Effect of initial phosphate concentration on cell growth and ginsenoside saponin production by suspended cultures of panax notoginseng

Effects of initial phosphate concentration on the growth, ginsenoside saponin production, and the consumption of sugar and nitrogen sources by suspended cells ofPanax notoginseng (Burk) F. H. Chen were investigated in a 250-mL shake flask. The results indicate that by increasing the initial phosphate concentration in the medium in the range of 0–1.25 mM, both the cell growth and the saponin accumulation were greatly improved, and the utilization of sugar and nitrogen sources was also increased. The highest production, productivity, and yield of ginsenosides obtained were 0.98 g/L, 45.5 mg/L/d, and 0.030 g/g at 1.25 mM of initial medium phosphate. At a relatively higher level of medium phosphate, i.e., 2.0 mM, the product accumulation was inhibited to some degree, although the cell growth was not.     

Production of triterpene acids by cell suspension cultures of Olea europaea

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.     

Production of exocellular polysaccharide by azotobacter chroococcwn

Environmental conditions affect the production of extracellular polysaccharide by Azotobacter chroococcum ATCC 4412. Production of exocellular polymer from a variety of carbon sources depended on the air flow rate. A high sucrose concentration in medium (8%) markedly favored expopolysaccharide production, which reached 14 g/L in about 72 h. In cell suspensions incubated in the presence of 8% sucrose in a nitrogen-free medium, biopolymer final concentration of 9 g/L corresponds to 68 g/g biomass. Maximum efficiency of sucrose conversion into exopolysaccharide peaked at 70% for initial disaccharide concentration of 6%. High performance liquid chromatography and gas liquid chromatography of acid hydrolysates of the exopolymer revealed the presence of mannuronosyl, guluronosyl, and acetyl residues, but not neutral sugars. The infrared spectrum corroborated the presence of carboxylate anions and O-acetyl groups in the exopolymer. Though the presence of more than one kind of polysaccharide cannot be ruled out, these data suggest that, under the experimental conditions used in this work, only a type of alginate-like exopolysaccharide is produced by A. chroococcum ATCC 4412.     

Effect of ginseng saponins on the survival of cerebral cortex neurons in cell cultures

The effects of nerve growth factor (NGF) and saponins isolated from Panax ginseng C.A. Mayer on the survival of chick and rat embryonic cerebral cortex neurons were examined. Ginsenoside Rg1 (GRg1) exerted a survival-promoting effect on both chick and rat cerebral cortex neurons in cell cultures. Ginsenoside Rb1 (GRb1) also had an effect in the rat and displayed some influence in the chick. NGF alone exerted no effect on both neurons, although it did potentiate the GRb1 effect on chick embryonic cerebral cortex neurons, but did not alter the GRb1 effect on rat embryonic cerebral cortex neurons. NGF did not alter the survival-promoting effect of GRg1 on either chick or rat embryonic cerebral cortex neurons. The other saponins alone or with NGF exerted no effect on the survival of cerebral cortex neurons in either the chick or rat.     

Production of Minor Ginsenosides from Panax notoginseng Flowers by Cladosporium xylophilum

Panax notoginseng flowers have the highest content of saponins compared to the other parts of Panax notoginseng, but minor ginsenosides have higher pharmacological activity than the main natural ginsenosides. Therefore, this study focused on the transformation of the main ginsenosides in Panax notoginseng flowers to minor ginsenosides using the fungus of Cladosporium xylophilum isolated from soil. The main ginsenosides Rb₁, Rb₂, Rb₃, and Rc and the notoginsenoside Fa in Panax notoginseng flowers were transformed into the ginsenosides F₂ and Rd₂, the notoginsenosides Fd and Fe, and the ginsenoside R₇; the conversion rates were 100, 100, 100, 88.5, and 100%, respectively. The transformation products were studied by TLC, HPLC, and MS analyses, and the biotransformation pathways of the major ginsenosides were proposed. In addition, the purified enzyme of the fungus was prepared with the molecular weight of 66.4 kDa. The transformation of the monomer ginsenosides by the crude enzyme is consistent with that by the fungus. Additionally, three saponins were isolated from the transformation products and identified as the ginsenoside Rd₂ and the notoginsenosides Fe and Fd by NMR and MS analyses. This study provided a unique and powerful microbial strain for efficiently transformating major ginsenosides in P. notoginseng flowers to minor ginsenosides, which will help raise the functional and economic value of the P. notoginseng flower.     

