Evaluation of phenolics and sugars as inducers of quercetinase activity in Penicillium olsonii

Quercetinase is produced by various filamentous fungi when grown on rutin as sole carbon and energy source. We investigated on the effect of 10 phenolics and two sugars, structurally related to substrates and products of the rutin catabolic pathway, on the induction of a quercetinase activity in Penicillium olsonii. Neither the sugars (glucose and rhamnose, two constituents of rutin), nor phenolics such as protocatechuic acid, salicylic acid, 4-hydroxy-benzoic acid and phloroglucinol were inducers. Rutin (maximum activity 150 nmol/min/mL after 5 days), quercetin (70 nmol/min/mL, 3 days), phloroglucinol carboxylic acid (60 nmol/min/mL, 3 days), 2-protocatechuoylphloroglucinolcarboxylic acid (50 nmol/min/mL, 5 days), 2,6-dihydroxy-carboxylic acid (90 nmol/min/mL, 7 days) and 2,4-dihydroxy-carboxylic acid (30 nmol/min/mL, 7 days) were demonstrated to be quercetinase inducers. We propose that rutin, quercetin and 2-protocatechuoyl-phloroglucinol carboxylic acid, the product of the reaction catalysed by quercetinase, act as inducers after their catabolic transformation in phloroglucinol carboxylic acid.     

加压毛细管电色谱法测定复方芦丁片中芦丁和维生素C的含量

目的:采用加压毛细管电色谱法测定复方芦丁片中芦丁与维生素C的含量。方法:采用Trisep~(TM)-2100加压毛细管电色谱仪,以C18毛细管色谱柱(300 mm×100μm i.d.,1.8μm)为固定相,流动相为甲醇-乙腈-0.4%磷酸(18:70:12),用三乙胺调节pH值至3.0,总流速为0.05 mL·min~(-1),检测波长为254 nm。结果:芦丁的线性范围为2.0~20μg·mL~(-1)(r~2=0.999);平均回收率为99.4%,RSD为1.2%(n=9);维生素C的线性范围为2.0~50μg·mL~(-1)(r~2=0.999);平均回收率为99.3%,RSD为1.4%(n=9)。另外,本方法与原国标方法进行了比较,结果基本一致。结论:加压毛细管电色谱法可用于复方芦丁片有效成分含量的测定,该方法简单、快速、准确,为复方芦丁片质量控制提供了新检测技术。     

Simultaneous effects of night-time temperature and an allelochemical on performance of an insect herbivore

One effect of global warming may be an increase in night-time temperatures with daytime temperatures remaining largely unchanged. We examined this potential effect of global warming on the performance of tobacco hornworm larvae, Manduca sexta (Sphingidae), by manipulating night-time temperature and dietary rutin levels simultaneously under a 12 light:12 dark photoregime. All four thermal regimes (26:14, 26:18, 26:22, and 26:26° C) had a daytime temperature of 26° C, with the night-time temperature increased from 14 to 26° C by increments of 4° C. Dietary rutin levels (0, 10 and 20 moles g^–1 fresh weight of diet) reflected those occurring naturally in the leaves of tomato, a preferred host plant of M. sexta. With low night-time temperatures (14 and 18° C), rutin had a negative linear effect on developmental rate, relative growth rate and relative consumption rate of the caterpillars. However, at a night-time temperature of 22° C, rutin had a negative non-linear effect. At a night-time temperature of 26° C, rutin had a negative linear impact but less so than at the other nightime temperatures. Likewise, the negative effect of rutin on molting duration was mitigated as night-time temperature increased. Final larval weight decreased linearly with increased dietary rutin concentrations. Total amount of food ingested was not affected by either rutin or thermal regime. As expected, the caterpillars developed faster under an alternating 26:14° C regime than a constant 20° C regime (the average temperature for the alternating regime), but the effect of rutin depended on the thermal regime. Switching daytime and night-time temperatures had no statistically significant effect on caterpillar performance. Overall, the effect of rutin on rates of larval performance was greater at some levels of warmer nights but damped at another level. These results indicate that the potential effect of warmer nights on insect performance is not a simple function of temperature because there can be interactions between night-time temperature and dietary allelochemicals.     

