Ellagic acid formation from galloylglucoses by a crude enzyme of Cornus capitata adventitious roots.

The aqueous extract of acetone powder, which had been prepared from Cornus capitata 'Mountain Moon' adventitious roots, cultured in MS medium with a high concentration of Cu2+(10 microM), showed strong oxidative activity toward galloylglucoses. A compound formed from galloyglucoses, such as 1,2,3,4,6-penta-O-galloyl-beta-D-glucose and tannic acid, by the reaction with the crude enzyme solution of the adventitious roots was isolated and characterized as ellagic acid by spectrometric analyses.

Distribution of verectin in Aloe vera leaves and verectin contents in clonally regenerated plants and the commercial gel powders by immunochemical screening.

Verectin antiserum raised in white rabbits was immunochemically applied to examine verectin distribution in Aloe vera leaves during growth and flowering seasons, and to quantify verectin in clonally regenerated plants and commercial A. vera gel products.

[Characteristics of Papaver somniferum L. cv. ikkanshu cultivated in Izu].

The seeds of Papaver somniferum L. cv. Ikkanshu were sown in November (Autumn sowing: AS) and March (Spring sowing: SS) in a field at Izu Experimental Station for Medicinal Plants of National Institute of Health Sciencs, and both AS and SS plants were cultivated to investigate their growth, opium yield and alkaloid content in the opium. Growing periods from the sowing to the opium harvest were approximately six months for AS plants and three months for SS plants. Sizes of plants and capsules in AS were bigger than those in SS, reflecting their growth period. Opium yields per an are in AS and SS were 212.09 g and 142.03 g, respectively. The opium was able to be collected four times in the AS plants though the SS plants ceased to exude opium after the second incision. Therefore higher yield of opium in AS plants seems to be attributed to an amount of opium in the third and fourth incision. Average morphine content in the total opium was 15.61% in AS plants and 15.04% in SS plants, and the estimated amounts of morphine per an are in AS and SS plants were 33.16 g and 21.38 g, respectively.

High production of ginsenosides by transformed root cultures of Panax ginseng: effect of basal medium and Agrobacterium rhizogenes strains.

Successful transformation of Panax ginseng was achieved when petiole segments were infected with Agrobacterium rhizogenes ATCC 15834 and MAFF 03-01724. Transformed roots were obtained after galls developed at infected sites. The root morphology, growth and ginsenoside productivity of roots transformed with different bacterial strains differed, and the roots from A. rhizogenes ATCC 15834 grew better and produced much more ginsenosides. Using the ATCC transformed root clone, various liquid culture media were tested to determine the optimum culture medium for ginsenoside production. The root growth was optimum in phytohormone-free Gamborg B5 liquid medium, however highest content of ginsenosides (a total of five ginsenosides 1.88% dry weight) was obtained when the roots were cultured in half-macro-salt strength Gamborg B5 liquid medium. Growth of the roots over a period of 8 weeks showed that their fresh and dry weight continued to increase. The ginsenoside Rb1 content was optimum after 5 weeks of culture. Ginsenoside Rc content began to decrease slightly after the third week of culture. Ginsenosides Rd and Rg1 contents fluctuated, while ginsenoside Re content continued to rise throughout the 8 weeks of culture. Ginsenoside production, however, did not peak within the 8 weeks of culture.

Somatic embryogenesis and ginsenoside production of Panax ginseng in phytohormone-free medium.

Embryogenic cultures of Panax ginseng were established without using phytohormones. Somatic embryos developed from the roots of an in vitro seedling and from excised leaf and petiole segments cultured in half-macro-salt strength Murashige and Skoog medium. Excised leaf and petiole segments were obtained from in vitro germinated seedlings. Plantlets were subsequently obtained from developing somatic embryos in phytohormone-free media. Shoot formation from somatic embryos was influenced by light intensity. The rate of growth and frequency of embryogenesis were improved when cut-up embryogenic tissues were inoculated into liquid media in the dark. The ginsenoside contents of a 4 year-old field-cultivated root, seedlings from zygotic embryos, somatic embryos and embryogenic tissues were determined and compared. Somatic embryos contained 1.7 times the amount of ginsenoside Rb1 and 2.3 times the amount of ginsenoside Re compared to seedlings from zygotic embryos. Ginsenoside Rd, which was absent in the seedlings derived from zygotic embryos, was detected in somatic embryos. Higher ginsenosides Rd and Rg1 levels were found in embryogenic tissues grown on solid media than in tissues grown in liquid media. The total ginsenoside yields, including the ginsenosides Rb1 and Rg1 levels, of cut-up embryogenic tissues, were higher than those of clump tissues.

