Objectives. Knee osteoarthritis (KOA) pain is caused by nociceptors, which are actually sensory nerve fiber endings that can detect stimuli to produce and transmit pain signals, and high levels of NGF in synovial tissue led to peripheral hyperalgesia in KOA. The purpose of this study is to investigate how sensory nerve fibers respond to the NGF/TrKA signal pathway and mediate the peripheral hyperalgesia in KOA rats. Methods. Forty SD male rats were randomly divided into 4 groups: normal, KOA, KOA + NGF, and KOA + siRNA TrKA. KOA model rats were induced by anterior cruciate ligament transection (ACLT). Mechanical and cold withdrawal thresholds (MWT and CWT) were measured 4 times in each group. The synovial tissues were harvested on day 28, and the expressions of NGF, TrKA, TRPV1, IL-1β, and PGP9.5 were determined using western blot, qPCR, and immunofluorescence staining. The primary rat fibroblast-like synoviocytes (FLSs) and DRG cells were divided into 4 groups as in vivo. The expressions of NGF, TrKA, TRPV1, and CGRP in vitro were determined using western blot and qPCR. Results. KOA and intra-articular injection with NGF protein increased both mRNA and protein levels, not only TRPV1, PGP 9.5, and IL-1β in the synovial tissue, but also TRPV1, PGP 9.5, and S100 in the DRG tissue, while above changes were partly reversed after siRNA TrKA intervention. Besides, siRNA TrKA could improve peripheral hyperalgesia and decreased the TRPV1 positive nerve fiber innervation in synovial tissue. The results in vitro were consistent with those in vivo. Conclusion. This study showed the activation of the NGF/TrKA signaling pathway in KOA promoted the release of pain mediators, increased the innervation of sensory nerve fibers in the synovium, and worsened peripheral hyperalgesia. It also showed increased TRPV1 positive sensory innervation in KOA was mediated by NGF/TrKA signaling and exacerbated peripheral hyperalgesia.

目的 对三色散凝胶贴膏原料药的提取工艺进行优化.方法 将SD大鼠分为空白组、模型组、传统工艺组、水提组和醇提组,每组5只,采用前交叉韧带横断术建立膝骨关节炎模型,考察不同提取方式所得凝胶贴膏对膝骨关节炎大鼠的药效学影响.分别以冷、热痛阈值与疼痛介质降钙素基因相关肽(CGRP)、环氧合酶2(COX-2)、P物质(SP)、前列腺素E2(PGE2)含量为指标进行镇痛实验;对大鼠滑膜组织进行HE染色,观察大鼠滑膜细胞增殖、炎症细胞浸润、血管入侵程度,并以白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)、IL-6蛋白与mRNA表达水平为指标进行抗炎实验,对比各组大鼠的抗炎镇痛效果差异.以加醇倍数、提取时间、提取次数作为正交试验考察因素,以蔓荆子黄素、士的宁和马钱子碱含量为评价指标并计算综合评分,优选出三色散凝胶贴膏原料药的提取工艺并进行验证实验.结果 与模型组比较,各给药组大鼠的冷痛阈值和热痛阈值(除传统工艺组外)均显著升高(P＜0.05),疼痛介质CGRP、COX-2、SP、PGE2含量均显著下降(P＜0.05);滑膜组织炎症细胞浸润减少、纤维化程度减轻,胶原沉积减少,可见少量毛细血管增生;滑膜组织中IL-1β、IL-6、TNF-α蛋白与mRNA表达水平均显著降低(P＜0.05).与传统工艺组比较,醇提组大鼠的大部分检测指标显著降低(P＜0.05),而水提组大鼠仅热痛阈值和滑膜组织中IL-6 mRNA表达水平显著降低(P＜0.05).三色散凝胶贴膏原料药的最佳提取工艺为取适量三色散细粉,加8倍量55%乙醇,加热回流提取90 min,提取2次;3次验证实验结果显示,蔓荆子黄素、士的宁与马钱子碱含量平均值分别为0.007%、0.092%、0.214%,RSD均小于5%.结论 优化的三色散凝胶贴膏原料药提取工艺稳定、可行,有利于提高该制剂药效.

