The cellular degradation of the low density lipoprotein (LDL) receptor, and its ligand, LDL, were investigated in order to clarify certain mechanistic aspects of these important processes. Long-term lymphoblastoid cell lines and cultured human skin fibroblasts were used to examine the fate of ¹²⁵I-LDL subsequent to its uptake via receptor-mediated endocytosis. In both cases, binding activity was saturable, depended on the presence of calcium ions in the medium, and was calculated to have an equilibrium dissociation constant at 4ᵒC of 2 μg ¹²⁵I-LDL/ml. No high-affinity binding was detected when the ligand was modified by acetylation. After incubating the monolayers at 37°C LDL/LDL receptor complexes were internalized, and the receptors were recycled back to the surface within about 10 minutes. Apolipo-protein B in the LDL particles was largely degraded to the amino acid level: chloroquine, a lysosomotropic agent, inhibited the formation of the ¹²⁵I-LDL degradation products. Cells obtained from a number of heterozygous and homozygous familial hypercholesterolemic patients, as expected, bound markedly reduced amounts of ligand. The half-life of ¹²⁵I-LDL was measured after it had been introduced into cultured fibroblasts by one of the following processes: (i) uptake via receptor-mediated endocytosis in human skin fibroblasts with normal LDL receptors, or (ii) incorporation via scrape-loading into fibroblasts defective in LDL receptor content. The half-lives obtained were about 1 hour and 50 hours, respectively, indicating that efficient degradation of LDL occurred only when it was deIivered to lysosomes via receptor-mediated endocytosis.

INTRODUÇÃO: Estudos mostraram que, após a injeção de LDE na corrente sangüínea de mulheres portadoras de câncer de mama, ela encontra-se mais concentrada em tecido neoplásico que no tecido normal. Recentemente, estudos pré-clínicos comprovaram que a associação LDE-oleato de paclitaxel é estável, menos tóxica e com mais atividade terapêutica quando comparada ao uso de paclitaxel comercial em animais. O presente estudo teve como objetivo verificar a estabilidade dessa associação na circulação, a sua capacidade em se concentrar no tecido neoplásico e determinar os parâmetros farmacocinéticos em relação ao paclitaxel isolado. MÉTODOS: Para determinar os parâmetros farmacocinéticos foram administrados, por via intravenosa, [3H]-oleato de paclitaxel associado a [14C]- oleato de colesterol-LDE em três pacientes e [3H]-paclitaxel comercial em duas pacientes 24 horas antes do procedimento cirúrgico. Todas as pacientes eram portadoras de neoplasia maligna da mama. Amostras de sangue foram colhidas durante 24 horas. A radioatividade foi medida por cintilação líquida e os parâmetros farmacocinéticos foram calculados usando um modelo multicompartimental. Fragmentos de tecido neoplásico e de tecido normal mamário foram coletados durante a cirurgia e submetidos à contagem radioativa. RESULTADOS: As taxas fracionais de remoção da LDE e do oleato de paclitaxel foram semelhantes (0,0296 ± 0,0264 e 0,0182 ± 0,0186, respectivamente, p = 0,5742). A captação tanto da LDE quanto do oleato de paclitaxel mostrou concentração 2,5 a 3 vezes maior no tecido tumoral do que no tecido mamário normal. O tempo de meia vida do oleato de paclitaxel foi maior do que o da formulação comercial (18,97 ± 7,7 horas e 7,34 ± 0,40 horas) e, a depuração plasmática, menor (1,51 ± 0,18 (L/h) e 7,95 ± 4,32 (L/h)). CONCLUSÃO: A maior parte do fármaco ficou retido na microemulsão até sua remoção da circulação e captação pelas células. O oleato de paclitaxel associado à LDE mostrou-se estável na circulação sangüínea e apresentou tempo de meia vida maior e a depuração plasmática menor do que a formulação comercial, além de se concentrar mais no tecido neoplásico da mama. Os resultados permitem sugerir que essa associação pode se constituir em uma estratégia útil no tratamento de mulheres portadoras de câncer da mama. / INTRODUCTION: Studies had shown that, after the injection of LDE in the circulation of women with breast cancer, it was more concentrate in neoplastic tissue that in the normal tissue. Recently, studies had proven that the LDE-paclitaxel oleate association is steady, less toxic and with more