Compounds leached from quinoa seeds inhibit matrix metalloproteinase activity and intracellular reactive oxygen species.

OBJECTIVE: Quinoa (Chenopodium quinoa Willd.) is a seed crop rich in bioactive compounds including phytoecdysones (especially 20-hydroxyecdysone, 20HE), polyphenols, proteins and essential fatty acids. We previously reported a method to leach and concentrate quinoa bioactives into a complex phytochemical mixture termed quinoa leachate (QL). Here, we aimed to determine the effect of QL and its chemically distinct fractions on five biochemical endpoints relevant to skin care applications: (i) cell viability, (ii) matrix metalloproteinase (MMP) mRNA expression, (iii) MMP enzymatic activity, (iv) tyrosinase enzymatic activity and (v) intracellular reactive oxygen species (ROS) production. METHODS: Quinoa leachate was fractionated and chemically characterized using column chromatography and liquid chromatography-mass spectrometry (LC-MS). Cell viability was determined using a MTT assay in four mammalian cell lines. MMP-1 mRNA expression was assessed in human dermal fibroblasts (HDF) via qRT-PCR. The enzymatic activity of MMP-9 and tyrosinase was measured using fluorometric and colorimetric in vitro assays, respectively. Lipopolysaccharide (LPS)-induced ROS production was determined in human dermal fibroblasts by fluorescence intensity of an oxidant-sensitive probe. RESULTS: Quinoa leachate was separated into three fractions: (i) carbohydrate-rich fraction (QL-C; 71.3% w/w of QL); (ii) phytoecdysone, polyphenol and protein-rich fraction (QL-P, 13.3% w/w of QL); (iii) oil-rich fraction (QL-O, 10.8% w/w of QL). QL did not reduce cell viability in any of the four cell lines tested. QL, QL-P and QL-O each significantly inhibited MMP-1 mRNA expression in HDF at a concentration of 5 µg mL(-1) . QL and QL-P also significantly inhibited MMP-9 enzymatic activity, whereas QL-P demonstrated significant tyrosinase enzymatic inhibition. Furthermore, QL, QL-P, QL-O and 20HE significantly inhibited intracellular ROS production. CONCLUSION: This study is the first to demonstrate the MMP, tyrosinase and ROS inhibiting properties of multiple different phytochemical components derived from quinoa seeds. Our work indicates that quinoa phytochemicals may play a role in the treatment and prevention of skin ageing through a multiplicity of effects.

Bioactive polyphenols from muscadine grape and blackcurrant stably concentrated onto protein-rich matrices for topical applications.

OBJECTIVES: Natural botanical agents that are antimicrobial, or that modulate skin hyperpigmentation via tyrosinase inhibition, are increasingly sought in the cosmetic industry. METHODS: In this study, an efficient tactic is demonstrated for concentrating and stabilizing skin-beneficial bioactive compounds from muscadine grape and blackcurrant juice or muscadine pomace, into hemp flour (HF), hemp protein isolate (HPI) and soy protein isolate (SPI) matrices suitable for cosmetic applications. RESULTS: Anthocyanins were most efficiently captured from blackcurrant juice into HF (8.39 mg g(-1) ). HPI most effectively captured total phenolics from muscadine pomace (72.32 and 77.32 mg g(-1) from Noble and Carlos, respectively), while the three matrices incorporated highest levels of ellagic acid, gallic acid, and PAC B1 from Noble muscadine grape juice. The enriched matrices demonstrated effective in vitro inhibition of tyrosinase (up to 57.29% for blackcurrant juice-HPI matrix), and in general, juice sources provided greater inhibition on L-dopamine oxidation by tyrosinase than pomace sources. The polyphenol-enriched matrices effectively inhibited microbial proliferation in a screening assay against Staphylococcus aureus bacteria, whereas untreated HF, HPI or SPI did not inhibit bacterial growth. CONCLUSION: The technology of combining and stably concentrating phytoactive polyphenols with proteins has potential use for cosmetic topical applications.

Potato protease inhibitors inhibit food intake and increase circulating cholecystokinin levels by a trypsin-dependent mechanism.

