Resveratrol is a naturally occurring phytoalexin of the stilbene family produced by various plants in response to stress, UV radiation, and fungal attack. It is primarily found in peanuts, berries, grape skin, and red wine. Resveratrol has been found to exhibit anti-cancer, anti-inflammatory, anti-aging, and anti-oxidant properties. Research indicates that diets enriched with resveratrol containing substances result in less incidence of cancer. Unfortunately, the low bioavailability and solubility has been a huge setback for its potential prospects. As a result, efforts have been made to synthesize derivatives of resveratrol with increased solubility and bioavailability. Three triester novel resveratrol derivatives 3, 4’, 5-tri (benzoyloxy) stilbene, 3, 4’, 5-tri (toluyloxy) stilbene and 3, 4’, 5-tri (2”-butenoyloxy) stilbene have been synthesized by esterification process that can further be subjected for biological evaluation. Structures and purities of all newly synthesized derivatives were confirmed by 1H, 13C NMR spectroscopy and infrared spectroscopy.

Recently, the compound resveratrol has had media attention as an anti carcinogen. However, the bioavailability of resveratrol is low in the human system due to its hydrophobic nature. Therefore, it must be administered in high dosages to be effective. A plethora of derivatives have been synthesized that have the potential of resveratrol but sadly share low bioavailability. The first effort of this research was an attempt to produce a more hydrophilic ester of resveratrol. Failing this, the final product was synthesized using a glycine derivative to produce 4-[(1E)-2-(3,5-diacetyloxyphenyl)ethenyl]phenyl N-[(1,1-dimethylethoxy)carbonyl]-glycinate.

Phytoestrogens are chemicals produced by plants that act like estrogens and have the ability to bind to the mammalian estrogen receptor system. The purpose of this study is to evaluate a new phytoestrogen analog called 4-acetoxy Resveratrol. Resveratrol is a phytoestrogen that has been found in the skin of grapes. Resveratrol has been shown to be able to bind to the estrogen receptors and has a similar molecular structure as estradiol. Resveratrol has been shown to have many positive health benefits such as improving cardiovascular health, serving as a neuroprotective agent, acting as an anti-inflammatory agent, working as an anti-cancer agent, increasing sperm output, acting as an anti-aging agent, and reducing incidence of prostatic adenocarcinoma. The challenge with using Resveratrol as an oral therapy is that it is quickly metabolized by the liver so for this study we used injections. The injections were 5mg/ Kg, 20 mg/Kg, and 90 mg/Kg of 4-acetoxy Resveratrol. We used intact 160 day old male Long-Evans rats and intact 90 day old female Long-Evans rats. The rats were given injections once a day for 21 days based on their treatment group. The animals were weighed daily and then tested in the Porsolt swim test at day 160 and 90 respectively. At the end of 21 days the rats were sacrificed and white adipose tissue, blood, brains, testis, and prostates were collected. Administration of 4-acetoxy Resveratrol decreased weight gain but not white adipose tissue in the male rats but has no effect in the females. In the male rat administration of 4-acetoxy resveratrol the high group also decreased testosterone, 5α-DHT , and prostate 5α-reductase activity. The high dose of 4-acetoxy Resveratrol also caused a change in prostate histology and decreased prostate weight. 4-acetoxy Resveratrol had no effect on testis weight and only showed a slight increase in depressive-like behaviors. In the females, 4-acetoxy resveratrol had no effect on white adipose tissue deposition, estrous cycle, hypothalamus aromatase activity, or depressive-like behaviors.