therapeutical activity when compared with the commercial paclitaxel in animals. The present study was designed to verify the stability of this association in the circulation, its capacity in concentrating in the neoplastic tissue and to determine the plasma kinetics of the association compared to that of paclitaxel isolated. METHODS: To determine the pharmacokinetic parameters, [3H]-paclitaxel oleate associated to LDE labeled with [14C]-cholesterol oleate was intravenously injected into three patients and [3H]-commercial paclitaxel into two patients 24 hours before the surgical procedure. All the patients had breast cancer. Blood samples were collected during 24 hours. Radioactivity was quantified in a scintillation solution and the pharmacokinetic parameters were calculated by compartmental analysis. Specimens of tumoral and normal breast were excised during the surgery and submitted to a radioactive counting. RESULTS: Fractional clearance rate of LDE and of the paclitaxel oleate were similar (0,0296 ± 0,0264 and 0,0182 ± 0,0186, respectively, p = 0,5742). The uptake of both [14C]-LDE and [3H]-paclitaxel oleate by breast malignant tissue was two and three fold greater than that of the normal breast tissue. The paclitaxel oleate plasma half-life (h) was greater than the commercial paclitaxel (T1/2 = 18,97 ± 7,7 and 7,34 ± 0,40) and the total plasma clearence (L/h) of paclitaxel oleate was lesser than the commercial (CL = 1,51 ± 0,18 and 7,95 ± 4,32). CONCLUSION: Most of the drug was restrained in the microemulsion until its removal from the circulation and captation by the cells. The paclitaxel oleate associated to LDE is stable in the bloodstream and has greater plasma half-life and lesser clearence than those for commercial paclitaxel. In addition, the association could be concentrated more in malignant breast tissue. The results allow to suggest that this association can consist in a useful strategy in the treatment of women with breast cancer.

7-cetocoleterol (7KC) é um oxisterol conhecido por inibir a proliferação celular e por ser citotóxico. Desenvolvemos uma nanoemulsão contendo 7KC (LDE/7KC) que demonstrou efeito anti-proliferativo sobre as linhagens RPMI 8226 (mieloma) e melanoma (B16F10), in vitro, sendo preferencialmente captada via receptores de LDL. No presente trabalho avaliamos, in vivo, a cinética plasmática, biodistribuição, ação anti-tumoral e parâmetros tóxico-hematológicos em camundongos portadores de melanoma. A cinética plasmática apresentou um decaimento estatisticamente igual entre os animais portadores de melanoma e não portadores. Em relação à biodistribuição da nanoemulsão, houve um acúmulo de seus componentes radioativamente marcados, principalmente no fígado e no tumor, sugerindo sua captação via receptores de LDL. LDE/7KC promoveu uma redução superior a cinqüenta por cento do tamanho do tumor, que apresentou maior área de necrose e menor quantidade de vasos. Nos camundongos tratados com LDE/7KC houve um aumento da sobrevida. As análises toxico-hematológicas demonstraram que a nanoemulsão apresentou pouca ou nenhuma toxicidade. Os resultados demonstram a possibilidade da utilização da nanoemulsão LDE/7KC como um agente no tratamento do câncer, com poucos efeitos colaterais, devido a sua seletividade aos receptores da LDL. / 7-ketocholesterol (7KC) is an oxysterol known to inhibit cell proliferation and to be cytotoxic. A nanoemulsion containing-7KC (LDE/7KC) was shown to have antiproliferative effects on RPMI 8226 myeloma cell line and melanoma (B16F10), in vitro. This particle is taken up mainly by LDL receptors. Here we have evaluated the plasma kinetic, biodistribution, anti-tumoral action and hematologic toxicity of LDE/7KC in melanoma bearing mice. The nanoemulsion accumulated in the liver and tumor, tissues with a high expression of LDL receptors. LDE/7KC promoted a tumor size reduction over fifty percent. An increased necrosis area and a decreased amount of blood vessels were found. An increased survival rate was observed. The hematolgic analyses demonstrated a lack of toxicicity. The results shows the possibility to use the LDE/7KC nanoemulsion as an agent for cancer treatment, with few collateral effects probably due to its internalization by LDL receptors.

