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Full-Text Articles in Biochemical and Biomolecular Engineering

The Biological Activity Of Dual-Responsive Glycopolymers For Intracellular Codelivery Of Antigen And Lipophilic Adjuvant, Micaela Shields May 2023

The Biological Activity Of Dual-Responsive Glycopolymers For Intracellular Codelivery Of Antigen And Lipophilic Adjuvant, Micaela Shields

Honors Theses

Subunit vaccines present themselves as an attractive alternative to traditional approaches to vaccines, which utilizes whole organisms to trigger an immune response. While these traditional approaches have proven themselves reliable within the history of vaccinations, they generally deliver subpar robust cellular-mediated immunity and pose many safety risks. Composed of proteins and /or peptides, subunit vaccines are safer and more precise alternatives to traditional approaches. However, the pharmacokinetic properties of the macromolecules that make up these vaccines hinder the subunit vaccine’s level of immunogenicity [d1] Examples of this hindrance include but are not limited to aggregation at the injection site and …


Investigating Myosin Ensemble Force Generation Using Optical Tweezers, Janie Watts Apr 2023

Investigating Myosin Ensemble Force Generation Using Optical Tweezers, Janie Watts

Honors Theses

Myosin is a motor protein that facilitates muscle contraction and movement by stepping along actin filaments using energy from ATP hydrolysis. If myosin or other motors are disrupted throughout the body, it can have many harmful effects. Hypertrophic cardiomyopathy is a disease caused by gene mutations that affect myosin heavy chains in the heart. The tissue of the heart becomes abnormally thick, which can make it more difficult to pump blood or block blood flow out of the heart. Our goal is to discern the mechanistic difference function of a healthy and diseased heart. To accomplish this, we construct model …


Design Of An Undergraduate Laboratory Experiment Utilizing A Stirred-Tank, Jacketed Bioreactor, Brennen Middleton May 2021

Design Of An Undergraduate Laboratory Experiment Utilizing A Stirred-Tank, Jacketed Bioreactor, Brennen Middleton

Honors Theses

Bioreactors are utilized in many industries, such as the food, alternative fuel, and pharmaceutical industries, to design and manufacture products. Unlike the similar chemical batch reactors utilized in many chemical processing facilities, bioreactor utilization requires engineers to consider a wider range of operating conditions and parameters. This results in a more complex reaction system and controls network. Due to this, it is imperative for chemical and biomedical engineering students to not only understand the theory surrounding these reactor systems, but also understand how to properly design and perform operating procedures with these systems. Thus, it was determined to create an …


Effects Of Predation On The Biochemical Constituents Of The Bleached Soft Coral Sinularia Polydactyla, Carley Elizabeth Cole Jan 2018

Effects Of Predation On The Biochemical Constituents Of The Bleached Soft Coral Sinularia Polydactyla, Carley Elizabeth Cole

Honors Theses

Climate change can indirectly affect the level of predation on soft corals. As sea temperatures rise, more and more bleaching events are occurring, which can affect the production of chemical defenses and the concentrations of the biochemical constituents that make up the soft corals. These changes may make the soft corals more attractive to predators. This phenomenon is suggested by the environmental stress theory (EST), which states that under stressful conditions, more of an organism's energy is allocated to survival rather than to other factors, such as reproduction or predator deterrence. To test the effect of predation on already bleached …


O-Glcnac Post Translational Modification Of Hypoxia-Inducible Factor (Hif) Proteins, Hailey Elisabeth Grisham Jan 2018

O-Glcnac Post Translational Modification Of Hypoxia-Inducible Factor (Hif) Proteins, Hailey Elisabeth Grisham

Honors Theses

O-linked β-N-acetylglucosamine (O-GlcNAc) post-translational modification (O- GlcNAcylation), plays a vital role in many metabolic pathways, including those of numerous diseases. New research methods and basic functional roles of O-GlcNAc post- translational modification on metabolism, human health, and cancer are quickly developing, but a vast amount of research remains on the specificity of this pathway towards specific proteins. In this study, T47D breast cancer cells were used as an in vitro model to investigate the effects of O-GlcNAc modification on hypoxia-inducible factor (HIF) proteins.


