Crude Plant Extracts Research Articles

Meeting technical challenges for protein characterization and surrogate equivalence studies that resulted from insecticidal protein co-expression in maize event MZIR098

Safety assessment of genetically modified plants includes protein characterization to confirm the intended trait protein expression. In addition, to conduct safety tests, the large amount of purified protein needed is usually met through the use of a surrogate, microbially produced protein source. Characterization of the eCry3.1Ab and mCry3A proteins as derived from Event MZIR098 maize was challenging because of the difficulty in purifying/isolating these proteins that are of similar molecular weight and have considerable shared sequence and immunogenicity. This also applies to establishing the biochemical equivalence to the microbially produced surrogate proteins, as highly-purified plant protein is required. While use of crude plant extracts facilitated functional equivalence testing with the surrogate proteins, a separate technical challenge had to be met. The eCry3.1Ab and mCry3A proteins display differentiated modes of action toward CRW pests, however, with the same overall target pest spectrum, no differential test organism existed to allow equivalence testing for one insecticidal protein in the presence of the other. To establish that the microbially produced proteins are suitable surrogates for the plant-produced proteins, the challenges in the protein purification and bioactivity testing had to be addressed. This article describes technical solutions to assess and characterize the insecticidal proteins in this new event and thereby confirm equivalence/suitability of the microbially produced protein surrogates.

Comparative Analysis of Phytochemical Constituents and Antibacterial Activity of Crude and Purified Ethanol and Ethyl-acetate Extracts of Euphorbia hirta L. Whole Plant

Aims: To study the phytochemical constituents and antibacterial efficacy of crude and purified Euphorbia hirta whole plant extract on Gram-positive bacteria isolated from otitis media.
 Study Design: Experimental Research design.
 Place and Duration of Study: Sample: Department of Microbiology (Mtech Laboratory) and Department of Chemistry (Organic Chemistry Laboratory), School of Sciences, Federal University of Technology, Akure, Ondo State, Nigeria. Between November 2018 and March 2019.
 Methods: The streak plate method was used for bacterial isolation, maceration method for Euphorbia hirta whole plant extraction using ethanol and ethyl-acetate as solvents. The E. hirta whole plant extracts were purified using column chromatography method. The extracts were assayed on the test bacterial isolates by agar diffusion technique. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the extracts were carried out by agar dilution and agar diffusion techniques, respectively.
 Results: The ethanolic extract had the highest extraction yield (19%). The Staphylococcus aureus was resistant to multiple antibiotics: amoxicillin (30 µg), gentamycin (10 µg), and streptomycin (30 µg). The phytochemical screening of crude plant extracts showed presence of flavonoids, glycosides, saponins, tannins and terpenoids. At 100 mg/ml, crude and purified ethanol extracts showed antibacterial activity with 18±0.57 mm and 14±0.57 mm respectively on Streptococcus pyogenes. The MIC and MBC of purified ethanol extract ranged between 6.25-50 mg/ml and 25 mg/ml-100 mg/ml respectively.
 Conclusion: This research showed that E. hirta whole plant extract possesses antibacterial activity. The purified E. hirta whole plant extract showed higher inhibitory effect compared to crude extracts. This is an indication that purified E. hirta whole plant extract can be used in the development of novel therapeutic drugs in the treatment of otitis media.

Anti-inflammatory activities of Waltheria indica extracts by modulating expression of IL-1B, TNF-α, TNFRII and NF-κB in human macrophages.

In Hawaiian traditional medicinal practices, the indigenous 'uhaloa, Waltheria indica var. Americana is one of the most recognized plants. Waltheria is also known in various cultures as a medicinal plant for the treatment of inflammatory conditions. Results in human subjects and cell and animal models supported anti-inflammatory activity for the Waltheria flavonoid quercetin, and for crude plant extracts, limited animal studies also confirmed anti-inflammatory effects. Yet no systematic studies have examined immune or inflammatory responses affected by these extracts. In order to gain insight into inflammatory cascades modulated by Waltheria extracts, and to uncover the mechanistic bases for the effective use of this medicinal plant as a natural anti-inflammatory agent, we have undertaken analyses of LPS and TNF-α/IF-γ-stimulated human macrophages treated with Waltheria extracts using targeted qRT-PCR and Inflammation Panels to test differential mRNA expression of two hundred immune-related genes, furthermore, ELISA assays and Inflammatory Protein arrays to determine extracts-modulated intracellular and secreted levels of prominent cytokines. Results demonstrate that Waltheria extracts inhibit key inflammatory cytokines and cytokine receptors including protein levels of IL-1B, IL-1ra, IL-8 and IL-6, reduce both mRNA and protein levels of TNF-α and protein levels of its receptor, TNF RII, predicting diminished TNF-α-associated inflammatory signaling that, together with significant reduction of NF-κB mRNA and protein, can effectively diminish activities of multiple pro-inflammatory signaling pathways and mitigate key processes in diseases with inflammatory components.

