Cocaine is a widely abused and addictive drug, resulting in serious medical and social problems in modern society. Currently, there is no FDA-approved medication specific for cocaine abuse treatment. The disastrous medical and social consequences of cocaine abuse have made the development of an anti-cocaine medication a high priority. However, despite decades of efforts, traditional pharmacodynamic approach has failed to yield a truly useful small-molecule drug due to the difficulties inherent in blocking a blocker like cocaine without affecting the normal functions of the transporters or receptors. An alternative approach, i.e. pharmacokinetic approach, is to interfere with the delivery of cocaine to its receptors/transporters and/or accelerate its metabolism in the body. It would be an ideal anti-cocaine medication to accelerate cocaine metabolism producing biologically inactive metabolites. Two natural enzymes may catalyze hydrolysis of cocaine: human butyrylcholinesterase (BChE) and bacterial cocaine esterase (CocE). However, the wild-type enzymes are not suitable as anti-cocaine therapeutics, due to the low catalytic activity, thermoinstability, or short biological half-life. In this investigation, we performed integrated computational-experimental studies to rationally design and discover mutants of these enzymes in order to improve the catalytic activity, thermostability, and/or biological half-life. To rationally design desirable mutants of the enzymes, we have successfully developed computational models, including those for BChE gating, glycosylated BChE structure, BChE-substrate complex structures, BChE dimer/tetramer structures, CocE monomer/dimer structures, and CocE-substrate complex structures. Development of the computational models enabled us to rationally design new amino-acid mutations that may improve the catalytic activity, thermostability, and/or prolonged biological half-life. The computational design was followed by wet experimental tests, including both in vitro and in vivo experiments, leading to discovery of new enzyme forms with not only a high catalytic efficiency against cocaine, but also an improved thermostability and/or prolonged biological half-life. The identified new mutants of BChE and CocE are expected to be valuable candidates for development of a more efficient enzyme therapy for cocaine abuse. The encouraging outcomes of the present study also suggest that the structure-and-mechanism-based design and integrated computational-experimental approach is promising for rational drug design and discovery.

Cocaine is a widely abused drug without an FDA-approved medication. It has been recognized as an ideal anti-cocaine medication to accelerate cocaine metabolism producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e. butyrylcholinesterase (BChE)-catalyzed hydrolysis. However, the native BChE has a low catalytic activity against cocaine. We recently designed and discovered a set of BChE mutants with a high catalytic activity specifically for cocaine. An ideal, therapeutically valuable mutant of human BChE should have not only a significantly improved catalytic activity against cocaine, but also certain selectivity for cocaine over neurotransmitter acetylcholine (ACh) such that one would not expect systemic administration of the BChE mutant to interrupt cholinergic transmission. Through integrated computational-experimental studies, several BChE mutants were identified to have not only a considerably improved catalytic efficiency against cocaine, but also the desirable selectivity for cocaine over ACh. Representative BChE mutants have been confirmed to be potent in actual protection of mice from acute toxicity (convulsion and lethality) of a lethal dose of cocaine (180 mg/kg, LD100). Pretreatment with the BChE mutant (i.e. 1 min prior to cocaine administration) dose-dependently protected mice against cocaine-induced convulsions and lethality. The in vivo data reveal the primary factor, i.e. the relative catalytic efficiency, determining the efficacy in practical protection of mice from the acute cocaine toxicity and future direction for further improving the efficacy of the enzyme in the cocaine overdose treatment. For further characterization in animal models, we successfully developed high-efficiency stable cell lines efficiently expressing the BChE mutants by using a lentivirus-based repeated-transduction method. The large-scale protein production enabled us to further characterize the in vivo profiles of the BChE mutant concerning the biological half-life and potency in accelerating cocaine clearance. In particular, it has been demonstrated that the BChE mutant can rapidly metabolize cocaine and completely eliminate cocaine-induced hyperactivity in rodents, implying that the BChE mutant may be developed as a promising therapeutic agent for cocaine abuse treatment.

