Junichi Shinozaki

Identification of medicinal Dendrobium species by phylogenetic analyses using matK and rbcL sequences.

Species identification of five Dendrobium plants was conducted using phylogenetic analysis and the validity of the method was verified. Some Dendrobium plants (Orchidaceae) have been used as herbal medicines but the difficulty in identifying their botanical origin by traditional methods prevented their full modern utilization. Based on the emerging field of molecular systematics as a powerful classification tool, a phylogenetic analysis was conducted using sequences of two plastid genes, the maturase-coding gene (matK) and the large subunit of ribulose 1,5-bisphosphate carboxylase-coding gene (rbcL), as DNA barcodes for species identification of Dendrobium plants. We investigated five medicinal Dendrobium species, Dendrobium fimbriatum, D. moniliforme, D. nobile, D. pulchellum, and D. tosaense. The phylogenetic trees constructed from matK data successfully distinguished each species from each other. On the other hand, rbcL, as a single-locus barcode, offered less species discriminating power than matK, possibly due to its being present with little variation. When results using matK sequences of D. officinale that was deposited in the DNA database were combined, D. officinale and D. tosaense showed a close genetic relationship, which brought us closer to resolving the question of their taxonomic identity. Identification of the plant source as well as the uniformity of the chemical components is critical for the quality control of herbal medicines and it is important that the processed materials be validated. The methods presented here could be applied to the analysis of processed Dendrobium plants and be a promising tool for the identification of botanical origins of crude drugs.

Squalene cyclase and oxidosqualene cyclase from a fern

Ferns are the most primitive vascular plants. The phytosterols of ferns are the same as those of higher plants, but they produce characteristic triterpenes. The most distinct feature is the lack of oxygen functionality at C‐3, suggesting that the triterpenes of ferns may be biosynthesized by direct cyclization of squalene. To obtain some insights into the molecular bases for the biosynthesis of triterpenes in ferns, we cloned ACX, an oxidosqualene cyclase homologue, encoding a cycloartenol synthase (CAS) and ACH, a squalene cyclase homologue, encoding a 22‐hydroxyhopane synthase from Adiantum capillus‐veneris. Phylogenetic analysis revealed that ACH is located in the cluster of bacterial SCs, while ACX is in the cluster of higher plant CASs.

Dammaradiene synthase, a squalene cyclase, from Dryopteris crassirhizoma Nakai ☆

Ferns produce a variety of cyclic triterpene hydrocarbons in large amount. Squalene cyclases (SCs) are responsible enzymes for formation of cyclic triterpene hydrocarbon skeletons. Although more than ten bacterial SCs have been cloned and four of them characterized for their enzymatic products, the only example of a fern SC is ACH, from Adiantum capillus-veneris, which produces hydroxyhopane. To obtain a deeper understanding of the molecular evolution of SCs and the origin of the structural diversity of fern triterpenes, further cloning and characterization of SCs have been pursued. In this study, a SC cDNA, DCD, was cloned from Dryopteris crassirhizoma by homology-based RT-PCR. DCD contains a 2058-bp open reading frame that encodes a 685 amino acid polypeptide exhibiting 66% identity to the previously identified fern SC, ACH, and 35-40% identity to bacterial SCs. Heterologous expression of DCD in yeast established it to be a dammaradiene synthase affording dammara-18(28),21-diene, a tetracyclic triterpene hydrocarbon. Although neither this compound nor any derived metabolites have been previously reported from D. crassirhizoma, re-investigation of the leaflets demonstrated the presence of dammara-18(28),21-diene. DCD represents the first SC that produces a tetracyclic triterpene hydrocarbon.

Water-soluble undenatured type II collagen ameliorates collagen-induced arthritis in mice.

Earlier studies have reported the efficacy of type II collagen (C II) in treating rheumatoid arthritis (RA). However, a few studies have investigated the ability of the antigenic collagen to induce oral tolerance, which is defined as active nonresponse to an orally administered antigen. We hypothesized that water-soluble undenatured C II had a similar effect as C II in RA. The present study was designed to examine the oral administration of a novel, water-soluble, undenatured C II (commercially known as NEXT-II) on collagen-induced arthritis (CIA) in mice. In addition, the underlying mechanism of NEXT-II was also identified. After a booster dose (collagen-Freunds complete adjuvant), mice were assigned to control CIA group, or NEXT-II treatment group, to which saline and NEXT-II were administered, respectively. The arthritis index in the NEXT-II group was significantly lower compared with the CIA group. Serum IL-6 levels in the NEXT-II group were significantly lower compared with the CIA group, while serum IL-2 level was higher. Furthermore, oral administration of NEXT-II enhanced the proportion of CD4+CD25+T (Treg) cells, and gene expressions of stimulated dendritic cells induced markers for regulatory T cells such as forkhead box p3 (Foxp3), transforming growth factor (TGF)-β1, and CD25. These results demonstrated that orally administered water-soluble undenatured C II (NEXT-II) is highly efficacious in the suppression of CIA by inducing CD4+CD25+ Treg cells.

