Linda S. M. Ooi

Antimicrobial Activities of Cinnamon Oil and Cinnamaldehyde from the Chinese Medicinal Herb Cinnamomum cassia Blume

Both Cinnamomum verum J.S. Presl. and Cinnamomum cassia Blume are collectively called Cortex Cinnamonmi for their medicinal cinnamon bark. Cinnamomum verum is more popular elsewhere in the world, whereas C. cassia is a well known traditional Chinese medicine. An analysis of hydro-distilled Chinese cinnamon oil and pure cinnamaldehyde by gas chromatography/mass spectrometry revealed that cinnamaldehyde is the major component comprising 85% in the essential oil and the purity of cinnamaldehyde in use is high (> 98%). Both oil and pure cinnamaldehyde of C. cassia were equally effective in inhibiting the growth of various isolates of bacteria including Gram-positive (1 isolate, Staphylococcus aureus), and Gram-negative (7 isolates, E. coli, Enterobacter aerogenes, Proteus vulgaris, Pseudomonas aeruginosa, Vibrio cholerae, Vibrio parahaemolyticus and Samonella typhymurium), and fungi including yeasts (four species of Candida, C. albicans, C. tropicalis, C. glabrata, and C. krusei), filamentous molds (4 isolates, three Aspergillus spp. and one Fusarium sp.) and dermatophytes (three isolates, Microsporum gypseum, Trichophyton rubrum and T. mentagraphytes). Their minimum inhibition concentrations (MIC) as determined by agar dilution method varied only slightly. The MICs of both oil and cinnamaldehyde for bacteria ranged from 75 microg/ml to 600 microg/ml, for yeasts from 100 microg/ml to 450 microg/ml, for filamentous fungi from 75 microg/ml to 150 microg/ml, and for dermatophytes from 18.8 microg/ml to 37.5 microg/ml. The antimicrobial effectiveness of C. cassia oil and its major constituent is comparable and almost equivalent, which suggests that the broad-spectrum antibiotic activities of C. cassia oil are due to cinnamaldehyde. The relationship between structure and function of the main components of cinnamon oil is also discussed.

Narcissus tazetta lectin shows strong inhibitory effects against respiratory syncytial virus, influenza A (H1N1, H3N2, H5N1) and B viruses

A mannose-binding lectin (Narcissus tazetta lectin [NTL]) with potent antiviral activity was isolated and purified from the bulbs of the Chinese daffodil Narcissus tazetta var. chinensis, using ion exchange chromatography on diethylaminoethyl (DEAE)-cellulose, affinity chromatography on mannose-agarose and fast protein liquid chromatography (FPLC)-gel filtration on Superose 12. The purified lectin was shown to have an apparent molecular mass of 26 kDa by gel filtration and 13 kDa by SDS-PAGE, indicating that it is probably a dimer with two identical subunits. The cDNA-derived amino acid sequence of NTL as determined by molecular cloning also reveals that NTL protein contains a mature polypeptide consisting of 105 amino acids and a C-terminal peptide extension. Three-dimensional modelling study demonstrated that the NTL primary polypeptide contains three subdomains, each with a conserved mannose-binding site. It shows a high homology of about 60%–80% similarity with the existing monocot mannose-binding lectins. NTL could significantly inhibit plaque formation by the human respiratory syncytial virus (RSV) with an IC50 of 2.30 µg/ml and exhibit strong antiviral properties against influenza A (H1N1, H3N2, H5N1) and influenza B viruses with IC50 values ranging from 0.20 µg/ml to 1.33 µg/ml in a dose-dependent manner. It is worth noting that the modes of antiviral action of NTL against RSV and influenza A virus are significantly different. NTL is effective in the inhibition of RSV during the whole viral infection cycle, but the antiviral activity of NTL is mainly expressed at the early stage of the viral cycle of influenza A (H1N1) virus. NTL with a high selective index (SI=CC50/IC50≥141) resulting from its potent antiviral activity and low cytotoxicity demonstrates a potential for biotechnological development as an antiviral agent.