目的 优化三色散凝胶贴膏处方.方法 根据 2020 年版《中国药典》四部通则项下规定进行初黏力、持黏力测定,对渗透情况、皮肤残留情况进行评分,流变仪进行振幅扫描、频率扫描、蠕变恢复扫描,分析储能模量(G')、复合黏度(ŋ∗)、蠕变柔量[J(t)]值,结合黏附力、流变学参数采用正交试验优化处方.结果 最佳处方为AH-106、甘油、甘羟铝、酒石酸比例 55 ∶ 300 ∶ 2 ∶ 3,G'、ŋ∗、J(t)值、初黏力、持黏力、剪切应力值、渗透情况评分、残留情况评分分别为 1 169.63 Pa、11 865.6 Pa·s、1.810×10-3/Pa、25 号、720 min、723.755 Pa、8 分、8 分.结论 本实验结合流变学参数、黏附力指标调节辅料用量,所得最优处方可使三色散凝胶贴膏形变程度、粘弹性等指标较好,固化时间大大缩短.

Apocyni Veneti Folium (AVF) is a salt-tolerant medicinal halophyte and soil salinity is a main stress affecting its quality. Molecular bases involved in quality evaluation and ecological adaptations to abiotic constraints can be explored using omics tools. In the study, AVF was treated with four levels of salt stress (control, 100, 200 and 300 mM NaCl, respectively) and subjected to de novo-based RNA-sequencing. We constructed GO and KEGG analysis on the obtained DEGs. After molecular phylogenetic analysis, we isolated and characterized one representative and up-regulated candidate gene AvUGT (Tr_AVENL_25169) encoding UDP-glucosyltransferase under salt stress. Results showed that the obtained clean reads were assembled into a total of 54,276 high-quality unigenes. Notably, specific genes related to flavonoid glucoside biosynthesis and salt-tolerant regulation, such as genes encoding transcription factors, transporters, glucosyltransferase, heat shock protein and plant hormone signal transduction-related protein, preferentially up-regulated by low level of salt. Combined with previous metabonomic analysis, the results revealed key genes that contribute to elucidate the reduced salt toxicity in AVF. Furthermore, the transcript profiles of UGT genes were consistent with the accumulation of flavonoid glycosides in AVF; the candidate gene AvUGT probably plays a critical role in biosynthesis of flavonoid glucosides in response to salt environment. These results provided a basis for future research on the regulatory mechanism of salt stress of medicinal halophyte AVF.

