Antioxidant efficacy of black pepper (Piper nigrum L.) and piperine in rats with high fat diet induced oxidative stress

The present study was aimed to explore the effect of black pepper (Piper nigrum L.) on tissue lipid peroxidation, enzymic and non-enzymic antioxidants in rats fed a high-fat diet. Thirty male Wistar rats (95-115 g) were divided into 5 groups. They were fed standard pellet diet, high-fat diet (20% coconut oil, 2% cholesterol and 0.125% bile salts), high-fat diet plus black pepper (0.25 g or 0.5 g/kg body weight), high-fat diet plus piperine (0.02 g/kg body weight) for a period of 10 weeks. Significantly elevated levels of thiobarbituric acid reactive substances (TBARS), conjugated dienes (CD) and significantly lowered activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and reduced glutathione (GSH) in the liver, heart, kidney, intestine and aorta were observed in rats fed the high fat diet as compared to the control rats. Simultaneous supplementation with black pepper or piperine lowered TBARS and CD levels and maintained SOD, CAT, GPx, GST, and GSH levels to near those of control rats. The data indicate that supplementation with black pepper or the active principle of black pepper, piperine, can reduce high-fat diet induced oxidative stress to the cells.     

Occurrence of symptomless source of Piper yellow mottle virus in black pepper (Piper nigrum L.) varieties and a wild Piper species

Symptomless nature of Piper yellow mottle virus (PYMoV) infection in three varieties of black pepper (Piper nigrum) (Panniyur 1, Panniyur 5 and Panchami) and a wild species of Piper (Piper colubrinum) was confirmed by polymerase chain reaction (PCR) using PYMoV specific primers. The virus could be transmitted from these PYMoV-infected symptomless plants onto symptom producing black pepper cv. Karimunda through mealybug vector, Ferrisia virgata and by graft transmission. About 20–50% seedlings showed typical symptoms of the PYMoV at 30 days after mealybug inoculations while it was 75–94% at 90 days after inoculation. PCR test of the inoculated seedlings confirmed the presence of PYMoV in 50–64%, 76–100% and 80–100% of plants in 30, 60 and 90 days after inoculation, respectively. Similarly, 50–66%, 91–100% and 100% of graft-transmitted plants showed typical symptoms of the disease at 30, 60 and 90 days after grafting. PCR test of the graft-transmitted plants showed 100% PYMoV infection at 60 days after grafting. The results clearly demonstrated the existence of PYMoV-infected symptomless plants that can act as source for secondary spread of the virus in the field.     

In silico Characterisation and Phylogenetic Analysis of Two Evolutionarily Conserved miRNAs (miR166 and miR171) from Black Pepper (Piper nigrum L.)

Among computationally predicted and experimentally validated plant miRNAs, several are conserved across species boundaries in the plant kingdom. In this study, a combined experimental–in silico approach was adopted for characterization of two conserved miRNAs, miR166 and miR171, from black pepper (Piper nigrum). A PCR-based detection and cloning strategy of miRNAs from tissues of black pepper was used. Conservation analysis of miR166 and miR171 along with their corresponding targets identified from P. nigrum revealed that these miRNAs are highly conserved with their counterparts in other plant species. miRNA-mediated cleavage of the conserved targets was also verified by RLM-RACE experiments. Real-time quantitative PCR revealed the differential expression patterns of these miRNAs in black pepper tissues. Our miRNA-based phylogenetic analysis of plants belonging to the Piperaceae family was in agreement with the typical paleoherb evolutionary scheme of primitive angiosperms. This method will help in the detection of evolutionarily conserved miRNAs in other plant species and provide a strategy for a novel phylogenetic reconstruction based on the evolutionary history of miRNA genes.     

Trichome density of main stem is tightly linked to PepMoV resistance in chili pepper (Capsicum annuum L.)

A relationship between pepper trichome and pepper mottle virus (PepMoV) resistance was examined. In an intraspecific F(2) mapping population from the cross between Capsicum annuum CM334 (trichome-bearing and PepMoV resistant) and Chilsungcho (glabrous and PepMoV susceptible), major QTLs for both traits were identified by composite interval mapping in linkage group (LG) 24 corresponding a telomere region on pepper chromosome 10. Ptel1 of putative trichome enhancing locus was a common major QTL for trichome density on the main stem and calyx. Ptel1 apart from HpmsE031 at a 1.03?cM interval was specifically associated to the trichome density on the main stem, whereas Ptel2 near m104 marker on LG2 was specific for the calyx trichome. Epistatic analysis indicated that Ptel1 engaged in controlling the trichome density by mutual interactions with the organ-specific QTLs. For PepMoV resistance, two QTLs (Pep1 and Pep2) were identified on the LG 24. Pep1 was located with Ptel1 in the R-gene cluster (RGC) for potyvirus resistance including Pvr4 with broad spectrum resistance to potyviruses. Pep1 flanking TG420 marker seemed to be the major factors determining correlation with PepMoV resistance. These results indicate that the level of trichome density on pepper main stem can be used as a morphological marker for Pvr4 in pepper breeding.     

