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13 C NMR data of licodione and its methylated derivatives in keto-enol tautomeric mixture are given, and evidence for correct assignment of α-proton and methyl signals in 1 H NMR is presented.

Intraparticle diffusion resistance was studied for Papaver somniferum cells immobilized by Ca alginate gel. In callus tissue, these plant cells convert codeinone to codeine. First, the diffusion rates of substrates in the gel were measured, followed by investigation of the consumption rates of the substrates by free cells. The consumption rate of sucrose was zero order in relation to sucrose concentration, whereas that of codeinone was first order in relation to its concentration. The oxygen consumption rate obeyed Michaelis-Menten type kinetics with respect to dissolved oxygen concentration. Combining the reaction rates and diffusion rates allows calculation of the extent of the effect of diffusion limitation on the overall reaction rates. The analysis showed that the effectiveness factor for each substrate was about unity and that the influence of diffusion resistance was negligible. However, the oxygen concentration decreased considerably inside the particle, and this may affect the activity of the plant cell for repeated use over a long time period. Thus, deactivation proceeds due to the oxygen deficit although the temporal reaction rate is not affected.

Constituents of the cultured cells of Glycyrrhiza echinata have been investigated. Echinatin (4,4′-dihydroxy-2-methoxychalcone), a biosynthetically unique retrochalcone, and licodione (1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)-1,3-propanedione), a dibezoylmethane derivative, which is the possible precursor of echinatin, were obtained. The structures were determined by spectroscopic methods and syntheses. 1H NMR of licodione revealed new features in chemical shifts of protons of diketonic and keto—enolic forms. 7,4′-Dihydroxyflavone, two of its prenyl derivatives and formononetin were also isolated. A discussion on retrochalcone biosynthesis is presented.

The callus tissues from 11 representative species of the Papaveraceae and the redifferentiated plantlets from four species were successfully derived and maintained. The alkaloids in the callus tissues and redifferentiated plantlets were examined in comparison with those of the original plants. All the callus tissues are similar in their alkaloid chemistry and contain benzophenanthridine, protopine and aporphine type alkaloids. By contrast, the plantlets have a more specific alkaloid pattern, being similar in content to the original plants.

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.

The structure of a new macrocyclic pyrrolizidine alkaloid, ligularidine(1), having significantly high mutagenic activity was elucidated and the configurations of the necic acid and its enantiomer reported by Edwards2 were also determined.

A new pyrrolizidine alkaloid, senecicannabine (4) which was isolated from Senecio cannabifolius was derived from seneciphylline (1) by oxidation with performic acid. The transformation of jacozine (6) to 4 was also carried out with performic acid, and the configuration of the epoxide ring of 6 was chemically determined to be 15S and 20S.

This review represents an attempt to provide an up-to-date introduction to the pyrrolizidine alkaloids which have highly cytotoxic activities.Numerous naturally occurring pyrrolizidine alkaloids are grouped systematically and the structural elucidation of a few alkaloids are discussed in detail. Their chemical synthesis and biosynthesis are also covered.

Ten biotransformation products have been isolated from Eucalyptus perriniana cultured cells administered (–)-menthol. The main product is (–)-menthol 3-O-β-D-gentiobioside (1) and the minor products are (–)-menthol 3-O-β-D-cellobioside (2), (+)-neomenthol 3-O-β-D-gentiobioside (3), (1R,3R,4R)-p-menthane-3,8-diol 3-O-β-D-glucoside (4) and 8-O-β-D-glucoside (5), (1R,3R,4R)-p-menthane-3,8-diol 3-O-β-D-gentiobioside (6) and 8-O-β-D-gentobioside (7), (1S,3R,4S)-p-menthane-1,3-diol 3-O-β-D-glucoside (8), (1R,2S,3S,4S)-p-menthane-2,3-diol 3-O-β-D-gentiobioside (9), and 2-O-β-D-gentiobioside (10).