Cocaine is one of the most reinforcing drugs of abuse and has caused serious medical and social problems. There is no FDA-approved medication specific for cocaine. It is of a high priority to develop an effective therapeutic treatment for cocaine abuse. Human butyrylcholinesterase (BChE) has been recognized as a promising candidate of enzyme therapy to metabolize cocaine into biologically inactive metabolites and prevent it from reaching central nervous system (CNS). However, the catalytic activity of wide-type human BChE against cocaine is not sufficiently high for treatment of cocaine abuse. Dr. Zhan’s lab has successfully designed and discovered a series of high-activity mutants of human BChE specific for cocaine metabolism. This dissertation is mainly focused to address the possible concerns in further development of promising human BChE mutants for cocaine detoxification, including whether the administration of this exogenous enzyme will affect the cholinergic system, whether it can efficiently hydrolyze cocaine’s toxic metabolites, and whether the commonly used therapeutic agents will significantly affect the catalytic activity of the BChE mutants against cocaine when they are co-administered. According to the results obtained, all of the examined BChE mutants have a considerably improved catalytic efficiency against (-)-cocaine, without significantly improving the catalytic efficiency against any of the other examined substrates, including neurotransmitter acetylcholine. Two representative mutants (including E12-7) also have a considerably improved catalytic activity against cocaethylene (formed from combined use of cocaine and alcohol) compared to wild-type BChE, and E12-7 can rapidly metabolize cocaethylene, in addition to cocaine, in rats. Further evaluation of possible drug-drug interactions between E12-7 and some other commonly used therapeutic agents revealed that all of the examined agents, except some tricyclic antidepressants, do not significantly inhibit E12-7. In addition, an effort to discover new mutants with further improved activity against cocaine led to the discovery of a new BChE mutant, denoted as E20-7, according to both the in vitro and in vivo assays. The encouraging outcomes of the present investigation suggest that it is possible to develop a more effective enzyme therapy for cocaine abuse treatment using one of the most promising BChE mutants, such as E12-7 or E20-7.

Todos os agrupamentos humanos que se organizam para o trabalho usam rios, lagos ou lagoas como depósitos para a decomposição de matéria indesejável. A contaminação do meio aquático por herbicidas e agrotóxicos derivados de práticas agrícolas se tornou, faz tempo, um problema de importância mundial. Precisamos de informações detalhadas sobre a bioquímica da intoxicação de peixes nativos para avaliarmos quais os efeitos de agrotóxicos sobre os processos bioquímicos que mantêm o ciclo de vida dos peixes em águas do Brasil. Organofosfatos, que são agrotóxicos de uso disseminado, podem interagir com as B-esterases butirilcolinesterase (EC 3.1.1.8) e carboxilesterase (EC 3.1.1.1) presentes no fígado e no plasma. Tanto a butirilcolinesterase (BChE) como a carboxilesterase (CarbE), se presentes em concentrações relativamente elevadas, agem como limpadores estequiométricos (scavengers moleculares) por ligarem o átomo de fósforo do grupo P=O com a hidroxila de uma serina presente nos seus sítios ativos. Em nossos resultados observamos que curimbatá possui a CarbE plasmática (IC50 74 ηM) mais sensível ao organofosfato metilparaoxon quando comparado ao pacu (IC50 691 ηM). Isolamos CarbE dos plasmas de curimbatá e pacu. Piavussu não possui uma atividade expressiva de CarbE no sangue, por isso não a isolamos. O tipo e a distribuição das esterases nos tecidos são particulares da espécie. Curimbatá tem alta atividade de CarbE no fígado (237,8 U.g-1) e no sangue (29,85 U.mL-1), pacu é dotado de alta atividade de BChE (134,0 U.g-1) e CarbE (149,6 U.g-1) no fígado, mas o piavussu conta apenas com a BChE do sangue (17,87 U.g-1). Este arsenal enzimático foi suficiente para proteger as AChE de cérebro, músculo e coração das três espécies e evitar a sua intoxicação leve por 0,2 mg metilparation/L. A abordagem cinético-bioquímica para conhecer a inibição das esterases presentes nos tecidos de diferentes espécies de peixes por / Every human group who organizes to work together uses rivers, lakes or ponds as