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.     

Mediation of cholino-piperine like receptors by extracts of Piper nigrum induces melanin dispersion in Rana tigerina tadpole melanophores

Aim: The present study was carried out to determine the effects of lyophilized dried fruit extracts of Piper nigrum and pure piperine on the tadpole melanophores of frog Rana tigerina which offer excellent in vitro opportunities for studying the effects of pharmacological and pharmaceutical agents. The nature of specific cellular receptors present on the neuro-melanophore junction and their involvement in pigmentary responses has been explored.

Material: Effects of lyophilized extracts of P. nigrum and pure piperine were studied on the isolated tail melanophores of tadpoles of the frog R. tigerina as per the modified method.

Results: The extract of P. nigrum and its active ingredient piperine caused significant melanin dispersal responses leading to darkening of the tail melanophores, which were completely antagonized by atropine and hyoscine. These per se melanin dispersal effects were also found to be markedly potentiated by neostigmine an anticholinesterase agent.

Conclusion: It appears that the melanin dispersal effects of the extracts of P. nigrum and pure piperine leading to skin darkening are mediated by cholinergic muscarinic or piperine-like receptors having similar properties.     

Bioactivity of Piper guineense Schum. & Thonn seed and Moringa oleifera Lam. leaf powder against Trogoderma granarium Everts (Coleoptera: Dermestidae)

Laboratory studies were carried out to investigate bioactivity of Piper guineense seeds and Moringa oleifera leaf powders applied singly or in a mixture against larvae and adult Trogoderma granarium Everts in airtight containers. Three levels (0.0 g, 0.5 g, and 1.0 g/20 g groundnut seeds) of the plant powders were used and pirimiphos-methyl was applied at 0.01 g/20 g seeds (recommended dose). Another control consisting of untreated seeds with aerated lids was included in the bioassay. Both larvae and adults were not killed in control with aerated lids throughout the experimental period and larvae were also tolerant to airtight storage conditions. Adults were more susceptible to plant powders than larvae and adult mortality recorded in P. guineense at 1.0 g, 0.5 g and M. oleifera at 1.0 g/20 g seeds were not significantly different from the mortality observed with the recommended dose of Pirimiphos methyl at five days after treatment (DAT). Larval mortality observed in a mixture of both plants (1:1; w/w) caused significantly higher mortality (77.5%) than other treatments at 5 DAT. All treatments (P. guineense and M. oleifera applied singly or in a mixture) were repellant to larvae T. granarium with 60% repellency recorded in the mixture of plants, 50% repellency in P. guineense and 30% repellency in M. oleifera slurry. The water absorption capacity of treated seeds was not affected by treatment with plant powders and ranged from 31.98% to 37.59%.     

Chemical Profile and Biological Activities of Fungal Strains Isolated from Piper nigrum Roots: Experimental and Computational Approaches

The current report describes the chemical investigation and biological activity of extracts produced by three fungal strains Fusarium oxysporum, Penicillium simplicissimum, and Fusarium proliferatum isolated from the roots of Piper nigrum L. growing in Vietnam. These fungi were namely determined by morphological and DNA analyses. GC/MS identification revealed that the EtOAc extracts of these fungi were associated with the presence of saturated and unsaturated fatty acids. These EtOAc extracts showed cytotoxicity towards cancer cell lines HepG2, inhibited various microbacterial organisms, especially fungus Aspergillus niger and yeast Candida albicans (the MIC values of 50–100 μg/mL). In α-glucosidase inhibitory assay, they induced the IC₅₀ values of 1.00-2.53 μg/mL were better than positive control acarbose (169.80 μg/mL). The EtOAc extract of F. oxysporum also showed strong anti-inflammatory activity against NO production and PGE-2 level. Four major compounds linoleic acid (37.346 %), oleic acid (27.520 %), palmitic acid (25.547 %), and stearic acid (7.030 %) from the EtOAc extract of F. oxysporum were selective in molecular docking study, by which linoleic and oleic acids showed higher binding affinity towards α-glucosidase than palmitic and stearic acids. In subsequent docking assay with inducible nitric oxide synthase (iNOS), palmitic acid, oleic acid and linoleic acid could be moderate inhibitors.     

