Thesis (Ph. D.)--Columbia University, 1940. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 102-124). Also available on the World Wide Web.

Graduation date: 1994

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains xiv, 139 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 129-138).

Kong Ka-lai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 126-149). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abstract (Chinese Version) --- p.vi / Table of Contents --- p.ix / List of Figures/Table --- p.xiii / List of Abbreviations --- p.xvi / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- An Overview on Cancer --- p.1 / Chapter 1.2 --- Biological Effects of Chlorophyllin --- p.7 / Chapter 1.2.1 --- CHL as Photosensitizer --- p.7 / Chapter 1.2.2 --- CHL as Antioxidant --- p.8 / Chapter 1.2.3 --- CHL as Anticarcinogenic Agent --- p.9 / Chapter 1.3 --- Regulation of Cell Cycle --- p.13 / Chapter 1.3.1 --- Cell-Cycle Checkpoints --- p.13 / Chapter 1.3.2 --- Cell-Cycle Regulatory Proteins --- p.15 / Chapter 1.4 --- Regulation of Mitogen-Activated Protein Kinase (MAPK) Signaling Cascade --- p.21 / Chapter 1.5 --- Programmed Cell Death (or Apoptosis) --- p.27 / Chapter 1.5.1 --- Regulation of Caspase-Dependent Apoptosis --- p.28 / Chapter 1.5.2 --- Regulation of Caspase-Independent Cell Death --- p.32 / Chapter 1.5.3 --- Bcl-2 Family Proteins in Modulation of Cell Death --- p.32 / Chapter 1.6 --- In Vivo Antitumor Screening System --- p.37 / Chapter 1.7 --- Aims of the Present Study --- p.38 / Chapter Chapter 2 --- In Vitro Studies of the Anticancer Effect of Chlorophyllin / Chapter 2.1 --- Introduction --- p.39 / Chapter 2.1.1 --- DNA-Flow Cytometric Analysis --- p.51 / Chapter 2.1.2 --- Western Blot Analysis --- p.54 / Chapter 2.2 --- Materials and Methods --- p.56 / Chapter 2.2.1 --- Maintenance of Cell Lines --- p.56 / Chapter 2.2.2 --- Cytotoxic and Cytostatic Effects on the Cancer Cells --- p.56 / Chapter 2.2.3 --- DNA-Flow Cytometric Analysis --- p.60 / Chapter 2.2.4 --- Western Blot Analysis --- p.61 / Chapter 2.2.5 --- JC-1 Mitochondrial Potential Sensor --- p.64 / Chapter 2.2.6 --- Caspase Inhibitors --- p.65 / Chapter 2.2.7 --- Statistical Analysis --- p.66 / Chapter 2.2.8 --- Densitometric Analysis --- p.66 / Chapter 2.3 --- Results --- p.67 / Chapter 2.3.1 --- Effects of CHL on the Growth of Human Cancer Cells by MTT Assay --- p.67 / Chapter 2.3.2 --- Effect of CHL on the Proliferation of MCF-7 Cells by Chemi-BrdU Incorporation --- p.69 / Chapter 2.3.3 --- Effect of CHL on Cell Cycle of MCF-7 Cells --- p.71 / Chapter 2.3.4 --- Effect of CHL on the Cyclin D1 Expression in MCF-7 Cells --- p.74 / Chapter 2.3.5 --- Effects of CHL on JNK and c-Jun Expressions and Their Phosphorylations in MCF-7 Cells --- p.76 / Chapter 2.3.6 --- Effect of CHL on DNA fragmentation in MCF-7 Cells --- p.78 / Chapter 2.3.7 --- Effect of CHL on Mitochondrial Membrane Potential of MCF-7 Cells --- p.80 / Chapter 2.3.8 --- Effects of CHL on the PARP Expression and Cleavage in MCF-7 Cells --- p.83 / Chapter 2.3.9 --- "Effects of CHL on Bcl-2, Bcl-xL and Bad Expressions in MCF-7 Cells" --- p.85 / Chapter 2.3.10 --- Effects of CHL on Caspase Activations in MCF-7 Cells --- p.88 / Chapter 2.3.11 --- Effects of Caspase Inhibitors on the CHL-Induced Apoptosis in MCF-7 Cells --- p.90 / Chapter 2.4 --- Discussion --- p.93 / Chapter Chapter 3 --- In Vivo Studies of the Anticancer Effect of Chlorophyllin / Chapter 3.1 --- Introduction --- p.104 / Chapter 3.2 --- Materials and Methods --- p.106 / Chapter 3.2.1 --- Transplantation of MCF-7 Cells into the Nude Mice and Treatment --- p.106 / Chapter 3.2.2 --- Western Blot Analysis --- p.107 / Chapter 3.2.3 --- Statistical Analysis --- p.107 / Chapter 3.3 --- Results --- p.108 / Chapter 3.3.1 --- In Vivo Antitumor Activity of CHL --- p.108 / Chapter 3.3.2 --- In Vivo Effects of CHL on Cyclin D1 and Bcl-2 Expressions in MCF-7 Solid Tumor --- p.111 / Chapter 3.4 --- Discussion --- p.113 / Chapter Chapter 4 --- General Discussion --- p.115 / References --- p.126