Activation of macrophages by crude polysaccharide fractions obtained from shoots of Glycyrrhiza glabra and hairy roots of Glycyrrhiza uralensis in vitro.

Many plant polysaccharide fractions have been reported as immunomodulatory agents. However, sometimes the possibility of contamination with bacterial lipopolysaccharide (LPS), a potent B cell mitogen and immune modulator, is discussed. In the present paper, we investigated the effects of crude polysaccharide fractions obtained from the shoot and hairy root of Glycyrrhizae sp. on murine peritoneal macrophage function, in order to clarify whether plants grown under aseptic conditions produce immunomodulatory polysaccharides. All crude polysaccharide fractions induced nitric oxide production by murine peritoneal macrophages in vitro. Chemical analysis revealed that LPS-like molecules were not present in all preparations. These results suggested that shoot and hairy root biosynthesized polysaccharides that could stimulate macrophages de novo.

ELISA for the determination of saikosaponin a, an active component of Bupleuri Radix.

In order to quantify saikosaponin a (SSA), one of the major active components of Bupleuri Radix, a competitive and indirect ELISA method was developed. High titer rabbit polyclonal antibodies (pAbs) were raised against a conjugate of SSA and bovine serum albumin, coupled with a periodate oxidation method. SSA competitively inhibited the binding of rabbit anti-SSA pAbs to SSA-ovalbumin on the solid phase, a coated antigen on the well. The quantity of pAbs bound to the well was monitored using a peroxidase-conjugated anti-rabbit IgG as a secondary antibody, and tetramethylbenzidine solution as a substrate. The measuring range extended from 50 pg/ml to 20 ng/ml of SSA, with a detection limit of 40 pg/ml (5.13 pM). Antibodies showed some cross-reactivity with saikosaponin c (12.74%). However, the antibodies showed only slight cross-reactivities with saikosaponin d (0.3%), which differs from SSA only in the stereochemistry of the 16-hydroxyl group, and the artificial saikosaponins, saikosaponin b1 (2.1%) and saikosaponin g (0.53%). The specific and sensitive ELISA is especially suited for determination of SSA in samples when only small quantities of materials can be extracted for analysis.

In vitro propagation, restriction fragment length polymorphism, and random amplified polymorphic DNA analyses of Angelica plants.

Angelica acutiloba, a medicinal plant used as a natural medicine Touki, was clonally propagated through axillary buds in vitro. No substantial differences were found in the random amplified polymorphic DNA (RAPD) pattern between the original A. acutiloba and the plant propagated in vitro, suggesting no changes in the DNA sequences and structure during in vitro propagation. The genetic similarities of several Angelica plants were investigated by restriction fragment length polymorphism (RFLP) and RAPD analyses. The RFLP and RAPD patterns of A. sinensis Diels were substantially different from those of A. acutiloba. Using ten different restriction enzymes, no RFLP was observed in the varieties of A. acutiloba. By RAPD analysis, A. acutiloba varieties can be classified into two major subgroups, i.e., A. acutiloba Kitagawa and A. acutiloba Kitagawa var. sugiyamae Hikino. The varieties of A. acutiloba Kitagawa in Japan and Angelica spp. in northeast China exhibited a very close genetic relationship.

[Immunosuppressive activity of a microbial product].

