Analysis of Chlorophylls/Chlorophyllins in Food Products Using HPLC and HPLC-MS Methods.

Of the different quality parameters of any food commodity or beverage, color is the most important, attractive and choice-affecting sensory factor to consumers and customers. Nowadays, food industries are interested in making the appearance of their food products attractive and interesting in order to appeal to consumers/customers. Natural green colorants have been accepted universally due to their natural appeal as well as their nontoxic nature to consumers. In addition, several food safety issues mean that natural green colorants are preferable to synthetic food colorants, which are mostly unsafe to the consumers but are less costly, more stable, and create more attractive color hues in food processing. Natural colorants are prone to degradation into numerous fragments during food processing, and thereafter, in storage. Although different hyphenated techniques (especially high-performance liquid chromatography (HPLC), LC-MS/HRMS, and LC/MS-MS are extensively used to characterize all these degradants and fragments, some of them are not responsive to any of these techniques, and some substituents in the tetrapyrrole skeleton are insensitive to these characterization tools. Such circumstances warrant an alternative tool to characterize them accurately for risk assessment and legislation purposes. This review summarizes the different degradants of chlorophylls and chlorophyllins under different conditions, their separation and identification using various hyphenated techniques, national legislation regarding them, and the challenges involved in their analysis. Finally, this review proposes that a non-targeted analysis method that combines HPLC and HR-MS assisted by powerful software tools and a large database could be an effective tool to analyze all possible chlorophyll and chlorophyllin-based colorants and degradants in food products in the future.

Copper(II) based low molecular weight collagen fragments-chlorin e6 nanoparticles synergize anti-cancer and anti-bacteria photodynamic therapy.

Copper-based photosensitizer nanoparticle has high potential clinic translation potency for its extensive physiological effects such as anti-cancer progression, anti-bacteria and accelerate tissue regeneration. However, copper excess or improper coordination can induce toxicity or reduce drug efficacy. To get proper copper-photosensitizer complex nanoparticle, a portion of chlorin e6 covalently conjugated with low molecular weight fish collagen fragments-collage tripeptides (CTPs), and Cu2+ subsequently triggers CTP-Ce6 conjugates assemble to Cu(II) based CTP-Ce6 nanosphere(CCeC-Ns). CCeC-Ns are 10-20 nm nanoparticles. CCeC-Ns quenched Ce6 fluorescence in aqueous solution and improved longer wavelength light absorbance. It exhibited dramatically higher cellular uptake rates and much more anticancer potency than those of free Ce6 under 660 nm irradiation without obvious dark toxicity in vitro. CCeC-Ns have longer retention time and higher penetrating rate than free Ce6 in tumor spheroid model. CCeC-Ns displayed extremely higher anti-bacterial potency than free Ce6 and sustainable efficacy. It provides a more potent and safer nanodrug for cancer and infection treatment and an idea for highly efficient metal-photosensitizers complexes design.

Water-soluble chlorin e6-hydroxypropyl chitosan as a high-efficiency photoantimicrobial agent against Staphylococcus aureus.

The development of new antimicrobial agents is important to combat infections caused by pathogenic bacteria. Herein, Hydroxypropyl chitosan (HPCS), a hydrophilic modified product of chitosan (CS), was employed as a carrier of the photosensitizer chlorin e6 (Ce6) through an amide bond to obtain the products (HPCS-Ce6 conjugates) with a degree of substitution (DS) ranging from 2.95% to 5.25%. The UV-vis absorption spectra and 1H NMR spectra confirmed the successful synthesis of the products. The products have a better and more stable reactive oxygen species (ROS) generation capacity and higher bacterial affinity than Ce6. At a very low dose (1.8 µg/mL), the highest DS product (HPCS-Ce6-3) can effectively kill Staphylococcus aureus (S. aureus) under 660 nm irradiation. In addition, the HPCS-Ce6 conjugates showed high biocompatibility in the CCK-8 test. The HPCS-Ce6 conjugates could be a photodynamic antibacterial agent with good water solubility, high biocompatibility, and antibacterial activity.