places in which undesirable substances are deposited for decomposition. Contamination of the aquatic environment with herbicides and pesticides derived from agricultural practices has become a problem of global importance since a long ago. We need detailed information on the biochemistry of the poisoning of native fish to assess the effects of pesticides on the biochemical processes that maintain fishes life cycle in waters of Brazil. Organophosphates, which are widely used pesticides, can interact with the B-esterases butyrylcholinesterase (EC 3.1.1.8) and carboxylesterase (EC 3.1.1.1) in plasma and liver. Butyrylcholinesterase (BChE) and carboxylesterase (CarbE) in relatively high concentrations act as stoichiometric scavengers by linking the phosphorus atom of the P=O group with the serines hydroxyl they have in their active site. Our results show that the CarbE of curimbata plasma (IC50 74 ηm) is more sensitive to the organophosphate metilparaoxon than CarbE of pacu plasma (IC50 691 ηm). We isolated CarbE from curimbata and pacus plasma. Piavussu does not have an expressive activity of CarbE in plasma, so we did not isolate it. The type and distribution of esterases in tissues are peculiar to a species. Curimbata has high CarbE activity in the liver (237.8 U.g-1) and blood (29.85 U.mL-1), pacu is equipped with high activities of BChE (134.0 U.g-1) and CarbE (149.6 U.g-1) in the liver and piavussu relies only on BChE of blood (17.87 U.g-1). This enzymatic arsenal was sufficient to protect AChE from brain, muscle and heart of the three species and protect them against mild intoxication by methylparathion (0.2 mg / L). The biochemical kinetic approach that allows understanding of the inhibition of the esterases in tissues of different fish species is a good tool capable of anticipating the harmful consequences of these drugs.

abstract: Cocaine abuse affects millions of people with disastrous medical and societal consequences. Despite this, there is still no FDA-approved treatment to decrease the likelihood of relapse in rehabilitated addicts, and acute cocaine toxicity (overdose) is only symptomatically treated. Studies have demonstrated a promising potential treatment option with the help of the human serum enzyme butyrylcholinesterase (BChE), an enzyme capable of breaking down cocaine into biologically inactive side products. This activity of wild-type BChE, however, is relatively low. This prompted the design of variants of BChE which exhibit significantly improved catalytic activity against cocaine. Plants were used as a sustainable, scalable, affordable platform system to produce large amounts of human biologics such as these cocaine hydrolase variants of BChE. Using a tobacco relative, Nicotiana benthamiana, recombinant enzymes can be produced at quantities relevant to clinical use with desired kinetic properties. Next, the ability of the most promising plant-produced cocaine super hydrolase, pCocSH, to counter the lethal effects of cocaine overdose in vivo was tested. These studies revealed that this plant-produced enzyme can protect mice from an otherwise lethal dose of cocaine. Most excitingly, it was found that pCocSH can rescue mice from overdose when given immediately after the onset of cocaine-induced seizures. These studies provide in vitro and in vivo proof-of-principle for a promising plant-derived biologic to be used as a pharmacokinetic-based treatment for cocaine addiction-related diseases such as overdose. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2015

Todos os agrupamentos humanos que se organizam para o trabalho usam rios, lagos ou lagoas como depósitos para a decomposição de matéria indesejável. A contaminação do meio aquático por herbicidas e agrotóxicos derivados de práticas agrícolas se tornou, faz tempo, um problema de importância mundial. Precisamos de informações detalhadas sobre a bioquímica da intoxicação de peixes nativos para avaliarmos quais os efeitos de agrotóxicos sobre os processos bioquímicos que mantêm o ciclo de vida dos peixes em águas do Brasil. Organofosfatos, que são agrotóxicos de uso disseminado, podem interagir com as B-esterases butirilcolinesterase (EC 3.1.1.8) e carboxilesterase (EC 3.1.1.1) presentes no fígado e no plasma. Tanto a butirilcolinesterase (BChE) como a carboxilesterase (CarbE), se presentes em concentrações relativamente elevadas, agem como limpadores estequiométricos (scavengers moleculares) por ligarem