Rapid and accurate identification of Gastrodia elata Blume species based on FTIR and NIR spectroscopy combined with chemometric methods.

Different varieties of Gastrodia elata Blume (G. elata Bl.) have different qualities and different contents of active ingredients, such as polysaccharide and gastrodin, and it is generally believed that the higher the active ingredients, the better the quality of G. elata Bl. and the stronger the medicinal effects. Therefore, effective identification of G. elata Bl. species is crucial and has important theoretical and practical significance. In this study, first unsupervised PCA and t-SNE are established for data visualisation, follow by traditional machine learning (PLS-DA, OPLS-DA and SVM) models and deep learning (ResNet) models were established based on the fourier transform infrared (FTIR) and near infrared (NIR) spectra data of three G. elata Bl. species. The results show that PLS-DA, OPLS-DA and SVM models require complex preprocessing of spectral data to build stable and reliable models. Compared with traditional machine learning models, ResNet models do not require complex spectral preprocessing, and the training and test sets of ResNet models built based on raw NIR and low-level data fusion (FTIR + NIR) spectra reach 100 % accuracy, the external validation set based on low-level data fusion reaches 100 % accuracy, and the external validation set based on NIR has only one sample classification error and no overfitting.

Integration of volatile and non-volatile metabolite profile, and in vitro digestion reveals the differences between different preparation methods on physico-chemical and biological properties of Gastrodia elata.

Gastrodia elata Blume (G. elata) is a traditional medicinal and edible plant whose quality is significantly influenced by post-harvest processing. To obtain an optimal post-harvest processing method for G. elata, this study employed sensory evaluation, scanning electron microscopy (SEM), gas chromatography-ion mobility spectrometry (GC-IMS), and non-targeted metabolomics, in conjunction with an in vitro digestion model, to assess the impact of different processing and drying methods on the quality of G. elata. The findings showed that the steam treatment followed by heat pump drying resulted in the highest levels of total phenols, total flavonoids, and polysaccharides in G. elata, and caused more pronounced damage to its microstructure. This treatment also maintained the highest antioxidant activities and optimal acetylcholinesterase (AChE) inhibition capacity throughout in vitro digestion, meanwhile, effectively eliminating the unpleasant odor and achieving the highest sensory scores. Furthermore, non-targeted metabolomic analysis revealed noteworthy alterations in the metabolite profile of G. elata, mainly related to purine metabolism and the biosynthesis of amino acids pathways. This study provides valuable insights into the post-harvest processing of G. elata.

Investigation of the Pharmacodynamic Components of Gastrodia elata Blume for Treatment of Type 2 Diabetes Mellitus through HPLC, Bioactivity, Network Pharmacology and Molecular Docking.

The occurrence of type 2 diabetes mellitus (T2DM), a worldwide chronic disease, is mainly caused by insufficient insulin production and places a huge burden on the health system. Gastrodia elata Blume (GE), a food of medicine-food homology, has been reported to have the ability to inhibit glycosidase activity, indicating its potential in the treatment of diabetes. However, the main pharmacological components of GE for the treatment of T2DM have not been fully clarified. Therefore, this study aims to clarify the pharmacological components changes of GE with different drying methods and the treatment of T2DM using HPLC, network pharmacology, molecular docking and experimental evaluations. The results showed that the GE samples processed by the steam-lyophilized method possessed the highest total content of the six marker components and the strongest antioxidant and α-glucosidase inhibitory abilities. Meanwhile, the six marker compounds had a total of 238 T2DM-related gene targets. Notably, these active compounds have good affinity for key gene targets associated with T2DM signaling pathways. In conclusion, this study revealed that different drying methods of GE affect the content of its major active compounds, antioxidant capacity, α-glucosidase inhibitory capacity and potential pharmacological effects on T2DM, indicating that it is a potential treatment of T2DM.

Metabolomics-Based Study of the Protective Effect of 4-Hydroxybenzyl Alcohol on Ischemic Astrocytes.

