The antimutagenic activity of biomass extracts from the cultured cells of medicinal plants in the Ames test

Antimutagenic activity of 20 and 40% ethanol extracts from the biomass of Rhodiola rosea, Polyscias filicifolia, Panax ginseng and Ungernia victoris cultured cells have been studied. DDDTDP, ethidium bromide, benz(a)pyrene, benzidine served as model mutagens for Salmonella typhimurium TA 98 strain (the latter two were tested in presence of metabolic activation system); for S. typhimurium TA 100 strain these were tio-tefa, bichromate potassium and sodium azide and heavy metal compounds (chlorides of manganese, zinc, cadmium, lead acetate) for both strains. Higher capacity of the extracts from the biomass of R. rosea and P. filicifolia to counteract gene mutations induced by various mutagens was demonstrated (ca. 90% inhibition in isolated cases). In the experiment with the metabolic activation most effective proved to be the extracts from the P. ginseng biomass (up to 34% and 47% mutagenicity inhibition).     

Screening substances derived from cultures of medicinal plants for antimutagenic activity in the Escherichia coli-bacteriophage lambda system

In Escherichia coli--bacteriophage lambda system protective properties of the extracts derived from the biomass of cultured Panax ginseng, Polyscias filicifolia, Rhodiola rosea, Ungernia victoris cells, and those from intact Rhodiola roots have been studied. Escherichia coli--bacteriophage lambda system responsiveness was found to vary with the test-object state, namely: the deleted bacteriophage form (lambda-4) as well as previously mutagenized bacteriophage were more sensitive to the mutagenic and antimutagenic influence versus the native bacteriophage lambda +. The contribution of extracts in the induction and realization of the lethal injuries in phages caused by nitrite acid in extracellular phage (conditions in vitro) was estimated thus enabling to discriminate between the protective and antimutagenic extract activities. Protective extract effect in the given test-system appeared to be higher their antimutagenic action. With the most responsive bacteriophage variant the extracts from the biomass of cultured Rh. rosea and P. filicifolia cells showed high protective and somewhat lower antimutagenic activities. With other phages significant antimutagenic potential of extracts was demonstrated, which by their protective effect could be arranged in a raw as follows U. victoris > P. ginseng > P. filicifolia. The primary screening for the antimutagenic effect of preparations in the prokaryotic systems could be reduced to the investigation of their effects on the object inactivation exposed to the mutagen in vitro.     

中国人参花化学成分研究

目的:对人参(Panax ginseng C.A.Mey.)花蕾的化学成分进行研究。方法:采用多种柱色谱技术进行分离纯化,并通过波谱分析方法鉴定化合物的结构。结果:分离鉴定了9个化合物,分别鉴定为咖啡碱(1)、胡萝卜苷(2)、豆甾醇3-O-葡萄糖苷(3)、α-菠甾醇(4)、7-豆甾烯-3β-醇(5)、人参皂苷Rk3(6)、人参皂苷Re(7)、人参皂苷Rg2(8)、谷甾醇(9)结论:其中化合物1为五加科植物中首次分离得到,化合物3、4、5为人参花中首次分离得到。     

Molecular authentication of Panax species

Using conserved plant sequences as primers, the DNA sequences in the ribosomal ITS1-5.8S-ITS2 region have been amplified and determined for six Panax species, P. ginseng C. A. Mey. (Oriental ginseng), P. quinquefolius L. (American ginseng), P. notoginseng (Burkill) F. H. Chen (Sanchi), P. japonicus C. A. Mey. (Japanese ginseng), P. trifolius L. and P. major Ting, as well as two common adulterants of ginseng, Mirabilis jalapa L. and Phytolacca acinosa Roxb. An authentication procedure based upon the restriction fragment length polymorphism (RFLP) in the region is able to differentiate between P. ginseng and P. quinquefolius, and to discriminate the ginsengs from the two common poisonous adulterants. Broader application of this approach to authenticate other morphologically similar Chinese medicinal materials is rationalised.     