It was shown that Panax ginseng callus produces the same kinds of saponins in considerable amounts as in intact plants, and the saponin content is significantly lower in habituated callus than in normal callus. Furthermore, it was found that semicarbazide and particularly thiosemicarbazide inhibit the production of phytosterols and promote the biosynthesis of saponins in the presence of mevalonic acid, also the biosyntheses of saponins and phytosterols are inhibited by the individual end products.

Three novel triterpene saponins named desacylmasonosides 1 (1), 2 (2), and 3 (3) have been isolated from the weak alkaline hydrolyzate of the crude saponin obtained from the corms of Crocosmia masonorum. On the basis of spectral and chemical evidence, their structures were characterized as 3-O-[α-L-arabinopyranosyl-(1→6)-β-D-glucopyranosyl]-28-O-{2-O-[β-D-apio-D-furanosyl-(1→4)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl]-3-O-(β-D-glucopyranosyl)-β-D-fucopyranosyl}polygalacic acid (1), 3-O-β-D-glucopyranosyl-28-O-{2-O-[β-D-apio-D-furanosyl-(1→4)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl]-3-O-(β-D-glucopyranosyl)-β-D-fucopyranosyl}polygalacic acid (2) and 3-O-[α-L-arabinopyranosyl-(1→6)-β-D-glucopyranosyl]-28-O-{2-O-[β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl]-3-O-(β-D-glucopyranosyl)-β-D-fucopyranosyl}polygalacic acid (3).

A callus was induced from the petiole of PANAX GINSENG C. A. M EYER by treatment with 2,4-D. The callus has been maintained on the synthetic media containing 2,4-D as growth factor. Habituated ginseng callus which required no auxin for its growth was induced from a 2,4-D callus by successive treatments with a low concentration of 2,4-D. The fact that the process of habituation is reversible suggests that its mechanism is based upon adaptation rather than rare mutation followed by selection. It was also shown that the capacity of saponin production is significantly lower in habituated callus than in normal callus. Habituated callus which was maintained on 2,4-D media gradually recovered its capacity for saponin production and the degree of this capacity seemed to parallel the degree of its requirement for auxin.

The mutagenicities of 7 pyrrolizidine alkaloids to Salmonella typhimurium TA100 were demonstrated by a modified Ames's method. The pyrrolizidine alkaloids found to be mutagenic were clivorine, fukinotoxin, heliotrine, lasiocarpine, ligularidine, LX201 and senkirkine. Pre-incubation of these alkaloids with S9 mix and bacteria in a liquid medium was essential for demonstration of their mutagenicities.

In order to clarify the O-methylation step in the biosynthesis of a retrochalcone, echinatin(4,4′-dihydroxy-2-methoxychalcone), methyl transfer from S-adenosyl- l -methionine (SAM) to licodione (1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)-1,3-propanedione) in the cell-free extract of the cultured cells of Glycyrrhiza echinata was examined. Time course of methyl transferring activity during culture cycle in 4 strains was correlated to echinatin content. The enzyme catalysing this reaction, licodione O-methyltransferase (LMT), was purified 135-fold. Substrate specificity studies implied that the hydroxy group ortho to the C3 linkage in licodione was methylated in this reaction. O-Methyl-licodiones were synthesized for comparison and the sole product of LMT-catalysed reaction was identified as 2′-O-methyl-licodione. A possible scheme for the last steps of echinatin biosynthesis is proposed.

From cultured cells of PAPAVER SOMNIFERUM L., green buds and shoots were formed at a high frequency under illumination at low temperatures, 16-18° C. Differentiation was induced from meristemoids which contained large amounts of lipids. The differentiated tissues completely recovered the potential to biosynthesize morphinan alkaloids that was lost in the undifferentiated cells, i.e. calli. Moreover, it was confirmed by enzyme immunoassay (EIA) for opiate that the tissues, which differentiated only a tracheary element, produced morphinan alkaloids, especially codeine as a main component.