Hydrogen Peroxide and Hypochlorite Responsive Fluorescent Nanoprobes for Sensitive Cancer Cell Imaging.

Accurate diagnosis of cancer cells directly affects the clinical treatment of cancer and can significantly improve the therapeutic effect of cancer patients. Cancer cells have a unique microenvironment with a large amount of peroxide inside, effectively differentiated from relevant microenvironment normal cells. Therefore, designing the high-sensitive probes to recognize and distinguish the special physiological microenvironment of cancer cells can shed light on the early diagnosis of cancers. In this article, we design and construct a fluorescence (FL) contrast agent for cancer cell recognition and imaging analysis. Firstly, luminol-gold NPs (Lum-AuNPs) have been initially built, and then successfully loaded with the fluorescent receptor Chlorin e6 (Ce6) to prepare the luminescent nanoprobes (Ce6@Lum-AuNPs) with green synthesis, i.e., with biocompatible agents and mild temperature. The as-prepared fluorescent Ce6@Lum-AuNPs can efficiently and sensitively realize FL bioimaging of cancer cells. The relevant bio-sensing mechanism pertains to the presence of hypochlorite (ClO-); hydrogen peroxide (H2O2) in cancer cells could readily interact with luminol to produce chemiluminescence, which can activate the Ce6 component to emit near-infrared (NIR) FL. Therefore, this raises the possibility of utilizing the Ce6@Lum-AuNPs as efficient fluorescent nanoprobes for promising cancer early diagnosis and other relevant disease bioanalysis.

Photoactive "Bionic Virus" Robustly Elicits the Synergy Anticancer Activity of Immunophotodynamic Therapy.

Coronavirus represents an inspiring model for designing drug delivery systems due to its unique infection machinery mechanism. Herein, we have developed a biomimetic viruslike nanocomplex, termed SDN, for improving cancer theranostics. SDN has a unique core-shell structure consisting of photosensitizer chlorin e6 (Ce6)-loaded nanostructured lipid carrier (CeNLC) (virus core)@poly(allylamine hydrochloride)-functionalized MnO2 nanoparticles (virus spike), generating a virus-mimicking nanocomplex. SDN not only prompted cellular uptake through rough-surface-mediated endocytosis but also achieved mitochondrial accumulation by the interaction of cationic spikes and the anionic mitochondrial surface, leading to mitochondria-specific photodynamic therapy. Meanwhile, SDN could even mediate oxygen generation to relieve tumor hypoxia and, consequently, improve macrophage-associated anticancer immune response. Importantly, SDN served as a robust magnetic resonance imaging (MRI) contrast agent due to the fast release of Mn2+ in the presence of intracellular redox components. We identified that SDN selectively accumulated in tumors and released Mn2+ to generate a 5.71-fold higher T1-MRI signal, allowing for effectively detecting suspected tumors. Particularly, SDN induced synergistic immunophotodynamic effects to eliminate malignant tumors with minimal adverse effects. Therefore, we present a novel biomimetic strategy for improving targeted theranostics, which has a wide range of potential biomedical applications.

Enhanced ROS-Boosted Phototherapy against Pancreatic Cancer via Nrf2-Mediated Stress-Defense Pathway Suppression and Ferroptosis Induction.