Mesostructure of the ginseng photosynthetic apparatus in relation to ecological strategy of the species

Mesostructure of the photosynthetic apparatus was investigated in the wild plants of ginseng (Panax ginseng C.A. Mey, Araliaceae) taken from various habitats. The revealed features of the structure of phototrophic tissues (large cells, small number of photosynthesizing cells and chloroplasts per leaf area unit, low values of membrane indices of the cells and chloroplasts) point to stress-tolerant type of ecological strategy ginseng pursues in Nature.     

人参毛状根生物合成熊果苷的分离与鉴定

熊果苷(arbutin),化学名称为对-羟基苯-β-D-吡喃葡萄糖苷,能够竞争性抑制酪氨酸酶的活性从而抑制黑色素的形成,被国际公认为高效祛斑美白剂,是化妆品中理想的添加成分.人参(Panax ginseng C. A. Mey.)自古以来就是名贵药材,由于人参在栽培过程中存在着栽培困难、周期过长、地域限制等难题,人参的组织培养受到了广泛的重视.本实验室已建立了人参细胞大量培养体系[1]和人参毛状根培养体系[2],并把熊果苷与人参细胞配伍应用到化妆品生产中,产品深受广大消费者青睐.用植物培养物对外源底物进行生物转化,从而对其结构进行修饰,以获得更有意义的产物的研究报道很多[3～9],也是当今研究的热点.本实验室已对人参生物转化熊果苷的基本条件进行了初步探讨[10],本文在此基础上,对转化产物进行了分离鉴定.     

Stimulation of pituitary-adrenocortical system by ginseng saponin.

Effects of preparations of saponin mixture and isolated ginsenosides, extracted from the root of Panax ginseng, on plasma corticotropin (ACTH) and corticosterone concentrations in rats were determined by the radioimmunoassay and competitive protein binding method. When ginseng saponin mixture was administered to rats intraperitoneally, plasma ACTH and corticosterone increased significantly 30, 60 and 90 min after the treatment. The kinetic pattern of the increase in plasma ACTH was almost parallel to that in plasma corticosterone. Isolated ginsenoside, protopanaxadiol or protopanaxatriol glycoside, also increased plasma corticosterone. The ginseng-induced increase in plasma corticosterone was suppressed by pretreatment with dexamethasone. Thus the ginseng saponin was found to act on the hypothalamus and/or hypophysis primarily, and stimulated ACTH secretion which resulted in increased synthesis of corticosterone in the adrenal cortex.     

Features of ginseng saponin-induced corticosterone secretion.

Ginseng saponin administered intraperitoneally to rats induced a significant rise in plasma corticosterone, while it tended to increase plasma glucose and to decrease plasma immunoreactive insulin. Oral or intraperitoneal administration of ginseng saponin increased plasma corticosterone in unanesthetized, pentobarbital-anesthetized or alloxan-diabetes rats. The histamine-induced rise in plasma corticosterone was suppressed by pretreatment with diphenhydramine, whereas the ginseng-induced rise was not. Ginseng saponin decreased rectal temperature while it increased plasma corticosterone. Ginseng-induced corticosterone secretion was superimposed on the basal levels of plasma corticosterone due to fasting and circadian rhythm. Thus ginseng saponin would be a kind of stressful agent and have different features associated with the stimulation of the pituitary-adrenocortical system from several other chemical agents.     

