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Quantitative determination of anti-fungal and insecticide amides in adult plants, plantlets and callus from Piper tuberculatum by reverse-phase high-performance liquid chromatography.

A rapid, sensitive and reliable reverse-phase HPLC method was used for the quantitative determination of the anti-fungal and insecticide amides, dihydropiplartine (1), piplartine (2), deltaalpha,beta-dihydropiperine (3) and pellitorine (4) in plants in natura, in plantlets in vitro and ex vitro, and in callus of Piper tuberculatum. Well-resolved peaks were obtained with good detection response and linearity in the range of 15.0-3000 microg/mL. The plants in natura contained compounds 1-4, the plantlets ex vitro and in vitro accumulated compounds 1-2 and 1-4, respectively, while only amide 4 was found in callus.

Amides↗

Circadian rhythm of anti-fungal prenylated chromene in leaves of Piper aduncum.

Leaves of Piper aduncum accumulate the anti-fungal chromenes methyl 2,2-dimethyl-2H-1-chromene-6-carboxylate (1) and methyl 2,2-dimethyl-8-(3'-methyl-2'-butenyl)-2H-1-chromene-6-carboxylate (2). The enzymatic formation of 2 from dimethylallyl diphosphate and 1 was investigated using cell-free extracts of the title plant. An HPLC assay for the prenylation reaction was developed and the enzyme activity measured in the protein extracts. The prenyltransferase that catalyses the transfer of the dimethylallyl group to C-2' of 1 was soluble and required dimethylallyl diphosphate as the prenyl donor. In the leaves, the biosynthesis of the prenylated chromene 2 was time-regulated and prenyltransferase activity depended upon circadian variation. Preliminary characterisation and purification experiments on the prenyltransferase from P. aduncum have been performed.

Antifungal Agents↗

Potential antiamoebic property of the roots of Piper longum Linn.

In an earlier study we reported the effect of ethanol extracts of Piper longum fruit against caecal amoebiasis in rats. A follow-up study to determine whether the roots possess similar antiamoebic activity was carried out. The ethanolic extract of roots was amoebicidal at 1,000 microg/mL in vitro and cured 88% of caecal amoebiasis cases, suggesting that both the root and fruit of P. longum possess antiamoebic activity to approximately the same extent.

Administration, Oral↗

Antioxidant and non-toxic properties of Piper betle leaf extract: in vitro and in vivo studies.

Piper betle leaves are used in folk medicine for the treatment of various disorders and is commonly chewed among Asians. The present study investigates the protective efficacy of P. betle leaf extract. The presence of the extract inhibited the radiation induced lipid peroxidation process effectively. This could be attributed to its ability to scavenge free radicals involved in initiation and propagation steps. Oral supplementation with extract (1, 5 and 10 mg/kg) was administered daily for 2 weeks to Swiss albino mice and the hepatic antioxidant status was analysed. The GSH content was enhanced and no appreciable change was found in the levels of oxidative damage in terms of lipid peroxidation. Also, the specific activity of SOD increased in a dose dependent manner. These factors indicate the elevation of antioxidant status in the animals. The effect on the glyoxalase system which is considered to be activated under stress conditions was also investigated. Our findings did not observe any significant change in gly I and gly II activities, implying a non-stress condition after oral treatment of the extract. The present study indicates the antioxidant activity of P. betle leaf extract and its potential to elevate the antioxidant status.

Animals↗

Essential oil composition of Piper guineense and its antimicrobial activity. Another chemotype from Nigeria.

The analysis of the essential oil of Piper guineense from Nigeria presents a new chemotype of constituents different from earlier reports with the absence of the usual myristicin. Ishwarane, a common constituent of Aristolochia indica and Bixa orellana, was also isolated from the essential oil of the fruit. The essential oil inhibited the growth of Pseudomonas aeruginosa UCH 655 strain at 5 mg/mL on which standard antibiotic drugs were ineffective.

Anti-Bacterial Agents↗

Genetic diversity amongst landraces of a dioecious vegetatively propagated plant, betelvine (Piper betle L.).

Betelvine (Piper betle L., family Piperaceae) is an important, traditional and widely cultivated crop of India. The cultivators and consumers recognize more than 100 cultivars (landraces) based on regional and organoleptic considerations, while in terms of phytochemical constituents only five groups have been identified for all the landraces. Since betelvine is an obligate vegetatively propagated species, genomic changes, if any, may have become 'fixed' in the landraces. We carried out random amplified polymorphic DNA (RAPD) analysis in several landraces considered in four groups, namely, 'Kapoori', 'Bangla', 'Sanchi' and 'Others' in order to ascertain their genetic diversity. On the basis of the data from eleven RAPD primers, we distinguished genetic variation within and among the four groups of landraces. The results indicate the 'Kapoori' group is the most diverse. The neighbour joining (NJ) tree after a bootstrap (500 replicate) test of robustness clearly shows the four groups to be well separated. Interestingly, all known male or female betelvine landraces have separated in the NJ tree indicating an apparent gender-based distinction among the betelvines.