Advances in MS Based Strategies for Probing Ligand-Target Interactions: Focus on Soft Ionization Mass Spectrometric Techniques.

The non-covalent interactions between small drug molecules and disease-related proteins (ligand-target interactions) mediate various pharmacological processes in the treatment of different diseases. The development of the analytical methods to assess those interactions, including binding sites, binding energies, stoichiometry and association-dissociation constants, could assist in clarifying the mechanisms of action, precise treatment of targeted diseases as well as the targeted drug discovery. For the last decades, mass spectrometry (MS) has been recognized as a powerful tool to study the non-covalent interactions of the ligand-target complexes with the characteristics of high sensitivity, high-resolution, and high-throughput. Soft ionization mass spectrometry, especially the electrospray mass spectrometry (ESI-MS) and matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), could achieve the complete transformation of the target analytes into the gas phase, and subsequent detection of the small drug molecules and disease-related protein complexes, and has exerted great advantages for studying the drug ligands-protein targets interactions, even in case of identifying active components as drug ligands from crude extracts of medicinal plants. Despite of other analytical techniques for this purpose, such as the NMR and X-ray crystallography, this review highlights the principles, research hotspots and recent applications of the soft ionization mass spectrometry and its hyphenated techniques, including hydrogen-deuterium exchange mass spectrometry (HDX-MS), chemical cross-linking mass spectrometry (CX-MS), and ion mobility spectrometry mass spectrometry (IMS-MS), in the study of the non-covalent interactions between small drug molecules and disease-related proteins.

Potential of medicinal plants to treat dengue

Dengue is a major public health challenge worldwide, particularly in tropical areas. Nearly 390 million infections and 22,000 deaths occur every year. At present, there are no specific therapeutics available to treat dengue; however, possible treatment procedures are explained in the traditional medical systems (TMSs), such as Sri Lankan TMS, Indian Ayurvedic, Unani, and Siddha TMS. In these TMSs, medicinal plants have been used in several ways against dengue, such as virocides, larvicides, and mosquito repellents. Therefore, medicinal plants inherit biologically active compounds/lead compounds that are yet to be identified chemically and physiologically. Herein, we discuss the possible applications of crude plant extracts and isolated phytochemicals from medicinal plants such as quercetin, sulfated galactomannans, flavonoids, and glabranine in controlling dengue. Moreover, medicinal plant-based therapeutics can be safer, cost-effective, and non-toxic. Therefore, this paper reviews the medicinal plants that are used in TMSs to manage dengue, the phytochemicals they contain, and mode of action of these phytochemicals such as virocides, larvicides, and mosquito repellents.

Potential effects of Psidium sp., Mangifera sp., Mentha sp. and its mixture (PEM) in reducing bacterial populations in biofilms, adherence and acid production of S. sanguinis and S. mutans

ObjectivePsidium sp., Mangifera sp. and Mentha sp. and its mixture (PEM) are known to have antimicrobial and anti-adherence effects. DesignHere, we have investigated these individual plant extracts and its synergistic mixture (PEM) for its anti-cariogenic effect to reduce populations of single and mixed-species of Streptococcus sanguinis and Streptococcus mutans in a planktonic or/and biofilm and their others reduced virulence. Bacterial populations in the biofilm after 24 h, hydrophobic cell surface activity to n-hexadecane and pH changes at 5 min’ intervals until 90 min of incubation were recorded. Total phenolic content and bioactive compounds in the crude aqueous plant extracts were analysed. Regulatory gene expressions of S. mutans adhesins genes (gtfB, gtfC, gbpB and spaP) upon treatment with PEM were investigated in planktonic and biofilm conditions. ResultsAll plant extracts strongly reduced S. mutans in the biofilm compared to S. sanguinis in single and mixed-species. PEM reduced S. mutans by 84% with S. sanguinis 87% in the mixed population. Psidium sp. and PEM highly reduced cell-surface hydrophobicity of the two bacteria thus reducing adherence and biofilm formation. PEM and Mangifera sp. lowered initial pH change in the mixed populations of S. sanguinis and S. mutans. PEM downregulated the S. mutans gtfB gene expression in the single species planktonic and mixed-species biofilms. ConclusionsThe effectiveness of PEM in reducing S. mutans within the biofilm, cell-surface hydrophobicity, acid production and adhesin gene (gtfB) expression in mixed-species with S. sanguinis indicates its potential as an antibacterial agent against dental caries. This is attributed to the phenolic content in the PEM.