Total Synthesis of 4'-ester Resveratrol Analogs and 8, 9-amido Geldanamycin Analog and toward the Total Synthesis of (-)-Englerin A Yong Wang Department of Chemistry and Biochemistry, BYU Doctor of Philosophy The phytoalexin resveratrol and its 4'-ester analogs have been prepared with a decarbonylative Heck reaction. The deprotecting step has been modified and improved to increase yield and avoid chromatography. A set of resveratrol analogs and resveratrol have been tested with melanoma and pancreatic cell assays. The 8, 9-amido Geldanamycin analog has been synthesized with a convergent route, involving 28 simplified steps in its longest linear sequence. Synthetic methodologies, such as Andrus auxiliary controlled asymmetric anti-glycolate Aldol and selective p-Quinone formation, were employed. The total synthesis of Englerin A starts from (R)-carvone, passed through the modified Farvoskii ring-contraction and ring closing metathesis to get the ring skeleton. Other routes involving isopropyl group installation before closure of the seven-member ring failed. Although there are still problems to build the isopropyl moiety and the bridged ether, several reasonable alternative routes to address the problems have been designed.

Cabernet sauvignon is the most widely planted red wine grape in California and is valued in the hundreds of millions of dollars. Cabernet sauvignon grapes, when severely vigorous or overcropped, can contain vegetal aromas and flavors when harvested. 3-alkyl-methoxypyrazines are the volatile compounds responsible for this effect and can lower the perceived quality of the wine. Resveratrol is a phytoalexin that has many medical and health benefits and can be found in red wines. An experiment was conducted in Paso Robles, CA to assess the effects of five yield levels, manipulated through cluster thinning, on methoxypyrazine and resveratrol concentrations. Berry weights and chemistry were also measured, in the form of ºBrix, pH, and TA. In 2009 and 2010, no significant statistical differences were found in methoxypyrazines in the harvested grapes. In 2009, resveratrol concentrations were below the detection limits in the wine produced. In 2010, berry weight and chemistry measurements were not significantly different, except for grapes from lightly- and greatly-thinned vines which varied in pH at harvest. The 2009 wines were subjected to discrimination and preference testing by trained tasters. No significant difference was found in the discrimination test and no difference was found using the Friedman and Kramer’s Rank test for the preference test. Only a very slight difference was found between the wines made from unthinned and greatly-thinned vines according to Tukey’s Multiple Comparison Test. The findings of this thesis suggest that cluster thinning does not affect methoxypyrazine and resveratrol concentrations or sensory analysis in Cabernet sauvignon grown on the east side of Paso Roble, CA.

Las especies vegetales están continuamente desafiadas por un extenso número de microorganismos patógenos en su ambiente natural, principalmente bacterias, hongos y virus. Las plantas cuentan con un amplio arsenal de mecanismos de defensa tanto constitutivos como inducibles frente a estos patógenos. Los mecanismos inducibles incluyen el estadillo oxidativo, muerte celular rápida y localizada, acumulación de fitoalexinas y la síntesis de proteínas relacionadas con la patogénesis (Proteínas PR). Centrándose en las fitoalexinas, estas son compuestos antimicrobianos de bajo peso molecular producidos por las plantas en respuesta a estrés biótico y abiótico. Las fitoalexinas poseen un amplio rango de propiedades antifúngicas en varias especies de plantas. Debido a esto, estos compuestos han sido objeto de numerosos estudios en los últimos 20 años. En la Vid, Vitis vinífera L., la respuesta mejor caracterizada y más frecuente frente a la infección fúngica es la extensa acumulación de fitoalexinas y de proteínas relacionadas con la patogénesis. Entre la gran diversidad de compuestos naturales bioactivos producidos por las especies de Vitis sp. las fitoalexinas están formadas por un grupo reducido de moléculas que pertenecen a la familia de los estilbenos. Las