Optimization of the proportions of advantageous components in the hypolipidemic "bioequivalent substance system" of Jiang-Zhi-Ning and its mechanism of action.

CONTEXT: Jiang-Zhi-Ning (JZN), a traditional Chinese medicinal formula, is used to treat hyperlipidemia in clinics. OBJECTIVE: To screen the hypolipidemic "bioequivalent substance system (BSS)" of JZN and elucidate the potential hypolipidemic mechanism. MATERIALS AND METHODS: In vitro, the TG content in HepG2 cells was determined after the intervention of the combination of advantageous components (CAC) by uniform design. In vivo, hyperlipidemia models were established by Triton WR-1339 (400 mg/kg; i.p.) in male ICR mice, and corresponding treatments were administered via oral administration once. The mice were divided into 12 groups (n = 5): control, hyperlipidemic model, simvastatin (positive control, 20 mg/kg), gradient doses of JZN granules (2, 4 and 8 g/kg) and the hypolipidemic effective extraction (HEE) of JZN (120, 240 and 480 mg/kg) and CAC groups (20, 40 and 160 mg/kg). Serum TC, TG, LDL-C and HDL-C were performed after 24 h. Transcriptomics and qRT-PCR technology were used to explore the mechanism of the "BSS" of JZN. RESULTS: In vitro, the ratio of CAC was determined. CAC could reduce the TG content in HepG2 cells (77.21%). Compared with the model group, the high dose of CAC could markedly decrease the levels of TC (61.86%), TG (105.54%) and LDL-C (39.38%) and increase the level of HDL-C (232.67%). CAC was proved to be the "BSS". Transcriptomics and qRT-PCR analysis revealed CAC regulated non-alcoholic fatty liver disease, bile secretion, PPAR and adipocytokine signalling pathway. DISCUSSION AND CONCLUSIONS: These findings provided new feasible ideas and methods for the elucidation of the pharmacodynamic material basis.

Discrimination and Prediction of Lonicerae japonicae Flos and Lonicerae Flos and Their Related Prescriptions by Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy Combined with Multivariate Statistical Analysis.

LJF and LF are commonly used in Chinese patent drugs. In the Chinese Pharmacopoeia, LJF and LF once belonged to the same source. However, since 2005, the two species have been listed separately. Therefore, they are often misused, and medicinal materials are indiscriminately put in their related prescriptions in China. In this work, firstly, we established a model for discriminating LJF and LF using ATR-FTIR combined with multivariate statistical analysis. The spectra data were further preprocessed and combined with spectral filter transformations and normalization methods. These pretreated data were used to establish pattern recognition models with PLS-DA, RF, and SVM. Results demonstrated that the RF model was the optimal model, and the overall classification accuracy for LJF and LF samples reached 98.86%. Then, the established model was applied in the discrimination of their related prescriptions. Interestingly, the results show good accuracy and applicability. The RF model for discriminating the related prescriptions containing LJF or LF had an accuracy of 100%. Our results suggest that this method is a rapid and effective tool for the successful discrimination of LJF and LF and their related prescriptions.

[Calibration on chromone reference extract and application on quality control of Saposhnikoviae Radix].

The calibration of chromone reference extract(CRE) was conducted and a quality control method of Saposhnikoviae Radix(SR) was established based on CRE. Meanwhile, the quality control system of SR was improved and the feasibility of using reference extract as a substitute for single reference substance in quality control of Chinese medicine was discussed. In this study, the content of the prepared CRE was calibrated with prim-O-glucosylcimifugin, cimifugin, 4'-O-ß-D-glucosyl-5-O-methylvisamminol, and secO-glucosylhamaudol as indicators. Subsequently, an HPLC analytical method was developed to determine the content of four chromones in 20 batches of SR samples based on the CRE with known content as the standard substance. T-test was used for the comparison of the determination results of the two methods(single chemical component and CRE as reference substances, respectively), and the P values of prim-O-glucosylcimifugin, cimifugin, 4'-O-ß-D-glucosyl-5-O-methylvisamminol, and sec-O-glucosylhamaudol were 0. 16,0. 39, 0. 14, and 0. 42. The results demonstrated that there was no significant difference between the two methods. This study initially verified the feasibility that the CRE could be used as a substitute for single reference substance in quality control of SR. In conclusion,this study is expected to provide a scientific basis and a new research model for the application of reference extract in the quality control of Chinese medicine.

Identification of Potential Drug Targets for Myopia Through Mendelian Randomization.

Purpose: The purpose of this study was to identify potential drug targets for myopia and explore underlying mechanisms. Methods: Mendelian randomization (MR) was implemented to assess the effect of 2684 pharmacologically targetable genes in the blood and retina on the risk of myopia from a genomewide association study (GWAS) for age-at-onset of spectacle wearing-inferred mean spherical equivalent (MSE; discovery cohort, N = 287,448, European), which was further validated in a GWAS for autorefraction-measured MSE (replication cohort, N = 95,619, European). The reliability of the identified significant potential targets was strengthened by colocalization analysis. Additionally, enrichment analysis, protein-protein interaction network, and molecular docking were performed to explore the functional roles and the druggability of these targets. Results: This systematic drug target identification has unveiled 6 putative genetically causal targets for myopia-CD34, CD55, Wnt3, LCAT, BTN3A1, and TSSK6-each backed by colocalization evidence in adult blood eQTL datasets. Functional analysis found that dopaminergic neuron differentiation, cell adhesion, Wnt signaling pathway, and plasma lipoprotein-associated pathways may be involved in myopia pathogenesis. Finally, drug prediction and molecular docking corroborated the pharmacological value of these targets with LCAT demonstrating the strongest binding affinity. Conclusions: Our study not only opens new avenues for the development of therapeutic interventions for myopia but may also help to understand the underlying mechanisms of myopia.