Inhibitory Effect on Lipid Absorption and Variability of Chemical Constituents from Capparis sicula subsp. sicula and Capparis orientalis

In continuation of our research program on Mediterranean dietary plants, a bioassay‐guided fractionation of extracts from several accessions of Capparis sicula subsp. sicula and Capparis orientalis aerial parts was carried out. Antilipidemic activity of samples was assayed using inhibition of pancreatic lipase. To study the metabolic variability in Capparis species, HPTLC analyses were performed in order to characterize the species through the detection, isolation, and quantitative evaluation of rutin taken as significant chemical marker. The best activity was exerted by C. orientalis accession no. C10 and C. sicula subsp. sicula accession no. C6. The bioactivity evaluation of specific chemical markers, rutin and glucocapparin, led to the identification of a potent antilipidemic compound rutin. The HPTLC analysis showed large variation among the different analyzed samples with respect to rutin concentration. The chemical investigation showed a different composition between the species and between the collection zones. The variations showed by the studied accessions of caper could be attributed to exogenous factors. Capparis species contained predominantly quercetin rutinoside (rutin), accompanied by other constituents such as the glucosinolate glucocapparin. These rutin‐rich extracts exhibited pronounced dose‐dependent enzyme inhibitory activities toward pancreatic lipase.     

Isolation and Indentification of Flavonoids from Aerial Part of Bupleurum tenue Buch-Ham-ex D. Don

The present paper deals with the flavonoid components of the aerial part of B. tenue Buch.-Ham. ex D. Don. Four flavonoid components were obtained by polyamide column chromatography method. On the basis of spectroscopic analysis, preparation of derivatives, acid hydrolysis and physicochemical constants, they were identified as rutin, narcissin, kaempferol and quercetin, respectively, which are not reported in this plant.     

Freeze-drying duration influences the amino acid and rutin content in honeybee-collected chestnut pollen

Honeybee-collected pollen is gaining attention as functional food for human consumption, due to antiproliferative, antiallergic, antibiotic, antidiarrheic and antioxidant activities. Among the different bioactive compounds, flavonoids from bee-collected pollen are currently recognised as powerful antioxidant and antiradical molecules. Traditional conservation methods influence pollen organoleptic properties as well as the contents of nutrients and nutraceutical compounds. Here, freeze-drying (FD) was proposed as a novel conservation method, estimating its adequacy as drying process by the evaluation of changes in free and total amino acids and proline as well as in their ratios. Honeybee-collected chestnut pollen was taken into consideration and the level of rutin, as main flavonoid, was considered as marker compound highlighting the maintenance of pollen nutraceutical properties. Results showed that FD influenced rutin level, depending on the FD duration. However, the free proline to free amino acid ratio was always below 80%, and the free amino acid to total amino acid ratio remained unaltered indicating the adequacy of the FD treatment, which did not affect the nutritional value of chestnut pollen. Overall, this study shed light on the nutraceutical profile of honeybee-collected chestnut pollen, highlighting the promising potential of FD as a novel method to treat pollen for human consumption.     

A Rapid Purification Procedure for Camel Kidney Glutathione S-Transferase

Glutathione S-transferase was isolated from supernatant of camel kidney homogenate centrifugation at 37, 000 xg by glutathione agarose affinity chromatography. The enzyme preparation has a specific activity of 44 μ;mol/min/mg protein and recovery was more than 85% of the enzyme activity in the crude extract. Glutathione agarose affinity chromatography resulted in a purification factor of about 49 and chromatofocusing resolved the purified enzyme into two major isoenzymes (pI 8.7 and 7.9) and two minor isoenzymes (pI 8.3 and 6.9). The homogeneity of the purified enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration on Sephadex G-100.

The different isoenzymes were composed of a binary combination of two subunits with molecular weight of 29, 000 D and 26, 000 D to give a native molecular weight of 55, 000 D.

The substrate specificities of the major camel kidney glutathione S-transferase isoenzymes were determined towards a range of substrates. l-chloro-2, 4-dinltrobenzene was the preferred substrate for all the isoenzymes. Isoenzyme III (pI 7.9) had higher specific activity for ethacrynic acid and isoenzyme II (pI 8.3) was the only isoenzyme that exhibited peroxidase activity. Ouchterlony double-diffusion analysis with rabbit antiserum prepared against the camel kidney enzyme showed fusion of precipitation lines with the enzymes from camel brain, liver and lung and no cross reactivity was observed with enzymes from kidneys of sheep, cow, rat, rabbit and mouse.

Different storage conditions have been found to affect the enzyme activity and the loss in activity was marked at room temperature and upon repeated freezing and thawing.     