o átomo de fósforo do grupo P=O com a hidroxila de uma serina presente nos seus sítios ativos. Em nossos resultados observamos que curimbatá possui a CarbE plasmática (IC50 74 ηM) mais sensível ao organofosfato metilparaoxon quando comparado ao pacu (IC50 691 ηM). Isolamos CarbE dos plasmas de curimbatá e pacu. Piavussu não possui uma atividade expressiva de CarbE no sangue, por isso não a isolamos. O tipo e a distribuição das esterases nos tecidos são particulares da espécie. Curimbatá tem alta atividade de CarbE no fígado (237,8 U.g-1) e no sangue (29,85 U.mL-1), pacu é dotado de alta atividade de BChE (134,0 U.g-1) e CarbE (149,6 U.g-1) no fígado, mas o piavussu conta apenas com a BChE do sangue (17,87 U.g-1). Este arsenal enzimático foi suficiente para proteger as AChE de cérebro, músculo e coração das três espécies e evitar a sua intoxicação leve por 0,2 mg metilparation/L. A abordagem cinético-bioquímica para conhecer a inibição das esterases presentes nos tecidos de diferentes espécies de peixes por / Every human group who organizes to work together uses rivers, lakes or ponds as places in which undesirable substances are deposited for decomposition. Contamination of the aquatic environment with herbicides and pesticides derived from agricultural practices has become a problem of global importance since a long ago. We need detailed information on the biochemistry of the poisoning of native fish to assess the effects of pesticides on the biochemical processes that maintain fishes life cycle in waters of Brazil. Organophosphates, which are widely used pesticides, can interact with the B-esterases butyrylcholinesterase (EC 3.1.1.8) and carboxylesterase (EC 3.1.1.1) in plasma and liver. Butyrylcholinesterase (BChE) and carboxylesterase (CarbE) in relatively high concentrations act as stoichiometric scavengers by linking the phosphorus atom of the P=O group with the serines hydroxyl they have in their active site. Our results show that the CarbE of curimbata plasma (IC50 74 ηm) is more sensitive to the organophosphate metilparaoxon than CarbE of pacu plasma (IC50 691 ηm). We isolated CarbE from curimbata and pacus plasma. Piavussu does not have an expressive activity of CarbE in plasma, so we did not isolate it. The type and distribution of esterases in tissues are peculiar to a species. Curimbata has high CarbE activity in the liver (237.8 U.g-1) and blood (29.85 U.mL-1), pacu is equipped with high activities of BChE (134.0 U.g-1) and CarbE (149.6 U.g-1) in the liver and piavussu relies only on BChE of blood (17.87 U.g-1). This enzymatic arsenal was sufficient to protect AChE from brain, muscle and heart of the three species and protect them against mild intoxication by methylparathion (0.2 mg / L). The biochemical kinetic approach that allows understanding of the inhibition of the esterases in tissues of different fish species is a good tool capable of anticipating the harmful consequences of these drugs.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Sporotrichosis is an implantation mycose, caused by species complex Sporothrix spp., considered the subcutaneous mycosis with higher occurrence and importance in the Americas, this thermally dimorphic fungus induces a chronic inflammatory granulomatous response in their hosts. The cholinesterase, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are the "key" enzimes signal in inflammatory and immune processes, for regulating the levels of ACh, the main neurotransmitter "cholinergic antiinflammatory pathway". The cholinesterase activity has been demonstrated in various inflammatory conditions; however, there is no evidence of his participation in sporotrichosis. The objective of this study was to report a case of feline sporotrichosis by natural infection; evaluate the activities of AChE in the lymphocytes and whole blood; evaluate the activity of BChE; cytokines (IL1, IL6, TNF, and IL-10 INFγ) and immunoglobulins (IgA, IgG, IgM, IgE) in serum of rats experimentally infected with S. schenckii. Sixty-three rats were divided into three groups, control group (CG), group by subcutaneous infection (GSC) and group by intraperitoneal infection (GIP). Blood collection for activities evaluation of cholinesterase, cytokines and immunoglobulins were held on 15, 30 and 40 post-infection (PI). The GIP, had an increase in the activity of AChE in the blood in all experimental times, the increase in lymphocytes occurred only on 30 and 