The Veterinary Clinics of North America

This article is an extension to our recently published article in Asian Journal of Pharmaceutical and Clinical Research, entitled “Β-Sitosterol: Isolation from Muntingia Calabura Linn. Bark Extract, Structural Elucidation, and Molecular Docking Studies as Potential Inhibitor of SARSCoV-2 Mpro (COVID-19)”[1]. The article describes detailed procedure for the isolation (by Column Chromatography) and structural characterization (by FTIR, UV-Visible Spectroscopy and HPTLC) of β-sitosterol from Muntingia Calabura bark. The β-sitosterol was docked on SARSCoV-2 Mpro to study the binding affinity (kcal/mol) in comparison with favipiravir. It has been found that favipiravir has a less binding affinity, i.e. 5.7 kcal/mol than β-sitosterol which has 6.9 kcal/mol. The number of hydrogen bonds formed by the favipiravir is much more, i.e., 4 than β-sitosterol which formed only 01 hydrogen bond with SARS-CoV-2 Mpro. As an extension to this published research work, we have investigated the role of β-sitosterol as immunostimulant, antioxidant and inhibitory potential against receptor binding domain (RBD) of SARS-CoV-2 Spike Glycoprotein with the aid of molecular docking. There are many studies which reveals the antioxidant and immune boosting role of β-sitosterol especially in viral infection including pneumoniae. This commentary emphasized on further potential of β-sitosterol in treatment of COVID-19 through molecular docking studies.

https://www.scientificarchives.com/admin/assets/articles/pdf/beta-sitosterol-as-immunostimulant-antioxidant-and-inhibitor-of-sars-cov-2-spike-glycoprotein-20210329100327.pdf

Beta-Sitosterol: As Immunostimulant, Antioxidant and Inhibitor of SARS-CoV-2 Spike Glycoprotein

Objective The study aimed to synthesize and characterize pyrimidine-linked benzimidazole hybrids, define their antimicrobial and antifungal activities in vitro, and determine their ability to inhibit the main protease and spike glycoprotein of SARS-CoV-2. Methods The ability of the synthesized compounds to inhibit the main protease and spike glycoprotein inhibitory of SARS-CoV-2 was investigated by assessing their mode of binding to the allosteric site of the enzyme using molecular docking. The structures of pyrimidine-linked benzimidazole derivatives synthesized with microwave assistance were confirmed by spectral analysis. Antibacterial and antifungal activities were determined by broth dilution. Results Gram-negative bateria (Escherichia coli and Pseudomonas aeruginosa) were more sensitive than gram-positive bateria (Staphylococcus aureus and Streptococcus pyogenes) to the derivatives. Candida albicans was sensitive to the derivatives at a minimal inhibitory concentration (MIC) of 250 μg/mL. The novel derivatives had better binding affinity (kcal/mol) than nelfinavir, lopinavir, ivermectin, remdesivir, and favipiravir, which are under investigation as treatment for SARS-CoV-2 infection. Compounds 2c, 2e, and 2g formed four hydrogen bonds with the active cavity of the main protease. Many derivatives had good binding affinity for the RBD of the of SARS-CoV-2 spike glycoprotein with the formation of up to four hydrogen bonds. Conclusion We synthesized novel pyrimidine-linked benzi-midazole derivatives that were potent antimicrobial agents with ability to inhibit the SARS-CoV-2 spike glycoprotein. Understanding the pharmacophore features of the main protease and spike glycoprotein offers much scope for the development of more potent agents. We plan to optimize the properties of the derivatives using models in vivo and in vitro so that they will serve as more effective therapeutic options against bacterial and SARS-CoV-2 infections.

Novel pyrimidine-benzimidazole hybrids with antibacterial and antifungal properties and potential inhibition of SARS-CoV-2 main protease and spike glycoprotein

Design and development of terbinafine hydrochloride ethosomal gel for enhancement of transdermal delivery: In vitro, in vivo, molecular docking, and stability study