Chlorophyllin (CHL), a water-soluble derivative of the green plant pigment, chlorophyll, is an effective antimutagen and anticarcinogen in various model systems when used as a modulator against a class of carcinogens that, in general, have a structure consisting of at least three fused rings. Dibenzo[a,l]pyrene (DBP), an extremely potent environmental carcinogen, has been isolated from urban air samples, tobacco smoke, and coal smoke condensate. A study was conducted to evaluate the complex interrelationships among dietary DBP doses with co-exposure to a range of CHL doses. In order to achieve adequate statistical power in the generation of multiple dose-response curves, this dose-dose matrix experiment utilized over 12,000 rainbow trout. The resulting DNA adducts were assessed and evaluated as biomarkers of exposure to discern their relationship with the final tumor outcome. CHL was highly effective in reducing DBP-initiated DNA adduct formation in the liver and stomach and strongly inhibited tumor formation in the liver (56-79% inhibition), stomach (30-68%), and swim bladder (over 80% at the highest DBP dose). Molecular dosimetry revealed adduct formation to be predictive of final tumor response in both organs regardless of CHL dose. Other parameters evaluated were consistent with CHL-mediated protection. A clinical CHL preparation, evaluated in a human population subsequent to the seminal demonstration of CHL chemopreventive properties against AFB��� in trout (1), revealed CHL to be just as effective in reducing biomarkers of alfatoxin exposure to humans (2). Dietary administration of this clinical preparation along with DBP in the rainbow trout demonstrated CHL protective capacity against DBP-initiated multi-organ DNA adduct formation and final tumor incidence. Sucrose was evaluated, deemed unlikely to be sequestered in a complex with CHL, and was used as a control in a pharmacokinetic study evaluating the biodistribution of DBP with and without CHL. The results provide evidence against a non-specific masking mechanism for CHL-mediated blocking of DBP (or aflatoxin)-initiated tumorigenesis. CHL at multiple doses provided significant protection against multi-dose DBP-initiated DNA adduction and tumor formation in multiple organs. CHL-mediated protection, primarily by reduced carcinogen biouptake and consistent with a complexation mechanism, is supported by these results. / Graduation date: 2003

Copper chlorophyllin (CuChl) is an antioxidant from renewable sources, which has shown as a potential active ingredient in agricultural crop sprays. The major objectives of this thesis are to understand the colloidal and interfacial behaviors of CuChl, and to develop strategies for improving its effectiveness in field applications. In this project, the following three areas are examined and analyzed. In practice, CuChl-based formulations are sprayed directly onto a plant’s foliage. As such, there is a need to understand how CuChl interacts with relevant plant surfaces. To this end, quartz crystal microbalance with dissipation (QCM-D) was used to quantify the adsorption of CuChl aqueous solutions onto four model surfaces: polystyrene, cellulose, pullulan, and silica. The results showed that cellulose adsorbed the highest amount of CuChl, followed by polystyrene and pullulan. In addition, the results also showed that the surfactants, SDS or DTAB, could alter the binding of CuChl to cellulose when used in concentrations above the critical micelle concentration. CuChl is composed of water-soluble and dispersed components, therefore it is not intrinsically rainfast, which limits its field application. To immobilize CuChl on leaves, a polymer combination of CMC (carboxymethyl cellulose) and PAE (polyamidoamine-epichlorohydrin) was designed for use as a spray adjuvant. The release behaviors of CuChl from dried spray deposits were investigated using varied polymer compositions and concentrations and compared with those of a water-soluble dye, brilliant sulfaflavine (BSF). The results indicated that a small amount of CuChl was immediately released upon exposure to water whereas BSF’s release behavior was dependent on the square root of time. The unusual behavior of CuChl was attributed to the presence of particles in the solution. These nanoparticles were coated with CMC:PAE complex, with the result of being immobilized on parafilm. Suspoemulsion is the most complex agricultural formulation that is composed of both dispersed particles and emulsion droplets. The objective of