Characterization of chitinases and β-1,3-glucanases in grapefruit flavedo during fruit development

Chitinase (EC 3.2.1.14) and β-1,3-glucanase (EC 3.2.1.39) activities in the flavedo of grapefruit ( Citrus paradisi cv. Marsh) were determined at 17 times during the course of fruit development. Chitinase activity is initially high in flavedo, but drops rapidly and is low, although fairly constant throughout the remainder of fruit development. In contrast to chitinase, β-1,3-glucanase activity is lowest in young fruit and increases during development. Western blots of crude flavedo extracts following SDS-PAGE were probed with antibodies raised against purified citrus chitinase and β-1,3-glucanase. Results of immunostaining revealed that changes in the activities of chitinase and β-1,3-glucanase were reflected in the amount of chitinase and glucanase protein present in the extracts. Only a single chitinase band was detected on western blots of crude flavedo extracts, whereas one glucanase band was present in young fruit and a second one appeared later in older fruit. Partial purification of flavedo chitinases and glucanases was performed using extracts prepared from immature and mature fruit for the two enzymes, respectively. Acidic and basic forms of both enzymes were present in the extracts; acidic and basic forms of chitinase were present in nearly equal amounts whereas basic glucanases predominated (91% of total activity). Acidic and basic chitinases differed in substrate specificity as well as products of degradation indicating the heterogeneous nature of the enzymes. Both acidic and basic glucanases required the presence of β-1,3 linkages for activity, were active against both soluble and insoluble β-1,3 glucans and generated similar products.     

Phytopesticidal and repellent efficacy of Litsea salicifolia (Lauraceae) against Aedes aegypti and Culex quinquefasciatus

Litsea salicifolia is one of the many plants used as phytopesticide, traditionally by various tribes of Assam. Of the five extracts of L. salicifolia tested for the bioactivity against Aedes aegypti and Culex quinquefasciatus, the aqueous extract was more effective compared to other extracts exhibiting bioactivity at 72 ppm against A. aegypti. The hexane extract (2000 ppm) exhibited 70% repellent activity for 3 hr against A. aegypti and 46% activity for 3 hr against C. quinquefasciatus. This is the first report on the insecticidal properties of L. salicifolia which can be further developed as an eco-friendly biopesticides of the future.     

In vitro examination of anti-parasitic,anti-Alzheimer,insecticidal and cytotoxic potential of Ajuga bracteosa Wallich leaves extracts

This research study is mainly focused to evaluate the anti-parasitic, insecticidal, cytotoxic and anti-alzheimer potential of various leaf extracts of Ajuga bracteosa Wallich ex Bentham. 04 different extracts were prepared using solvent of different polarity to determine the best candidate for potent bioactivity i.e. n-hexane (NH), Ethyl acetate (EA), Ethanol (EL) and Chloroform (CH). Concentrations of each extracts were made specified for all activities. All extracts were exploited for broad range of biomedical applications including leishmaniasis, in vitro anti-Alzheimer, insecticidal and cytotoxic studies. Our results showed that A. bracteosa n-hexane extract was highly active against Leishmania Tropica with significant inhibition of 58 ± 1.61 for promastigote and 63 ± 2.29 for amastigote at 1000 μg/mL. Furthermore, promising anti-alzheimer activity acetylcholinesterase (AChE) 46 ± 0.83 and butrylcholineterase (BChE) 49 ± 1.17 was noted for n-hexane. The insecticidal potential of these extracts were test against five different insects (Rhyzopertha dominica, Trogoderma granarium, Tribolium castaneum, Sitophilus oryze, and Callosobruchus analis). The higest mortality rate of insecticidal activity was recorded by n-hexane followed by Ethyl acetate whereas ethanol extract was found to be less effective against all the test species. Significant cytotoxic potential of each plant sample against Artemia salina thus aware us for further detailed research to find out novel drugs. Based on our results we believe that Ajuga bracteosa could be used to develop as a potential botanical insecticide against different insect and pests, such as aphids as well as an excellent source for the compound isolation as anti-tumor agent.     