Ischemic stroke is a common and dangerous disease in clinical practice. Astrocytes (ASs) are essential for maintaining the metabolic balance of the affected regions during the disease process. 4-Hydroxybenzyl alcohol (4HBA) from Gastrodia elata Bl. has potential neuroprotective properties due to its ability to cross the blood-brain barrier. In an in vitro experiment, we replicated the oxygen-glucose deprivation/reoxygenation model, and used methyl thiazoly tertrazolium, flow cytometry, kits, and other technical means to clarify the protective effect of 4HBA on primary ASs. In in vivo experiments, the 2VO model was replicated, and immunofluorescence and immunohistochemistry techniques were used to clarify the protective effect of 4HBA on ASs and the maintenance of the blood-brain barrier. Differential metabolites and related pathways were screened and verified using metabolomics analysis and western blot. 4HBA noticeably amplified AS cell survival, reduced mitochondrial dysfunction, and mitigated oxidative stress. It demonstrated a protective effect on ASs in both environments and was instrumental in stabilizing the blood-brain barrier. Metabolomic data indicated that 4HBA regulated nucleic acid and glutathione metabolism, influencing purines, pyrimidines, and amino acids, and it activated the N-methyl-D-aspartate/p-cAMP-response element binding protein/brain-derived neurotrophic factor signaling pathway via N-methyl-D-aspartate R1/N-methyl-D-aspartate 2C receptors. Our findings suggest that 4HBA is a potent neuroprotective agent against ischemic stroke, enhancing AS cell survival and function while stabilizing the blood-brain barrier. The N-methyl-D-aspartate/p-cAMP-response element binding protein/brain-derived neurotrophic factor signaling pathway is activated by 4HBA.

Multivariate HPLC system assessment and optimization for traditional Chinese medicine: a case study of Gastrodia elata.

The development of HPLC analytical methods for traditional Chinese medicine is intricate and time-consuming, influenced by factors such as column wear, solvent purity, and instrumental settings. A comprehensive evaluation of the HPLC system is crucial to mitigate potential variability and ensure the reliability of data. This is especially important given the complex and synergistic nature of the chemical components in traditional Chinese medicine, necessitating a multivariate measurement system analysis (MSA) to assess multiple correlated quality characteristics effectively. This study introduced a multivariate MSA method based on weighted principal components (WPC) to evaluate the HPLC system for the determination of metabolites in Gastrodia elata. By integrating multiple principal components and assigning weights according to their eigenvalues, the WPC method significantly enhanced both accuracy and robustness. It demonstrated a repeatability and reproducibility (% R&R) of 26.43% and a number of distinct categories (ndc) index of 5, confirming the system's acceptability. A full factorial experimental design was employed to identify key performance factors, leading to the recommendation to use five reference solutions for the standard curve and to triple sample preparations for improved precision and accuracy. Monte Carlo simulations confirmed the reliability of the system, showing % R&R and ndc values that follow a normal distribution, ranging from 19% to 22% and 6.07 to 7.38, respectively. Chromatographic conditions were optimized using a Box-Behnken experimental design. Subsequent validation experiments verified the method's high accuracy and reliability, with all relative standard deviation values for analytical precision, repeatability, and stability below 5%. The method also exhibited high recovery rates, exceeding 91% across three concentration levels, with RSD values under 4%. In conclusion, the application of a WPC-based multivariate MSA enabled a detailed evaluation of the HPLC system, ensuring accurate and reliable measurement of quality attributes. This method exemplified a scientifically rigorous approach for developing analytical methods in traditional Chinese medicine, enhancing both precision and reliability.

Characteristics and functions of volatile organic compounds in the tripartite symbiotic system of Gastrodia elata-Armillaria gallica-Rahnella aceris HPDA25.

