Anti-adipogenic effects of extracts of Ficus deltoidea var. deltoidea and var. angustifolia on 3T3-L1 adipocytes简

Objective

This study examined the anti-adipogenic effects of extracts of Ficus deltoidea var. deltoidia and var. angustifolia, a natural slimming aid, on 3T3-L1 adipocytes.

Methods

Methanol and water extracts of leaves of the F. deltoidea varieties were analyzed to determine their total flavonoid content (TFC) and total phenolic content (TPC), respectively. The study was initiated by determining the maximum non-toxic dose (MNTD) of the methanol and water extracts for 3T3-L1 preadipocytes. Possible anti-adipogenic effects were then examined by treating 2-d post confluent 3T3-L1 preadipocytes with either methanol extract or water extract at MNTD and half MNTD (1/2MNTD), after which the preadipocytces were induced to form mature adipocytes. Visualisation and quantification of lipid content in mature adipocytes were carried out through oil red O staining and measurement of optical density (OD) at 520 nm, respectively.

Results

The TFCs of the methanol extracts were 1.36 and 1.97 g quercetin equivalents (QE)/100 g dry weight (DW), while the TPCs of the water extracts were 5.61 and 2.73 g gallic acid equivalents (GAE)/100 g DW for var. deltoidea and var. angustilofia, respectively. The MNTDs determined for methanol and water extracts were (300.0±28.3) and (225.0±21.2) μg/ml, respectively, for var. deltoidea, while much lower MNTDs [(60.0±2.0) μg/ml for methanol extracts and (8.0±1.0) μg/ml for water extracts] were recorded for var. angustifolia. Studies revealed that the methanol extracts of both varieties and the water extracts of var. angustifolia at either MNTD or 1/2MNTD significantly inhibited the maturation of preadipocytes. Conclusions: The inhibition of the formation of mature adipocytes indicated that leaf extracts of F. deltoidea could have potential anti-obesity effects.     

In vitro assay for the anti-brucella activity of medicinal plants against tetracycline-resistant Brucella melitensis

Brucellosis, a zoonosis caused by four species of brucella, has a high morbidity. Brucella melitensis is the main causative agent of brucellosis in both human and small ruminants. As an alternative to conventional antibiotics, medicinal plants are valuable resources for new agents against antibiotic-resistant strains. The aim of this study was to investigate the usage of native plants for brucellosis treatment. For this purpose, the anti-brucella activities of ethanolic and methanolic extracts of Salvia sclarea, Oliveria decumbens, Ferulago angulata, Vitex pseudo-negundo, Teucrium polium, Plantago ovata, Cordia myxa, and Crocus sativus were assessed. The activity against a resistant Br. melitensis strain was determined by disc diffusion method at various concentrations from 50–400 mg/ml. Antibiotic discs were also used as a control. Among the evaluated herbs, six plant (Salvia sclarea, Oliveria decumbens, Ferulago angulata, Vitex pseudo-negundo, Teucrium polium, and Crocus sativus) showed anti-brucella activity. Oliveria decumbens was chosen as the most effective plant for further studies. A tested isolate exhibited resistance to tetracycline, nafcillin, oxacillin, methicillin, and colistin. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values for Oliveria decumbens against resistant Br. melitensis were the same (5 mg/ml), and for gentamicin they were both 2 mg/ml. Time-kill kinetics for a methanolic extract of Oliveria decumbens was 7 h whereas for an ethanolic extract it was 28 h. Also, Oliveria decumbens extracts showed a synergistic effect in combination with doxycycline and tetracycline. In general, the similar values of MIC and MBC for Oliveria decumbens suggest that these extracts could act as bactericidal agents against Br. melitensis. In addition to Oliveria decumbens, Crocus sativus and Salvia sclarea also had good anti-brucella activity and these should be considered for further study.     

Green tea polyphenol,epigallocatechin-3-gallate,possesses the antiviral activity necessary to fight against the hepatitis B virus replication in vitro

Although several antiviral drugs and vaccines are available for use against hepatitis B virus (HBV), hepatitis caused by HBV remains a major public health problem worldwide, which has not yet been resolved, and new anti-HBV drugs are in great demand. The present study was performed to investigate the anti-HBV activity of epigallocatechin-3-gallate (EGCG), a natural-origin compound, in HepG2 2.2.15 cells. The antiviral activity of EGCG was examined by detecting the levels of HBsAg and HBeAg in the supernatant and extracellular HBV DNA. EGCG effectively suppressed the secretion of HBsAg and HBeAg from HepG2 2.2.15 cells in a dose- and time-dependent manner, and it showed stronger effects at the level of 0.11–0.44 μmol/ml (50–200 μg/ml) than lamivudine (3TC) at 0.87 μmol/ml (200 μg/ml). EGCG also suppressed the amount of extracellular HBV DNA. The data indicated that EGCG possessed anti-HBV activity and suggested the potential of EGCG as an effective anti-HBV agent with low toxicity.     