this work is to understand the relationship between the solution properties of suspoemulsions and the resulting dried deposits on hydrophobic surfaces. The results showed that the distribution of polychlorinated Cu (II) phthalocyanine (PG7) particles in dried deposits was related to the extent to which PG7 particles were adsorbed on or entrained in oil droplets. The PG7 particles that mainly ended up in the center (dome) area after drying were bound to the oil/water interface in the suspoemulsion, whereas individually dispersed particles ended up in the annulus. / Thesis / Doctor of Philosophy (PhD) / Agricultural formulations have been developed and widely applied to crops in an effort to maximize yields to keep up with the food demands of the world’s ever-growing population. However, there are still many challenges associated with the application of these formulations, such as huge losses due to spray drift, wash-off, and degradation during spraying. These issues can reduce the formulation’s overall efficacy and pose serious risks to the environment and human health. The primary objective of this thesis is to explore the agricultural application of a new environmentally-friendly active ingredient, copper chlorophyllin (CuChl). To this end, this work begins by determining CuChl’s colloidal and adsorption behaviors, with a particular focus on its binding tendencies for relevant plant surfaces. Next, a polymer combination was designed as a spray adjuvant to enhance CuChl’s rainfastness performance and CuChl’s release from dried deposits was characterized. Finally, the distribution of dispersed particles in dried suspoemulsion deposits was determined.

A ingestão de clorofilina cupnca (Chl-Cu), na forma de corante ou suplemento alimentar tem sido associada a potenciais efeitos benéficos e à prevenção de doenças crônicas. Contudo, são poucos os estudos relativos à sua absorção in vivo, atividade antioxidante e impacto no estado nutricional. Diante desses fatos, foram realizados três experimentos com ratos, nos quais foram fornecidas dietas suplementadas com o 1 e 3% de Chl-Cu. No primeiro experimento foi comprovada a absorção da Chl-Cu para o sangue e distribuição para fígado e rins. A análise da Chl-Cu comercial por CLAE evidenciou a presença de dois componentes principais, a clorina cúprica e4 e clorina cúprica e6. A clorina cúprica e4 foi efetivamente absorvida e detectada no soro, fígado e rins, enquanto a clorina cúprica e6 não foi detectada. Esses resultados indicam que a clorina cúprica e6 foi degradada durante a passagem pelo trato gastrointestinal ou não foi absorvida e distribuída para os órgãos. A análise histológica do intestino não indicou qualquer alteração morfológica. No segundo experimento foi avaliado o potencial antioxidante da Chl-Cu em ratos sob estresse oxidativo induzido por deficiência de vitamina E, e administração concomitante de Chl-Cu na dieta. A Chl-Cu foi capaz de proteger o tecido cerebral contra o aumento nos níveis de TBARS, mas o mesmo não foi observado no tecido hepático. Não houve diferença significativa na atividade das enzimas antioxidantes (CAT, SOD, GPx e GR) entre os grupos controle, deficiente em vitamina E e suplementado com Chl-Cu. Desta forma, o efeito antioxidante da Chl-Cu parece ser relacionado a uma proteção contra a peroxidaçãolipídica in situ, mas não com a modulação enzimática. Foi possível observar ainda, que os animais que receberam suplementação com Chl-Cu apresentaram ganho de peso menor que os respectivos controles. Um terceiro experimento foi realizado para investigar se o reduzido ganho de peso dos animais foi causado pela baixa aceitação da dieta ou estaria relacionado a algum efeito antinutricional da Chl-Cu. Em ensaio biológico pair-fed, o efeito negativo da Chl-Cu no desenvolvimento dos animais foi confirmado. A ingestão do pigmento levou também a uma diminuição na digestibilidade protéica aparente indicando ter havido comprometimento na absorção do N protéico e possivelmente de algum outro nutriente..A literatura indica a formação de complexos da Chl-Cu com diferentes componentes alimentares apoiando a hipótese de que a Chl-Cu possa ter interferido na utilização de proteínas e possivelmente de outros nutrientes pelo organismo animal. / The ingestion copper chlorophyllin (Cu-Chl) in form of as food colorant or supplement has been associated with potential beneficiai effects and the prevention of chronic diseases. However, little is known about Cu-Chl absorption in vivo, its antioxidant activity and its impact on status nutritional. In view of these facts, three experiments were carried out on rats fed 1 and 3% Cu-Chl supplemented diets. In the first experiment, the absorption of the pigment into the bloodstream and distribution to the liver and kidneys was comproved. HPLC analysis of food-grade Cu-Chl evidenced two main components, copper-chlorin e6 and copper-chlorin e4. The Cu-chlorin e4 was effectively absorbed and found in serum, liver and kidneys, while Cu-chlorin e6 was not detected. This result indicates that Cu-chlorin e6 was either not absorbed and distributed to organs or was degraded during its passage through the gastrointestinal tract. Histological analyses of the intestine did not show any morphological alteration. In a second experiment the antioxidant potential of Cu-Chl in rats under oxidative stress induced by vitamin E deficiency and simultaneous Cu-Chl administration in diet was investigated. The presence of chlorophyllin protected brain tissue from increased TBARS leveis, however the same effect was not observed in liver. There was no significant differences in activities of antioxidant enzymes (CAT, SOD, GPX and GR) among control, vitamin E deficient and Cu-Chl-supplemented groups. Therefore, any antioxidant effect of Cu-Chl seems to be related to in situ protection against peroxidation but not to antioxidant enzyme modulation. Weight gains of the animais receiving Cu-Chl supplemented diets were lower than those of the control group. A third experiment was conducted to evaluate whether the reduced weight gain was caused by low food acceptance or by an antinutritional effect of Cu-Chl. This biological pair-fed test confirmed the negative effect of CuChl on the development of rats. The pigment ingestion also decreased the apparent protein digestibility indicating that N protein and possibly other nutrients had itsabsorption compromised. Literature evidenced formation of Cu-Chl complexes with different food components supporting the hypothesis that Cu-Chl might have interfered in the utilization of proteins and possibly other nutrients by the animal organism.

A ingestão de clorofilina cupnca (Chl-Cu), na forma de corante ou suplemento alimentar tem sido associada a potenciais efeitos benéficos e à prevenção de doenças crônicas. Contudo, são poucos os estudos relativos à sua absorção in vivo, atividade antioxidante e impacto no estado nutricional. Diante desses fatos, foram realizados três experimentos com ratos, nos quais foram fornecidas dietas suplementadas com o 1 e 3% de Chl-Cu. No primeiro experimento foi comprovada a absorção da Chl-Cu para o sangue e distribuição para fígado e rins. A análise da Chl-Cu comercial por CLAE evidenciou a presença de dois componentes principais, a clorina cúprica e4 e clorina cúprica e6. A clorina cúprica e4 foi efetivamente absorvida e detectada no soro, fígado e rins, enquanto a clorina cúprica e6 não foi detectada. Esses resultados indicam que a clorina cúprica e6 foi degradada durante a passagem pelo trato gastrointestinal ou não foi absorvida e distribuída para os órgãos. A análise histológica do intestino não indicou qualquer alteração morfológica. No segundo experimento foi avaliado o potencial antioxidante da Chl-Cu em ratos sob estresse oxidativo induzido por deficiência de vitamina E, e administração concomitante de Chl-Cu na dieta. A Chl-Cu foi capaz de proteger o tecido cerebral contra o aumento nos níveis de TBARS, mas o mesmo não foi observado no tecido hepático. Não houve diferença significativa na atividade das enzimas antioxidantes (CAT, SOD, GPx e GR) entre os grupos controle, deficiente em vitamina E e suplementado com Chl-Cu. Desta forma, o efeito antioxidante da Chl-Cu parece ser relacionado a uma proteção contra a peroxidaçãolipídica in situ, mas não com a modulação enzimática. Foi possível observar ainda, que os animais que receberam suplementação com Chl-Cu apresentaram ganho de peso menor que os respectivos controles. Um terceiro experimento foi realizado para investigar se o reduzido ganho de peso dos animais foi