Olive fruit fly (Diptera: Tephritidae) populations in relation to region, trap type, season, and availability of fruit

Olive fruit fly, Bactrocera oleae (Gmelin), was monitored with adult captures by season and trap type, and was related to fruit volume and nonharvested fruit to elucidate the occurrence of the newly introduced pest in California. The highest numbers of adults captured in ChamP traps in olive trees, Olea europaea, were in October in an inland valley location, and in September in a coastal location. Comparisons of trap types showed that the number of olive fruit fly adults captured in Pherocon AM traps in a commercial orchard was significantly greater than in ChamP traps. A significantly greater number of females were captured in Pherocon AM traps with bait packets and pheromone lures than traps with pheromone lures alone, while a significantly greater number of adults and males were captured in traps with pheromone lures alone. Significantly more adults were captured in ChamP traps with bait packets and pheromone lures versus traps with bait packets alone. Fruit volume increased by four times from mid-June to mid-November. Olive fruit fly was found to oviposit on small olive fruit <1 cm3 shortly after fruit set, the maximum number of ovipositional sites per fruit occurred in October, and the greatest number of pupae and adults were reared from fruit collected in September and October. The highest numbers of pupae were collected from nonharvested fruit in March when high numbers of adults were captured in the same orchard.     

Partial purification and insecticidal activity of toxic metabolites secreted by a Mucor hiemalis strain (SMU-21) against adults of Bactrocera oleae and Ceratitis capitata (Diptera: Tephritidae)

The secondary metabolites present in the methanol extract of a Mucor hiemalis strain (SMU-21) mycelia, cultured in liquid medium, were evaluated for toxicity to Bactrocera oleae (Gmelin) and Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) adults. Feeding and contact bioassays revealed that the methanol fraction of the crude supernatant was strongly toxic to both species. Symptoms of toxicity developed quickly; lethargy occurred 1-2 h posttreatment, and mortality reached 82-97% after 24 h. Both feeding and contact bioassays showed that B. oleae was more susceptible than C. capitata. Concentrations producing 50% mortality (LC50) to B. oleae and C. capitata after 24 h in feeding bioassays were 0.52 and 1.28 mg/ 0.1 ml diet, respectively, but 34.8 and 64.0 microg/ cm2, respectively, after 4 h in contact bioassays. Solvent partition, precipitation, and chromatographic procedures were used to isolate the active principles from the crude supernatant. These resulted in the isolation of one high-pressure liquid chromatography fraction with insecticidal activity on B. oleae flies equal to the initial crude supernatant.     

Carbohydrate metabolism and fruit quality are affected in frost-exposed Valencia orange fruit

Exposure of citrus fruit to frost often results in the development of freeze injury during their maturation in planta. This work was aimed to analyze changes in the biochemistry and enzymology of carbohydrate metabolism in freeze-injured orange fruit ( Citrus sinensis var. Valencia late) and the involvement of oxidative stress in frost damage. The activities of pyrophosphate-dependent phosphofructokinase, phosphoenolpyruvate carboxylase and several fermentative enzymes increased in frost-exposed (FE) fruit, while NADP-malic enzyme and the mitochondrial isoform of NAD-malate dehydrogenase showed a reduction in their activities. Western blot analysis indicated a correlation between activity levels and protein content. Respiration rate in whole fruit was reduced by 40%, whereas the flavedo showed a more pronounced decline (53%). Volatile compound (i.e. ethanol and acetaldehyde) content was significantly higher in FE fruit than in control, as was that of l -malate (three-fold). Additionally, FE fruit showed a marked decrease in the maturity index (24%) because of a higher titratable acidity (39%). Evidence is presented that oxidative stress is involved in freeze-induced damage of orange fruit, where oxidative damage to lipids and proteins, and a greater electrolyte leakage in the flavedo were also observed. The results suggest that freezing temperatures provoke a notable metabolic switch in citrus fruit toward a fermentative stage, resulting in low-quality fruits.     

Radical scavenging activities of Rio Red grapefruits and Sour orange fruit extracts in different in vitro model systems

Antioxidant fractions from two different citrus species such as Rio Red (Citrus paradise Macf.) and Sour orange (Citrus aurantium L.) were extracted with five different polar solvents using Soxhlet type extractor. The total phenolic content of the extracts was determined by Folin-Ciocalteu method. Ethyl acetate extract of Rio Red and Sour orange was found to contain maximum phenolics. The dried fractions were screened for their antioxidant activity potential using in vitro model systems such as 1,1-diphenyl-2-picryl hydrazyl (DPPH), phosphomolybdenum method and nitroblue tetrazolium (NBT) reduction at different concentrations. The methanol:water (80:20) fraction of Rio Red showed the highest radical scavenging activity 42.5%, 77.8% and 92.1% at 250, 500 and 1000 ppm, respectively, while methanol:water (80:20) fraction of Sour orange showed the lowest radical scavenging activity at all the tested concentrations. All citrus fractions showed good antioxidant capacity by the formation of phosphomolybdenum complex at 200 ppm. In addition, superoxide radical scavenging activity was assayed using non-enzymatic (NADH/phenaxine methosulfate) superoxide generating system. All the extracts showed variable superoxide radical scavenging activity. Moreover, methanol:water (80:20) extract of Rio Red and methanol extract of Sour orange exhibited marked reducing power in potassium ferricyanide reduction method. The data obtained using above in vitro models clearly establish the antioxidant potential of citrus fruit extracts. However, comprehensive studies need to be conducted to ascertain the in vivo bioavailability, safety and efficacy of such extracts in experimental animals. To the best of our knowledge, this is the first report on antioxidant activity of different polar extracts from Rio Red and Sour oranges.     