目的 优化三色散凝胶贴膏剂的基质处方工艺,研究其对膝骨关节炎(KOA)大鼠滑膜炎症的干预效应.方法 以综合感官、初黏力、持黏力、剥离强度为评价指标,以聚丙烯酸钠、甘羟铝、酒石酸、甘油为考察因素,采用正交实验筛选最优基质处方.随后,将大鼠随机分为空白组、模型组、三色散凝胶贴膏剂组、三色散传统制剂组、三色散凝胶贴膏剂(含促渗剂)组.采用改良的Hulth法构建KOA模型,造模成功后,各给药组外敷膏药28 d.末次给药后,提取各组大鼠滑膜组织.HE染色评估滑膜炎症,天狼星红染色评估滑膜胶原沉积情况,Krenn评分评价滑膜炎症情况.Western blot和qPCR检测滑膜IL-1β、TNF-α、IL-6蛋白和mRNA表达.结果 经过筛选得到三色散凝胶贴膏剂的最优基质处方为聚丙烯酸钠50 g,甘羟铝2.5 g,酒石酸3 g,甘油500 g,三色散150 g,纯水50 mL,卡波姆120 g,薄荷脑36 g.大鼠给药28 d后,HE及天狼星红染色结果显示,与模型组比较,三色散凝胶贴膏剂组和三色散传统制剂组炎性细胞浸润、胶原沉积减少,纤维化程度减轻,Krenn评分显著降低(P<0.01),炎症因子IL-1β、TNF-α、IL-6蛋白和mRNA表达降低(P<0.01);与三色散传统制剂组比较,三色散凝胶贴膏剂组Krenn评分,IL-1β、TNF-α 和IL-6蛋白表达无显著差异,IL-1β、IL-6 mRNA表达显著降低(P<0.05),TNF-αmRNA表达无显著差异.与三色散凝胶贴膏剂组比较,三色散凝胶贴膏剂(含促渗剂)组Krenn评分显著降低(P<0.05),IL-1β蛋白表达显著下降(P<0.01),TNF-α 和IL-6蛋白表达无显著差异,IL-1β、TNF-α、IL-6 mRNA表达显著下降(P<0.05,P<0.01).结论 优化的基质处方工艺稳定可行,操作方法简便,制备得到的三色散凝胶贴膏剂外观及黏附性良好,且能有效减轻KOA大鼠滑膜炎症,效果不亚于三色散传统制剂.加入促渗剂能增强三色散凝胶贴膏剂的抗炎效果,为进一步研究提供参考.

Background: Frankincense and myrrh (FM) are often used together to treat knee osteoarthritis (KOA). However, the underlying mechanism of its treatment of KOA remains unclear. Objective: To analyze the active components of FM through network pharmacology and in vitro experiments, and to explore its potential therapeutic mechanism in the treatment of KOA. Materials and methods: The protein mapping relationship between potential drug targets and disease targets was screened and constructed through the database. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using R software. Discovery Studio software was used to perform molecular docking. The active components of FM were identified using liquid chromatography–mass spectrometry (LC-MS). In addition, experimental verification was carried out by Cell Counting Kit-8 detection, Western blot, and immunofluorescence analysis. Results: Combining the results of network pharmacology and LC-MS, 31 active compounds and 94 target genes of FM were identified. The common genes of FM and KOA suggest that FM exerts anti-KOA effect by regulating genes such as Transcription factor Jun (JUN), Interleukin-6 (IL-6), Interleukin-1 beta (IL-1β), C-X-C motif chemokine ligand 8 (CXCL8), Transcription factor p65 (RELA), and Mitogen-activated protein kinase 1 (MAPK1). GO enrichment analysis showed that FM exerted therapeutic effects on KOA by regulating biological processes such as cell proliferation, cell migration, and apoptosis. In addition, KEGG enrichment analysis involved signaling pathways such as fluid shear stress, the TNF, PI3K-Akt, NF-κB, and MAPK. Consistently, in vivo experiments showed that FM inhibited IL-1β-induced MAPK activation and attenuated inflammation in mouse chondrocytes. Furthermore, FM inhibited IL-1β-induced phosphorylation of p65 and the process of p65 translocation from the cytoplasm into the nucleus. Conclusions: Our results provide conclusive evidence and deepen the current understanding of FM in the treatment of KOA and further support its clinical application.