Algorithms↗

The tumorigenic characteristics of lime-piper betel quid-transformed JB6 cells.

Betel quid chewing is a general oral habit in Taiwan, India, southeastern Asian and South Africa with or without the additive of tobacco, alcohol or lime. In this study, the tumor-promoting neoplastic transformation effect of Lime-Piper betel quid (LPB) was examined on JB6 cells. The treatment of LPB at a high dose (1.0 mg/ml) for over 5 days or at lower doses (0.1, 0.5 mg/ml) for over 15 days induced the formation of transformed foci. The transformed cells showed the characteristics of colony formation in soft agar, higher growth rate and multilayer on culture dish. A two-fold induction of the protein levels of c-fos and c-jun proto-oncogenes was observed in the cells from the 50th passage (Cl1/p50, Cl2/p50 and Cl3/p50), suggesting that LPB-transformed cells were oncogenic. In addition, the LPB-transformed cells possessed an elevated level of c-Myc and an increased cell population distributed in the S phase of the cell cycle. These results demonstrated the promotion effect of LPB and indicate that it could be a tumor promoter.

Alkaloids↗

Increased sensitivity of Hep G2 cells toward the cytotoxicity of cisplatin by the treatment of piper betel leaf extract.

Piper betel leaves (PBL) are used in Chinese folk medicine for the treatment of various disorders. PBL has the biological capabilities of de-toxication, anti-oxidation and anti-mutation. In this study we first examined the effect of PBL extract on the activity of Glutathione S-transferase (GST) isoforms, and found that it inhibited total GST and the alpha class of GST (GSTA), but not the pi class of GST (GSTP), and the mu class of GST (GSTM), activity in Hep G2 cells. RT-PCR results verified a reduction in the expression of GSTA1. Next, we examined whether PBL extract could increase the sensitivity of Hep G2 cells to anti-cancer drugs. The data showed that the cytotoxicity of cisplatin was significantly enhanced by the presence of PBL extract, accompanied by a reduction in the expression of multidrug resistance protein 2 (MRP2). These effects of PBL extract were compared to its major constitute, eugenol. Although eugenol decreased MRP2 level more effectively than PBL extract, it exhibited less sensitizing effect. In conclusion, we demonstrated that PBL extract was able to increase the sensitivity of Hep G2 cells to cisplatin via at least two mechanisms, reducing the expression of MRP2 and inhibiting the activity of total GST and the expression of GSTA. The data of this study support an application of PBL as an additive to reduce drug resistance.

Antineoplastic Agents↗

Protection effect of piper betel leaf extract against carbon tetrachloride-induced liver fibrosis in rats.

Piper betel leaves (PBL) are used in Chinese folk medicine for the treatment of various disorders. PBL has the biological capabilities of detoxication, antioxidation, and antimutation. In this study, we evaluated the antihepatotoxic effect of PBL extract on the carbon tetrachloride (CCl(4))-induced liver injury in a rat model. Fibrosis and hepatic damage, as reveled by histology and the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were induced in rats by an administration of CCl(4) (8%, 1 ml/kg body weight) thrice a week for 4 weeks. PBL extract significantly inhibited the elevated AST and ALT activities caused by CCl(4) intoxication. It also attenuated total glutathione S-transferase (GST) activity and GST alpha isoform activity, and on the other hand, enhanced superoxide dismutase (SOD) and catalase (CAT) activities. The histological examination showed the PBL extract protected liver from the damage induced by CCl(4) by decreasing alpha-smooth muscle actin (alpha-sma) expression, inducing active matrix metalloproteinase-2 (MMP2) expression though Ras/Erk pathway, and inhibiting TIMP2 level that consequently attenuated the fibrosis of liver. The data of this study support a chemopreventive potential of PBL against liver fibrosis.

Actins↗

Biocontrol of collar rot disease of betelvine (Piper betle L.) caused by Sclerotium rolfsii by using rhizosphere-competent Pseudomonas fluorescens NBRI-N6 and P. fluorescens NBRI-N.