Inhibitory effects of Piper umbellatum and Piper peltatum extracts towards myotoxic phospholipases A2 from Bothrops snake venoms: isolation of 4-nerolidylcatechol as active principle

Phospholipases A(2) (PLA(2)) are important constituents of snake venoms, being responsible for several of their toxic actions. Extracts from plants used in folk medicine were screened for inhibition of the enzymatic activity of myotoxin I, a PLA(2) from Bothrops asper. Piper umbellatum and Piper peltatum extracts tested positive, and their fractionation resulted in the isolation of 4-nerolidylcatechol. Its inhibitory effects towards toxic activities of two Bothrops myotoxins, representing catalytically active (Asp49) and catalytically inactive (Lys49) types of group II PLA(2)s, respectively, were characterized. The enzyme activity of B. asper myotoxin I was completely inhibited by 4-nerolidylcatechol at an inhibitor:toxin ratio of 10:1 (wt/wt) with an IC50 of approximately 1mM. In addition, 4-nerolidylcatechol inhibited representatives of groups I and III of PLA(2)s. Its preincubation with Bothrops myotoxins significantly reduced their myotoxic and edema-inducing activities in animal experiments. However, when 4-nerolidylcatechol was administered in situ, immediately after toxin injection, its inhibitory ability was substantially lower or negligible. This might be explained by the rapid action of these toxins in vivo, together with the slow inactivation of PLA(2) activity observed in vitro. Electrophoretic and chromatographic analyses of myotoxins ruled out major changes in protein charge, hydrophobicity, or gross molecular mass being involved in the inhibition mechanism. Mass spectrometry determinations are consistent with the covalent modification of myotoxin by one molecule of 4-nerolidylcatechol. Finally, a novel compound was isolated from both Piper species, sharing the nerolidyl skeleton, but nevertheless not being inhibitory towards the PLA(2)s studied.     

紫茎泽兰提取物对四种储粮害虫的杀虫活性

紫茎泽兰提取物对米象、玉米象、绿豆象和蚕豆象等 4种储粮害虫成虫具有强烈的熏杀活性 ,用 4 4.4 4mg L的浓度熏蒸处理 4 8小时后 ,每种成虫死亡率均达到 1 0 0 .0 0 %。熏蒸毒力测定结果表明 ,紫茎泽兰提取物对上述 4种成虫 2 4小时的LC50 分别为 1 4 .65,1 2 .80 ,2 5.0 7和 1 2 .2 0mg L ,4 8小时的LC50 分别为 1 1 .79,9.67,1 3 .2 9和 9.76mg L。     

Effects of Piper cubeba L. essential oil on methicillin‐resistant Staphylococcus aureus: an AFM and TEM study

The increasing prevalence of antibiotic‐resistant bacteria is creating a real challenge for health care systems worldwide, making the development of novel antibiotics a necessity. In addition to the development of new antibiotics, there is an urgent need for in‐depth characterization of the mechanisms of bacterial resistance toward new drugs. Here, we used essential oils extracted in our laboratory from Piper cubeba against methicillin‐resistant Staphylococcus aureus ATCC 43300, one of the most prominent antibiotic‐resistant bacteria. Effects of the essential oils extracted from P cubeba on bacteria were mainly evaluated using 2 powerful microscopy techniques: atomic force microscopy and transmission electron microscopy. High‐resolution atomic force microscopy images of the cells were obtained close to their native environment by immobilizing the cells on porous Polyether sulfone membranes, which were prepared in our laboratory with a wide range and distribution of pore sizes and depth. Inhibition zones (mm) and minimum inhibitory concentrations were determined. Two different concentrations of the oil were used to treat the cells: 50 μg/mL minimum inhibitory concentration and 25 μg/mL. The 50 μg/mL oil solution caused severe damage to the bacterial cells at microscopic levels while the 25 μg/mL solution showed no effects compared to the control. However, at nanoscopic levels, the 25 μg/mL oil solution caused significant changes in the cell wall, which could potentially impair bacterial activities. These results were also confirmed by transmission electron microscopy micrographs. Our results indicate that the extract has a good biological activity against methicillin‐ and oxacillin‐resistant S aureus and that it acts on the cell wall and plasma (cytoplasmic) membrane.     

