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Olive (Olea europaea) contains large quantity of triterpene acids including oleanolic acid (6) as a major one. Varieties of biological activities exhibited by triterpene acids attracted our attentions, especially from pharmaceutical viewpoints. Cell culture of olive plant was induced and its triterpene constituents were studied. From the cell suspension cultures, six ursane type triterpene acids; ursolic acid (9), pomolic acid (10), rotundic acid (11), tormentic acid (12), 2alpha-hydroxyursolic acid (13) and 19alpha-hydroxyasiatic acid (14), and two oleanane type acids; oleanolic acid and maslinic acid (7), have been isolated. Quantity of ursane type triterpene acids produced by cell cultures was larger than that of oleanane type. Further, a multifunctional oxidosqualene cyclase (OSC) named OEA was cloned by homology based PCRs from the same cultured cells. Major product of OEA is alpha-amyrin (ursane skeleton), showing good accordance to higher content of ursane-type triterpene acids in the cultured cells, and strongly suggesting OEA to be a major contributor OSC for their production.

The Eucalyptus perriniana cultured cells are widely used to biotransform a variety of compounds. The glucosyltransferase activity of a crude protein extract of E. perriniana cultured cells was maximized when cell growth was in the pre-logarithmic to logarithmic phase. We cloned a cDNA encoding glucosyltransferase (EPGT) from E. perriniana cultured cells by RT-PCR using a degenerated primer and RACE-PCR. The cDNA contained an open reading frame encoding 467 amino acids with a calculated molecular mass of 51.6 kDa. The consensus sequence of the plant glucosyltransferases was included in the deduced amino acid sequence. The amino acid sequence of EPGT showed a high identity to glucosyltransferases from tobacco and petunia. The recombinant EPGT was expressed in Escherichia coli and its substrate specificity was examined using UDP-(U- 14 C) glucose. Cinnamic acid was the best sugar acceptor in the compounds tested.

Two new polyacetylenes, 1-hydroxydihydropanaxacol (3) and 17-hydroxypanaxacol (4), were isolated from Panax ginseng hairy root culture, along with dihydropanaxacol (1), panaxacol (2) and ginsenoyne D (5). Highly hydroxylated compounds 1-4 were isolated from the medium and compound 5, which was a biosynthetic precursor of compound 1, was isolated from the roots. Compounds 1-4 showed antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Cryptococcus neoformans and Aspergillus fumigatus. It is suggested that P. ginseng plants release antimicrobial polyacetylenes into the surrounding soil from the roots as defense compounds.

Cinobufacini (huachansu), an aqueous extract from the skin of the toad Bufo bufo gargarizans Cantor, is a traditional Chinese medicinal preparation widely used in clinical cancer therapy in China. Here, we screened and identified active compounds of cinobufacini and investigated their apoptosis-inducing effect on HepG2 cells. Screening was performed using bioassay-guided isolation. The effects of different fractions on the proliferation of HepG2 cells were detected by the MTT assay. The extraction and isolation of active fractions were performed by chloroform extraction, silica column chromatography, preparative thin-layer chromatography and high-performance liquid chromatography. Nuclear magnetic resonance (NMR) imaging and electron ionization-mass spectrometry (EI-MS) were used to identify the structure of the active compounds. The extent of cell apoptosis was detected by Hoechst 33258 staining and flow cytometric analysis. Western blot analysis was used to detect the expression of the apoptosis-related proteins Bax and Bcl-2. Through bioassay-guided isolation, two compounds were isolated from cinobufacini. NMR and EI-MS data revealed these compounds to be resibufogenin and cinobufagin. Cinobufagin was determined to be the more efficient of the two in inhibiting the proliferation of HepG2 cells. Hoechst 33258 staining and flow cytometric analysis indicated that cinobufagin induced marked changes in apoptotic morphology and significantly increased the proportion of apoptotic cells in HepG2 cells. Western blot analysis showed that cinobufagin up-regulated Bax expression and down-regulated Bcl-2 expression. In conclusion, we screened and identified two anti-proliferation compounds of cinibufacini, resibufogenin and cinobufagin. The most effective compound, cinobufagin, inhibited cell proliferation by inducing the apoptosis of HepG2 cells. This was potentially triggered by regulation of the Bax/ Bcl-2 ratio.