Evaluation of the dust and methanol extracts of Garcinia kolae for the control of Callosobruchus maculatus (F.) and Sitophilus zeamais (Mots)

Insecticidal effects of different doses of the dust and methanol extracts of Garcinia kolae on Collosobruchus maculatus and Sitophilus zeamais were tested. The dust had no significant effect on the two insects; none of them died even at 3 d after treatment. The methanol extracts, however, had rapid lethal effects on both C. maculatus and S. zeamais. The mortality of C. maculatus by the lowest concentration of methanol extracts ranged from 95%∼100% whereas in S. zeamais, the mortality ranged from 87.5%∼100% and 70%∼100% in concentrations of 1 g extract+3 ml methanol and 1 g extract+5 ml methanol, respectively, from 24 to 48 h. The least concentration of 1 g extract+15 ml methanol had no significant lethal effect on Sitophilus zeamais.      

Alternaria toxin-induced resistance against rose aphids and olfactory response of aphids to toxin-induced volatiles of rose plants

The search for active toxins for managing weeds or plant diseases is believed to be a promising avenue of investigation. However, the effects of Alternaria toxins on insects have just begun to be investigated. Bioactivities of toxins from four strains of Alternaria alternata on Rosa chinensis and rose aphid Macrosiphum rosivorum were tested in the present study. At a concentration of 50.0 μg/ml, the crude extract (toxin) of strain 7484 was found not to be harmful to rose plants with excised leaf-puncture method (P≥0.079), and rose plants showed enhanced resistance to rose aphids when this Alternaria toxin was sprayed on the plants (P≤0.001). However, this toxin caused no detrimental effects on aphids in insecticidal bioassay at a concentration of 10.0 to 160.0 μg/ml (P≥0.096). Therefore, the Alternaria toxin had significantly induced the resistance of rose plants against rose aphids, demonstrating that the resistance mechanism triggered by the Alternaria toxin in the rose plant may also be used by the plant to defend itself against insects. Further bioassays aimed to discover the olfactory responses of aphids to the toxin-induced volatiles of host plants. The aphids were significantly more attracted to both volatiles emitted and collected from control rose plants than to both volatiles emitted and collected from the toxin-treated rose plants (P≤0.014). This result showed that the toxin-induced resistance related to the volatile changes of host plants.     

Acaricidal activities of whole cell suspension,cell-free supernatant,and crude cell extract of <Emphasis Type="Italic">Xenorhabdus stokiae</Emphasis> against mushroom mite (<Emphasis Type="Italic">Luciaphorus</Emphasis> sp.)

Xenorhabdus bacterium has been used as a biological control agent against Luciaphorus sp., a mushroom mite endemic in Thailand. To develop an effective formulation of Xenorhabdus stokiae, treatments using different parts of X. stokiae isolate PB09 culture, including whole cell suspension, cell-free supernatant, and crude cell extract, were performed. The results show that different parts of X. stokiae isolate PB09 culture could induce variable effects on mite mortality and fecundity. Application with cell-free supernatant of X. stokiae culture resulted in both the highest mite mortality rate [(89.00±3.60)%] and the lowest mite fecundity [(41.33±23.69) eggs/gravid female]. Whole cell suspension of X. stokiae isolate PB09 culture was found to be slightly less effective than its cell-free supernatant, suggesting that X. stokiae was more likely to release its metabolites with acaricidal activities to the surrounding culture media. Crude cell extract of X. stokiae was not effective against mites. Cell-free supernatant of X. stokiae isolate PB09 was the most effective biological control agent and it could be conveniently used in future formulations instead of live bacteria.     

