Selective inhibition of vascular smooth muscle cell proliferation by coptisine isolated from Coptis rhizoma, one of the crude drugs composing Kampo medicines Unsei-in.

Acceleration of vascular smooth muscle cell (VSMC) proliferation is closely linked to the pathogenesis of vascular diseases. We, therefore, focused on traditional Japanese herbal medicines (Kampo medicines) used to ameliorate the impairment of microcirculation or blood stasis and screened them for their ability to inhibit rat VSMC proliferation. Among them, Unsei-in was found to effectively suppress VSMC proliferation, and Coptis rhizome was the responsible constituent crude drug. The extract of Coptis rhizome inhibited VSMC proliferation with the GI(50) value of 4.4 microg/ml, which was much lower than those against the proliferation of 3Y1, dRLh-84, B16, and HeLa cells. The Coptis rhizome extract inhibited the progression of VSMC arrested at G(0)/G(1) phase from G(0)/G(1) to S phase, but not that of 3Y1 cells. Biological assay-guided fractionation revealed that an alkaloid of Coptis rhizome, coptisine, was the active ingredient in selectively preventing VSMC proliferation with GI(50) of 3.3 microM (1.2 microg/ml). When the structurally-related isoquinoline alkaloids of protoberberine class were studied for their inhibitory activities, berberine decreased the VSMC proliferation with GI(50) of 95.1 microM (35.4 microg/ml), about 30 times higher concentration than coptisine, while palmatine failed to show any activity. This study provides evidence that coptisine, an ingredient of Unsei-in, prevents VSMC proliferation selectively at lower concentrations compared with various cells or other structurally related alkaloids.

Identification of the crude drug atractylodes rhizome (Byaku-jutsu) and atractylodes lancea rhizome (So-jutsu) using chloroplast TrnK sequence as a molecular marker.

Novel methods for molecular authentication of Atractylodes-derived crude drugs (Jutsu) were established based on PCR-restriction fragment length polymorphism (RFLP) and direct sequencing of chloroplast trnK. Two regions inside the chloroplast trnK were selected as molecular markers for identification and discrimination of Atractylodes Rhizome (Byaku-jutsu) and Atractylodes Lancea Rhizome (So-jutsu). The Region 1 fragment (260 bp) amplified from So-jutsu and Wa-byaku-jutsu (Atractylodes Rhizome derived from A. japonica) gave 2 bands of 180 bp and 80 bp on agarose gel electrophoresis after digestion with a restriction endonuclease HinfI, whereas the fragment amplified from Kara-byaku-jutsu (Atractylodes Rhizome derived from A. ovata) remained undigested, which allowed unambiguous identification of Kara-byaku-jutsu. By direct sequencing of Region 2 (436 bp) and comparison of the nucleotide sequence data sets we could not only discriminate Byaku-jutsu and So-jutsu but also identify the original plant species of each crude drug specimen. A simple and reliable protocol for rapid preparation of DNA suitable for PCR from as little as 1 mg of Atractylodes-derived crude drugs was also described.

Sesquiterpenoid derivatives from Ferula ferulaeoides [correction of ferulioides]. IV.

Four novel prenyl-furocoumarin type sesquiterpenoid derivatives, 2,3-dihydro-7-hydroxy-2S*,3R*-dimethyl-3-[4,8-dimethyl-3(E),7-nonadie nyl]-furo[3,2-c]coumarin, 2,3-dihydro-7-hydroxy-2R*,3R*-dimethyl-3-[4,8-dimethyl-3(E),7-nonadie nyl]-furo[3,2-c]coumarin, 2,3-dihydro-7-hydroxy-2S*,3R*-dimethyl-3-[4-methyl-5-(4-methyl-2-furyl)- 3(E)-pentenyl]-furo[3,2-c]coumarin, and 2,3-dihydro-7-methoxy-2S*,3R*-dimethyl-3-[4,8-dimethyl-3(E),7-nonadie nyl]-furo]3,2-c]coumarin were isolated from the roots of Ferula ferulaeoides [corrected]. Their structures were established by detailed spectral analysis and the biosynthetic pathway leading to these prenyl-furocoumarin type sesquiterpenoids is proposed based on these structures.

A simple and rapid protocol for preparation of crude drug DNA suitable for PCR.