OBJECTIVE: To investigate the mechanisms underlying the satiety-promoting effects of a novel protease inhibitor concentrate derived from potato (PPIC). METHODS: The acute and prolonged effects of oral PPIC administration (100 mg kg(-1) per day) on food intake, body weight and gastric emptying were evaluated in healthy rats. Parameters of body weight, food intake, plasma glucose, insulin and cholecystokinin (CCK) were measured. Duodenal proteolytic activity and CCK expression were determined in tissue extracts. Intestinal STC-1 cell culture model was used to investigate the direct effect of PPIC on CCK transcript level and secretion. RESULTS: Acute oral administration of PPIC reduced immediate food intake during the first 2 h following the treatment, delayed gastric emptying and decreased proteolytic activity in the duodenum. Repeated oral ingestion of PPIC reduced weight gain in male rats and significantly elevated the plasma CCK levels. Although duodenal mucosal CCK mRNA levels increased in response to PPIC administration, the concentrate failed to elevate CCK expression or release in STC-1 cells. The 14-day ascending dose range study (33-266 mg kg(-1) PPIC per day) showed no adverse side effects associated with PPIC administration. CONCLUSION: These findings provided evidence that PPIC is effective in reducing food intake and body weight gain in healthy rats when administered orally by increasing circulating CCK levels through a trypsin-dependent mechanism.

New Strategy for the Search of Natural Biologically Active Substances.

The modern pharmaceutics actively screens an immense diversity of substances occurring in plants and other natural resources in the search for new effective medicinal agents. The Global Institute for Bioexploration (GIBEX) established by joint efforts of Rutgers University and the University of Illinois (United States) represents the organizational core of international scientific community whose activity is directed towards the search and development of new medicinal preparations from natural raw materials. The basis of GIBEX activity is the transfer of modern screening technologies to countries and geographical regions characterized by remarkable biodiversity. The GIBEX goals are to encourage the search for new natural biologically active substances, to maintain biodiversity, and to monitor the natural resources conservation.

Antihyperglycemic activity of Tarralin, an ethanolic extract of Artemisia dracunculus L.

The studies reported here were undertaken to examine the antihyperglycemic activity of an ethanolic extract of Artemisia dracunculus L., called Tarralin in diabetic and non-diabetic animals. In genetically diabetic KK-A(gamma) mice, Tarralin treatment by gavage (500 mg/kg body wt./day for 7 days) lowered elevated blood glucose levels by 24% from 479+/-25 to 352+/-16 mg/dl relative to control animals. In comparison, treatment with the known antidiabetic drugs, troglitazone (30 mg/kg body wt./day) and metformin (300 mg/kg body wt./day), decreased blood glucose concentrations by 28% and 41%, respectively. Blood insulin concentrations were reduced in the KK-A(gamma) mice by 33% with Tarralin, 48% with troglitazone and 52% with metformin. In (STZ)-induced diabetic mice, Tarralin treatment, (500 mg/kg body wt./day for 7 days), also significantly lowered blood glucose concentrations, by 20%, from 429+/-41 to 376+/-58 mg/dl relative to control. As a possible mechanism, Tarralin was shown to significantly decrease phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression by 28% in STZ-induced diabetic rats. In non-diabetic animals, treatment with Tarralin did not significantly alter PEPCK expression, blood glucose or insulin concentrations. The extract was also shown to increase the binding of glucagon-like peptide (GLP-1) to its receptor in vitro. These results indicate that Tarralin has antihyperglycemic activity and a potential role in the management of diabetic states.

A quick and efficient system for antibiotic-free expression of heterologous genes in tobacco roots.

Requirement for antibiotic-resistance selection markers and difficulty in identifying transgenes with the highest expression levels remain the major obstacles for rapid production of recombinant proteins in plants. An alternative approach to producing transgenic plants free of antibiotic-resistance markers is the phenotypic-based selection with root-proliferation genes (rol genes) of Agrobacterium rhizogenes. By using Agrobacterium tumefaciens harboring the pRYG transformation vector with a cluster of rol genes linked to a heterologous gene of interest, we have developed a rapid transformation tool using hairy root formation as a selection marker. The expression of beta-glucuronidase in newly induced transgenic tobacco roots could be detected as early as 12 days after inoculation. Higher levels of transgene expression in the roots correlated positively with the rates of root elongation on hormone-free medium and thus could be used for positive selection. When tobacco plants were transformed with pRYG harboring the expression cassette for secreted alkaline phosphatase (SEAP), the release of SEAP from roots of the fully regenerated transgenic plants could be quantified at rates as high as 28 microg/g root dry weight per day.

Rhizosecretion of recombinant proteins from plant hairy roots.