Effects of Melia azedarach, (Meliaceae) fruit extracts on the leafminer Liriomyza huidobrensis, (Diptera, Agromyzidae): Assessment in laboratory and field experiments

Control of the widely distributed pest Liriomyza huidobrensis, is complicated due to the protected habit of the leafmining larvae, and their resistance to insecticides. The effects of Melia azedarach, (Meliaceae) fruit extracts against adults and larvae of L. huidobrensis, were investigated. In the laboratory, leaves of Cucurbita, sp. infested with first and third instar larvae were treated with different extract solutions. Larval and pupal survival, as well as wing‐spread of adults, were ssessed. Female adult behaviour towards the extract was also analysed in terms of number of feeding punctures and number of offspring left on treated/untreated leaves. In the field, an infested Vicia faba, crop was sprayed four times at weekly intervals with plant extract, water, and a blank solution. The number of adult leafminers and parasitoids emerging from sampled leaves from each treatment were compared. The laboratory tests showed translaminar action of the extracts, which negatively affected leafminer pupal survival, while body size was not affected. The extracts also deterred feeding by adult females and may also have caused reduction in oviposition rates. All solutions and concentrations tested had similar effects. In the field, extract effects were consistent with those from laboratory trials, number of pupae and pupal survival being lower on treated plants. Percentage parasitism was not affected by plant extract treatment, suggesting a selective activity.     

Insecticidal and insect growth regulatory activities of secondary metabolites from entomopathogenic fungi,Lecanicillium attenuatum

Insect growth regulators (IGRs) are effective alternatives to chemical insecticides because of their specificity and low environmental toxicity. Entomopathogenic fungi are an important natural pathogen of insects and have been developed as biological control agents. They produce a wide range of secondary metabolites such as antibiotics, pesticides, growth-promoting or inhibiting compounds and insect attracting agents. In this study, to explore novel IGR substances from entomopathogenic fungi, culture extracts of 189 entomopathogenic fungi isolated from Korean soil samples were investigated for their juvenile hormone (JH)-based IGR activities. Whereas none of the culture extracts exhibited JH agonist (JHA) activity, 14 extracts showed high levels of JH antagonist (JHAN) activity. Among them, culture extract of JEF-145 strain, which was identified as Lecanicillium attenuatum, showed the highest insecticidal against Aedes albopictus and Plutella xylostella. At liquid culture condition, JHAN activity was observed in culture soup rather than mycelial cake, indicating that substances with JHAN activity are released from the JEF-145 strain during culture. Furthermore, while extract from solid cultured JEF-145 strain showed insecticidal activities against both A. albopictus and P. xylostella, that from liquid cultured fungi showed insecticidal activity only against A. albopictus, indicating that L. attenuatum JEF-145 strain produces different kinds of secondary metabolites with JHAN activity depending on culture conditions. These results suggested that JHAN substances derived from entomopathogenic fungi could be usefully exploited to develop novel eco-friendly IGR insecticides.     

Efficacy of Melia azedarach L. extract on the malarial vector Anopheles stephensi Liston (Diptera: Culicidae)

Methanolic extracts of leaves and seeds from the chinaberry tree, Melia azedarach L. (Meliaceae) was tested against mature and immature mosquito vector Anopheles stephensi Liston (Diptera) under laboratory condition. The extract showed strong larvicidal, pupicidal, adulticidal, antiovipositional activity, repellency and biting deterency. The M. azedarach seed and leaf extracts were used to determine their effect on A. stephensi adults and their corresponding oviposition and consequent adult emergence in comparison with the control. The seed extracts showed high bioactivity at all doses, while the leaf extracts proved to be active, only in the higher dose. Results obtained from the laboratory experiment showed that the seed extracts suppressed the pupal and adult activity of A. stephensi even at low dose. In general, first and second instar larvae were more susceptible to both leaves and seed extracts. Clear dose-response relationships were established with the highest dose of 2% plant extract evoking 96% mortality. Entire development of A. stephensi was inhibited by M. azedarach treatment. Less expensive (less than 0.50 US dollars per 1 kg seed), naturally accruing bio-pesticide could be an alternative for chemical pesticides.     