A protocol for rapid purification of DNA from a variety of herbal crude drugs, starting from samples as small as 1 mg, has been developed. Neither phenol extraction nor alcohol precipitation is involved in the procedure. Total nucleic acids are first extracted with the lysis buffer containing SDS. After CHCl3 extraction, DNA is bound to glass fiber fleece in the presence of guanidine thiocyanate, while unbound impurities are washed away. The entire procedure can be carried out in 1.5-ml microtubes. The purified DNA obtained serves as a template for PCR.

DNA profiling of Acorus calamus chemotypes differing in essential oil composition.

The phylogenetic relationship of Acorus gramineus and three types of Acorus calamus was analyzed by comparing the 700 bp sequence of a 5S-rRNA gene spacer region. Although there was no intra-specific variation in the essential oil profile of A. gramineus which contained a phenylpropanoid (Z-asarone) as a predominant constituent, A. calamus was classified into two chemotypes: chemotype A in which Z-asarone is a major essential oil constituent and chemotype B which contained sesquiterpenoids predominantly. An intermediate type (M) of these two chemotypes in various ratios was also observable. The NJ tree constructed based on the sequences revealed that A. gramineus was clearly distinguished from any of the chemotypes of A. calamus and that the phylogenetic relationship predicted by the spacer region data correlated well with the essential oil chemotype pattern of A. calamus.

Phylogenetic analysis of Atractylodes plants based on chloroplast trnK sequence.

The phylogenetic relationship of Atractylodes lancea, A. chinensis, A. koreana, A. ovata and A. japonica were analyzed by comparing the 2.6 kb sequence in a chloroplast gene trnK encoding tRNALys (UUU). The dried rhizomes of the former three species have been used as the crude drug "So-jutsu" and those of the latter two as "Byaku-jutsu" in Chinese and Japanese traditional medicine ("Kampo-medicine"). The trnK phylogenetic tree revealed that A. ovata is an outgroup of the five Atractylodes species examined and that A. japonica and A. lancea are most closely related. PCR amplification of trnK with HinfI digestion provided us with a simple method to distinguish A. ovata from other Atractylodes species at the molecular level.

Induction of furanocoumarin biosynthesis in Glehnia littoralis cell suspension cultures by elicitor treatment.

Cell suspension cultures were established from Glehnia littoralis plants belonging to two different geographic strains. When the cells were treated with yeast extract, they started to produce and excrete furanocoumarins into the culture medium; a major component, bergapten, and a minor one, xanthotoxin, were detected and identified by HPLC and GC/MS. Changes in phenylalanine ammonia-lyase (PAL) activity and furanocoumarin production after elicitor treatment were traced, showing that PAL activity increased rapidly, reached a maximum after 24 h, and then declined to the normal level after 96 h which preceded the induced bergapten production. The induced-PAL activity of the cultured cells established from an S-type plant which accumulated trace amounts of furanocoumarins was about 50% of that in the cultured cells from an N-type plant that accumulated more than 0.1% furanocoumarins in the underground parts. However, the elicited production of bergapten was about six times higher in the cell cultures from the S-type plant. Addition of the PAL inhibitor 2-aminoindan-2-phosphoric acid (AIP) at 10 microM suppressed the induction of PAL activity and furanocoumarin production.

Long-term administration of "sho-saiko-to" increases cytochrome P-450 mRNA level in mouse liver.

Simplified differential display of mRNA was applied to isolate and identify genes transcriptionally regulated in mouse liver by sho-saiko-to administration. A cDNA fragment up-regulated by sho-saiko-to was isolated and characterized. cDNA sequencing and subsequent database analysis revealed that the fragment showed significant sequence similarity with mouse testosterone 16-alpha-hydroxylase (cytochrome P-450[16alpha]) cDNA. The increased level of mRNA expression of cytochrome P-450(16alpha) in association with sho-saiko-to administration suggests the molecular mechanism of the chemopreventive effect of sho-saiko-to. This result indicates the usefulness of the mRNA differential display technique to investigate the molecular mechanism of Kampo medicine.

Cloning and bacterial expression of a gene encoding ribosome-inactivating proteins, karasurin-A and karasurin-C, from Trichosanthes kirilowii var. japonica.

