Applications of chitosan to the roots and shoots change the accumulation pattern of cadmium in Talinum patens (Talinaceae) cuttings.

Chitosan induces tolerance to abiotic stress agents in plants. However, studies on the different application forms of this biopolymer are limited. This study evaluated the effect of two forms of chitosan application on the morphophysiology of and metal accumulation by Talinum patens cuttings subjected to Cd to develop new cadmium (Cd) decontamination technologies. Cuttings from 75-day-old plants were transferred to a hydroponic system. For 30 days, three Cd concentrations (0, 7, and 14 mg L-1) and three forms of chitosan application (without application, root, and foliar) were applied. The cuttings were tolerant to Cd because the metal did not influence biomass production or photosynthetic efficiency. Neither chitosan application nor Cd increased the modified chlorophyll content and fluorescence parameters. However, foliar chitosan reduced the transpiration rate. At the highest concentration of Cd, the application of chitosan in the root reduced the Mg content of the root system and shoots. The root application of chitosan increased the surface area and volume of thicker roots at the expense of finer ones. The foliar application resulted in greater total root length and surface area, mainly those finer. Furthermore, chitosan applied to the leaves activated catalase in the roots and leaves. In contrast to the root application, foliar application increased the accumulation of Cd in the roots. The action of catalase and the increase of fine roots may have favored a greater absorption of the nutrient solution and Cd in the chitosan foliar application treatment. It is concluded that chitosan foliar spraying can improve Cd rhizofiltration with T. patens.

Identification of chlorophyll molecules with peroxyl radical scavenger capacity in microalgae Phormidium autumnale using ultrasound-assisted extraction.

The identification of chlorophyll molecules with peroxyl radical scavenger capacity in microalgae Phormidium autumnale was determined. The ultrasound-assisted extraction was utilized for obtaining the chlorophyll compounds from biomass. A total of eleven molecules were separated in microalgae chlorophyll extract, with pheophytin a' (371µg·g-1) and chlorophyll a (159.3µg·g-1) as the major ones. The chlorophyll extract was shown to be a potent scavenger of peroxyl radical, being almost 200 times more potent than α-tocopherol. These facts suggest the microalgae Phormidium autumnale as potential source of bioactive tetrapyrrole compounds.

Semi-synthesis and PDT activities of a new amphiphilic chlorin derivative.

An amphiphilic chlorin derivative (CHL-T) was prepared from methylpheophorbide a (CHL) and 2-Amino-2-(hydroxymethyl)-1,3-propanediol (TRISMA®). The new chlorin was compared to other dyes (CHL and Hypericin) in relation to photophysical and photobiological activities in tumor and non-tumor cell lines. Cytotoxicity and cell death target were determined to evaluate the CHL-T efficiency, comparing to the precursor CHL and to the well-known dye hypericin (HY). All of the studied compounds exhibited absorption bands in the therapeutic window and presented a small fluorescence quantum yield compared to the reference dye (rhodamine B). CHL-T was about three times more efficient on singlet oxygen generation than the others photosensitizers. The lipophilicity order of the photosensitizers was CHL>HY>CHL-T. The tumoral HeLa cells presented improved accumulation for CHL and CHL-T compared to HY. The phototoxicity presented by the CHL-T was about ten times higher than by CHL, as demonstrated by the MTT assay. CHL-T showed more cytotoxicity to tumoral cell, comparing to non-tumoral cell in short incubation time. The cell death rises proportionally with increasing PSs concentrations, mainly by necrosis. These findings suggest that CHL-T is a potential new photosensitizer for PDT.

Antioxidant, antimutagenic and antiproliferative activities in selected seaweed species from Sinaloa, Mexico.