Rhizosecretion of a target protein in the hydroponic medium provides an alternative manufacturing platform that simplifies the downstream purification procedure and increases protein yield. In order to increase the production rates of rhizosecreted proteins, we have exploited the ability of Agrobacterium rhizogenes to induce the formation of large amounts of root tissue on transgenic tobacco plants engineered to secrete a model recombinant protein, human secreted alkaline phosphatase (SEAP). The secretion of SEAP from hairy roots induced on the stems of transgenic tobacco plants was 5-7 times higher than that from adventitious transgenic roots.

Tobacco ribosomal DNA spacer element stimulates amplification and expression of heterologous genes.

Here we show that the cis-acting genetic element aps (amplification-promoting sequence), isolated from the nontranscribed spacer region of tobacco ribosomal DNA (rDNA), increases the level of expression of recombinant proteins. Transgenic tobacco plants, transformed with expression cassettes containing the herbicide-resistant acetolactate synthase (hr-ALS) gene or the green fluorescent protein (GFP) gene fused to the aps sequence, had greater levels of corresponding messenger RNAs (mRNAs) and proteins compared to transformants lacking aps. Analysis of transgenic plants showed that aps increased the copy number and transcription of the adjacent heterologous genes and, in the case of hr-ALS, enhanced the herbicide resistance phenotype. Both the increased transgene copy number and enhanced expression were stably inherited. These data provide the first evidence that the aps sequence can be used for gene amplification in transgenic plants and possibly other multicellular organisms.

Production of recombinant proteins in tobacco guttation fluid.

Guttation, the loss of water and dissolved materials from uninjured plant organs, is a common phenomenon in higher plants. By using endoplasmic reticulum signal peptides fused to the recombinant protein sequences, we have generated transgenic tobacco (Nicotiana tabacum L. cv Wisconsin) plants that secrete three heterologous proteins of different genetic backgrounds (bacterial xylanase, green fluorescent protein of jellyfish [Aequorea victoria], and human placental alkaline phosphatase) through the leaf intercellular space into tobacco guttation fluid. Production rates of 1.1 microg/g of leaf dry weight per day were achieved for alkaline phosphatase with this protein comprising almost 3% of total soluble protein in the guttation fluid. Guttation fluid can be collected throughout a plant's life, thus providing a continuous and nondestructive system for recombinant protein production. Guttation fluid has the potential of increasing the efficiency of recombinant protein production technology by increasing yield, abolishing extraction, and simplifying its downstream processing.

Characterization of Arabidopsis acid phosphatase promoter and regulation of acid phosphatase expression.

The expression and secretion of acid phosphatase (APase) was investigated in Indian mustard (Brassica juncea L. Czern.) plants using sensitive in vitro and activity gel assays. Phosphorus (P) starvation induced two APases in Indian mustard roots, only one of which was secreted. Northern-blot analysis indicated transcriptional regulation of APase expression. Polymerase chain reaction and Southern-blot analyses revealed two APase homologs in Indian mustard, whereas in Arabidopsis, only one APase homolog was detected. The Arabidopsis APase promoter region was cloned and fused to the beta-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS expression was first evident in leaves of the P-starved Arabidopsis plants. In P-starved roots, the expression of GUS initiated in lateral root meristems followed by generalized expression throughout the root. GUS expression diminished with the addition of P to the medium. Expression of GFP in P-starved roots also initiated in the lateral root meristems and the recombinant GFP with the APase signal peptide was secreted by the roots into the medium. The APase promoter was specifically activated by low P levels. The removal of other essential elements or the addition of salicylic or jasmonic acids, known inducers of gene expression, did not activate the APase promoter. This novel APase promoter may be used as a plant-inducible gene expression system for the production of recombinant proteins and as a tool to study P metabolism in plants.

Subcellular localization and speciation of nickel in hyperaccumulator and non-accumulator Thlaspi species.

The ability of Thlaspi goesingense Hálácsy to hyperaccumulate Ni appears to be governed by its extraordinary degree of Ni tolerance. However, the physiological basis of this tolerance mechanism is unknown. We have investigated the role of vacuolar compartmentalization and chelation in this Ni tolerance. A direct comparison of Ni contents of vacuoles from leaves of T. goesingense and from the non-tolerant non-accumulator Thlaspi arvense L. showed that the hyperaccumulator accumulates approximately 2-fold more Ni in the vacuole than the non-accumulator under Ni exposure conditions that were non-toxic to both species. Using x-ray absorption spectroscopy we have been able to determine the likely identity of the compounds involved in chelating Ni within the leaf tissues of the hyperaccumulator and non-accumulator. This revealed that the majority of leaf Ni in the hyperaccumulator was associated with the cell wall, with the remaining Ni being associated with citrate and His, which we interpret as being localized primarily in the vacuolar and cytoplasm, respectively. This distribution of Ni was remarkably similar to that obtained by cell fractionation, supporting the hypothesis that in the hyperaccumulator, intracellular Ni is predominantly localized in the vacuole as a Ni-organic acid complex.