Root cultures of Panax ginseng are able to convert ( RS )-2-phenylpropionic acid into ( RS )-2-phenylpropionyl β- d -glucopyranoside, (2 RS )-2- O -(2-phenylpropionyl)- d -glucose, (2 S )-2-phenylpropionyl 6- O -β- d -xylopyranosyl-β- d -glucopyranoside and a myo -inositol ester of ( R )-2-phenylpropionic acid. The primeverosyl ester and myo -inositol ester were formed stereoselectively at C-2 from the racemic mixture. The conversion into glucosyl esters was very high at ca 90% and the total conversion reached 100% at day three. About a half of the products were excreted into the medium. Compared with the root culture, the callus showed lower glycosylation ability.

Two high saponin-producing cell lines were selected from the original callus induced in 1967 and maintained in good state. For saponin production and growth in two cell lines, the comparison of static and suspension cultures, static cultures in the dark or in the light and rotary or reciprocal shaking cultures were made in detail. It was shown that the growth in rotary shaking cultures were 1.8 times higher than in reciprocal cultures, while the saponin production was almost the same, and the most effective hormonal condition was the combination of indole-3-butyric acid with kinetin. The saponin content per fresh weight was almost the same as that of the crude drug, whereas that per dry weight was 4 times higher.

Seven new triterpenoids, ganoderic acid T, ganoderic acid S, ganoderic acid R, ganoderic acid P, ganoderic acid Q, ganoderic acid 0, 7-O-methyl-ganoderic acid 0 and a known ergosterol derivative, ergosta-4,7,22-triene-3,6-dione were isolated from the cultured mycelium of Ganoderma lucidum. The structure of the first compound was determined using spectroscopic and X-ray analysis, and the structures of the other compounds were elucidated by spectroscopic data.

Four new pyrrolizidine alkaloids, ligularidine, neoligularidine, ligularizine and ligularinine, were isolated together with the known compound clivorine from the roots and aerial parts of Ligularia dentata. Their structures were elucidated on the basis of the spectral data and chemical conversion of clivorine into these alkaloids. A useful epoxidation of pyrrolizidine alkaloids was achieved by using performic acid.

Nine novel triterpenoid saponins, named crocosmiosides A-I, were isolated from the corms of montbretia (Crocosmia crocosmiiflora N. E. BR., Iridaceae). Among those saponins, the structures of crocosmiosides A (1), B (2) and H (3) were determined on the basis of spectral and chemical evidence. They are unique acylated saponins which have 2, 9, 16-trihydroxypalmitic acid glycoside as the acyl moiety, and their structures were elucidated as polygalacic acid-3-{α-L-arabinopyranosyl-(1→6)-β-D-glucopyranosido}-28-{2-O-[β-D-apio-D-furanosyl-(1→4)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl]-4-O-(9-hydroxy-16-α-L-rhamnopyranosyloxy-2-β-D-xylopyranosyloxyhexadecanoyl)-β-D-fucopyranoside} (1), polygalacic acid-3-{α-L-arabinopyranosyl-(1→6)-β-D-glucopyranosido}-28-{2-O-[β-D-apio-D-furanosyl-(1→4)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl]-4-O-(9, 16-dihydroxy-2-β-D-xylopyranosyloxyhexadecanoyl)-β-D-fucopyranoside} (2) and polygalacic acid-3-{α-L-arabinopyranosyl-(1→6)-β-D-glucopyranosido}-28-{2-O-[β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl]-4-O-(9-hydroxy-16-α-L-rhamnopyranosyloxy-2-β-D-xylopyranosyloxyhexadecanoyl)-β-D-fucopyranoside} (3), respectively.

Two new perenniporiol derivatives, lanostane-type triterpenoids, were isolated from the benzene extract of cultured mycelia of Perenniporia ochroleuca. The structures of the two new compounds were elucidated by spectral data to be 3-O-acetyl-7,11-dihydroperenniporiol and 12β-acetoxyperenniporiol.

