Active Compound of Pharbitis Semen (Pharbitis nil Seeds) Suppressed KRAS-Driven Colorectal Cancer and Restored Muscle Cell Function during Cancer Progression.

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.

New octadecanoid derivatives from the seeds of Ipomoea nil.

Four new octadecanoid derivatives (1-4) including a pair of enantiomers (1/2), along with 12 known analogues (5-16), were isolatedfrom the seeds of Ipomoea nil. Their structures were determined by detailed spectroscopic analyses and comparison with reported data of structurally related compounds, with the absolute configurations of 1 and 2 being assigned by an in situ dimolybdenum ECD method. Our bioassays revealed that these isolates did not show ABTS radical scavenging activity while 10 and 13 displayed better α-glucosidase inhibitory activity than the positive control acarbose (IC50 167.7 ± 1.55 µmol·L-1), with IC50 of 92.73 ± 3.12 and 11.39 ± 2.18µmol·L-1, respectively.

Renal-protective effects of n-hexane layer from morning glory seeds ethanol extract.

Nephrotoxicity is a main problem in cancer patients using cisplatin. Oxidative stress, inflammation and apoptosis are the important mechanisms of cisplatin induced nephrotoxicity. In the present study, we investigated the effect of the extracts of morning glory on nephrotoxicity by cisplatin in human embryonic kidney cells 293 (HEK-293) and mice. Previous studies have reported that morning glory extracts showed potent activity on anti-inflammatory and anti-oxidant. However, the protective effects of the n-hexane layer of morning glory seed (MGs-Hx) on nephrotoxicity and its mechanisms have not been clearly understood. Oral administration with MGs-Hx showed protective effects in vivo experiments test and the treatment of MGs-Hx in a concentration of 100mg/kg/day had significant effect both of decreasing serum creatinine, BUN, serum uric acid level and reduced iNOS, COX-2 mRNA expressions with low side-effect. Moreover, cell viability was restored by MGs-Hx treatment compared to cisplatin-induced nephrotoxic HEK-293 cells. Co-treatment with MGs-Hx and cisplatin showed the significant effect to reduce inflammatory enzyme, iNOS expression and continuous production of NO. In addition, it exhibited a tendency to decreasing expression of apoptosis-related proteins, caspase-3, 8 and 9, and NF-κB translocation to nucleus as well as phosphorylation of p38, JNK, ERK in cisplatin-induced nephrotoxic HEK-293 cells. Our study provides insight into the underlying mechanisms of MGs-Hx and suggests that MGs-Hx might be a potential therapeutic agent to modulate inflammation and apoptosis in nephrotoxicity.

Antibacterial Activity of Pharbitin, Isolated from the Seeds of Pharbitis nil, against Various Plant Pathogenic Bacteria.

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.

Identification of antitumor lignans from the seeds of morning glory (Pharbitis nil).

In the search for antitumor compounds from Korean natural resources, activity-guided fractionation and purification processes were used on seeds of morning glory (Pharbitis nil). Air-dried P. nil seeds were extracted with ethanol and separated into n-hexane, chloroform, ethyl acetate, and n-butanol. Four new lignans, pharbilignans A-D (1-4) were isolated from the most active ethyl acetate fraction of the ethanol extract. Their structures were characterized on the basis of spectroscopic methods, including one- and two-dimensional nuclear magnetic resonance (NMR) techniques, high resolution mass spectrometry (HRMS), and circular dichroism (CD) spectroscopy. The cytotoxic activities of the isolates (1-4) were evaluated by determining their inhibitory effects on four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT15) using a sulforhodamine B (SRB) bioassay. Pharbilignan C (3) showed potent cytotoxicity against A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines with IC50 values of 1.42, 0.16, 0.20, and 0.14 µM, respectively. On the basis of the expanded understanding that inflammation is a crucial cause in tumor progress, we also evaluated anti-inflammatory activity of the isolates (1-4). Pharbilignan C (3) strongly inhibited nitric oxide (NO) production in the lipopolysaccharide (LPS)-activated BV-2 microglia cell line with an IC50 value of 12.8 µM.

Pharbinilic acid, an allogibberic acid from morning glory (Pharbitis nil).

Pharbinilic acid (1), the first naturally occurring allogibberic acid, was isolated from ethanol extracts of morning glory (Pharbitis nil) seeds. Its absolute configuration was determined by NOESY NMR and ECD experiments. Compound 1 showed weak cytotoxicity against A549, SK-OV-3, SK-MEL-2, and HCT-15 cells and weakly inhibited nitric oxide production in lipopolysaccharide-activated BV-2 microglia cells.

