RESUMO
Kirsten rat sarcoma viral oncogene homolog (KRAS)-driven colorectal cancer (CRC) is notorious to target with drugs and has shown ineffective treatment response. The seeds of Pharbitis nil, also known as morning glory, have been used as traditional medicine in East Asia. We focused on whether Pharbitis nil seeds have a suppressive effect on mutated KRAS-driven CRC as well as reserving muscle cell functions during CRC progression. Seeds of Pharbitis nil (Pharbitis semen) were separated by chromatography and the active compound of Pharbitis semen (PN) was purified by HPLC. The compound PN efficiently suppressed the proliferation of mutated KRAS-driven CRC cells and their clonogenic potentials in a concentration-dependent manner. It also induced apoptosis of SW480 human colon cancer cells and cell cycle arrest at the G2/M phase. The CRC related pathways, including RAS/ERK and AKT/mTOR, were assessed and PN reduced the phosphorylation of AKT and mTOR. Furthermore, PN preserved muscle cell proliferation and myotube formation in cancer conditioned media. In summary, PN significantly suppressed mutated KRAS-driven cell growth and reserved muscle cell function. Based on the current study, PN could be considered as a promising starting point for the development of a nature-derived drug against KRAS-mutated CRC progression.
Assuntos
Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Ipomoea nil/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Humanos , Células Musculares/efeitos dos fármacos , Células Musculares/patologia , Mutação/efeitos dos fármacos , Sementes/químicaRESUMO
CONTEXT: Antiacetylcholinesterase (AChE) drugs have been a main therapeutic treatment for Alzheimer's disease because increased AChE levels play a key role in reducing neurotransmission. OBJECTIVES: Extracts from 35 Korean plants were selected and screened for antioxidant and anti-cholinesterase activity to explore new sources derived from Korean natural resources that could be used as AD therapeutic agents. MATERIALS AND METHODS: The antioxidant effect of extracts from 35 selected Korean plants was determined using two most common free radical scavenging assays using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS). Additionally, the effect of extracts, identified as antioxidants, on acetylcholinesterase inhibition was assessed by an acetylcholinesterase assay kit. RESULTS: Out of 36 extracts of 35 plants tested, Oenothera biennis L. (9.09 µg/mL), Saururus chinensis (Lour.) Baill. (9.52 µg/mL) and Betula platyphylla var. japonica (9.85 µg/mL) showed strong DPPH scavenging activity. Twelve other extracts also exerted moderate free radical scavenging activities with IC50 values ranging from 10 to 50 µg/mL. Antioxidant capacity detected by ABTS assay was only significant in O. biennis (23.40 µg/mL), while the other extracts were weak or unable to reduce the production of ABTS. Based on the antioxidant activities of these plant extracts, 19 extracts with IC50 values less than 100 µg/mL in DPPH assay were selected for further AChE inhibition assay. Among the extracts tested, the IC50 value for Prunella vulgaris var. lilacina NAKAI (18.83 µg/mL) in AChE inhibitory activity was the lowest, followed by O. biennis (20.09 µg/mL) and Pharbitis nil Chosy (22.79 µg/mL). CONCLUSIONS: Considering complex multifactorial etiology of AD, the extracts of P. vulgaris var. lilacina (aerial part), O. biennis (seed) and P. nil (seed) may be safe and ideal candidates for future AD modifying therapies.
Assuntos
Antioxidantes/farmacologia , Inibidores da Colinesterase/farmacologia , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Acetilcolinesterase/efeitos dos fármacos , Acetilcolinesterase/metabolismo , Antioxidantes/administração & dosagem , Antioxidantes/isolamento & purificação , Inibidores da Colinesterase/administração & dosagem , Inibidores da Colinesterase/isolamento & purificação , Sequestradores de Radicais Livres/isolamento & purificação , Sequestradores de Radicais Livres/farmacologia , Técnicas In Vitro , Concentração Inibidora 50 , Extratos Vegetais/administração & dosagem , Extratos Vegetais/isolamento & purificação , República da CoreiaRESUMO
Pharbitidis Semen, the seed of Morning glory (Pharbitis nil), is a medicinal agent that has traditionally been used as a purgative in Korea. Pharbilignan C (PLC) is a dihydro[b]-benzofuran-type neolignan from Pharbitidis Semen, which reportedly exhibited the most potent cytotoxicity against human tumor cells. To further study the antiproliferative activity of PLC, its molecular mechanisms of action in two breast adenocarcinoma cells, MCF-7 and MDA-MB 231 cells were investigated. PLC inhibited the proliferation of MDA-MB 231 and MCF-7 cells, in order of sensitivity (IC50 of MDA-MB 231 cells: 7.0±2.0µM). PLC induced apoptosis in MDA-MB 231 cells with down regulation of Bcl-2 and up-regulation of Bax expression. It also decreased mitochondrial membrane potential accompanied with increasing initiator caspase, caspase-9 activation and executioner caspase, caspase-3 activation. This study demonstrates that PLC inhibited proliferation of MDA-MB 231 cells by inducing apoptosis via the mitochondria-mediated intrinsic pathway.