causado pela baixa aceitação da dieta ou estaria relacionado a algum efeito antinutricional da Chl-Cu. Em ensaio biológico pair-fed, o efeito negativo da Chl-Cu no desenvolvimento dos animais foi confirmado. A ingestão do pigmento levou também a uma diminuição na digestibilidade protéica aparente indicando ter havido comprometimento na absorção do N protéico e possivelmente de algum outro nutriente..A literatura indica a formação de complexos da Chl-Cu com diferentes componentes alimentares apoiando a hipótese de que a Chl-Cu possa ter interferido na utilização de proteínas e possivelmente de outros nutrientes pelo organismo animal. / The ingestion copper chlorophyllin (Cu-Chl) in form of as food colorant or supplement has been associated with potential beneficiai effects and the prevention of chronic diseases. However, little is known about Cu-Chl absorption in vivo, its antioxidant activity and its impact on status nutritional. In view of these facts, three experiments were carried out on rats fed 1 and 3% Cu-Chl supplemented diets. In the first experiment, the absorption of the pigment into the bloodstream and distribution to the liver and kidneys was comproved. HPLC analysis of food-grade Cu-Chl evidenced two main components, copper-chlorin e6 and copper-chlorin e4. The Cu-chlorin e4 was effectively absorbed and found in serum, liver and kidneys, while Cu-chlorin e6 was not detected. This result indicates that Cu-chlorin e6 was either not absorbed and distributed to organs or was degraded during its passage through the gastrointestinal tract. Histological analyses of the intestine did not show any morphological alteration. In a second experiment the antioxidant potential of Cu-Chl in rats under oxidative stress induced by vitamin E deficiency and simultaneous Cu-Chl administration in diet was investigated. The presence of chlorophyllin protected brain tissue from increased TBARS leveis, however the same effect was not observed in liver. There was no significant differences in activities of antioxidant enzymes (CAT, SOD, GPX and GR) among control, vitamin E deficient and Cu-Chl-supplemented groups. Therefore, any antioxidant effect of Cu-Chl seems to be related to in situ protection against peroxidation but not to antioxidant enzyme modulation. Weight gains of the animais receiving Cu-Chl supplemented diets were lower than those of the control group. A third experiment was conducted to evaluate whether the reduced weight gain was caused by low food acceptance or by an antinutritional effect of Cu-Chl. This biological pair-fed test confirmed the negative effect of CuChl on the development of rats. The pigment ingestion also decreased the apparent protein digestibility indicating that N protein and possibly other nutrients had itsabsorption compromised. Literature evidenced formation of Cu-Chl complexes with different food components supporting the hypothesis that Cu-Chl might have interfered in the utilization of proteins and possibly other nutrients by the animal organism.

A clorofilina cúprica de sódio (CuChl) é um corante semissintético derivado da clorofila. Quimicamente é constituído de diversas clorinas, em especial a clorina cúprica e4 (CuCe4), a clorina cúprica e6 (CuCe6), e possíveis clorinas e porfirinas não cúpricas em proporções variáveis. Além do seu uso como corante alimentar, são atribuídas atividades biológicas à CuChl, tais como, antimutagênica, anticarcinogênica e antioxidante. Em decorrência destes potenciais efeitos benéficos, sua comercialização sob a forma de suplementos é crescente. Todavia, curiosamente, informações sobre a absorção e biodisponibilidade da CuChl são escassas. Além disso, até o momento nenhum estudo avaliou o impacto da composição da CuChl em sua bioatividade e eficácia. Assim, o presente estudo teve como objetivo identificar e caracterizar quimicamente duas amostras de CuChl (Sigma® e Chr. Hansen®) e o padrão de CuCe6 (Frontier Scientific®). Para tanto, empregou-se técnicas cromatográficas e espectrofotométricas, determinou-se a lipofilicidade em modelos miméticos de membrana, cinética de degradação e avaliou-se a interação CuCe6/BSA. A análise elementar da CuChl resultou em teores de cobre total inferiores aos recomendados pela United States Pharmacopeia (U.S.P.). Os elementos (CHN) e