Isolation, characterization, molecular cloning and modeling of a new lipid transfer protein with antiviral and antiproliferative activities from Narcissus tazetta

A fetuin-binding peptide with a molecular mass of about 9kDa (designated NTP) was isolated and purified from the bulbs of Chinese daffodil, Narcissus tazetta var. chinensis L., by gel filtration and high-performance liquid chromatography, after removing the mannose-binding proteins by mannose-agarose column. Molecular cloning revealed that NTP contained an open reading frame of 354bp encoding a polypeptide of 118 amino acids which included a 26-amino-acid signal peptide. An analysis of the deduced amino acid sequence of NTP shows considerable sequence homology to the non-specific lipid transfer proteins (nsLTPs) of certain plants. Model of the three-dimensional (3D) structure of NTP exhibits an internal hydrophobic cavity which can bind lipid-like molecules and transfer a wide range of ligands. As a member of the putative non-specific lipid transfer protein of N. tazetta, NTP did not possess hemagglutinating activity toward rabbit erythrocytes. In a cell-free system, it could arrest the protein synthesis of rabbit reticulocytes. Using the in vitro antiviral assays, NTP could significantly inhibit the plaque formation by respiratory syncytial virus (RSV) and the cytopathic effect induced by influenza A (H1N1) virus, as well as the proliferation of human acute promyelocytic leukemia cells (HL-60).

Purification and characterization of non-specific lipid transfer proteins from the leaves of Pandanus amaryllifolius (Pandanaceae).

Two proteins were isolated from the saline extract of mature leaves of Pandanus amaryllifolius, using affinity chromatography on fetuin-agarose and Affi-gel Blue gel, anion exchange chromatography as well as gel filtration. The proteins were demonstrated as non-glycoproteins, with molecular mass of 18 and 13 kDa, respectively, comprising of peptide subunits from 6.5 to 9 kDa in the forms of heterodimer and homodimer. All of them have similar N-terminal amino acid sequences with only minor variations and are matched to non-specific lipid transfer proteins (nsLTPs) of the other plants such as wheat LTP using NCBI Blast searching for short, nearly exact matches. Furthermore, they explicated each other as isoforms originated putatively from a multigene family with various molecular weight, binding affinity, ionic strength, and subunits. However, the potencies for antiproliferation of HL-60 cell line and inhibition of the growth of the bacteria Pseudomonas aeruginosa are different in that those of the fetuin-binding protein are greater than non-fetuin binding proteins. The non-specific lipid transfer proteins of P. amaryllifolius exhibit weak to moderate hemagglutinating activity toward rabbit erythrocytes, but, this activity could not be reversed by mannose. They thus could be easily differentiated from the previously reported mannose-binding lectin isolated from this plant, which has subunits with similar molecular weight.

Molecular Cloning and the cDNA-Derived Amino Acid Sequence of Narcissus tazetta Isolectins

Recently several complete cDNAs encoding the Narcissus tazetta lectins (NTL) were cloned. The sequence analyses of the cloned DNAs reveal that there are at least three unidentical positive clones for NTLs. The primary structure of the three NTL clones contains a mature polypeptide consisting of 105 amino acids and a C-terminal peptide extension beyond the C-terminal amino acids Thr-Gly. There are two fixed-position cysteines within the protein domain (amino acids 29 and 52), which are probably involved in the disulfide-bond linkage within the molecules to confer the secondary structure of the mature lectin. One third of the deduced amino acid composition consisted of glycine, leucine, and asparagine. From the cDNA-derived amino acid sequences the three NTL clones are not identical and are suggested to be isolectins present in N. tazetta var. chinensis. This study further confirms the previous isolation of mannose-specific isolectins from Chinese daffodil leaves [Ooi et al. (2000), J. Protein Chem.19, 163-168].

Mannose-Specific Isolectins with Different Hemagglutinating Potencies Isolated from Chinese Daffodil (Narcissus tazetta var. chinensis) Leaves

Three mannose-specific lectins exhibiting considerable similarities in NH2-terminal amino acid sequence were isolated from leaves of the Chinese daffodil Narcissus tazetta (Family Amaryllidaceae). The purification protocol involved extraction with an aqueous buffer, anion exchange chromatography on DEAE-cellulose using stepwise elution with increasing salt concentrations, affinity chromatography on mannose-agarose, and FPLC-gel filtration on Superose 12. From the peak unadsorbed on DEAE-cellulose, and two peaks adsorbed on the ion exchanger and eluted respectively with 0.2 M Tris-HCl buffer and 0.5 M NaCl, were prepared fractions which yielded isolectins 1, 2, and 3 after adsorption on mannose-agarose and FPLC-gel filtraton. All three isolectins were homodimers with a molecular weight of 26 kDa. The lectin unadsorbed on DEAE-cellulose had the lowest, while the most strongly adsorbed lectin had the highest hemagglutinating activity.