水稻花药特异启动子Osg6B的序列及功能分析

对水稻花药特异表达基因Ｏｓｇ６启动子简称Ｏｓｇ６Ｂ的全序列进行了测定,结果表明,Ｏｓｇ６Ｂ含有１．７３ｋｂ个核苷酸,与文献报道的该启动子相差仅８个核苷酸,两者核苷酸同源性为９９．５％。将Ｏｓｇ６Ｂ同ＧＵＳ基因编码区相连,将构建成的融合基因用基因抢轰击烟草的花药和幼叶,荧光分析结果为含Ｏｓｇ６Ｂ的ＧＵＳ融合基因在大于２ｍｍ烟草花药中的表达量比对照和幼叶均高出８倍,在小于或等于２ｍｍ的花药中,则高达到     

Flower and nectar attributes of pepper (Capsicum annuum L.) plants in relation to their attractiveness to honeybees (Apis mellifera L.)

Flower morphology, nectary structure, nectar traits and rates of honeybee foraging on pepper plants were studied. The nectary appears as swellings on the basal part of the ovary. The nectariferous cells are smaller and denser than the neighbouring parenchyma. Stomata are present in the nectary epidermis, but do not appear on the other parts of the ovary epidermis. Seven pepper breeding lines were grown near a bee yard in Rehovot. Five to six fold differences in nectar volume were found between the extreme genotypes. Nectar volumes were higher during noon and afternoon hours, as compared with morning hours. High correlation coefficients between nectar volume and sugar concentration were found. These were significant for the four high nectar yielding genotypes, ranging between r = 0.65 to r = 0.94. Male-fertile flowers produced more nectar and higher sugar concentration than sterile ones. Skewed distribution was observed in nectar volume of F₂ populations, but relatively low heritability values were calculated. Pepper nectar contains fructose and glucose only. The former occupies 52–82 % of the total sugar content. Pepper genotypes varied in frequency of honeybee visits and significant correlation between sugar quantity and number of honeybee visits per flower was evident. Fertile pepper flowers are not very attractive to honeybees and male-sterile flowers are even less so. The considerable variation in nectar characteristics can be exploited to increase attractiveness to honeybees, thus facilitating bee pollination in commercial production of F₁hybrid seeds and improve fruit quality.     

High-quality embryo production and plant regeneration using a two-step culture system in isolated microspore cultures of hot pepper (Capsicum annuum L.)

We developed an efficient culture system for producing cotyledonary embryos from isolated microspores of hot pepper (Capsicum annuum L.) and analyzed the ploidy levels of regenerated plants. Three culture protocols were studied: liquid, double-layer, and two-step culture. In the double-layer culture, cotyledonary embryos were produced more efficiently when the same medium composition was used for the liquid upper-layer and the solid under-layer. The two-step culture system, in which microspores were first incubated on liquid medium and then subcultured on double-layer medium, was most effective for producing cotyledonary embryos. Cotyledonary embryos were produced more efficiently when the isolated microspores were cultured in liquid medium for 1 week in 60 × 15-mm plates at a density of 8–10 × 10⁴/mL and microspore suspensions from two liquid culture plates were combined into a single 100 × 20-mm plate containing solid medium, and the culture was continued for an additional 3 weeks. When cotyledonary embryos obtained from this two-step culture were transplanted into regeneration medium, more than 95 % developed into plants. Only 31 of the 190 analyzed plants (16.3 %) generated by this method were spontaneous doubled haploids. This two-step culture system outperforms all previously reported culture protocols for isolated microspores of hot pepper, and appears to be a promising tool for the production of haploid plants for hot pepper breeding.     

BAC-derived markers converted from RFLP linked to Phytophthora capsici resistance in pepper (Capsicum annuum L.)