Production of conjugated linoleic acids by Lactobacillus plantarum strains isolated from naturally fermented Chinese pickles

Naturally fermented pickles harbour many lactic acid bacteria (LAB). Forty-three LAB strains with conjugated linoleic acid (CLA)-producing ability were isolated from three naturally fermented pickle brines. Of these isolates, lp15 identified as Lactobacillus plantarum by API 50 CHL system and full-length 16S rDNA sequence analysis exhibited the highest CLA-producing ability (26.1% conversion) at 48 h in de Man Rogosa Sharpe (MRS) broth in the presence of 100 μg/ml of linoleic acid (LA). Compared to other strains, L. plantarum strain lp15 showed the highest tolerance upon increased levels of LA in the medium, i.e., up to 600 μg/ml. This strain converted about 25% of LA into CLA isomers [predominantly cis-9, trans-11 CLA (9-CLA) and trans-10, cis-12 CLA (10-CLA)], of which 75% was 9-CLA. Interestingly, though the conversion rate of LA into CLA by lp15 remained stable between 100 to 600 μg/ml LA levels in the medium, it dropped sharply at 1000 μg/ml. Taken together, the lp15 strain displayed relatively high LA tolerance with higher conversion rate, which implies that this strain is a valuable candidate for enhancing the CLA content in food-sources like pickles.     

Effect of lipopolysaccharide on the hemocyte apoptosis of Eriocheir sinensis

In the present study, we investigated the possible toxicity mechanism of lipopolysaccharide (LPS) extracted from Gram-negative bacteria in Eriocheir sinensis hemocytes. Apoptotic hemocytes and reactive oxygen species (ROS) production induced by the LPS were monitored by the combination of flow cytometry and microscope observation. It was shown that LPS induced serious damage on the DNA and morphological changes in hemocytes, including cell shrinkage, fracture of nucleus membrane, margination, condensation and fragmentation of chromatin, and formation of apoptotic bodies indicating obvious hemocyte apoptosis. As compared with the control group, the apoptotic cell ratio increased to 30.61% and 39.01% after 1-h exposure and 57.72% and 75.01% after 2-h exposure to 1 and 10 μg/ml LPS, respectively (P<0.05). Significant outburst of ROS production was observed in LPS-treated hemocytes with approximately 176.6% of relative dichlorofluorescein mean fluorescence at 1-h exposure, followed by a drastic decline (P<0.05). These results indicated that LPS would induce oxidative stress on hemocytes from E. sinensis and cause ROS burst, DNA damage, and subsequently apoptosis. The process of ROS-mediated apoptosis might be one of the potential toxicity mechanisms of LPS on crustacean hemocytes.     

Cytotoxicity and effect of extraction methods on the chemical composition of essential oils of Moringa oleifera seeds

Renewed interest in natural materials as food flavors and preservatives has led to the search for suitable essential oils. Moringa oleifera seed essential oil was extracted by solvent-free microwave and hydrodistillation. This study assessed its chemical constituents. Cytotoxicity of the oils was investigated using hatchability and lethality tests on brine shrimps. A total of 16 and 26 compounds were isolated from the hydrodistillation extraction (HDE) and solvent-free microwave extraction (SME) oils, respectively, which accounted for 97.515% and 97.816% of total identifiable constituents, respectively. At 24 h when the most eggs had hatched, values of the SME (56.7%) and HDE (60.0%) oils were significantly different (P<0.05) from those of sea water (63.3%) and chloramphenicol (15.0%). Larva lethality was different significantly (P<0.05) between HDE and SME oils at different concentrations and incubation periods. The median lethal concentration (LC₅₀) of the oils was >1000 mg/ml recommended as an index for non-toxicity, which gives the oil advantage over some antioxidant, antimicrobial, therapeutic, and preservative chemicals.     

题目：遗传改造细菌基因组的策略：基因定点突变、基因失活和基因过表达

With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators.     

A comparative study of conventional and supercritical fluid extraction methods for the recovery of secondary metabolites from <Emphasis Type="Italic">Syzygium campanulatum</Emphasis> Korth

Syzygium campanulatum Korth is a plant, which is a rich source of secondary metabolites (especially flavanones, chalcone, and triterpenoids). In our present study, three conventional solvent extraction (CSE) techniques and supercritical fluid extraction (SFE) techniques were performed to achieve a maximum recovery of two flavanones, chalcone, and two triterpenoids from S. campanulatum leaves. Furthermore, a Box-Behnken design was constructed for the SFE technique using pressure, temperature, and particle size as independent variables, and yields of crude extract, individual and total secondary metabolites as the dependent variables. In the CSE procedure, twenty extracts were produced using ten different solvents and three techniques (maceration, soxhletion, and reflux). An enriched extract of five secondary metabolites was collected using n-hexane:methanol (1:1) soxhletion. Using food-grade ethanol as a modifier, the SFE methods produced a higher recovery (25.5%?84.9%) of selected secondary metabolites as compared to the CSE techniques (0.92%?66.00%).     