INTRODUÇÃO: Estudos mostraram que, após a injeção de LDE na corrente sangüínea de mulheres portadoras de câncer de mama, ela encontra-se mais concentrada em tecido neoplásico que no tecido normal. Recentemente, estudos pré-clínicos comprovaram que a associação LDE-oleato de paclitaxel é estável, menos tóxica e com mais atividade terapêutica quando comparada ao uso de paclitaxel comercial em animais. O presente estudo teve como objetivo verificar a estabilidade dessa associação na circulação, a sua capacidade em se concentrar no tecido neoplásico e determinar os parâmetros farmacocinéticos em relação ao paclitaxel isolado. MÉTODOS: Para determinar os parâmetros farmacocinéticos foram administrados, por via intravenosa, [3H]-oleato de paclitaxel associado a [14C]- oleato de colesterol-LDE em três pacientes e [3H]-paclitaxel comercial em duas pacientes 24 horas antes do procedimento cirúrgico. Todas as pacientes eram portadoras de neoplasia maligna da mama. Amostras de sangue foram colhidas durante 24 horas. A radioatividade foi medida por cintilação líquida e os parâmetros farmacocinéticos foram calculados usando um modelo multicompartimental. Fragmentos de tecido neoplásico e de tecido normal mamário foram coletados durante a cirurgia e submetidos à contagem radioativa. RESULTADOS: As taxas fracionais de remoção da LDE e do oleato de paclitaxel foram semelhantes (0,0296 ± 0,0264 e 0,0182 ± 0,0186, respectivamente, p = 0,5742). A captação tanto da LDE quanto do oleato de paclitaxel mostrou concentração 2,5 a 3 vezes maior no tecido tumoral do que no tecido mamário normal. O tempo de meia vida do oleato de paclitaxel foi maior do que o da formulação comercial (18,97 ± 7,7 horas e 7,34 ± 0,40 horas) e, a depuração plasmática, menor (1,51 ± 0,18 (L/h) e 7,95 ± 4,32 (L/h)). CONCLUSÃO: A maior parte do fármaco ficou retido na microemulsão até sua remoção da circulação e captação pelas células. O oleato de paclitaxel associado à LDE mostrou-se estável na circulação sangüínea e apresentou tempo de meia vida maior e a depuração plasmática menor do que a formulação comercial, além de se concentrar mais no tecido neoplásico da mama. Os resultados permitem sugerir que essa associação pode se constituir em uma estratégia útil no tratamento de mulheres portadoras de câncer da mama. / INTRODUCTION: Studies had shown that, after the injection of LDE in the circulation of women with breast cancer, it was more concentrate in neoplastic tissue that in the normal tissue. Recently, studies had proven that the LDE-paclitaxel oleate association is steady, less toxic and with more therapeutical activity when compared with the commercial paclitaxel in animals. The present study was designed to verify the stability of this association in the circulation, its capacity in concentrating in the neoplastic tissue and to determine the plasma kinetics of the association compared to that of paclitaxel isolated. METHODS: To determine the pharmacokinetic parameters, [3H]-paclitaxel oleate associated to LDE labeled with [14C]-cholesterol oleate was intravenously injected into three patients and [3H]-commercial paclitaxel into two patients 24 hours before the surgical procedure. All the patients had breast cancer. Blood samples were collected during 24 hours. Radioactivity was quantified in a scintillation solution and the pharmacokinetic parameters were calculated by compartmental analysis. Specimens of tumoral and normal breast were excised during the surgery and submitted to a radioactive counting. RESULTS: Fractional clearance rate of LDE and of the paclitaxel oleate were similar (0,0296 ± 0,0264 and 0,0182 ± 0,0186, respectively, p = 0,5742). The uptake of both [14C]-LDE and [3H]-paclitaxel oleate by breast malignant tissue was two and three fold greater than that of the normal breast tissue. The paclitaxel oleate plasma half-life (h) was greater than the commercial paclitaxel (T1/2 = 18,97 ± 7,7 and 7,34 ± 0,40) and the total plasma clearence (L/h) of paclitaxel oleate was lesser than the commercial (CL = 1,51 ± 0,18 and 7,95 ± 4,32). CONCLUSION: Most of the drug was restrained in the microemulsion until its removal from the circulation and captation by the cells. The paclitaxel oleate associated to LDE is stable in the bloodstream and has greater plasma half-life and lesser clearence than those for commercial paclitaxel. In addition, the association could be concentrated more in malignant breast tissue. The results allow to suggest that this association can consist in a useful strategy in the treatment of women with breast cancer.