40 days PI. The increase of the enzyme suggests a decrease in the levels of ACh with consequently increased IL-1, IL-6, TNF-α, INF-γ, as observed in this study. In the acute phase of infection wants an intense inflammatory response in an attempt to remove the agent. However, with the chronicity of infection if the stimulus is maintained can lead to remarkable tissue injury. Regarding the chronic process observed in GIP, 30 and 40 days PI, that IL-10 was shown to be increased, but not enough to contain inflammation, as pro cytokines, continued high levels. Conversely, what happened in the GIP, the SC group had reduced levels of IL-10 at all times PI, this decrease can lead to tissue damage by not blocking the inflammatory response. The activity of AChE in the GSC only statistically increased in lymphocytes, at 40 days PI. There was no significant activity when assessing the BChE, in both groups, except in the GSC 40 days PI. The BChE is not efficient in Ach s hydrolysis in low concentrations, but it can replace the AChE when itself is inhibited. High levels of immunoglobulins in all groups and at all times, shows the induction of humoral immune response in sporotrichosis. Thus, the increase in the AChE activity in whole blood and lymphocytes, together with high levels of serum immunoglobulins and cytokines, suggesting the involvement of cholinesterases, and in immune cell response against infection by S. schenckii. / A esporotricose é uma micose de implantação, causada pelo complexo de espécies Sporothrix spp., considerada a micose subcutânea com a maior ocorrência e importância nas Américas, esse fungo termodimórfico induz uma resposta inflamatória granulomatosa crônica em seus hospedeiros. As colinesterases, acetilcolinesterase (AChE) e butirilcolinesterase (BChE) desempenham um papel chave na sinalização de processos inflamatórios e imunes, por regular os níveis de ACh, o principal neurotransmissor da via colinérgica antiinflamatória . A atividade das colinesterases tem sido demonstrada em várias condições inflamatórias, contudo, não há evidencias de sua participação na esporotricose. O objetivo deste estudo foi relatar um caso de esporotricose felina por infecção natural; avaliar as atividades da AChE em linfócitos e no sangue total; avaliar a atividade da BChE; citocinas (IL1, IL6, TNFα, INFγ e IL10) e imunoglobulinas (IgA, IgG, IgM, IgE) no soro de ratos infectados experimentalmente por S. schenckii. Foram utilizados sessenta e três ratos distribuídos em três grupos, grupo controle (GC), grupo por infecção subcutânea (GSC) e grupo por infecção intraperitoneal (GIP). A coleta de sangue para avaliação das atividades das colinesterases, citocinas e imunoglobulinas foram realizadas nos dias 15, 30 e 40 pós-infecção (PI). No GIP, ocorreu um aumento na atividade das AChE no sangue em todos os tempos experimentais, nos linfócitos o aumento ocorreu apenas nos dias 30 e 40 dias PI. O aumento dessa enzima sugere uma diminuição nos níveis de ACh, com consequentemente aumento das IL-1, IL-6, TNFα, INFγ, como observado neste estudo. Na fase aguda da infecção deseja-se uma resposta inflamatória intensa na tentativa de eliminar o agente. Porém, com a cronicidade da infecção, se o estímulo for mantido pode acarretar marcante lesão tecidual. Com relação ao processo crônico observou no GIP, aos 30 e 40 dias PI, que a IL-10 mostrou-se aumentada, porém não sendo suficiente para conter a inflamação, pois as citocinas pró, continuaram com altos níveis. Inversamente o que aconteceu no GIP, o grupo SC obteve níveis reduzidos de IL 10 em todos s tempos PI, este decréscimo pode levar a danos teciduais pelo não bloqueio da resposta inflamatória. A atividade da AChE, no GSC só se mostrou estatisticamente aumentada nos linfócitos, aos 40 dias PI. Não houve atividade estatística significativa ao avaliar a BChE, em ambos os grupos, exceto no GSC 40 dias PI. A BChE não é eficiente em hidrolisar ACh em baixas concentrações, mas ela pode substituir a AChE na hidrolise da ACh quando a mesma estiver inibida. Os altos níveis evidenciados das imunoglobulinas em ambos os grupos e em todo os tempos, demonstra a indução da resposta imune humoral na esporotricose. Assim, o aumento na atividade da AChE em sangue total e em linfócitos, juntamente com os elevados níveis de citocinas e imunoglobulinas séricas, sugere-se o envolvimento dessa colinesterase, na resposta imune e celular frente a infecção por S. schenckii.