Comparative titration of ginsenosides by different techniques in commercial ginseng products and callus cultures

The ginsenoside content of different ginseng species (Panax ginseng, P. quinquefolium, and P. vietnamensis) from different sources (roots from field-grown plants or from in vitro cultures, cells from solid calluses or from liquid cultures, commercial powders, and suspensions) is evaluated by means of a new high-performance thin-layer chromatography (HPTLC) technique combining an automatic TLC sampler and scanner. The results are compared with those obtained through more classical gross spectrometric and high-performance liquid chromatography (HPLC) techniques. HPTLC and HPLC allow the separation and estimation of the different ginsenosides. For this, HPTLC is faster and simpler than HPLC. Both techniques determine less amounts of ginsenosides than spectrophotometry, which displays overestimated values caused by light absorption by contaminating osides. In vitro cultured cells and roots contain the same ginsenosides as those produced by their mother plants, although at quite lower levels. The culture media also accumulates ginsenosides.     

Identification of ginsenoside metabolites in plasma related to different bioactivities of Panax notoginseng and Panax ginseng

Although the chemical components of Panax notoginseng (PN) and Panax ginseng (PG) are similar, their bioactivities are different. In this study, the differential bioactivities of PN and PG were used as the research object. First, the different metabolites in the plasma after oral administration of PN and PG were analyzed using a UPLC-Q/TOF-MS-based metabolomics approach. Afterward, the metabolite-target- pathway network of PN and PG was constructed, and thus the pathways related to different bioactivities were analyzed. As a result, 7 different metabolites were identified in PN group, and 10 different metabolites were identified in the PG group. In the PN group, the metabolite N1 was related to hemostasis, N1 and N3 were related to inhibiting the nerve center, antihypertensive, and abirritation. The metabolites N1, N3, N4, N5, and N6 were related to liver protection. The results showed that the metabolites G1, G2, G3, G5, and G6 in PG group were related to heart protection, and G1, G2, G6, and G9 were related to increased blood pressure. There were 13 signaling pathways related to different biological activities of PN (8 pathways) and PG (5 pathways). These pathways further clarified the mechanism of action that caused the different bioactivities between PN and PG. In summary, metabolomics combined with network pharmacology could be helpful to clarify the material basis of different bioactivities between PN and PG, promoting the research on PN and PG.     

Compatible stability study of panax notoginseng saponin injection (xueshuantong®) in combination with 47 different injectables

Panax Notoginseng Saponin (PNS) injection (Xueshuantong®, XST) plays an important role in acute and chronic treatment of cardiovascular disease in China. XST, a freeze‐dried powder injection of PNS, contains ginsenosides, Rg1, Rb1, Re, Rd. and notoginsenoside, R1. Study of the stability of herbal medicine injections is limited. The degradation products may contribute to adverse drug reactions (ADRs). The stability kinetics and degradation mechanisms of the five ingredients of PNS in XST injection were systematically explored in aqueous solution. The compatible stability of XST injection in combination with 47 injectables was evaluated using biophysical analysis. A principal components analysis model provided a good clustering result after compatibility. The degradation of the five ingredients in aqueous solution was found to be acid, temperature, oxidation and trivalent ion‐dependent. All ingredients were much more stable in XST injection than in aqueous solutions. Nine degradation products were identified by adopting LC‐Q‐TOF/MS. Standards exceeding the osmotic pressure and/or particle size were found to be the probable causes of the ADR, which were related to drug combination. To minimize the risk of ADRs, drug combinations should be avoided until the complicated chemical matrices of the Chinese medicine injections are more clearly understood. Copyright © 2016 John Wiley & Sons, Ltd.     

Separation and purification of both tea seed polysaccharide and saponin from camellia cake extract using macroporous resin

A novel method to separate and purify tea seed polysaccharide and tea seed saponin from camellia cake extract by macroporous resin was developed. Among four kinds of resins (AB‐8, NKA‐9, XDA‐6, and D4020) tested, AB‐8 macroporous resin possessed optimal separating capacity for the two substances and thus was selected for the separation, in which deionized water was used to elute tea seed polysaccharide, 0.25% NaOH solution to remove the undesired pigments, and 90% ethanol to elute tea seed saponin. Further dynamic adsorption/desorption experiments on AB‐8 resin‐based column chromatography were conducted to obtain the optimal parameters. Under optimal dynamic adsorption and desorption conditions, 18.7 and 11.8% yield of tea seed polysaccharide and tea seed saponin were obtained with purities of 89.2 and 96.0%, respectively. The developed method provides a potential approach for the large‐scale production of tea seed polysaccharide and tea seed saponin from camellia cake.     