Collar rot disease of betelvine (Piper betle L.) caused by Sclerotium rolfsii is difficult to control by conventional means by use of chemicals; therefore, use of biocontrol agents is desirable. In the present study, 186 bacterial strains of different morphological types were screened for their biocontrol activity against S. rolfsii under in vitro conditions. Two strains, Pseudomonas fluorescens NBRI-N6 and P. fluorescens NBRI-N, were selected for further studies because of their ability to inhibit the mycelial growth of the pathogen significantly. Spontaneous rifampicin-resistant (Rif) derivatives of P. fluorescens NBRI-N6 and P. fluorescens NBRI-N showing growth rate and membrane protein composition comparable to the wild type were selected to facilitate their monitoring in the rhizosphere. Field trials demonstrated that strain P. fluorescens NBRI-N6 was better than P. fluorescens NBRI-N in increasing the yield of betelvine significantly, whereas a consortium of the two strains controlled the disease more than either of the strains. The screening method should prove useful in identifying rhizosphere bacteria with the greatest potential for controlling diseases caused by phytopathogenic fungi.

Ascomycota↗

High-frequency plant regeneration through cyclic secondary somatic embryogenesis in black pepper (Piper nigrum L.).

A high-frequency plantlet regeneration protocol was developed for black pepper (Piper nigrum L.) through cyclic secondary somatic embryogenesis. Secondary embryos formed from the radicular end of the primary somatic embryos which were originally derived from micropylar tissues of germinating seeds on growth regulator-free SH medium in the absence of light. The process of secondary embryogenesis continued in a cyclic manner from the root pole of newly formed embryos resulting in clumps of somatic embryos. Strength of the medium and sucrose concentration influenced the process of secondary embryogenesis and fresh weight of somatic embryo clumps. Full-strength SH medium supplemented with 1.5% sucrose produced significantly higher fresh weight and numbers of secondary somatic embryos while 3.0 and 4.5% sucrose in the medium favored further development of proliferated embryos into plantlets. Ontogeny of secondary embryos was established by histological analysis. Secondary embryogenic potential was influenced by the developmental stage of the explanted somatic embryo and stages up to "torpedo" were more suitable. A single-flask system was standardized for proliferation, maturation, germination and conversion of secondary somatic embryos in suspension cultures. The system of cyclic secondary somatic embryogenesis in black pepper described here represents a permanent source of embryogenic material that can be used for genetic manipulations of this crop species.

Germination↗

Phenolic acid amides: a new type of DNA strand scission agent from Piper caninum.

In a survey of the active components of crude plant extracts for their ability to cleave DNA, a crude extract prepared from Piper caninum was found to induce the relaxation of supercoiled pBR322 plasmid DNA in the presence of Cu(2+). Bioassay-guided fractionation was carried out on this extract, guided by an in vitro DNA strand scission assay. Three active principles were isolated and identified as N-cis-feruloyl tyramine (1),N-trans-feruloyl tyramine (2), and 1-cinnamoylpyrrolidine (3). Compounds 1-3 represent a structurally new type of DNA strand scission agent.

Amides↗

Comparison of soxhlet, ultrasound-assisted and pressurized liquid extraction of terpenes, fatty acids and Vitamin E from Piper gaudichaudianum Kunth.

This paper describes a comparative study of extraction methods of terpenes (terpenic alcohols and phytosterols), fatty acids and Vitamin E from leaves of Piper gaudichaudianum Kunth. The analysis of extracts was done by gas chromatography with mass spectrometric detection. The identification and quantification was made by co-injections of the extract with certified standards. The use of pressurized liquid extraction (PLE; Dionex trade name: ASE, for accelerated solvent extraction) decrease significantly the total time of extraction, the amount of solvent and the manipulation of sample and solvents in comparison with soxhlet (SE) and ultrasound-assisted (USE). In addition, PLE was more effective for the extractions of terpenes (terpenic alcohols and phytosterols), fatty acids and Vitamin E.

Chemical Fractionation↗

Optimization of pressurized liquid extraction of Piper gaudichaudianum Kunth leaves.

Piperaceae family is original from tropical regions and it shows more than 700 species around the world. Piper gaudichaudianum Kunth is the specie more abundant in Brazil, occurring from Northeast to South Brazil. In this paper, it was investigated the influence of some experimental parameters on the pressurized liquid extraction (PLE) of P. gaudichaudianum Kunth leaves, using petroleum ether as extractor solvent. The optimization of the main variables involved in the PLE process (extraction temperature and time) has been done by response surface methodology (RSM) using, as responses, the extraction yield and the chromatographic profile (GC/MS) of the extracts. The optimized procedure employed 3 g of ground leaves, 10 min of extraction and one cycle of extraction at 85 degrees C. The major compounds present in the petroleum ether extracts were: palmitic acid, stearic acid and nerolidol. The results presented in this work show the possibility of using a fast and easy process to recover compounds from P. gaudichaudianum Kunth.

Alkanes↗

Protective effect of Piper longum fruit ethanolic extract on radiation induced damages in mice: a preliminary study.