Phytophthora capsici Leonian, an oomycete pathogen, is a serious problem in pepper worldwide. Its resistance in pepper is controlled by quantitative trait loci (QTL). To detect QTL associated with P. capsici resistance, a molecular linkage map was constructed using 100 F(2) individuals from a cross between Capsicum annuum 'CM334' and C. annuum 'Chilsungcho'. This linkage map consisted of 202 restriction fragment length polymorphisms (RFLPs), 6 WRKYs and 1 simple sequence repeat (SSR) covering 1482.3 cM, with an average interval marker distance of 7.09 cM. QTL mapping of Phytophthora root rot and damping-off resistance was performed in F(2:3) originated from a cross between resistant Mexican landrace C. annuum 'CM334' and susceptible Korean landrace C. annuum 'Chilsungcho' using composite interval mapping (CIM) analysis. Four QTL explained 66.3% of the total phenotypic variations for root rot resistance and three 44.9% for damping-off resistance. Of these QTL loci, two were located close to RFLP markers CDI25 on chromosome 5 (P5) and CT211A on P9. A bacterial artificial chromosome (BAC) library from C. annuum 'CM334' was screened with these two RFLP probes to obtain sequence information around the RFLP marker loci for development of PCR-based markers. CDI25 and CT211 probes identified seven and eight BAC clones, respectively. Nine positive BAC clones containing probe regions were sequenced and used for cytogenetic analysis. One single-nucleotide amplified polymorphism (SNAP) for the CDI25 locus, and two SSRs and cleaved amplified polymorphic sequence (CAPS) for CT211 were developed using sequences of the positive BAC clones. These markers will be valuable for rapid selection of genotypes and map-based cloning for resistance genes against P. capsici.     

Endophytic bacterial flora in root and stem tissues of black pepper (Piper nigrum L.) genotype: isolation, identification and evaluation against Phytophthora capsici

Aim:  To isolate and identify black pepper ( Piper nigrum L) associated endophytic bacteria antagonistic to Phytophthora capsici causing foot rot disease.
Methods and Results:  Endophytic bacteria (74) were isolated, characterized and evaluated against P. capsici . Six genera belong to Pseudomonas spp (20 strains), Serratia (1 strain), Bacillus spp. (22 strains), Arthrobacter spp. (15 strains), Micrococcus spp. (7 strains), Curtobacterium sp. (1 strain) and eight unidentified strains were isolated from internal tissues of root and stem. Three isolates, IISRBP 35, IISRBP 25 and IISRBP 17 were found effective for Phytophthora suppression in multilevel screening assays which recorded over 70% disease suppression in green house trials. A species closest match (99% similarity) of IISRBP 35 was established as Pseudomonas aeruginosa ( Pseudomonas EF568931), IISRBP 25 as P. putida ( Pseudomonas EF568932), and IISRBP 17 as Bacillus megaterium ( B. megaterium EU071712) based on 16S rDNA sequencing.
Conclusion:  Black pepper associated P. aeruginosa , P. putida and B. megaterium were identified as effective antagonistic endophytes for biological control of Phytophthora foot rot in black pepper.
Significance and Impact of the Study:  This work provides the first evidence for endophytic bacterial diversity in black pepper stem and roots, with biocontrol potential against P. capsici infection.     

Individual and multi-environment combined analyses identify QTLs for morphogenetic and reproductive development traits in white clover (Trifolium repens L.)

White clover (Trifolium repens L.) is a key component legume of temperate pasture agriculture and an important target for molecular marker-assisted plant breeding. A genetic map of white clover has been used to assess genetic control of agronomically important traits that vary in the F₂(I.4R×I.5J) mapping family. Phenotypic analysis was performed for a range of vegetative morphogenesis traits (such as leaf area, internode length, plant height and plant spread) and reproductive morphogenesis and development traits (such as flowering date, floral intensity and seed yield), with both spatial and temporal replication. A multi-environment combined analysis (combined analysis) has been performed for traits assessed across multiple experimental datasets in order to identify consistent genetic effects. Quantitative trait locus (QTLs) were detected for the majority of traits, and the locations and magnitudes of QTL effects were compared between individual and combined analyses. This molecular genetic dissection of agronomic traits in white clover provides the basis for equivalent studies in more complex populations, design of marker-assisted selection strategies and comparative genetics with model legume species. Selection for QTLs derived from the combined analysis will permit robust improvement of phenotypic traits over different environments.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Production of wine from mango (Magnifera indica L.) using Saccharomyces and Schizosaccharomyces species isolated from palm wine

Juice extracted from pulp of the mature ripe tropical fruit, mango ( Magnifera indica L.), containing 15.9% soluble solids, was fermented with four strains of yeast isolated from palm wine. Two of the strains belonged to the genus Schizosaccharomyces (T1 and T2) while the other two were Saccharomyces (B2 and M1). The two strains of Schizosaccharomyces were found to be suitable for the production of sweet, table mango wine with alcohol contents of 8.0 and 9.0% for T1 and T2, respectively. The two strains of Saccharomyces were found suitable for the production of dry mango fruit wines containing 10.0% alcohol.     