Assuntos
Apoptose/efeitos dos fármacos , Benzofuranos/farmacologia , Neoplasias da Mama/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , HumanosRESUMO
BACKGROUND: Pharbitidis Semen (the seeds of Pharbitis nil), traditionally used as a purgative in Japan, China and Korea, contains a resin glycoside fraction named pharbitin, which is known as a purgative ingredient. Due to the complex nature of pharbitin, little is known about either the action on intestinal tension caused by resin glycoside itself or by its components. METHODS: In this study, we investigated the effects of pharbitin, the glycosidic acid fraction (pharbitic acid) and the aglycone fraction (phar-genin) generated from pharbitin on peristalsis of colon and ileum isolated from mice with the Magnus method. RESULTS: We demonstrated that pharbitin (3-30 µg/mL) concentration-dependently increased tonus of mice colon via acetylcholine receptors, its components phar-genin (1.27-12.7 µg/mL) and pharbitic acid (10-1000 µg/mL) also had the increment on colon tonus. On the other hand, ileum tension decreased in the presence of pharbitin. CONCLUSIONS: The effects of resin glycoside of Pharbitidis Semen on colon tonus are different with those on ileum tonus isolated from mice. In the next step it is necessary to investigate details of its pharmacological mechanism.
Assuntos
Glicosídeos , Sementes , Animais , Colo , Glicosídeos/farmacologia , Íleo , Camundongos , Resinas VegetaisRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Pharbitis nil (L.) Choisy is a medicinal herb, and herbal remedies based on its seeds have been used to treat of obesity and liver diseases, including fatty liver and liver cirrhosis in East Asia. AIM OF THE STUDY: Liver fibrosis is a major cause of morbidity and mortality in patients with chronic liver inflammation such as that caused by non-alcoholic steatohepatitis. However, no effective pharmaceutical treatment for liver fibrosis has been approved. In this study, we aimed to investigate that ethanol extract of pharbitis nil (PNE) alleviates the liver fibrosis. MATERIALS AND METHODS: We studied the effects of PNE on two preclinical models. Six-week-old male C57BL/6 mice were intraperitoneally injected with CCl4 twice weekly for 6 weeks and then treated with 5 or 10 mg/kg PNE daily from week 3 for weeks. Secondly, mice were fed HFD for 41 weeks and at 35 weeks treated with 5 mg/kg PNE daily for the remaining 6 weeks. In addition, we examined the antifibrotic effects of PNE in primary mouse hepatic stellate cells and LX-2 cells. RESULTS: PNE treatment ameliorated hepatocyte necrosis, inflammation, and liver fibrosis in CCl4-treated mice and inhibited the progression of liver fibrosis in mice with HFD-induced fibrosis. PNE reduced the expressions of fibrosis markers and SMAD2/3 activations in mouse livers and in TGFß1-treated primary mouse hepatic stellate and LX-2 cells CONCLUSIONS: This study demonstrates that PNE attenuates liver fibrosis by downregulating TGFß1-induced SMAD2/3 activation.
Assuntos
Ipomoea nil , Hepatopatia Gordurosa não Alcoólica , Animais , Etanol/farmacologia , Fibrose , Células Estreladas do Fígado , Humanos , Inflamação/patologia , Ipomoea nil/metabolismo , Fígado/metabolismo , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , Extratos Vegetais/metabolismo , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
The circadian clock is synchronized by the day-night cycle to allow plants to anticipate daily environmental changes and to recognize annual changes in day length enabling seasonal flowering. This clock system has been extensively studied in Arabidopsis thaliana and was found to be reset by the dark to light transition at dawn. By contrast, studies on photoperiodic flowering of Pharbitis nil revealed the presence of a clock system reset by the transition from light to dark at dusk to measure the duration of the night. However, a Pharbitis photosynthetic gene was also shown to be insensitive to this dusk transition and to be set by dawn. Thus Pharbitis appeared to have two clock systems, one set by dusk that controls photoperiodic flowering and a second controlling photosynthetic gene expression similar to that of Arabidopsis. Here, we show that circadian mRNA expression of Pharbitis homologs of a series of Arabidopsis clock or clock-controlled genes are insensitive to the dusk transition. These data further define the presence in Pharbitis of a clock system that is analogous to the Arabidopsis system, which co-exists and functions with the dusk-set system dedicated to the control of photoperiodic flowering.