Design And Synthesis Of Novel Cannabinoid Receptor Modulators, Mary Paige Thrash Jan 2018

Design And Synthesis Of Novel Cannabinoid Receptor Modulators, Mary Paige Thrash

Honors Theses

Cannabinoid (CB) receptors are validated drug targets in the endocannabinoid signaling system associated with a number of human pathologies, and the development of novel and selective small molecule CB ligands is warranted. A lead molecule HL-010, a member of the 4H-thieno[3,2-b]pyrrole-5-carboxamide class of compounds, was previously identified through CB homology modeling and virtual screening protocols as a potential high affinity ligand for cannabinoid receptors. Validation of the in silico data was realized with the evaluation of both CB1 and CB2 receptor binding and functional activity assessment: HL-010 was found to be a potent (~10 nM) and selective CB2 receptor agonist …


Inhibition Of Blood Coagulation Factor Xii: Evaluation Of Flavonoids As A Drug Candidate, Betsy Crosswhite Jan 2018

Inhibition Of Blood Coagulation Factor Xii: Evaluation Of Flavonoids As A Drug Candidate, Betsy Crosswhite

Honors Theses

This study investigated natural product inhibitors of blood coagulation Factor XII (FXII). FXII initiates the Kallikrein Kinin-System (KKS) within the intrinsic coagulation pathway. Increased expression of this system leads to proinflammatory and procoagulant activities. Inflammation occurs through downstream activation in the KKS. Following the activation of FXII-dependent pathway, bradykinin (BK) binds to the BK receptor 2 (B2) and produces inflammatory signaling. Abnormally increased BK can result in life threatening Hereditary Angioedema (HAE), characterized by severe reoccurring swelling. FXII is critical for pathogenic thrombosis, but is dispensable for hemostasis. This unique property of FXII makes it an attractive drug target. We …


Technique For The Synthesis Of Difluorinated Organic Molecules Using Magnesium Bases, Cassidy S. Baldwin Jan 2018

Technique For The Synthesis Of Difluorinated Organic Molecules Using Magnesium Bases, Cassidy S. Baldwin

Honors Theses

The presence of fluorine in organic molecules can confer biological advantages which are often exploited in drug development. For example, fluorine has been shown to limit drug metabolism and enhance drug distribution. However, the development of fluorinated pharmaceutics poses synthetic challenges. These challenges are especially pronounced when developing molecules with a difluoromethyl group. One difluoromethyl functional group of particular interest is the α,α-difluoro-β-amino carbonyl group, which has been seen in potential drug leads. The previous methods of accessing this group are inefficient and complex. The goal of this project was to develop a new technique to access this group by …


Neurotoxicity Of Asimina Triloba, Myriam Sanders Jan 2018

Neurotoxicity Of Asimina Triloba, Myriam Sanders

Honors Theses

Neurodegenerative diseases are thought to be brought on by genetic and environmental factors including exposure to compounds and extracts found in plants. Many substances extracted from plants have been shown to induce cell death in neurons. This experiment tests different plant compounds to determine if they induce death in neurons in cell culture. In particular, we focused on extracts and compound from the Asimina Triloba plant- a plant that had anecdotally been connected with Parkinson's Disease-like effects. Cultured neurons were treated with increasing concentrations of the compounds and extracts. Twenty-four hours after treatment, viability was assessed. Results show that while …


Development Of A Scn1a Null Zebrafish Model For Screening Potential Anti-Epileptic Natural Products, Patricia Ann Lipson Jan 2018

Development Of A Scn1a Null Zebrafish Model For Screening Potential Anti-Epileptic Natural Products, Patricia Ann Lipson

Honors Theses

Epilepsy affects around 50 million people in the world. Thus, it is imperative that new more effective and safe treatments be found. This study was designed to use zebrafish to screen potential anti-epileptic natural products. We utilized two scn1Lab null zebrafish models (morpholino-injected fish where scn1Lab was transiently knocked down and a transgenic scn1Lab -/- fish line). Alternatively, we used AB wildtype fish to test compounds in a chemically-induced seizure model. Natural compounds including cannabidiol, CBD, and Tapinanthus globiferous, TG, extracts were screened for their anti-seizure activity. When scn1Lab -/- fish were treated with CBD (0.075 and 0.3 mg/L) from …


Targeting Metastatic Breast Cancer With Statin Drugs And Coq10, Jacob Thrasher Jan 2018