Effect of acaricidal compound extracted from Arachis hypogaea Linn against Tetranychus cinnabarinus

AbstractSix plant crude extracts were chosen to evaluate their acaricidal activity against Tetranychus cinnabarinus adults. The crude extract from the stems and leaves of Arachis hypogaea L. presented the highest activity in leaf‐dip bioassays, with an LC50 value of 3,545.98 mg/L. Further extraction using four different solvents (petroleum ether, ethyl acetate, n‐butyl alcohol and distilled water) demonstrated that the active components mainly existed in the petroleum ether phase. Then, the active compound, palmitic acid, was isolated from the petroleum ether phase via two‐step column chromatography using columns filled with silica gel and C‐18 and identified via mass spectrometry and nuclear magnetic resonance analyses. The LC50 value of active palmitic acid against T. cinnabarinus was 534.58 mg/L. The present study demonstrated that the active compound extracted from A. hypogaea is a potential novel botanical acaricide for controlling T. cinnabarinus.

Bactericidal Efficacy of Selected Medicinal Plant Crude Extracts and their Fractions against Common Fish Pathogens

The emergence of new diseases and the increased use of antibiotics have led to the development of resistant bacterial strains. Thus, there is greater attention to seek new antibacterial agents from the natural sources for combating fish diseases in the aquaculture industry. The present study evaluated the bactericidal efficacy of crude methanolic and aqueous extracts from Polygonum chinense, Syzygium polyanthum, Premna foetida, Pimenta dioica, Brucea javanica, Vitex negundo, Alpinia conchigera and Clinacanthus nutans against Vibrio harveyi, Vibrio alginolyticus, Vibrio parahaemolyticus and Aeromonas hydrophila using disc diffusion method. The results showed that methanolic extracts of P. dioica, P. foetida and P. chinense, and aqueous extracts of P. dioica and S. polyanthum showed moderate to strong activity (10.8 to 17.2 mm) against all the tested bacteria. These five potential crude extracts were fractionated using liquid-liquid extraction method to obtain the methanol, dichloromethane and ethyl acetate fractions. Among the fractions, ethyl acetate fraction showed the highest activity against all tested bacteria, with minimum inhibition concentration (MIC) values between 0.625 and 10.000 mg/mL. In addition, the five potential crude extracts had low to moderate toxicity with LC50 >100 μg/mL using brine shrimp cytotoxicity assays. The results of this study indicated that methanolic extracts of P. chinense and P. foetida that showed high bactericidal activity and low toxicity could be good potentials for use in fish culture.

Melodamide A from Melodorum fruticosum - Quantification using HPLC and one-step-isolation by centrifugal partition chromatography.

Melodamide A, a phenolic amide from the leaves of Melodorum fruticosum Lour., has previously shown pronounced anti‐inflammatory activity. In order to rapidly isolate larger quantities for biological testing, a fast, one‐step isolation method by centrifugal partition chromatography was developed within this study. Fractionation of the dichloromethane extract was performed with a two‐phase solvent system consisting of n‐hexane, ethyl acetate, methanol, and water (3:7:5:5, v/v), leading to the isolation of melodamide A with a purity of >90% and a yield of 6.7 w% within 32 min. The developed method can also be used in dual mode for the enrichment of further constituents like flavonoids or chalcones. In order to support the centrifugal partition chromatography method development, additionally, a high‐performance liquid chromatography method was established and validated to determine quantities of melodamide A in plant material and crude extracts. Analysis of M. fruticosum leaves and a dichloromethane extract obtained from this plant material showed a total melodamide A content of 0.19 ± 0.008 and 8.9 ± 0.249 w%, respectively.