Context Seaweeds from the Mexican Pacific Ocean have not been evaluated as a source of chemoprotectants. Objective The objective of this study is to evaluate chemopreventive activities of the seaweeds Phaephyceae - Padina durvillaei (Dictyotaceae) - Rodhophyceae - Spyridia filamentosa (Spyridiaceae), Gracilaria vermiculophylla (Gracilariaceae) - and Chlorophyceae - Ulva expansa (Ulvaceae), Codium isabelae (Codiaceae), Rhizoclonium riparium (Cladophoraceae) and Caulerpa sertularioides (Caulerpaceae). Materials and methods Methanol, acetone and hexane seaweed extracts were assessed at 30 and 3 mg/mL on antioxidant capacity (DPPH and ABTS assays), 0.003-3.0 mg/plate on antimutagenic activity against AFB1 using Salmonella typhimurium TA98 and TA100 tester strains in Ames test, and 12.5 to 100 µg/mL on antiproliferative activity on Murine B-cell lymphoma. Phenols, flavonoids and pigments content were also assessed as antioxidant compounds. Results Extraction yield was higher in methanol than in acetone and hexane extracts (6.4, 2.7 and 1.4% dw). Antioxidant capacity was higher in brown and green than in red seaweed species, particularly in P. durvillaei extracted in acetone (EC50 value= 16.9 and 1.56 mg/mL for DPPH and ABTS). Flavonoids and chlorophylls were identified as mainly antioxidant components; particularly in hexane extracts, which were correlated with the antioxidant capacity. Highest mutagenesis inhibition (> 40%) occurred in R. riparium at the lowest concentration assayed (0.003 mg/plate), while highest antiproliferative inhibition (37 and 72% for 12.5 and 25 µg/mL) occurred in C. sertularioides. Discussion and conclusion Flavonoids and chlorophylls explained the chemopreventive activities assessed in S. filamentosa, R. riparium and C. sertularioides. These seaweeds have a high potential as a source of novel chemoprotectants.

Photobiological characteristics of chlorophyll a derivatives as microbial PDT agents.

Chlorin-e6 (chl-e6) and a hydrogenated derivative (chl-e6H) were semi-synthesized, and their photophysical properties and photodynamic activity against Escherichia coli, Staphylococcus aureus and Candida albicans evaluated. Methyl pheophorbide-a (Mepheo-a) was obtained from S. maxima using methanolic extraction with acid catalysis (CH3OH­H2SO4). Chlorin-e6 was prepared from Mepheo-a by basic hydrolysis with H2O­acetone and NaOH. Hydrogenated Chlorin-e6 was synthesized by a similar procedure starting from the hydrogenated methyl pheophorbide-a (Mepheo-aH). Photophysical studies were performed in order to determine the singlet oxygen quantum yield of chl-e6H which is higher than that of chl-e6. The microorganism inactivation of chl-e6 and chl-e6H was investigated at two concentrations and three fluence levels. Both chl-e6 and chl-e6H showed microorganism inactivation against Gram-positive bacteria and a fungus.

Effects of lutein and chlorophyll b on GSH depletion and DNA damage induced by cisplatin in vivo.

Recent studies have proposed the use of low concentrations of phytochemicals and combinations of phytochemicals in chemoprevention to reduce cytotoxicity and simulate normal ingestion through diet. The purpose of the present study was to evaluate whether the DNA damage, chromosome instability, and oxidative stress induced by cisplatin (cDDP) are modulated by a combination of the natural pigments lutein (LT) and chlorophyll b (CLb). The protective effects observed for synergism between phytochemicals have not been completely investigated. The comet assay and micronucleus test were performed and the catalase activities and glutathione (GSH) concentrations were measured in the peripheral blood, bone marrow, liver, and kidney cells of mice. The comet assay and micronucleus test results revealed that the pigments LT and CLb were not genotoxic or mutagenic and that the pigments presented antigenotoxic and antimutagenic effects in the different cell types evaluated. This protective effect is likely related to antioxidant properties in peripheral blood cells through the prevention of cDDP-induced GSH depletion. Altogether our results show that the combination of LT and CLb, which are both usually present in the same foods, such as leafy green vegetables, can be used safely.

Antigenotoxic properties of chlorophyll b against cisplatin-induced DNA damage and its relationship with distribution of platinum and magnesium in vivo.