For the treatment and maintenance of postprandial blood glucose increases (i.e., diabetes mellitus), alpha (α)-amylase is a well-known therapeutic target. In this paper, we report an initial exploration of the work, i.e., in vitro alpha-amylase activity of the hydroalcoholic polyherbal extract of the selected plants. After drying, the plant material is ground individually, and at least 100 gm of the crude powder is prepared from each plant. 100 gm of each plant was combined, and a total of 500 gm of the crude powder (Ichnocarpus frutescens (100 gm) + Ficus dalhousie (100 gm) + Crateva magna (100 gm) + Alpinia galangal (100 gm) + Swertia chirata (100 gm)) was prepared to carry out the extraction. This obtained extract was subjected to preliminary phytochemical screening and in vitro alpha-amylase activity. At 16 mg/mL, acarbose displayed 78.40 ± 0.36% inhibition, whereas the extract exhibited 72.96 ± 0.70% inhibition, which is significantly comparable. The IC50 value of acarbose was 12.9 ± 1.12, whereas the extract exhibited 13.31 ± 1.12 mg/mL. The extract possesses numerous classes of chemicals such as alkaloids, glycosides, tannins, polyphenols, and terpenoids, which can contribute to the antidiabetic activity through alpha-amylase inhibition. This was an initial exploration of the work as a proof of concept for the development of polyherbal tea bag formulation for the treatment of diabetes. In the future, we are aiming to investigate the effectiveness of polyherbal tea bags in the treatment of diabetes using more in vitro and in vivo models. From the present investigation, we have concluded that this extract can be used for the treatment of diabetes.

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In Vitro Alpha-Amylase Enzyme Assay of Hydroalcoholic Polyherbal Extract: Proof of Concept for the Development of Polyherbal Teabag Formulation for the Treatment of Diabetes

Objective: A novel human coronavirus, labeled as SARS-CoV-2 (COVID-19), causing pneumonia is spreading around the world. At present, there are no specific treatments for COVID-19. β-sitosterol is well known for its multiple biological actions. This research aims to isolate and study the binding affinity of β-sitosterol for SARS-CoV-2 (COVID-19) main protease (Mpro). Methods: Extraction and column chromatography was performed to isolate the β-sitosterol from an n-hexane extract of Muntingia calabura bark followed by thin-layer chromatography (TLC), high-performance TLC (HPTLC), Fourier-Transform infrared (FTIR), and ultraviolet-visible spectroscopy. The molecular docking studies were performed on SARS-CoV-2 Mpro to determine the binding affinity of the β-sitosterol using PyRx Virtual Screening Tool. Results: In the present study, preliminary phytochemical screening showed the presence of carbohydrate, steroid, terpenoid, and flavonoid compounds. A total of 115 fractions was collected from column chromatography using benzene as a solvent by an isocratic elution technique. HPTLC fingerprinting analysis showed the presence of β-sitosterol under 366 nm. FTIR characterization was performed of the same fraction which gives the absorption peaks which resembles the β-sitosterol structure. Conclusion: The overall study concludes this method can be considered as a standard method for isolation of β-sitosterol from M. calabura bark. Favipiravir has a less binding affinity, i.e. -5.7 kcal/mol than β-sitosterol which has -6.9 kcal/mol. The number of hydrogen bonds formed by the favipiravir is much more, i.e., 4 than β-sitosterol which formed only 01 hydrogen bonds with SARS-CoV-2 Mpro.

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B-sitosterol: Isolation from muntingia calabura linn. bark extract, structural elucidation, and molecular docking studies as potential inhibitor of sars-cov-2 mpro (COVID-19)