Molecular Evolution of Fern Squalene Cyclases

Triterpenes, a diverse group of natural products comprising six isoprene units, are distributed across various organisms from bacteria to higher plants. Ferns are sporophytes that produce triterpenes and are lower on the evolutionary scale than higher plants. Among ferns that produce triterpenes analogous to bacterial hopanoids, Polypodiodes niponica produces migrated dammaranes and oleananes, which are also widely found in higher plants. Because the study of terpene‐producing ferns could help us to understand the molecular basis of triterpene biosynthesis, cDNA cloning of squalene cyclases (SCs) from P. niponica was carried out. Two SCs (PNT and PNG) were obtained. The heterologously expressed PNT produces tirucalla‐7,21‐diene (67 % major), and PNG produces germanicene (69 %). Phylogenetic analysis revealed that PNT and PNG, which produce higher‐plant‐type migrated dammaranes and oleananes, are closely related to bacterial‐type SCs. Furthermore, analysis of the minor products indicated that fern SCs gained the ability to directly form dammarenyl cations, which are key intermediates in oleanane formation during molecular evolution.

Cyclization of all-E- and 2Z-geranylfarnesols by a bacterial triterpene synthase: insight into sesterterpene biosynthesis in Aleuritopteris ferns.

Aleuritopteris ferns produce triterpenes and sesterterpenes with tricyclic cheilanthane and tetracyclic 18-episcalarane skeletons. The structural and mechanistic similarities between both classes of fern terpene suggest that their biosynthetic enzymes may be closely related. We investigate here whether a triterpene synthase is capable of recognizing geranylfarnesols as a substrate, and is able to convert them to cyclic sesterterpenes. We found that a bacterial triterpene synthase converted all-E-geranylfarnesol (1b) into three scalarane sesterterpenes with 18αH stereochemistry (5, 7 and 8), as well as mono- and tricyclic sesterterpenes (6 and 9). In addition, 2Z-geranylfarnesol (4) was converted into an 18-episcalarane derivative (10), whose skeleton can be found in sesterterpenes isolated from Aleuritopteris ferns. These results provide insight into sesterterpene biosynthesis in Aleuritopteris ferns.

Migrated Hopene Synthases from Colysis pothifolia and Identification of a Migration Switch Controlling the Number of 1,2-Hydride and Methyl Shifts.

Ferns are known to produce triterpenes, derived from squalene, that are synthesized by squalene cyclases (SCs). Among these, Colysis species produce onoceroids, the bis‐cyclic skeleton of which can be cyclized from both termini of squalene. To gain insight into the molecular basis of triterpene structural diversity, cDNA cloning of SCs from C. elliptica and C. pothifolia was performed; this leads to the isolation of five SC cDNAs. Functional analysis of these clones revealed their enzymatic products to be hop‐22(29)‐ene, α‐polypodatetraene, and hop‐17(21)‐ene. One of these clones (CPF) is a transcribed pseudogene with a 22‐nucleotide deletion causing a nonsense mutation. To predict the inherent function of CPF, we constructed an insertion mutant of CPF that successfully converted inert CPF to the active SC, the product of which is fern‐9(11)‐ene. Subsequent mutations identified active‐site residues that control the number of 1,2‐hydride and methyl shifts.

Squalene Cyclases and Cycloartenol Synthases from Polystichum polyblepharum and Six Allied Ferns

Ferns are the most primitive of all vascular plants. One of the characteristics distinguishing them from flowering plants is its triterpene metabolism. Most cyclic triterpenes in ferns are hydrocarbons derived from the direct cyclization of squalene by squalene cyclases (SCs). Both ferns and more complex plants share sterols and biosynthetic enzymes, such as cycloartenol synthases (CASs). Polystichum belongs to Dryopteridaceae, and is one of the most species-rich of all fern genera. Several Polystichum ferns in Japan are classified as one of three possible chemotypes, based on their triterpene profiles. In this study, we describe the molecular cloning and functional characterization of cDNAs encoding a SC (PPH) and a CAS (PPX) from the type species Polystichum polyblepharum. Heterologous expression in Pichia pastoris revealed that PPH and PPX are hydroxyhopane synthase and CAS, respectively. By using the PPH and PPX sequences, we successfully isolated SC- and CAS-encoding cDNAs from six Polystichum ferns. Phylogenetic analysis, based on SCs and oxidosqualene cyclase sequences, suggested that the Polystichum subclade in the fern SC and CAS clades reflects the chemotype—but not the molecular phylogeny constructed using plastid molecular markers. These results show a possible relation between triterpenes and their biosynthetic enzymes in Polystichum.

PCR-RFLP Method for Rapid Discrimination of Toxic Plants Involved in Food Poisoning

Food poisoning caused by natural toxins, especially poisonous plants, is characterized by severe symptoms and a relatively high mortality rate. Therefore, rapid and accurate identification of the causative agent is extremely important. From plant toxin food poisoning data published by the Ministry of Health, Labour and Welfare of Japan from 1989 to 2015, we selected five plants (Veratrum spp., Datura spp., Aconitum spp., Narcissus spp. and Colchicum spp.) that are frequently involved in poisoning outbreaks, and developed a PCR-RFLP assay to discriminate them. Separation of the PCR-RFLP products by electrophoresis resulted in detection of two fragments from poisonous plants and one from edible plants. The PCR-RFLP method is rapid and straightforward and does not require expensive analytical devices. This assay was also confirmed to be applicable to cooked samples.