Tripartite interactions among plants, fungi, and bacteria are critical for maintaining plant growth and fitness, and volatile organic compounds (VOCs) play a significant role in these interactions. However, the functions of VOCs within the niche of mycoheterotrophic plants, which represent unique types of interactions, remain poorly understood. Gastrodia elata, a mycoheterotrophic orchid species, forms a symbiotic relationship with specific Armillaria species, serving as a model system to investigate this intriguing issue. Rahnella aceris HPDA25 is a plant growth-promoting bacteria isolated from G. elata, which has been found to facilitate the establishment of G. elata-Armillaria symbiosis. In this study, using the tripartite symbiotic system of G. elata-Armillaria gallica-R. aceris HPDA25, we investigate the role of VOCs in the interaction among mycoheterotrophic plants, fungi, and bacteria. Our results showed that 33 VOCs of HPDA25-inducible symbiotic G. elata elevated compared to non-symbiotic G. elata, indicating that VOCs indeed play a role in the symbiotic process. Among these, 21 VOCs were accessible, and six active VOCs showed complete growth inhibition activities against A. gallica, while R. aceris HPDA25 had no significant effect. In addition, three key genes of G. elata have been identified that may contribute to the increased concentration of six active VOCs. These results revealed for the first time the VOCs profile of G. elata and demonstrated its regulatory role in the tripartite symbiotic system involving G. elata, Armillaria, and bacteria.

Gastrodin, a Promising Natural Small Molecule for the Treatment of Central Nervous System Disorders, and Its Recent Progress in Synthesis, Pharmacology and Pharmacokinetics.

Gastrodia elata Blume is a traditional medicinal and food homology substance that has been used for thousands of years, is mainly distributed in China and other Asian countries, and has always been distinguished as a superior class of herbs. Gastrodin is the main active ingredient of G. elata Blume and has attracted increasing attention because of its extensive pharmacological activities. In addition to extraction and isolation from the original plant, gastrodin can also be obtained via chemical synthesis and biosynthesis. Gastrodin has significant pharmacological effects on the central nervous system, such as sedation and improvement of sleep. It can also improve epilepsy, neurodegenerative diseases, emotional disorders and cognitive impairment to a certain extent. Gastrodin is rapidly absorbed and widely distributed in the body and can also penetrate the blood-brain barrier. In brief, gastrodin is a promising natural small molecule with significant potential in the treatment of brain diseases. In this review, we summarised studies on the synthesis, pharmacological effects and pharmacokinetic characteristics of gastrodin, with emphasis on its effects on central nervous system disorders and the possible mechanisms, in order to find potential therapeutic applications and provide favourable information for the research and development of gastodin.

[Main nutrients content in different specifications of Gastrodiae Rhizoma].

Gastrodiae Rhizoma is a valuable traditional Chinese medicine(TCM) and was newly approved as a catalogue species of medicinal and food homologous substances in 2023. The consumption of Gastrodiae Rhizoma as a food has been increasing year by year, and its nutrients content has become a public concern. However, there is a lack of systematic research on its nutrients content. Gastrodiae Rhizoma is widely distributed and exhibits various specifications. The quality of Gastrodiae Rhizoma varies among different varieties, origins, and grades. In this paper, 76 batches of samples were selected, involving 2 varieties(G. elata f. elata and G. elata f. qlauca), 6 origins(Anhui, Shaanxi, Hubei, Yunnan, Henan and Northeast China) and 5 grades(special grade, first grade, second grade, third grade, and fourth grade). The content of main nutrients of the above samples was determined and analyzed to explore the differences in the content of different specifications of Gastrodiae Rhizoma. The results show that Gastrodiae Rhizoma is rich in a variety of nutrients, including protein, fat, starch, crude fiber, total saponins, moisture, polysaccharides, mineral elements, amino acids, and volatile oils. The total mass of volatile oils reached about 96.00%. The percentages of starch, moisture and polysaccharides werethe highest, accounting for 64.52%, 10.45%, and 8.32%, respectively. There were also differences in nutrient content among different specifications, especially the polysaccharide content of different varieties. Therefore, the research direction of Gastrodiae Rhizoma medicinal and food homologous products can be inclined to the development of meal replacement staple food or polysaccharide functional food. This study provides a reference for the research of Gastrodiae Rhizoma in the field of medicinal and food homologous products.

