不同地区草乌种子中生物碱含量的测定与分析

测定不同产地野生草乌（Aconitum kusnezoffii Reichb.）种子中生物碱类含量，并与草乌入药部分（块根、叶）生物碱含量做对比。采用HPLC法测定了内蒙古通辽市扎鲁特旗、赤峰市阿鲁科尔沁旗和赤峰市巴林右旗野生草乌的种子，色谱柱Supersil ODS2 5 μm（250 mm×4.6 mm，5 μm），流动相为甲醇-0.1 mol·L^-1：三乙胺（75：25），流速为0.8 mL·min^-1，检测波长为235 nm，柱温为35℃。草乌种子中检测到生物碱类，以苯甲酰新乌头原碱含量最高。通辽市扎鲁特旗种子含量为7.647 9 mg·g^-1；赤峰市阿鲁科尔沁旗种子含量为8.256 3 mg·g^-1；赤峰市巴林右旗种子含量为7.512 6 mg·g^-1，双酯型生物碱含量较低。不同产地的总乌头碱含量通辽市的高于赤峰市的，同为赤峰市含量之间显著差异。相关文献报道草乌药材（块根）中双酯型生物碱的含量明显高于单酯型生物碱，本研究实验表明，不同地区草乌种子中单酯型生物碱的含量明显高于双酯型生物碱，为草乌种子的综合利用开发及筛选不同含量种质材料提供理论依据。     

Production of Lysergic Acid Derivatives in Submerged Culture: IV. Inorganic Nutrition Studies with Claviceps paspali

The inorganic requirements for growth and alkaloid production by submerged cultures of a lysergic acid alkaloid-producing isolate of Claviceps paspali were determined in a defined medium. The requirement for peak growth was essentially the same as the requirement for maximal alkaloid production in the case of potassium and magnesium. With phosphorus, however, maximal growth was reached at lower levels than those required for maximal alkaloid production. Sulfur was required in smaller amounts for peak alkaloid production than for maximal growth. In the case of iron and zinc, the requirement for maximal alkaloid production was greater than for growth. Manganese and copper could be sufficiently depleted to demonstrate their essentiality for alkaloid production, but their requirements for growth could not be demonstrated.     

Methyl jasmonate vapor increases the developmentally controlled synthesis of alkaloids in Catharanthus and Cinchona seedlings

Shortly after germination, alkaloids are rapidly synthesized in seedlings of both Catharanthus roseus L.G. Don and Cinchona ledgeriana Moens. The effect of low-level, atmospheric methyl jasmonate on this developmentally controlled process was studied. In both species, about 1 p.p.m. of methyl jasmonate vapor significantly enhanced alkaloid synthesis during germination, resulting in a doubling of alkaloid content in seedlings. Treatment with methyl jasmonate resulted in increased allocation of alkaloid precursors and in enhanced enzyme activities in alkaloid biosynthesis. The ability of methyl jasmonate to increase alkaloid biosynthesis decreased with age of the seedlings. Susceptibility of the process to methyl jasmonate was confined to a narrow time interval where the developmentally regulated onset of alkaloid synthesis occurred. When methyl jasmonate was applied at later developmental stages, its ability to enhance alkaloid content in the seedlings declined sharply.     

Regulation of product synthesis in cell cultures of Catharanthus roseus. V. Long-term maintenance of cells on a production medium

Three unselected cell lines of C. roseus maintained on a growth-associated alkaloid production medium were studied over a period of 2 to 5.5 years for the stability of alkaloid production (serpentine and ajmalicine). Large fluctuations in the total alkaloid content of 20-day-old cells were found for all three cell lines at each subculture over a two-year period. Growth rates increased during prolonged subculture and one cell line became unproductive after five years culture. By selection of small autofluorescent aggregates, high alkaloid production was restored in this cell line, while the parent line was found to be unresponsive to alkaloid induction treatments. The instability in both alkaloid production and spectrum and the loss of alkaloid productivity are discussed in relation to the selection pressures present during long-term maintenance of cell suspension cultures.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - nHS n-heptane sulphonate     

Saprophytic production of Clavine alkaloids and activity of Hydroxymethylglutaryl-CoA reductase

In submerged Claviceps cultures the activity of hydroxymethylglutaryl-CoA reductase preceded the increase of alkaloid production and of sterol content. During the first alkaloid phase, cell mevalonate was involved in the biosynthesis of both alkaloids and steroids. In the second production phase, it was predominantly used for alkaloid synthesis. Hydroxymethylglutaryl-CoA reductase appears to be a suitable target for physiological manipulation to increase clavine alkaloid yields.     

