Yeast-extract improved biosynthesis of lignans and neolignans in cell suspension cultures of <Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.

Lignans and neolignans are important biologically active ingredients (BAIs) biosynthesized by Linum usitatissimum. These BAIs have multi-dimensional effects against cancer, diabetes and cardio vascular diseases. In this study, yeast extract (YE) was employed as an elicitor to evaluate its effects on dynamics of biomass, BAIs and antioxidant activities in L. usitatissimum cell cultures. During preliminary experiments, flax cultures were grown on different concentrations of YE (0–1000 mg/L), and 200 mg/L YE was found to be optimum to enhance several biochemical parameters in these cell cultures. A two-fold increase in fresh (FW) and dry weight (DW) over the control was observed in cultures grown on MS medium supplemented with 200 mg/L YE. Similarly, total phenolic (TPC; 16 mg/g DW) and flavonoids content (TFC; 5.1 mg/g DW) were also positively affected by YE (200 mg/L). Stimulatory effects of YE on biosynthesis of lignans and neolignans was also noted. Thus, 200 mg/L of YE enhanced biosynthesis of secoisolariciresinol diglucoside (SDG; 3.36-fold or 10.1 mg/g DW), lariciresinol diglucoside (LDG; 1.3-fold or 11.0 mg/g DW) and dehydrodiconiferyl alcohol glucoside (DCG; 4.26-fold or 21.3 mg/g DW) in L. usitatissimum cell cultures with respect to controls. This elicitation strategy could be scaled up for production of commercially feasible levels of these precious metabolites by cell cultures of Linum.     

Role of bifidobacteria in the activation of the lignan secoisolariciresinol diglucoside

Lignans are ubiquitous plant polyphenols, which have relevant health properties being the major phytoestrogens occurring in Western diets. Secoisolariciresinol (SECO) is the major dietary lignan mostly found in plants as secoisolariciresinol diglucoside (SDG). To exert biological activity, SDG requires being deglycosylated to SECO and transformed to enterodiol (ED) and enterolactone (EL) by the intestinal microbes. The involvement of bifidobacteria in the transformation of lignans glucosides has been investigated for the first time in this study. Twenty-eight strains were assayed for SDG and SECO activation. They all failed to transform SECO into reduced metabolites, excluding any role in ED and EL production. Ten Bifidobacterium cultures partially hydrolyzed SDG, giving both SECO and the monoglucoside with yields < 25%. When the cell-free extracts were assayed in SDG transformation, seven additional strains were active in the hydrolysis. Cellobiose induced β-glucosidase activity and caused the enhancement of both the rate of SDG hydrolysis and the final yield of SECO only in the strains capable of SDG bioconversion. The highest SDG conversion to SECO was achieved by Bifidobacterium pseudocatenulatum WC 401, which exhibited 75% yield in cellobiose-based medium after 48 h. These results indicate that SDG hydrolysis is not a common feature in Bifidobacterium genus, but selected probiotic strains can be combined to β-glucoside-based prebiotics to enhance the release of SECO, thus improving its bioavailability for absorption by colonic mucosa and/or the biotransformation to ED and EL by other intestinal microorganisms.     

Salicylic acid‐enhanced biosynthesis of pharmacologically important lignans and neo lignans in cell suspension culture of Linum ussitatsimum L

Linum usitatsimum L. (flax) is a perennial herb with magnitude of medicinal and commercial applications. In the present study, we investigated the effects of salicylic acid (SA) on biosynthesis of lignans (secoisolariciresinol diglucoside (SDG) and lariciresinol diglucoside (LDG)) and neolignans (dehydrodiconiferyl alcohol glucoside (DCG) and guaiacylglycerol‐β‐coniferyl alcohol ether glucoside (GGCG)) in cell cultures of flax. Moderate concentration of SA (50 μM) enhanced biomass accumulation (10.98 g/L dry weight (DW)), total phenolic content (37.81 mg/g DW), and antioxidant potential (87.23%) to two‐fold than their respective controls after 72 h of exposure. However, higher levels of total flavonoid content (5.32 mg/g DW) were noted after 48 h of exposure to 50 μM of SA. HPLC analyses revealed that 50 μM SA, significantly enhanced biosynthesis of SDG (7.95 mg/g DW), LDG (7.52 mg/g DW), DCG (54.90 mg/g DW), and GGCG (16.78 mg/g DW), which was almost 2.7, 1.8, 3.88, and 3.98 fold higher than their respective controls after 72 h of exposure time, respectively. These results indicated that moderate concentrations of SA had significant effects on biosynthesis and productivity of lignans and neolignans in cell culture of L. usitatissimum.     