Abscess forming ability of streptococcus milleri group: synergistic effect with Fusobacterium nucleatum.

The abscess forming abilities of "Streptococcus milleri" strains (Streptococcus constellatus, Streptococcus anginiosus, and Streptococcus intermedius) isolated from dentoalveolar abscesses and the synergistic effect of Fusobacterium nucleatum co-inoculated with the isolates were examined on a mouse subcutaneous abscess model. Five days after inoculation, all S. milleri strains formed abscesses, which showed less pathological spread to surrounding connective tissues than those formed by Staphylococcus aureus 209P strain and were similar to those by F. nucleatum ATCC25586. When each S. milleri strain and F. nucleatum were co-inoculated, abscess sizes and each bacterial number recovered from abscesses increased in comparison to those treated by bacterial mono-inoculation of each S. milleri strain or F. nucleatum alone. The strongest synergistic effect was observed in the combination of S. constellatus and F. nucleatum. In a time course experiment with this combination, the recovery of S. constellatus subsequently decreased after the decrement of F. nucleatum, and it appeared that the association with F. nucleatum maintained the bacterial number of S. constellatus in the abscess. The cell-free supernatant of F. nucleatum had a tendency to increase the abscess size caused by S. constellatus in this model. When S. constellatus was cultured with F. nucleatum culture supernatant in vitro, growth enhancement in the early phase was observed. Furthermore, the phagocytic killing of S. constellatus by human polymorphonuclear leukocytes (PMNs) was significantly suppressed and the PMN membranes appeared to be injured by addition of the F. nucleatum culture supernatant. These results suggest that the pathogenicity of S. milleri strains in odontogenic infections may be enhanced by the co-existence of F. nucleatum.     

14种菊科植物提取物对菜青虫的杀虫活性

用胜红蓟、黄花蒿、青蒿、艾蒿、鬼针草、三叶鬼针草、鱼眼草、小鱼眼草、紫茎泽兰、千里光、希签草、万寿菊、肿柄菊、苍耳等１４种菊科植物的石油醚、氯仿、丙酮、乙醇、甲醇和水提取物对菜青虫５龄幼虫的生测结果表明,黄花蒿、青蒿、艾蒿、鱼眼草、小鱼眼草、紫茎泽兰、万寿菊等７种菊科植物甲醇和乙醇提取物具有较高的杀虫活性。此结果为进一步从菊科植物中提取,分离和筛选高活性杀虫活性成分奠定了基础。     

The Piperaceae Amides I: Structure of Pipercide,A New Insecticidal Amide from Piper nigrum L.