A new method for the production of tocopherols by safflower ( Carthamus tinctorius ) cell culture has been developed. The main tocopherol produced is α-tocopherol which has the strongest vitamin E activity among tocopherol analogues. In a time course experiment tocopherol production showed a secondary metabolic pattern rather than a primary one. Better cell lines in terms of both growth rate and amounts of tocopherols produced were obtained by selection using various growth regulators and media additives. In addition, tocopherol production was effectively stimulated by administration of biosynthetic precursors. In particular, phytol increased the total tocopherol content by some 18-fold, i.e. 63.6 mg per 100 g dry weight, and the α-tocopherol content by some 11- fold i.e. 28.8 mg per 100 g dry weight.

オタネニンジン (Panax ginseng C. A. Meyer) カルスからの幼苗への再分化のうち, とくにシュートからの発根条件について統計的手法を用いて考察を行った. Murashige and Skoog (MS) 基本培地中の主要無機塩類のうち, 発根要因を直交配列実験計画法を用いて選び出した後, 主要2要因と発根率の関係を Box-Wilson 法を用いて, 2次多項近似式でシミュレートした. その結果, MS基本培地から, NH4NO3を除き, MgSO4濃度を185mg/lとしたホルモンフリーの培地が完全に発根した植物体を得るのに最適であると推定された. この培地で得られた幼植物体は, 現在鉢上げし, 栽培を試みている.

Digitoxigenin was oxidized to digitoxigenone which was reduced to epidigitoxigenin and then glucosylated to epidigitoxigenin glucoside by cell cultures of Digitalis purpurea . The epidigitoxigenin glucoside, together with digitoxigenone and epidigitoxigenin, was isolated in considerable amounts, whereas digitoxigenin glucoside could only be detected in low concentration. Furthermore, it was confirmed by TLC and HPLC that digitoxigenin was hydroxylated to periplogenin.

Suspension cultures of Nicotiana tabacum and Dioscoreophyllum cumminsii converted 2-(RS)-phenylpropionic acid and its ethyl ester into 2-(RS)-phenylpropionyl β- D -glucopyranoside, 2-(RS)-phenylpropionyl 6-O-β- D -glucopyranosyl-β- D -glucopyranoside and 6-O-β- D - glucopyranosyl-2-O-[2-(RS)-phenylpropionyl]- D -glucose which accumulated in the cells. A suspension culture of Aconitum japonicum converted these substrates into ethyl 6-O-[2-(RS)-phenylpropionyl]- β- D -glucopyranoside which was mostly excreted into the medium. The diastereomeric mixture of the glucosyl esters of 2-(RS)-phenylpropionic acid was resolved by HPLC to show the ratio of R:S was 1:1.

Suspension cultures of Coffea arabica are able to convert phenylacetic acid and 2-( R , S )-phenylpropionic acid and its ethyl ester into their sucrose esters, β- d -fructofuranosyl 6- O -phenylacetyl-α- d -glucopyranoside and β- d -fructofuranosyl 6- O -[2-( R , S )-phenylpropionyl]-α- d -glucopyranoside, which are accumulated in the cells. The diastereomeric mixture of the sucrose ester of 2-( R , S )-phenylpropionic acid (R: S::1:1) was resolved by HPLC.

Six new triterpenoids, (26 S )-26- O -methylperenniporiol, (26 S )-3-acetyl-26- O -methylperenniporiol, perenniporiol, 3-acetylperenniporiol, (26 S -15-deacetoxy-7,11-dihydro-26- O -methylperenniporiol and 15-deacetoxy-7,11-dihydroperenniporiol were isolated from the cultured mycelia of Perenniporia ochroleuca . The structures of the first two compounds were determined using spectroscopic and X-ray analyses, and the structures of the other compounds were elucidated by spectroscopic data.