Elucidation of rutin’s role in inducing caspase-dependent apoptosis via HPV-E6 and E7 down-regulation in cervical cancer HeLa cells

Over the recent few years rutin has gained wider attention in exhibiting inhibitory potential against several oncotargets for inducing apoptotic and antiproliferative activity in several human cancer cells. Several deregulated signaling pathways are implicated in cancer pathogenesis. Therefore we have inclined our research towards exploring the anticancerous efficacy of a very potent phytocompound for modulating the incontinent expression of these two crucial E6 and E7 oncogenes. Further, inhibitory efficacy of rutin against human papillomavirus (HPV)-E6 and E7 oncoproteins in cervical cancer has not been elucidated yet. This research addresses the growth inhibitory efficacy of rutin against E6 and E7 oncoproteins in HeLa cells, which is known to inactivate several tumor suppressor proteins such as p53 and pRB. Rutin treatment exhibited reduced cell viability with increased cell accumulation in G₀/G₁ phase of cell cycle in HeLa cell lines. Additionally, rutin treatment has also led to down-regulation of E6 and E7 expression associated with an increased expression of p53 and pRB levels. This has further resulted in enhanced Bax expression and decreased Bcl-2 expression releasing cytochrome c into cytosol followed by caspase cascade activation with cleavage of caspase-3, caspase-8 and caspase-9. Further, in silico studies have also supported our in vitro findings by exhibiting significant binding energy against selected target oncoproteins. Therefore, our research findings might recommend rutin as one of the potent drug candidate in cervical cancer management via targeting two crucial oncoproteins associated with viral progression.     

芦丁通过激活NRF2/HO-1通路抑制Ang II诱导的血管平滑肌细胞表型转化和炎症反应

摘要 目的：探讨芦丁（Rutin）对血管紧张素II（Angiotensin II，Ang II）诱导的小鼠主动脉血管平滑肌细胞表型转化和炎症反应的影响及机制。方法：采用Ang II处理小鼠主动脉平滑肌细胞构建表型转化和炎症反应模型。将对数生长期的小鼠主动脉血管平滑肌细胞分为以下4组：正常对照组（Control组），Rutin组（100 μM），Ang II组（1μM），Rutin+ Ang II组（100 μM，1 μM）。采用细胞增殖实验（Cell Counting Kit-8，CCK8）检测芦丁对小鼠主动脉平滑肌细胞活力的影响，采用蛋白免疫印迹实验检测α平滑肌肌动蛋白（α-Smooth muscle actin，α-SMA）、平滑肌蛋白22α（Smooth muscle protein 22-alpha，SM22α）、骨桥蛋白（Osteopontin，OPN）、基质金属蛋白酶2（Matrix metalloproteinase 2，MMP2）、基质金属蛋白酶9（Matrix metalloproteinase 9，MMP9）、白细胞介素6（Interleukin 6，IL6）、肿瘤坏死因子-α（Tumor necrosis factor-alpha，TNF-α）、核因子E2相关因子2（nuclear factor erythroid 2-related factor 2，Nrf2）、血红素氧合酶-1（Heme oxygenase-1，HO-1）、核因子活化B细胞κ轻链增强子（nuclear factor kappa-light-chain-enhancer of activated B cells，NF-κB）的蛋白表达和磷酸化水平，采用双抗体一步夹心法酶联免疫吸附试验（Enzyme-linked immunosorbent assay，ELISA）检测细胞上清中TNF-α和IL6细胞因子水平，采用荧光显微镜和流式细胞术检测小鼠主动脉血管平滑肌细胞活性氧水平。结果：与对照组相比，Ang II组收缩型标志物α-SMA和SM22α表达水平降低、合成型标志物OPN表达水平升高，炎症相关因子MMP2、MMP9、IL6和TNF-α表达水平升高，NRF2和HO-1表达水平降低，NRF2及NF-κB的磷酸化增加。此外，相较于Ang II组，Rutin+ Ang II组收缩型标志物α-SMA和SM22α表达水平升高、合成型标志物OPN表达水平降低，炎症相关因子MMP2、MMP9、IL6和TNF-α表达水平降低，NRF2和HO-1表达水平升高，NRF2及NF-κB的磷酸化水平降低。结论：芦丁可以抑制Ang II诱导的小鼠主动脉血管平滑肌细胞表型转化和炎症反应，可能与其激活NRF2/HO-1通路和抑制活性氧的产生有关。