Purification, cloning, and expression of a pathogen inducible UDP-glucose:Salicylic acid glucosyltransferase from tobacco.

Salicylic acid (SA) plays an important role in plant disease resistance. Inoculation of tobacco leaves with incompatible pathogens triggers the biosynthesis of SA which accumulates primarily as the SA 2-O-beta-D-glucoside (SAG) and glucosyl salicylate (GS). The tobacco UDP-glucose:salicylic acid glucosyltransferase (SA GTase) capable of forming both SAG and GS was purified, characterized, and partially sequenced. It has an apparent molecular mass of 48 kDa, a pH optimum of 7.0, and an isoelectric point at pH 4.4. UDP-glucose was the sole sugar donor for the enzyme. However, SA and several phenolics served as glucose acceptors. The apparent K(m) values for UDP-glucose and SA were 0.27 and 1-2 mM, respectively. Zn(2+) and UDP inhibited its activity. The corresponding cDNA clone which encoded a protein of 459 amino acids was isolated from an SA-induced tobacco cDNA library and overexpressed in Escherichia coli. The recombinant protein catalyzed the formation of SAG and GS, and exhibited a broad specificity to simple phenolics, similar to that of the purified enzyme. Northern blot analysis showed that the SA GTase mRNA was induced both by SA and incompatible pathogens. The rapid induction timing of the mRNA by SA indicates that it belongs to the early SA response genes.

Production of recombinant proteins in plant root exudates.

The large-scale production of recombinant proteins in plants is limited by relatively low yields and difficulties in extraction and purification. These problems were addressed by engineering tobacco plants to continuously secrete recombinant proteins from their roots into a simple hydroponic medium. Three heterologous proteins of diverse origins (green fluorescent protein of jellyfish, human placental alkaline phosphatase [SEAP], and bacterial xylanase) were produced using the root secretion method (rhizosecretion). Protein secretion was dependent on the presence of the endoplasmic reticulum signal peptide fused to the recombinant protein sequence. All three secreted proteins retained their biological activity and, as shown for SEAP, accumulated in much higher amounts in the medium than in the root tissue.

Use of plant roots for phytoremediation and molecular farming.

Alternative agriculture, which expands the uses of plants well beyond food and fiber, is beginning to change plant biology. Two plant-based biotechnologies were recently developed that take advantage of the ability of plant roots to absorb or secrete various substances. They are (i) phytoextraction, the use of plants to remove pollutants from the environment and (ii) rhizosecretion, a subset of molecular farming, designed to produce and secrete valuable natural products and recombinant proteins from roots. Here we discuss recent advances in these technologies and assess their potential in soil remediation, drug discovery, and molecular farming.

Isolation and partial characterization of a lead-accumulating Brassica juncea mutant.

A new screening method for non-destructive, high-sensitivity, high-throughput isolation of plant mutants capable of accumulating large amounts of heavy metals has been developed. This method is based on incubating seedlings in a solution containing radioisotopes of the metals of interest and visualizing the tissue accumulation of these metals with a phosphorimager. We used this technique to isolate mutants of Brassica juncea (L.) Czern with increased accumulation of Cd and Pb for use in phytoremediation, an emerging technology using plants to remediate polluted soil and water. Approximately 50,000 M2 seedlings were screened and 21 mutants were recovered that retained increased accumulation through the third generation. Mutant 7/15-1 is characterized by enhanced Pb accumulation per unit of root fresh weight, stunted root growth, and decreased root cell size. Data indicate that roots of 7/15-1 contain more cell-wall material on a fresh-weight basis than roots of the wild-type, which may at least partially explain its ability to accumulate more Pb.