In situ oxygen generation is the most common strategy to boost reactive oxygen species (ROS) for enhancing the efficacy of phototherapy in cancer, including photodynamic therapy (PDT) and photothermal therapy (PTT). However, hyperoxidation or hyperthermia often triggers stress-defense pathways and promotes tumor cell survival, thus severely limiting the therapeutic efficacy. To overcome the tumor hypoxia and thermal resistance existing in phototherapy, we constructed a self-synergistic nanoplatform for tumors by incorporating brusatol, a nuclear factor erythroid 2-related factor (Nrf2) inhibitor, into the silica nanonetwork. It was then sequentially decorated with MnO2 and the photosensitizer chlorin e6 (Ce6) and then coated with poly(ethylene glycol)-folate (PEG-FA)-functionalized polydopamine (PDA) (designated as brusatol/silica@MnO2/Ce6@PDA-PEG-FA). As an oxygen generator, MnO2 can promote ROS production, which not only directly enhances Ce6-mediated PDT but also strengthens PDA-mediated PTT by attacking heat shock proteins (HSPs). Particularly, brusatol could efficiently inhibit the activation of Nrf2 defense pathway under hyperoxidation and hyperthermia and cause glutathione peroxidase 4 (GPX4) and ferritin heavy chain (FTH) inactivation, thereby inducing ferroptosis and ultimately enhancing the phototherapeutic effects. By exploiting these features, brusatol/silica@MnO2/Ce6@PDA-PEG-FA exhibited excellent antitumor efficacy with enhanced PDT and PTT both in in vitro and in vivo studies. Overall, our work highlights a promising strategy against hypoxia- and hyperthermia-associated resistance in phototherapy via suppressing stress-defense system and inducing ferroptosis.

Dual-Responsive and ROS-Augmented Nanoplatform for Chemo/Photodynamic/Chemodynamic Combination Therapy of Triple Negative Breast Cancer.

Integrating chemodynamic therapy (CDT) and photodynamic therapy (PDT) into one nanoplatform can produce much more reactive oxygen species (ROS) for tumor therapy. Nevertheless, it is still a great challenge to selectively generate sufficient ROS in tumor regions. Meanwhile, CDT and PDT are restricted by insufficient H2O2 content in the tumor as well as by the limited tumor tissue penetration of the light source. In this study, a smart pH/ROS-responsive nanoplatform, Fe2+@UCM-BBD, is rationally designed for tumor combination therapy. The acidic microenvironment can induce the pH-responsive release of doxorubicin (DOX), which can induce tumor apoptosis through DNA damage. Beyond that, DOX can promote the production of H2O2, providing sufficient materials for CDT. Of note, upconversion nanoparticles at the core can convert the 980 nm light to red and green light, which are used to activate Ce6 to produce singlet oxygen (1O2) and achieve upconversion luminescence imaging, respectively. Then, the ROS-responsive linker bis-(alkylthio)alkene is cleaved by 1O2, resulting in the release of Fenton reagent (Fe2+) to realize CDT. Taken together, Fe2+@UCM-BBD exhibits on-demand therapeutic reagent release capability, excellent biocompatibility, and remarkable tumor inhibition ability via synergistic chemo/photodynamic/chemodynamic combination therapy.

Assembly Transformation Jointly Driven by the LAP Enzyme and GSH Boosting Theranostic Capability for Effective Tumor Therapy.

Developing intelligent and morphology-transformable nanomaterials that can spatiotemporally undergo stimulus-responsive size transformation holds great promise for improving the tumor delivery efficiency of drugs in vivo. Here, we report a smart size-transformable theranostic probe Ce6-Leu consisting of a leucine amino peptidase (LAP) and glutathione (GSH) dual-responsive moiety, an 1,2-aminothiol group, and a clinically used photosensitizer Ce6. This probe tends to self-assemble into uniform nanoparticles with an initial size of ∼80 nm in aqueous solution owing to the amphiphilic feature. Surprisingly, taking advantage of the biocompatible CBT-Cys condensation reaction, the large nanoprobes can be transformed into tiny nanoparticles (∼23 nm) under the joint action of LAP and GSH in a tumor microenvironment, endowing them with great tumor accumulation and deep tissue penetration. Concomitantly, this LAP/GSH-driven disassembly and size shrinkage of Ce6-Leu can also activate the fluorescence/magnetic resonance signals and the photodynamic effect for enhanced multimodal imaging-guided photodynamic therapy of human liver HepG2 tumors in vivo. More excitingly, the Mn2+-chelating probe (Ce6-Leu@Mn2+) was demonstrated to have the capability to catalyze endogenous H2O2 to persistently release O2 at the hypoxic tumor site, as a consequence improving the oxygen supply to boost the radiotherapy effect. We thus believe that this LAP/GSH-driven size-transformable nanosystem would offer a novel advanced technology to improve the drug delivery efficiency for achieving precise tumor diagnosis and treatment.