Targeting Metastatic Breast Cancer With Statin Drugs And Coq10, Jacob Thrasher

Honors Theses

Statin drugs are commonly prescribed to lower cholesterol levels in patients, but recently statins have been under investigation as a potential anti-cancer drug. Statins are HMG-CoA reductase inhibitors and subsequently inhibit the mevalonate pathway and cholesterol synthesis. Since cholesterol trafficking is vital in the growth and metastasis of cancer, statin drugs are being studied to explore their effects on cancer cells. CoQ10, one of the products from the mevalonate pathway, also has shown some potential anti-cancer properties, but its use to combat cancer is controversial. While some studies have shown that CoQ10 supplements can be synergistic with other cancer treatment …


Ca2+/Calmodulin-Dependent Protein Kinase Ii Activity And Expression In The Aged Mouse Hippocampus, James Mitchum Jan 2018

Ca2+/Calmodulin-Dependent Protein Kinase Ii Activity And Expression In The Aged Mouse Hippocampus, James Mitchum

Honors Theses

CaM Kinase II (Ca2+/calmodulin-dependent protein kinase II) is a serine/threonine-specific protein kinase enzyme that acts as a critical regulator of learning and memory. It has previously been shown that the inability of CaMKII to maintain activation is connected to memory deficits within the aging brain. While the connection between CaMKII dysfunction and learning and memory impairments is well established, little is known about the changes that cause CaMKII dysfunction. Our research set out to investigate if CaMKII is altered during the process of aging. Specifically, we looked at activity, regulation, and stability by conducting experiments to quantify CaMKII levels of …


Perceptions Regarding Concussions In Collegiate Football At The University Of Mississippi, Madison Frerker Jan 2018

Perceptions Regarding Concussions In Collegiate Football At The University Of Mississippi, Madison Frerker

Honors Theses

Concussions are a form of mild traumatic brain injury (mTBI) that can occur from any force to the head or body that causes the brain to move within the skull. Concussions are common in the sport of football due to the highly physical nature of the sport, and injured athletes can experience a wide array of symptoms. Like other players participating with powerhouse football programs, those at the University of Mississippi are at risk of concussion throughout their collegiate career. Whether players and fans perceive the risks of concussion equally has not been previously studied. Considering the passionate history of …


Cardiolipin Profiling In Metastatic Breast Cancer Cells, Meghan Houston Mcneely Jan 2018

Cardiolipin Profiling In Metastatic Breast Cancer Cells, Meghan Houston Mcneely

Honors Theses

Six human breast cancer cell lines (MDA-MB-231, LM, BoM, T47D, MCF7, and MCF7-BoM) were treated with three differentially targeted phospholipase inhibitors (bromoenol lactone, tricyclodecan-9-yl-xanthogenate, and halopemide) as well as various dietary oils (canola oil, avocado oil, coconut oil, olive oil, sesame oil, and soybean oil). Cell viability assays were performed to examine the effects of each inhibitor and oil in varying concentrations on the proliferation of each breast cancer cell line. The goal was to detect changes in cardiolipin concentration within each cell line after exposure to a series of various phospholipase inhibitors and oils. Liquid chromatography-mass spectrometry (LC-MS) was …


Cm-Dii And Mcf-7 Breast Cancer Cell Responses To Chemotherapeutic Agents, Ashten M.C. Anderson Jan 2018

Cm-Dii And Mcf-7 Breast Cancer Cell Responses To Chemotherapeutic Agents, Ashten M.C. Anderson

Honors Theses

CM-DiI is a lipophilic, red fluorescent dye used for staining and tracking the migration of cells. CM-DiI makes it possible to visualize cells in histological regions and can therefore be very useful for the tracking of cancer cell proliferation and metastasis in vivo. The ability to track and quantitate cancer cell proliferation in vivo is essential for cancer drug discovery. If CM-DiI labeled cancer cells respond to chemotherapy agents similar to unlabeled cancer cells, it facilitates screening of potential anti-cancer compounds using CM-DiI labeled cells in a xenotransplanted, transgenic zebrafish model (Danio rerio). To investigate whether CM-DiI labeling would affect …


Synthesis Of Difluoromethyl And Monofluoromethyl Ketones For Biological Evaluation At The Gaba-B Receptor, Mallory Maier Jan 2017