Breathing activity of suspension culture of cells of Polyscias filicifolia Bailey, Stephania glabra (Roxb.) Miers, and Dioscorea deltoidea Wall

Peculiarities of breathing of cultures of cells producing biologically active compounds (isoprenoids and alkaloids) were investigated in order to optimize productivity of culture growth and biosynthesis. It had been revealed that studied cultures of cells of Dioscorea deltoidea Wall (producer of furistanol glycosides), Stephania glabra (Roxb.) Miers (producer of stepharin alkaloid) and Polyscias filicifolia Bailey (complex of biologically active agents) differ both in joint breathing activity and in ratio between cytochrome and cyanide-resistant breathing, while changes of rate of total oxygen consumption and activity of alternative oxidase during growth were found to be individual for every investigated culture. Maximum rate of oxygen consumption for cells of D. deltoidea and S. glabra was marked in the period preceding active synthesis of secondary metabolites (lag phase for D. deltoidea and exponential phase for S. glabra). The revealed trends can be used for further monitoring and regulation of growth and biosynthesis of secondary metabolites in producing cell cultures during deep cultivation.     

Defense response mechanisms of ginseng callus cultures induced by Yersinia pseudotuberculosis, a human pathogen

The effect of Yersinia pseudotuberculosis, the bacterial pathogen affecting humans and animals, on growth of ginseng (Panax ginseng C.A. Mey) cell cultures was studied. The bacteria strongly induced the expression of phenylalanine ammonia-lyase and β-1,3-glucanase, the proteins encoded by the defense-related genes of ginseng and inhibited the normal ginseng callus growth but did not affect the resistant cell cultures. The thermostable and thermolabile protein toxins of these bacteria are lethal to mice when induced parentherally, and they also induced the expression of the defense-related genes in ginseng callus cultures. At the same time, the ginseng cells completely suppressed the bacterial cell growth. These data suggest that the ginseng cells recognized the yersinia and developed the immune response to this pathogen. The interaction between the ginseng cells and Y. pseudotuberculosis is similar to the hypersensitive response of plants to plant pathogens.     

Effects of Different Light Qualities on Structure of Chloroplasts and Photosynthetic Physiological Properties in Panax ginseng

The ultrastructure of chloroplasts and the photosynthetic physiological properties of Panax ginseng C. A. Mey. grown under different light qualities with same light transmission rate (25 % of sun light) were investigated. The results showed that the thylakoid membranes of Panax ginseng chloroplasts are well deve lopted, and the number of grana and granal lame llae under green and violet film are more than that under red and blue film. The content of chlorophyll in the same area of leaves and the absorption spectra area of chlorophylls and leaves under violet and green film are higher than those under other films. All the photosynthetic rates are very low, and their sequence from high to low are violet, green, red and blue . Green film is advantageous to the accumulation of chlorophylls and the development of thylakoid membranes and red film is advantageous to the accumulation of chlorophyll b. Blue film reduced granal thylakoid staking and decreased the photosynthetic rate. A superior trend of the photosynthetic physiologic properties as well as the structure of chloropiasts of Panasc ginseng leaves under violet film,being composed with red and blue film is significant.     

Influence of salicylic acid on biosynthesis of nucleic acids in Polyscias filicifolia cell culture under the action of unfavorable temperatures

Amounts of DNA and RNA was increased (from 20 to 50%) in the presence of salicylic acid in cells of Polyscias filicifolia tissue culture grown in Murachige-Skoog modified medium. Treatment of the tissue culture with salicylic acid resulted in a significant increase of intracellular protein and decrease of proteolytic activity. In cells treated with salicylic acid, the amounts of DNA and RNA was higher in conditions of heat (3 h, 45 degrees C) and cold (24 h, 7 degrees C) stress in comparison with cells exposed to unfavorable temperatures without the initial treatment with salicylic acid.     

Effect of Hormones on Embryo After-Ripening of Panax ginseng G. A. Mey. Seeds

Panax ginseng C. A. Mey seed has a deep dormancy for the morphologica and physiological after-ripening of embryo. This paper described the changes of some enzymic activities and of soluble-protein content during after-ripening of seeds, in the first stage of morphological after-development the activities of amylase, peroxidase and catalase increased gradually, whereas the soluble-protein content decresed rapidly. A high level of enzymatic activities and increasing protein content were also observed, indicating vigorous metabolism in physiological after-ripening of seeds. Hormone treatment increased the activities of the above-mentional enzymes, promoted the development and differentiation of embryo, causing the time of seeds germination one year earlier than that of untreated ones.     