A genomic DNA clone of karasurin was isolated using the polymerase chain reaction from Trichosanthes kirilowii var. japonica (Cucurbitaceae). The amino acid sequence deduced from the nucleotide sequence was consistent with previously reported sequences of karasurin-A and karasurin-C except for a putative signal peptide and extra amino acids at the C-terminus, neither of which is present in the natural protein. Recombinant karasurin was synthesized in Escherichia coli, in which the cloned karasurin gene was expressed under the control of the trc promoter.

Random amplified polymorphic DNA analysis and variation of essential oil components of Atractylodes plants.

Total DNAs were prepared from the leaves of Atractylodes lancea DE CANDOLLE, A. chinensis KOIDZUMI, A. lancea var. simplicifolia KITAMURA, A. japonica KOIDZUMI ex KITAMURA and A. ovata DE CANDOLLE. The DNAs were subjected to random amplified polymorphic DNA (RAPD) analysis. Some primers showed the definitive polymorphic DNA patterns in A. lancea, A. japonica and A. ovata. The RAPD of A. lancea var. simplicifolia and one of A. chinensis gave similar patterns to those of A. lancea, but one of the other A. chinensis gave a similar pattern to A. japonica. Furthermore, quantitative analysis of atractylon, hinesol, beta-eudesmol and atractylodin in the rhizomes was done using gas chromatography. Though atractylon was detected not only in A. japonica and A. ovata but also in some of A. lancea, their RAPD profiles revealed the presence of intraspecific variation with A. lancea.

Effect of karasurin-A on nitric oxide production by murine macrophages and mitogenic response of murine splenocytes in vitro.

We studied the effect of karasurin-A, a type-I ribosome-inactivating protein (RIP) isolated from fresh root tubers of Trichosanches kirilowii MAX. var. japonica KITAMURA, on the mitogenic response of murine splenocytes and nitric oxide (NO) production by murine peritoneal macrophages in vitro. Karasurin-A inhibited the lymphocyte proliferation by LPS, ConA and PHA and NO production by LPS at non-cytotoxic concentrations (10-1000 ng/ml for splenocytes and 10-100 ng/ml for macrophages, respectively). These data suggest that karasurin-A has immunosuppressive activity in vitro.

Amino acid sequences and ribosome-inactivating activities of karasurin-B and karasurin-C.

Complete amino acid sequences of karasurin-B and karasurin-C purified from root tubers of Trichosanthes kirilowii Maxim.var.japonica Kitamura (Cucurbitaceae) were determined. Karasurin-B consist of 247 amino acid residues with a calculated molecular weight of 27,214 Da and karasurin-C of 249 amino acid residues with a calculated molecular weight of 27,401 Da. Their amino acid sequences are quite similar to that of karasurin-A, a major basic protein from the same root tubers, but the pI values are slightly different among them. All karasurins strongly inhibited in vitro translation in the rabbit reticulocyte system. The amino acid sequences of karasurins were compared with those of the ribosome-inactivating proteins (RIPs) from nine different species by UPGMA method, and the RIPs fro Cucurbitaceae plants phylogenetically formed one cluster clearly distinguishable from those of other origin.

Restriction fragment length polymorphisms of rDNA and variation of essential oil composition in Atractylodes plants.

Total DNA was extracted from the leaves of Atractylodes lancea DE CANDOLLE, A. ovata DE CANDOLLE and A. japonica KOIDZUMI ex KITAMURA of various origins and hybridized with digoxigenin-labeled rice ribosomal DNA after digestion with eight different restriction endonucleases. The resulting restriction fragment length polymorphism (RFLP) profiles allowed us to distinguish the three Atractylodes species when DNA was digested with Sac I. Although atractylon was detected in the rhizomes of some of the cultivated strains of A. lancea, their RFLP profiles clearly indicate that these plants are not hybrids of A. ovata or A. japonica. RFLP analysis also revealed the presence of intraspecific variation in DNA sequence of rRNA locus among A. lancea as well as A. japonica.

Amplification and sequence of a 5s-rRNA gene spacer region from the crude drug "angelica root".