The chemotherapeutic agent cisplatin (cDDP) is widely used to treat a variety of solid and hematological tumors. However, cDDP exerts severe side effects, such as nephrotoxicity, neurotoxicity, and bone-marrow suppression. The use of some dietary compounds to protect organs that are not targets in association with chemotherapy has been encouraged. This study evaluated the protective effects of chlorophyll b (CLb) on DNA damage induced by cDDP by use of single-cell gel electrophoresis (SCGE) assays. Further, this investigation also determined platinum (Pt) and magnesium (Mg) bioaccumulation in mice tissues after treatment with CLb alone and/or in association of cDDP (simultaneous treatment) by inductively coupled plasma-mass spectroscopy (ICP-MS). All parameters were studied in peripheral blood cells (PBC), kidneys, and liver of mice after administration of CLb (0.2 or 0.5 mg/kg of body weight [b.w.]), cDDP (6 mg/kg b.w.), and the combination CLb 0.2 plus cDDP or CLb 0.5 plus cDDP. Pt accumulation in liver and kidneys was higher than that found in PBC, while DNA damage was higher in kidneys and liver than in PBC. Further, treatment with CLb alone did not induce DNA damage. Evidence indicates that genotoxic effects produced by cDDP may not be related to Pt accumulation and distribution. In combined treatments, CLb decreased DNA damage in tissues, but the PT contents did not change and these treatments also showed that CLb may be an important source of Mg. Thus, our results indicate that consumption of CLb-rich foods may diminish the adverse health effects induced by cDDP exposure.

An evaluation, using the comet assay and the micronucleus test, of the antigenotoxic effects of chlorophyll b in mice.

We investigated the effects of the dietary pigment chlorophyll b (CLb) on cisplatin (cDDP)-induced oxidative stress and DNA damage, using the comet assay in mouse peripheral blood cells and the micronucleus (MN) test in bone marrow and peripheral blood cells. We also tested for thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH) in liver and kidney tissues, as well as catalase (CAT) activity and GSH in total blood. CLb (0.2 and 0.5mg/kg b.w.) was administrated by gavage every day for 13 days. On the 14th day of the experiment, 6 mg/kg cDDP or saline was delivered intraperitoneally. Treatment with cDDP led to a significant decrease in DNA migration and an increase in MN frequency in both cell types, bone marrow and peripheral blood cells. In the kidneys of mice treated with cDDP, TBARS levels were increased, whereas GSH levels were depleted in kidney and liver. In mice that were pre-treated with CLb and then treated with cDDP, TBARS levels maintained normal concentrations and GSH did not differ from cDDP group. The improvement of oxidative stress biomarkers after CLb pre-treatment was associated with a decrease in DNA damage, mainly for the highest dose evaluated. Furthermore, CLb also slightly reduced the frequency of chromosomal breakage and micronucleus formation in mouse bone marrow and peripheral blood cells. These results show that pre-treatment with CLb attenuates cDDP-induced oxidative stress, chromosome instability, and lipid peroxidation.

Effects of metal and the phytyl chain on chlorophyll derivatives: physicochemical evaluation for photodynamic inactivation of microorganisms.

Chlorophyll compounds and their derivatives containing metal or phytyl chain can be used as photosensitizer in photodynamic inactivation of microorganisms (PDI). So, the physicochemical properties and antimicrobial effect of chlorophyll derivatives were investigated: Mg-chlorophyll (Mg-Chl), Zn-chlorophyll (Zn-Chl), Zn-chlorophyllide (Zn-Chlde), Cu-chlorophyll (Cu-Chl), pheophytin (Pheo) and pheophorbide (Pheid). The photobleaching experiments showed photostability according to Cu-Chl > Pheo ∼ Pheid ≫ Zn-Chl ∼ Zn-Chlde > Mg-Chl. This order was discussed in terms of metal and the phytyl chain presences. Pheid and Zn-Chl in aqueous Tween 80 solution exhibited highest singlet oxygen yield compared with the other derivatives. Chlorophyll derivatives (CD) with phytyl chain was limited by the self-aggregation phenomenon at high concentrations, even in micellar systems (Tween 80 and P-123). The antimicrobial effect of CD derivatives was investigated against Staphylococcus aureus, Escherichia coli, Candida albicans and Artemia salina. Pheid showed the best results against all organisms tested, Zn-Chlde was an excellent bactericide in the dark and Cu-Chl had no PDI effect. No correlation with CD uptake by microorganisms and darkness cytotoxicity was found. The physicochemical properties allied to bioassays results indicate that Mg-Chl, Pheo, Zn-Chl and Pheid are good candidates for PDI.