Chlorin e6-Biotin Conjugates for Tumor-Targeting Photodynamic Therapy.

To improve the tumor-targeting efficacy of photodynamic therapy, biotin was conjugated with chlorin e6 to develop a new tumor-targeting photosensitizer, Ce6-biotin. The Ce6-biotin had good water solubility and low aggregation. The singlet-oxygen generation rate of Ce6-biotin was slightly increased compared to Ce6. Flow cytometry and confocal laser scanning microscopy results confirmed Ce6-biotin had higher binding affinity toward biotin-receptor-positive HeLa human cervical carcinoma cells than its precursor, Ce6. Due to the BR-targeting ability of Ce6-biotin, it exhibited stronger cytotoxicity to HeLa cells upon laser irradiation. The IC50 against HeLa cells of Ce6-biotin and Ce6 were 1.28 µM and 2.31 µM, respectively. Furthermore, both Ce6-biotin and Ce6 showed minimal dark toxicity. The selectively enhanced therapeutic efficacy and low dark toxicity suggest that Ce6-biotin is a promising PS for BR-positive-tumor-targeting photodynamic therapy.

Photo-Driven Delivery of 125I-Labeled Nanomicelles for Nucleus-Targeted Internal Conversion Electron-Based Cancer Therapy.

As a kind of high linear energy transfer (LET) radiation, internal conversion electrons are emitted from some radionuclides, such as 125I, triggering severe DNA damage to tumor cells when transported into the nucleus. Herein, we develop a curcumin-loaded nanomicelle composed of a photosensitizer chlorin e6 (Ce6) and amphiphilic poly(ethylene glycol) (poly(maleic anhydride-alt-1-octadecene)-poly(ethylene glycol) (C18-PMH-PEG)) to deliver 125I into the nucleus under 660 nm laser irradiation, leading to the optimized imaging-guided internal conversion electron therapy of cancer. Ce6-containing nanomicelles (Ce6-C18-PEG) self-assemble with nucleus-targeted curcumin (Cur), obtaining Ce6-C18-PEG/Cur nanoparticles. After labeling Cur with 125I, Ce6-C18-PEG/Cur enables single-photon emission computed tomography and fluorescence imaging of the tumor, serving as a guide for follow-up laser irradiation. Notably, the 660 nm laser-triggered photodynamic reaction of Ce6 optimizes the delivery of Ce6-C18-PEG/125I-Cur at various stages, including tumor accumulation, cellular uptake, and lysosome escape, causing plenty of 125I-Cur to enter the nucleus. By this strategy, Ce6-C18-PEG/125I-Cur showed optimal antitumor efficacy and high biosafety in mice treated with local 660 nm laser irradiation using efficient energy deposition of internally converted electrons over short distances. Therefore, our work provides a novel strategy to optimize 125I delivery for tumor treatment.

Photodynamic Therapy of Melanoma B16 with Chlorin E6 Conjugated with a PSMA-Ligand.

