Three diarylheptanoids were isolated from the n-butanol fraction of Zingiber officinale peel by MCI Gel CHP-20, Sephadex LH-20, ODS and semipreparative high performance liquid chromatography. Their structures were identified by MS and NMR spectroscopy techniques: (2S, 2'S, 3R, 3'R, 4R, 4'R, 6R, 6'R)-6, 6'-bis((S)-1-hydroxy-2-(4-hydroxyphenyl)ethyl)-2, 2'-bis(4-hydroxy-3-methoxyphenyl)octahydro-2H, 2'H-[3, 3'-bipyran]-4, 4'-diol (1), (E)-7-(4-hydroxy-3-methoxyphenyl)-1-(4-hydroxyphenyl)hept-4-en-3-one (2), and alpinin B (3). Compound 1 is a new compound, and compounds 2-3 were obtained from Zingiber officinale peel for the first time.

The objective of this work was to explore the content and composition of aristolochic acid compounds in Chinese medicinal materials containing toxic aristolochic chemicals, so as to ensure the safety of these medicinal materials and their related products. Nine Chinese medicinal materials were selected for study, including the tuber of Aristolochia cinnabarina, the herbs of Asarum forbesii, the stems of Aristolochia manshuriensis., the fruits of Aristolochia debilis, the roots of Aristolochia debilis, the stems and leaf of Aristolochia debilis, the herbs of Aristolochia mollissima, the roots of Aristolochia fangchi, and the roots of Asarum heterotropoides var. mandshuricum. The aristolochic acid components in the nine Chinese medicinal materials were analyzed by high performance liquid chromatography-quadrupole time of flight mass spectrometry (HPLC-Q-TOF-MS) combined with high performance liquid chromatography diode-array detection. The separation was performed on an Agilent ZORBAX SB-Aq column (250 mm×4.6 mm, 5 μm) with gradient elution using a mobile phase consisting of acetonitrile and 0.2% acetic acid. ESI positive ion mode MS was used to investigate the ionization pathways of aristolochic acid Ⅰ, Ⅱ, Ⅲa, Ⅳa, Ⅶa, and aristololactam Ⅰ, Ⅱ using seven reference standards, and the structures of the components with UV spectrasimilar to those of the seven reference standards in the selected medicinal materials were qualitatively analyzed by following the investigated ionization pathways. The identified aristolochic acid components were quantified using an external standard method by HPLC-UV with detection at 254 nm. Twenty-two aristolochic acid components including 11 aristolochic acids and 11 aristololactams were identified from the nine selected medicinal materials; 15 aristolochic acids were found in the tuber of Aristolochia cinnabarina and the roots of Aristolochia debilis, followed by 14 aristolochic acids in the fruits of Aristolochia debilis and the stems of Aristolochia manshuriensis. The greatest content of aristolochia components was found in the tuber of Aristolochia cinnabarina and the stems of Aristolochia manshuriensis, ranging from 8.91 mg·g^-1 to 13.40 mg·g^-1, and the least amount was in the herbs of Asarum forbesii, at less than 0.10 mg·g^-1 and containing only two aristolochia components. This study systematically explored the quantity and composition of aristolochic acid components in selected Chinese medicinal materials believed to contain toxic aristolochic compounds, providing a basis for follow-up studies on the toxicity of these substances that can lead to safety standards for their use.

Gemcitabine (GEM) is a commonly used drug in the clinical treatment of non-small cell lung cancer. Due to the accumulation of cells mediating immune escape and T cell depletion after chemotherapy, tumor microenvironment (TME) tends to be immunosuppressive status, which ultimately leads to tumor metastasis. The experimental protocol was approved by the Medical Laboratory Animal Ethics Committee of Jiangsu Provincial Academy of Chinese Medicine. Therefore, we observed the immunomodulatory effects of micro-particulate Ganoderma lucidum spore β-glucan (PGSG) on macrophages in vitro experiments. Next, mice subcutaneous Lewis lung cancer models were established to observe the anti-tumor effects of PGSG through oral administration of PGSG combined with GEM. Flow cytometry analysis was used to analyze the ratio of anti-tumor T cells in tumors and spleen, as well as the proportion of myeloid-derived suppressor cells (MDSC), tumor-associated macrophages (TAM) and regulatory cells (Tregs). The results showed that PGSG can up-regulate the expression of major histocompatibility antigens (MHC-Ⅱ), CD40, CD86 and CD80 on the surface of macrophages, enhance the ability to phagocytosis of neutral red and further mediate the release of interleukin-6 (IL-6), tumor necrosis factor-α(TNF-α), interleukin-4 (IL-4) and interleukin-10 (IL-10). In vivo experiments, combined administration can significantly decrease the volume and weight of tumors, reduce the ratio of MDSC (CD11b⁺Gr-1⁺), M-MDSC (CD11b⁺Ly6G^-Ly6C^high) and Treg (CD4⁺Foxp3⁺). At the same time, PGSG promoted the conversion of M₂ (F4/80⁺CD206⁺) to M1 (F4/80⁺MHC-Ⅱ⁺) and enhanced the response of helper T cell-1 (Th1) (CD4⁺IFN-γ⁺) and cytotoxic T lymphocyte (CTL) (CD8⁺IFN-γ⁺), which is of great significance for killing tumors. These results suggest that PGSG can regulate innate and adaptive antitumor immune responses, reshape the immunosuppressive microenvironment and enhance the anti-lung cancer effect of GEM.