Production and optimisation of rosmarinic acid by Satureja hortensis L. callus cultures

In this study, production and optimisation of rosmarinic acid, a phenolic acid and an economically important metabolite, was investigated in the callus cultures established from the mature seeds of Satureja hortensis L. (summer savory) plant. Gamborg's B5 basal medium, supplemented with indol butyric acid (IBA) (1.00 mg L(-1)), N6-benzyl aminopurine (6-BA) (1.00 mg L(-1)) and sucrose (2.5%, w/v), was employed for the establishment and maintenance of the callus cultures. Applications were individually prepared by preparing the media containing different IBA/6-BA combinations and sucrose concentrations. All of the applications were carried out in the continuous dark. In the applications, where the effects of IBA/6-BA combinations on the growth and rosmarinic acid accumulation were assayed (1-15 applications), the highest biomass yield was obtained from the medium supplemented with 1.00 mg L(-1) IBA and 5.00 mg L(-1) 6-BA. In the case of the rosmarinic acid accumulation, an opposite relationship was determined between the growth and rosmarinic acid production. While the highest biomass yield was obtained from the medium containing 1.00 mg L(-1) IBA and 5.00 mg L(-1) 6-BA, the highest rosmarinic acid accumulation was obtained from the medium supported with 1.00 mg L(-1) IBA and 1.00 mg L(-1) 6-BA. In the applications where the effects of sucrose concentrations on the growth and rosmarinic acid accumulation were examined, the highest biomass yield was obtained from the medium which is supplemented with 5.0% (w/v) sucrose. In this category, the highest rosmarinic acid accumulation was obtained from the medium which is supported with 3.0% (w/v) sucrose. According to the experiments carried out with the wild S. hortensis, it is found to have 25.02+/-1.21 mg g(-1) rosmarinic acid. No differentiation was observed in any callus during the course of this study.     

Production of antioxidant compounds by culture of Panax ginseng C.A. Meyer hairy roots

Ginseng (Panax ginseng C.A. Meyer) hairy root cultures, established by infecting ginseng root discs with Agrobacterium rhizogenes, were used for secondary metabolite production. In this study, several elicitors [salicylic acid (SA), acetylsalicylic acid (ASA), yeast elicitor, and bacterial elicitor] were used to improve the productivity of useful metabolite in P. ginseng hairy root cultures. In SA elicitation, total ginseng saponin content increased slightly at lower elicitor dosages (0.1 to 0.5 mM). Also, the use of ASA as an elicitor resulted in the inhibition of biomass growth and an increase in total ginseng saponin content at every elicitor dosage (0.1 to 1.0 mM) by about 1.1 times. With yeast elicitor addition, hairy root growth was inhibited about 0.8-fold on a dry weight basis compared to the control, but total ginseng saponin content increased by about 1.17 times when compared to the control. The bacterial elicitor showed a slight inhibition of biomass growth, but total ginseng saponin content increased by about 1.23 times upon the addition of 1 mL.     

Monitoring chemical impacts on cell cultures by means of image analyses

Many chemical processes are involved in the interactions of living cells with their environment; however, monitoring such processes often requires sophisticated analyzers. In this study, a sensing strategy based on imaging techniques has been developed to (i) enable cell discrimination based on their physical appearance such as size and shape and (ii) to build predictive models that relate the measured cell appearance to chemical parameters in their environment. Both goals aim at innovative and straightforward sensing strategies for analyzing cell–environment interactions. Image analyses offer several advantages such as the use of simpler, more robust sensors and the omission of extensive sample/sensor preparations. Imaging can analyze numerous cells and thus gains a culture representative insight rather than a potentially nonrepresentative single‐cell response. As a proof‐of‐principle application, different species of microalgae cells have been exposed to various nutrient conditions. Microalgae are known to sensitively adapt to changing nutrient conditions and could potentially become biological “probes” for chemical shifts in ecosystems. Because of considerable spreads of cell size and shapes within one class, size and shape distributions have been derived from visible images of cell cultures. It is shown that the novel image analyses are capable of discriminating different cell species based on their cell shapes and sizes. It is also demonstrated that in conjunction with the recently introduced, nonlinear multivariate “predictor surfaces”, the nutrient availability has a quantifiable impact on the cell size distributions. In this application, predictor surfaces are somewhat more precise than partial least squares. Copyright © 2012 John Wiley & Sons, Ltd.