The efficacy of a new photosensitizer of chlorin E6 conjugated with a prostate-specific membrane antigen (PSMA) in photodynamic therapy of murine melanoma B16 was studied in in vivo experiments. The dynamics of photosensitizer accumulation in the tumor and surrounding tissues was evaluated and antitumor efficacy of photodynamic therapy was assessed by parameters of regression and morphological characteristics of experimental transplanted melanoma B16. The inhibitory effect of photodynamic therapy on melanoma was evaluated by complete regression of the tumor, absolute tumor growth coefficient in animals with continuation of tumor growth, and the increase in life span in comparison with the control; the criterion of cure was the absence of signs of tumor recurrence in mice within 90 days after therapy. The therapeutic potential of photodynamic therapy was determined by devitalization of tumor cells (histological examination of the zones of laser exposure on day 21 after treatment). The photosensitizer with PSMA-ligand exhibited high antitumor activity in photodynamic therapy for melanoma B16. Photodynamic therapy carried out at the optimum time after photosensitizer injection with experimentally determined parameters of laser exposure allows achieving the maximum inhibitory effect on melanoma. Pathomorphological study in the zones of exposure detected no survived tumor cells.

Hitchhiking Nanoparticles: Mesenchymal Stem Cell-Mediated Delivery of Theranostic Nanoparticles.

Nanotechnology has emerged as a promising solution to permanent elimination of cancer. However, nanoparticles themselves lack specificity to tumors. Due to enhanced migration to tumors, mesenchymal stem cells (MSCs) were suggested as cell-mediated delivery vehicles of nanoparticles. In this study, we have constructed a complex composed of photoluminescent quantum dots (QDs) and a photosensitizer chlorin e6 (Ce6) to obtain multifunctional nanoparticles, combining cancer diagnostic and therapeutic properties. QDs serve as energy donors-excited QDs transfer energy to the attached Ce6 via Förster resonance energy transfer, which in turn generates reactive oxygen species. Here, the physicochemical properties of the QD-Ce6 complex and singlet oxygen generation were measured, and the stability in protein-rich media was evaluated, showing that the complex remains the most stable in protein-free medium. In vitro studies on MSC and cancer cell response to the QD-Ce6 complex revealed the complex-loaded MSCs' potential to transport theranostic nanoparticles and induce cancer cell death. In vivo studies proved the therapeutic efficacy, as the survival of tumor-bearing mice was statistically significantly increased, while tumor progression and metastases were slowed down.

Nanoliposomes co-encapsulating Ce6 and SB3CT against the proliferation and metastasis of melanoma with the integration of photodynamic therapy and NKG2D-related immunotherapy on A375 cells.

Purpose. To overcome the insufficiency of conventional photodynamic therapy (PDT) for treating metastatic melanoma, the combination of smart nanoparticles and PDT with immunotherapy was used to achieve a higher efficiency by accumulating more photosensitizers in tumor areas and triggering stronger immune responses against tumors after PDT.Methods. In this study, we designed a nanoliposome co-encapsulation of chlorin E6 (Ce6) and SB-3CT to realize significant antitumoral proliferation and metastasis efficacy after laser irradiation in A375 cells. The morphology, size distribution, and loading efficiency of Ce6-SB3CT@Liposome (Lip-SC) were characterized. The reactive oxygen species (ROS) generation and cytotoxicity were evaluated in A375 cells, and the mechanisms of natural killer (NK) cell-mediated killing were assessed.Results. Lip-SC showed good stability and was well-dispersed with a diameter of approximately 140 nm in phosphate-buffered saline. The nanoliposomes could accumulate in tumor areas and induce apoptosis in cancer cells upon 660 nm light irradiation, which could trigger an immune response and induce the expression of NK group 2 member D (NKG2D) ligands. The subsequently released SB-3CT could further activate NK cells effectively and strengthen the immune system by inhibiting the shedding of soluble NKG2D ligands.Discussion. Taken together, the synergistic effects of SB-3CT on nanoliposomes for Ce6-mediated PDT were analyzed in detail to provide a new platform for future anti-melanoma treatment.

Efficient Delivery of Chlorin e6 by Polyglycerol-Coated Iron Oxide Nanoparticles with Conjugated Doxorubicin for Enhanced Photodynamic Therapy of Melanoma.