Synthesis Of Difluoromethyl And Monofluoromethyl Ketones For Biological Evaluation At The Gaba-B Receptor, Mallory Maier

Honors Theses

The central nervous system communicates using neurotransmitters. Most are excitatory neurotransmitters, such as acetylcholine, dopamine, and serotonin, but


Synthesis And Biological Evaluation Of Cannabinoid Receptor Ligands, Mariah Leigh Cole Jan 2017

Synthesis And Biological Evaluation Of Cannabinoid Receptor Ligands, Mariah Leigh Cole

Honors Theses

The cannabinoid receptors, members of the G-protein coupled receptor (GPCR) superfamily, have been implicated in numerous human physiological functions and diseases. These receptors, cannabinoid type 1 (CB1) and cannabinoid type 2 (CB2), are most concentrated in the central nervous system and immune cells, respectively, and have each become a target of therapeutic interest. Dual CB1/CB2 agonists such as delta-9-tetrahydrocannbinol (THC) have demonstrated efficacy in the treatment of nausea, pain, and glaucoma, but suffer from psychotropic effects mediated by CB1, motivating the search for CB2 selective therapeutic agents. Selective modulation of the CB2 receptor has therapeutic potential in human health disorders …


Utilizing The Zebrafish Model To Determine Anti-Epileptic Properties Of Mistletoe And Cannabis, Collin Dietrich Jan 2017

Utilizing The Zebrafish Model To Determine Anti-Epileptic Properties Of Mistletoe And Cannabis, Collin Dietrich

Honors Theses

Epilepsy affects around 50 million people in the world, therefore improving treatment efficacy and safety for epileptics is imperative. In this study we sought to screen the effectiveness and safety of cannabis constituents (delta-9-tetrahydrocannabinol and cannabidiol) and Tapinanthus globiferus extracts in treating epilepsy. We used a zebrafish model wherein seizures were induced by treatment with pentylenetetrazol. Our results showed that Tapinanthus globiferus dose-dependently reduced seizure activity, and no toxicities were seen at the concentrations used. In contrast delta-9-tetrahydrocannabinol and cannabidiol did not significantly reduce seizure activity and some toxicities were seen at the higher concentrations tested. We conclude that extracts …


Impacts Of Aplysina Red Band Syndrome On Secondary Metabolite Profiles And Antibacterial Activity Of The Caribbean Sponge Genus Aplysina, Mafie Claire Vickers Jan 2017

Impacts Of Aplysina Red Band Syndrome On Secondary Metabolite Profiles And Antibacterial Activity Of The Caribbean Sponge Genus Aplysina, Mafie Claire Vickers

Honors Theses

Coral reef ecosystems continue to decline due to abiotic and biotic factors, including disease. Although much of the existing scientific literature has focused on coral diseases, reports of sponge diseases have been increasing. This is a cause for concern, as sponges are increasingly dominant components of coral reef communities, where they are responsible for a wide range of ecological functions. A sponge disease of particular interest is Aplysina Red Band Syndrome (ARBS), which impacts the Caribbean's most abundant genus of sponges, Aplysina. Three morphotypes of branching sponges within this genus are affected, including Aplysina fulva, and the thin and thick …


Biochemical Variation In Caribbean Sponges Of The Genus Aplysina, Sarah L. Criddle Jan 2017

Biochemical Variation In Caribbean Sponges Of The Genus Aplysina, Sarah L. Criddle

Honors Theses

Sponges are among the simplest multicellular organisms, consisting of groupings of cells with similar functions rather than true tissues. Nonetheless, sponges are ecologically significant in many marine environments, including coral reefs. They are sedentary organisms that feed by filtration from the water column, which contributes to the numerous roles they play on coral reefs, including upkeep of the reef, production of usable energy, and recycling of nutrients. Like all multicellular organisms, sponges are made up of biologically significant macromolecules. These macromolecules provide the energy for the sponge, and their relative concentrations determine how sponges can store and access the energy …


The Pharmacodynamic Functions Of Low-Dose Rapamycin As A Model For Universal Influenza Protection, William Gust Jan 2017

The Pharmacodynamic Functions Of Low-Dose Rapamycin As A Model For Universal Influenza Protection, William Gust