[Influence of Tis108 on GA content and expression of key enzyme GeCYP714A1 involved in GA deactivation of Gastrodia elata].

To investigate the influence of the strigolactone inhibitor Tis108 on the growth of Gastrodia elata, this study treated G. elata tuber with Tis108 solution of 10 µmol·L~(-1) and measured the content of endogenous hormone gibberellin(GA) in the tuber. By using reverse transcription-polymerase chain reaction(RT-PCR) technology, the key enzyme GeCYP714A1 gene involved in GA deactivation was cloned. Bioinformatics analysis on the GeCYP714A1 gene was carried out by using ExPASy, SWISS-MODEL, MEGA, etc., and its expression levels in different parts of G. elata were determined. The results showed that after Tis108 treatment, GA content in G. elata tuber was significantly increased, and the transcription level of the GeCYP714A1 gene was significantly decreased. The full length of the coding region of the GeCYP714A1 gene is 1 173 bp, encoding 390 amino acids. The protein has a molecular weight of 44.85 kDa, a theoretical isoelectric point of 9.83, an instability index of 49.20, an aliphatic index of 89.03, and a grand average of hydropathicity of-0.235, classifying it as an unstable, basic, hydrophilic protein, and the GeCYP714A1 protein was localized in the mitochondria, lacking a signal peptide and a transmembrane structure. Phylogenetic tree analysis revealed that GeCYP714A1 was most closely related to the DcCYP714C2(PKU78454.1) protein from Dendrobium candidum, with a sequence identity of 67.25%. The qRT-PCR analysis of the expression patterns of the GeCYP714A1 gene indicated that GeCYP714A1 had the highest transcription level in G. elata tuber, followed by stem and inflorescence. The study represented that Tis108 inhibited the transcription level of GeCYP714A1 involved in GA deactivation in G. elata tuber, thereby increasing the accumulation of GA and affecting the growth of G. elata tuber. These results provided a basis for further studies of strigolactone regulation of GA signal and tuber development in G. elata.

Genome-wide identification of glycoside hydrolase family 1 members reveals GeBGL1 and GeBGL9 for degrading gastrodin in Gastrodia elata.

KEY MESSAGE: We revealed the intrinsic transformation molecular mechanism of gastrodin by two ß-d-glucosidases (GeBGL1 and GeBGL9) during the processing of Gastrodia elata. Gastrodia elata is a plant resource with medicinal and edible functions, and its active ingredient is gastrodin. However, the intrinsic transformation molecular mechanism of gastrodin in G. elata has not been verified. We speculated that ß-d-glucosidase (BGL) may be the key enzymes hydrolyzing gastrodin. Here, we identified 11 GeBGL genes in the G. elata genome. These genes were unevenly distributed on seven chromosomes. These GeBGL proteins possessed motifs necessary for catalysis, namely, TF(I/M/L)N(T)E(Q)P and I(V/L)T(H/S)ENG(S). These GeBGLs were divided into five subgroups together with homologous genes from Arabidopsis thaliana, rice, and maize. Quantitative real-time PCR analysis showed GeBGL genes expression was tissue-specific. Gene cloning results showed two mutation sites in the GeBGL1 gene compared with the reference genome. And, the GeBGL4 gene has two indel fragments, which resulted in premature termination of translation and seemed to turn into a pseudogene. Furthermore, protein expression and enzyme activity results proved that GeBGL1 and GeBGL9 have the activity of hydrolyzing gastrodin into 4-hydroxybenzyl alcohol. This study revealed the function of ß-d-glucosidase in degrading active compounds during the G. elata processing for medicinal purposes. These results offer a theoretical foundation for elevating the standard and enhancing the quality of G. elata production.