células de vid son capaces de acumular estilnenos en respuesta a estrés biótico y abiótico. Los estilbenos de la vid derivan primariamente del trans-resveratrol (tR) (3,4`,5 -trihidroxiestilbeno), el cual puede sufrir modificaciones químicas, como glicosilación, metilación, hidroxilación y oligomerización, dando lugar a varios derivados bioactivos. tR es producido como producto final de la ruta fenilpropanoide mediante la condensación de tres moléculas de malonil-CoA con una molécula de 4-cumaroil-CoA en una reacción catalizada por la Estilbeno Sintasa (STS). Los estilbenos de la vid han sido ampliamente estudiados ya que poseen unas propiedades en la salud humana sorprendentes. Se han observado beneficios del tR en las principales enfermedades humanas, como agente cardioprotector, como inhibidor de la carcinogénesis, neuro protector y antienvejecimiento entre otros, ya que tR posee un alto efecto antioxidante. Por lo tanto, el interés farmacéutico, nutracéutico y biotecnológico del tR y por tanto de la vid, han ido en aumento desde los últimos 20 años. De la misma manera, la respuesta de defensa ha sido estudiada en cultivos celulares de vid utilizando inductores naturales de esta respuesta. En este sentido, la 2,6 dimetil-ꞵ-ciclodextrina (MBCD) ha demostrado poseer un gran poder elicitor, promoviendo la producción de tR y su acumulación extracelular. Estos estudios han permitido profundizar en los diferentes mecanismos de regulación de la producción de estos compuestos a través de análisis transcriptómicos y proteómicos. Sin embargo, estos análisis revelaron algunos genes y proteínas cuyo papel en esta respuesta es desconocida hasta hoy. Este es el caso de dos parálogos de Fosfoenolpiruvato carboxilasa quinasa (PPCK). La presente tesis doctoral está centrada en el estudio del posible papel regulador de la enzima fosfoenolpiruvato carboxilasa quinasa (PPCK) en la producción de resveratrol y otros metabolitos secundarios en cultivos celulares de vid. La hipótesis principal de esta tesis es que dicha enzima podría estar regulando el flujo del carbono entre el metabolismo primario y secundario, con la finalidad de potenciar este último en condiciones de estrés. Para demostrar esta hipótesis, el contenido de la tesis se divide en 4 capítulos principales. El primer capítulo consiste en el desarrollo de un método analítico basado en espectrometría de masas de triple cuadrupolo para la detección precisa y la cuantificación exacta de resveratrol y sus derivados bioactivos, producidos por la vid. El segundo capítulo es un estudio transcripcional de 2 isoformas de la enzima PPCK, así como el sustrato de esta última (la enzima fosfoenolpiruvato carboxilsa, PPC), en condiciones de estrés, además de estudiar la coexpression con enzimas y factores de transcripción cuyo papel en la producción de resveratrol se conoce con anterioridad. En este capítulo se demuestra tanto en cultivos celulares de vid, como en hojas intactas, que los genes PPCK son fuertemente reprimidos en condiciones de estrés, mientras que los genes PPC permanecen invariables. En cambio se produce un descenso en torno al 20-30% en la actividad específica de la enzima PPC en tales condiciones. Por otro lado, en tales condiciones se observa la correlación entre la represión de los genes PPCK y el incremento en la producción de resveratrol y otros estilbenos. Es conocido que los cambios de actividad en la enzima PPC son debidos a cambios en la fosforilación de la misma, los cuales a su vez son regulados por la PPCK. Por lo tanto el capítulo 3 consiste en el desarrollo de un método para detectar y cuantificar cambios en el patrón de fosforilación de la enzima PPC, adoptando una estrategia de proteómica dirigida. El desarrollo de esté método para la proteína PPC ha permitido observar como el estrés inducido por MBCD provoca un descenso en el grado de fosforilación de la enzima PPC, lo cual correlaciona directamente con el descenso en su actividad específica, y ambos efectos a su vez están correlacionados con la represión génica de su proteína reguladora PPCK. Por último el capítulo 4 es el análisis funcional de los genes PPCK, a través de transformación genética estable de cultivos celulares de vid, con