Enhanced production of phenolic compounds in hairy root cultures of Polygonum multiflorum and its metabolite discrimination using HPLC and FT-IR methods.

Hairy root cultures (HRCs) are characterized by fast and unlimited root growth, and they have greater genetic stability than other cultivation methods. HRCs are known to accumulate phytochemical levels comparable to those of intact plant. In this study, HRCs of Polygonum multiflorum were established from leaf explants infected with Agrobacterium rhizogenes strain KCCM 11879. Over 60% of the explants showed hairy root induction after 21 days of cultivation on hormone-free MS (Murashige and Skoog Physiol Plant 15:473-479, 1962) medium; induced roots were confirmed by PCR using a rolC-specific primer. Of the six lines of HRCs selected for further analysis, line HR-01 performed best, producing a root biomass (105.2 g L-1 of FW, 9.7 g L-1 of DW), which is 10-fold higher than that of non-transgenic roots. The HR-01 line also showed a significant increase in its total phenolic content (26.64 mg g-1 DW), while non-transgenic roots accumulated 8.36 mg g-1 DW of total phenolic. The levels of phenolic compounds in the HRCs increased more than 2.5-fold following exposure to 50 µM methyl jasmonate for 5 days. Fourier transform infrared (FT-IR) spectroscopic analysis of bioactive accumulation in P. multiflorum enabled discrimination between hairy root and adventitious root cultures. Thus, it is evident from this study that HRCs could be an attractive proposition for large-scale production of root biomass and secondary metabolites of P. multiflorum in bioreactors.

Improvement of biosynthesis and accumulation of bioactive compounds by elicitation in adventitious root cultures of Polygonum multiflorum.

We examined the effects of abiotic (methyl jasmonate [MeJA] and salicylic acid [SA]) and biotic (yeast extract and chitosan) elicitors for improvement of bioactive compounds production on adventitious root cultures in Polygonum multiflorum. The application of yeast extract resulted in significantly (p ≤ 0.05) higher dry root biomass (9.98 g/L) and relative growth rate versus the control. Cultures treated with abiotic elicitors showed higher percentage of dry weight than the other samples. Low concentrations of all elicitors (50 µM MeJA and SA, and 50 mg/L yeast extract) improved secondary metabolite production except for chitosan, whose performance was worse than that of the control. HPLC analysis of various bioactive compounds revealed significantly higher elicitation efficiency for MeJA than for the other treatments, with an approximately 2-fold increase in root dry weight (22.08 mg/g DW) under 50 µM MeJA treatment versus the control (10.35 mg/g DW). We also investigated the feasibility of scaling up the production process by comparing shake flask cultures with 3- and 5-L balloon type bubble bioreactors (BTBB) using 50 µM MeJA as an elicitor. Growth and metabolite accumulation increased in BTBB compared with shake flask cultures. We detected a non-significant difference in biomass productivity between 3 and 5-L BTBB, but the efficiency of bioactive compound accumulation decreased with increasing volume. These findings will be useful for developing a pilot-scale P. multiflorum adventitious root cultivation process for high biomass and bioactive compound production to meet the demands for natural ingredients by the pharmaceutical and cosmetic industries without affecting the natural habitat of this plant.

Metabolomics Reveals Rubiadin Accumulation and the Effects of Methyl Jasmonate Elicitation in Damnacanthus major Calli.

Callus suspension techniques have been considered attractive for improving bioactive metabolite productivity; methyl jasmonate (MeJA) is a widely used elicitor for stimulating synthetic pathways. In this study, a multivariate analysis-based metabolomics approach was employed to investigate the primary and specialized metabolites in the leaves, unelicited calli, and 100 or 200 µM MeJA elicited calli of Damnacanthus major. Rubiadin, a powerful anthraquinone with various therapeutic properties, was only identified in D. major calli, accumulating in a MeJA elicitation concentration-dependent manner. Callus cultures also contained high levels of amino acids, sugars, and phenolic compounds, indicating energy metabolism and metabolic adaptation responses for proliferation and stabilization. Regarding MeJA application, elicited calli contained higher amounts of quinic acid, kaempferol, and glucose with lower amounts of sucrose and raffinose than those in the unelicited control, which were closely related to protective mechanisms against MeJA. Moreover, excessive elicitation increased the asparagine, fructose, and raffinose levels and decreased the glucose and sucrose levels, which was ascribed to increased activation of the aminoacyl-tRNA biosynthesis pathway and wider utilization of glucose than of fructose after sucrose degradation. These results will be useful for optimizing plant cell culture techniques to achieve high production rates for valuable specialized metabolites.

Proliferation and Metabolic Profiling of Cynanchum wilfordii Adventitious Roots Using Explants from Different Cultivation Methods.