Salinity stress significantly affects the contents of bioactive constituents in licorice Glycyrrhiza uralensis. To elucidate the molecular mechanism underlying the difference in the accumulation of these constituents under sodium chloride (NaCl, salt) stress, licorice seedlings were treated with NaCl and then subjected to an integrated transcriptomic and metabolite profiling analysis. The transcriptomic analysis results identified 3,664 differentially expressed genes (DEGs) including transcription factor family MYB and basic helix-loop-helix (bHLH). Most DEGs were involved in flavonoid and terpenoid biosynthesis pathways. In addition, 121 compounds including a triterpenoid and five classes of flavonoids (isoflavone, flavone, flavanone, isoflavan, and chalcone) were identified, and their relative levels were compared between the stressed and control groups using data from the ultrafast liquid chromatography (UFLC)-triple quadrupole-time of flight-tandem mass spectrometry (TOF-MS/MS) analysis. Putative biosynthesis networks of the flavonoids and triterpenoids were created and combined with structural DEGs such as phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase [4CL], cinnamate 4-hydroxylase [C4H], chalcone synthase [CHS], chalcone-flavanone isomerase [CHI], and flavonoid-3 ',5 ' hydroxylase (F3 ',5 ' H) for flavonoids, and CYP88D6 and CYP72A154 for glycyrrhizin biosynthesis. Notably, significant upregulation of UDP-glycosyltransferase genes (UGT) in salt-stressed licorice indicated that postmodification of glycosyltransferase may participate in downstream biosynthesis of flavonoid glycosides and triterpenoid saponins. Accordingly, the expression trend of the DEGs is positively correlated with the accumulation of glycosides. Our study findings indicate that key DEGs and crucial UGT genes co-regulate flavonoid and saponin biosynthesis in licorice under salt stress.

Purpose: Our recent research is dedicated to finding effective drugs for the treatment of knee osteoarthritis (KOA) from traditional Chinese medicine and trying to make full use of modern science and technology to uncover the mechanisms and targets behind them. Synovial inflammation is one of the key pathological features of KOA, and a growing number of researchers realize that early intervention of synovial inflammation may be able to reverse disease progression. The close association of traditional natural products with modern nanotechnology may be important for improving the anti-synovitis efficacy. The purpose of our research was to explore the anti-synovitis mechanism of NEs-SP-EO that might be associated with the ERS/TXNIP/NLRP3 signalling axis. Methods: Chemical composition of "Sanse Powder" essential oil (SP-EO) and NEs-SP-EO were analyzed by GC-MS. NEs-SP-EO were prepared and characterized by nanoparticle tracking analysis, polydispersity index (PDI), zeta potential (ZP), ultraviolet-visible spectroscopy, and transmission electronic microscopy. The CCK8 assay for cell viability of NEs-SP-EO was performed on fibroblast-like synovial cells (FLSs) and the inflammatory environment was stimulated by LPS to explore the therapeutic mechanisms in vitro. Experiments of NEs-SP-EO in vivo were performed in male SD rats. Results: The GC-MS results showed that 30 compounds were present in SP-EO and 11 components of NEs-SP-EO were identified. The results also showed that the formulation of NEs-SP-EO exhibited suitable particle size, negative charge, and stable system. In vitro and vivo testing, NEs-SP-EO produced anti-synovitis efficacy by reduced the induction of the ERS/TXNIP/NLRP3 signaling axis as well as regulating the overproduction of IL-1β, IL-18. Conclusion: We have developed a new type of essential oil nanoemulsion from "Sanse Powder" and demonstrated that it can managing synovitis of KOA. Besides, we have initially explored the anti-inflammatory mechanism that may be related to the ERS/TXNIP/NLRP3 signaling axis.

目的 建立三色散凝胶贴膏剂的HPLC指纹图谱,同时对其多种成分进行含量测定,为其质量控制提供依据.方法 采用Hypersil Gold C18色谱柱(250 mm×4.6 mm,5μm),流动相为乙腈-0.1％磷酸水溶液,梯度洗脱,流速为1.0 mL/min,柱温为30℃,检测波长为240 nm,进样量为20μL,建立三色散凝胶贴膏剂的HPLC指纹图谱,运用《中药色谱指纹图谱相似度评价系统》2012A版对其进行相似度评价;采用外标法,利用混合对照品测定12个成分的含量.结果 在选定的色谱条件下建立了三色散凝胶贴膏剂的HPLC指纹图谱,共标定16个共有峰,与对照品比对出12个成分,10批三色散凝胶贴膏剂的相似度在0.943～0.994之间;12个成分在一定浓度范围内均成良好的线性,相关系数均大于0.9991,平均加样回收率在95.35％～99.43％之间,RSD均小于3.0％,12个成分在10个批次中的含量基本一致.结论 建立的三色散凝胶贴膏剂指纹图谱及含量测定方法,准确可靠,重复性好,可为进一步完善三色散凝胶贴膏剂的质量控制提供参考.