It was evidenced that mutagenic principles in tryptophan pyrolysate, 3-amino-1,4-dimethyl-5H pyrido(4,3-b) indole and 3-amino-1-methyl-5H pyrido(4,3-b) indole (abbreviated as Trp-P-1 and Trp-P-2, respectively) bind to DNA without activation by rat liver microsomes. The bindings of Trp-P-1 and Trp-P-2 were not random and did not introduce strand scissions into DNA. Trp-P-1 bound more easily than Trp-P-2. The bindings of these mutagenic principles to DNA were concluded by using negatively superhelical simian virus 40 (SV40) DNA from following experimental data. (1) The intensity of ethidium bromide (EtBr)-DNA fluorescence by illumination with UV light and the electrophoretic mobility of superhelical DNA in agarose gel decreased as a function of the amounts of Trp-P-1 and Trp-P-2. (2) In vitro RNA synthesis catalyzed by Escherichia coli DNA-dependent RNA polymerase and nick-translation catalyzed by Escherichia coli DNA polymerase I (Kornberg enzyme) were inhibited significantly on DNA treated with Trp-P-1 and Trp-P-2. (3) The negative superhelicity of SV40 DNA introduces unpaired regions into DNA. These regions can be cleaved by single-strand-specific S1 endonuclease to generate unit length linear duplex molecules. It was found that this S1-sensitivity of DNA decreased by treatment with Trp-P-1. (4) The cleavage patterns of Trp-P-1 treated DNA with five restriction endonucleases were investigated. The protection of the cleavage site by the drug was observed against HincII, HindIII and EcoRII, whereas not against HaeIII and HinfI. These results show that the binding of Trp-P-1 to DNA is not random. Identical results were also obtained in Trp-P-2.

However, the bindings of Trp-P-1 and Trp-P-2 were not so tight, and phenol extraction of the complex dissociated these drugs from DNA.     

Eimeria tenella: synergistic interaction of sulfaquinoxaline and t-butylaminoethanol in the chicken.

A significant (P < 0.001) synergistic interaction between sulfaquinoxaline and t-butylaminoethanol was demonstrated against a sulfaquinoxaline-resistant strain of Eimeria tenella in chickens. The t-butylaminoethanol is a dimethylaminoethanol- and choline-reversible anticoccidial whereas sulfaquinoxaline is a classic PABA-reversible anticoccidial.     

黄皮种子提取物对斜纹夜蛾幼虫的杀虫活性及有效成分鉴定

【目的】测定黄皮Clausena lansium种子提取物对斜纹夜蛾Spodoptera litura幼虫的拒食活性,对有效成分进行分离、鉴定,并进一步测定其拒食活性、生长发育抑制活性及细胞毒性,为筛选斜纹夜蛾生态防治植物源杀虫剂提供依据。【方法】采用叶蝶法测定黄皮种子甲醇提取物及其石油醚、乙酸乙酯、正丁醇和水4种溶剂萃取物对斜纹夜蛾3龄幼虫的非选择性拒食活性和生长发育抑制活性;用硅胶柱层析进行石油醚萃取物和乙酸乙酯萃取物拒食活性成分分离;用核磁共振谱(NMR)进行拒食有效成分结构鉴定;用MTT法测定拒食活性成分对SL细胞毒性。【结果】黄皮种子甲醇提取物对斜纹夜蛾3龄幼虫具有较强的拒食活性,活性成分主要分布于石油醚相和乙酸乙酯相中;石油醚相和乙酸乙酯相经硅胶柱层析分离得10个馏分,以黄色晶体馏分2的拒食活性最强;经分离纯化及核磁共振氢谱和碳谱鉴定,黄皮新肉桂酰胺B(lansiumamide B)为主要的拒食活性成分,对斜纹夜蛾3龄幼虫48 h拒食中浓度为214.95μg/m L,同时具有较强的细胞毒性,对SL细胞48 h抑制中浓度为10.09μg/m L。【结论】黄皮种子提取物对斜纹夜蛾幼虫具有较强的拒食活性及细胞毒性,黄皮新肉桂酰胺B是主要活性成分之一,作为植物源杀虫剂开发具有较好的发展前景。     

Morphological and immunohistochemical study of the midgut and fat body of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: noctuidae) treated with essential oils of the genus Piper