Evaluation of Antimalarial Properties of Ficus platyphylla Del Leaf Extract in Mice

Aim: This study was aim at investigating the effect of crude petroleum ether leaf extract of Ficus platyphylla Del on Plasmodium berghei infected mice.
 Place and Duration of Study: This research was carried out at the department of biochemistry, Federal university of technology minna, Niger state Nigeria in 2014.
 Methodology: The crude plant extract of F. platyphylla was administered 72 hours at different doses post and pre infection for both the curative and prophylactic study respectively against residual infection. Mice were divided into 5 groups of 5 mice each, 3 of the groups where administered crude plants extract of F. platyphaylla at different doses (200, 400 and 600 mg/kg body weight) while the other two serve as negative and positive control group and were administered 0.5 ml and 50 mg/kg body weight respectively.
 Results: The extract at all doses produced significant (P<0.05) dose dependent chemo-suppressive activity with % inhibition of 38%, 61%, 74% and 81.8% for curative studies and 36.0%, 38.5%, 49.5% and 63.4% for prophylactic studies against the parasites at doses of 200 mg/kgbw, 400 mg/kgbw, 600 mg/kgbw of the extract and 50 mg/kgbw of Artesunate. All doses of the extract increased the survival time of the infected mice compared to the negative control group that was administered 0.5 ml normal saline. The variation in the values of Packed Cell Volume (PCV) for treated group before and after extract administration was not significant at (P<0.05). The phytochemical screening of the plant extract showed the presence of tannin, saponin, flavonoids, terpenoids, steroids, anthroquinone and phenol.
 Conclusion: The result of this study shows that F. Platyphylla leaf extract exhibited some antiplasmodial activity that could be exploited for safe, effective and affordable antimalaria regimen.

Molluscicidal and Feeding Deterrent Activity of Crude Plant Extracts on <em>Pomacea maculata</em> Perry

This study was focused on molluscicidal effect and feeding deterrent activity of saponin from five different plant materials; <em>Andrographis paniculata</em>, <em>Entada spiralis</em>, <em>Ficus deltoidea</em>, <em>Furcraea selloa</em> and <em>Ipomoea batatas</em>. Crude plant extracts were prepared from plant powders using methanol. The crude extracts were then tested on <em>Pomacea maculata</em> using five different concentrations (5, 10, 15, 20 and 25 ppm, respectively) against niclosamide (control). After 72 h of exposure, the highest percentage of mortality of 80% was achieved from 15 ppm of <em>F. selloa</em>. Two analyses were conducted to observe the feeding deterrent activity and after 24 h, both analyses demonstrated the feeding deterrent activity in both crude extracts (<em>F. selloa</em> and <em>E. spiralis</em>) similar in niclosamide.

Moringa oleifera Lam. and derived phytochemicals as promising antiviral agents: A review

Frequent use of medicinal plants is common in healthcare needs of mankind since ancient ages. Among these Moringa oleifera Lam. is one of the vastly used plant whose various parts (leaf, fruit, seeds etc.) are included in regular diet for their multiple ability of combating several health issues. WHO has highlighted on the proper utilization of natural products and marked plant-based medicines as prime study candidates. Taking initiative to explore bioactive leads other than conventional chemotherapeutic agents (with undesirable side effects) have drawn attention of the scientists involved in viral research due to the lack of effective vaccines and insufficient supply of existing costly drugs to socio-economic demands. Several studies were reported regarding the antiviral activity of M. oleifera plant, a pronounced bioprospective aspirant. The plant is known to be used in many traditional medicines and pharmacopeias against an array of medical conditions that include malaria, diabetes, skin infection, tuberculosis, anemia, headaches, epilepsy, sexually transmitted diseases and so on. In African traditional medicine, the plant is popularly used against AIDS and related secondary infections associated with HIV. It showed significant activities against viruses like HIV, HSV, HBV, EBV, FMDV and NDV. In some cases active molecules with mode of actions were documented by authors. On the other hand, there is a number of reports where neither lead compounds nor the relevant mechanisms were clarified regarding the viral inhibitory activities of crude plant extract. Immense studies should be going on to resolve those unanswered motifs along with the well planned trials of clinical application of already discovered potent phytochemicals. For conducting the continuous investigational groundwork in this field, the respective plant should be preserved with proper maintenance, necessarily.

