13,13a-seco-protoberberines from the tubers of Corydalis yanhusuo and their anti-inflammatory activity.

Six undescribed protoberberine derivatives including two pairs of enantiomers, named yanhusanines G-L, along with fifteen reported protoberberine alkaloids, were isolated from the tubers of Corydalis yanhusuo. Among them, yanhusanines H-L feature a unique 13,13a-seco skeleton which is rare in nature. Their structural elucidations were achieved by extensive spectroscopic analysis and quantum chemistry calculations. A biogenetic route for yanhusanines H-L was proposed. Bioassay results showed that yanhusanine J exhibited potent inhibitory effect against the nitric oxide (NO) production in lipopolysaccharide (LPS) induced RAW 264.7 cells (IC50 = 2.25 ± 1.32 µM). Western blot analysis demonstrated that yanhusanine J exerted its anti-inflammatory effect via suppressing the nuclear factor kappa B (NF-κB) pathway, together with the decrease of the inflammatory factors TNF-α, IL-6 and IL-1ß. Furthermore, molecular simulation docking indicated that yanhusanine J had strong interaction with the active site of the inducible nitric oxide synthase (iNOS) protein.

Optimization of the extraction and purification of Corydalis yanhusuo W.T. Wang based on the Q-marker uniform design method.

Corydalis yanhusuo W.T. Wang alkaloids are mainly divided into three categories: protoberberine, prototropine and aporphine alkaloids. Therefore, we have taken into account these three alkaloid contents when extracting and purifying crude drugs, which is essential for the quality control of C. yanhusuo and its derivative products. Herein, we investigated the feasibility of the Q-marker uniform design method in the optimization of the extraction and purification of C. yanhusuo. In this study, Q-marker-based comprehensive scoring (CS) and uniform design methods were used to optimize the extraction and purification of C. yanhusuo. The inspective factors included the solvent concentration, pH, liquid-solid ratio, extraction time and frequency. Then 8 Q-markers, the total alkaloid extraction and the extraction rate were considered as the evaluating indicators during the process. The results indicated that the optimal reflux extraction process of C. yanhusuo was as follows: a total amount of 20 times 70% ethanol (pH = 10 of diluted ammonia), heating and refluxing twice, and extracting each time for 60 min. The results of nine-resin screening exhibited that NKA-9 macroporous adsorption resin had the best separation and purification effect on 8 kinds of C. yanhusuo alkaloids with stronger enrichment. During the optimal enrichment process and elution conditions, the water-soluble impurities were washed off with 5 BV distilled water at a volume flow rate of 2 BV/h, and the elution solvent was 70% ethanol with an elution volume flow rate of 1.5 BV/h and an elution dosage of 12 BV. Additionally, the total alkaloids of the obtained product were over 50%, of which eight quality markers were (+)-corydaline 3.55%, tetrahydropalmatine 3.13%, coptisine 2.79%, palmatine hydrochloride 2.24%, dehydrocorydaline 13.11%, (R)-(+)-corypalmine 2.37%, protopine 2.71% and glaucine 14.03%. Our data demonstrated that the optimal extraction and purification process was stable and feasible, which was expected to provide an experimental basis and reference for the industrial production of C. yanhusuo.

Diversity and community structure of endophytic fungi in Corydalis yanhusuo tuber and their correlations with tetrahydropalmatine content / 中草药

Objective: To analyze the diversity and community structure of endophytic fungi in Corydali yanhusuo tuber and their correlations with tetrahydropalmatine content. Methods The endophytic fungi in C. yanhusuo tuber from eight localities (Pan’an of Zhejiang Province, Dongyang of Zhejiang Province, Jinyun of Zhejiang Province, Sanhe of Shanxi Province, Dongjiaying of Shanxi Province, Kaixian of Chongqing, Xuancheng of Anhui Province and Xinyang of Henan Province) were isolated and identified according to the morphology and ITS sequences, so as to analyze the community structure of endophytic fungi; HPLC was used to determine tetrahydropalmatine content, and Excel and SPSS were used to analyze their correlations and establish multiple linear regression equations at different levels of dominant genera and species. Results: A total of 1 742 endophytic fungi were isolated and divided into 19 taxa (14 were identified to species level and five to genera level), belonging to 3 phyla, 5 classes, 10 orders, 14 families and 15 genera; The tetrahydropalmatine content in C. yanhusuo tuber from eight localities was higher than the standard of Chinese Pharmacopoeia (2015 edition); The richness (S) and diversity index (H’) of endophytic fungi in C. yanhusuo tuber and tetrahydropalmatine content in Zhejiang Province were extremely significant or significantly higher than those in the other localities. The diversity index (H’) was significantly positive correlated with the tetrahydropalmatine content; There was the largest positive correlation between the tetrahydropalmatine content and T83 (Trichoderma sp.) (correlation coefficient: 0.793). Conclusion: There are abundant endophytic fungi resources in C. yanhusuo; T83 as a dominant endophytic fungus probably related to the accumulation of tetrahydropalmatine.

