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Jiao J.,Northeast Forestry University | Gai Q.-Y.,Northeast Forestry University | Gai Q.-Y.,Collaborative Innovation Center for Development and Utilization of Forest Resources | Luo M.,Northeast Forestry University | And 9 more authors.
RSC Advances | Year: 2015

A direct analysis approach for plant in vitro cultures, namely, high speed homogenization coupled with cavitation-accelerated extraction (HSH-CAE) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), was developed for the simultaneous determination of six astragalosides and five isoflavonoids in Astragalus membranaceus hairy root cultures (AMHRCs). In comparison to reported Soxhlet extraction (SE) and ultrasound-assisted extraction (UAE) methods, the proposed sample preparation procedure (HSH-CAE) offers significant improvements with regard to simplicity in operation (elimination of biomass drying and grinding), high efficiency, enhanced yield and green aspects in terms of saving energy cost and minimizing the generation of waste. In addition, the HSH-CAE mechanism was clarified via cytohistological studies of samples at cellular/tissular levels. Moreover, the established LC-MS/MS method provided linearity with correlation coefficients above 0.9991, limit of detections (LODs) below 1.77 ng mL-1, relative standard deviations (RSDs) below 6.01%, and recoveries above 96.84%. Furthermore, the proposed HSH-CAE-LC-MS/MS method was also successfully applied for screening high-productive AMHRCs. Overall, this study opened up a new avenue for the direct determination of secondary metabolic profiles from fresh plant in vitro cultures, which was valuable for improving the quality control of plant cell/organ cultures and shed light on the metabolomics analysis from biological samples. © The Royal Society of Chemistry 2015. Source


Gai Q.-Y.,Northeast Forestry University | Gai Q.-Y.,Collaborative Innovation Center for Development and Utilization of Forest Resources | Jiao J.,Northeast Forestry University | Luo M.,Northeast Forestry University | And 9 more authors.
Biochemical Engineering Journal | Year: 2015

For the first time, Isatis tinctoria hairy root cultures (ITHRCs) induced by Agrobacterium rhizogenes, were established as alternative sources for the production of bioactive alkaloids (AK). The highest transformation rate (76.67%) was obtained when 3 week-old petiole explants were co-cultured with A. rhizogenes for 2 days by the aid of 125. μM acetosyringone and 1.5. mM arginine. Among eight I. tinctoria hairy root lines (ITHRLs), ITHRL III was screened as the lead line and was confirmed by the PCR amplification of rolB, rolC and aux1 genes. Culture conditions of ITHRCs were optimized by Box-Behnken design, and six main AK constituents were qualitatively and quantitatively determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Under the culture temperature (24.7. °C), inoculum size (0.75%), sucrose concentration (3.14%) and initial pH (5.8), ITHRCs (23 day-old) in MS/2 medium gave the maximum biomass dry weight (DW) of 12.85. g/L and the optimal total AK content of 521.77. μg/g DW. Results showed that the proposed ITHRCs system possessed higher ability of AK production as compared to that of 2 year-old field-grown roots (464.69. μg/g DW). Overall, this work offered a promising, sustainable and high-productive biosynthesis platform that was capable of augmentation production of valuable naturally-derived AK. © 2014 Elsevier B.V. Source


Gai Q.-Y.,Northeast Forestry University | Gai Q.-Y.,Collaborative Innovation Center for Development and Utilization of Forest Resources | Jiao J.,Northeast Forestry University | Luo M.,Northeast Forestry University | And 9 more authors.
PLoS ONE | Year: 2015

In this work, Isatis tinctoria hairy root cultures (ITHRCs) were established as an alternative source for flavonoids (FL) production. I. tinctoria hairy root line V was found to be the most efficient line and was further confirmed by the PCR amplification of rol B, rol C and aux1 genes. Culture parameters of ITHRCs were optimized by Box-Behnken design (BBD), and eight bioactive FL constituents (rutin, neohesperidin, buddleoside, liquiritigenin, quercetin, isorhamnetin, kaempferol and isoliquiritigenin) were quali-quantitatively determined by LC-MS/MS. Under optimal conditions, the total FL accumulation of ITHRCs (24 day-old) achieved was 438.10 μg/g dry weight (DW), which exhibited significant superiority as against that of 2 year-old field grown roots (341.73 μg/g DW). Additionally, in vitro antioxidant assays demonstrated that ITHRCs extracts exhibited better antioxidant activities with lower IC50 values (0.41 and 0.39, mg/mL) as compared to those of field grown roots (0.56 and 0.48, mg/mL). To the best of our knowledge, this is the first report describing FL production and antioxidant activities from ITHRCs. © 2015 Gai et al. Source


Jiao J.,Northeast Forestry University | Gai Q.-Y.,Northeast Forestry University | Gai Q.-Y.,Collaborative Innovation Center for Development and Utilization of Forest Resources | Wang W.,Northeast Forestry University | And 9 more authors.
Biochemical Engineering Journal | Year: 2016

In this work, the elicitation of Astragalus membranaceus hairy root cultures (AMHRCs) with methyl jasmonate (MJ), salicylic acid (SA) and acetylsalicylic acid (ASA) was conducted to boost astragaloside (AG) biosynthesis. MJ was found to be the most effective inducer for AG production among all elicitor treatments. The highest enhancement of AG accumulation was achieved in 34 day-old AMHRCs elicited by 157.4 μM MJ for 18.4 h. The resulting AG yield was 5.5 ± 0.13 mg/g dry weight (DW), which was 2.1-fold higher than that of non-treated control (2.7 ± 0.05 mg/g DW). Moreover, transcriptional analyses revealed that MVD, IDI, FPS and SS were the potential key regulated genes in AG biosynthetic pathway. Overall, this study offered a feasible approach of utilizing MJ elicitation for the enhanced production of valuable AGs in AMHRCs, and also provided a basis for AG biosynthesis via metabolic engineering strategies in the future. © 2015 Elsevier B.V. Source


Gai Q.-Y.,Northeast Forestry University | Gai Q.-Y.,Collaborative Innovation Center for Development and Utilization of Forest Resources | Jiao J.,Northeast Forestry University | Luo M.,Northeast Forestry University | And 9 more authors.
Industrial Crops and Products | Year: 2016

Astragalosides (AGs) are triterpene saponins that uniquely occur in Astragalus species with multiple pharmacological activities. For the first time, we reported an elicitation strategy, using UV radiation (UV-A, UV-B, and UV-C), to enhance AG production in Astragalus membranaceus hairy root cultures (AMHRCs). The treatment of 32 day-old AMHRCs with 54 kJ/m2 dose of UV-B radiation was capable of achieving the maximum production of AGs, i.e. 3.431 ± 0.092 mg/g dry weight (DW), which was 1.30-fold higher as compared to the level in non-treated control (2.645 ± 0.073 mg/g DW). Expressions of eleven genes involved in AG biosynthetic pathway were monitored by quantitative real-time PCR (qRT-PCR). Transcriptional results showed that only the HMGR gene was significantly up-regulated in response to UV-B elicitation. The present study demonstrated that UV-B elicitation is a feasible strategy to promote AG production in AMHRCs and that HMGR represents a potential key gene in AG biosynthetic pathway. © 2016 Elsevier B.V. Source

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