A suspension culture of Glycyrrhiza echinata converted benzoic acid into its glucosyl ester. Suspension cultures of Aconitum japonicum , Coffea arabica , Dioscoreophyllum cumminsii and Nicotiana tabacum , transformed benzoic acid into its gentiobiosyl ester in addition to the glucosyl ester. The suspension cultures of A.japonicum and G. echinata converted phenylacetic acid into the esters attached to the C-6 position of glucose, that is, 6- O -phenylacetyl- d -glucose and ethyl 6- O -phenylacetyl-β- d -glucopyranoside. That of D. cumminsii converted phenylacetic acid into the glucose ester and also into phenethyl β- d -glucopyranoside showing glucosylation after the reduction of the carboxylic group. These suspension cultures converted cinnamic acid into p -coumaric acid and its glucosyl ester and p -coumaric acid into its glucosyl ester. However, the conversion of caffeic acid was not observed. The suspension cultures of A.japonicum and C. arabica converted 3-phenylpropionic acid into its gentiobiosyl ester. On the other hand, the culture of D. cumminsii did not produce the glycosyl ester but instead 3-(4-hydroxyphenyl)propionic acid was formed, thus showing hydroxylation capability.

Eight triterpenes were isolated from the methanol extracts of Eucalyptus perriniana cultured cells as their methyl esters. The structures were elucidated by spectral data to be oleanolic acid, ursolic acid, maslinic acid, 2α-hydroxyursolic acid, hederagenin, 23-hydroxyursolic acid, arjunolic acid and asiatic acid.

A root culture of Panax ginseng was able to convert 3,5-dimethoxyphenol (taxicatigenin) into its glucoside (taxicatin), primeveroside and gentiobioside, methyl salicylate into its glucoside and gentiobioside, p-hydroxy-acetophenone into its glucoside (picein), and coniferyl alcohol into dihydroconiferin [3-(3-methoxy-4-O-β- d -glucopyranosylphenyl)propan-1-ol]. The conversion ratio and the excretion ratio of the conversion products from methyl salicylate were higher than those from the more polar p-hydroxyacetophenone, 3,5-dimethoxyphenol and coniferyl alcohol. Among the conversion products, in particular, the excretion ratio (ER) of a glucoside into the medium decreased in proportion to the quotient of substrate's Mr divided by its RRt on reversed phase HPLC ( - ER α Mr/RRt).

The production of a retrochalcone, echinatin, by isoflavone-rich Glycyrrhiza echinata (M-2) cultured cells was stimulated by the addition of yeast extract or calcium alginate beads to the culture medium. Combined addition of yeast extract and cycloheximide suppressed the formation of retrochalcone, suggesting de novo synthesis. A new metabolite was isolated from the induced cells and its structure was determined to be 1-[2,4-dihydroxy-5-(3-methyl-2-butenyl)phenyl]-3-(4-hydroxyphenyl)-1,3-propanedione (5′-prenyl-licodione).

Two novel triterpenoid saponins, named crocosmiosides A (1) and B (2) were isolated from montbretia, and their structures were elucidated on the basis of spectral and chemical evidence. They are the first examples of saponins containing hydroxylated palmitic acid derivatives as acyl moieties.

Chalcone synthase activity catalyzing the formation of naringenin (5-hydroxyflavanone) was detected in cell suspension cultures of Glycyrrhiza echinata. This activity rapidly increased by treatment of the cells with yeast extract, while non-treated cells showed a constant low activity. Isolated G. echinata protoplasts accumulated retrochalcone (echinatin) and its biosynthetic intermediate (licodione) during 24 h of culture. When the protoplasts were incubated with [(14)C(U)]phenylalanine, liquiritigenin (5-deoxyflavanone) was transiently labeled, indicating the induction of 6'-deoxychalcone synthase. The formation of liquiritigenin, in addition to naringenin, was observed when the crude extracts from the protoplasts were assaved for CHS activity.