Cloning,expression, and characterization of the β-glucosidase hydrolyzing secoisolariciresinol diglucoside to secoisolariciresinol from Bacteroides uniformis ZL1

Previously, from the human intestinal flora we isolated the bacterial strain Bacteroides uniformis ZL1, which could convert secoisolariciresinol diglucoside (SDG) to its aglycone secoisolariciresinol (SECO) in vivo. In this study, 24 putative β-glucosidase genes were screened from the genome of B. uniformis ATCC 8492, which were used as templates to design PCR primers for the target genes in B. uniformis ZL1. Fifteen genes (bgl1–bgl15) were amplified from strain ZL1, and among them we identified bgl8 as the gene encoding the SDG-hydrolyzing β-glucosidase. We sequenced the bgl8 gene, cloned it into the expression vector and then transformed Escherichia coli to construct the recombinant bacteria that could synthesize the target β-glucosidase (BuBGL8). We purified and characterized BuBGL8, which showed maximal activity and stability under the culture conditions of pH 6.0 and 30 °C. SDG (2.0 mg/ml) was converted to SECO by both the purified BuBGL8 (0.035 mg/ml) and crude enzyme extract (0.23 mg crude protein/ml) with the efficiency of more than 90 % after 90 min at the reaction conditions. This is, to our knowledge, the first report of using recombinant bacteria to synthesize the SDG-hydrolyzing β-glucosidase, which could be used to produce SECO from SDG conveniently and highly efficiently.     

Structural determinants of plant lignans for the formation of enterolactone in vivo

The quantity of mammalian lignans enterolactone (ENL) and enterodiol (END) and of plant lignans secoisolariciresinol (SECO) and 7-hydroxymatairesinol (HMR) excreted in a 24-h rat urine sample was measured after a single p.o. dose of an equivalent quantity of secoisolariciresinol diglycoside (SDG), secoisolariciresinol (SECO), matairesinol (MR), 7-hydroxymatairesinol (HMR) and ENL. Plant lignans (SECO and HMR) were partially absorbed as such. The aglycone form of SECO was more efficiently converted into mammalian lignans END and ENL than the glycosylated form, SDG. Of plant lignans, MR produced the highest quantities of ENL: the quantity was over twofold compared with HMR or SDG. The majority of the animals, which had been given SECO, excreted higher quantities of END than ENL into urine, but ENL was the main lignan metabolite after SDG. The highest quantities of ENL in urine were measured after the administration of ENL as such. The (-)SECO isolated from Araucaria angustifolia was converted into (-)ENL only. The administration of (-)SDG, which was shown to produce (+)SECO, resulted in excretion of (+)ENL only and (-)HMR was converted into (-)ENL only. This confirmed that the absolute configurations at C8 and C8' are not changed during the microbial metabolism. Whether the biological effects are enantiomer-specific, remains to be resolved.     

Phylogeny of human intestinal bacteria that activate the dietary lignan secoisolariciresinol diglucoside

The human intestinal microbiota is essential for the conversion of the dietary lignan secoisolariciresinol diglucoside (SDG) via secoisolariciresinol (SECO) to the enterolignans enterodiol (ED) and enterolactone (EL). However, knowledge of the species that catalyse the underlying reactions is scant. Therefore, we focused our attention on the identification of intestinal bacteria involved in the conversion of SDG. Strains of Bacteroides distasonis, Bacteroides fragilis, Bacteroides ovatus and Clostridium cocleatum, as well as the newly isolated strain Clostridium sp. SDG-Mt85-3Db, deglycosylated SDG. Demethylation of SECO was catalysed by strains of Butyribacterium methylotrophicum, Eubacterium callanderi, Eubacterium limosum and Peptostreptococcus productus. Dehydroxylation of SECO was catalysed by strains of Clostridium scindens and Eggerthella lenta. Finally, the newly isolated strain ED-Mt61/PYG-s6 catalysed the dehydrogenation of ED to EL. The results indicate that the activation of SDG involves phylogenetically diverse bacteria, most of which are members of the dominant human intestinal microbiota.     