Microbial products have amazing diversity and complexity in their chemical structures as well as in their biological activities. The successful discovery and development of FK506 (Tacrolimus) adds a new example showing that microbial products are agents with potential therapeutic benefits. In 1984, we found a novel immunosuppressant FK506 during the course of our research program for discovery of specific inhibitors on IL-2 production in the microbial products. FK506, a new class of 23-membered macrolide lactone, was isolated the fermentation broth of Streptomyces tsukubaensis No.9993. This agent strongly inhibited the proliferative response of lymphocytes to alloantigen stimulation, and a variety of T cell associated immune reaction. FK506 also suppressed immune responses in vivo as well as in vitro and this immunosuppressive effect was more highly potent than cyclosporin, a well-known fungal metabolite. The profound immunosuppressive properties of FK506 were subsequently explored in organ transplantation in animal models. Clinical trials of FK506 were begun in 1989 by Professor Stazl and his colleagues at the University of Pittsburgh. Since then wider evaluation of FK506 in a variety of transplantation fields has been performed globally; in North America, Europe and Japan. In 1993, FK506 was commercialized in Japan for prevention of liver transplant rejection. FK506 is also undergoing extensive evaluation as an agent for the treatment of various autoimmune diseases.

Potent inhibitors of tyrosinase activity and melanin biosynthesis from Rheum officinale.

Thirty-three crude drug extracts were screened for their tyrosinase inhibitory activity. Among them, the acetone extract of the rhizomes of Rheum officinale Baillon showed the strongest inhibitory activity. Tyrosinase inhibitory activity-guided fractionation and chemical analysis led to the isolation of two potent compounds, 3,4',5-trihydroxystilbene-4'-O-beta-D-(2"-O-galloyl)glucopyr anoside (1) and 3,4',5-trihydroxystilbene-4'-O-beta-D-(6"-O-galloyl)glucopyr anoside (2). These compounds showed a competitive inhibition against tyrosinase and also inhibited the melanin biosynthesis.

Tissue cultures of Taxus baccata as a source of 10-deacetylbaccatin III, a precursor for the hemisynthesis of taxol.

Callus cultures of Taxus baccata L. cv. stricta were induced from hypocotyl and leaf explants on Woody Plant medium (hormone-free or additionated with phytohormones). Under continuous dark condition, adventitious roots were regenerated from hypocotyl- and leaf-derived callus cultures. Antibodies raised in rabbits against 10-succinyl-10-deacetylbaccatin III were used for the detection and the semi-quantitative determination of 10-deacetylbaccatin III in Taxus cultures. The presence of 10-deacetylbaccatin III in callus extracts was confirmed by TLC, HPLC using a photodiode array detector and mass spectrometry (CI-MS). The highest equivalent content of the taxoid derivatives (7.83 mg/100 g dry wt.) was detected in an extract from leaf-derived callus.

Two flavone 2'-glucosides from Scutellaria baicalensis.

Two new flavone glucosides, 5,2',6'-trihydroxy-6,7,8-trimethoxyflavone 2'-O-glucoside and 5,2',6'-trihydroxy-6,7-dimethoxyflavone 2'-O-glucoside were isolated from the aqueous methanol extract of the roots of Scutellaria baicalensis. From the extract, seven phenolics, 5,7,2',6'-terahydroxyflavone, 5,7,2',5'-tetrahydroxy-8,6'-dimethoxyflavone, skullcapflavone II, baicalin, baicalin methyl ester, wogonin 7-glucuronide and 3,5,7,2',6'-pentahydroxyflavanone were also isolated.

Genetic relationships among Glycyrrhiza plants determined by RAPD and RFLP analyses.

The genetic similarities of four species of Glycyrrhiza plants were determined by DNA analyses of random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP). The phylogenic trees were constructed from the genetic similarities estimated from RAPD and RFLP profiles. These results indicated that G. glabra and G. uralensis, rich in glycyrrhizin, are more closely related to each other than to G. echinata or to G. pallidiflora.

Determination of cardenolides in hairy root cultures of Digitalis lanata by enzyme-linked immunosorbent assay.