Preparation and evaluation of gastrodin microsphere-loaded Gastrodia elata polysaccharides composite hydrogel on UVB-induced skin damage in vitro and in vivo.

Skin damage from sun exposure is a common issue among outdoor workers and is primarily caused by ultraviolet rays. Upon absorption of these rays, the skin will experience inflammation and cell apoptosis. This study explored the concept of 'Combination of medicine and adjuvant' by utilizing Gastrodia elata polysaccharide, a key component of Gastrodia elata Bl.|, to develop a new hydrogel material. Oxidized Gastrodia elata polysaccharide (OGEP) and carboxymethyl chitosan (CMCS) was use to prepare a biocompatible, biodegradable and self-healing hydrogel OGEP/CMCS (OC). And this hydrogel was further loaded with Gastrodin-containing microspheres (GAS/GEL) to create GAS/GEL/OGEP/CMCS (GGOC) hydrogel. Characterization studies revealed that OC and GGOC hydrogels exhibited favorable mechanical properties, antioxidant activity and biocompatibility. The experiments showed that OC and GGOC hydrogels could regulate mitochondrial membrane potential, prevent mitochondrial breakage, inhibit proinflammatory factors, prevent NF-κB protein activation and regulate apoptosis-related pathways. This study highlighted the application potential of Gastrodia elata polysaccharide as a 'Combination of medicine and adjuvant' and the anti-UVB damage effect of the prepared hydrogel.

[Effects of soil relative water content on growth characteristics of Armillaria spp. and seedling production of Gastrodia elata f. glauca].

The seedling survival rate, yield, and individual weight of Gastrodia elata is closely related to the soil relative water content(RWC) and the growth characteristics of the associated fungi Armillaria spp. This study explored the effects of the soil RWC on the growth characteristics of Armillaria spp. and the seedling production of G. elata f. glauca, aiming to provide guidance for breeding G. elata f. glauca and selecting elite strains of Armillaria. According to the growth characteristics on the medium for activation, thirty strains of Armillaria were classified into 4 clusters. Two strains with good growth indicators were selected from each cluster and cultiva-ted with immature tuber(Mima) and the branches of the broad-leaved trees in a water-controlled box. The results showed that the Armillaria clusters with uniaxial branches of rhizoid cords, such as clusters Ⅲ and Ⅳ, were excellent clusters in symbiosis with G. elata f. glauca. The soil RWC had significant effects on the growth characteristics of Armillaria strains and the seedling survival rate, yield, and individual weight of G. elata f. glauca. The growth characteristics of Armillaria strains and the seedling survival rate, yield, and individual weight of G. elata f. glauca in the case of the soil RWC being 75% were significantly better than those in the case of other soil RWC. Cultivating Mima with elite strains of Armillaria, together with branches of broad-leaved trees, in the greenhouses with the artificial control of the soil RWC, can achieve efficient seedling production and Mima utilization of G. elata f. glauca.

Anti-hypertensive effect and potential mechanism of gastrodia-uncaria granules based on network pharmacology and experimental validation.

Hypertension has become a major contributor to the morbidity and mortality of cardiovascular diseases worldwide. Despite the evidence of the anti-hypertensive effect of gastrodia-uncaria granules (GUG) in hypertensive patients, little is known about its potential therapeutic targets as well as the underlying mechanism. GUG components were sourced from TCMSP and HERB, with bioactive ingredients screened. Hypertension-related targets were gathered from DisGeNET, OMIM, GeneCards, CTD, and GEO. The STRING database constructed a protein-protein interaction network, visualized by Cytoscape 3.7.1. Core targets were analyzed via GO and KEGG using R package ClusterProfiler. Molecular docking with AutodockVina 1.2.2 revealed favorable binding affinities. In vivo studies on hypertensive mice and rats validated network pharmacology findings. GUG yielded 228 active ingredients and 1190 targets, intersecting with 373 hypertension-related genes. PPI network analysis identified five core genes: AKT1, TNF-α, GAPDH, IL-6, and ALB. Top enriched GO terms and KEGG pathways associated with the anti-hypertensive properties of GUG were documented. Molecular docking indicated stable binding of core components to targets. In vivo study showed that GUG could improve vascular relaxation, alleviate vascular remodeling, and lower blood pressure in hypertensive animal models possibly through inhibiting inflammatory factors such as AKT1, mTOR, and CCND1. Integrated network pharmacology and in vivo experiment showed that GUG may exert anti-hypertensive effects by inhibiting inflammation response, which provides some clues for understanding the effect and mechanisms of GUG in the treatment of hypertension.

