Revealing the mechanisms of Arisaema cum Bile on allergic asthma with systematic pharmacology approach-experimental validation.

Arisaema cum Bile (Dan Nanxing in Chinese, DNX) have been employed to treat allergic asthma. However, the active components and its mechanisms remain unknown. Therefore, the systematic pharmacology approach-experimental validation was performed in this study. Each 5, 6, and 10 compounds of DNX were obtained by HPLC analysis, TCMSP, and literature report, respectively. A total of 379 targets on all these compounds were acquired from Swiss Target Prediction, and 1973 targets on allergic asthma were predicated. The KEGG enrichment analysis was performed. Furthermore, a rat model of allergic asthma was established and DNX (450 mg/kg, p.o.) was given for 2 weeks. DNX treatment prevented OVA-induced pathological changes in lung cell of irregular arrange and necrotic bronchial epithelial. It also decreased inflammatory cytokines IL-4, IL-5, and IL-13 of serum and BALF, and increased IL-12 and IFN-γ. The main MAPK signaling pathway predicted by KEGG enrichment was verified, as indicated by the decreased protein expression of JNK (p < 0.05 & p < 0.01), ERK (p < 0.05), and p38 MAPK (p < 0.01) in lung tissue. These findings indicated that DNX attenuated OVA-induced allergic asthma mainly by decreasing the MAPK signaling pathway.

Exploration of carbohydrate binding behavior and anti-proliferative activities of Arisaema tortuosum lectin.

BACKGROUND: Lectins have come a long way from being identified as proteins that agglutinate cells to promising therapeutic agents in modern medicine. Through their specific binding property, they have proven to be anti-cancer, anti-insect, anti-viral agents without affecting the non-target cells. The Arisaema tortuosum lectin (ATL) is a known anti-insect and anti-cancer candidate, also has interesting physical properties. In the present work, its carbohydrate binding behavior is investigated in detail, along with its anti-proliferative property. RESULTS: The microcalorimetry of ATL with a complex glycoprotein asialofetuin demonstrated trivalency contributed by multiple binding sites and enthalpically driven spontaneous association. The complex sugar specificity of ATL towards multiple sugars was also demonstrated in glycan array analysis in which the trimannosyl pentasaccharide core N-glycan [Manα1-6(Manα1-3)Manß1-4GlcNAcß1-4GlcNAcß] was the highest binding motif. The high binding glycans for ATL were high mannans, complex N-glycans, core fucosylated N-glycans and glycans with terminal lactosamine units attached to pentasaccharide core. ATL induced cell death in IMR-32 cells was observed as time dependent loss in cell number, formation of apoptotic bodies and DNA damage. As a first report of molecular cloning of ATL, the in silico analysis of its cDNA revealed ATL to be a ß-sheet rich heterotetramer. A homology model of ATL showed beta prism architecture in each monomer with 85% residues in favoured region of Ramachandran plot. CONCLUSIONS: Detailed exploration of carbohydrate binding behavior indicated ATL specificity towards complex glycans, while no binding to simple sugars, including mannose. Sequence analysis of ATL cDNA revealed that during the tandem evolutionary events, domain duplication and mutations lead to the loss of mannose specificity, acquiring of new sugar specificity towards complex sugars. It also resulted in the formation of a two-domain single chain polypeptide with both domains having different binding sites due to mutations within the consensus carbohydrate recognition sites [QXDXNXVXY]. This unique sugar specificity can account for its significant biological properties. Overall finding of present work signifies anti-cancer, anti-insect and anti-viral potential of ATL making it an interesting molecule for future research and/or theragnostic applications.

Tuber extract of Arisaema flavum eco-benignly and effectively synthesize silver nanoparticles: Photocatalytic and antibacterial response against multidrug resistant engineered E. coli QH4.

Metal nanoparticles, synthesized using Phyto-constituents are the most economically and environmentally benign materials ever. Biogenic silver nanoparticles (AgNPs) from three fractions of Arisaema flavum tuber extract were synthesized and characterized by UV-visible spectroscopy, XRD (X-rays diffraction), FT-IR (Fourier transform infrared spectroscopy) TEM (transmission electron microscopy) and EDX (Energy dispersive Microscopy). XRD pattern show the face centred cubic crystalline (Fcc) structure of AgNPs. FTIR spectra confirmed the presence of different Polyphenolic compounds capping the AgNps. UV-visible spectroscopy result confirmed the presence of Ag because of the particular surface plasmon Resonance (SPR) in the area of 400-430 nm. The electron microscope studies revealed the formation of spherical AgNPs with diameter ranging from 12 nm to 20 nm. Strong signals of AgNPs were confirmed with EDX analysis. The antibacterial properties of the AgNPs prepared with various extracts were tested against multi-drug resistant bacteria. Which showed significant antibacterial activity against all the multidrug resistant bacterial strains and especially multidrug resistant engineered E.ColiQH4. AgNPs synthesized by methanolic, Ethyl Acetate and aqueous Extracts of Areseama Flavum exhibited significant Photocatalytic activity to reduce methylene blue. Small size, spherical shape and high dispersion are the key properties due to which the AgNPs are having significant biological and photocatalytic activity. To the best of our knowledge, it is the first report of biogenic AgNPs regarding antibacterial activity against multidrug resistant Engineered E.Coli QH4.

De novo sequencing and transcriptome assembly of Arisaema heterophyllum Blume and identification of genes involved in isoflavonoid biosynthesis.