金钗石斛生物碱的组织化学定位研究

利用组织化学定位方法对金钗石斛不同生长年限和不同营养器官的生物碱分布和积累进行比较分析。结果表明,金钗石斛茎生物碱主要分布于基本组织,三年生金钗石斛的生物碱含量高于一年生和二年生金钗石斛。金钗石斛根部生物碱含量同样较多,主要分布于皮层薄壁细胞中;金钗石斛叶片生物碱含量较低,存在于上下表皮细胞中,与铁皮石斛和铜皮石斛完全不同。说明三年生金钗石斛作为最适宜采收期,而且金钗石斛根部具有很好的开发利用价值。金钗石斛叶片表皮细胞中呈现生物碱的原因尚未清楚,需要进一步研究。     

Production of alkaloids and differentiation in a submerged culture ofClaviceps purpurea (Fr.) Tul

The submerged culture of the Claviceps purpurea strain studied was polymorphous. In the process of alkaloid synthesis, cytodifferentiation preceded biochemical differentiation. The onset of the alkaloid phase was characterized by: predominance of chlamydospores in the culture, the presence of vegetative cells with reduced or arrested proliferation, maximum acetylCoA carboxylase activity, the maximum amount of total fatty acids, an over-average cell pool tryptophan level and minimum tryptophan synthetase activity. Intracellular ricinoleic acid was an indicator of differentiation of the culture towards alkaloid formation and also of alkaloid synthesis. The cytodifferentiation period in the initial phases of fermentation, when the cell has several alternative possibilities of development, is regarded as the most sensitive sector of the regulatory mechanisms of alkaloid formation.     

Comparison of Cell Growth and Alkaloid Production of Catharanthus roseus Cells Cultured in Shake Flask and in Bioreactor

The cell growth and alkaloid production of Catharanthus roseus (L.) G. Don cells cultured in the shake flasks with different volumes and in the stirred tank bioreactor (10 L) were compared. Cell dry weight and alkaloid production showed no significant difference in the small volume scale-up shake flasks. When more broths were added to a certain volume in the shake flask, both cell weight and alkaloid production were decreased. The maximum cell dry weight was similar between the cell cultures in the shake flask and the bioreactor, but the alkaloid production of cells was much less in the bioreactor. Gas regime and shear stress were recognized to be the main factors contributing the important effect on alkaloid production during the scale-up processes.     

The Structure of △"-Dehydroaloperine

A new alkaloid named ll-dehydroaloperine was isolated from the leaves and stalks of Sophora alopecuroides L. during flowering. Total alkaloid was obtained by diluted acid percolation and cation-exchange resin, then the new alkaloid was obtained via various polar solvent extraction under various pH value and Al2O3 column chromatography. The new alkaloid was proved to be analogous to aloperine through UV, IR, and mass spectrometry. Its structure was further identified by 1HNMR, 13CNMR, 1H-1H cosy spectrum, NOE as well as by some other chemical methods.     

Induction and in vitro alkaloid yield of calluses and protocorm-like bodies (PLBs) from Pinellia ternata

This study investigated the induction and in vitro alkaloid yield of calluses and protocorm-like bodies (PLBs) from Pinellia ternata (Thunb.) Berit (Araceae). We planned to use this material in future studies related to the mass production of medicinally valuable compounds and regulation of alkaloid metabolism. Different combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyladenine (6-BA), kinetin (Kin), and α-naphthaleneacetic acid (NAA) were used to induce callus and PLB formation from P. ternata tuber explants. The results showed that three physiologically distinct calluses were induced by different combinations of 2,4-D, 6-BA, and Kin used in this study. The calluses differed in color, texture, differentiation status, and alkaloid content. The alkaloid content of the three calli types ranged from 0.0175% to 0.0293%. In comparison, the alkaloid content of field-grown tubers was 0.0072%. Many reports have indicated that 2,4-D suppresses the biosynthesis of secondary metabolites; however, our results show that 2,4-D promoted alkaloid production in Pinellia calluses. The combination of NAA?+?6-BA induced PLB formation. The PLB alkaloid content of 0.0321% was 1.1 to 1.8 times higher than the alkaloid content of the calluses and 4.5 times higher than the field-grown tubers. In conclusion, the induction of calluses and PLBs with alkaloid content greater than that of field-grown tubers indicates the potential use of these tissue culture materials for bioprocessing alkaloids from P. ternata and for the study of alkaloid metabolism.     

