RESUMO
Flavonoids and chalcones are known for their manifold biological activities, of which many affect the central nervous system. Pyranochalcones were recently shown to have a great neurogenic potential, which is partly due to a specific structural motif-the pyran ring. Accordingly, we questioned if other flavonoid backbones with a pyran ring as structural moiety would also show neurogenic potential. Different semi-synthetic approaches starting with the prenylated chalcone xanthohumol, isolated from hops, led to pyranoflavanoids with different backbones. We identified the chalcone backbone as the most active backbone with pyran ring using a reporter gene assay based on the promoter activity of doublecortin, an early neuronal marker. Pyranochalcones therefore appear to be promising compounds for further development as a treatment strategy for neurodegenerative diseases.
Assuntos
Chalcona , Chalconas , Humulus , Propiofenonas , Chalcona/química , Flavonoides/química , Propiofenonas/química , Humulus/químicaRESUMO
Isatis tinctoria and its indigo dyes have already provided highly active anti-leukaemic lead compounds, with the focus mainly being on indirubin, whereas indigo itself is inactive. There are many more indigoids to find in this plant extract, for example, quingdainone, an indigoid derived from tryptanthrin. We present here a new synthesis of hitherto neglected substituted quingdainones, which is very necessary due to their poor solubility behaviour, and a structure-dependent anti-leukaemic activity study of a number of compounds. Substituted α-phenylaminoacrylic acid was synthesised by hydrogen sulfide extrusion from an analogue mercaptoacetic acid, available from the condensation of rhodanin and a substituted tryptanthrin. It is shown that just improving water solubility does not increase anti-leukaemic activity, since a quingdainone carboxylic acid is inactive compared to dihydroxyquingdainone. The most effective compound, dihydroxyquingdainone with an AC50 of 7.5 µmole, is further characterised, revealing its ability to overcome multidrug resistance in leukaemia cells (Nalm-6/BeKa) with p-glycoprotein expression.
Assuntos
Citostáticos , Leucemia , Linfoma , Apoptose , Caspase 3 , Índigo Carmim , Leucemia/tratamento farmacológico , Folhas de PlantaRESUMO
Loss of neuronal tissue is a hallmark of age-related neurodegenerative diseases. Since adult neurogenesis has been confirmed in the human brain, great interest has arisen in substances stimulating the endogenous neuronal regeneration mechanism based on adult neural stem cells. Medicinal plants are a valuable source of neuroactive small molecules. In the structure-activity study presented here, the activities of prenyl- and pyranochalcones were compared to each other, using a differentiation assay based on the doublecortin promoter sequences. The latter revealed that the pyrano ring is a crucial structural element for the induction of neuronal differentiation of adult neural stem cells, while compounds with a prenyl group show significantly lower activities. Furthermore, a decrease of pro-differentiation activity was observed following structural modifications, such as substitutions on the pyrano ring and on the B-ring of the chalcone. We also initiated the elucidation of the structural characteristics of the newly discovered lead substance xanthohumol C, which correlated with the activation of the doublecortin promoter during neuronal differentiation.
Assuntos
Chalconas/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese , Regeneração , Animais , Diferenciação Celular/efeitos dos fármacos , Humulus/química , Camundongos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Microbial cyclic lipopeptides are an important class of antifungal compounds with applications in pharmacology and biotechnology. However, the cytotoxicity of many cyclic lipopeptides limits their potential as antifungal drugs. Here we present a structure-activity relationship study on the puwainaphycin/minutissamide (PUW/MIN) family of cyclic lipopeptides isolated from cyanobacteria. PUWs/MINs with variable fatty acid chain lengths differed in the dynamic of their cytotoxic effect despite their similar IC50 after 48 hours (2.8 µM for MIN A and 3.2 µM for PUW F). Furthermore, they exhibited different antifungal potency with the lowest MIC values obtained for MIN A and PUW F against the facultative human pathogen Aspergillus fumigatus (37 µM) and the plant pathogen Alternaria alternata (0.6 µM), respectively. We used a Grignard-reaction with alkylmagnesium halides to lengthen the lipopeptide FA moiety as well as the Steglich esterification on the free hydroxyl substituents to prepare semi-synthetic lipopeptide variants possessing multiple fatty acid tails. Cyclic lipopeptides with extended and branched FA tails showed improved strain-specific antifungal activity against A. fumigatus (MIC = 0.5-3.8 µM) and A. alternata (MIC = 0.1-0.5 µM), but with partial retention of the cytotoxic effect (â¼10-20 µM). However, lipopeptides with esterified free hydroxyl groups possessed substantially higher antifungal potencies, especially against A. alternata (MIC = 0.2-0.6 µM), and greatly reduced or abolished cytotoxic activity (>20 µM). Our findings pave the way for a generation of semi-synthetic variants of lipopeptides with improved and selective antifungal activities.