ABSTRACT

Essential oils are a promising alternative to insecticides. We investigated the LD₅₀ of oils extracted from Piper corcovadensis, P. marginatum, and P. arboreum after 48 h topical contact with Spodoptera frugiperda larvae using morphometry, histochemistry and immunohistochemistry of the midgut and fat body. Chromatography revealed that E-caryophyllene was the principal compound common to the Piper species. The essential oils of P. corcovadensis, P. marginatum and P. arboreum caused deleterious changes in the midgut of S. frugiperda larvae. P. corcovadensis oil produced the lowest LD₅₀ and significant histopathological alterations including elongation of the columnar cells, formation of cytoplasmic protrusions, reduction in carbohydrate, increased apoptotic index and decreased cell proliferation. P. arboreum oil caused histopathological alterations similar to P. corcovadensis, but caused the highest rate of cell proliferation and increased regenerative cells, which indicated rapid regeneration of the epithelium. Our findings demonstrated the insecticidal potential of P. corcovadensis for control of S. frugiperda owing to the significant damage it inflicted on S. frugiperda midgut.     

Gender-specific responses of Piper betle L. to low temperature stress: changes in chlorophyllase activity

Gender based differences in response to low temperature stress in leaf chlorophyll (Chl), and carotenoids (Car) contents and chlorophyllase (Chlase) activity were monitored in male (Kapoori Vellaikodi and Madras Pan Kapoori) and female (Bangla Mahoba, Desi Bangla and Kaker) betel vine landraces. Although female plants contained nearly two fold more Chl than male counterparts, the low temperature induced Chl loss was comparable, however, male plants showed higher Chl a/b ratio than females. Chlase activity increased due to cold stress in all the landraces. Male plants always showed higher activities of Chlase, which may be one of the reasons for the rather low Chl contents in male plants.     

18种中草药提取物对褐飞虱和甜菜夜蛾的杀虫活性分析

为了筛选对农产品和环境安全、对害虫高效的植物源杀虫剂,分别采用喷雾法和饲毒法,测定了18种中草药的3种溶剂（水、乙醇、石油醚）提取物对褐飞虱Nilaparvata lugens Stål的触杀活性和对甜菜夜蛾Spodoptera exigua Hübner的胃毒作用。结果表明,各种提取物（0.8 g/mL）对甜菜夜蛾的胃毒作用不明显,校正死亡率最高为33.33%;但对褐飞虱的杀虫活性较高,其中百部、黄柏、榧子、生山栀、鹤虱、木香的乙醇或石油醚提取物,对褐飞虱3龄若虫的24 h校正死亡率均在80%以上,其石油乙醚提取物的LC50值依次为：0.0826,0.0455,0.0145,0.0047,0.0038,0.0033 g/mL。采用系统溶剂法分析发现,木香和鹤虱的主要杀虫活性成分均在氯仿萃取物中,表明其杀虫成分易溶于较弱极性溶剂中。进一步通过活性追踪法,对鹤虱提取物的柱层析各流分的活性检测表明,其杀虫活性成分的Rf值为0.5556~0.5926。该研究为进一步纯化新型植物源杀虫物质并确定其分子结构,从而开发其应用前景及发现新的杀虫活性先导化合物奠定了基础。     

Control of flea beetles Podagrica spp. (Coleoptera: Chrysomelidae) infestation on okra (Abelmoschus esculentus (L.) Moench) using Piper guineense seed extracts

Crops in the Family Malvaceae are attacked by various insect pests especially Podagria species (Coleoptera: Chrysomelidae) at different stages of their development. The emergence of biodegradable pesticides (botanicals) as safe option has reduced the problems that result from the use of synthetic insecticides, thus creating a renewed interest in their development and use in integrated pest management of crops. Therefore, field experiments were conducted to investigate the control capability of water extracts of Piper guineense seeds and Cypermethrin on flea beetles (Podagrica spp.) infesting okra (Abelmoschus esculentus (L.) Moench). The experiments were conducted in two locations in the rain forest zone of Nigeria. The first experiment was conducted at the Teaching and Research Farm of The Federal University of Technology Akure, Ondo State (2011 Early cropping season), while the second experiment was carried out at the Teaching and Research Farm of Rufus Giwa Polytechnic, Owo, Ondo State (2011 Late cropping season). A synthetic insecticide (Cypermethrin) was included in the treatments as a standard check alongside the untreated (control). The experimental design was laid out in a randomised complete block design with five treatments replicated three times. The effectiveness of the treatments was determined based on reduction in the population of the adult insects. The results showed that water extract of P. guineense seeds and the synthetic insecticide significantly (p?<?0.05) reduced the population of the flea beetles as compared with the control. The synthetic insecticide-treated plots produced the highest standard check, but this was not significantly different from the dried yield recorded in the plots sprayed with aqueous extracts of P. guineense seeds. The results of field trials in both cropping seasons showed that all the treatments significantly suppressed the infestation by the flea beetles throughout the study period. In view of the ability of water extract of P. guineense seeds to control the infestation of Podagrica spp. its use by farmers is therefore recommended.     