Chlorin e6 (Ce6) is a promising photosensitizer for tumor photodynamic therapy (PDT). However, the efficacy of Ce6 PDT is limited by Ce6's poor water solubility, rapid blood clearance, and inadequate accumulation in the tumor tissue. This problem is tackled in this work, wherein functionalized superparamagnetic iron oxide nanoparticles (IO-NPs) were used as carriers to deliver Ce6 to melanoma. The IO-NPs were coated with polyglycerol (PG) to afford good aqueous solubility. The chemotherapeutic agent doxorubicin (DOX) was attached to the PG coating via the hydrazone bond to afford affinity to the cell membrane and thereby promote the cell uptake. The hydrophobic nature of DOX also induced the aggregation of IO-NPs to form nanoclusters. Ce6 was then loaded onto the IO nanoclusters through physical adsorption and coordination with surface iron atoms, yielding the final composites IO-PG-DOX-Ce6. In vitro experiments showed that IO-PG-DOX-Ce6 markedly increased Ce6 uptake in mouse melanoma cells, leading to much-enhanced photocytotoxicity characterized by intensified reactive oxygen species production, loss of viability, DNA damage, and stimulation of tumor cell immunogenicity. In vivo experiments corroborated the in vitro findings and demonstrated prolonged blood clearance of IO-PG-DOX-Ce6. Importantly, IO-PG-DOX-Ce6 markedly increased the Ce6 distribution and retention in mouse subcutaneous melanoma grafts and significantly improved the efficacy of Ce6-mediated PDT. No apparent vital organ damage was observed at the same time. In conclusion, the IO-PG-DOX NPs provide a simple and safe delivery platform for efficient tumor enrichment of Ce6, thereby enhancing antimelanoma PDT.

Assessing the photodynamic efficacy of different photosensitizer-light treatments against foodborne bacteria based on the number of absorbed photons.

Increasing interests in photodynamic treatment (PDT) for food preservation require a holistic method to evaluate and compare different photosensitizer (PS)-light treatments. In this report, the absorbed photons were used as the basis to assess the antimicrobial photodynamic efficacy of two PSs, chlorophyllin sodium magnesium salt (Chl-Mg) and chlorophyllin sodium copper salt (Chl-Cu), under blue and white light against two typical foodborne pathogens, Gram-negative Escherichia coli, and Gram-positive Staphylococcus aureus. The results showed that the phototoxicity of a PS was predominantly decided by the absorbed photons rather than the characteristics of light sources. Photosensitized Chl-Mg exhibited superior antimicrobial activity as compared to that of ChlCu. The applied treatments were found to be more effective against S. aureus than E. coli. Bacterial inactivation kinetics as a function of the number of absorbed photons could be described by Weibull model with R2 from 0.947-0.962, and kinetics constants D in the range of 0.202 × 1017 photons/cm2-2.409 × 1018 photons/cm2. The kinetics models may find promising applications in the design, assessment, and optimization of PDT processes.

Photodynamic therapy using self-assembled nanogels comprising chlorin e6-bearing pullulan.

With minimal invasiveness and spatiotemporal therapeutic effects, photodynamic therapy is one of the most promising candidates for cancer treatment. Here, we developed a facile self-assembled nanogel using photosensitizer-grafted polysaccharides called chlorin e6-bearing pullulan. Chlorin e6 is used as a photosensitizer in cancer therapy. The anti-cancer effect of photodynamic therapy with our nanogel system was 780 times higher than that of the commercially available photosensitizer Photofrin. Finally, we demonstrated that actively growing cancer cell spheroids can be completely suppressed after treatment. Our system could efficiently induce tumor regression in tumor xenograft mice.

Chitosan modified ultra-thin hollow nanoparticles for photosensitizer loading and enhancing photodynamic antibacterial activities.