Synovitis is the primary driving factor for the occurrence and development of knee osteoarthritis (KOA) and fibroblast-like synoviocytes (FLSs) and plays a crucial role during this process. Our previous works revealed that transient receptor potential ankyrin 1 (TRPA1) ion channels mediate the amplification of KOA synovitis. In recent years, essential oils have been proved to have blocking effect on transient receptor potential channels. Meanwhile, the therapeutic effect of Sanse Powder on KOA synovitis has been confirmed in clinical trials and basic studies; although, the mechanism remains unclear. In the present study, Sanse Powder essential oil nanoemulsion (SP-NEs) was prepared, and then chemical composition, physicochemical properties, and stability were investigated. Besides, both in MIA-induced KOA rats and in LPS-stimulated FLSs, we investigated whether SP-NES could alleviate KOA synovitis by interfering with AMP-activated protein kinase- (AMPK-) mammalian target of rapamycin (mTOR), an energy sensing pathway proved to negatively regulate the TRPA1. Our research shows that the top three substances in SP-NEs were tumerone, delta-cadinene, and Ar-tumerone, which accounted for 51.62% of the total, and should be considered as the main pharmacodynamic ingredient. Less inflammatory cell infiltration and type I collagen deposition were found in the synovial tissue of KOA rats treated with SP-NEs, as well as the downregulated expressions of interleukin (IL)-1β, IL-18, and TRPA1. Besides, SP-NEs increased the phosphorylation level of AMPK and decreased the phosphorylation level of mTOR in the KOA model, and SP-NEs also upregulated expressions of peroxisome proliferator-activated receptor-gamma (PPARγ) and PPARγ coactivator-1α and downstream signaling molecules of AMPK-mTOR in vivo and in vitro. To conclude, a kind of Chinese herbal medicine for external use which is effective in treating synovitis of KOA was extracted and prepared into essential oil nanoemulsion with stable properties in the present study. It may alleviate synovitis in experimental KOA through the negative regulation of TRPA1 by AMPK-mTOR signaling.

Prunella vulgaris L. is a moderately salt tolerant plant commonly found in China and Europe, whose spica (Prunellae Spica) has been used as a traditional medicine. The scant transcriptomic and genomic resources of Prunellae Spica have greatly hindered further exploration of the underlying salt tolerance mechanism of this species. To clarify the genetic basis of its salt tolerance, high-throughput sequencing of mRNAs was employed for de novo transcriptome assembly differential expression analysis of Prunellae Spica under salt stress. 118,664 unigenes were obtained by assembling pooled reads from all libraries with 68,119 sequences annotated. A total of 3857 unigenes were differentially expressed under low, medium and high salt stress, including 2456 up-regulated and 1401 down-regulated DEGs, respectively. Gene ontology analysis revealed that salt stress-related categories involving 'catalytic activity', 'binding', 'metabolic process' and 'cellular process' were highly enriched. KEGG pathway annotation showed that the DEGs from different salt stress treatment groups were mainly enriched in the pathways of translation, signal transduction, carbohydrate metabolism, energy metabolism, lipid metabolism and amino acid metabolism, accounting for over 60% of all DEGs. Finally, it showed that the results of quantitative real-time polymerase chain reaction (qRT-PCR) analysis for 10 unigenes that randomly selected were significantly consistent with RNA-seq data, which further assisted in the selection of salt stress-responsive candidate genes in Prunellae Spica. This study represents a significant step forward in understanding the salt tolerance mechanism of Prunellae Spica, and also provides a significant transcriptomic resource for future work.