Charité, Universiẗat-Med., Diss--Berlin, 2005.

An egg yolk is a common component of diluents used as protectants of sperm cells during the process of cryopreservation. Its substitution by low density lipoproteins (LDL) has shown that it is the LDL that provides the egg yolk with its cryoprotective characteristics: it protects sperms against cold shock and other changes, thus helping to preserve their fertilization ability even after the freezing-thawing process. However, the sperm quality is affected by many other internal and external factors. Therefore, the aim of this study was to evaluate if the effect of the addition of LDL to the diluent of the ejaculate is significantly influenced by the following selected factors - the type of used diluent, the bull´s breed and its individuality and the date of the sampling. Experimental insemination doses were repetitively (four times) obtained from a group of six bulls (three Holstein bulls and three Czech Fleckvieh bulls) at the Natural Hradišťko insemination service s.r.o. The samples of semen were diluted with two types of non-egg diluents containing soybean lecithin extracts (AndoMed and BioXCell). To each of these diluents, LDL at 4, 6, and 8% concentration was added; a non-LDL diluent served as a check. All the insemination doses were frozen by a standard procedure and then stored in liquid nitrogen. The CASA system (Computer Assisted Sperm Analysis) was selected for the evaluation of the sperm motility. Immediately after thawing and then after two hours of incubation in water bath (37 °C), the values of kinematic parameters were obtained from the samples - the total percentage of motile sperm and the percentage of progressively motile sperm. These data were then statistically processed; based on the outputs, VAP, VCL, ALH and the percentage of progressively motile sperm (PMOT) were chosen as representative kinematic parameters. The values of the parameters were higher in almost all evaluated samples diluted with BioXCell, compared to those diluted with AndroMed. Even though we demonstrated the existence of a high variability of results depending on the time of incubation, bull breed, the individuality of the bull and the date of the sampling, it could be said that the best concentration of added LDL is 6 % for BioXCell and 8 % for AndroMed. It would be useful to perform more experiments evaluating the effect of adding LDL to non-egg diluents on the quality of the thawed sperm. In these experiments, more sperm quality parameters should be examined and factors influencing the variability of results demonstrated in this work should be taken into consideration.

7-cetocoleterol (7KC) é um oxisterol conhecido por inibir a proliferação celular e por ser citotóxico. Desenvolvemos uma nanoemulsão contendo 7KC (LDE/7KC) que demonstrou efeito anti-proliferativo sobre as linhagens RPMI 8226 (mieloma) e melanoma (B16F10), in vitro, sendo preferencialmente captada via receptores de LDL. No presente trabalho avaliamos, in vivo, a cinética plasmática, biodistribuição, ação anti-tumoral e parâmetros tóxico-hematológicos em camundongos portadores de melanoma. A cinética plasmática apresentou um decaimento estatisticamente igual entre os animais portadores de melanoma e não portadores. Em relação à biodistribuição da nanoemulsão, houve um acúmulo de seus componentes radioativamente marcados, principalmente no fígado e no tumor, sugerindo sua captação via receptores de LDL. LDE/7KC promoveu uma redução superior a cinqüenta por cento do tamanho do tumor, que apresentou maior área de necrose e menor quantidade de vasos. Nos camundongos tratados com LDE/7KC houve um aumento da sobrevida. As análises toxico-hematológicas demonstraram que a nanoemulsão apresentou pouca ou nenhuma toxicidade. Os resultados demonstram a possibilidade da utilização da nanoemulsão LDE/7KC como um agente no tratamento do câncer, com poucos efeitos colaterais, devido a sua seletividade aos receptores da LDL. / 7-ketocholesterol (7KC) is an oxysterol known to inhibit cell proliferation and to be cytotoxic. A nanoemulsion containing-7KC (LDE/7KC) was shown to have antiproliferative effects on RPMI 8226 myeloma cell line and melanoma (B16F10), in vitro. This particle is taken up mainly by LDL receptors. Here we have evaluated the plasma kinetic, biodistribution, anti-tumoral action and hematologic toxicity of LDE/7KC in melanoma bearing mice. The nanoemulsion accumulated in the liver and tumor, tissues with a high expression of LDL receptors. LDE/7KC promoted a tumor size reduction over fifty percent. An increased necrosis area and a decreased amount of blood vessels were found. An increased survival rate was observed. The hematolgic analyses demonstrated a lack of toxicicity. The results shows the possibility to use the LDE/7KC nanoemulsion as an agent for cancer treatment, with few collateral effects probably due to its internalization by LDL receptors.