Intermediates of salicylic acid biosynthesis in tobacco

Salicylic acid (SA) is an important component of systemic-acquired resistance in plants. It is synthesized from benzoic acid (BA) as part of the phenylpropanoid pathway. Benzaldehyde (BD), a potential intermediate of this pathway, was found in healthy and tobacco mosaic virus (TMV)-inoculated tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaf tissue at 100 ng/g fresh weight concentrations as measured by gas chromatography-mass spectrometry. BD was also emitted as a volatile organic compound from tobacco tissues. Application of gaseous BD to plants enclosed in jars caused a 13-fold increase in SA concentration, induced the accumulation of the pathogenesis-related transcript PR-1, and increased the resistance of tobacco to TMV inoculation. [13C6]BD and [2H5]benzyl alcohol were converted to BA and SA. Labeling experiments using [13C1]Phe in temperature-shifted plants inoculated with the TMV showed high enrichment of cinnamic acids (72%), BA (34%), and SA (55%). The endogenous BD, however, contained nondetectable enrichment, suggesting that BD was not the intermediate between cinnamic acid and BA. These results show that BD and benzyl alcohol promote SA accumulation and expression of defense responses in tobacco, and provide insight into the early steps of SA biosynthesis.

The role of EDTA in lead transport and accumulation by indian mustard

Indian mustard (Brassica juncea) plants exposed to Pb and EDTA in hydroponic solution were able to accumulate up to 55 mmol kg-1 Pb in dry shoot tissue (1.1% [w/w]). This represents a 75-fold concentration of Pb in shoot tissue over that in solution. A threshold concentration of EDTA (0.25 mm) was found to be required to stimulate this dramatic accumulation of both Pb and EDTA in shoots. Below this threshold concentration, EDTA also accumulated in shoots but at a reduced rate. Direct measurement of a complex of Pb and EDTA (Pb-EDTA) in xylem exudate of Indian mustard confirmed that the majority of Pb in these plants is transported in coordination with EDTA. The accumulation of EDTA in shoot tissue was also observed to be directly correlated with the accumulation of Pb. Exposure of Indian mustard to high concentrations of Pb and EDTA caused reductions in both the transpiration rate and the shoot water content. The onset of these symptoms was correlated with the presence of free protonated EDTA (H-EDTA) in the hydroponic solution, suggesting that free H-EDTA is more phytotoxic than Pb-EDTA. These studies clearly demonstrate that coordination of Pb transport by EDTA enhances the mobility within the plants of this otherwise insoluble metal ion, allowing plants to accumulate high concentrations of Pb in shoots. The finding that both H-EDTA and Pb-EDTA are mobile within plants also has important implications for the use of metal chelates in plant nutritional research.

Glucosylation of Salicylic Acid in Nicotiana tabacum Cv. Xanthi-nc.

ABSTRACT Salicylic acid (SA) is a key regulatory component of disease resistance in plants. In tobacco mosaic virus (TMV)-inoculated tobacco (Nicotiana tabacum cv. Xanthi-nc NN genotype), newly synthesized SA is converted primarily to SA 2-O-beta-D-glucoside (SAG) and glucosyl salicylate (GS), a relatively minor metabolite. Similar patterns in the formation of GS and SAG were observed in tobacco inoculated with Pseudomonas syringae pv. phaseolicola, suggesting the accumulation of two glucosylated metabolites is a general phenomenon in tobacco plants. After SA infiltration, GS was synthesized rapidly, reached a maximal level at 6 h, declined, and remained relatively constant for at least 24 h. In contrast, SAG content increased gradually after SA treatment. Our in vitro and in vivo data suggest that a high concentration of free SA triggers transient formation of GS and continuous accumulation of SAG, which is a more stable metabolite of SA. The two distinct SA glucosyltransferases catalyzed the formation of GS and SAG, respectively. The activities of these enzymes were enhanced by TMV or P. syringae pv. phaseolicola inoculation or SA treatment and were found in different fractions of gel filtration chromatography.

PHYTOREMEDIATION.

Contaminated soils and waters pose a major environmental and human health problem, which may be partially solved by the emerging phytoremediation technology. This cost-effective plant-based approach to remediation takes advantage of the remarkable ability of plants to concentrate elements and compounds from the environment and to metabolize various molecules in their tissues. Toxic heavy metals and organic pollutants are the major targets for phytoremediation. In recent years, knowledge of the physiological and molecular mechanisms of phytoremediation began to emerge together with biological and engineering strategies designed to optimize and improve phytoremediation. In addition, several field trials confirmed the feasibility of using plants for environmental cleanup. This review concentrates on the most developed subsets of phytoremediation technology and on the biological mechanisms that make phytoremediation work.