RESUMO
The isomers 8-prenylnaringenin and 6-prenylnaringenin, both secondary metabolites occurring in hops, show interesting biological effects, like estrogen-like, cytotoxic, or neuro regenerative activities. Accordingly, abundant sources for this special flavonoids are needed. Extraction is not recommended due to the very low amounts present in plants and different synthesis approaches are characterized by modest yields, multiple steps, the use of expensive chemicals, or an elaborate synthesis. An easy synthesis strategy is the demethylation of xanthohumol, which is available due to hop extraction industry, using lithium chloride and dimethylformamide, but byproducts and low yield did not make this feasible until now. In this study, the demethylation of xanthohumol to 8-prenylnaringenin and 6-prenylnaringenin is described the first time and this reaction was optimized using Design of Experiment and microwave irradiation. With the optimized conditions-temperature 198 °C, 55 eq. lithium chloride, and a reaction time of 9 min, a final yield of 76% of both prenylated flavonoids is reached.
Assuntos
Desmetilação , Flavanonas/síntese química , Flavonoides/química , Flavonoides/síntese química , Micro-Ondas , Propiofenonas/química , Projetos de Pesquisa , Flavanonas/química , Cloreto de Lítio/química , Temperatura , Fatores de TempoRESUMO
Bacopa monnieri is an Ayurvedic plant with rising interest in the pharmacological effects of its extract and constituents, including flavonoids, saponins, and triterpenes such as cucurbitacins, betulinic acid, and bacosine. The latter two compounds are isomeric 3-hydroxy lupenoic acids, which vary only in the arrangement of the carboxylic acid group and the methyl group at C-27 and C-28 and the orientation of the hydroxy group at C-3. In this study, we have reinvestigated the contents of betulinic acid and bacosine, respectively, in extracts from various commercially available B. monnieri powders and food supplements. To our surprise, HPLC-ion trap time-of-flight analyses identified only betulinic acid, but not bacosine, in all extracts under study, which was verified by GC-MS, HPLC-ELSD, 1D NMR (1H,13C), and 2D NMR (1H,1H COSY, 1H,13C HMBC, 1H,13C HSQC, 1H,1H NOESY) experiments. Moreover, it turned out that commercially available reference samples of bacosine were structurally identical with betulinic acid.
Assuntos
Bacopa , Saponinas , Triterpenos , Cromatografia Líquida de Alta Pressão , Extratos VegetaisRESUMO
Prenylated flavonoids from hops (Humulus lupulus) have become of interest in recent years due to a range of bioactivities. The potential health benefits of prenylated flavonoids include anti-cancerous activities and treatment of the metabolic syndrome among others. Since prenylated flavonoids from hops have shown pharmaceutical potential in clinical trials, robust analytical methods to determine their concentrations in food, supplements, and beverages are required. One such, the gold standard of analytical methods, is stable isotope dilution analysis due to its ability to compensate matrix effects and losses during sample work-up. As no commercial standards were available, the synthesis of seven different prenylated flavonoid isotopes utilizing various strategies (microwave assistance, acid base catalyst in the presence of deuterated substance and lastly, the use of Strykers catalyst) is described. The produced prenylated flavonoid isotopes were then applied in the first stable isotope dilution analysis method that quantified six natural prenylated flavonoids (Isoxanthohumol, Isoxanthohumol-C, 8-Prenylnaringenin, 6- Prenylnaringenin, Xanthohumol, and Xanthohumol-C) in beer, hop tea and hops to prove its applicability. The SIDA-LC-MS/MS method was validated resulting in LODs and LOQs for all analytes between 0.04 and 3.2 µg/L. Moreover, due to the simple clean-up the developed method allows the prospect for measuring clinical samples in the future.