Ganciclovir, foscarnet, vidarabine and ribavirin, which are used to treat viral infections in humans, inhibited the proliferation of a baculovirus (Bombyx mori nucleopolyhedrovirus) in BmN4 cells, a cultured silkworm cell line. These antiviral agents inhibited the proliferation of baculovirus in silkworm body fluid and had therapeutic effects. Using the silkworm infection model, the antiviral activity of Kampo medicines was screened and it was found that cinnamon bark, a component of the traditional Japanese medicine Mao-to, had a therapeutic effect. Based on the therapeutic activity, the antiviral substance was purified. Nuclear magnetic resonance analysis of the purified fraction revealed that the antiviral activity was due to cinnzeylanine, which has previously been isolated from Cinnamomum zeylanicum. Cinnzeylanine inhibits the proliferation of herpes simplex virus type 1 in Vero cells. These results suggest that the silkworm–baculovirus infection model is useful for screening antiviral agents that are effective for treating humans infected with DNA viruses.

Thiarubrine A, thiarubrine A epoxide, thiarubrine A diol, and their precursor pentayneene were produced by hairy roots of Ambrosia maritima. The molluscicidal activity of the lipophilic extract of the hairy roots and isolated polyacetylenes were investigated. The activity was studied against Schistosome intermediate host Biomphalaria glabrata. Thiarubrine A diol, the main form detected in the medium, showed the highest activity with LC50 = 0.37 ppm. This activity is 23 times higher than that of ambrosin, a sesquiterpene lactone that has long been regarded as the main molluscicidal agent of the plant. Thiarubrine A diol can be an environmentally accepted molluscicidal agent in areas endemic with schistosomiasis.

コーヒーノキ (Coffea arabica L.) 培養細胞は, プリンアルカロイドであるカフェインおよびテオブロミンを生産し, その70～90%を培地中に放出した. カフェイン生産量はオーキシンの種類によって影響を受け, 3-indole butyric acid; 2mg/l kinetin; 0.1mg/lを含むMS基本培地 (B2K培地) において最大であり, 全体で43.1mg/flask, 培地中の濃度は131mg/lであった. アルギン酸カルシウムゲルで包括固定化した細胞は4代目まで培養可能であり, この場合は成長培地として用いた2,4-D; 1mg/l, kinetin; 0.1mg/lを含むMS基本培地 (DK培地) で4代目に最も高い生産量を示した.

Six types of cultured tissues at various stages of differentiation were induced from rhizome segments of Wasabia japonica Matsum. (Japanese name: wa-sa-bi); NC callus, rootlets, embryoids, differentiated tissues I and II, and plantlets. Myrosinase activity was detected at all stages of differentiation, but sinigrin could not be detected from NC callus, rootlets and embryoids, so that pungency could be felt from differentiated tissues I, II and plantlets. It is supposed that the process of differentiation stages (the increase of the rhizome weight) is parallel to the sinigrin content in the tissues.

Hairy roots of Ambrosia maritima L. were cultured in MS in the dark. Time course production was studied for pentayneene and thiarubine A after addition of 40 μ M methyl jasmonate to the 13 ‐ day ‐ old hairy roots. The levels of pentayneene and thiarubrine A showed maximum increase after 72 hrs adding methyl jasmonate. Lipoxygenase activity was increased in the hairy roots elicited with 40 μ M methyl jasmonate. The increase in activity was 28 ‐ fold higher than control cultures 24 hrs after elicitor addition. The increase in enzyme activity was concomitant with the increase in pentayeneene, the precursor for polyacetylene biosynthesis. N ‐ propyl gallate, a potent in vivo and in vitro lipoxygenase inhibitor, reduced polyacetylene production by the hairy roots at concentration of 100 μ M. It is suggested that lipoxygenase may be at least partially involved in polyacetylene biosynthesis in A. maritima hairy roots. Plant Tissue Cult. & Biotech. 23 (1): 31 ‐ 38, 2013 (June) DOI: http://dx.doi.org/10.3329/ptcb.v23i1.15557