A competitive ELISA technique using digoxin-specific antibody has been developed to determine digoxin and its related compounds in hairy root cultures of Digitalis lanata. The ELISA could detect 0.2-2 nM digoxin and closely related cardenolides. Hairy roots cultured in the dark accumulated very small amounts of cardenolides (0.02-0.07 micrograms/g dry wt), while the content of cardenolides in green hairy roots cultured in the light was increased maximally 600-fold (16.5 micrograms/g dry wt) compared to those in the dark.

Production of tropane alkaloids by cultured cells of a Duboisia hybrid.

The production of scopolamine-rich calli was investigated by the quantification of tropane alkaloids in a gas chromatograph equipped with a flame thermoionic detector (FTD). Stem and leaf segments from a selected strain, M-II-8-6, a hybrid between Duboisia myoporoides R. Br. and D. leichhardtii F. Muell, were used for this experiment. Stem-derived callus subcultured for over one year in the dark on Murashige-Skoog (MS) medium containing 0.1 mg/l indole-3-acetic acid (IAA) produced hyoscyamine ane scopolamine with a yield of 0.006 and 0.005% dry weight, respectively. Leaves of shoot cultures did not show any detectable levels of alkaloids. However, the leaf callus subcultured for over one year in the dark on MS medium containing 0.1 mg/l IAA produced hyoscyamine and scopolamine with a yield of 0.007 and 0.009% dry weight, respectively. These results indicate that the ability to synthesize tropane alkaloids in stem- and leaf-derived calli can be maintained on MS medium containing IAA.

Abnormal heart rate control in vasospastic angina: effects of calcium antagonists.

We examined the effects of administration of calcium antagonists on the heart rate response to treadmill exercise in 11 patients with vasospastic angina and 8 healthy young volunteers. The exercise test was performed by walking on a treadmill at a constant speed and grade according to a scheme of pseudo-randomized sequence for 19 min. The dynamic property of heart rate response to exercise was evaluated by using a frequency analytic procedure. The exercise test was also studied in 21 age-matched normal controls without drug administration. Administration of calcium antagonists revealed no significant effects on heart rate and blood pressure at rest in young healthy subjects or in patients with vasospastic angina. Young volunteers showed the same normal properties of heart rate response to exercise before and after calcium antagonists. Vasospastic angina showed abnormal heart rate response to exercise and revealed characteristically different transfer function from that in normal controls. These characteristics were not affected by treatment with calcium antagonists except for a slight, uniform decrease of gain of the system over the whole frequency range. Accordingly, the present exercise test can feasibly be used in the diagnosis and management of vasospastic angina even when calcium antagonists are administered to the patients.

An unusual glucocerebroside in the crustacean nervous system.

High concentrations of glucocerebroside (glucosylceramide) were found in the ventral nerve cord, brain, optic nerve, and antenna, but not in the nonneural tissue, of the brown shrimp Penaeus aztecus aztecus. This lipid contained unusual sphingoid bases consisting of 14-, 15-, and 16-carbon sphinganines and sphingenines. The fatty acids were mainly nonhydroxylated homologs 22 carbons long and longer, similar to those found in galactocerebroside but differing from those in glucocerebroside in mammalian nervous systems.

A simple histochemical screening method for amine uptake.

A simple histochemical method for screening amine uptake in vitro is described. The filum terminale and the Vibratome sections of the caudate nucleus of reserpinized rats were used as the sources of noradrenaline (NA) and 5-hydroxytryptamine (5-HT), and as the sources of dopamine (DA) containing neurons, respectively. When incubated in an oxygenated medium (Krebs-Ringer) containing the corresponding amines, these preparations effectively accumulated the amines. 6-Hydroxytryptamine (6-HT) was used instead of 5-HT because of its stronger fluorescence and lower photo-decomposition of beta-carboline formed from 6-HT. The inhibitory patterns of representative uptake inhibitors obtained by the present method agreed well with those reported previously by biochemical methods. Effects of other types of drugs were also studied. The present method is a simple, rapid and non-radioisotopic method for determining amine uptake inhibiting action of a drug and the degree and selectivity in which it acts on the three kinds of amines.