a razão Cu/N não foram coerentes com os valores teóricos da molécula de CuChl. Apenas uma amostra de CuChl apresentou razão Soret/Q dentro dos valores preconizados pela U.S.P. A titulação base-ácido da CuCe6 revelou dois valores de pkas (10,62 e 6,41) que foram similares para as amostras de CuChl. A determinação de log P da CuCe6 mostrou que a hidrofobicidade é máxima em pH 3 (log P = 1,49±0,09) e sua hidrofilicidade ocorre em pHs > 7. Esse comportamento foi confirmado nos ensaios de incorporação em lipossomas em função do pH. A degradação térmica da CuChl (25 a 95 °C) avaliada por HPLC foi drástica a partir de 75 °C. A energia necessária para que ocorra a degradação da CuChl e CuCe6 é Ea = 16,1 e 9,3kcal/mol, respectivamente. A meia-vida a 35 °C é de 6 horas para a CuChl e 2 horas e meia para a CuCe6. A separação mais eficiente dos componentes da CuChl por HPLC foi conseguida utilizando coluna C30 e a identificação dos principais constituintes CuCe6, CuCe4 e a clorina cúprica p6 (CuCp6), ocorreu por HPLC/MSMS. No estudo da ligação entre CuCe6 e proteína BSA foram obtidos os valores de KD = 0,38 ± 0,07 µM, KA = 3,3 ± 0,28 x 106 M-1 e número de sítios de ligação ~1 (N = 0,75 ± 0,09), indicativo de alta afinidade entre a clorina e a proteína. Assim, o comportamento químico dos principais componentes da CuChl e sua interação com os componentes do soro tornaram inviáveis a identificação e quantificação destas moléculas em ensaios in vivo. Os resultados aqui apresentados servem de subsídio para o desenvolvimento de outras pesquisas que visem o estudo específico da associação e dissociação da CuChl em material biológico. / Sodium copper chlorophyllin (CuChl) is a semisynthetic derivative of chlorophyll dye. It is composed chemically by several chlorins, especially copper chlorin e4 (CuCe4), copper chlorin e6 (CuCe6), and possible others no copper porphyrins and chlorins in different proportions. In addition to its use as a food coloring, CuChl may have interesting biological effects as antimutagenic, anticarcinogenic and antioxidant. Because of these potential benefits, its use as a dietary supplement is increasing. However, information on the absorption and bioavailability of CuChl is scarce. Furthermore, no studies have evaluated the impact of CuChl composition in its bioactivity and efficacy. Thus, the present study aimed to identify and chemically characterize two samples of CuChl (Sigma® and Hansen®) and the standard of CuCe6 (Frontier Scientific®). Chromatographic and spectrometric techniques as well as mimetics models membrane were used. The CuCe6/BSA interaction was also evaluated. The elemental analysis of CuChl showed that the total copper content of it was smaller that the one recommended by United States Pharmacopeia (USP). The elements (CHN) and the ratio Cu / N were not consistent with the theoretical values of the molecule CuChl. Only one CuChl sample showed Soret / Q ratio within the range recommended by USP. The acid-base titration of CuCe6 revealed two pKas values (10.62 and 6.41), which were similar for CuChl samples. The log P determination of CuCe6 showed that its hydrophobicity is maximal at pH 3 (log P = 1.49 ± 0.09) and its hydrophilicity occurs at pH> 7. These results were confirmed using the incorporation into liposomes assay in function of pH. Using HPLC, it was observed that thermal degradation of CuChl (25 to 95 °C) hardly occurred from 75 °C. The energy necessary for CuChl and CuCe6 degradation is Ea = 16.1 and 9.3 kcal/mol, respectively. The half-life at 35°C for CuChl and CuCe6 is 6 hours and 2 ½ hours, respectively. A more efficient separation of the CuChl components by HPLC was achieved using a C30 column while its major constituents CuCe6, CuCe4 and copper chlorin p6 (CuCp6) were identified by HPLC / MS-MS. In binding analysis of CuCe6 and BSA, it was observed KD = 0.38 ± 0.07 mM, KA = 3.3 ± 0.28 x 106 M-1, and number of binding sites ~ 1 (N = 0.75 ± .09), indicating high affinity between BSA and chlorine. Thus, due to the chemical characteristics of the main components of CuChl and their interaction with serum components the identification and quantification of these molecules in vivo is unviable. Future studies should investigate the association and dissociation of CuChl in biological samples.