Soil salinity is a major abiotic stress that limits plant growth and productivity. Understanding the mechanisms of plant salinity tolerance can facilitate engineering for quality improvement. Apocynum venetum L. exhibits tolerance to salinity. Due to the lack of a genomic database, RNA-seq based transcriptomics and isobaric tag for relative and absolute quantitation (iTRAQ) based proteomic profiles of Apocyni Veneti Folium (leaves of Apocynum venetum L.) exposure to four levels of salt treatments (0, 100, 200 and 300 mM NaCl, respectively) were performed. A total of 143, 162 and 167 differentially expressed proteins (DEPs) were found between salt-treated Apocyni Veneti Folium compared with control, respectively. They were mainly involved in carbohydrate and energy metabolism, biosynthesis of metabolites and signal transduction. Furthermore, results showed that carbon and nitrogen metabolisms were altered under salt stress; low and moderate levels of salt stress enhanced photosynthetic functions and ramped up carbohydrate metabolism. However, severe salt stress depressed biosynthesis of secondary metabolites, consistent with the metabolomics results. It is worth emphasizing that some key salt-responsive proteins, such as dehydrin 1, annexin, pathogenesis-related protein, prolyl oligopeptidase, peroxidase, cinnamyl alcohol dehydrogenase, 4-hydroxycinnamoyl-CoA ligase 3, cytochrome P450 CYP73A120, were screened. These novel proteins provide a good starting point for further research into their functions using genetic or other approaches. In addition, a weak correlation between the abundance of DEPs and the corresponding differentially expressed genes highlighted the effect of post-transcriptional modifications and the importance of employing proteomics and transcriptomics to analyze global protein level changes. In conclusion, the protein profiles indicate that halophyte uses a multipronged approach to overcome salt stress, and provides some novel information on revealing the mechanisms of adaption and quality formation.

Licorice (Glycyrrhiza uralensis Fisch) possesses a substantial share of the global markets for its unique sweet flavor and diverse pharmacological compounds. Cultivated licorice is widely distributed in northwest regions of China, covered with land with a broad range of salinities. A preliminary study indicated that suitable salt stress significantly increased the content of bioactive constituents in licorice. However, the molecular mechanisms underlying the influence of salinity on the accumulation of these constituents remain unclear, which hinders quality breeding of cultivated licorice. In our study, flavonoid-related structural genes were obtained, and most of them, such as phenylalanine ammonia-lyases, cinnamate 4-hydroxylases, 4-coumarate: CoA ligases, chalcone synthases, chalcone-flavanone isomerase, and flavonol synthase, showed high levels after salt treatment. In the biosynthesis of glycyrrhizin, three key enzymes (bAS, CYP88D6, and CYP72A154) were identified as differentially expressed proteins and remarkably upregulated in the salt-stressed group. Combining these results with the contents of 14 bioactive constituents, we also found that the expression patterns of those structural proteins were logically consistent with changes in bioactive constituent profiles. Thus, we believe that suitable salt stress increased the accumulation of bioactive constituents in licorice by upregulating proteins involved in the related biosynthesis pathways. This work provided valuable proteomic information for unraveling the molecular mechanism of flavonoid and glycyrrhizin metabolism and offered fundamental resources for quality breeding in licorice.

Licorice is extensively applied in food as well as herbal medicine across the world, possessing a substantial share in the global market. It has made great progress in chemical and pharmacological research in recent years. Currently, Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat., and Glycyrrhiza glabra L. were officially used as Gan-Cao according to the Chinese Pharmacopoeia. Accumulating evidence demonstrated three varieties of licorice have their own special compounds except for two quality markers set by Pharmacopoeia, providing great possibility for better understanding their characteristics, evaluating quality of each species and studying biosynthesis mechanisms of species-specific compounds. As a special "guide drug" in clinic, licorice plays an important role in Chinese herbal formulas. The interaction between licorice with other ingredients and their metabolism in vivo should also be taken into consideration. In addition, draft genome annotation, and success of the final step of glycyrrhizin biosynthesis have paved the way for biosynthesis of other active constituents in licorice, a promising beginning of solving source shortage. Accordingly, we comprehensively explored the nearly 400 chemical compounds found in the three varieties of licorice so far, systematically excavated various pharmacological activities, including metabolism via CYP450 system in vivo, and introduced the complete biosynthesis pathway of glycyrrhizin in licorice. The review will facilitate the further research toward this herbal medicine.