la finalidad de sobreexpresar los genes PPCK, y del silenciamiento genético de los mismos. Los cultivos celulares transformates que sobreexpresaban alguno de los dos parálogos de PPCK resultaron acumular menos resveratrol en condiciones de estrés en comparación con la estirpe silvestre (WT) avalando de esta manera nuestra hipótesis inicial. Además, este efecto no afectaba únicamente a la producción de resveratrol, si no a una gran variedad de metabolitos secundarios producidos en la ruta fenilpropanoide (Flavonoides, taninos condensados, ácidos hidroxicinámicos etc.) sugiriendo que el papel de la enzima PPCK está implicado en la regulación del suministro de carbono hacia esa vía en condiciones de estrés. Por último, el silenciamiento genético tuvo una eficiencia inadecuada, sugiriendo que los genes PPCK pueden ser esenciales, y difícilmente manipulables por en la región metabólica en la que participan. / La presente tesis doctoral ha sido financiada gracias a la subvención de Ministerio de economía y competitividad (BIO2014-51861-R) y (BIO2017- 82374-R). Así mismo, las dos estancias de investigación realizadas en el Integrative Biology Institute, Liverpool University, (Liverpool) han sido financiadas por la Escuela de Doctorado de la Universidad de Alicante, a través del programa de subvenciones para facilitar la obtención de la mención de Doctorado internacional en el título de Doctor y por la European Molecular Biology Organization (EMBO) a través de su programa Short-term Fellowships (ASTF No 7754).

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The purported chemopreventive and chemotherapeutic properties of the dietary phytochemical resveratrol continue to undergo active investigations. Systemic pharmacokinetics of this compound revealed that it was rapidly and extensively metabolized into its sulfate and glucuronide conjugates. This extensive metabolism leads to high plasma levels of resveratrol sulfates and glucuronides and very low levels of the parent compound (low bioavailability). These observations raised many questions, some of which this body of work examined and has helped to explain. Chapter 1 presents a detailed introduction to resveratrol and its role in colorectal cancer chemoprevention. It also lays the foundation for the hypotheses generated and the studies presented in succeeding chapters. In chapter 2, we explored the possibility that resveratrol metabolites possess intrinsic activity and thus contribute to the observed effects of the parent. The mono-sulfated and glucuronidated conjugates of trans-resveratrol were synthesized and tested for antiproliferative activity in a panel of mammalian cell lines. Their activity was then compared with the parent compound. Resveratrol was shown to be antiproliferative in all cell lines studied while no discernible antiproliferative activity was observed for the metabolites. Chapter 3 details the results of the glucuronidation kinetics of cis and trans-resveratrol isomers across a wide concentration range chosen to mimic blood levels following high dose consumption. Human tissue microsomes and recombinant supersomes over-expressing the enzymes (UGTs) of interest were used for these studies. Our results show the presence of atypical kinetics for the formation of resveratrol glucuronides across most of the protein sources used. Prior to this study, the full glucuronidation kinetics of total resveratrol had not been conducted. In chapter 4, we examined the association between genetic polymorphisms in the major enzymes (UGT1A1 and UGT1A6) and rates of glucuronidation of trans and cis-resveratrol. We set out to correlate functional genetic variations in these UGTs with their catalytic rates and a positive association was made for cis-resveratrol and UGT1A6 where the UGT1A6 variants mediated higher glucuronidation rates compared to the reference genotype. Chapter 5 explored the inherent ability of resveratrol to induce its own glucuronidation upon chronic dosing. Enzyme induction has been proposed as a mechanism that may contribute to the low bioavailability of resveratrol. Since dietary polyphenols like resveratrol are not consumed in isolation, we also studied the effects of combining resveratrol