Structural characterization of the glucan from Gastrodia elata Blume and its ameliorative effect on DSS-induced colitis in mice.

The polysaccharide glucan was extracted from Gastrodia elata Blume, and its structural characterizations and beneficial effects against acute dextran sulfate sodium (DSS)-induced ulcerative colitis were investigated. The results showed that a polysaccharide GP with a molecular weight of 811.0 kDa was isolated from G. elata Blume. It had a backbone of α-D-1,4-linked glucan with branches of α-d-glucose linked to the C-6 position. GP exhibited protective effects against DSS-induced ulcerative colitis, and reflected in ameliorating weight loss and pathological damages in mice, increasing colon length, inhibiting the expression of inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), decreasing the levels of inflammatory related proteins NLRP3 and ASC, and elevating the anti-inflammatory cytokine interleukin-10 (IL-10) level in mouse colon tissues. GP supplementation also reinforced the intestinal barrier by promoting the expression of ZO-1, Occludin, and MUC2 of colon tissues, and positively regulated intestinal microbiota. Thus, GP treatment possessed a significant improvement in ulcerative colitis in mice, and it was expected to be developed as a functional food.

Experimental study on the detection of Gastrodia elata by enzymatic recombinase amplification and immunochromatography.

OBJECTIVE: The objective of this research is to develop two methodologies, Enzymatic recombinase amplification (ERA) and Polymerase Chain Reaction (PCR) coupled with Lateral Flow Dipstick (LFD), for the swift authentication of Gastrodia elata. METHODOLOGY: Primers and nfo probes for the ERA of Gastrodia elata were developed based on the ITS2 genome sequences of Gastrodia elata and its counterfeits. Specific primers for the PCR analysis of Gastrodia elata were generated using the NCBI (National Center for Biotechnology Information) online platform. Through experimental validation, the optimal reaction system and conditions for both methodologies were established, and their efficacy was assessed. RESULTS: The methodologies developed herein are applicable for the targeted analysis of the medicinal species, Gastrodia elata. The sensitivity of the ERA-LFD detection method matched that of the conventional PCR-LFD approach, recorded at 1 ng µL-1. Consistency was observed in the results across three replicates of visualization test strips for both techniques. Upon evaluation, both the PCR-LFD and ERA-LFD methods demonstrated a total compliance rate of 100 %. CONCLUSION: The ERA-LFD and PCR-LFD methods facilitate reduced detection times and offer visual results. These techniques are particularly effective for on-site detection and quality control in the authentication of Gastrodia elata within traditional Chinese medicine markets and at the primary level of healthcare provision.

Identification of geographical origins of Gastrodia elata Blume based on multisource data fusion.