Spectrum-effect relationship and mechanism of anti-inflammatory effects of different extracts of Corydalis yanhusuo / 中草药

Objective: To establish a spectrum-effect relationship betweent anti-inflammation effects and extracts of Corydalis yanhusuo, in order to provide ideas and methods for study of material basis of efficacy. Methods UPLC-Q-TOF/MS was used to establishe fingerprints of different extracts of C. yanhusuo, and the flurescent enzyme was used as a marker to perform the anti-inflammation activity test. Finally, the relationships between characteristic peaks and anti-inflammation activity was established by partial least squares regression analysis (PLSR) and gray relational analysis (GRA). The anti-inflammatory component obtained by spectral effect analysis was predicted by molecular docking technology, and its anti-inflammatory mechanism was preliminarily studied. Results:The 95% ethanol extract had significant anti-inflammatory activity. The characteristic peaks of No. 5 and 8-11 were significantly affected in PLSR and GRA. Molecular docking results showed that C. yanhusuo exerted anti-inflammatory effects by acting on PKC, ERK2, IKKβ, JAK1, PI3K-α, PI3K-γ, TNF-α, affecting the transmission of inflammatory signals. Conclusion: The anti-inflammatory effect of C. yanhusuo is the result of the combination of various components. The main anti-inflammatory components are coptisine, berberine, palmatine, dihydrogenine, and dehydrocryptine, which exert anti-inflammatory effects by affecting PI3K, JAK, PKC, ERK, IKKβ, and TNF-α signaling pathways.

Identification of Alkaloids from Corydalis yanhusuo W. T. Wang as Dopamine D1 Receptor Antagonists by Using CRE-Luciferase Reporter Gene Assay.

Corydalis yanhusuo W. T. Wang (C. yanhusuo) has been traditionally used for drug addiction and pain relief in China. In our previous study, we showed that the extract of C. yanhusuo blocks dopamine receptors, demonstrating that its pharmacological activities are mostly due to the antagonistic effects of some of its components at dopamine receptors. As part of our ongoing project on C. yanhusuo, the aim of the present study is to establish a high-throughput and low-cost screening assay system and test the abilities of the isolated alkaloids from C. yanhusuo to inhibit dopamine-induced dopamine D1 receptor activity. By using our established cyclic adenosine monophosphate (cAMP)-response element (CRE)-luciferase reporter gene assay system, we identified eight alkaloids from C. yanhusuo with D1 receptor antagonistic activities. We next validated the activities of these compounds using fluorometric imaging plate reader (FLIPR) assay by measuring the intracellular Ca2+ change. Six out of eight compounds, including tetrahydropalmatine, corydaline, 13-methyldehydrocorydalmine, dehydrocorybubine, dehydrocorydaline, and columbamine, can be confirmed for their inhibitory activities. The dopamine-receptor-antagonistic effects of four compounds, including 13-methyldehydrocorydalmine, dehydrocorydaline, columbamine, and corydaline, are reported for the first time. The present study provides an important pharmacological basis to support the traditional use of C. yanhusuo in China.

An Ultra-High Performance Liquid Chromatography with Tandem Mass Spectrometry Method for Determination of 10 Alkaloids in Beagle Dog Plasma after the Oral Administration of the Corydalis yanhusuo W.T. Wang Extract and Yuanhuzhitong Tablets.

This study has developed a sensitive and simple ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometry method for the simultaneous determination of corydaline, dehydrocorydaline, tetrahydropalmatine, protopine, palmatine, tetrahydroberberine, columbamine, berberine, coptisine and berberrubine in beagle dog plasma after the oral administration of the Corydalis yanhusuo W.T. Wang and Yuanhuzhitong tablets. Chromatographic separation was achieved on an Agilent Eclipse Plus C18 RRHD column (1.8 µm, 50 × 2.1 mm) using a gradient elution program with a mobile phase consisting of acetonitrile and water containing 0.1% formic acid at a flow rate of 0.3 mL/min. A tandem mass spectrometric detection was conducted by multiple reaction monitoring (MRM) mode via an electrospray ionization source in the positive mode. The calibration curves of all analytes showed good linear (r² > 0.9800). The intra-day and inter-day precisions were less than 15% and the accuracies were within ±15%. The extraction recoveries conformed to the acceptable range. And there was no interference of endogenous substances in the sensitive assay method. All analytes were proven to be stable during sample storage and analysis procedures. The pharmacokinetic study indicated that the Yuanhuzhitong tablets could get a better absorption than Corydalis yanhusuo W.T. Wang.