HPLC method with fluorescence detection for the quantitative determination of flaxseed lignans

We report a rapid and simple HPLC method with fluorescence detection for the quantification of the major flaxseed lignan, secoisolarisiresinol diglucoside (SDG) and its major metabolites. The method is specific for SDG, secoisolarisiresinol (SECO), enterodiol (ED) and entrolactone (EL) in rat serum. The assay procedure involves chromatographic separation using a Waters Symmetry C₁₈ reversed-phase column (4.6 mm × 150 mm, 5 μm) and mobile phase gradient conditions consisting of acetonitrile (0.1% formic acid) and water (0.1% formic acid). SDG extraction from serum requires the use of Centrifuge filters while SECO, ED and EL are extracted with diethyl ether. The organic layer is evaporated and reconstituted in 100 μL of mobile phase and 50 μL of reconstituted sample or filtrate is injected onto the column. Total run time is 25 min. Calibration curves are linear (r² ≥ 0.997) from 0.05 to 10 μg/mL for SDG and EL and 0.01–10 μg/mL for SECO and ED. Precision and accuracy are within USFDA specified limits. The stability of all lignans is established in auto-injector, bench-top, freeze–thaw and long-term stability at −80 °C for 30 days. The method's reasonable sensitivity and reliance on more widely available HPLC technology should allow for its straightforward application to pharmacokinetic evaluations of lignans in animal model systems such as the rat.     

Bacterial conversion of secoisolariciresinol and anhydrosecoisolariciresinol

Aims:  It has been investigated whether secoisolariciresinol (SECO) and anhydrosecoisolariciresinol (AHS), an acid degradation product of SECO, could be fermented in a similar way, and to a similar extent, by members of the intestinal microbiota.
Methods and Results:  AHS and SECO were demethylated by Peptostreptococcus productus , Eubacterium limosum and Clostridium methoxybenzovorans . These bacteria have been identified as members of the human intestinal flora or closely related species. Demethylated AHS and demethylated SECO were purified by preparative RP-HPLC, and subsequently subjected to fermentation with Eggerthella lenta , Clostridium scindens and Clostridium hiranonis . Eggerthella lenta efficiently dehydroxylated demethylated SECO to enterodiol, whereas the other bacteria showed no dehydroxylation activity.
Conclusions:  The conversion of the diol structure of SECO into the furan ring in AHS did not influence the demethylation capability of the tested bacteria. The results also showed that the extent of dehydroxylation of demethylated AHS was much lower than that of demethylated SECO.
Significance and Impact of the Study:  Plant lignans are converted into bioactive mammalian lignans by the human intestinal bacteria. This study showed that the modification of plant lignans resulted in the formation a new type of mammalian lignan.     

Transcriptomics analysis investigates sesquiterpenoids accumulation pattern in different tissues of <Emphasis Type="Italic">Atractylodes lancea</Emphasis> (Thunb.) DC. plantlet

Linum usitatissimum: L. is well-known for production of pharmacologically important secondary metabolites. Due to their tremendous beneficial effects on human health, these compounds are receiving greater attention throughout the World, especially in the treatment of various types of cancers. In present study, we have developed an efficient protocol for production of lignans like secoisolariciresinol diglucoside (SDG) and lariciresinol diglucoside (LDG) and neo-lignans like dehydrodiconiferyl alcohol glucoside (DCG) and guaiacylglycerol-β-coniferyl alcohol ether glucoside (GGCG) by exploiting in vitro callus cultures of Flax. These cultures were established from stem and leaf explants, inoculated on Murashige and Skoog (MS) media supplemented with various concentrations of α-naphthalene acetic acid (NAA), thidiazuron (TDZ) and 6-benzyl adenine (BA). Results revealed that the leaf-derived calli (1.0 mg/l NAA) accumulated highest levels of biomass (DW; 15.7 g/l) and antioxidant activity, while highest production of total phenolics (111.09 mg/l) and flavonoids (45.02 mg/l) were observed in stem-derived calli (1.0 mg/l NAA). The high-performance liquid chromatography (HPLC) analysis revealed that the stem-derived calli (1.0 mg/l NAA) accumulated optimum concentrations of SDG (2.7?±?0.021 mg/g DW), LDG (9.8?±?0.062 mg/g DW) and DCG (13.8?±?0.076 mg/g DW), while leaf-derived calli (1.0 mg/l NAA) showed optimum accumulation of GGCG (3.8?±?0.022 mg/g DW) as compared to all other treatments. These results provided definite evidence that the NAA differentially influence the production of lignans and neo-lignans in callus culture of Flax. This study opens new dimensions to devise strategies to enhance the production of these valuable metabolites.     