Relationship between epistasis and aggressiveness in resistance of pepper (Capsicum annuum L.) to Phytophthora nicotianae

This study evaluated the types of gene action governing the inheritance of resistance to Phytophthora nicotianae necrosis in populations derived from two crosses involving two susceptible (Beldi and Nabeul II) and one resistant (CM334) cultivars of pepper (Capsicum annuum L.). Populations, composed of Pr, Ps, F(1) , F (2) , BC (1) Pr, and BC (1) Ps generations, were inoculated with six P. nicotianae isolates. Generation means analysis indicated that an additive-dominance model was appropriate for P. nicotianae isolates Pn (Ko1) , Pn (Ko2) and Pn (Kr1) , which showed low aggressiveness in the two crosses. For the more aggressive isolates Pn (Bz1) , Pn (Bz2) and Pn (Kr2) , epistasis was an integral component of resistance in the two crosses. The presence of epistasis in the resistance of pepper to P. nicotianae was dependent on the level of aggressiveness of the isolates. Selection in pepper with less aggressive isolates was efficient, but not with more aggressive isolates; on the other hand, selection with more aggressive isolates was more stable. The minimum number of genes controlling resistance was estimated at up to 2.71. In the majority of cases, the additive variance was significant and greater than the environmental and dominance variance.     

Control of stored grain pest,Callosobruchus maculatus (F.) (Coleoptera: Bruchidae), using the essential oils isolated from Zingiber officinale (L.) and Mentha pulegium (L.) in laboratory condition

Fumigant activity of essential oil vapours distilled from Zingiber officinale (L.) and Mentha pulegium (L.) was tested against eggs, larvae and adults of Callosobruchus maculatus (F.). Fumigant toxicity was assessed at 27?±?1?°C and 60?±?5% RH, in dark condition. The influence of different concentrations of the essential oil vapours on egg hatchability, larval and adult mortality was significant. Data probit analysis showed that lethal concentration of the essential oil to kill 50% of the population (LC₅₀) for egg, larvae and adult was found to be 1.151, 2.336 and 2.183?μl/l air of Z. officinale, followed by 0.072, 0.113 and 0.093?μl/l air essential oil of M. pulegium, respectively. Between these essential oils, Z. officinale is almost more toxic than M. pulegium on all growth stages of C. maculatus. The present study suggests that essential oils from these medicinal plants may be potential grain protectants as botanical alternative fumigants and could be used in the management of various life stages of C. maculatus.     

Spectrum of resistance to root-knot nematodes and inheritance of heat-stable resistance in in pepper (Capsicum annuum L.)

Capsicum annuum L. has resistance to root-knot nematodes (RKN) (Meloidogyne spp.), severe polyphagous pests that occur world-wide. Several single dominant genes confer this resistance. Some are highly specific, whereas others are effective against a wide range of species. The spectrum of resistance to eight clonal RKN populations of the major Meloidogyne species, M. arenaria (2 populations), M. incognita (2 populations), M. javanica (1 population), and M. hapla (3 populations) was studied using eight lines of Capsicum annuum. Host susceptibility was determined by counting the egg masses (EM) on the roots. Plants were classified into resistant (R; EM ≤ 5) or susceptible (H; EM >5) classes. The french cultivar Doux Long des Landes was susceptible to all nematodes tested. The other seven pepper lines were highly resistant to M. arenaria, M. javanica and one population of M. hapla. Variability in resistance was observed for the other two populations of M. hapla. Only lines PM687, PM217, Criollo de Morelos 334 and Yolo NR were resistant to M. incognita. To investigate the genetic basis of resistance in the highly resistant line PM687, the resistance of two progenies was tested with the two populations of M. incognita: 118 doubled-haploid (DH) lines obtained by androgenesis from F₁ hybrids of the cross between PM687 and the susceptible cultivar Yolo Wonder, and 163 F₂ progenies. For both nematodes populations, the segregation patterns 69 R / 49 S for DH lines and 163 R / 45 S for F₂ progenies were obtained at 22°C and at high temperatures (32°C and 42°C). The presence of a single dominant gene that totally prevented multiplication of M. incognita was thus confirmed and its stability at high temperature was demonstrated. This study confirmed the value of C. annuum as a source of complete spectrum resistance to the major RKN. Received: 2 July 1998 / Accepted: 11 March 1999     

Morphological markers to correlate bud and anther development with microsporogenesis and microgametogenesis in pepper (Capsicum annuum L.)