RESUMO
The chroman-like chalcone Xanthohumol C, originally found in hops, was demonstrated to be a potent neuroregenerative and neuroprotective natural product and therefore constitutes a strong candidate for further pharmaceutical research. The bottleneck for in vivo experiments is the low water solubility of this chalcone. Consequently, we developed and validated a suitable formulation enabling in vivo administration. Cyclodextrins were used as water-soluble and nontoxic complexing agents, and the complex of Xanthohumol C and 2-hydroxypropyl-ß-cyclodextrin was characterized using HPLC, HPLC-MS, NMR, and differential scanning calorimetry. The water solubility of Xanthohumol C increases with increasing concentrations of cyclodextrin. Using 50 mM 2-hydroxypropyl-ß-cyclodextrin, solubility was increased 650-fold. Furthermore, in vitro bioactivity of Xanthohumol C in free and complexed form did not significantly differ, suggesting the release of Xanthohumol C from 2-hydroxypropyl-ß-cyclodextrin. Finally, a small-scaled in vivo experiment in a rat model showed that after i.âp. administration of the complex, Xanthohumol C can be detected in serum, the brain, and the cerebrospinal fluid at 1 and 6 h post-administration. Mean (± SD) Xanthohumol C serum concentrations after 1, 6, and 12 h were determined as 463.5 (± 120.9), 61.9 (± 13.4), and 9.3 (± 0.8) ng/mL upon i.âv., and 294.3 (± 22.4), 45.5 (± 0.7), and 13 (± 1.0) ng/mL after i.âp. application, respectively. Accordingly, the formulation of Xanthohumol C/2-hydroxypropyl-ß-cyclodextrin is suitable for further in vivo experiments and further pharmaceutical research aiming for the determination of its neuroregenerative potential in animal disease models.
Assuntos
2-Hidroxipropil-beta-Ciclodextrina/administração & dosagem , Benzopiranos/administração & dosagem , 2-Hidroxipropil-beta-Ciclodextrina/química , 2-Hidroxipropil-beta-Ciclodextrina/farmacocinética , Animais , Benzopiranos/química , Benzopiranos/farmacocinética , Disponibilidade Biológica , Varredura Diferencial de Calorimetria , Cromatografia Líquida de Alta Pressão , Estabilidade de Medicamentos , Espectroscopia de Ressonância Magnética , Ratos , SolubilidadeRESUMO
Restoration of neuronal connectivity after lesion of the central nervous system, such as spinal cord injury, is one of the biggest challenges in modern medicine. In particular, the accumulation of axon growth inhibitory factors at the site of injury constitutes a major obstacle to structural and thus functional repair. We previously investigated a group of prenylflavonoids derived from hops for their capacity to promote neuroregeneration. We identified a molecule called ENDF1 that was very potent to enhance regrowth and branching of neurites from dorsal root ganglion neurons in culture on growth promoting substrates. In the present study, we investigated ENDF1's capacity to promote regeneration of rat dorsal root ganglion neurons in vitro in the presence of three main components of the extracellular matrix acting as axon growth inhibitors: Semaphorin 3A, Ephrin A4 and mixed chondroitin sulfate proteoglycans. We report that ENDF1 application significantly promoted the percentages of sensory neurons able to regrow their neurites regardless of the presence of those inhibitors, and this to an extent similar to the one obtained after NGF treatment. Moreover, ENDF1 strongly enhanced the total neurite length and the complexity of neurites extending from neurons challenged with axon growth inhibitors. Although the impact of NGF and ENDF1 on the regeneration of neurons was similar, the activity of ENDF1 was not mediated by signaling through the TrkA receptor, indicating that each molecule act through different signaling pathways. In addition, ENDF1 did not decrease the phosphorylation of cofilin, a downstream effector of the regeneration-associated RhoA/ROCK signaling pathway. Hence, ENDF1 is a potent pro-neuroregenerative factors that could help in identifying new efficient targets for regenerative therapies of the nervous system.