In this paper, the effects of the blend of nanoparticles and microneedle matrix materials on the mechanical properties of dissolving microneedles were studied mainly, so as to construct microneedles with excellent mechanical properties. Different kinds of nanoparticles (calcium carbonate, hydroxyapatite, silica), particle sizes (20, 60, 100 nm) and the proportion of prescription (2%, 6%, 10%) were blended with the matrix material[polyvinyl pyrrolidone (PVP), poly(1-vinylpyrrolidone-co-vinyl acetate)(PVP/VA)] to form dissolving microneedles. The effects of nanoparticles on the elastic modulus and hardness of the microneedles were investigated using a nanoindenter. The results showed that the elastic modulus and hardness of PVP microneedles were significantly improved by nano-calcium carbonate (P < 0.001), and the elastic modulus and hardness of PVP/VA microneedles were significantly improved by nano-hydroxyapatite (P < 0.001). When the particle size of hydroxyapatite was 20 nm, the elastic modulus of PVP/VA microneedles was (10.6±1.0) GPa, and the hardness was (0.47±0.06) GPa. As the size of the nanoparticles increases, the mechanical performance of the microneedles decreases. When the mass proportion of nano-hydroxyapatite increased from 2% to 6%, the elastic modulus and hardness of the microneedles were significantly improved (P < 0.001), but the effect of continue increasing the proportion of nanoparticles on the microneedles was not significant. The nano-enhanced PVP/VA dissolving microneedles has no irritant effect on intact skin and has a slight irritation to damaged skin, but they disappear completely after 72 h. Animal experiments have been approved by the Laboratory Animal Welfare and Ethics Committee of Zhejiang University of Technology. Therefore, the nano-enhanced dissolving microneedles has good biological safety. To sum up, it is necessary to select the appropriate kind of nanoparticle, particle size, and prescription ratio when microneedles constructing with a given matrix material, so as to effectively improve its mechanical performance.

Gentiana crassicaulis Duthie ex Burk. in Gentiana (Sect. Cruciata), Gentianaceae, is one of the original plants of both Gentianae Macrophyllae Radix and Tibetan herb Jie-Ji Na-Bao, which contain such bioactive iridoids as gentiopicroside, loganic acid and others. In this study, based on previous work, the transcriptome of G. crassicaulis was sequenced and analyzed to construct transcriptome databases of roots, stems, leaves and flowers. qRT-PCR verification was conducted for parts of unigenes that may be key enzymes in the pathway of iridoid biosynthesis. The results are as follows: ① a total of 159 534 unigenes were obtained, with an average length of 679 bp. According to the functional classification of GO, unigenes can be divided into 3 categories with 67 branches. The unigenes were aligned in the KOG database and were classified into 25 categories according to function. ② In the KEGG database, 215 unigenes were implicated in 20 standard secondary metabolism pathways. The analysis shows that 305 unigenes encoded 28 key enzymes in the pathway of iridoid biosynthesis, and their expression in different organs is different; and ③ qRT-PCR was approximately consistent with RNA-Seq results. The 7 annotated unigenes identified in this study, HMGS, DXS, MCS, GPPS, G10H, 7-DLNGT and STR, all had higher relative expression levels in the above-ground parts (stem, leaf and flower) than in the underground part (root). Iridoids are common active and index components of such traditional Chinese medicines as Qinjiao, Longdan, Dangyao, and Qingyedan, among others. Therefore, this work provides basic scientific data for further development including obtaining active components or intermediates through biotechnology, exploring the accumulation of effective components, evaluating the quality of different ecotype varieties, and identifying authentic biosynthesis pathways of medicinal materials.