Investigation of in vitro and in vivo antioxidant potential of secoisolariciresinol diglucoside

The present study was designed to evaluate the in vitro and in vivo ameliorative antioxidant potential of secoisolariciresinol diglucoside (SDG). In vitro antioxidant activity of synthetic SDG was carried out using DPPH, reducing power potency, and DNA protection assays. Wistar albino rats weighing 180–220 g were used for in vivo studies and liver damage was induced in the experimental animals by a single intraperitoneal (I.P.) injection of CCl₄ (2 g/kg b.w.). Intoxicated animals were treated orally with synthetic SDG at (12.5 and 25 mg/kg b.w.) and Silymarin (25 mg/kg) for 14 consecutive days. The levels of catalase (CAT), superoxide dismutase (SOD), peroxidase (POX), and lipid peroxidase (LPO) were measured in liver and kidney homogenates. The synthetic SDG exerts high in vitro antioxidant potency as it could scavenge DPPH at a IC₅₀ value of 78.9 μg/ml and has dose-dependent reducing power potency and protected DNA at 0.5 mg/ml concentration. Oral administration of synthetic SDG at 12.5 and 25 mg/kg b.w. showed significant protection compared to Silymarin (25 mg/kg) and the activities of CAT, SOD, and POX were markedly increased (P < 0.05), whereas LPO significantly decreased (P < 0.001) in a dose-dependent manner in liver and kidney in both pre- and post-treatment groups when compared to toxin-treated group. The results of in vitro and in vivo investigations revealed that synthetic SDG at 25 mg/kg b.w. is associated with beneficial changes in hepatic enzyme activities and thereby plays a key role in the prevention of oxidative damage in immunologic system.     

Seasonal incidence of aphid,Amphorophora ampullata Bukton (Homoptera: Aphididae) on fern,Hypolepis polypodioides (Blume) Hook (Hypolepidaceae) from Western Himalaya

Seasonal incidence of aphid, Amphorophora ampullata on Hypolepis polypodioides was recorded throughout the year from November 2012 to November 2013 at weekly interval. Peak incidence of aphid was recorded during third week of November 2012 (25.94 ± 2.39 aphids/pinna), later the aphid population gradually decreased from December 2012 onwards and reached below threshold level during the last week of January (0.1 ± 0 aphids/pinna). The aphid population starts building up again from first week of February 2013 (1.6 ± 0.13 aphids/pinna) and attained its peak in the last week of August (32.17 ± 1.22 aphids/pinna) then decreased gradually in the first week of September (20.82 ± 4.70 aphids/pinna). Aphid densities again increased gradually from second week of September (21.62 ± 3.02 aphids/pinna) to November 2013 and reached maximum aphid densities during November (56.55 ± 4.34 aphids/pinna). Among weather parameters, aphid population showed significant positive correlation with relative humidity during morning hours.     

Spirit-based distillers’ grain as a promising raw material for succinic acid production

Spirit-based distillers’ grain (SDG) is the main by-product of the Chinese liquor industry, with an annual output of approx. 100 million tons. The economical potential of fermentative production of succinic acid from SDG was investigated using Actinobacillus succinogens. Use of pretreated SDG (PSDG) as the sole source of C and N yielded succinic acid at 35.5 g l^?1 with a yield of 19.7 % (g per 100 g PSDG) after 48 h in a 3 l stirred bioreactor. SDG is thus a promising feedstock for the economical production of succinic acid.     

Design,synthesis, and bioactivities of novel oxadiazole-substituted pyrazole oximes

A series of novel pyrazole oxime derivatives containing a substituted oxadiazole group were designed and synthesized. The bioassay results indicated that some title compounds displayed good acaricidal and insecticidal activities against Tetranychus cinnabarinus, Aphis medicaginis, Oriental armyworm, and Nilaparvata lugens. Especially, compounds 7a, 7b, and 7c had 80%, 90%, and 90% insecticidal activities against A. medicaginis at 20 μg/mL, respectively. Interestingly, some of the designed compounds displayed wonderful fungicidal activities in vivo against cucumber Pseudoperonospora cubensis. Furthermore, compounds 7a (EC₅₀ = 4.97 μg/mL) and 7h (EC₅₀ = 0.51 μg/mL) showed excellent fungicidal activity against P. cubensis comparable or better than that of the control Pyraclostrobin (EC₅₀ = 4.59 μg/mL).     