Seasonal characterization of antioxidant responses in plants of Ipomoea nil cv. Scarlet O'Hara / Caracterização sazonal de respostas antioxidativas em plantas de Ipomoea nil cv. Scarlet O'Hara

Reactive oxygen species can be produced in leaf cells during normal aerobic metabolism or in a variety of exogenous factors, which may cause oxidative damage to plants, unless they have an efficient antioxidant defense system, consisting of enzymatic and non-enzymatic substances. This work raised the hypothesis that plants of Ipomoea nil cv. Scarlet O'Hara, a native species and ornamental vine of the tropics, might tolerate oxidative stress factors imposed by natural fluctuations in weather conditions through changes in the antioxidant profile.The objective of this study was to determine the variations in three leaf antioxidants in plants growing inside a greenhouse without air pollutants and exposed to varying meteorological conditions throughout the four seasons of the year and to observe if such variations are related to the oscillations in meteorological factors. Four experimental campaigns were carried out, one in each season of 2006. Each campaign lasted 28 days and started with 45 plants. Ascorbic acid (AA) concentrations and superoxide dismutase (SOD) and peroxidase (POD) activities were determined in leaves of five plants in nine sampling days of each campaign. The antioxidant responses oscillated throughout the year. The highest values were found during the spring. This seasonal antioxidant profile was associated to variations in temperature, relative humidity and global radiation. Plants of this cultivar may then tolerate oxidative stress naturally imposed by meteorological conditions.

As espécies reativas de oxigênio podem ser produzidas em células de folhas durante o metabolismo aeróbico normal ou o sob uma diversidade de fatores exógenos que, por sua vez, podem causar danos oxidativos às plantas, a menos que estas tenham um eficiente sistema de defesa antioxidativo, formado por substâncias enzimáticas e não enzimáticas. Neste trabalho, levantou-se a hipótese de que as plantas de Ipomoea nil cv. Scarlet O'Hara, uma espécie trepadeira ornamental e nativa dos trópicos, podem tolerar fatores de estresse oxidativo imposto por oscilações naturais nas condições meteorológicas por meio de mudanças no perfil antioxidativo. Assim, este trabalho objetivou determinar as variações em três espécies antioxidantes foliares em plantas crescidas em casa de vegetação sob ar filtrado e expostas a condições meteorológicas variáveis ao longo das quatro estações do ano de 2006, bem como verificar se tais variações estão relacionadas às oscilações de fatores meteorológicos. Para tanto, realizaram-se quatro campanhas experimentais. Cada campanha durou 28 dias e começou com 45 plantas. Concentrações de ácido ascórbico (AA) e as atividades de superóxido dismutase (SOD) e peroxidase (POD) foram determinadas em folhas de cinco plantas distintas e retiradas da casa de vegetação em nove dias de amostragem de cada campanha. As respostas antioxidativas oscilaram durante todo o ano, sendo os maiores valores encontrados durante a primavera. Este perfil sazonal de antioxidantes foi associado às variações de temperatura, umidade relativa e radiação global. As plantas desta cultivar podem, então, tolerar o estresse oxidativo naturalmente imposto pelas condições meteorológicas.

Seasonal characterization of antioxidant responses in plants of Ipomoea nil cv. Scarlet O'Hara.

Reactive oxygen species can be produced in leaf cells during normal aerobic metabolism or in a variety of exogenous factors, which may cause oxidative damage to plants, unless they have an efficient antioxidant defense system, consisting of enzymatic and non-enzymatic substances. This work raised the hypothesis that plants of Ipomoea nil cv. Scarlet O'Hara, a native species and ornamental vine of the tropics, might tolerate oxidative stress factors imposed by natural fluctuations in weather conditions through changes in the antioxidant profile.The objective of this study was to determine the variations in three leaf antioxidants in plants growing inside a greenhouse without air pollutants and exposed to varying meteorological conditions throughout the four seasons of the year and to observe if such variations are related to the oscillations in meteorological factors. Four experimental campaigns were carried out, one in each season of 2006. Each campaign lasted 28 days and started with 45 plants. Ascorbic acid (AA) concentrations and superoxide dismutase (SOD) and peroxidase (POD) activities were determined in leaves of five plants in nine sampling days of each campaign. The antioxidant responses oscillated throughout the year. The highest values were found during the spring. This seasonal antioxidant profile was associated to variations in temperature, relative humidity and global radiation. Plants of this cultivar may then tolerate oxidative stress naturally imposed by meteorological conditions.

Reversal of multidrug resistance by morning glory resin glycosides in human breast cancer cells.

Reversal of multidrug resistance (MDR) by thirty resin glycosides from the morning glory family (Convolvulaceae) was evaluated in vinblastine-resistant human breast carcinoma cells (MCF-7/Vin). The effects of these amphipathic compounds on the cytotoxicity and P-glycoprotein (P-gp)-mediated MDR were estimated with the sulforhodamine B colorimetric assay. Active noncytotoxic compounds exerted a potentiation effect of vinblastine susceptibility by 1- to over 1906-fold at tested concentrations of 5 and 25 µg/mL. Murucoidin V (1) enhanced vinblastine activity 255-fold when incorporated at 25 µg/mL and also, based on flow cytometry, significantly increased the intracellular accumulation of rhodamine 123 with the use of reserpine as a positive control for a MDR reversal agent. Incubation of MCF-7/Vin cells with 1 caused an increase in uptake and notably lowered the efflux rate of rhodamine 123. Decreased expression of P-glycoprotein by compound 1 was detected by immunofluorescence flow cytometry after incubation with an anti-P-gp monoclonal antibody. These results suggest that resin glycosides represent potential efflux pump inhibitors for overcoming MDR in cancer therapy.

