[Identification of Cervi Cornu (Cervus elaphus) and its formula granules based on PCR-RFLP].

Cervi Cornu is the ossified antler, or the base antler that falls off in the spring of the following year after the pilose antler is sawn off from Cervus elaphus or C. nippon, as a precious traditional Chinese medicine, has been recognized for its medicinal value and widely used in clinical practice. However, the origins of Cervi Cornu are miscellaneous, and Cervi Cornu is even mixed with adulterants in the market. Currently, there is a shortage of ways to identify Cervi Cornu and no standard to control the quality of Cervi Cornu. So it is valuable to develop a way to effectively identify Cervi Cornu from the adulterants. In this study, the differences in the mitochondrial barcode cytochrome b(Cytb) gene sequences of C. elaphus, C. nippon and their related species were compared and the specific single nucleotide polymorphism(SNP) sites on the Cytb sequences of Cervi Cornu were screened out. According to the screened SNPs, Cervi Cornu-specific primers dishmy-F and dishmy-R were designed. The PCR system was established and optimized, and the tolerance and feasibility of Taq polymerases and PCR systems affecting the repeatability of the PCR method were investigated. The amplification products of C. elaphus and C. nippon were digested using the restriction enzyme MseⅠ. The results showed that after electrophoresis of the product from PCR with the annealing temperature of 56 ℃ and 35 cycles, a single specific band at about 100 bp was observed for C. elaphus samples, and the product of C. elaphus samples was 60 bp shorter than that of C. nippon samples. There was no band for adulterants from other similar species such as Alces alces, Rangifer tarandus, Odocoileus virginianus, O. hemionus, Cap-reolus pygargus, Przewalskium albirostis and negative controls. The polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method established in this study can quickly and accurately identify Cervi Cornu originated from C. elaphus in crude drugs, standard decoctions, and formula granules, and distinguish the origins of Cervi Cornu products, i.e., C. nippon and similar species. This study can be a reference for other studies on the quality standard of other formula granules of traditional Chinese medicines.

Exploring the Q-markers of Angelica sinensis (Oliv.) Diels of anti-platelet aggregation activity based on spectrum-effect relationships.

The radix of Angelica sinensis (Oliv.) Diels (RAS) is widely used in medicinal and dietary applications in China, and has the function for replenishing and invigorating the blood, stopping pain and moistening the intestines. In this study, RAS from the main geoherb regions showed better efficacy in inhibiting Adenosine diphosphate- or arachidonic acid-induced platelet aggregation than those from non-geoherb regions. In addition, the HPLC fingerprints of 30 batches of RAS, as part of the comprehensive evaluation of RAS, were established and used for spectral efficiency to screen the quality markers for anti-platelet aggregation activities. Five compounds in RAS-senkyunolide I, uridine, guanine, ferulic acid and adenosine-were demonstrated to contribute significantly to the anti-platelet aggregation activity. These bioactive compounds, especially senkyunolide I and ferulic acid with stronger activities, could be used as quality markers of RAS for quality control of RAS.

[Research progress on chemical constituents and pharmacological activities of seabuckthorn and prediction of its Q-markers].

Seabuckthorn contains flavonoids, tannins, terpenoids, polysaccharides, and vitamins, which have anti-inflammation,anti-oxidation, liver protection, anti-cardiovascular disease, anti-aging, immune enhancing, anti-tumor, and anti-bacterial activities.We reviewed the papers focusing on the chemical constituents, pharmacological activities, and utilization of seabuckthorn. The quality markers(Q-markers) of seabuckthorn were predicted and analyzed based on original plant phylogeny, chemical composition correlation, traditional medicinal properties, pharmacodynamic correlation, traditional and extended efficacy, pharmacokinetics, metabolic processes, and measurable components. With this review, we aim to provide theoretical reference for the quality control and quality standard establishment of seabuckthorn, so as to promote the rational exploitation and utilization of seabuckthorn resources, and improve the healthy and sustainable development of seabuckthorn industry.

Constituents Isolated from the Leaves of Glycyrrhiza uralansis and Their Anti-Inflammatory Activities on LPS-Induced RAW264.7 Cells.

Licorice, the root and rhizome of Glycyrrhiza uralansis Fisch, is one of the most frequently used Traditional Chinese Medicines in rigorous clinical trials to remove toxins and sputum, and to relieve coughing. However, the aerial parts are not used so widely at present. It has been reported that the aerial parts have many bioactivities such as anti-microbial and anti-HIV activities. In this study, we aimed to discover the bioactive compounds from the leaves of G. uralensis. Four new compounds, licostilbene A-B (1-2) and licofuranol A-B (3-4), together with eight known flavonoids (5-12), were isolated and identified from the leaves of G. uralensis. Their structures were elucidated mainly by the interpretation of high-resolution electrospray mass spectrometry (HR-ESI-MS) and nuclear magnetic resonance (NMR) spectroscopic data. Compared with quercetin, which showed a 50% inhibitory concentration (IC50) value of 4.08 µg/mL, compounds 1-9 showed significant anti-inflammatory activities by inhibiting lipopolysaccharide (LPS)-induced nitric oxide (NO) production with IC50 values of 2.60, 2.15, 3.21, 3.25, 2.00, 3.45, 2.53, 3.13 and 3.17 µg/mL, respectively. The discovery of these active compounds is important for the prevention and treatment of inflammation.