Antioxidant activity and protective effect of Clitoria ternatea flower extract on testicular damage induced by ketoconazole in rats

Background

Ketoconazole (KET), an antifungal drug, has adverse effects on the male reproductive system. Pre-treatments with antioxidant plant against testicular damage induced by KET are required. The flowers of Clitoria ternatea (CT) are proven to have hepatoprotective potential. However, the protective effect on KET-induced testicular damage has not been reported.

Objective

To investigate the protective effect of CT flower extracts with antioxidant activity on male reproductive parameters including sperm concentration, serum testosterone level, histopathology of the testis, and testicular tyrosine phosphorylation levels in rats induced with KET.

Methods

The antioxidant activity of CT flower extracts was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. Male rats were treated with CT flower extracts (10, 50, or 100 mg/kg BW) or distilled water via a gastric tube for 28 d (preventive period: Days 1–21) and induced by KET (100 mg/kg BW) via intraperitoneal injection for 7 d (induction period: Days 22–28). After the experiment, all animals were examined for the weights of the testis, epididymis plus vas deferens and seminal vesicle, serum testosterone levels, sperm concentration, histological structures and diameter of testis, and testicular tyrosine phosphorylation levels by immunoblotting.

Results

The CT flower extracts had capabilities for DPPH scavenging and high reducing power. At 100 mg/kg BW, the extract had no toxic effects on the male reproductive system. Significantly, in CT+KET groups, CT flower extracts (50 and 100 mg/kg BW) alleviated the reduction of reproductive organ weight parameters, testosterone levels, and sperm concentration. In addition, CT flower extracts gave protection from testicular damage in KET-induced rats. Moreover, in the CT100+KET group, CT flower extracts significantly enhanced the expression of a testicular 50-kDa tyrosine phosphorylated protein compared with that of other groups.

Conclusions

C. ternatea flower extracts possessing antioxidant activity are not harmful to the male reproductive system and can protect against testicular damage in KET-induced rats.     

茶树中咖啡酰辅酶A氧甲基转移酶的基因克隆及其催化活性研究(英文)

目的:从茶树中克隆一种可以催化表没食子儿茶素没食子酸酯(EGCG)生成甲基化EGCG的酶——咖啡酰辅酶A氧甲基转移酶(CCo AOMT),实现甲基化EGCG的酶学合成,为甲基化EGCG的进一步开发利用提供理论依据和技术指导。创新点:本研究首次从茶树中克隆了一条CCo AOMT基因组序列;分析了CCo AOMT基因在不同茶树品种和不同成熟度茶鲜叶中的基因表达规律;证明了CCo AOMT具有催化合成甲基化EGCG的生物活性。方法:采用聚合酶链式反应(PCR)和序列分析获得CCo AOMT的编码序列和基因组序列;采用高效液相色谱-四级杆-飞行时间串联质谱技术(HPLC-QTOF-MS)分析酶促反应生成的甲基化EGCG产物(图4);采用实时荧光定量PCR分析CCo AOMT基因的表达差异(图5)。结论:本研究从茶树中克隆了CCo AOMT基因的编码序列(738 bp)和基因组序列(2678 bp),明确了该基因具有4个内含子和5个外显子;揭示了CCo AOMT可以催化EGCG生成EGCG4"Me、EGCG3"Me和EGCG3’Me等多种甲基化产物;证明了CCo AOMT具有催化生成甲基化EGCG的活性;并发现该基因的表达量高低与茶鲜叶的成熟度呈正相关关系。     

Effects of organic solvents on two retinal pigment epithelial lipofuscin fluorophores,A2E and all-trans-retinal dimer

Gene and drug therapies are being developed to alleviate vision loss in patients with Stargardt’s disease and age-related macular degeneration (AMD). To evaluate the therapeutic effects of these treatments, organic solvents are routinely used to extract and quantify bisretinoid lipofuscin constituents, such as N-retinylidene-N-retinyl-ethanolamine (A2E) and all-trans-retinal dimer (ATR-dimer). By high-performance liquid chromatography (HPLC), we found that A2E and ATR-dimer were both altered by tetrahydrofuran (THF) and chloroform, but were stable in dimethyl sulfoxide (DMSO) or methanol (MeOH). In addition, cyclohexane and ethanol (EtOH) did not alter ATR-dimer, whereas an alteration of A2E occurred in EtOH. On the basis of these findings, we designed processes II–IV, generated by modifications of process I, a routine method to measure bisretinoid compounds in vivo. Extra amounts of either ATR-dimer or A2E in mouse eyecups were released by processes II–IV versus process I. Efforts to clarify the effects of organic solvents on lipofuscin pigments are important because such studies can guide the handling of these fluorophores in related experiments.     