with two dietary polyphenols (curcumin and chrysin) on two chemoprevention endpoints - i) antiproliferation and ii) UGT enzyme induction. Our results indicate that resveratrol is capable of inducing UGT1A1 expression and activity in a non-concentration dependent manner and this induction as well as its antiproliferative effects are enhanced by both curcumin and chrysin. In summary, en route to probing the activity of resveratrol metabolites, we optimized two synthetic routes and generated measurable quantities of these compounds for future use. While the in vitro kinetics of resveratrol did not allow for any in vivo predictions, we were able to show alterations in resveratrol metabolism with respect to genotypic differences and enzyme induction that may contribute to the observed low bioavailability profile. / Pharmaceutical Sciences

Introdução: A nefropatia induzida por contraste iodado (NIC) é um efeito adverso comum em pacientes com doença renal crônica (DRC). Caracteriza-se por uma síndrome de doença renal crônica agudizada. Resultado da vasoconstrição renal, hipóxia, ativação da cascata inflamatória, lesão celular oxidativa, a NIC deteriora a função renal, aumenta os dias de hospitalização, os custos hospitalares e a mortalidade do paciente portador de DRC. Objetivo: Avaliar o efeito renoprotetor do resveratrol, um polifenol com propriedades vasodilatadoras e anti-inflamatórias em ratos com doença renal crônica que receberam contraste iodado. Métodos: Ratos Wistar, machos, adultos randomizados em quatro grupos: SHAM: controle do modelo de doença renal crônica; Nx: ratos com nefrectomia 5/6 (modelo experimental de DRC); Nx+C: ratos Nx que receberam 6 ml/Kg de contraste iodado; Nx+C+R: animais Nx que foram pré-medicados com resveratrol 25 mg/Kg e 6 ml/Kg de contraste iodado. Foram avaliadas a função (clearance de inulina) e hemodinâmica renal (fluxo sanguíneo renal e resistência vascular renal), estresse oxidativo (peróxidos, óxido nítrico e substâncias reativas ao ácido tiobarbitúrico urinário e tióis no tecido renal) e análise histológica. Resultados: O uso de contraste resultou em deterioração da função renal, redução do fluxo sanguíneo renal, aumento da resistência vascular renal, lesão oxidativa e lesão túbulo intersticial dos ratos com DRC. O uso do resveratrol resultou na manutenção da taxa de filtração glomerular, do fluxo sanguíneo e resistência vascular renal, reduziu a lesão oxidativa e a lesão túbulo intersticial após a exposição ao contraste. Conclusões: O resveratrol apresentou efeito renoprotetor, hemodinâmico e antioxidante, na NIC em ratos com doença renal crônica. / Introduction: Contrast-Induced acute kidney injury (CI-AKI) is a common adverse effect in patients with chronic kidney disease (CKD). Result of renal vasoconstriction, hypoxia, activation of inflammatory cascade, oxidative cell damage, CI-AKI promote impaired renal function, increased length of hospitalization, hospital costs and mortality. Objective: This study evaluated the renoprotection of resveratrol, a polyphenol with vasodilating and anti-inflammatory properties in rats with chronic kidney disease receiving iodinated contrast media. Methods: Wistar, adult, male rats randomized into four groups; Sham: control of chronic renal injury model; Nx: rats with 5/6 nefrectomy (experimental model of CKD); Nx+IC: Nx rats that received 6 ml/kg of iodinated contrast; Nx+IC+R: Nx rats that received 6 ml/kg of iodinated contrast and resveratrol 25 mg/Kg. Renal function (inulin clearance), renal hemodynamics (renal blood flow and renal vascular resistance), oxidative profile (peroxides, urinary nitric oxide, reactive substances to thiobarbituric acid in urine, thiols in renal tissue) and histological analysis were evaluated. Results: Iodinated contrast led to impaired renal function, reduced renal blood flow, intensified renal vascular resistance, and promoted oxidative and tubular injury in CKD rats. Resveratrol maintained glomerular filtration rate, renal blood flow and vascular resistance, reduced oxidative and tubular injury after contrast exposition. Conclusion: Resveratrol showed a renoprotective effect, with antioxidant and hemodynamics impact, on CI-AKI in rats with CKD.