Assessment of inter- and intra-population variation in quinoline alkaloid profile of juvenile shoot cultures of Cinchona ledgeriana: effect of method of nutrient delivery on alkaloid profile

Intra-population quinoline alkaloid profiles surveying quinine, quinidine, cinchonine and cinchonidine were determined for each of five populations of Cinchona ledgeriana grown as shoot-culture for 125 days. No significant difference in respect of mean alkaloid content between populations was detected. In contrast, there was considerable between-seedling variation in alkaloid content within each population. When nutrients were delivered to shoot-cultures in droplet form by means of an aerosol spray (as compared to the supply of nutrients direct from agar-or liquid-based reservoirs) alkaloid profile was greatly perturbed; most notable in this respect was a four-fold increase in the production of cinchonidine concomitant with a four-fold decrease in the production of cinchonine. These data are discussed with reference to the optimisation of quinoline alkaloid production by juvenile shoot-cultures of Cinchona ledgeriana.     

滇产对叶百部中一个新的百部生物碱

从对叶百部（Stemona tuberosa）根的乙醇提取物中分离鉴定1个新的百部生物碱,命名为去氢异滇百部碱（1）,以及1个已知化合物：异滇百部碱（2）。它们的化学结构通过现代波谱解析得以鉴定。     

Contribution of indole alkaloids to Sitobion avenae (F.) resistance in wheat

Abstract:  Indole alkaloid contents of five winter wheat varieties at different growth stages, and the relationship between indole alkaloids and carboxylesterase activities of Sitobion avenae (F.) from wheat ears of different varieties were studied. The results indicated that KOK1679 and My295, highly and moderately aphid-resistant varieties, had a high indole alkaloid content during vegetative growth. During their reproduction growth, resistant varieties had a high alkaloid content in penultimate leaves, and indole alkaloid contents of KOK1679 were significantly higher than that of other varieties in wheat ears. The indole alkaloid contents of all varieties were low in flag leaves except for Han4564 . A significant correlation was shown between carboxylesterase activity in aphids collected from wheat ears and indole alkaloid contents in ears of the wheat varieties ( r  = 0.9646).     

Some Accounts on Culture Conditions of Callus Tissues of Solanum laciniatum Ait. for Producing Solasodine

Production of solasodine in callus cultures of Solanum laciniatum Ait. was examined under several culture conditions. The steroidal alkaloid was produced more actively in rapidly proliferating callus tissues cultured on PN medium. The alkaloid concentration in the tissue was about 0.05% (dry weight basis) during the first 5 weeks’ culture. The highest accumulation of the alkaloid per culture was obtained with 2,4-d concentration in the medium at 1~2 ppm. It is noteworthy that the alkaloid production was not inhibited by such high concentration of 2,4-d as up to 10 ppm in the medium. Supplementation of kinetin slightly increased the alkaloid production.     

The Effects of Certain Environmental Factors on Alkaloid Contents of Rauvolfia

The effects of light intensity, mineral nutrients and seasonal climatic changes on the alkaloid contents of Rauvolfia yunnanensis Tsiang were studied and the experimental results were summarized as follows: The alkaloid contents of Rauvolfia roots increased with the light intensity decreased, and a maximum was reached at about 28% of the normal day light. The supply of the mineral nutrients of P, K especially N would raise the root production and alkaloid contents respectively. Determinations were carried out about the variations of alkaloid contents of the roots, stems and leaves on the samples collected every month of the year from both the wild and the cultivated Rauvolfia. The alkaloid contents in the roots of both wild and cultivated Rauvolfia appeared with only one high peak-August to September (end of the raining season), and a period with low contents appeared from March to May (dry season). The alkaloid contents in stems appeared with two high peaks, one was in coincidence with that of the roots, the other appeared between March and May (dry season) just as the period with low alkaloid contents in root. The alkaloid contents of leaves of wild Rauvolfia appeared with two high peaks, both were the same as those of the stems, but in the cultivated Rauvolfia, only one peak with high contents from July to August (raining season) was observed.     

Biochemical strategy of sequestration of pyrrolizidine alkaloids by adults and larvae of chrysomelid leaf beetles