Flavonoid composition of fruit tissues of citrus species

An HPLC analysis was performed on the concentrations of flavonoids in 42 species and cultivars of the Citrus genus and those of two Fortunella and one Poncirus species according to the classification system established by Tanaka. The composition of 8 flavanones and 9 flavone/ols for these species was determined in the albedo, flavedo, segment epidermis and juice vesicle tissues, and those in the fruit and peel tissues were calculated from the composition data of the tissues. A principal component analysis showed that such neohesperidosyl flavonoids as neoeriocitrin, naringin, neohesperidin, and rhoifolin had large factor loading values in the first principal component for each tissue. The flavonoid composition of citrus fruits was approximately the same within each section of Tanaka's system, except for the species in the Aurantium section and those with a peculiar flavonoid composition such as Bergamot (C. bergamia), Marsh grapefruit (C. paradisi), Sour orange (C. aurantium), and Shunkokan (C. shunkokan). The Aurantium section included both naringin-rich and hesperidin-rich species.     

Insect growth regulatory effects of some extracts and sterols from Myrtillocactus geometrizans (Cactaceae) against Spodoptera frugiperda and Tenebrio molitor

A methanol extract from the roots and aerial parts of Myrtillocactus geometrizans (Cactaceae) yielded peniocerol 1, macdougallin 2, and chichipegenin 3. The natural products 1, 2 their mixtures, MeOH and CH(2)Cl(2) extracts showed insecticidal and insect growth regulatory activity against fall armyworm [Spodoptera frugiperda J. E. Smith (Lepidoptera: Noctuidae)], an important insect pest of corn, and [Tenebrio molitor (Coleoptera)], a pest of stored grains in Mexico. The most active compounds were 1, 2, and a mixture (M(2)) of 1 and 2 (6:4). All these extracts, compounds and the mixture had insect growth regulating (IGR) activity between 5.0 and 50.0 ppm and insecticidal effects between 50 and 300 ppm in diets. The extracts were insecticidal to larvae, with lethal doses between 100 and 200 ppm. These compounds appear to have selective effects on the pre-emergence metabolism of Coleoptera, because in all treatments of the larvae of T. molitor, pupation were shortened and this process show precociousness in relation to controls. In contrast to S. frugiperda larvae, onset of pupation was noticeably delayed. Emergence in both cases was drastically diminished. In both pupae and in the few adults that were able to emerge, many deformations were observed. The results of these assays indicated that the compounds were more active than other known natural insect growth inhibitors such as gedunin and methanol extracts of Cedrela salvadorensis and Yucca periculosa. Peniocerol, macdougallin and chichipegenin, as well as mixtures of these substances, may be useful as natural insecticidal agents.     

Phytogenic compounds from avocado (Persea americana L.) extracts; antioxidant activity,amylase inhibitory activity,therapeutic potential of type 2 diabetes

Diabetes is a worldwide public health disease. Currently, the most effective way to treat diabetes is to mitigate postprandial hyperglycemia by inhibiting carbohydrate hydrolysis enzymes in the digestive system. Plant extracts are rich in bioactive compounds, which can be used in diabetes treatment. This study aims to evaluate the polyphenols content in ethanolic extracts of avocado fruit and leaves (Persea americana Mill.). Additionally, their antioxidant activity using DPPH, while the inhibition ability of α-amylase was examined by reacting different amounts of the extracts with α-amylase compared to acarbose as standard inhibitor. The active compounds were detected in the extracts by LC/MS. The obtained results showed that the leaf extract recorded a significant content of total phenolic compounds compared to the fruit extract (178.95 and 145.7 mg GAE /g dry weight, respectively). The total flavonoid values ??ranged from 32.5 to 70.08 mg QE/g dry weight of fruit and leaves extracts, respectively. Twenty-six phytogenic compounds were detected in leaf and fruit extract by LC/MS. These compounds belong to fatty acids, sterols, triterpenes, phenolic acids, and flavonoids. The antioxidant activity of the extracts is due to the exist of phytogenic compounds, i.e., polyphenols and flavonoids. The antioxidant activity increased in a concentration dependant manner. Avocado fruit extract (1000 µg/mL) scavenged 95% of DPP? while leaf extract rummaged 91.03% of free radicals compared with Vit C and BHT. Additionally, higher α-amylase inhibitory activity was observed in fruit extract than the leaf extract, where the fruit and leaf extract (1000 μg/ml) inhibited the enzyme by 92.13% and 88.95%, respectively. The obtained results showed that the ethanolic extracts of avocado could have a significant impact on human health due to their high content of polyphenols.     

