Sofosbuvir (Sovaldi: brand name) currently approved as new anti-HCV drug, was used as standard in current study for comparative analysis in computational docking screening. HCV NS5B polymerase as name indicates plays key role in viral genome replication. On the basis of which NS5B gene is targeted for determining antiviral role of T. officinale extract and 65% inhibition of NS5B expression was documented at nontoxic dose concentration (200µg/ml) using Real-time PCR. In addition, 57% inhibition of HCV replication was recorded when incubating Huh-7 cells with high titer serum of HCV infected patients along with leaves extract. Phytochemicals for instance d-glucopyranoside (-31.212 Kcal/mol), Quercetin (-29.222 Kcal/mol), Luteolin (-26.941 Kcal/mol) and some others displayed least binding energies as compared to standard drug Sofosbuvir (-21.0746 Kcal/mol). Results of our study strongly revealed that T. officinale leaves extract potentially blocked the viral replication and NS5B gene expression without posing any toxic effect on normal fibroblast cells of body.

Antiviral activity of Acacia nilotica against Hepatitis C Virus in liver infected cells.

Hepatitis C virus (HCV) belonging to the family Flaviviridae has infected 3% of the population worldwide and 6% of the population in Pakistan. The only recommended standard treatment is pegylated INF-α plus ribavirin. Due to less compatibility of the standard treatment, thirteen medicinal plants were collected from different areas of Pakistan on the basis of undocumented antiviral reports against different viral infections. Medicinal plants were air dried, extracted and screened out against HCV by infecting HCV inoculums of 3a genotype in liver cells. RT-PCR results demonstrate that acetonic and methanolic extract of Acacia nilotica (AN) showed more than 50% reduction at non toxic concentration. From the above results, it can be concluded that by selecting different molecular targets, specific structure-activity relationship can be achieved by doing mechanistic analysis. So, additional studies are required for the isolation and recognition of antiviral compound in AN to establish its importance as antiviral drug against HCV. For further research, we will scrutinize the synergistic effect of active antiviral compound in combination with standard PEG INF-α and ribavirin which may be helpful in exploring further gateways for antiviral therapy against HCV.

Inhibition of HCV 3a core gene through Silymarin and its fractions.

UNLABELLED: Hepatitis C is a major health problem affecting 270 million individuals in world including Pakistan. Current treatment regimen, interferon alpha and ribavirin only cure half of patients due to side effects and high cost. RESULTS: In the present study Silybum marianum (Milk thistle) seeds were collected, extracted and analyzed against HCV 3a core gene by transiently transfecting the liver cells with HCV core plasmid. Our results demonstrated that Silymarin (SM) dose dependently inhibit the expression or function of HCV core gene at a non toxic concentration while the GAPDH remained constant. To identify the active ingredient, SM was fractioned by thin layer chromatography (TLC), column chromatography and HPLC. Purified fractions were tested for HCV core gene and western blotting results showed that two factions of SM (S1 and S2) inhibit HCV 3a core expression or function in liver cells CONCLUSION: Our results suggest SM and its fractions (S1 and S2) inhibit HCV core gene of 3a genotype and combination of SM and its fractions with interferon will be a better option to treat HCV infection.

In-vitro antiviral activity of Solanum nigrum against Hepatitis C Virus.

BACKGROUND: Hepatitis C is a major health problem causes liver cirrhosis, hepatocellular carcinoma and death. The current treatment of standard interferon in combination with ribavirin, has limited benefits due to emergence of resistant mutations during long-term treatment, adverse side effects and high cost. Hence, there is a need for the development of more effective, less toxic antiviral agents. RESULTS: The present study was designed to search anti-HCV plants from different areas of Pakistan. Ten medicinal plants were collected and tested for anti-HCV activity by infecting the liver cells with HCV 3a innoculum. Methanol and chloroform extracts of Solanum nigrum (SN) seeds exhibited 37% and more than 50% inhibition of HCV respectively at non toxic concentration. Moreover, antiviral effect of SN seeds extract was also analyzed against HCV NS3 protease by transfecting HCV NS3 protease plasmid into liver cells. The results demonstrated that chloroform extract of SN decreased the expression or function of HCV NS3 protease in a dose- dependent manner and GAPDH remained constant. CONCLUSION: These results suggest that SN extract contains potential antiviral agents against HCV and combination of SN extract with interferon will be better option to treat chronic HCV.