Tracer feeding experiments with (14)C-labeled senecionine and senecionine N-oxide were carried out to identify the biochemical mechanisms of pyrrolizidine alkaloid sequestration in the alkaloid-adapted leaf beetle Oreina cacaliae (Chrysomelidae). The taxonomically closely related mint beetle (Chrysolina coerulans) which in its life history never faces pyrrolizidine alkaloids was chosen as a 'biochemically naive' control. In C. coerulans ingestion of the two tracers resulted in a transient occurrence of low levels of radioactivity in the hemolymph (1-5% of radioactivity fed). With both tracers, up to 90% of the radioactivity recovered from the hemolymph was senecionine. This indicates reduction of the alkaloid N-oxide in the gut. Adults and larvae of O. cacaliae sequester ingested senecionine N-oxide almost unchanged in their bodies (up to 95% of sequestered total radioactivity), whereas the tertiary alkaloid is converted into a polar metabolite (up to 90% of total sequestered radioactivity). This polar metabolite, which accumulates in the hemolymph and body, was identified by LC/MS analysis as an alkaloid glycoside, most likely senecionine O-glucoside. The following mechanism of alkaloid sequestration in O. cacaliae is suggested to have developed during the evolutionary adaptation of O. cacaliae to its alkaloid containing host plant: (i) suppression of the gut specific reduction of the alkaloid N-oxides, (ii) efficient uptake of the alkaloid N-oxides, and (iii) detoxification of the tertiary alkaloids by O-glucosylation. The biochemical mechanisms of sequestration of pyrrolizidine alkaloid N-oxides in Chysomelidae leaf beetles and Lepidoptera are compared with respect to toxicity, safe storage and defensive role of the alkaloids.     

An N-formyl cyclopeptide alkaloid from the bark of zizyphus sativa

From the bark of Zizyphus sativa, previously undescribed cyclopeptide alkaloid, sativanine-F has been isolated. The structure was deduced by spectroscopic methods and chemical degradation. It is a 13-membered cyclopeptide alkaloid and provides the first example of such a naturally occurring N-formyl cyclopeptide alkaloid.     

Alkaloid production by plant cell cultures of Holarrhena antidysenterica: II. Effect of precursor feeding and cultivation in stirred tank bioreactor

Precursor feeding strategy for increasing the yield of conessine, a steroidal alkaloid of Holarrhena antidysenterica, was established in cell suspension culture. A total of 50 mg/L added cholesterol was converted into 43 mg/L of alkaloid, 90% of which constituted the conessine. By applying the precursor feeding policy to the cell suspension culture in modified Murashige and Skoog (MS) medium, a total of 143 mg/L of alkaloid was produced in 8 days. In this way the alkaloid content of the cells was increased more than six times compared to that obtained in the standard MS medium. The steps leading to biotransformation of cholesterol into alkaloids were unaffected by phosphate. The shake flask data were successfully transferred to a bench scale 6-L stirred tank bioreactor in which the specific biosynthetic rate of alkaloid production was 110 mg/100 g dry cell weight per day, about 160 times higher than that of whole plant.     

Variability in tissue cultures of Choisya ternata. Alkaloid accumulation in protoclones and aggregate clones obtained from established strains

Protoclones and aggregate clones have been prepared from 5 callus strains of C. ternata chosen for their dihydrofuroquinoline-accumulating capacities in a population of well-established strains. The results show that it is possible to obtain cell lines which accumulate higher alkaloid contents than the highest alkaloid-producing strain; the probability of obtaining a high-producing clone is greater if a high-producing strain is chosen as the parent strain for cloning. Compared to the alkaloid content of the whole plant, one alkaloid (platydesminium) could be obtained in greater amounts in some clones, but another alkaloid (balfourodinium) was always found in lesser quantities, even in the clones which accumulated most alkaloids. Aggregate clones were easier to obtain than protoclones and alkaloid production was rather unstable in all the clones. The protoplast-cloning procedure was not more efficient than the aggregate-cloning procedure for the selection of high-alkaloid producing lines.     

Effect of Light on Alkaloid Accumulation in Cell Cultures of Nicotiana Species

The effect of light on alkaloid accumulation in a range of cellcultures of tobacco was determined. Cell suspension culturesof Nicoriana rabacwn L. cv. Wisconsin-38 with differing degreesof photosynthetic activity, callus cultures of N. glauca Graham,root cultures of N. rustica L. and shoot cultures of N. tabacumwere used. The alkaloid content of green illuminated cultureswas greatly reduced compared with non-green cultures grown inthe dark, but decreased accumulation did not correlate withincreasing photosynthetic activity. The accumulation of allof the major alkaloids was affected, regardless of the speciesof tobacco used. Transfer of N. glauca callus from the darkinto the light caused a decrease in alkaloid accumulation, whilemoving cultures from the light into the dark resulted in anincrease in alkaloid content. In root cultures light causeda reduction in growth, which affected alkaloid synthesis. Inshoot cultures there were only traces of alkaloid detectable,regardless of whether or not cultures were illuminated. Lightappeared to cause a non-photosynthetic suppression of alkaloidaccumulation in visibly undifferentiated cultures, and thiseffect was modified in visibly differentiated cultures. Key words: Nicoriana spp, tobacco, alkaloid accumulation, cell culture