Effect of growth regulators on ginsenoside production in the cell culture of two ginseng species

Plants belonging to the genus Panax produce ginsenosides that possess pharmacological properties. The ability to synthesize these compounds is preserved in some cultured cells of ginseng. In this work, we used suspension cell cultures of two species of ginseng: Panax japonicus var. repens C. A. Mey and P. ginseng C. A. Mey. The first culture was grown on MS medium supplemented with α-NAA. After one subculturing, cell biomass increased 5–6 times with the level of ginsenosides being equal to 2.5–3.0% of dry weight. The second culture was grown on the same medium supplemented with 2,4-D. In this case, biomass increment was 3–5-fold, and ginsenosides were produced in trace amounts. Substitution of 2,4-D for NAA in suspension cell culture of P. japonicus brought about deterioration of growth characteristics, but the content and composition of ginsenosides was not changed. In the suspension cell culture of P. ginseng, substitution of NAA for 2,4-D decreased the rate of biomass accumulation and increased the extent of cell aggregation, with total content of ginsenosides increasing 25 times and their assortment being complete.     

Improvement of insulin resistance by panax ginseng in fructose-rich chow-fed rats.

In an attempt to probe a new target for handling insulin resistance, we used Panax ginseng root to screen the effect on insulin resistance induced by fructose-rich chow in rats. Insulin action on glucose disposal rate was measured using the glucose-insulin index, which is the product of the areas under the curve of glucose and insulin during the intraperitoneal glucose tolerance test. Oral administration of Panax ginseng root (125.0 mg/kg) into rats three times daily for three days after receiving fructose-rich chow for four weeks reversed the increased glucose-insulin index, indicating that Panax ginseng root has the ability to improve insulin sensitivity. In addition, the plasma glucose concentrations in rats repeatedly treated with Panax ginseng root were not elevated as markedly as those of the vehicle-treated group during the fructose-rich chow-feeding period. Also, the time in which the plasma glucose-lowering response to tolbutamide (10.0 mg/kg, i. p.) receded in fructose-rich chow fed rats was markedly delayed by repeated Panax ginseng root treatment compared to the vehicle-treated group. The plasma glucose-lowering activity of tolbutamide is believed to depend on the secretion of endogenous insulin, which is widely used as an indicator of insulin resistance development. Thus, it provided supportive data that oral administration of Panax ginseng root could delay the development of insulin resistance in rats. In conclusion, our results suggest that oral administration of Panax ginseng root improves insulin sensitivity and may be used as an adjuvant therapy for treating diabetic patients with insulin resistance.     

Ginseng modifies the diabetic phenotype and genes associated with diabetes in the male ZDF rat

Asian ginseng (Panax ginseng) and its close relative North American ginseng (Panax quinquefolius) are perennial aromatic herbs that are widely used in Oriental medicine and have been acclaimed to have various health benefits including diabetes treatment. In this study, we compared the effects of a diet containing rosiglitazone to a diet containing ginseng (Panax quinquefolius) in male Zucker diabetic fatty (ZDF) rats. Animals were assigned to one of three diets: control, rosiglitazone (0.1 g/1 kg diet), or ginseng (10 g/1 kg diet). During the 11-week study, body weight, food intake, organ weight, blood glucose, plasma cholesterol, and plasma triglyceride levels were evaluated. Animals treated with rosiglitazone or ginseng exhibited increased body weight (p<0.05) and decreased kidney weight (p<0.05) compared to control animals. The rosiglitazone group demonstrated decreased food intake and plasma triglyceride levels versus the other groups (p<0.05). The ginseng group revealed decreased cholesterol levels relative to the control group (p<0.05). Furthermore, ginseng and rosiglitazone had marked effects on the expression of genes involved in PPAR actions and triglyceride metabolism compared to controls. In conclusion, ginseng modified the diabetic phenotype and genes associated with diabetes in the male ZDF rat. These data are encouraging, and warrant further research to determine the therapeutic value of this medicinal herb in treating human diabetes.     