Discovery of Lipid Peroxidation Inhibitors from Bacopa Species Prioritized through Multivariate Data Analysis and Multi-Informative Molecular Networking

A major goal in the discovery of bioactive natural products is to rapidly identify active compound(s) and dereplicate known molecules from complex biological extracts. The conventional bioassay-guided fractionation process can be time consuming and often requires multi-step procedures. Herein, we apply a metabolomic strategy merging multivariate data analysis and multi-informative molecular maps to rapidly prioritize bioactive molecules directly from crude plant extracts. The strategy was applied to 59 extracts of three Bacopa species (B. monnieri, B. caroliniana and B. floribunda), which were profiled by UHPLC-HRMS2 and screened for anti-lipid peroxidation activity. Using this approach, six lipid peroxidation inhibitors 1–6 of three Bacopa spp. were discovered, three of them being new compounds: monnieraside IV (4), monnieraside V (5) and monnieraside VI (6). The results demonstrate that this combined approach could efficiently guide the discovery of new bioactive natural products. Furthermore, the approach allowed to evidence that main semi-quantitative changes in composition linked to the anti-lipid peroxidation activity were also correlated to seasonal effects notably for B. monnieri.

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study.

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

α-Costic acid, a plant sesquiterpene with acaricidal activity against Varroa destructor parasitizing the honey bee

The organic extract of the aerial parts of Dittrichia viscosa, a perennial native plant of the Mediterranean basin, showed a significant acaricidal activity against Varroa destructor, the parasite mite of Apis mellifera, commonly called honey bee. Among the metabolites isolated from the organic extract of this Asteraceae, α-costic acid showed to be one of the compounds responsible for the toxic activity exhibited by the crude plant extract on this parasite mite species. In addition to the toxic effect a clear acaricidal response has been recorded when the parasitic mite was exposed to 1 mg/mL concentration of α-costic acid while no effects have been showed on honey bees using the same compound at the same concentration. This finding suggests a potential use of α-costic acid to control Varroa mites. The possibility to reliably achieve absolute configuration of α-costic acid by DFT computational analysis of chiroptical spectra has been also demonstrated.†

AGO-unbound cytosolic pool of mature miRNAs in plant cells reveals a novel regulatory step at AGO1 loading.

RNA interference (RNAi) is mediated by small, 20-24-nt-long, non-coding regulatory (s)RNAs such as micro (mi) and small interfering (si) RNAs via the action of ARGONAUTE (AGO) proteins. High-throughput sequencing of size-separated sRNA pools of plant crude extracts revealed that the majority of the canonical miRNAs were associated with high molecular weight RNA-induced silencing complexes co-migrating with AGO1 (HMW RISC). In contrast, the majority of 24-nt-long siRNAs were found in association with low molecular weight complexes co-migrating with AGO4 (LMW RISC). Intriguingly, we identified a large set of cytoplasmic sRNAs, including mature miRNA sequences, in the low molecular size range corresponding to protein-unbound sRNAs. By comparing the RISC-loaded and protein-unbound pools of miRNAs, we identified miRNAs with highly different loading efficiencies. Expression of selected miRNAs in transient and transgenic systems validated their altered loading abilities implying that this process is controlled by information associated with the diverse miRNA precursors. We also showed that the availability of AGO proteins is a limiting factor determining the loading efficiency of miRNAs. Our data reveal the existence of a regulatory checkpoint determining the RISC-loading efficiencies of various miRNAs by sorting only a subset of the produced miRNAs into the biologically active RISCs.

IN GEL DETECTION OF A HIS-TAGGED TRANSGENE FOLLOWING THE SEPARATION OF CRUDE PLANT PROTEIN EXTRACTS WITH SDS PAGE

In the present study, we visualised sundew His6-tagged chitinase protein in crude protein extracts of transgenic tobacco plants following protein separation with sodium dodecyl sulfate polyacrylamide gel electrophoresis by detecting chitinase activity as well as the His-tag. A short sequence encoding six histidines was fused downstream of the DNA sequence encoding the last amino acid of the mature protein. Following binary vector construction and plant transformation, a set of 10 transgenic plants was analysed for transgene expression. Except for one, all transgenic plants exhibited the presence of sundew chitinase protein of ~52 kDa, which was different from the calculated molecular weight of ~32 kDa. Clear identification of DrChitHis protein was performed with a Ni2+:NTA complex conjugated to a fluorescent dye and visualized using light laser-based scanner. A subsequent endochitinolytic activity assay using a N-fluorescein-labelled chitin substrate confirmed that the two transgenic lines with the strongest expression of DrChitHis protein had endochitinolytic activity 6.4 and 6.7 times higher than non-transgenic control.