RESUMO
BACKGROUND/AIMS: Prenylnaringenins are natural prenylflavonoids with anticancer properties. However, the underlying mechanisms have not been elucidated yet. Here we report a novel mode of action of 6- and 8-prenylnaringenin (PN) on human melanoma cells: Inhibition of cellular histone deacetylases (HDACs). METHODS: We performed in silico and in vitro analyses using 6-PN or 8-PN to study a possible interaction of 6-PN or 8-PN with HDAC as well as Western blot and FACS analyses, real-time cell proliferation and cell viability assays to assess the impact of 6-PN and 8-PN on human metastatic melanoma cells. RESULTS: In silico, 6-PN and 8-PN fit into the binding pocket of HDAC2, 4, 7 and 8, binding to the zinc ion of their catalytic center that is essential for enzymatic activity. In vitro, 100 µmol/L of 6-PN or 8-PN inhibited all 11 conserved human HDAC of class I, II and IV. In clinical oncology HDAC inhibitors are currently investigated as new anticancer compounds. In line, treatment of SK-MEL-28 cells with 6-PN or 8-PN induced a hyperacetylation of histone complex H3 within 2 h. Further, 6-PN or 8-PN mediated a prominent, dose-dependent reduction of cellular proliferation and viability of SK-MEL-28 and BLM melanoma cells. This effect was apoptosis-independent and accompanied by down-regulation of mTOR-specific pS6 protein via pERK/pP90 in SK-MEL-28 cells. CONCLUSION: The identification of a broad inhibitory capacity of 6-PN and 8-PN for HDAC enzymes with antiproliferative effects on melanoma cells opens the perspective for clinical application as novel anti-melanoma drugs and the usage as innovative lead structures for chemical modification to enhance pharmacology or inhibitory activities.
Assuntos
Apoptose/efeitos dos fármacos , Flavanonas/farmacologia , Flavonoides/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Humulus/química , Acetilação/efeitos dos fármacos , Sítios de Ligação , Domínio Catalítico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Flavanonas/química , Flavanonas/isolamento & purificação , Flavonoides/química , Flavonoides/isolamento & purificação , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/isolamento & purificação , Histona Desacetilases/química , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Humulus/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Simulação de Acoplamento Molecular , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Proteínas Quinases S6 Ribossômicas/genética , Proteínas Quinases S6 Ribossômicas/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Malaria remains one of the most deadly diseases threatening humankind and is still affecting a significant proportion of the world population, especially in Africa. Chemotherapy is a vital component of the fight against the disease and new antimalarial agents are urgently needed to curb the spread of malaria parasites that are resistant to existing drugs. The natural product tryptanthrin is known for its wide range of activities, including antiplasmodial activity, but its poor solubility has undermined its development as potent antimicrobial and antiprotozoan agent. The aim of this work was to synthesize analogues of tryptanthrin and to evaluate their antiplasmodial activity against the asexual and sexual blood stages of Plasmodium falciparum. Our results suggest that most tryptanthrin analogues retained their antiplasmodial activity against chloroquine-sensitive and chloroquine-resistant malaria parasites in the nanomolar range (30-100 nM). The antiplasmodial activity of the most active compound NT1 (IC50: 30 nM; SI: 155.9) was similar in both strains and close to that of chloroquine (IC50: 20 nM) on the sensitive strain. The antiplasmodial activity was improved with derivatization, thus pointing out the necessity to explore tryptanthrin using medicinal chemistry approaches. Ten (10) of the tested derivatives met the criteria, allowing for advancement to animal testing, i.e., SI > 100 and IC50 < 100 nM. In addition to their activity on the asexual stages, tryptanthrin and two selected derivatives (NT1 and T8) prevented the maturation of gametocytes at their IC90 concentrations, indicating a transmission-blocking potential. Moreover, NT1 was able to impair gametogenesis by reducing the exflagellation of microgametes by 20% at IC90, while tryptanthrin and T8 had no influence on exflagellation. The results of this study confirm that tryptanthrin and its derivatives are potential antimalarial candidates with abilities to kill the intraerythrocytic asexual stages and prevent the formation of sexual stages of the parasite.