Cynanchum wilfordii root is used in traditional herbal medicine owing to its various pharmacological activities. However, C. wilfordii roots are misused owing to their morphological similarities with C. auriculatum. Adventitious root (AR) culture can prevent such misuse, and the selection of plant materials is an important procedure for producing high-quality ARs. This study aimed to compare the proliferation and metabolic profiles of C. wilfordii ARs in two types of explants from different cultivation methods (either cultivated in open field (ECF) or cultivated on a heap of C. wilfordii (ECH)). After 4 weeks of culture, the proliferation rate and number and length of secondary ARs were determined, and 3/4 Murashige and Skoog (MS) salt medium, 4.92 µM indole-3-butyric acid (IBA), and 5% sucrose were suggested as the best proliferation conditions for ARs originating from both ECF and ECH. Through metabolic profiling, ARs from ECH were found to show higher accumulation patterns for flavonoids, polysaccharides, hydroxyacetophenones, aromatic amino acids, and mono-unsaturated fatty acids, which were ascribed to the activation of flavonoid biosynthesis, the phenylpropanoid pathway, and fatty acid desaturase, stimulated by abiotic stresses. In contrast, ARs from ECF had higher levels of TCA cycle intermediates, amino acids in the aspartate-glutamate pathway, and saturated and polyunsaturated fatty acids, indicating energy metabolism and plant development. Overall, the current study provided information on the optimal conditions for inducing C. wilfordii ARs with higher amounts of bioactive compounds.

18F-fluorodeoxyglucose-positron emission tomography in the staging of malignant lymphoma compared with CT and 67Ga scan.

The accurate staging of Hodgkin's disease (HD) and non- Hodgkin's lymphoma (NHL) is an important aspect of treatment. In this study, the authors undertook to prospectively evaluate the clinical value of 2-(fluorine-18)fluoro-2-deoxy-D- glucose position emission tomography (FDG-PET) for the staging of malignant lymphoma as compared with computed tomography and 67Ga scan. Thirty consecutive cases with biopsy-proven lymphoma (4 HD, 26 NHL) were examined by FDG-PET for the initial staging and the restaging work-up between September 2000 and April 2001. The FDG-PET and conventional study, including a CT of the neck, chest, abdomen, and of the pelvis, a bone scan, a 67Ga scan, and a bone marrow study were undertaken to investigate nodal/extranodal manifestations and bone marrow infiltration. In terms of the detection of nodal lymphoma manifestation, the sensitivities and specificities of the PET, CT, and 67Ga scan were determined to be 93.3%, 98.9%, and 25.8%, and 100%, 99.1%, and 99.8%, respectively. In terms of the detection of extranodal lymphoma manifestation, the sensitivities and specificities of the PET, CT, and 67Ga scan were 87.5%, 87.5%, and 37.5%, and 100%, 100%, and 100%, respectively. The FDG-PET proved to be very accurate for the staging of malignant lymphoma and superior to Ga-67 scan. Although the results of PET and CT were substantially comparable, both imaging studies were found to complement each other in some cases with respect to the evaluation of lymphomatous involvement.

Early biliary complications of laparoscopic cholecystectomy: evaluation on T2-weighted MR cholangiography in conjunction with mangafodipir trisodium-enhanced 3D T1-weighted MR cholangiography.

OBJECTIVE: Our aim was to assess preliminary experience with combined conventional T2-weighted and mangafodipir trisodium (MnDPDP)-enhanced T1-weighted MR cholangiography in evaluating early biliary complications of laparoscopic cholecystectomy. SUBJECTS AND METHODS: Conventional heavily T2-weighted MR cholangiography with MnDPDP-enhanced T1-weighted MR cholangiography and ERCP were performed in seven patients with high clinical suspicion of biliary complications after laparoscopic cholecystectomy. The final diagnoses of complications were classified according to the presence and degree of bile duct injury, bile leakage, and retained stones. RESULTS: The diagnoses on MR cholangiography were as follows: complete transection and occlusion of the common bile duct with bile leakage (n = 3), partial strictures of the common bile duct with bile leakage (n = 1), cystic duct leakage (n = 1), partial ligation of an aberrant right hepatic duct (n = 1), and hemorrhage without biliary complication (n = 1). The final diagnoses at surgery (n = 2) and ERCP (n = 5) were as follows: complete transection and occlusion of the common bile duct with bile leakage (n = 2), partial strictures of the common bile duct with bile leakage (n = 2), cystic duct leakage (n = 1), partial ligation of an aberrant right hepatic duct (n = 1), and hemorrhage without biliary complication (n = 1). MR cholangiography accurately yielded the same findings as the final diagnoses, except in one case with partial stricture of the bile duct with bile leakage (overdiagnosed as complete occlusion on MR cholangiography). CONCLUSION: Combined conventional T2-weighted and MnDPDP-enhanced T1-weighted MR cholangiography may eliminate the use of other studies for the imaging of biliary complications after cholecystectomy if this preliminary data can be verified in a larger study.