The radioprotective property of an ethanolic extract of Piper longum fruits (EEPLF) was investigated in Swiss mice. The white blood cell (WBC) count in irradiated control mice was drastically reduced to 1900 cells/mm3 on third day but in treated animals the count was 2783.3 cells/mm3. The number of bone marrow cells and alpha-esterase positive cells was also enhanced by the EEPLF administration (16.7 x 10(6) cells/femur and 946.5/4000 cells, respectively) when compared to the radiation exposed control animals (12.2 x 10(6) cells/femur and 693.5/4000 cells, respectively). EEPLF reduced the elevated levels of glutathione pyruvate transaminase (GPT), alkaline phosphatase (ALP), and lipid peroxidation (LPO) in liver and serum of radiation treated animals. The extract administration also increased the reduced glutathione (GSH) production to offer the radioprotection.

Alkaline Phosphatase↗

Piper longum inhibits VEGF and proinflammatory cytokines and tumor-induced angiogenesis in C57BL/6 mice.

The antiangiogenic activity of Piper longum was studied using in vivo as well as in vitro models. In vivo, antiangiogenic activity was studied using B16F-10 melanoma cell-induced capillary formation in C57BL/6 mice. Intraperitoneal administration of the extract (10 mg/dose/animal) significantly inhibited (50.6%) the number of tumor-directed capillaries induced by injecting B16F-10 melanoma cells on the ventral side of C57BL/6 mice. The cytokine profile in the serum of these animals showed a drastically increased level of proinflammatory cytokines such as IL-1beta, IL-6, TNF-alpha, GM-CSF and the direct endothelial cell proliferating agent, VEGF. Administration of the methanolic extract of P. longum could differentially regulate the level of these cytokines. The level of IL-2 and tissue inhibitor of metalloprotease-1 (TIMP-1) was increased significantly when the angiogenesis-induced animals were treated with the extract. The extract of P. longum at non-toxic concentrations (10 microg/ml, 5 microg/ml, 1 microg/ml) inhibited the VEGF-induced vessel sprouting in rat aortic ring assay. Moreover, P. longum was able to inhibit the VEGF-induced proliferation, cell migration and capillary-like tube formation of primary cultured human endothelial cells. Hence, the observed antiangiogenic activity of the plant P. longum is related to the regulation of these cytokines and growth factors in angiogenesis-induced animals.

Angiogenesis Inhibitors↗

Preliminary pharmacological studies on Piper chaba stem bark.

Piper chaba Hunter (Piperaceae) is a common pepper in the southern part of Bangladesh. Various parts of this plant have been extensively used in different traditional formulations including ayurveda. In order to rationalize the ethnomedical uses of this plant in a number of ailments, the methanol extract of the stem bark was subjected to preliminary evaluation for analgesic, anti-inflammatory, diuretic, anti-diarrhoeal, effect on gastrointestinal motility and CNS depressant activity in mice and rat at 125, 250 and 500 mg/kg body weight doses. The extract at given doses significantly and dose dependently reduced the frequency of acetic acid induced writhing in mice, prolonged the tail flicking latency in mice, reduced Carrageenan-induced paw edema volume in rat, delayed the onset as well as reduced the frequency of castor oil induced diarrhoeal episodes in mice, decreased gastrointestinal motility as assessed by the charcoal motility test in mice and prolonged pentobarbitone induced sleeping time in mice. However at the same doses, the extract exhibited moderate diuretic activity only at the highest dose.

Acetic Acid↗

HPLC assisted chemobiological standardization of alpha-glucosidase-I enzyme inhibitory constituents from Piper longum Linn-An Indian medicinal plant.

Formulations of traditional medicines are usually made up of complex mixture of herbs. However, effective quality control methods in order to select right quality materials are lacking. Though Piper longum is a widely used herb in several Ayurvedic formulations prescribed for various diseases, there is no analytical method in the literature so far which can help in selecting the right quality material with proper proportions of the active ingredients (alpha-glucosidase-I enzyme inhibitory principles). We employed a systematic bioassay guided fractionation method and isolated pipataline, pellitorine, sesamin, brachystamide B and guineensine as active principles. A reversed-phase high-performance liquid chromatography method was developed to quantify these active principles in the plant material, which can serve as an effective quality control tool. The separation was carried out using a Discovery HS F5 C-18 (ODS) column and the solvent system used was a gradient comprising of (A) acetonitrile and (B) water with a flow rate of 1 ml/min. The detection was performed using a PDA detector. Regression equation pertaining to all the bioactive isolates revealed a linear relationship (r2 > 0.9995). The detection limits (S/N = 3) ranged from 0.005 to 0.001 microg/ml. Of all the active isolates, sesamin was identified to be present in maximum quantities (0.91%) where as brachystamide B was found in minimum quantity (0.01%).

Calibration↗