Purpose: The human cytochrome P450 3A4 (CYP3A4) is the biggest individual from the CYP3A subfamily and records for 30–60% of the total for CYP450 adult liver. Hereditary varieties in CYP3A4 are a noteworthy hotspot for inter-patient changeability in plasma concentration, adverse effects and pharmacological response to medications. This research was done to discover naturally occurring novel CYP3A4 inhibitors from flavonoids. Methods: The molecular docking method was used to optimize the inhibiting activity of flavonoids against CYP3A4. PyRx Virtual Screening Tool 0.8 and BIOVIA Discovery Studio 2019 was used for simulation. Results: Flavonoids like Pongamoside A, Pongamoside B, and Pongamoside D have more binding affinity (kcal/mol) i.e. -11.6, -10.9, -10.8 respectively than Doxorubicin which have -10.7 against CYP3A4. Although, Daidzein, Genistein, and Luteolin form more hydrogen bonds than doxorubicin. Conclusion: The rational synthesis of natural analogues in reference to synthetic drugs, could generate drugs with improved therapeutic effect for chemoprevention. CYP3A4 plays a major role in the metabolism of various drugs; by the help of flavonoids, we can control the selective drug metabolism by inhibiting CYP3A4. Despite this, these molecules are not marketed for cancer treatment because of high polarity. If we could overcome this problem, these molecules can acts as effective anticancer agents in the future. Still, if we want to use these compounds clinically, there is a need to generate more scientific evidence and quality data by using in vivo and in vitro models. © 2020 iGlobal Research and Publishing Foundation. All rights reserved. Cite this article as: Khan, S.L.; Sonwane, G.M.; Siddiqui, F.A.; Jain, S.P.; Kale, M.A.; Borkar, V.S. Discovery of naturally occurring flavonoids as human cytochrome P450 (CYP3A4) inhibitors with the aid of computational chemistry. Indo Global J. Pharm. Sci., 2020; 10(4): 58-69. DOI: http://doi.org/10.35652/IGJPS.2020.10409 . Indo Global Journal of Pharmaceutical Sciences, 2020; 10(4): 58-69 59 inflammatory, antiallergic, modulators of enzymatic activities and anti-cancer activity [3]. Apigenin, luteolin, quercetin and kaempferol, the hydroxylated flavonoids are the main constituents of various dietary products and beverages and have been the focus of extensive research over the last years. Apigenin exerts anticancer effects through the modulation of various pathways namely, apoptosis, Reactive oxygen species (ROS) and DNA damage and repair[4]. Malignant growth is one of the terrible illnesses caused by unusual cell growth and can attack different tissues. They shape a subset of neoplasms. It speaks of the greatest social insurance issues for humankind and requests a proactive system for cure [5]. It is accounted for the rate of malignancy that has been expanding in developing nations and has turned into the fourth driving reason for death around the world. Chemoprevention by phytoconstituents has advanced as a powerful procedure to control the prevalence of malignant growth. The journey of anticancer agents from plant sources began in the 1950s with the discovery of the vinca alkaloids, vincristine, vinblastine, combretastatin, and colchicine. These Phytochemicals act explicitly on tumor cells without influencing non-cancerous cells. Carcinogenesis is a mindboggling marvel that includes many signaling cascades. Phytochemicals are viewed as reasonable candidates for anticancer medication advancement due to their pleiotropic activities on target. The examination is in advancement for creating potential competitors (those can square or back off the development of disease cells without any side effects) from these phytochemicals. Numerous phytochemicals and their determined analogs have been distinguished as potential candidates for anticancer treatment. Plants serve as a source of novel compound elements and give a promising line to investigate on malignant growth. The plant and plant metabolites are the reforming sources as these are simple, more secure, easy, quick, and less dangerous as contrasted to traditional treatment methods. There is a positive relationship set up by the epidemiological examinations between expanded utilization of common items with diminished danger of disease. The mechanism responsible for chemoprevention remains essentially unidentified, however, it is likely identified with the closeness of phytochemicals related to plants. Consequently, the search for powerful and more secure natural anticancer agents have attracted the researchers throughout the world. [6,7]. The human cytochrome P450 3A4 (CYP3A4) is the biggest individual from the CYP3A subfamily and records for 30– 60% of the total for CYP450 adult liver. The CYP3A4 gene is limited on chromosome 7q21 and up to now, 41 CYP3A4 alleles have been recognized. The human CYP3A locus contains the three CYP3A gene (CYP3A4, CYP3A5, and CYP3A7), three pseudogenes, and a novel CYP3A gene named CYP3A43. Hereditary varieties in CYP3A4 are a noteworthy hotspot for inter-patient changeability in plasma concentration, adverse effects and pharmacological response to medications, for example, paclitaxel, fentanyl, tamoxifen, tacrolimus, and statins. Moreover, existing investigations have announced the role of CYP3A4 inadequate alleles in the disease susceptibility to prostate malignant growth, estrogen receptor-negative breast cancer, and type-2 diabetes [8,9]. Protein-ligand docking is a fundamental part of computeraided drug design, and it distinguishes the coupling pattern of proteins and ligands by computer simulation. Molecular docking results decide a general binding mode of a ligand. Varieties of compounds from plant sources have been accounted to have significant anticancer properties; in any case, their modes of activity have not been characterized. Molecular docking studies were performed on some flavonoids by using Autodock vina 1.1.2 in PyRx 0.8. [10]. The docking was performed utilizing receptor proteins required with cell cycle, cell development, and DNA replication, i.e., cyclin-subordinate protein kinase 2 (CDK-2), CDK-6, DNA topoisomerases I and II, B-cell lymphoma 2 (Bcl-2), vascular endothelial development factor receptor 2 (VEGFR-2), and the telomere: G-quadruplexes. By molecular docking, the bound confirmations and the coupling attachment among flavonoid and CYP3A4 as the target could be anticipated [11]. Doxorubicin, sold under the brand names adriamycin, used to treat breast malignant growth, bladder cancer, lymphoma, and intense lymphocytic leukemia was utilized for docking studies whose binding interactions were compared with the flavonoids [12]. Docking of the small molecule into the binding site of a receptor and guessing the binding interaction of the complex is a noteworthy part of the structure-based drug design process. By molecular docking, the bound conformations and the binding affinity between Flavonoids and human cytochrome P450 3A4 as the target could be predicted [13]. Table 1 represents the names and structures of doxorubicin and the flavonoids used for molecular docking. The structures of all the compounds were generated by using ChemDraw Ultra 8.0 with the help of IUPAC name took from the official website of U.S. National Library of Medicine PubChem (https://pubchem.ncbi.nlm.nih.gov/). Indo Global Journal of Pharmaceutical Sciences, 2020; 10(4): 58-69 60 Table 1. Name and Structures of compounds used for molecular docking Doxorubicin Pongamoside A Pongamoside B