Honors Theses

One of the most sought after breakthroughs in modern influenza research is the creation of a universal influenza vaccine that would protect against all strains of avian influenza. An unlikely model for this kind of protection might be found in the mTOR inhibitor rapamycin. Traditionally used to prevent organ transplant rejection, low doses of rapamycin have been found to protect mice against infection with distinct subtypes of influenza. However, it is not understood how a partial block in this pathway provides optimal heterosubtypic immunity. Interestingly, previous experiments have shown that low dose rapamycin inhibits germinal center formation to reduce antibody …


Evaluation Of Sigma Ligand Az66 Analgesic Potentiation Of Cannabinoids, Jacqueline Morris Jan 2017

Evaluation Of Sigma Ligand Az66 Analgesic Potentiation Of Cannabinoids, Jacqueline Morris

Honors Theses

Sigma receptors have become a popular subject for research the past few decades, but there is still much mystery behind these receptors and how they work. Sigma receptors, like cannabinoid-1 (CB1) receptors, have effects on the human body including appetite regulation, depression, and analgesia. AZ66 is a highly selective sigma receptor ligand that has antagonistic properties. Sigma receptor antagonists have been shown to potentiate the analgesic effects of opiates; however, there are no known literature reports about the interaction between the sigma receptor system and the endocannabinoid system, including CB1 receptors. The purpose of this study is to evaluate AZ66 …


The Evaluation Of Az66, A Highly Selective Sigma-1 And Sigma-2 Receptor Antagonist, For Its Anti-Convulsive Effects In C57bl/6 Mice, Jamie Stone Jan 2017

The Evaluation Of Az66, A Highly Selective Sigma-1 And Sigma-2 Receptor Antagonist, For Its Anti-Convulsive Effects In C57bl/6 Mice, Jamie Stone

Honors Theses

The Evaluation of AZ66, A Highly Selective Sigma-1 and Sigma-2 Antagonist, for Anticonvulsive Effects in C57Bl/6 Mice (Under the direction of Lisa Wilson and Dr. Christopher McCurdy) Sigma receptors have become a popular subject for research the past few decades, but there is still much mystery behind these receptors and how they work. Researchers have found that sigma receptor antagonists can attenuate cocaine induced convulsions, however, limited research has been conducted on the effects of these antagonists on convulsions that mimic the types of seizures associated with epilepsy. Therefore, the aim of the current study was to evaluate AZ66 (20 …


Exploration Of Compound Viability As An Organ-Selective Anti-Metastatic Therapeutic Treatment For Triple Negative Breast Cancer, Ashley E. Noblin Jan 2016

Exploration Of Compound Viability As An Organ-Selective Anti-Metastatic Therapeutic Treatment For Triple Negative Breast Cancer, Ashley E. Noblin

Honors Theses

Phenformin and metformin are compounds that are currently used to treat Type II Diabetes and have been shown to suppress breast cancer metastasis. These compounds were tested, after extensive research and screening of current approved oncology drugs, in various experiments with the cell lines MDA-MB-231, LM, and BoM, with MDA-MB-231 being the parental cell line, LM being the lung metastatic line, and BoM being the bone metastatic line. The cells were treated with compound addition at various concentrations and subject to a two-day concentration response viability study in order to determine any inhibitory effects, followed by a five-day assay in …


An Evaluation Of The Relationship Between Sigma-1 And Sigma-2 Receptors And The Endocannabinoid System, Jenn Miller Jan 2016

An Evaluation Of The Relationship Between Sigma-1 And Sigma-2 Receptors And The Endocannabinoid System, Jenn Miller

Honors Theses

Within the past two decades, sigma receptors have become a popular area for research. Although much has been learned about their structure, subtypes, and functions; there is still much to be learned. Consisting of two receptor subtypes (sigma-1 and sigma-2), it has been discovered that sigma receptor ligands potentiate the analgesic effects of both opiates and cannabinoids, though the exact mechanism and sigma subtype on which this occurs is still unknown. The purpose of this study is to determine if potentiation of opiates and cannabinoids occurs through sigma-1 or sigma-2 receptor signaling. Tetrad assays were performed for: 1. Morphine, an …


Design And Synthesis Of Dual Opioids/Npff Ligands As Novel Analgesics With Reduced Tolerance Potential, John N. Yi Jan 2016

Design And Synthesis Of Dual Opioids/Npff Ligands As Novel Analgesics With Reduced Tolerance Potential, John N. Yi