Assuntos
Ritmo Circadiano/efeitos da radiação , Escuridão , Flores/metabolismo , Flores/efeitos da radiação , Ipomoea nil/metabolismo , Ipomoea nil/efeitos da radiação , Luz , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Proteínas de Plantas/genéticaRESUMO
This study aimed to isolate and characterize antibacterial metabolites from Pharbitis nil seeds and investigate their antibacterial activity against various plant pathogenic bacteria. The methanol extract of P. nil seeds showed the strongest activity against Xanthomonas arboricola pv. pruni (Xap) with a minimum inhibition concentration (MIC) value of 250 µg/ml. Among the three solvent layers obtained from the methanol extract of P. nil seeds, only the butanol layer displayed the activity with an MIC value of 125 µg/ml against Xap. An antibacterial fraction was obtained from P. nil seeds by repeated column chromatography and identified as pharbitin, a crude resin glycoside, by instrumental analysis. The antibacterial activity of pharbitin was tested in vitro against 14 phytopathogenic bacteria, and it was found to inhibit Ralstonia solanacearum and four Xanthomonas species. The minimum inhibitory concentration values against the five bacteria were 125-500 µg/ml for the n-butanol layer and 31.25-125 µg/ml for pharbitin. In a detached peach leaf assay, it effectively suppressed the development of bacterial leaf spot, with a control value of 87.5% at 500 µg/ml. In addition, pharbitin strongly reduced the development of bacterial wilt on tomato seedlings by 97.4% at 250 µg/ml, 7 days after inoculation. These findings suggest that the crude extract of P. nil seeds can be used as an alternative biopesticide for the control of plant diseases caused by R. solanacearum and Xanthomonas spp. This is the first report on the antibacterial activity of pharbitin against phytopathogenic bacteria.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Agentes de Controle Biológico/farmacologia , Glicosídeos/farmacologia , Ipomoea nil/química , Doenças das Plantas/microbiologia , Extratos Vegetais/farmacologia , Resinas Vegetais/farmacologia , Sementes/química , 1-Butanol , Glicosídeos/química , Solanum lycopersicum/microbiologia , Testes de Sensibilidade Microbiana , Folhas de Planta/microbiologia , Ralstonia solanacearum/efeitos dos fármacos , Resinas Vegetais/química , Sementes/microbiologia , Xanthomonas/efeitos dos fármacosRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Pharbitis Nil (PN) is used as a main component of the existing drug, DA-9701, which was developed to treat functional dyspepsia (FD) in Korea. PN extracts isolated from its seeds have been reported to have anticancer effects. AIM OF THE STUDY: The purpose of this study was to investigate the underlying mechanism of the chemotherapeutic effects of PN in lung cancer cells. MATERIALS AND METHODS: We performed MTT assays, colony formation assays, flow cytometry assays, Western blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence analysis, and cell counting assays to study the molecular mechanism of chemotherapeutic effects of PN in lung cancer cells. RESULTS: Our results indicate that PN induced autophagy as well as apoptosis. PN inhibited cell proliferation and survival by inducing apoptosis in several lung cancer cell lines. PN-treated cells also exhibited induction of autophagy, as evidenced by increased protein expression levels and punctuate patterns of LC3 II. Moreover, activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which plays an important role in autophagy activation, was shown to be related with PN-induced autophagy. Interestingly, pharmacological blockade of autophagy activation with wortmannin and inhibition of ERK1/2 phosphorylation by U0126 markedly enhanced PN-induced apoptosis and reduced cell viability, suggesting that autophagy induced by PN may have a cytoprotective effect by suppressing apoptosis. PN- induced apoptosis was regulated by signal transducer and activator of transcription 3 (STAT3) deactivation. Moreover, decrease of STAT3 activation in PN-treated cells was associated with reduced survivin expression, further demonstrating that PN-induced apoptosis was regulated by STAT3 deactivation. CONCLUSION: We believe that PN, which is already proven to treat human patients with FD, might be a potential anticancer drug for human lung cancer. In addition, our data suggest that the combination of PN treatment with an autophagy inhibitor or traditional anticancer agents may be an effective anticancer therapy.
Assuntos
Antineoplásicos/farmacologia , Ipomoea nil , Extratos Vegetais/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Fator de Transcrição STAT3/metabolismo , Sementes , SurvivinaRESUMO
CDPK kinases are a unique class of calcium sensor/responders that regulate many growth and developmental processes as well as stress responses of plants. PnCDPK1 kinase from Pharbitis nil is regulated by light and contributes to seed germination, seedling growth and flower formation. Following an earlier work in which we identified the PnCDPK1 coding sequence and a 330bp long 3'UTR (untranslated region), we present for the first time the genomic organization of PnCDPK1, including intron analysis and the gene copy number designation. We completed the research by identifying the 5'-flanking region of PnCDPK1 and analyzed it in silico, which led to the discovery of several cis-regulatory elements involved in light regulation, embryogenesis and seed development. The functional analysis of P. nil CDPK showed characterization of the PnCDPK1 transcript and PnCDPK protein level during seed formation and fruit maturation. The greatest amount of PnCDPK1 mRNA was present in the last stages of seed maturation. Moreover, two PnCDPK proteins of different molecular masses were discovered during fruit development, showing various protein accumulation and activity profile. The 56kDa protein dominated in the early stages of fruit development, whereas the smaller protein (52kDa) was prominent in the latter stages.