A 5S-ribosomal gene spacer region was amplified by the polymerase chain reaction using a DNA preparation from the crude drug "Angelica Root" as a template. The nucleotide sequence of the amplified product was identical to that of Angelica acutiloba, the source plant of "Angelica Root", but different from that of Bupleurum falcatum or Glehnia littoralis. The possibility of discriminating "Angelica Root" from other umbelliferous crude drugs based on a Hin dIII restriction site inside the spacer region was suggested.

Cytotoxic activity of bryonolic acid isolated from transformed hairy roots of Trichosanthes kirilowii var. japonica.

Bryonolic acid was isolated in high yield from transformed hairy root cultures of Trichosanthes kirilowii var. Japonica. Bryonolic acid exhibited cytotoxic activity to various tumor cells in vitro, independent of cell type. Normal cells such as rat hepatocytes are less sensitive to bryonolic acid. The appearance of a DNA ladder was detected in the bryonolic acid-treated HL-60RG cells, indicating that cell death triggered by bryonolic acid is due to apoptosis.

Bryonolic acid production in hairy roots of Trichosanthes kirilowii Max. var Japonica Kitam. Transformed with Agrobacterium rhizogenes and its cytotoxic activity.

The hairy roots of Trichosanthes kirilowii Max, var. japonica Kitam. were induced by Agrobacterium rhizogenes (TCC 15834) on sterile shoots. The axenic culture of hairy roots proliferated 30 to 60-fold based on the initial fresh weight after three weeks of culture in Murashige & Skoog liquid media. Bryonolic acid as the main triterpenoid was isolated in a high yield, together with chondrillasterol from the hairy roots of this plant. Bryonolic acid showed strong inhibition of the growth of B-16 melanoma cells.

Bupleurum falcatum L. in northern Kyushu and Yamaguchi Prefecture are genetically distinguished from other populations, based on DNA fingerprints.

Total DNA was extracted from leaves of Bupleurum falcatum originating from eight different habitats in Japan and hybridized with digoxigenin-labeled rice ribosomal DNA after digestion with various restriction enzymes. The resulting DNA fingerprints allowed us to distinguish the geographic populations of B. falcatum when the DNA was digested with Dra I, Kpn I or Taq I. A dendrogram based on the RFLP profiles indicated that B. falcatum distributing in northern Kyushu and Yamaguchi Prefecture is classified into a taxon or subtaxon that can be differentiated from the plants grown in other regions.

Restriction fragment length polymorphisms of medicinal plants and crude drugs. II. Analysis of Glehnia littoralis of different geographical origin.

Total DNA was extracted from leaves of Glehnia littoralis belonging to various geographical strains with different furanocoumarin composition, and digested with restriction enzymes. Hybridization of digoxigenin-labeled probe containing rice ribosomal DNA to the digested DNA showed no difference in the restriction fragment length polymorphism (RFLP) profiles among the plants of different geographical origin. It is concluded that genetic diversity among the geographical strains of G. littoralis is so narrow as to be incapable of detecting RFLPs using rDNA as a probe.

Restriction fragment length polymorphisms (RFLPs) of medicinal plants and crude drugs. I. RFLP probes allow clear identification of Duboisia interspecific hybrid genotypes in both fresh and dried tissues.

A simple and efficient method for identification of Duboisia leichhardtii F. Muell, D. myoporoides R. BR. and their interspecific hybrid was established by analyzing restriction fragment length polymorphism (RFLP) profiles using non-radioactive rice ribosomal DNA as a probe. This procedure was shown to be applicable to both fresh and dried samples, and also suitable for detection of somatic hybrid at the callus stage after protoplast fusion.

Method of Harvesting the Crude Drug Based on Distribution of Alkaloids in the Hook and in the Stem with Hook of Uncaria rhynchophylla1.

The distribution of the content of the pharmacologically active oxindole alkaloids in the hook and in the stem of the crude drug "Cho-to-ko" (dried stem with hooks of UNCARIA RHYNCHOPHYLLA) prepared from the cultivated plants were investigated. The total oxindole alkaloid content of the stem was similar to that of the hook portion. It was also found that the area closest to the hook showed a slightly higher alkaloid content than the hook, and that the content of the oxindole alkaloid tended to decrease as the distance from the hook increased. Therefore, the presence of the hook does not affect the quality of the crude drug as far as the oxindole alkaloid content is concerned.