Purification and characterization of SDG-β-d-glucosidase hydrolyzing secoisolariciresinol diglucoside to secoisolariciresinol from Aspergillus oryzae

The SDG-β-d-glucosidase that hydrolyzes the glucopyranoside bond of secoisolariciresinol diglucoside (SDG) to release secoisolariciresinol (SECO) was isolated from Aspergillus oryzae 39 strain and the enzyme was purified and characterized. The enzyme was purified to one spot in SDS polyacrylamide gel electrophoresis, and its molecular weight was about 64.9 kDa. The optimum temperature of the SDG-β-d-glucosidase was 40 °C, and the optimum pH was 5.0. The SDG-β-d-glucosidase was stable at less than 65 °C, and pH 4.0–6.0. Ca²⁺, K⁺, Mg²⁺ and Na⁺ ions have no significant effect on enzyme activity, Zn²⁺ and Cu²⁺ ions have weakly effect on enzyme activity, but Fe³⁺ ion inhibits enzyme activity strongly. The K_m value of SDG-β-d-glucosidase was 0.14 mM for SDG.     

Relationships between soybean shoot nitrogen components and soybean aphid populations

Defining the relationships between soybean (Glycine max [L.] merr.) shoot nitrogen (N) components and soybean aphid (Aphis glycines Matsumura) populations will increase understanding of the biology of this important insect pest. In this 2-year field study, caged soybean plants were infested with soybean aphids (initial infestation of 0, 10, 50, or 100 aphids plant^?1) at the fifth node developmental stage. Soybean aphid populations, soybean shoot dry weight, and shoot concentrations of nitrate-N, ureide-N, and total N were measured starting at full bloom through full seed soybean development stages. Soybean aphid population as well as shoot concentration of ureide-N increased rapidly starting at full bloom, peaked at beginning seed, and dramatically decreased by full seed soybean reproductive stages. Regression analysis indicated significant relationships (P = 0.01; r = 0.71) between soybean aphid populations and shoot ureide-N concentration. Thus, soybean aphid population levels appear to coincide with shoot ureide-N concentrations in the soybean plant.     

Intestinal bacterial communities that produce active estrogen-like compounds enterodiol and enterolactone in humans

Lignans are dietary diphenolic compounds which require activation by intestinal bacteria to exert possible beneficial health effects. The intestinal ecosystem plays a crucial role in lignan metabolism, but the organisms involved are poorly described. To characterize the bacterial communities responsible for secoisolariciresinol (SECO) activation, i.e., the communities that produce the enterolignans enterodiol (ED) and enterolactone (EL), a study with 24 human subjects was undertaken. SECO activation was detected in all tested fecal samples. The intestinal bacteria involved in ED production were part of the dominant microbiota (6 x 10(8) CFU g(-1)), as revealed by most-probable-number enumerations. Conversely, organisms that catalyzed the formation of EL occurred at a mean concentration of approximately 3 x 10(5) CFU g(-1). Women tended to have higher concentrations of both ED- and EL-producing organisms than men. Significantly larger amounts of EL were produced by fecal dilutions from individuals with moderate to high concentrations of EL-producing bacteria. Two organisms able to demethylate and dehydroxylate SECO were isolated from human feces. Based on 16S rRNA gene sequence analyses, they were named Peptostreptococcus productus SECO-Mt75m3 and Eggerthella lenta SECO-Mt75m2. A new 16S rRNA-targeted oligonucleotide probe specific for P. productus and related species was designed and further used in fluorescent in situ hybridization experiments, along with five additional group-specific probes. Significantly higher proportions of P. productus and related species (P = 0.012), as well as bacteria belonging to the Atopobium group (P = 0.035), were typical of individuals with moderate to high concentrations of EL-producing communities.     

Determination the population trends of cereal aphids and associated parasitoids by yellow sticky traps with reference to aphid management on wheat

Population trends of cereal aphids and their associated parasitoids inhabiting wheat plantations were monitored by yellow sticky traps. The identified aphids exhibited one seasonal peak for each and were found to be active during the first half of March. Data revealed that, Rhopalosiphum padi L peak occurred one week later than both of Schizaphis graminum (Rondani) and Rhopalosiphum maidis (Fitch). The numbers of aphid complex was recorded more at 90 than at 30 and 60 cm height. Hymenopterous parasitoids activity is synchronised with aphid species, whereas they appeared in small numbers at early and late wheat season and showed their peak on March, with a positive correlation coefficient with aphid populations. The tested compounds (Karate, Biscaya, Match 5% EC, Tracer 24% SC and Neem Azal T/S) showed 100% reduction in aphid numbers after 24 h post application. However, the general reduction percentages indicated 99.31 > 98.32 > 97.97 > 97.06 > 95.66%, by using the abovementioned compounds, respectively.     