Bulvová, L.: Alkaloids of Papaver rhoeas L. (Papaveraceae) and their biological activity related to Alzheimerʼs disease I. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany and Ecology, Hradec Králové 2017. The aim of this study was to process the summary alkaloidal extract of aerial parts of Papaver rhoeas L.; to isolate contained alkaloids using chromatographical methods; to identify them and to determine their inhibitory activity towards human enzymes acetylcholinesterase, butyrylcholinesterase and prolyloligopeptidase. Two alkaloids (+)-rhoeagenine and LB-2 were isolated, and the structure of LB-2 (its absolute configuration) is being determined nowadays. In vitro biological assays of these alkaloids found the following results: (+)-rhoeagenine (IC50 AChE ˃ 1000 μM, IC50 BuChE = 230 ± 10 μM, IC50 POP = 878 ± 45 μM) and LB-2 (IC50 AChE ˃ 1000 μM, IC50 BuChE = 314 ± 13 μM, IC50 POP = 706 ± 2 μM). The determined IC50 values of isolated alkaloids were compared with inhibitory standards of cholinesterases galanthamine (IC50 AChE = 1,71 ± 0,065 μM, IC50 BuChE = 42,30 ± 1,30 μM), huperzine A (IC50 AChE = 0,033 ± 0,001 μM, IC50 BuChE > 1000 μM, IC50 POP > 1000 μM) and rivastigmine (IC50 AChE = 0,037 ± 0,001 μM, IC50 BuChE = 0,0033 ± 0,0003...

Čakurdová, M.: Alkaloids of Papaver rhoeas L. (Papaveraceae) and their biological activity related to Alzheimer's disease II. Diploma thesis, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany and Ecology, Hradec Králové, 2017. The aim of this work was focused on isolation of alkaloids from the fraction 1 (joined fractions 20-27), which was obtained from the summary alkaloid extract of Papaver rhoeas L. (Papaveraceae). The alkaloids were identified by structural analysis NMR, GC-MS, optical activity and melting point. Two alkaloids were identified as (-)-stylopine and (+)-rhoeadine. Isolated substances were tested on ability to inhibit the enzymes acetylcholinesterase, butyrylcholinesterase and prolyloligopeptidase. Obtained data were expressed as IC50 values: (-)-stylopine (IC50 AChE = 522 ± 67 µM, IC50 BuChE = >1000 µM, IC50 POP = >790 µM); (+)-rhoeadine (IC50 AChE = 915 ± 64 µM, IC50 BuChE = >1000 µM, IC50 POP = >790 µM). None of the isolated substances showed so potent cholinesterase inhibitory activity as the alkaloids galanthamine (IC50 AChE = 1.71± 0.065 µM, IC50 BuChE =42.30 ± 1.30 µM) and huperzine A (IC50 AChE = 0.033 ± 0.001 µM, IC50 BuChE = >1000 µM) or rivastigmine (IC50 AChE = 0.037 ± 0.001 µM, IC50 BuChE = 3.3 ± 0.3 nM)....

Miklová V.: Cytotoxic and cholinesterase inhibitory activity of extracts from selected species of the Centaurea L. genus II. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany, Hradec Králové, 2020. This Diploma Thesis was carried out at the Department of Pharmaceutical Botany FAF UK and it is a part of a screening of biologically active secondary metabolites of selected on taxa of the genera Centaurea L. and Psephellus L. in the family Asteraceae. Secondary metabolites are responsible for various effects on the human body. The study is focused on the phytochemical research of extracts prepared from aerial parts (cauloms with leaves) of Centaurea cyanus L., Centaurea stoebe L., Cyanus montanus (L.) Hill, Centaurea benedicta L., Centaurea jacea L., Centaurea macrocephala Muss. Puschk. ex. Willd, Centaurea solstitialis L., Centaurea nigra L., Centaurea scabiosa L., Psephellus bellus (Trautv.) Wagenitz, Centaurea pannonica (Heuff.) Hayek and Psephellus dealbatus (Willd) K. Koch. Both ethyl acetate and summary ethanolic extracts were prepared for detection of individual groups of secondary metabolites by thin-layer chromatohraphy. Consistently with previously published results, sesquiterpenes, flavonoids, coumarins, phenolic compounds,...