Licorice is one of the ancient and most frequently applied herbs for its diverse phytochemicals. At present, wild resources of licorice have rapidly declined with increasing demand and the proportion of cultivated products in the market is quickly growing. However, the different level in chemical composition between the wild and cultivated licorice may result in the discrepancy in quality and pharmacological activity. Therefore, an ultra-fast liquid chromatography coupled with triple quadrupole-time of flight tandem mass spectrometry (UFLC-Triple TOF-MS/MS) method combined with multivariate statistical analysis technology was employed to explore chemical composition differences. The result showed that total 63 components were identified from licorice samples. The wild and the cultivated licorice are obviously classified into two groups according to principal component analysis (PCA). PCA and partial least squared discrimination analysis (PLS-DA) were also introduced to rapidly find 14 candidate compounds between two ecotypes of licorice. Apart from glycyrrhizin, licorice saponin J2/G2, glyasperin D and dehydroglyasperin D also could be selected as chemical markers based on t-test and variable importance in the projection (VIP) value. Our study successfully established an effective method for exploring metabolite profiling between two ecotypes of licorice and laying the foundation for distinguishing wild and cultivated licorice.

The demand for licorice and its natural product derivatives in domestic and foreign market is considerably huge. The core production areas of licorice are covered with salinity and drought land in northwestern China. Studies have shown that suitable environmental stress can promote the accumulation of glycyrrhizin and liquiritin to improve its quality as medicinal materials. However, there are few reports on other bioactive constituents of licorice, not to mention their dynamic accumulation under stressed conditions. To explore the quality formation of licorice from the perspective of salt influence, a reliable method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-MS/MS) was established for simultaneous determination of sixteen bioactive constituents, including triterpenoids, flavonoids, chalcones and their glycosides. Physiological experiments were performed to investigate salt tolerance of licorice under different salinity treatments. The expressions of crucial genes (bAS and CHS), key enzymes of triterpenoid and flavonoid synthesis, were also tested by qRT-PCR. Our study found that 50 mM NaCl treatment (low stress) was the most favorable to promote the accumulation of bioactive constituents in the long term, without harming the plants. Flavonoid accumulation of non-stressed and low-stressed groups became different in the initial synthesis stage, and glycosyltransferases may have great influence on their downstream synthesis. Furthermore, bAS and CHS also showed higher levels in low-stressed licorice at harvest time. This work provides valuable information on dynamic variations in multiple bioactive constituents in licorice treated by salt and insight into its quality formation under stressed conditions.

Apocyni Veneti Folium (AVF) has been raised great interest in the antioxidant properties recently for the preservation of human health. However, little research was found on the integrate metabolites except our previous investigation on the variations of the bioactive constituents. To understand the salt-tolerant mechanisms of the halophyte, metabolomic platform based on ultra-fast liquid chromatography tandem triple time-of-flight mass/mass spectrometer was applied in this study. The results showed that metabolic profiles were separated and differentiated among groups based on multivariate statistical analysis; different metabolites belonged to various chemical classes. Besides, phenylpropanoid pathway and terpenoid biosynthesis were disturbed in all salt-stressed AVF and low salt-treated group appeared to be better than other samples in terms of relative contents (peak areas) of the wide variety of bioactive components and physiological variations of photosynthetic pigments, osmotic homeostasis, lipid peroxidation product and antioxidative enzymes. This study may provide additional insight into the salt-tolerant mechanisms and the quality assessment of AVF in a holistic level based on the plant metabolomics.