Chemical diversity and biological activities of the saponins isolated from Astragalus genus: focus on Astragaloside IV

Astragalus is a cosmopolitan genus characterised by a large number of species with an exceptional rate of diversification. Many of these species are used since ancient times in the Traditional Chinese Medicine (TCM) to cure a condition of general weakness known as “qi”-deficiency. The main phytochemical constituents of these species have been identified as imidazoline alkaloids, nitro toxins, selenium derivatives, polysaccharides, phenols and saponins. Among these, oleanane saponins and cycloartane glycosides are considered as the main responsible for the bioactivity of the plant crude extracts. However, although a plethora of literature data regarding the pharmacological activities of the Astragalus saponins is available, a clear understanding of their mode of action is still missing. In this review, a brief outline of the taxonomy, distribution and morphology of the Astragalus genus is presented along with a comprehensive description of the chemical features and the biological activities of the the saponins from it derived. Moreover, in an attempt to summarise the current mechanistic knowledge on these metabolites, we elaborated a detailed overview of the key signalling pathways that are known to be affected by Astragalus saponins. Finally, we dedicated a section to Astragaloside IV, the best-characterised cycloartane glycoside from Astragalus, to summarise its potentiality as a pharmacological agent. The ultimate goal of this review is to provide the foundations for future investigations, which would eventually enable the introduction of Astragalus saponins in the clinical setting.

Column chromatography for preparing rosmarinic acid rich extract from Orthosiphon aristatus

Chromatographic techniques were used to prepare rosmarinic acid rich extract from Orthosiphon aristatus. Such extract is highly required for herbal product development because of its remarkable bioactivities. Therefore, the plant crude extract was fractionated using solvent mixture, ethyl acetate and ethanol in column chromatography. The solvent system was developed using thin layer chromatography. The results found that 60-99.9% ethyl acetate in ethanol could separate rosmarinic acid with the retention factor (Rf) ranged from 0.2–0.3. Of the solvent systems, 70% ethyl acetate could produce the highest yield of rosmarinic acid, 4.5% w/w with 100% purity. The concentration of rosmarinic acid was quantified by high performance liquid chromatography integrated with tandem mass spectrometry. The subsequent combined fractions could recover 70% of total rosmarinic acid from crude extract. The bed height of the silica gel packed column increased from 16 to 24 and 32 cm did not affect the performance of rosmarinic acid elution in column chromatography. The silica gel absorbent could be repeatedly used up to five times to recover rosmarinic acid from crude extract. Column chromatography can be used to recover rosmarinic acid (>85%) using the binary solvent system (70% ethyl acetate:30% ethanol) effectively.

Effect of plant extracts onPseudomonas syringaepv.actinidiaegene expression, motility and virulence

Pseudomonas syringae pv. actinidiae (Psa), the causative agent of bacterial canker of kiwi, was firstly isolated in 1992, but the second outbreak dating back to 2008 is responsible for the current pandemics that has led to major economic losses worldwide. Despite the high economic impact of Pseudomonas syringae pv. actinidiae (Psa) on kiwifruit industry, and the intense research efforts, the molecular mechanisms involved in pathogenesis are still not fully characterized. In particular, the precise mechanism of recognition/communication between Psa and its host plant has not been elucidated yet. Our findings show that plant crude extracts or xylem juices of Actinidia deliciosa and A. chinensis were able to increase Psa virulence in comparison with artificial growing media such as Luria Broth or Nutrient agar. The study of gene expression in response to plant-based or artificial growing medium was performed by qPCR and showed a differential expression of many virulence-related genes. In addition, genes related to biofilm formation, motility and inter-cell signaling (e.g. quorum sensing in the broad sense) were differentially expressed according to the type of growing media. Interestingly, the expression of many of the studied genes responded differentially both to plant extracts and Psa cell density. The molecular data were corroborated by phenological studies testing the effect of plant-based medium on biofilm formation, motility and symptom development in micro-cuttings of Actinidia spp. The present study demonstrates the existence of a not yet identified, host-specific group of molecules able to activate the pathogenesis machinery of Psa. Moreover, these molecules could lead to different physiological and molecular behavior depending on Psa population density. Finally, it was also demonstrated that these molecules were differentially present in leaves and xylem sap collected by different Actinidia species.