Assuntos
Antimaláricos/síntese química , Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Quinazolinas/síntese química , Quinazolinas/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistência a Medicamentos , Humanos , Estrutura MolecularRESUMO
Neurodegenerative diseases are an increasing burden for our ageing societies; there is an as yet unmet need for the development of effective therapies. Neurogenesis, i.e., the generation of new neurons in the adult brain from neural stem cells, has received increasing attention since it offers the potential for endogenous brain repair and functional regeneration. Adult neurogenesis is partially under the control of sex hormones such as estradiol, and boosting neurogenesis with estradiol in animals correlates with cognitive improvement. 8-Prenylnaringenin imitates as highly potent phytoestrogen the effects of estradiol. Here, we studied the potential of 8-prenylnaringenin, 6-prenylnaringenin, and related compounds on differentiation induction in vitro using neural precursor cells transiently transfected with a doublecortin promoter luciferase construct, which was recently shown to indicate neuronal fate and differentiation. The flavanones 8-prenylnaringenin and 6-prenylnaringenin showed slight activity in this assay but significant activity by immunostaining. Although the estrogen-like activities of 8-prenylnaringenin and 6-prenylnaringenin are very different, the activity in differentiation induction is similar. Interestingly, also some prenylflavonoids with extended prenyl groups, e.g., a geranyl group, showed increased differentiation activity, while estrogen-like activity is decreased. This allows the conclusion that estrogen-like activity of prenylflavanones does not correlate directly with the activity of differentiation induction in neural precursor cells.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Flavanonas/farmacologia , Humulus/química , Células-Tronco Neurais/efeitos dos fármacos , Fitoestrógenos/farmacologia , Extratos Vegetais/farmacologia , Animais , Annonaceae/química , Linhagem Celular , Estradiol/metabolismo , Estrogênios/metabolismo , Flavonoides/farmacologia , Camundongos , Células-Tronco Neurais/fisiologia , NeurôniosRESUMO
Flavonoids target a variety of pathophysiological mechanisms and are therefore increasingly considered as compounds encompassed with therapeutic potentials in diseases such as cancer, diabetes, arteriosclerosis, and neurodegenerative diseases and mood disorders. Hops (Humulus lupulus L.) is rich in flavonoids such as the flavanone 8-prenylnaringenin, which is the most potent phytoestrogen identified so far, and the prenylchalcone xanthohumol, which has potent tumor-preventive, anti-inflammatory and antiviral activities. In the present study, we questioned whether hops-derived prenylflavonoids and synthetic derivatives thereof act on neuronal precursor cells and neuronal cell lines to induce neuronal differentiation, neurite outgrowth and neuroprotection. Therefore, mouse embryonic forebrain-derived neural precursors and Neuro2a neuroblastoma-derived cells were stimulated with the prenylflavonoids of interest, and their potential to activate the promoter of the neuronal fate-specific doublecortin gene and to stimulate neuronal differentiation and neurite outgrowth was analyzed. In this screening, we identified highly "neuroactive" compounds, which we termed "enhancement of neuronal differentiation factors" (ENDFs). The most potent molecule, ENDF1, was demonstrated to promote neuronal differentiation of neural stem cells and neurite outgrowth of cultured dorsal root ganglion neurons and protected neuronal PC12 cells from cobalt chloride-induced as well as cholinergic neurons of the nucleus basalis of Meynert from deafferentation-induced cell death. The results indicate that hops-derived prenylflavonoids such as ENDFs might be powerful molecules to promote neurogenesis, neuroregeneration and neuroprotection in cases of chronic neurodegenerative diseases, acute brain and spinal cord lesion and age-associated cognitive impairments.