Assuntos
Regulação da Expressão Gênica de Plantas , Genômica , Ipomoea nil/enzimologia , Proteínas Quinases/genética , Regiões 3' não Traduzidas/genética , Flores/enzimologia , Flores/genética , Flores/fisiologia , Flores/efeitos da radiação , Frutas/enzimologia , Frutas/genética , Frutas/fisiologia , Frutas/efeitos da radiação , Regulação da Expressão Gênica no Desenvolvimento , Germinação , Íntrons/genética , Ipomoea nil/genética , Ipomoea nil/fisiologia , Ipomoea nil/efeitos da radiação , Luz , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Quinases/metabolismo , Plântula/enzimologia , Plântula/genética , Plântula/fisiologia , Plântula/efeitos da radiação , Sementes/enzimologia , Sementes/genética , Sementes/fisiologia , Sementes/efeitos da radiaçãoRESUMO
Poor nutrition and low temperature stress treatments induced flowering in the Japanese morning glory Pharbitis nil (synonym Ipomoea nil) cv. Violet. The expression of PnFT2, one of two homologs of the floral pathway integrator gene FLOWERING LOCUS T (FT), was induced by stress, whereas the expression of both PnFT1 and PnFT2 was induced by a short-day treatment. There was no positive correlation between the flowering response and the homolog expression of another floral pathway integrator gene SUPPRESSOR OF OVEREXPRESSION OF CO1 and genes upstream of PnFT, such as CONSTANS. In another cultivar, Tendan, flowering and PnFT2 expression were not induced by poor nutrition stress. Aminooxyacetic acid (AOA), a phenylalanine ammonia-lyase inhibitor, inhibited the flowering and PnFT2 expression induced by poor nutrition stress in Violet. Salicylic acid (SA) eliminated the inhibitory effects of AOA. SA enhanced PnFT2 expression under the poor nutrition stress but not under non-stress conditions. These results suggest that SA induces PnFT2 expression, which in turn induces flowering; SA on its own, however, may not be sufficient for induction.
Assuntos
Flores/efeitos dos fármacos , Flores/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Genes de Plantas/genética , Ipomoea nil/efeitos dos fármacos , Ipomoea nil/metabolismo , Ácido Salicílico/farmacologiaRESUMO
In the present study, polysaccharides were extracted from Pharbitis nil seeds (PNSs) by ultrasonic extraction method for the first time. Response surface methodology with Box-Behnken design was used to optimize the extraction process of PNS polysaccharides (PNSPs). Highest PNSPs yield 6.01 ± 0.15% that agreed closely with the predicted yield 5.99% were obtained under the optimal conditions as follows: ratio of water to raw material 6.5 mL/g, extraction temperature 49.0 °C, ultrasonic power 61.6 W, and extraction time 32.6 min. Preliminary characterization indicated that the sugar, uronic acid and protein contents of the product were 83.6 ± 1.61, 21.8 ± 1.25 and 16.4 ± 0.88% (w/w), respectively. FT-IR analysis revealed the general characteristic absorption peaks of polysaccharides. Besides, PNSPs showed a remarkable antioxidant activity in vitro in a dose-dependent manner. These may provide theoretical basis for further system research and rational development and utilization of PNS resources.
Assuntos
Antioxidantes/isolamento & purificação , Ipomoea nil/embriologia , Polissacarídeos/isolamento & purificação , Sementes/química , Antioxidantes/química , Antioxidantes/farmacologia , Polissacarídeos/química , Polissacarídeos/farmacologia , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The role of endogenous gibberellin (GA) in the flowering of the short-day plant, Pharbitis nil, was investigated by using uniconazole, which is a specific inhibitor of GA biosynthesis. Both the endogenous GA level and flowering response decreased with increasing concentration of uniconazole applied via the roots. The strongest inhibition of flowering was observed when uniconazole was applied one day before a 15-h dark treatment. The inhibition by uniconazole was overcome by an application of GAs to the plumules, the order of effectiveness of the endogenous GAs in P. nil being GA1 â§GA20>GA19â§GA44>GA53¼GAH. This is the first report of the correlation between the endogenous GA level and flowering response in P. nil. It was found that endogenous GAs were required for the flowering of P. nil during or just after the dark period.