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Discovery of Naturally Occurring Flavonoids as Human Cytochrome P450 (CYP3A4) Inhibitors with the Aid of Computational Chemistry

One of the most significant challenges of diabetes health care is diabetic foot ulcers (DFU). DFUs are more challenging to cure, and this is particularly true for people who already have a compromised immune system. Pathogenic bacteria and fungi are becoming more resistant to antibiotics, so they may be unable to fight microbial infections at the wound site with the antibiotics we have now. This article discusses the dressings, topical antibacterial treatment, medications and debridement techniques used for DFU and provides a deep discussion of DFU and its associated problems. English-language publications on DFU were gathered from many different databases, such as Scopus, Web of Science, Science Direct, Springer Nature, and Google Scholar. For the treatment of DFU, a multidisciplinary approach involving the use of diagnostic equipment, skills, and experience is required. Preventing amputations starts with patient education and the implementation of new categorization systems. The microbiota involved in DFU can be better understood using novel diagnostic techniques, such as the 16S-ribosomal DNA sequence in bacteria. This could be achieved by using new biological and molecular treatments that have been shown to help prevent infections, to control local inflammation, and to improve the healing process.

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An Overview of Diabetic Foot Ulcers and Associated Problems with Special Emphasis on Treatments with Antimicrobials

Ethosomal systems are newer lipid vesicular carriers that have been around for 20 years, but over that period they have grown significantly as a means of transdermal drug delivery. They have a sizable amount of ethanol in them. These nanocarriers carry medicinal substances with various physicochemical qualities throughout the skin and deep skin layers. Since they were created in 1996, ethosomes have undergone substantial investigation; new substances have been added to their original composition, creating new varieties of ethosomal systems. These innovative carriers, which can be added to gels, patches, and lotions, are prepared using several novel methods. In addition to clinical trials, many in vivo models are employed to assess the effectiveness of dermal/transdermal administration. This review focuses on different generation of ethosomes and their comparison with other conventional liposomes. 

https://doi.org/10.1016/j.amsu.2022.104595

Sharuk L. Khan

Papers

B-sitosterol: Isolation from muntingia calabura linn. bark extract, structural elucidation, and molecular docking studies as potential inhibitor of sars-cov-2 mpro (COVID-19)

Discovery of Naturally Occurring Flavonoids as Human Cytochrome P450 (CYP3A4) Inhibitors with the Aid of Computational Chemistry

Novel pyrimidine-benzimidazole hybrids with antibacterial and antifungal properties and potential inhibition of SARS-CoV-2 main protease and spike glycoprotein

An Overview of Diabetic Foot Ulcers and Associated Problems with Special Emphasis on Treatments with Antimicrobials

Ethosomes: A novel drug carrier