Evaluation of antipyretic activity of leaf extracts of Mallotus peltatus (Geist) Muell. arg. var acuminatus: a folk medicine.

A study was carried out to evaluate the anti-pyretic potential of the methanol extract of Mallotus peltatus (Geist) Muell. Arg. var acuminatus leaf, a folk medicine of Onge tribes of Bay Islands, on normal body temperature and yeast-induced pyrexia in Wister albino rats. The leaf extract at oral doses of 100, 200 and 300 mg kg(-1), p.o., showed significant reduction in normal body temperature and yeast-provoked elevated temperature in a dose-dependent manner and the anti-pyretic effect was comparable to that of standard anti-pyretic agent paracetamol (150 mg kg(-1), p.o.). The effect also extended up to 5 hours after the drug administration.     

Parallel and antiparallel dimers of magainin 2: their interaction with phospholipid membrane and antibacterial activity.

Magainin 2 (M2) forms pores by associating with several other M2 molecules in lipid membranes and shows antibacterial activity. To examine the effect of M2 dimerization on biological activity and membrane interaction, parallel and antiparallel M2 dimers were prepared from two monomeric precursors. Antibacterial and haemolytic activities were enhanced by dimerization. CD measurements showed that both dimers and monomers have an alpha-helical structure in the presence of lipid vesicles. Tryptophan fluorescence shift and KI quenching studies showed that all the peptides were more deeply embedded in acidic liposomes than in neutral liposomes. Experiments on dye-leakage activity and membrane translocation of peptides suggest that dimers and monomers form pores through lipid membranes, although the pore formation may be accompanied by membrane disturbance. Although dimerization of M2 increased the interaction activity with lipid membranes, no appreciable difference between the activities of parallel and antiparallel M2 dimers was observed.     

Cytotoxic activity induced by crude extracts of Ganoderma lucidum (W. Curt.: Fr.) P. Karst. on mouse myeloma cancer cell-line

Ganoderma lucidum powder using hot water and methanol extraction methods indicated a twofold more active cytotoxic activity with IC₅₀ of 44 ± 3.8 μg/ml in the latter method. The representative dose-response curves of the G. lucidum crude extracts on J558 cell-lines revealed that there were great similarities between the curves which reflected rapid killing activities. The percentage viability of the J558 cell exposed to these crude extracts was dose dependent only up to 150 μg/ml. After which, there was no significant reduction when the dose was increased to 200 or 400 μg/ml. The morphological alterations induced by the crude extract were examined under the phase contrast, fluorescent and electron microscopy. When J558 cells were treated with doses higher than 50 μg/ml of the crude extract, obvious morphological changes and apoptosis occurred after 72 h. At 400 μg/ml, most of the cells showed necrosis characterized as small fragments with uniformly stained red nuclei. The apoptotic and necrotic cells increased by 16.5 and 29.1%, respectively whereas the viable cells decreased by as much as 45.6. The mode of cell death via apoptosis was 3.6% higher than necrosis. However, these morphological changes were not observed in the case of 3T3 cells. Results obtained from scanning electron microscopy and transmission electron microscopy further confirmed the occurrence of various apoptotic and necrotic features.