No description available.

OSM, ein Vertreter der IL-6-Typ-Zytokine, ist nicht nur für entzündliche, sondern auch für metabolische Prozesse von Bedeutung. Vorarbeiten der Arbeitsgruppe GEIER/HERMANNS und Studien von KOMORI et al. legen protektive Eigenschaften des Zytokins nahe, da Mäuse, denen OSMR fehlte, Charakteristika des metabolischen Syndroms aufwiesen. Zur eingehenderen Untersuchung der von OSM vermittelten Wirkung auf den murinen Lipidstoffwechsel wurden zwei für die NAFLD und Atherosklerose anfällige Modelle herangezogen und jeweils in Gegenwart und Abwesenheit des Osmr studiert: Weibliche Apoe-/-(Osmr-/-) und Ldlr-/-(Osmr-/-) Mäuse wurden über einen Zeitraum von zwölf Wochen mit westlicher Diät gefüttert, wöchentlich gewogen, am Ende der Diät geopfert und geerntet. Wildtypische C57Bl/6-Mäuse erfuhren die gleiche Behandlung und dienten als Referenzgruppe. Im Rahmen des Promotionsprojektes wurden Leberfettgehalt, Serumlipidspiegel, Lipoproteinfraktionen und Stuhllipide von Apoe-deletierten Mäusen bestimmt und mit bereits vorhandenen Daten der Ldlr-/-(Osmr-/-) und wildtypischen Mäuse in Beziehung gesetzt. Expressionsanalysen von am Lipidstoffwechsel beteiligten Genen in Darm-, Leber- und Fettgewebe trugen dazu bei, OSM-abhängige Regulationen aufzudecken. Ldlr-/- Tiere nahmen unter der Diät exzessiv zu, hatten hohe Serumspiegel an Leptin, Gluco-se und Lipiden, eine Lebersteatose und, begleitet von einer Induktion des Vldlr, erhöhte inflammatorische Marker im visceralen Fettgewebe. Der zusätzliche Knockout des Osmr ging mit einer geringeren Vldlr-Expression im Fettgewebe und einer hepatozytären Induktion von Cyp7a1 einher und resultierte in einem metabolisch günstigeren Phänotyp. Apoe-defiziente Tiere unterschieden sich hinsichtlich ihrer Gewichtszunahme nicht von Ldlr-/-Osmr-/- und C57Bl/6-Mäusen. Überraschenderweise zeigten sich im Serum von Apoe-/-Osmr-/- jedoch gegenüber Apoe-/- Mäusen erhöhte Konzentrationen des Gesamt- und VLDL-Cholesterins, der Triglyceride und freien Fettsäuren. Obwohl Lebern der Apoe-/-Osmr-/- Mäuse geringere Ldlr- und Lrp1-mRNA-Spiegel als die der Apoe-/- Mäuse aufwiesen, hatten sie einen höheren hepatischen Cholesteringehalt. Bei gesteigerter Cpt1a-Expression fiel der hepatische Tri-glyceridgehalt Apoe-deletierter Mäuse geringer aus als in Ldlr-/-(Osmr-/-) und wildtypischen Tieren. Unter Umgehung einer Fettgewebsentzündung präsentierten Apoe-defiziente Mäuse Hinweise einer inflammatorischen Leberschädigung, die pathogenetisch am ehesten mit einer gestörten Cholesterinhomöostase in Verbindung zu bringen war. Abhängig vom genetischen Hintergrund des Mausmodells hatte OSM schützende oder schädliche Effekte auf den Lipidmetabolismus. Die Ergebnisse der vorliegenden Arbeit betonen die entscheidende Bedeutung entzündlicher, von OSM modulierter Prozesse für den Fettstoffwechsel in Leber- und Fettgewebe. Weiterführende Experimente sind nötig, um die den Beobachtungen zugrunde liegenden molekularen Mechanismen zu entschlüsseln. / OSM, a member of the IL-6-type family, plays a pivotal role not only in inflammatory pro-cesses, but also in the regulation of metabolism. In line with studies conducted by KOMORI et al., findings obtained by GEIER/HERMANNS revealed characteristics of the metabolic syndrome in mice lacking the OSMR. Therefore, protective properties of OSM were suggested. In order to further investigate OSM-mediated effects on murine lipid metabolism, two models prone to NAFLD and atherosclerosis were employed and studied in the presence and absence of Osmr: Female Apoe-/-(Osmr-/-) and Ldlr-/-(Osmr-/-) mice were fed a Western-type diet for twelve weeks, weighed weekly, sacrificed and harvested at the end of the diet. Wild-type C57Bl/6 mice underwent the same procedure and were used as a reference group. Thereafter, lipid levels and