Arisaema heterophyllum Blume (AhBl) is one of the valued medicinal plants. However, its genetic information is limited, which impedes further studies of this valuable resource. To investigate the genes involved in the isoflavonoid biosynthesis, we deeply performed transcriptome sequencing for AhBl. An average of 10.98 Gb clean reads were obtained based on root, tuber and leaf tissues, and 109,937 unigenes were yielded after de novo assembly. In total, 72,287 of those unigenes were annotated in at least one public database. The numbers of expressed unigenes in each tissue were 35,686, 43,363 and 47,783, respectively. The overall expression levels of transcripts in leaf were higher than those in root and tuber. Differentially expressed genes analysis indicated that a total of 12,448 shared unigenes were detected in all three tissues, 10,215 of which were higher expressed in tuber than that in root and leaf. Besides, 87 candidate unigenes that encode for enzymes involved in biosynthesis of isoflavonoid were identified and analyzed, and some key enzyme genes were experimentally validated by quantitative Real-Time PCR (qRT-PCR). This study provides a unique dataset for the systematic analysis of AhBl functional genes and expression characteristics, and facilitates the future study of the pharmacological mechanism of AhBl.

Tuber Lectins with Potentially Exploitable Bioactivities.

Lectins are a group of proteins or glycoproteins with various potentially exploitable bioactivities and have been capturing more interest recently. They have been isolated and reported from various tissues of a diversity of plant species. Tubers are modified and enlarged plant structures derived from stems or roots that are used for nutrient storage and asexual reproduction. A number of plants such as yam, taro and potato are grown for their edible tubers, and lectins are found to be one of the major storage proteins. These lectins exhibit potent bioactivities encompassing mitogenic, antitumor, antimicrobial, immunomodulatory, antioxidative, hypoglycemic, insecticidal and nematicidal activities. They are potential resources for development into functional or healthy foods and targets for food protein researchers.

Cloning and characterization of an agglutinin gene from Arisaema lobatum.

A novel agglutinin gene was cloned from Arisaema lobatum using SMART RACE-PCR technology. The full-length cDNA of Arisaema lobatum agglutinin (ala) was 1078 bp and contained a 774 bp open reading frame encoding a lectin precursor (proproprotein) of 258 amino acid residues with a 23 aa signal peptide. ALA contained three mannose-binding sites (QXDXNXVXY) with two-conserved domains of 45% identity, ALA-DOM1 and ALA-DOM2. The three-dimensional structure of ALA was very similar to that of GNA (Galanthus nivalis agglutinin). ALA shared varying identities, ranging from 40% to 85%, with mannose-binding lectins from other species of plant families, such as Araceae, Alliaceae, Iridaceae, Lillaceae, Amaryllidaceae and Bromeliaceae. Genomic sequence of ala was also cloned using genomic walker technology, and it was found to contain three putative TATA boxes and eight possible CAAT boxes in the 5'-flanking region. No intron was found within the region of genomic sequence. Southern blot analysis indicated that the ala belonged to a multi-copy gene family. Expression pattern analysis revealed that the ala preferentially expressed in the tissues with the higher expression being found in spadix, bud, leaf, spathe and tuber. The cloning of the ala gene not only provides a basis for further investigation of its structure, expression and regulation mechanism, but also enables us to test its potential role in controlling pests and fungal diseases by transferring the gene into plants in the future.

An efficient method for rapid amplification of Arisaema heterophyllum agglutinin gene using a genomic walking technique.

The genomic sequence of Arisaema heterophyllum agglutinin (AHA), a mannose-binding lectin (MBL), was cloned through a novel genomic walking technique. Adaptor ligation reactions and subsequent amplifications with adaptor primer and multiple specific primers were used to generate specificity in this method. The method allowed for the amplification of over 1 kb of genomic DNA sequence immediately upstream and downstream from the 5' and 3' ends of full-length cDNAs. For aha gene, the upstream regions contained a putative transcription initiation start site and other sequences commonly found in eukaryotic promoters. The downstream regions of aha contained two polyadenylation signals. Our study demonstrated that aha had no intron like mannose-binding lectin genes cloned from other plant species so far. This efficient method, based on a genomic walking technique, was useful for the cloning of promoters, insertion sites, and other sequences of interest without constructing and screening genomic libraries.

Isolation and characterization of two N-acetyl-D-lactosamine specific lectins from tubers of Arisaema intermedium Blume and A. wallichianum Hook f.

Two new lectins were purified from the tubers of Arisaema intermedium Blume and A. wallichianum Hook. f. (family: Araceae) by affinity chromatography on asialofetuin-linked amino activated silica beads. The bound lectins were eluted with 0.1 M glycine-HCl, pH 2.5. They gave a single band corresponding to subunit M(r) 13.4 kDa in SDS-PAGE, pH 8.3. On gel filtration chromatography, the lectins showed a M(r) of 51.2 kDa, suggesting a homotetrameric structure. Both the lectins gave a single peak on size exclusion HPLC and cation-exchange columns and a single band on PAGE, pH 4.5. However, like other monocot lectins, they gave multiple bands in isoelectric focusing and at PAGE 8.3. The lectins were inhibited by N-acetyl-D-lactosamine (LacNAc), a disaccharide and asialofetuin, a complex desialylated serum glycoprotein. They had no requirement for divalent metal ions i.e., Ca2+ and Mn2+ for their activity and were found to be mitogenic towards human lymphocytes. A. intermedium showed antiproliferative effect against various human cancer cell lines in vitro.