INTRODUCTION: Identifying the geographical origin of Gastrodia elata Blume contributes to the scientific and rational utilization of medicinal materials. In this study, infrared spectroscopy was combined with machine learning algorithms to distinguish the origin of G. elata BI. OBJECTIVE: Realization of rapid and accurate identification of the origin of G. elata BI. MATERIALS AND METHODS: Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectra and Fourier transform near-infrared (FT-NIR) spectra were collected for 306 samples of G. elata BI. SAMPLES: Firstly, a support vector machine (SVM) model was established based on the single-spectrum and the full-spectrum fusion data. To investigate whether feature-level fusion strategy can enhance the model's performance, the sequential and orthogonalized partial least squares discriminant analysis (SO-PLS-DA) model was established to extract and combine two types of spectral features. Next, six algorithms were employed to extract feature variables, SVM model was established based on the feature-level fusion data. To avoid complicated preprocessing and feature extraction processes, a residual convolutional neural network (ResNet) model was established after converting the raw spectral data into spectral images. RESULTS: The accuracy of the feature-level fusion model is better as compared to the single-spectrum model and the fusion model with full-spectrum, and SO-PLS-DA is simpler than feature-level fusion based on the SVM model. The ResNet model performs well in classification but requires more data to enhance its generalization capability and training effectiveness. CONCLUSION: Sequential and orthogonalized data fusion approaches and ResNet models are powerful solutions for identifying the geographic origin of G. elata BI.

Gastrodin relieves Parkinson's disease-related motor deficits by facilitating the MEK-dependent VMAT2 to maintain dopamine homeostasis.

BACKGROUND: Dysfunction of dopamine homeostasis (DAH), which is regulated by vesicular monoamine transporter 2 (VMAT2), is a vital cause of dopamine (DA) neurotoxicity and motor deficits in Parkinson's disease (PD). Gastrodin (4-hydroxybenzyl alcohol 4-O-ß-D-glucoside; GTD), a natural active compound derived from Gastrodia elata Blume, can be used to treat multiple neurological disorders, including PD. However, whether GTD regulates VMAT2-mediated DAH dysfunction in PD models remains unclear. PURPOSE: To explore whether GTD confers dopaminergic neuroprotection by facilitating DA vesicle storage and maintaining DAH in PD models. METHODS: Mice were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and PC12 cells with 1-methyl-4-phenyl-pyridinium (MPP+) to induce PD characteristics. Multiple behavioural tests were performed to evaluate the motor functions of the mice. HPLC was used to measure DA and 3,4-dihydroxyphenylacetic acid (DOPAC) levels. Transmission electron microscopy was used to observe synaptic vesicles. Molecular docking and molecular dynamics were used to determine the binding affinity of GTD to the target protein. Reserpine (Res, a VMAT2 inhibitor) and PD0325901 (901, a MEK inhibitor) were employed to investigate the mechanism of GTD. Western blotting and immunohistochemistry were used to assess the expression of the target proteins. RESULTS: GTD attenuated motor deficits and dopaminergic neuronal injury, reversed the imbalance of DAH, and increased VMAT2 levels and vesicle volume in MPTP-induced mice. GTD ameliorated cell damage, ROS release, and dysfunction of DAH in MPP+-induced PC12 cells. Moreover, the neuroprotective effects of GTD were reversed by Res in vitro and in vivo. Furthermore, GTD can activate the MEK/ERK/CREB pathway to upregulate VMAT2 in vitro and in vivo. Interestingly, 901 reversed the effects of GTD on VMAT2 and dopaminergic neuronal impairment. CONCLUSION: GTD relieved PD-related motor deficits and dopaminergic neuronal impairment by facilitating MEK-depended VMAT2 to regulate DAH, which offers new insights into its therapeutic potential.

Identification of key anti-neuroinflammatory components in Gastrodiae Rhizoma based on spectrum-effect relationships and its mechanism exploration.

Gastrodiae Rhizoma was proven to have anti-inflammatory activity based on its main component of 4-hydroxybenzyl alcohol (4-HBA) and gastrodin (GAS). However, the anti-inflammatory activity of other phenols has been less reported. In this study, the n-BuOH extract was selected as the active anti-inflammatory part of Gastrodiae Rhizoma based on the LPS-induced inflammatory BV-2 cells. The spectral-effect relationship analysis of the n-BuOH extract showed the main effective components were GAS, 4-HBA, parishin A (PA), parishin B (PB), and parishin C (PC). Among them, PB could reduce LPS-induced expression of nitric oxide (NO), intracellular ROS, TNF-α, IL-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Molecular docking predicted that PB had a good binding capacity to AMPKα and SIRT1 proteins of -12.1 kJ/mol and -7.6 kJ/mol, respectively. The Western Blot results further demonstrated that PB could inhibit NF-κB pathway by activating AMPK/SIRT1 pathway, thus exerting anti-LPS-induced neuroinflammatory effects. This study provides a referable idea for solving the problem of unclear action of TCM with complex compositions and is of great significance for the development of innovative medicines of traditional Chinese medicine.