高产人参寡糖素培养细胞克隆系的筛选

人参（ＰａｎａｘｇｉｎｓｅｎｇＣ．Ａ．Ｍｅｙ．）培养细胞经细胞平板克隆,获得近３００个克隆系。克隆系在细胞生长速率和寡糖素含量及产率上均存在显著差异,且寡糖素产率和细胞生长之间有明显的相关性。经１１代连续继代培养观察及过氧化物酶同工酶谱特征分析,筛选到一株寡糖素产率高且稳定的克隆系ＰＧ－１８０,其平均生长速率是０．４９５ｇ于重／Ｌ·天,为原始株系的１．３９倍,平均寡糖素含量为１４．６９％干重,是亲本的１．６５倍,平均寡糖素产率是２．１８３ｇ／Ｌ,为原始株系的２．３２倍。比较克隆系ＰＧ－１８０和原始株系细胞悬浮培养时间进程发现,由人参培养细胞生产寡糖素的最佳细胞收获期为３周左右。     

Screening of Clone Lines with High Yield of Oligosaccharins from Culture Cells of Panax ginseng

Cell plating clone technique was employed to screen clone lines with high yield of oligosaccharins from culture cells of Panax ginseng C. A. Mey. Near 300 clone lines were obtained. The results from some clone lines analysed implied that these clone lines were significantly different in cell growth rate, oligosaccharins content and yield. Furthermore, there was a distinct correlation between oligosaccharins productivity and cell growth. A more stable high-yield oligosaccharin clone line PG-180 had been selected according to the characteristics of growth rate, oligosaccharin yield and peroxidases isozyme patterns during successive subculturing of 11 generations of clone lines. The mean growth rate of clone line PG-180 was 0. 495 g dry wt/L · d, and was 1.39 folds higher than to the original strain. Its mean content and yield of oligosaccharins were 14. 69 % dry wt and 2.183 g/L, which were 65 % and 132% respectively higher than those of the original strain. In comparing the time course of cell suspension culture between clone line PG-180 and the original strain, the optimal period for high oligosaccharin production from P. ginseng culture cells was approximately three weeks.     

细胞培养生产人参寡糖素降低成本的途径

在人参（Ｐａｎａｘｇｉｎｓｅｎｇ）细胞悬浮培养中,以无离子水代替重蒸馏水,细胞生长速率和寡糖素产率分别降低２．３％和２．９％。用白糖代替蔗糖,细胞生长速率和寡糖素产生率分别降低１．７４％和１．２３％。综合上述两方面结果,以无离子水和白糖分别替代原培养基中的重蒸馏水和蔗糖组成替代培养基,用替代培养基培养人参培养细胞,其生长速率可达０．５０９ｇＤＷ／Ｌ．ｄ．寡糖素产率可达１．４４３ｇ／Ｌ,和原培养基相     

Ethephon- and Jasmonate-Elicited Pathogenesis-Related Ribonucleases in Cultured Ginseng Cells

Cultured ginseng cells (Panax ginseng C.A. Mey strain R-1) produce two proteins exhibiting RNase activity (Pg1 and Pg2), which, on the basis of their amino acid sequences, have been earlier referred to intracellular pathogenesis-related proteins. An immunoenzyme technique for estimation of these proteins was developed. A close correlation was found between the content of these proteins and the RNase activity of the cultured cells. Ethephon and jasmonic acid activated the RNase activity, ethephon being more efficient. Salicylic acid did not activate Pg1 and Pg2; high concentrations of salicylic acid suppressed the RNase activity of the culture. The protein kinase inhibitor, H-7, reduced the content and activity of RNases both in the presence and absence of ethephon. The results obtained permit a suggestion that ethylene and jasmonic acid signaling pathways, which include protein phosphorylation, are involved in the induction of PR-10 proteins.