Application of Laser-Induced Breakdown Spectroscopy Coupled With Spectral Matrix and Convolutional Neural Network for Identifying Geographical Origins of Gentiana rigescens Franch

Accurate geographical origin identification is of great significance to ensure the quality of traditional Chinese medicine (TCM). Laser-induced breakdown spectroscopy (LIBS) was applied to achieve the fast geographical origin identification of wild Gentiana rigescens Franch (G. rigescens Franch). However, LIBS spectra with too many variables could increase the training time of models and reduce the discrimination accuracy. In order to solve the problems, we proposed two methods. One was reducing the number of variables through two consecutive variable selections. The other was transforming the spectrum into spectral matrix by spectrum segmentation and recombination. Combined with convolutional neural network (CNN), both methods could improve the accuracy of discrimination. For the underground parts of G. rigescens Franch, the optimal accuracy in the prediction set for the two methods was 92.19 and 94.01%, respectively. For the aerial parts, the two corresponding accuracies were the same with the value of 94.01%. Saliency map was used to explain the rationality of discriminant analysis by CNN combined with spectral matrix. The first method could provide some support for LIBS portable instrument development. The second method could offer some reference for the discriminant analysis of LIBS spectra with too many variables by the end-to-end learning of CNN. The present results demonstrated that LIBS combined with CNN was an effective tool to quickly identify the geographical origin of G. rigescens Franch.

A Novel Multi-Preprocessing Integration Method for the Qualitative and Quantitative Assessment of Wild Medicinal Plants: Gentiana rigescens as an Example

Until now, the over-exploitation of wild resources has increased growing concern over the quality of wild medicinal plants. This led to the necessity of developing a rapid method for the evaluation of wild medicinal plants. In this study, the content of total secoiridoids (gentiopicroside, swertiamarin, and sweroside) of Gentiana rigescens from 37 different regions in southwest China were analyzed by high performance liquid chromatography (HPLC). Furthermore, Fourier transform infrared (FT-IR) was adopted to trace the geographical origin (331 individuals) and predict the content of total secoiridoids (273 individuals). In the traditional FT-IR analysis, only one scatter correction technique could be selected from a series of preprocessing candidates to decrease the impact of the light correcting effect. Nevertheless, different scatter correction techniques may carry complementary information so that using the single scatter correction technique is sub-optimal. Hence, the emerging ensemble approach to preprocessing fusion, sequential preprocessing through orthogonalization (SPORT), was carried out to fuse the complementary information linked to different preprocessing methods. The results suggested that, compared with the best results obtained on the scatter correction modeling, SPORT increased the accuracy of the test set by 12.8% in qualitative analysis and decreased the RMSEP by 66.7% in quantitative analysis.

The Insulin Receptor: A Potential Target of Amarogentin Isolated from Gentiana rigescens Franch That Induces Neurogenesis in PC12 Cells

Amarogentin (AMA) is a secoiridoid glycoside isolated from the traditional Chinese medicine, Gentiana rigescens Franch. AMA exhibits nerve growth factor (NGF)-mimicking and NGF-enhancing activities in PC12 cells and in primary cortical neuron cells. In this study, a possible mechanism was found showing the remarkable induction of phosphorylation of the insulin receptor (INSR) and protein kinase B (AKT). The potential target of AMA was predicted by using a small-interfering RNA (siRNA) and the cellular thermal shift assay (CETSA). The AMA-induced neurite outgrowth was reduced by the siRNA against the INSR and the results of the CETSA suggested that the INSR showed a significant thermal stability-shifted effect upon AMA treatment. Other neurotrophic signaling pathways in PC12 cells were investigated using specific inhibitors, Western blotting and PC12(rasN17) and PC12(mtGAP) mutants. The inhibitors of the glucocorticoid receptor (GR), phospholipase C (PLC) and protein kinase C (PKC), Ras, Raf and mitogen-activated protein kinase (MEK) significantly reduced the neurite outgrowth induced by AMA in PC12 cells. Furthermore, the phosphorylation reactions of GR, PLC, PKC and an extracellular signal-regulated kinase (ERK) were significantly increased after inducing AMA and markedly decreased after treatment with the corresponding inhibitors. Collectively, these results suggested that AMA-induced neuritogenic activity in PC12 cells potentially depended on targeting the INSR and activating the downstream Ras/Raf/ERK and PI3K/AKT signaling pathways. In addition, the GR/PLC/PKC signaling pathway was found to be involved in the neurogenesis effect of AMA.

Amarogentin from Gentiana rigescens Franch Exhibits Antiaging and Neuroprotective Effects through Antioxidative Stress

In the present study, the replicative lifespan assay of yeast was used to guide the isolation of antiaging substance from Gentiana rigescens Franch, a traditional Chinese medicine. A compound with antiaging effect was isolated, and the chemical structure of this molecule as amarogentin was identified by spectral analysis and compared with the reported data. It significantly extended the replicative lifespan of K6001 yeast at doses of 1, 3, and 10 μM. Furthermore, amarogentin improved the survival rate of yeast under oxidative stress by increasing the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx), and these enzymes’ gene expression. In addition, this compound did not extend the replicative lifespan of sod1, sod2, uth1, and skn7 mutants with K6001 background. These results suggested that amarogentin exhibited antiaging effect on yeast via increase of SOD2, CAT, GPx gene expression, enzyme activity, and antioxidative stress. Moreover, we evaluated antioxidant activity of this natural products using PC12 cell system, a useful model for studying the nervous system at the cellular level. Amarogentin significantly improved the survival rate of PC12 cells under H2O2-induced oxidative stress and increased the activities of SOD and SOD2, and gene expression of SOD2, CAT, GPx, Nrf2, and Bcl-x1. Meanwhile, the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) of PC12 cells were significantly reduced after treatment of the amarogentin. These results indicated that antioxidative stress play an important role for antiaging and neuroprotection of amarogentin. Interestingly, amarogentin exhibited neuritogenic activity in PC12 cells. Therefore, the natural products, amarogentin from G. rigescens with antioxidant activity could be a good candidate molecule to develop drug for treating neurodegenerative diseases.

Gentiopicroside, a Secoiridoid Glycoside from Gentiana rigescens Franch, Extends the Lifespan of Yeast via Inducing Mitophagy and Antioxidative Stress

Gentiopicroside (GPS), an antiaging secoiridoid glycoside, was isolated from Gentiana rigescens Franch, a traditional Chinese medicine. It prolonged the replicative and chronological lifespans of yeast. Autophagy, especially mitophagy, and antioxidative stress were examined to clarify the mechanism of action of this compound. The free green fluorescent protein (GFP) signal from the cleavage of GFP-Atg8 and the colocation signal of MitoTracker Red CMXRos and GFP were increased upon the treatment of GPS. The free GFP in the cytoplasm and free GFP and ubiquitin of mitochondria were significantly increased at the protein levels in the GPS-treated group. GPS increased the expression of an essential autophagy gene, ATG32 gene, but failed to extend the replicative and chronological lifespans of ATG32 yeast mutants. GPS increased the survival rate of yeast under oxidative stress condition; enhanced the activities of catalase, superoxide dismutase, and glutathione peroxidase; and decreased the levels of reactive oxygen species and malondialdehyde. The replicative lifespans of Δsod1, Δsod2, Δuth1, and Δskn7 were not affected by GPS. These results indicated that autophagy, especially mitophagy, and antioxidative stress are involved in the antiaging effect of GPS.