Background: Pseudostellaria heterophylla is an important tonic traditional Chinese medicine. However, the molecular changes in the herb from geo-authentic habitat and cultivated bases remain to be explored. Objective: The purpose of this research was to study differences in P. heterophylla from geo-authentic habitat and cultivated bases. Methods: High-throughput technologies of transcriptomic and proteomic were used to identify proteins. Isobaric Tags for Relative and Absolute Quantification (iTRAQ) MS/MS has been utilized to evaluate changes in P. heterophylla from geo-authentic habitat and cultivated bases. Results: In this study, a total of 3775 proteins were detected, and 140 differentially expressed proteins were found in P. heterophylla from geo-authentic habitat and cultivated bases. 44 significantly differential expressed proteins were identified based on functional analysis classified into nine categories. Five differentially expressed proteins were confirmed at the gene expression level by Quantitative realtime PCR. Catabolic metabolism, carbohydrate metabolism, and response to stress of oxidoreductases and transferases in P. heterophylla from geo-authentic habitat were stronger than in those from cultivated bases, but protein folding and response to stress of heat shock proteins, isomerases, rubisco large subunit-binding proteins, chaperone proteins, and luminal-binding proteins in herbs from cultivated bases were more active. ADG1 and TKTA could be the critical proteins to regulate sucrose; MFP2 and CYS may be the crucial proteins that control the metabolism of fatty acids and amino acids. Conclusion: These results will provide the basic information for exploring the differences in secondary metabolites in P. heterophylla from geo-authentic habitat and cultivated bases and the protein mechanism of its quality formation.

Spica Prunellae is an important Chinese herbal medicine. Because of its good curative effect on various diseases, this herb is consumed in large quantities in clinical applications. The metabolites of Spica Prunellae are known to change under salt stress; however, the difference in protein levels of Spica Prunellae between saline and normal conditions is unclear. In this study, we used proteomics techniques to identify differentially expressed proteins in Spica Prunellae under different saline conditions. (iTRAQ) MS/MS was used to detect statistically significant changes in protein between salt stress and normal conditions. Ultimately, we detected 1,937 proteins, 89 of which were detected in two different comparison. Based on GO, STRING and KEGG analyses, 35 significantly differentially expressed proteins were selected for further analysis. The results of functional and signal pathway analyses indicated that the cellular protein and carbohydrate metabolism of Spica Prunellae was weaker, calcium ion transport was higher, photosynthesis was higher, and protein production was faster under saline conditions than under normal conditions. This study provides useful information for studying the causes of differences in secondary metabolites in Spica Prunellae under salt stress and the protein mechanisms related to their quality.

Licorice is a famous Chinese medicinal material widely applied worldwide in food and drugs. It possesses a substantial share in the international and domestic markets. While the demand is continuously increasing, wild-type of licorice is gradually disappearing. Its cultivated-type is significantly different compared to its wild-type, especially the bioactive compounds in dried roots and rhizomes (the medicinal parts of wild and cultivated licorice) are responsible for their quality difference. In this study, a total of thirty-two constituents, including seventeen secondary compounds and fifteen primary metabolites, were simultaneously analyzed by UFLC coupled with triple quadrupole-linear ion trap mass spectrometry (UFLC-QTRAP-MS/MS) in different medicinal parts of licorice. Our findings indicated that the content of major bioactive compounds in wild licorice were significantly higher than those in its cultivated-type. The metabolites in rhizomes showed totally different outcomes between wild and cultivated licorice. Remarkably high level of some important amino acids related to abiotic stress (drought and salt) were found in wild licorice. Notably, the high contents of phenylalanine and compounds in upstream of flavonoid synthesis in cultivated licorice suggested that the synthesis may differ at the initial stage. The distribution pattern of metabolites in different medicinal parts of wild and cultivated licorice will not only provide a novel clue in agricultural breeding but also facilitate the further study on their quality formation.