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Cromanos/farmacologia , Flavonoides/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Neuritos/fisiologia , Neurogênese , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Embrião de Galinha , Cobalto/toxicidade , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Flavanonas/farmacologia , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Neuropeptídeos/metabolismo , Células PC12 , Propiofenonas/farmacologia , Ratos , Células Tumorais CultivadasRESUMO
Polyphenols or phenolic compounds are groups of secondary metabolites widely distributed in plants and found in olive mill wastewater (OMW). Phenolic compounds as well as OMW extracts were evaluated in vitro for their antimicrobial activity against Gram-positive (Streptococcus pyogenes and Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae). Most of the tested phenols were not effective against the four bacterial strains when tested as single compounds at concentrations of up to 1000 µg mL(-1). Hydroxytyrosol at 400 µg mL(-1) caused complete growth inhibition of the four strains. Gallic acid was effective at 200, and 400 µg mL(-1) against S. aureus, and S. pyogenes, respectively, but not against the gram negative bacteria. An OMW fraction called AntiSolvent was obtained after the addition of ethanol to the crude OMW. HPLC analysis of AntiSolvent fraction revealed that this fraction contains mainly hydroxytyrosol (10.3%), verbascoside (7.4%), and tyrosol (2.6%). The combinations of AntiSolvent/gallic acid were tested using the low minimal inhibitory concentrations which revealed that 50/100-100/100 µg mL(-1) caused complete growth inhibition of the four strains. These results suggest that OMW specific fractions augmented with natural phenolic ingredients may be utilized as a source of bioactive compounds to control pathogenic bacteria.
RESUMO
The identification of effective cancer preventive compounds from hops has become an important issue in public health-related research. We compared the antiproliferative and apoptosis-inducing effects of side chain variants of prenylflavanones, e. g., 8-prenylnaringenin (7) and 8-geranylnaringenin (10), which have been identified in hops (Humulus lupulus), and their synthetic variations 8-furanmethylnaringenin (8) and 8-cinnamylnaringenin (9). These were accessible by a Mitsunobu reaction and Claisen rearrangement. Flavanones 9 and 10 showed cytotoxic and apoptotic activities. Apoptosis was induced in a mitochondrial dependent manner. 8-Cinnamylnaringenin (9) displayed noticeably improved apoptotic effects when compared to 8-prenylnaringenin. The potential of 8-prenylnaringenin (7) is shown in an ex vivo experiment on a multi-drug resistant leukaemia blast.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Humulus , Fitoterapia , Extratos Vegetais/farmacologia , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/uso terapêutico , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Flavanonas/administração & dosagem , Flavanonas/farmacologia , Flavanonas/uso terapêutico , Humanos , Extratos Vegetais/administração & dosagem , Extratos Vegetais/uso terapêuticoRESUMO
Starting from commercially available phloracetophenone (= 1-(2,4,6-trihydroxyphenyl)ethanone), we synthesized demethylxanthohumol (4), a derivative of xanthohumol, devoid of 6'-O-methyl group. Both are prenylchalcones derived from hops (Humulus lupulus). The synthesis was accomplished by an aldol condensation between MOM-protected acetophenone 2 and MOM-protected benzaldehyde 3. The resulting demethylxanthohumol (4) displayed antiproliferative properties. Demethylxanthohumol (4) induced also apoptosis via the mitochondrial pathway in BJAB cells (Burkitt lymphoma cell line) and in primary lymphoblasts of childhood acute lymphoblastic leukemia (ALL).