Histidine 352 (His352) and Tryptophan 355 (Trp355) Are Essential for Flax UGT74S1 Glucosylation Activity toward Secoisolariciresinol

Flax secoisolariciresinol diglucoside (SDG) lignan is a natural phytoestrogen for which a positive role in metabolic diseases is emerging. Until recently however, much less was known about SDG and its monoglucoside (SMG) biosynthesis. Lately, flax UGT74S1 was identified and characterized as an enzyme sequentially glucosylating secoisolariciresinol (SECO) into SMG and SDG when expressed in yeast. However, the amino acids critical for UGT74S1 glucosyltransferase activity were unknown. A 3D structural modeling and docking, site-directed mutagenesis of five amino acids in the plant secondary product glycosyltransferase (PSPG) motif, and enzyme assays were conducted. UGT74S1 appeared to be structurally similar to the Arabidopsis thaliana UGT72B1 model. The ligand docking predicted Ser³⁵⁷ and Trp³⁵⁵ as binding to the phosphate and hydroxyl groups of UDP-glucose, whereas Cys³³⁵, Gln³³⁷ and Trp³⁵⁵ were predicted to bind the 7-OH, 2-OCH₃ and 17-OCH₃ of SECO. Site-directed mutagenesis of Cys³³⁵, Gln³³⁷, His³⁵², Trp³⁵⁵ and Ser³⁵⁷_, and enzyme assays revealed an alteration of these binding sites and a significant reduction of UGT74S1 glucosyltransferase catalytic activity towards SECO and UDP-glucose in all mutants. A complete abolition of UGT74S1 activity was observed when Trp³⁵⁵ was substituted to Ala³⁵⁵ and Gly³⁵⁵ or when changing His³⁵² to Asp³⁵²_, and an altered metabolite profile was observed in Cys335Ala, Gln337Ala, and Ser357Ala mutants. This study provided for the first time evidence that Trp³⁵⁵ and His³⁵² are critical for UGT74S1’s glucosylation activity toward SECO and suggested the possibility for SMG production in vitro.     

Salicylic acid-mediated reductions in yield in Nicotiana attenuata challenged by aphid herbivory

Aphid herbivory decreases primary production in natural ecosystems and reduces crop yields. The mechanism for how aphids reduce yield is poorly understood as some studies suggest aphid feeding directly impedes photosynthesis, whereas other studies suggest a change in allocation of resources from growth to defense compounds reduces yield. To determine the mechanisms underlying reduced plant growth by aphids, Nicotiana attenuata plants, native tobacco, were infested with Myzus persicae ssp. nicotianae, tobacco-adapted green peach aphids, at low and high densities, and plant performance including fitness was assessed. To test the direct defense capacity of salicylic acid (SA) on aphid performance, we fed aphids an artificial diet with varying levels of SA and measured their survivorship and fecundity. There was no detectable effect of aphid herbivory on net photosynthesis, yet herbivory reduced plant growth, final biomass (43 % at high aphid density), and seed set (18 % at high aphid density) at both low and high aphid infestation levels. High-density aphid attack during the rosette and flowering stage caused an increase in SA levels, but caused only a transient decrease in jasmonic acid concentration at low aphid density. SA concentrations similar to those found in infested flowering plants decreased aphid fecundity, suggesting that SA was an effective chemical defense response against aphids. These results suggest that as aphid densities increased the proximal cause of reduced growth and yield was not reduced photosynthesis, but instead resources may have been mobilized for defense via the SA pathway, decreasing the availability of resources for building plant biomass.     

The in vitro genotoxicity of benzoic acid in human peripheral blood lymphocytes

The chromosomal aberration (CA), sister chromatid exchange (SCE) and micronucleus test (MN) were employed to investigate the in vitro effect of antimicrobial food additive benzoic acid on human chromosomes. Lymphocytes were incubated with various concentrations (50, 100, 200 and 500 μg/mL) of benzoic acid. The results of used assays showed that benzoic acid significantly increased the chromosomal aberration, sister chromatid exchange and micronucleus frequency (200 and 500 μg/mL) without changing the pH of the medium in a dose-dependent manner. Also this additive significantly decreased the mitotic index (MI) at the highest concentration for 24 h and 100, 200 and 500 μg/mL for 48 h. This decrease was dose-dependent as well. However, it did not effect the replication (RI) and nuclear division (NDI) indices.