UDP glucosyltransferase (UDPGT) catalyzes the synthesis of secondary metabolites and plant hormones to regulate plant growth and development, pathogen defense and environmental adaptability. In this study 18 members of the RcUDPGT gene family were cloned from Tibetan Rhodiola crenulata and analyzed using bioinformatics. The tissue-specific expression, abiotic stresses and plant hormones (abscisic acid, auxin, methyl jasmonate) induced expression patterns were identified by real-time quantitative PCR. The bait vector of RcUDPGT (JX228125.1) was constructed to select interacting proteins from an Arabidopsis yeast library. The results of the bioinformatics analysis revealed that RcUDPGT nucleotide sequences were about 1 400 bp and encoded 452-498 amino acids. In the primary protein sequences, C-terminal sequences were more conserved compared with N-terminal regions, which held a PSPG (plant secondary product glycosyltransferase) domain. In the tertiary structures, RcUDPGTs contained a UDP sugar donor recognition binding site. In addition, all genes had multiple phosphorylation sites. The results of qRT-PCR showed that RcUDPGTs genes were expressed in root, stem and leaf. The expression levels were regulated by low temperature/ultraviolet light and various plant hormones (ABA, IAA, MeJA), but the expression patterns were quite different among them. For example, RcUDPGT6, RcUDPGT11, and RcUDPGT17 had the highest expression in leaves and were induced by all three hormones, suggesting that the functions of these genes might be to respond to environmental changes. RcUDPGT9, RcUDPGT10, RcUDPGT14 were most abundantly expressed in roots and were significantly induced by ABA and MeJA hormones, indicating that these genes may be involved in the synthesis and accumulation of salidroside. Yeast two-hybrid results showed that RcUDPGT did not exhibit autoactivation and cell toxicity, and two significant interactional genes were identified, AtKCR1 (AT1G67730.1) and AtSNL4 (AT1G70060). The AtKCR1 gene encodes a β-ketoacyl reductase (KCR) involved in synthesis of very long chain fatty acids. The AtSNL4 gene encodes a homolog of the transcriptional repressor SIN3, which could participate in the ABA hormone signaling pathway and enhance the transcriptional repression of AP2/EREBP class factors in Arabidopsis. These results suggest that the accumulation of the secondary metabolite salidroside in Rhodiola crenulata might be affected by several regulatory mechanisms. The above results may lay the foundation for understanding the adaptive mechanism of Rhodiola crenulata in a high altitude environment and stimulate an in-depth study of the synthesis and accumulation of secondary metabolites in this species.

1-Deoxy-D-xylulose-5-phosphate synthase (DXS) is a rate-limiting enzyme involved in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for terpenoid precursor biosynthesis. DXS plays an essential role in glycyrrhizic acid (GA) biosynthesis. Based on our previous transcriptome study, there was a negative correlation between DXS expression and GA content. Therefore, we explored the regulatory role of DXS in GA biosynthesis using both gene overexpression and gene knockout in a hairy root culture system. DXS was cloned from Glycyrrhiza glabra L. (GenBank Accession No. MN158121). A plant binary expression vector pCA-DXS was constructed by a gene fusion method. The sgRNA sequence was designed based on the first exon of DXS to construct the gene editing vector pHSE-DXS. Hairy roots overexpressing or knocking out DXS were generated through an Agrobacterium-mediated method with licorice hypocotyls as explants. Wild-type hairy roots and negative control hairy roots containing empty plasmids were also evaluated. UPLC was used to determine the GA content in each licorice hairy root line. Results showed that the content of GA in the hairy root group knocking out DXS was significantly higher than that in the wild-type and negative control groups, while in the hairy root group overexpressing DXS was significantly lower, suggesting that DXS plays a negative role in GA biosynthesis. This study provides a foundation for determining the function of DXS in terpenoid metabolism and for further establishment of a molecular regulatory network of GA biosynthesis.

Neurodegenerative diseases (ND) mainly include Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, ataxia, and other diseases. The number of patients with ND is increasing, but the proportion of patients who can be diagnosed and treated early is less than 30% and the cause of ND is still unclear. In order to intervene in the disease as early as possible, researchers are committed to finding biomarkers that facilitate the early diagnosis of ND. Among them, cerebrospinal fluid (CSF) closely reflects the composition of the extracellular space of the brain, and may be the most sensitive biomarker for evaluating ND. However, the method of taking cerebrospinal fluid is more complicated, and it is not a common method in primary care or elderly medical institutions for the treatment of ND patients. Imaging examinations are expensive and difficult to spread among the community. The peripheral blood collection is convenient and less traumatic, which is a potential early screening and follow-up method. There are many components in the blood for analysis and research. This article reviews the research progress of the changes of apolipoprotein in the blood of ND patients as markers.

Chemokines are small cytokines with chemotactic activity, they are involved in regulating immune responses and inflammatory responses. In the development of tumors, chemokines are multi-functional mediators that not only affect the infiltration of immune cells into the tumor, but also have an important impact on tumor growth, angiogenesis, invasion, and metastasis. Besides, they are important targets of tumor therapy. Here we review chemokines involved in the regulation of signaling pathways, analyze the mechanism of chemokines in the development of breast cancer, summarize the chemokines targeted drugs for breast cancer in recent years and make a prospect about the role of chemokines in anti-breast cancer therapy.

Torpor refers to a state in which the metabolic activity in the body of the living animal is greatly reduced during the period of reduced food supply, which is manifested as a substantial decrease in body temperature, metabolic level, and exercise level. Mammals have a strict body temperature regulation system to maintain a constant body temperature. When the energy supply is insufficient for a long time, some mammals will enter a hibernation state. Torpor is very similar to the hibernation state. The research on the mechanism of torpor state is of great significance in aerospace, military medicine and other fields. This review summarizes the specific mechanisms regulating the occurrence of torpor from four aspects: adenylate cyclase activating polypeptide (adcyap) neurons, leptin, pyroglutamylated RFamide peptide (QRFP) neurons, and sympathetic nervous system, aiming to provide ideas for further research on the mechanism of torpor.