Identification of Chemical Markers for the Discrimination of Radix Angelica sinensis Grown in Geoherb and Non-Geoherb Regions Using UHPLC-QTOF-MS/MS Based Metabolomics.

This research aimed to discover chemical markers for discriminating radix Angelica sinensis (RAS) from different regions and to explore the differences of RAS in the content of four active compounds and anti-inflammatory activities on lipopolysacchride (LPS)-induced RAW264.7 cells and calcium antagonists on the HEK 293T cells of RAS. Nine compounds were selected as characteristic chemical markers by ultra-high-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UHPLC-QTOF-MS/MS), based on metabolomics, in order to rapidly discriminate RAS from geoherb and non-geoherb regions. The contents of senkyunolide I and butylidenephthalide in geoherb samples were higher than those in non-geoherb samples, but the contents of ferulic acid and levistolide A were lower in the geoherb samples. Furthermore, the geoherbs showed better nitric oxide (NO) inhibitory and calcium antagonistic activities than the non-geoherbs. These results demonstrate the diversity in quality of RAS between geoherbs and non-geoherbs.

[Transcriptomics of therapeutic effect of Huangqi Liuyi decoction in treating type 2 diabetes].

In this study, Illumina sequencing platform was applied in sequencing rat pancreas, counting expression of target points, analyzing expression differences among blank group, model group and Huangqi Liuyi decoction group and exploring the therapeutic effect and mechanism of Huangqi Liuyi decoction on type 2 diabetes mellitus. According to the result, 24.25% of these genes belonged to the unknown functional class, which was the largest classification unit according to the classification analysis of genes by eggNOG. The rest were classified as energy conversion, amino acid transport and metabolism, nucleotide transport and metabolism, carbohydrate transport and metabolism, coenzyme transport and metabolism, and lipid transport and metabolism, etc.Huangqi Liuyi decoction may play a therapeutic role in the treatment of type 2 diabetes mellitus through four metabolic pathways, namely environmental information processing, cellular process, organismal system and human diseases according to KEGG enrichment analysis. This study shows that, Huangqi Liuyi decoction can significantly improve the fasting blood glucose and glycosylated hemoglobin in type 2 diabetic rats.

[Model Predicts of Astragalus membranceus Transcriptional Gene].

Objective: Astragali Radix, one of the most widely used traditional Chinese medicines, has been extracted a variety of efficiently ingredients. However, the formation mechanism of the secondary metabolites is still unclear and the genome of Astragali Radix has not been sequenced yet. This study aimed at predicting gene model according to transcriptome data, which can be helpful to the use of Astragalus membranceus in the medicine field. Methods: Based on the transcriptome data of Astragalus membranceus and the existing whole genome sequence of its closely related species, the unigene of transcriptome of Astragalus membranceus was predicted and analyzed. Results: Astragalus membranceus had close genetic relationship with Glycine max,Medicago truncatula and Lotus japonicus. Astragalus membranceus shared 2 039 sequences with Medicago truncatula, and their base similarity was 90. 14%. There were 1 948 sequences matched up with Glycine max, and their base similarity was 90. 40%. In the aligement of Astragalus membranceus and Lotus corniculatus, the matched sequences dropped to 1 003,and the base similarity dropped to 89. 77%. Conclusion: The matched gene with three species discributed in chromosome genomes and extrachromosomal genome.

Ligustilide covalently binds to Cys703 in the pre-S1 helix of TRPA1, blocking the opening of channel and relieving pain in rats with acute soft tissue injury.

ETHNOPHARMACOLOGICAL RELEVANCE: The natural anodyne Ligustilide (Lig), derived from Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort., has been traditionally employed for its analgesic properties in the treatment of dysmenorrhea and migraine, and rheumatoid arthritis pain. Despite the existing reports on the correlation between TRP channels and the analgesic effects of Lig, a comprehensive understanding of their underlying mechanisms of action remains elusive. AIM OF THE STUDY: The objective of this study is to elucidate the mechanism of action of Lig on the analgesic target TRPA1 channel. METHODS: The therapeutic effect of Lig was evaluated in a rat acute soft tissue injury model. The analgesic target was identified through competitive inhibition of TRP channel agonists at the animal level, followed by Fluo-4/Ca2+ imaging on live cells overexpressing TRP proteins. The potential target was verified through in-gel imaging, colocalization using a Lig-derived molecular probe, and a drug affinity response target stability assay. The binding site of Lig was identified through protein spectrometry and further analyzed using molecular docking, site-specific mutation, and multidisciplinary approaches. RESULTS: The administration of Lig effectively ameliorated pain and attenuated oxidative stress and inflammatory responses in rats with soft tissue injuries. Moreover, the analgesic effects of Lig were specifically attributed to TRPA1. Mechanistic studies have revealed that Lig directly activates TRPA1 by interacting with the linker domain in the pre-S1 region of TRPA1. Through metabolic transformation, 6,7-epoxyligustilide (EM-Lig) forms a covalent bond with Cys703 of TRPA1 at high concentrations and prolonged exposure time. This irreversible binding prevents endogenous electrophilic products from entering the cysteine active center of ligand-binding pocket of TRPA1, thereby inhibiting Ca2+ influx through the channel opening and ultimately relieving pain. CONCLUSIONS: Lig selectively modulates the TRPA1 channel in a bimodal manner via non-electrophilic/electrophilic metabolic conversion. The epoxidized metabolic intermediate EM-Lig exerts analgesic effects by irreversibly inhibiting the activation of TRPA1 on sensory neurons. These findings not only highlight the analgesic mechanism of Lig but also offer a novel nucleophilic attack site for the development of TRPA1 antagonists in the pre-S1 region.