Investigation of genotoxic and antigenotoxic activities of chlorophylls and chlorophyllin in cultured V79 cells.

Chlorophyll and its derivatives are examples of plant compounds (purified and/or extracted) which appear to protect DNA from damage caused by chemical or physical agents, although some studies have identified clastogenic activity of these compounds. This study was carried out to assess the genotoxic activity of chlorophyll-a (Chl-a), -b (Chl-b) and chlorophyllin (Chl) and their antigenotoxic activity against the DNA damage induced by methyl methanesulphonate (MMS) under conditions of simultaneous, pre-, post-treatment, and simultaneous treatment after pre-incubation of the chemical with MMS. The micronucleus (MN) test was used in binucleated cells (induced by cytochalasin-B) of a mammalian cell line (V79). The three concentrations of Chl-a, Chl-b or Chl (0.1375, 0.275, 0.55microM) were not genotoxic and the genotoxic action of MMS (400microM) decreased (74-117%) under all treatment conditions. The results showed that there was no significant difference among the treatment types, the concentration or the nature of chlorophyll used. The data obtained suggest that Chl-a, Chl-b and Chl when associated with the DNA damaging agent, MMS, may protect the DNA by desgenotoxic action and/or by bio-antigenotoxic mechanisms, with the similar efficiency.

Inactivation of cell surface receptors by pheophorbide a, a green pigment isolated from Psychotria acuminata.

The inhibition of cytokine and monoclonal antibody binding cell surfaces caused by an extract of Psychotria acuminata, a medicinal plant used in the traditional medicine of the people of Belize (Central Africa), was attributed to the presence of pheophorbide a and pyropheophorbide a. Since the binding of tumor necrosis factor-alpha, interleukin-8, complement factor 5a as well as epidermal growth factor to target cells was dramatically reduced, the inhibition was not receptor or cytokine specific. In addition, the respective binding of radiolabeled monoclonal antibodies CL203 and R15.7 to the cell surface antigens intracellular cell adhesion molecule-1 and lymphocyte function-associated antigen-1 beta-chain was decreased by pretreatment of cells with pheophorbide a as well. In all cases, the inhibition by pheophorbides was dependent on the simultaneous presence of light, indicating causative involvement of a photodynamic process. These observations are not unique to pheophorbides and can be extended to porphyrins as well as to other photodynamic agents. Cytotoxicity resulting from photodynamic therapy (PDT) has been documented by many studies. Our investigations suggest that the inactivation of cell surface receptors contributes not only to an antitumor effect of PDT but also to the systemic immunosuppression, a serious side effect of PDT.

Horseradish peroxidase-catalyzed aerobic oxidation of indole-3-acetic acid. II. Oxygen uptake and chemiexcitation.

Light emission from the horseradish peroxidase-catalyzed aerobic or anaerobic oxidation of indole-3-acetic acid has been investigated under opposite extreme conditions of enzyme/substrate ratio. The O2-dependent chemiluminescent processes represent a minor part of the total oxygen consumption. Superoxide is involved in chemiexcitation as is evident from the observed inhibitory effect of superoxide dismutase. At high enzyme/substrate ratio, only a part of the emission is dependent on superoxide ion; at low ratio the dependence is extensive. At high ratio, some of the emission is independent of superoxide and O2. The identical quenching effects of D- and L-tryptophan are consistent with the formation of the quenching species only in bulk solution. The similarity of the emission spectra under extreme conditions indicates that the same main emitters are formed. This is also supported by the effect of quenchers. Possibly some of the emitters originate in the oxidative cleavage of the 2,3-double bond of the indole ring.