The identification of microspores or pollen grains at particular developmental stages during microsporogenesis or microgamentogenesis is an important step for different basic and applied purposes. Among them, the most relevant example from a biotechnological perspective is the production of androgenic doubled haploids. For this and other techniques, precise, fast, easy and reliable criteria to identify flower buds carrying microspores or pollen at particular stages are essential. In anthocyanin-producing pepper types, the particularities of flower development allow for the identification of several morphological markers potentially useful as criteria for such an identification. In this work, our aim was to determine the easiest and more accurate criterion to correlate visible, measurable traits of bud and anther development with each of the individual stages of microsporogenesis and microgametogenesis. For this, we used three Spanish sweet pepper F1 hybrids (‘Herminio’, ‘Gacela’ and ‘Águila’). We analyzed and discussed the accuracy and practical usefulness of using anther length, bud length, anther purple pigmentation and the ratio between calyx length and bud length (calyx/bud ratio) as predictors of individual microspore/pollen developmental stages. According to our results, we propose a combination of calyx/bud ratio and anther pigmentation as an easy, fast and accurate criterion potentially applicable to anthocyanin-producing pepper cultivars to determine their particular markers.     

Simultaneous UFLC-ESI-MS/MS determination of piperine and piperlonguminine in rat plasma after oral administration of alkaloids from Piper longum L.: application to pharmacokinetic studies in rats

The alkaloids from Piper longum L. showed protective effects on Parkinson's disease models in our previous study and piperine and piperlonguminine were the two main constituents in the alkaloids. The present study aimed at developing a rapid, sensitive, and accurate UFLC-ESI-MS/MS method and validating it for the simultaneous determination of piperine and piperlonguminine in rat plasma using terfenadine as the internal standard. The analytes and internal standard (IS) were extracted from rat plasma using a simple protein precipitation by adding methanol/acetonitrile (1:1, v/v). A Phenomenex Gemini 3 u C18 column (20 mm × 2.00 mm, 3 μm) was used to separate the analytes and IS using a gradient mode system with a mobile phase consisting of water with 0.1% formic acid (mobile phase A) and acetonitrile with 0.1% formic acid (mobile phase B) at a flow rate of 0.4 mL/min and an operating column temperature of 25°C. The total analytical run time was 4 min. The detection was performed using the positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode with transitions at m/z 286.1-201.1 for piperine, m/z 274.0-201.1 for piperlonguminine, and m/z 472.4-436.4 for the IS. The calibration curves were both linear (r>0.995) over a concentration range of 1.0 to 1000 ng/mL; the lower limit of quantification (LLOQ) was 1.0 ng/mL for both piperine and piperlonguminine. The intra-day and inter-day precisions (RSD %) were <12.1%, accuracies ranged from 86.6 to 120%, and recoveries ranged from 90.4 to 108%. The analytes were proven stable in the short-term, long-term, and after three freeze-thaw cycles. The method was successfully applied to pharmacokinetic studies of piperine and piperlonguminine in rats after oral administration of alkaloids from P. longum L.     

Blossom-end rot in relation to growth rate and calcium content in fruits of sweet pepper (Capsicum annuum L.)

The relative importance of growth rate and calcium concentration in sweet pepper fruits (Capsicum annuum L.) for the induction of blossom-end rot (BER) was investigated in (1) four pollination treatments in one cultivar, (2) four cultivars with the same fruit load and (3) three fruit load treatments in four cultivars. For fruits with the same pollination treatment those eventually developing BER had a higher initial fruit growth rate than those not developing BER. Within the same experiment both the growth rate of the young fruit and BER increased with the number of seeds. The Ca concentration of the pericarp in mature fruits was negatively related to both fruit size and BER incidence. Differences in levels of BER between different pollination experiments could not be explained solely by differences in growth rate of the young fruit, but related to different Ca concentrations in the mature fruits. In the spring, but not in the summer, cultivars more susceptible to BER had a larger final size but lower Ca concentration in the young fruit than the resistant ones. By lowering the fruit load in the summer both the final fruit size and the BER incidence increased, but the Ca concentrations of both proximal and distal pericarp in the young fruit of all cultivars were not consistently affected. Despite a correlation between growth rate and low Ca concentration in the fruit, the incidence of BER may only be predicted from separate effects of fruit growth and of Ca concentration of fruit. The data indicated that at a higher growth rate a higher Ca concentration is required to prevent the induction of BER. The usefulness of the total Ca concentration of the fruit for determining the critical Ca concentration in the induction of BER is discussed.Key words: Capiscum annuum L., sweet pepper, blossom-end rot, calcium, growth rate, pollination, fruit load.