Ligustilide covalently binds to Cys129 of HMGCS1 to ameliorate dyslipidemia.

Dyslipidemia is characterized by elevated levels of total cholesterol and triglycerides in serum, and has become the primary human health killer because of the major risk factors for cardiovascular diseases. Although there exist plenty of drugs for dyslipidemia, the number of patients who could benefit from lipid-lowering drugs still remains a concern. Ligustilide (Lig), a natural phthalide derivative, was reported to regulate lipid metabolic disorders. However, its specific targets and underlying molecular mechanism are still unclear. In this study, we found that Lig alleviated high fat diet-induced dyslipidemia by inhibiting cholesterol biosynthesis. Furthermore, a series of chemical biological analysis methods were used to identify its target protein for regulating lipid metabolism. Collectively, 3-hydroxy-3-methylglutaryl coenzyme A synthetase 1 (HMGCS1) of hepatic cells was identified as a target for Lig to regulate lipid metabolism. The mechanistic study confirmed that Lig irreversibly binds to Cys129 of HMGCS1 via its metabolic intermediate 6,7-epoxyligustilide, thereby reducing cholesterol synthesis and improving lipid metabolism disorders. These findings not only systematically elucidated the lipid-lowering mechanism of Lig, but also provided a new structural compound for the treatment of dyslipidemia.

Clinical effect of flunarizine combined with duloxetine in the treatment of chronic migraine comorbidity of depression and anxiety disorder.

BACKGROUND: Migraine is common in primary headaches, and with the development of social economy and the increase in living pressure, the prevalence of migraine has an upward trend. OBJECTIVE: To observe the clinical effect of flunarizine combined with duloxetine in the treatment of chronic migraine with comorbid depression and anxiety disorders and to provide a reference for clinical treatment. METHODS: A total of 118 patients with chronic migraine complicated with depression and anxiety disorder admitted to our hospital from June 2018 to August 2020 were selected and divided into two groups according to treatment methods, 59 cases in each group. The control group was treated with flunarizine combined with loxoprofen sodium, and the observation group was treated with flunarizine combined with duloxetine. The changes of electroneurophysiological indexes, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), high sensitivity-C reactive protein (hs-CRP), Hamilton depression scale (HAMD) score, and Hamilton anxiety scale (HAMA) score before and after treatment in the two groups were recorded, and the total effective rate of clinical treatment in the two groups was counted. RESULTS: After treatment, TNF-α, IL-6, and hs-CRP in the two groups decreased gradually (p < .05). Further comparison between groups showed that TNF-α, IL-6, and hs-CRP in the observation group were lower than those in the control group (p < .05). After treatment, the HAMD score and the HAMA score of the two groups decreased gradually (p < .05). Further comparison between the two groups showed that HAMD score and HAMA score of the observation group were lower than those of the control group (p < .05). CONCLUSION: Flunarizine combined with duloxetine in the treatment of chronic migraine with depression and anxiety disorder can effectively improve neuroelectrophysiological indexes, reduce inflammation, and reduce depression and anxiety.

Effect of Light Treatment on Chemical Composition of Andrographis paniculata Seedlings.

Light quality consists of a spectrum of different bands, which not only affects plant, development, and primary metabolism but also affects the secondary metabolism of plants. It is an important factor affecting the content of active components of medicinal plants. The A. paniculata seedlings planted in the laboratory, as materials, were tested with red light, far red light, blue light, and ultraviolet light separately. The study assays the content of six main chemical components separately by LC-MS, observes the changes in the content, and analyzes the relationship between the light quality and the active ingredient of A. paniculata. Using the ointment yield and pH value, the fingerprint analysis method of A. paniculata standard decoction was established, and we discussed the selection of index components of A. paniculata standard decoction. It was suggested to select andrographolide as the index component. It will provide a theoretical basis for the large area cultivation of A. paniculata and optimize the quality of medicinal materials to ensure the quality of standard decoction.