lipoprotein fractions in the sera of Apoe-deleted mice were deter-mined. In addition, their lipid content in liver tissue and stool was measured. Findings were compared with data from Ldlr-/-(Osmr-/-) and wild-type mice. To reveal OSM-dependent regulations of genes playing a key role in lipid metabolism, gene expression analyses were performed in intestinal, liver, and adipose tissue samples from all mice groups. Ldlr-/- animals excessively gained weight during the diet, had high serum levels of leptin, glucose, and lipids, hepatic steatosis, and, accompanied by induction of Vldlr, increased inflammatory markers in visceral adipose tissue. The additional knockout of Osmr was accom-panied by a lower Vldlr expression in adipose tissue and an induction of liver Cyp7a1, resulting in a metabolically favorable phenotype. In terms of weight gain, Apoe-deficient animals were not different from Ldlr-/-Osmr-/- and C57Bl/6 mice. Surprisingly, however, serum from Apoe-/-Osmr-/- mice showed increased concentrations of total and VLDL cholesterol, triglyc-erides, and free fatty acids when compared to Apoe-/- animals. Despite lower hepatic Ldlr and Lrp1 mRNA levels, Apoe-/-Osmr-/- mice had a higher hepatic cholesterol content than Apoe-/- mice. Fitting to an increased Cpt1a expression, the hepatic triglyceride content of Apoe-deleted mice was lower than in Ldlr-/-(Osmr-/-) and wild-type mice. Most likely due to an impaired hepatic cholesterol homeostasis, liver sections of Apoe-deleted mice displayed features of inflammation, whereas the adipose tissues of these animals remained rather unscathed. Depending on the genetic background of the mouse model, OSM had protective or deleterious effects on lipid metabolism. The results of this project emphasize the significance of OSM regarding both inflammation and metabolism in liver and adipose tissue. Further ex-periments are needed to unravel the molecular mechanisms underlying these observations.

Atherosclerosis is thought to be initiated by endothelial cell dysfunction. Research described in this dissertation is focused on interactions of nutrients, cytokines and pharmaceutical compounds in the intracellular signaling pathways leading to endothelial cell activation. The flavonoid quercetin could significantly downregulate the inflammatory pathways induced by linoleic acid as determined by DNA binding assays of the proinflammatory transcription factors nuclear factor-kappaB and activator protein-1 as well as by gene expression studies of interleukin-6 and vascular adhesion molecule-1. Interestingly, quercetin and vitamin E also prevented the linoleic acid-induced activation of PPAR DNA binding - suggesting a role of oxidation in the fatty acid-mediated induction of PPAR. In addition, we studied an interaction of zinc with the antiinflammatory transcription factors, peroxisome proliferator activated receptors (PPARs) alpha and gamma. Our data suggest that PPAR alpha and gamma and their synthetic agonists require zinc for their antiinflammatory properties in endothelial cells. We could confirm the importance of zinc in PPAR gamma signaling in vivo by a decreased PPAR DNA binding activity in livers of zinc deficient mice. Furthermore, zinc had dramatic lipid lowering effects in LDL-receptor deficient mice on a diet rich in corn oil. Triglycerides, phospholipids and cholesterol levels were significantly elevated in mice receiving a zinc deficient diet when compared to control and where decreased in zinc supplemented animals. Zinc deficiency also increased oxidative stress as determined by quantitation of plasma isoprostanes and mRNA expression of glutathione reductase. In conclusion, our data show novel interactions of proinflammatory nutrients, such as linoleic acid, with antioxidant and anti-inflammatory nutrients, such as quercetin and zinc.