Digitalis purpurea normal callus suspension culture is capable of metabolizing 5β-pregnane-3,20-dione (1) to 3β-hydroxy-5β-pregnan-20-one (2), 3α-hydroxy-5β-pregnan-20-one (3), 3β-hydroxy-5β-pregnan-20-one glucoside (7) and 3α-hydroxy-5β-pregnan-20-one glucoside (8). Digitalis purpurea habituated callus suspension culture is also capable of metabolizing 1 to 2, 3, 5β-pregnane-3β,20β-diol (5), (7), (8), 5β-pregnane-3β,20α-diol monoglucoside (9) and 5β-pregnane-3α,20α-diol monoglucoside (11). Furthermore, it was observed that 3β-hydroxy-5β-pregnan-20-one (2) is converted to 7, 9 and 11 by both suspension cultures. At the same time, 1, 3, 5 and 8 were detected in normal callus, while 5β-pregnane-3β,20α-diol (4) and 5β-pregnane-3β,20β-diol monoglucoside (10) were present in the habituated callus culture.

It has been shown that the cultured cells of Nicotiana tabacum “Bright Yellow” are capable of transforming testosterone to Δ 4 -androstene-3, 17-dione, 5α-androstan-17β-ol-3-one, 5α-androstane-3β, 17β-diol, its dipalmitate and 3- and 17-monoglucosides, epiandrosterone, its palmitate and glucoside, testosterone glucoside. 5α-Androstane-3β, 17β-diol dipalmitate and 3- and 17-monoglucosides, epiandrosterone palmitate and glucoside, and testosterone glucoside have been found for the first time as metabolites of testosterone in plant systems. Δ 4 -Androstene-3,17-dione was converted to testosterone. 5α-Androstan-17β-ol-3-one, which has been recognized as an active form of testosterone in mammals, was also detected. It has also been demonstrated that [4- 14 C]testosterone is actively incorporated in these transformations.

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.

Two new acylated flavonols, montbretins A and B were isolated from the corms of Crocosmia crocosmiiflora. Their structures were established as myricetin 3-O-[β- d -glucopyranosyl(1→2)-6-O-caffeoyl-β- d -glucopyranosyl (1→2)-α- l -rhamnopyranoside]-4′-O-[α- l -rhamnopyranosyl(1→4)-β- d -xylopyranoside] and 3-O-[β- d -glucopyranosyl (1→2)-6-O-p-coumaroyl-β- d -glucopyranosyl(1→2)-α- l -rhamnopyranoside]-4′-O-[α- l -rhamnopyranosyl(1→4)-β- d -xylopyranoside] by spectral and chemical methods.

Root culture of Panax ginseng is able to convert aromatic carboxylic acids into glucose and/or sophorose substituted conjugates. 2-(4-hydroxyphenyl) Propionic acid is converted into its R and S glucosyl esters and glucosides; its ethyl ester into a glucoside and a small amount of a glucosyl ester; (4-hydroxyphenyl)acetatic acid into its glucoside; tropic acid into its glucose esters and glucosides; and 2-(3-benzoylphenyl)propionic acid and 2-[2-(6-methoxy)-naphthyl] propionic acid, into their glucosyl and sophorosyl esters. These conversion products are all new compounds. The conversion ratio and binding sugars depend on the properties of the administered substrate, in particular, its polarity and toxicity. Unless the root culture is injured by substrate toxicity,the conversion ratio from substrates having smaller polarities are higher and in some cases 100%. Among the conversion products, the excretion ratio of glycosyl esters into the medium decreased in proportion to the quotient of the M r divided by the RR t of substrate in a reversed phase HPLC. In the conversion of ( RS )-2-(4-hydroxyphenyl)propionic acid into its glucoside [(2 R )-2-(4-O-β- d -glucopyranosylphenyl)propionic acid], the root culture selectivity converted the R isomer, although the formation of glucosyl esters was not selectively performed.