Effect of applying diammonium phosphate on yield and quality of Corydalis Yanhusuo / 中草药

Objective: To study the effect of applying diammonium phosphate on the yield and quality of Corydalis yanhusuo. Methods: By designing different gradient diammonium phosphate amount, the main growth indicators at different fertilizer amount were observed and recorded under the same planting condition. The contents of total ash, alcohol soluble extract, and tetrahydropalmatine were measured and analyzed after harvest according to the requirement of 2015 edition Pharmacopeia. Results: The tubers growth of C. yanhusuo was the most flourishing with the highest drying rate of 33.7% at diammonium phosphate application amount of 375 kg/hm2. Tuber diameter was the largest (2.57 cm), the fresh yield was the highest (5 482.2 kg/hm2), and the yield was increased to 38.2% than that without fertilizer cultivation. The content of total ash (3.0%) was the lowest. The contents of alcohol soluble extract and tetrahydropalmatine were 24.8% and 0.128%, respectively. Conclusion: Applying diammonium phosphate could increase the yield and quality of C. yanhusuo.

Research progress on Corydalis yanhusuo in opioid addiction / 中草药

Corydalis yanhusuo and its active ingredients dl-THP have clear efficacy of analgesia, as well as the effect on vasodilatation, cardiac muscle protection, antibiosis, and anti-inflammation, thus they are used for the treatment of various disease. In recent years, C. yanhusuo is widely used for the treatment of drug addiction. In this paper, the research progress in the chemical constituents and pharmacologic actions of C. yanhusuo are reviewed, which provide the scientific basis for its modernization research and comprehensive utilization.

Discovery of N-methyltetrahydroprotoberberines with κ-opioid receptor agonists-opioid receptor agonist activities from corydalis yanhusuo W. T. Wang by using two-dimensional liquid chromatography.

ETHNOPHARMACOLOGICAL RELEVANCE: The need for an efficacious analgesic without unwanted side effects is urgent. κ-opioid receptor agonists are known to exhibit potent analgesic effects and elicited fewer side effects than other opioid agonists. Thus in this study we chose the κ-opioid receptor as the target to identify the active components from traditional Chinese medicines (TCMs). MATERIALS AND METHODS: The κ-opioid receptor was expressed in human embryonic kidney-293 T cells (HEK293T). Fluorometric Imaging Plate Reader (FLIPR) assay was used for the determination of Ca(2+) response when κ-opioid receptor was activated. A novel 2D separation system employing C18HCE as the first dimension and a strong cation exchange column (SCX) as the second dimension was conducted for the purification of the active principles. RESULTS: With the aid of HPLC-based activity profiling, activities could be linked to two peaks from Corydalis yanhusuo W. T. Wang (C. yanhusuo) extract. Two N-methyltetrahydroprotoberberines with κ-opioid receptor agonist activities were isolated for the first time from C. yanhusuo by using 2D-LC. CONCLUSIONS: Our study suggests that N-methyltetrahydroprotoberberines may serve as a new scaffold for κ-opioid receptor ligands. The strategy that we adopted can be applied to other naturally-occurring active alkaloids acting at different receptors.

Cytotoxicity enhancement in MDA-MB-231 cells by the combination treatment of tetrahydropalmatine and berberine derived from Corydalis yanhusuo W. T. Wang.

AIM: Our previous works have demonstrated that Chinese herb medicine yanhusuo (Corydalis yanhusuo W. T. Wang) has strong anti-cancer proliferation effect in MDA-MB-231 cells. The goal of this study was to find out the synergic cytotoxicity effect of three natural compounds, tetrahydropalmatine (THP), berberine (Ber), and dehydrocorydaline (DHC), isolated from C. yanhusuo W. T. Wang. MATERIALS AND METHODS: The IC50 of THP Ber and DHC in single use, as well as in combination use at fixed ratios and doses was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Isobologram, combination index and modified coefficient of drug interaction (CDI) methods were used for evaluation the combination effects of THF! Ber, and DHC in different ratio and concentration. RESULTS: The results indicated that the combination of THP and Ber shown the strongest anti-cancer cell proliferation effect at the ratio of 2:3 (Ber: THF the average CDI value was 0.5795). DHC and THP have additive cytotoxicity in MDA-MB-231 cells. However, there wasn't any synergistic effect between Ber and DHC, and it even exhibited antagonistic effect when the percentage of DHC was >50%. CONCLUSION: Our findings suggested that the combination of THP and Ber might be beneficial for anti-proliferation of MDA-MB-231 breast cancer cells through a significant synergy effect.

Supercritical CO_2 Fluid Extraction of Tetrahydropalmatine from Corydalis yanhusuo W.T.Wang by Orthogonal Design / 中药新药与临床药理

Objective To find out the optimal supercritical CO2 fluid extr ac tion prosess for Tetrahydropalmatine. Methods The extraction process was optimiz ed by orthogonal design with the yield of Tetrahydropalmatine as markers. Three factors including extraction pressure, temperature and the volume of entrainer were observed. Results The optimal condition of supercritical CO2 fluid extrac tion process was: under the pressure of 15Mpa at 40 ℃ , 1.5 fold of 95 % EtO H with Corydalis yanhusuo W.T.Wang as the entrainer. Conclusion The optimal supe rcritical process is feasible, with the advantage of low temperature and energy consumption, short time, high production and no organic residua.