杨梅果实提取物抗小鼠腹泻作用的研究

目的研究杨梅果实提取物的抗小鼠腹泻作用,包括预防和治疗作用。方法给小鼠腹腔注射甲型副伤寒沙门菌造成急性腹泻模型,经口灌胃杨梅果实提取物溶液,通过腹泻次数研究提取物对小鼠腹泻的预防作用;先灌胃杨梅果实提取物溶液再给小鼠注射菌液致泻,记录腹泻次数以研究提取物对小鼠腹泻的治疗作用。结果灌胃杨梅果实提取物的小鼠的腹泻次数均有明显的下降,且醇提物的作用效果要好于水提物。结论杨梅果实提取物对甲型副伤寒沙门菌引起的小鼠腹泻具有一定的预防和治疗作用。     

Insecticidal activity of five Chinese medicinal plants against Plutella xylostella L. larvae

Twenty-five extracts of five Chinese medicinal plant species were screened for insecticidal activity against the diamondback moth, Plutella xylostella L., larvae by a leaf dipping bioassay method. The ethyl acetate, ethanol, and acetone extracts of Veratrum nigrum L. root and rhizome, the ethyl acetate extract of Phytolacca americana L. root, and the petroleum ether extract of Pseudolarix kaempferi Gord. root bark were effective against P. xylostella. Among them, the ethyl acetate extract of V. nigrum showed the strongest insecticidal activity against the second and third instar larvae of P. xylostella, with LC₅₀ values of 225 and 335 ppm, respectively. The other extracts gave little or no insecticidal activity against P. xylostella. These results indicated that V. nigrum may be a promising naturally occurring agent for P. xylostella larval control, and that the organic solvent used for plant extraction can affect its insecticidal activity.     

Toxicity of Bacillus thuringiensis to laboratory populations of the olive fruit fly (Dacus oleae).

A survey of Bacillus thuringiensis recovered from the environments of olive groves in Greece was carried out. Of 80 soil samples, 24 were found to contain B. thuringiensis with parasporal crystal inclusions; these were tested for toxicity against the olive fruit fly (Dacus oleae). Mortality levels of larvae caused by the different isolates varied from 7 to 87%. Higher levels of mortality were observed if a mixture of relatively pure crystals and spores was used compared with the mortality resulting from either fraction alone. We were able to show that the toxicity of the most active isolate is likely to be specific for D. oleae.     

Toxicity of Bacillus thuringiensis to laboratory populations of the olive fruit fly (Dacus oleae).

A survey of Bacillus thuringiensis recovered from the environments of olive groves in Greece was carried out. Of 80 soil samples, 24 were found to contain B. thuringiensis with parasporal crystal inclusions; these were tested for toxicity against the olive fruit fly (Dacus oleae). Mortality levels of larvae caused by the different isolates varied from 7 to 87%. Higher levels of mortality were observed if a mixture of relatively pure crystals and spores was used compared with the mortality resulting from either fraction alone. We were able to show that the toxicity of the most active isolate is likely to be specific for D. oleae.     

嗜线虫致病杆菌HB310菌株杀虫蛋白的纯化及活性鉴定

嗜线虫致病杆菌Xenorhabdus nematophila HB310是从河北省土壤中筛选出的一株昆虫病原线虫体内分离纯化获得的共生菌,该菌的发酵液对多种昆虫有较高的杀虫活性。利用85％饱和度的硫酸铵盐析分别获得胞内蛋白提取物和上清液中胞外蛋白提取物,生测结果表明这两种蛋白提取物中都含有胃毒素和血腔毒素。通过制备型非变性凝胶电泳对蛋白提取物进行分离和纯化,得到了3种有杀虫活性的毒素蛋白（毒素Ⅰ、毒素Ⅱ和毒素Ⅲ）,胞内的毒素蛋白与分泌到胞外上清液中的毒素蛋白是同种蛋白。毒素Ⅰ和毒素Ⅱ对棉铃虫初孵幼虫有明显的胃毒活性,但没有血腔毒性;毒素Ⅲ对大蜡螟幼虫有很强的血腔毒性,LD₅₀为0.18 μg/头。SDS-PAGE图谱显示毒素Ⅰ和毒素Ⅱ是由多个多肽组成的复合蛋白,而毒素Ⅲ只分离出一条多肽。毒素Ⅱ在50℃处理10 min,其杀虫活性没有显著变化;70℃处理10 min对毒素Ⅲ杀虫活性没有显著影响。