Bioactive phenolic constituents from the seeds of Pharbitis nil.

Two new lignans, termed pharsyringaresinol (1) and pharbilignoside (2), a new phenylethanoid glycoside, termed pharbiniloside (3), and 22 known compounds, were isolated from the ethanol extract of the seeds of Pharbitis nil. The structures of the new compounds (1-3) were determined on the basis of spectroscopic analyses, including 2D-NMR and circular dichroism (CD) spectroscopy studies. Among the isolates, compounds 2, 11, 12, and 24 exhibited significant cytotoxicity against human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15) with IC(50) values ranging from 8.07 to 28.30 µM. In addition, compounds 11, 12 and 24 potently inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV-2 cells, a microglia cells with IC(50) values ranging from 14.7 to 19.9 µM.

Two new phenolic amides from the seeds of Pharbitis nil.

Two new phenolic amides, pharnilatins A (1) and B (2), were isolated from the seeds of Pharbitis nil. These new compounds possess a p-coumaroyl unit with a structurally unique side chain, (2S,3S)-2,3-dihydroxyputrescine. The chemical structures and absolute stereochemistries of the new compounds were determined on the basis of spectroscopic analyses including 1D- and 2D-NMR experiments and chemical reactions. Compounds 1 and 2 exhibited cytotoxicity against A549, SK-OV-3, SK-MEL-2, and HCT-15 human tumor cells. However, none of the compounds inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-activated microglia cells.

Acylated glycosides of hydroxy fatty acid methyl esters generated from the crude resin glycoside (pharbitin) of seeds of Pharbitis nil by treatment with indium(III) chloride in methanol.

Treatment of the crude ether-insoluble resin glycoside (convolvulin) from seeds of Pharbitis nil (Pharbitis Semen), called pharbitin, with indium(III) chloride in methanol provided seven oligoglycosides of hydroxy fatty acid methyl esters partially acylated by 2-methyl-3-hydroxybutyric (nilic) and 2S-methylbutyric acids. Their structures were elucidated on the basis of NMR and MS data and chemical conversions.

Diterpene glycosides from the seeds of Pharbitis nil.

Six new ent-kaurane diterpene glycosides, pharbosides A-F (1-6), and a new ent-gibbane diterpene glycoside, pharboside G (7), together with three known ent-kaurane diterpenoids, 7beta,16beta,17-trihydroxy-ent-kauran-6alpha,19-olide (8), 6beta,7beta,16alpha,17-tetrahydroxy-ent-kauranoic acid (9), and 6beta,7beta,16beta,17-tetrahydroxy-ent-kauranoic acid (10), were isolated from an ethanolic extract of the seeds of Pharbitis nil. The structures of the new compounds were determined by spectroscopic methods including 1D and 2D NMR analysis. The absolute configurations of the compounds were clarified by CD spectroscopic studies. Full NMR data assignments of the three known compounds (8-10) are reported. The isolated compounds were evaluated for their cytotoxic activities against four human cancer cell lines.

Triterpenoid saponins from the seeds of Pharbitis nil.

From the seeds of Pharbitis nil (Convolvulaceae), two new oleanene-type triterpene glycosides, pharbitosides A (1) and B (2), together with beta-sitosterol, beta-sitosterol glucoside (daucosterol), caffeic acid, and methyl caffeate were isolated. The structure of pharbitoside A (1) was elucidated to be queretaroic acid 3-O-alpha-L-rhamnopyranosyl-(1-->2)-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranoside (1). Pharbitoside B (2) is a 21alpha-hydroxyoleanolic acid saponin carrying the same sugar moiety as that of pharbitoside A (1).

Preparation of proteins from different organs of Japanese morning glory by an in vivo electro-elution procedure.

An electro-elution procedure has been employed efficiently to collect proteins from stem segments, young leaves and roots of the Japanese morning glory. Electrophoretic conditions were optimised, including the size of segments (10-30 mm), the strength of the current for electro-elution (2.5-10 mA), and the exposure time of electro-elution (2-12 h). From the same quantity of organs, the in vivo electro-elution procedure permitted the collection of an amount of protein up to six times greater than that obtained with an earlier-reported centrifugation procedure. Both preparations were analysed by SDS-PAGE and showed similar protein profiles. This new technique provided an interesting insight into the large differences in both the quality and quantity of proteins between different organs of the plants. The average amount of protein collected from organs was 0.1 mg/g of tissue fresh weight. It is expected that this procedure may facilitate the discovery of new proteins with unique functions in extracellular matrices involved in the response of plants to various external stimuli.