Honors Theses

Opioid use has become prevalent in today's culture. As a treatment for acute and chronic pain, opioids permeate in the public sphere as a seemingly easy solution. However, problems that come from the increased use of opiates, especially in the last fifteen years, have shown detrimental effects in society. Not only are all opioid-based analgesics considered addictive, but the nature of such painkillers also enables a phenomenon known as tolerance, where the body adapts to the use of exogenous opioids. This creates a situation where a more potent dose of painkiller is required for the same analgesic effect. The focus …


Biological Activity Of Garcinia Kola On Hepatitis C, Madeline Dwyer Jan 2016

Biological Activity Of Garcinia Kola On Hepatitis C, Madeline Dwyer

Honors Theses

Hepatitis C is a liver disease caused by the hepatitis C virus (HCV). According to the World Health Organization, hepatitis C infections affect between 130-150 million people worldwide. It is a major cause of liver cirrhosis and liver cancer, and approximately 500,000 people die from hepatitis C infections or related complications each year. Although new treatments are 90% effective at curing chronic hepatitis C infections, they cost tens of thousands of dollars. Available treatments are incredibly cost prohibitive, especially considering that the regions most affected by hepatitis C are Africa and central and eastern Asia (Hepatitis C). This creates a …


G-Quadruplex Mediated Splicing Of P53 Mrna, Melissa Holy Jan 2016

G-Quadruplex Mediated Splicing Of P53 Mrna, Melissa Holy

Honors Theses

The protein p53 is a master regulator of the cell cycle and its mutation/inactivation is associated with over 50% of cancer cases. Mutations in p53 are associated with worse survival rates, resistance to therapies, and other risk factors in breast cancer specifically. p53 is encoded by the TP53 gene and is expressed as several transcripts. In the G-rich portion of intron 3, secondary DNA structures known as G-quadruplexes are able to form. The stabilization of these G-quadruplexes leads to correct splicing of intron 2, thus making a functional p53 protein. A known quadruplex-stabilizing compound (TMPyP4) was tested to determine its …


The Effects Of Genistein, Thymoquinone, Epigallocatechin-3-Gallate,5-Fluorouracil, And Low Level Laser Therapy On Fadu Hypopharyngeal And Laryngeal Carcinoma Cells, Osasu Adah Jan 2016

The Effects Of Genistein, Thymoquinone, Epigallocatechin-3-Gallate,5-Fluorouracil, And Low Level Laser Therapy On Fadu Hypopharyngeal And Laryngeal Carcinoma Cells, Osasu Adah

Honors Theses

Laryngeal and hypopharyngeal carcinoma are two types of head and neck squamous cell carcinomas. The location of the tumor makes it hard to detect without complex tests. Overall, these types of cancer are not common, and there are no simple screening tests for such cancers. Due to this issue, laryngeal and hypopharyngeal carcinoma are usually diagnosed when signs and symptoms occur, such as sore throat, trouble swallowing, and constant coughing. With the use of FaDu, a head and neck cancer cell line of the hypopharyngeal origin, experimentation can be performed in order to initiate possible treatments. The standard treatments for …


Secondary Metabolites Isolated From Perovskia Atriplicifolia And Their In Vitro Binding Affinity For Human Opioid And Cannabinoid Receptors, Ashli Fitzpatrick Jan 2015

Secondary Metabolites Isolated From Perovskia Atriplicifolia And Their In Vitro Binding Affinity For Human Opioid And Cannabinoid Receptors, Ashli Fitzpatrick

Honors Theses

Plants and their natural products have been utilized for thousands of years in the treatment of many human diseases. Because of their structural diversity, natural products are ideal lead compounds for drug development. In this thesis, the natural products of the plant Perovskia atriplicifolia were investigated, specifically their in vitro binding affinity to receptors in opioid and cannabinoid classes. Extracts that demonstrated good selective opioid and cannabinoid receptor binding were subjected to bioassay-guided fractionation. Four active compounds were identified from the crude extract in Perovskia atriplicifolia: 1) 5-hydroxy-3',4',6,7-tetramethoxy flavone, 2) 5,7-dihydroxy-3',4',6,-trimethoxy flavone, 3) 5-hydroxy-4',6,7-trimethoxy flavone, and 4) 5,7-dihydroxy-4',6,-dimethoxy flavone. Each …