Diverse Mycena Fungi and Their Potential for Gastrodia elata Germination.

It remains to be determined whether there is a geographical distribution pattern and phylogenetic signals for the Mycena strains with seed germination of the orchid plant Gastrodia elata. This study analyzed the community composition and phylogenetics of 72 Mycena strains associated with G. elata varieties (G. elata. f. glauca and G. elata. f. viridis) using multiple gene fragments (ITS+nLSU+SSU). We found that (1) these diverse Mycena phylogenetically belong to the Basidiospore amyloid group. (2) There is a phylogenetic signal of Mycena for germination of G. elata. Those strains phylogenetically close to M. abramsii, M. polygramma, and an unclassified Mycena had significantly higher germination rates than those to M. citrinomarginata. (3) The Mycena distribution depends on geographic site and G. elata variety. Both unclassified Mycena group 1 and the M. abramsii group were dominant for the two varieties of G. elata; in contrast, the M. citrinomarginata group was dominant in G. elata f. glauca but absent in G. elata f. viridis. Our results indicate that the community composition of numerous Mycena resources in the Zhaotong area varies by geographical location and G. elata variety. Importantly, our results also indicate that Mycena's phylogenetic status is correlated with its germination rate.

Composition and diversity of soil microbial communities change by introducing Phallus impudicus into a Gastrodia elata Bl.-based soil.

BACKGROUND: The Gastrodia elata Bl. is an orchid, and its growth demands the presence of Armillaria species. The strong competitiveness of Armillaria species has always been a concern of major threat to other soil organisms, thus disrupting the equilibrium of soil biodiversity. Introducing other species to where G. elata was cultivated, could possibly alleviate the problems associated with the disequilibrium of soil microenvironment; however, their impacts on the soil microbial communities and the underlying mechanisms remain unclear. To reveal the changes of microbial groups associated with soil chemical properties responding to different cultivation species, the chemical property measurements coupled with the next-generation pyrosequencing analyses were applied with soil samples collected from fallow land, cultivation of G. elata and Phallus impudicus, respectively. RESULTS: The cultivation of G. elata induced significant increases (p < 0.05) in soil pH and NO3-N content compared with fallow land, whereas subsequent cultivation of P. impudicus reversed these G. elata-induced increases and was also found to significantly increase (p < 0.05) the content of soil NH4+-N and AP. The alpha diversities of soil microbial communities were significantly increased (p < 0.01) by cultivation of G. elata and P. impudicus as indicated with Chao1 estimator and Shannon index. The structure and composition of soil microbial communities differed responding to different cultivation species. In particular, the relative abundances of Bacillus, norank_o_Gaiellales, Mortierella and unclassified_k_Fungi were significantly increased (p < 0.05), while the abundances of potentially beneficial genera such as Acidibacter, Acidothermus, Cryptococcus, and Penicillium etc., were significantly decreased (p < 0.05) by cultivation of G. elata. It's interesting to find that cultivation of P. impudicus increased the abundances of these genera that G. elata decreased before, which contributed to the difference of composition and structure. The results of CCA and heatmap indicated that the changes of soil microbial communities had strong correlations with soil nutrients. Specifically, among 28 genera presented, 50% and 42.9% demonstrated significant correlations with soil pH and NO3-N in response to cultivation of G. elata and P. impudicus. CONCLUSIONS: Our findings suggested that the cultivation of P. impudicus might have potential benefits as result of affecting soil microorganisms coupled with changes in soil nutrient profile.