Using chimeric piggyBac transposase to achieve directed interplasmid transposition in silkworm Bombyx mori and fruit fly Drosophila cells

The piggyBac transposon has been long used to integrate foreign DNA into insect genomes. However, undesirable transgene expression can result from random insertions into the genome. In this study, the efficiency of chimeric Gal4-piggyBac transposase in directing integration onto a DNA target plasmid was evaluated in cultured silkworm Bombyx mori Bm-12 and fruit fly Drosophila Schneider 2 (S2) cells. The Gal4-piggyBac transposase has a Gal4 DNA-binding domain (DBD), and the target plasmid has upstream activating sequences (UAS) to which the Gal4 DBD can bind with high affinity. The results indicate that, in the Bm-12 and S2 cells, transpositional activity of Gal4-piggyBac transposase was increased by 4.0 and 7.5 times, respectively, compared to controls, where Gal4-UAS interaction was absent. Moreover, the Gal4-piggyBac transposase had the ability of directing piggyBac element integration to certain sites of the target plasmid, although the target-directing specificity was not as high as expected. The chimeric piggyBac transposase has the potential for use in site-directed transgenesis and gene function research in B. mori.     

Anethum graveolens Linn. (dill) extract enhances the mounting frequency and level of testicular tyrosine protein phosphorylation in rats

Objective:To investigate the effect of Anethum graveolens(AG) extracts on the mounting frequency,histology of testis and epididymis,and sperm physiology.Methods:Male rats induced by cold immobilization before treating with vehicle or AG extracts [50,150,and 450 mg/kg body weight(BW)] via gastric tube for consecutive 1,7,and 14 d were examined for mounting frequency,testicular phosphorylation level by immunoblotting,sperm concentration,sperm acrosome reaction,and histological structures of testis and epididymis,respectively.Results:AG(50 mg/kg BW) significantly increased the mounting frequency on Days 1 and 7 compared to the control group.Additionally,rat testis treated with 50 mg/kg BW AG showed high levels of phosphorylated proteins as compared with the control group.In histological analyses,AG extract did not affect the sperm concentration,acrosome reaction,and histological structures of testis and epididymis.Conclusions:AG extract enhances the aphrodisiac activity and is not harmful to sperm and male reproductive organs.     

Effects of the crude extract of Polygala tenuifolia Willd on human sperm in vitro

The aim of the present study is to analyze sperm membrane changes and the spermicidal effect in treatment with the crude extract from Polygala tenuifolia Willd (PTW) in vitro. The root of PTW was extracted in distilled water. Normal human spermatozoa were used to assess the spermicidal activity (Sander-Cramer assay) of the extract from the PTW root. The hypo-osmotic swelling (HOS) test and the eosin Y (EY) staining were used to detect the integrity of sperm membrane and vitality. The sperm chromatin dispersion (SCD) test was performed to determine sperm DNA integrity. N-9 was used as a reference standard and semen added to physiological saline was used as the control. Semen samples were donated by 42 healthy fertile men. The crude extract from the root of PTW could immobilize and kill 100% spermatozoa within 20 s in vitro at the concentrations of 20.0 and 10.0 mg/ml; at the concentration of 5.0 mg/ml, spermatozoa were immobilized in (39.5±3.2) s. In the groups of the crude extract from the root of PTW and N-9 solution, the rate of the normal HOS (tails swollen) and the white head (unstained) was 0%, and the rate of the abnormal HOS (tails unswollen) and red head (stained) was 100%. Sperm DNA fragmentation showed no change in exposure to the crude extract from the root of PTW and N-9 solution. The sperm revival test did not show any spermatozoa that recovered their motilities. The rapid spermicidal activity of the crude extract from the root of PTW in vitro may occur by the disruption of the sperm membrane integrity.