Antibacterial photodynamic therapy (PDT) has attracted extremely attention due to not inducing bacteria to generate resistance. However, the poor utilization and low reactive oxygen species (ROS) field of photosensitizers hinder their further application for antibacterial. Here, we designed ultra-thin hollow silica nanoparticles (UHSN), followed by pore-engineering including covalent anchoring of chitosan (UHSN@CS) for enhanced loading and photodynamic property of photosensitizer. The UHSN@CS exhibit high loading efficiency (80.6%, pH = 6.0) and controllable pH-responsive release for Ce6. Additionally, UHSN@CS can enhance the ROS yield of photosensitizers and effectively adhere to S. aureus, thus enormously enhancing antibacterial performance toward bacteria. Moreover, UHSN@CS-Ce6 can obliterate mature S. aureus biofilm and cause an 81% decrease in the biomass, showing a better therapeutic effect than Ce6 (59.2%) under laser irradiation. In vivo results confirm that UHSN@CS-Ce6 is effective to promote infectious wound regeneration. As photodynamic-based nanoplatforms, UHSN@CS-Ce6 are potential antibacterial agents for skin infection therapy.

Photodynamic inactivation of planktonic Staphylococcus aureus by sodium magnesium chlorophyllin and its effect on the storage quality of lettuce.

Photodynamic inactivation (PDI) is a fast and effective non-heat sterilization technology. This study established an efficient blue light-emitting diode (LED) PDI with the photosensitizer sodium magnesium chlorophyllin (SMC) to eradicate Staphylococcus aureus in food. The antibacterial mechanisms were determined by evaluating DNA integrity, protein changes, morphological alteration, and the potency of PDI to eradicate S. aureus on lettuce was evaluated. Results showed that planktonic S. aureus could not be clearly observed on the medium after treatment with 5.0 µmol/L SMC for 10 min (1.14 J/cm2). Bacterial cell DNA and protein were susceptible to SMC-mediated PDI, and cell membranes were found to be disrupted. Moreover, SMC-mediated PDI effectively reduced 8.31 log CFU/mL of S. aureus on lettuce under 6.84 J/cm2 radiant exposure (30 min) with 100 µmol/L SMC, and PDI displayed a potent ability to restrain the weight loss as well as retard the changes of color difference of the lettuce during 7 day storage. The study will enrich our understanding of the inactivation of S. aureus by PDI, allowing for the development of improved strategies to eliminate bacteria in the food industry.

Facile production of chlorophyllides using recombinant CrCLH1 and their cytotoxicity towards multidrug resistant breast cancer cell lines.

The purity of chlorophylls plays one of the key role for the production of chlorophyllides. We have designed a facile method for chlorophyll purification by twice solvent extraction. Twice extraction causes the loss of chlorophylls, but the purity of total chlorophylls can be enhanced 182%. Then, the purified chlorophylls can be converted to relatively pure chlorophyllides facilely. The results show that higher purity of chlorophyllides could be obtained when purified chlorophylls (ethanol-hexane extract) was used as starting materials than that of crude chlorophylls (ethanol-only extract). In biocompatibility test, the results showed that the prepared chlorophyllides can be applied as biomaterials. When the prepared chlorophyllides were applied to anticancer tests, they were active both in MCF7 and MDA-MB-231 (multidrug resistant breast cancer cells) cell lines. In addition, the results suggested that the prepared chlorophyllides could be a potential candidate of combination therapy with doxorubicin to breast cancers.

Detection of 132-carboxy-chlorin produced by the in vitro BciC enzymatic hydrolysis of zinc chlorophyllide.

Green photosynthetic bacteria with an efficient light-harvesting system contain special chlorophyll molecules, called bacteriochlorophylls c, d, e, in their main antennae. In the biosynthetic pathway, a BciC enzyme is proposed to catalyze the hydrolysis of the C132-methoxycarbonyl group of chlorophyllide a, but the resulting C132-carboxy group has not been detected yet because it is spontaneously removed due to the instability of the ß-keto-carboxylic acid. In this study, the in vitro BciC enzymatic reactions of zinc methyl (131R/S)-hydroxy-mesochlorophyllides a were examined and a carboxylic acid possessing the C132S-OH was first observed as the hydrolyzed product of the C132-COOCH3.