A triple combination strategy of UHPLC-MSn, hypolipidemic activity and transcriptome sequencing to unveil the hypolipidemic mechanism of Nelumbo nucifera alkaloids.

ETHNOPHARMACOLOGICAL RELEVANCE: Nelumbo nucifera (N. nucifera), a kind of edible Chinese herbal, has been studied in treating hyperlipidemia. However, the hypolipidemic mechanism of N. nucifera remains unknown. Aims of this review: We aimed to screen the effective constituent of N. nucifera alkaloids and elucidated the potential mechanism for treating hyperlipidemia. A triple combination strategy of UHPLC-MSn, hypolipidemic activity and transcriptome sequencing was built to unveil the hypolipidemic mechanism of Nelumbo nucifera alkaloid. MATERIALS AND METHODS: We comprehensively investigated the characterization of N. nucifera alkaloids by using UHPLC-LTQ-Orbitrap MSn. And the hypolipidemic activity of candidate active ingredients were evaluated on sodium oleate-induced HepG2 cell. Finally, O-nornuciferine and N. nucifera alkaloid extraction were analyzed by RNA sequence (RNA-seq) to decipher the underlying hypolipidemic mechanism and were verified by qRT-PCR. RESULTS: 35 compounds were identified from N. nucifera alkaloid extraction by UHPLC-LTQ-Orbitrap MSn. Among them, O-nornuciferine and N. nucifera alkaloid extraction which showed significant hypolipidemic activity were analyzed by transcriptome sequencing. After the intervention of O-nornuciferine and N. nucifera alkaloid extraction, 1 and 158 differentially expressed genes (DEGs) were identified, severally. The enrichment analysis indicated that the hypolipidemic effect was adjusted by the expression of numerous key DEGs involved in bile secretion, glycerolipid and sphingolipid metabolism, PPAR signaling pathway. CONCLUSIONS: O-nornuciferine and N. nucifera alkaloids had exibited significant effects in hyperlipidemia. The candidate genes were LDLR, LPL and ANGPTL4, etc. It was most likely that they adjusted lipid metabolism by modulating expression levels of various key factors which were involved in bile secretion, glycerolipid metabolism, sphingolipid metabolism and PPAR signaling pathway, and so on. This study clarified the hypolipidemic mechanism of the alkaloids in N. nucifera, and laid a foundation for the subsequent development of clinical application and better quality of N. nucifera.

Screening of hypolipidemic active components in Jiang-Zhi-Ning and its preliminary mechanism research based on "active contribution value" study.

ETHNOPHARMACOLOGICAL RELEVANCE: Jiang-Zhi-Ning (JZN) is a traditional Chinese medicine formula, which has the effect of lowering blood lipid level and softening blood vessels. It is clinically used in the treatment of hyperlipidemia with significant curative effect. AIM OF THE STUDY: This study aims to screen the active components of JZN that are responsible for its blood lipids lowering effect and lay the foundation for elucidating pharmacodynamic material basis of the hypolipidemic effect of the formula. MATERIALS AND METHODS: The hyperlipidemia model was used to evaluate the efficacy of the JZN effective extraction with the TC and TG of rat plasma as evaluation index. Then the established ultra-high performance liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry (UPLC-ESI-Q-TOF-MSn) method was utilized to analyze the components of JZN effective extraction and the absorbed components in rat plasma, the potential active components were screened by using the combined analysis results of in vivo and in vitro component identification. Then an established ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QqQ-MSn) method was used to determine the content of potential active components and its natural ratio in JZN effective extraction, and a potential active components combination (PACC) was formed accordingly. Then a HepG2 cell hyperlipidemia model induced by sodium oleate was used to study the hypolipidemic activity of PACC by detecting the content of TG level in the model. Meanwhile, the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to conduct preliminary research on its hypolipidemic mechanism. Then combined with the concept of "combination index" in the "median-effect principle", to calculate the half inhibitory concentration (IC50) values of PACC and each monomer component on inhibiting the TG level in the cell model. Subsequently, the "activity contribution study" was carried out, and the components with the sum of the "activity contribution value" of 85% were finally selected as the hypolipidemic active components of JZN. RESULTS: The pharmacodynamics results showed that JZN effective extraction has displayed a good hypolipidemic effect. 45 components were identified in vitro, 108 components were identified from rat plasma, and 17 potential active components were screened out. The content determination result showed that the ratio of each potential active components in PACC as following: cassiaside C: rubrofusarin-6-O-gentiobioside: aurantio-obtusin-6-O-glucoside: hyperoside: isoquercitrin: quercetin-3-O-glucuronide: (E)-2,3,5,4'-tetrahydroxystilbene-2-O-glucoside: rutin: emodin-8-O-glucoside: astragalin: armepavine: N-nornuciferine: coclaurine: O-nornuciferine: nuciferine: N-norarmepavine: higenamine = 3.30: 16.06: 9.15: 23.94: 98.40: 417.45: 189.68: 8.62: 1.28: 5: 3.51: 14.57: 1.06: 1.35: 1: 5.64: 6.06, and the activity study results showed that it has displayed a good hypolipidemic activity. Finally, the hypolipidemic active components screened out by the "activity contribution study" were: quercetin-3-O-glucuronide, (E)-2,3,5,4'-tetrahydroxystilbene-2-O-glucoside, isoquercitrin, O-nornuciferine, hyperoside and rubrofusarin-6-O-gentiobioside. CONCLUSIONS: A scientific and rational approach of screening the hypolipidemic active ingredients of JZN has been developed in the current study. In addition, the research revealed the blood lipid lowering mechanism of those ingredients, which provide a solid basis for further elucidating the hypolipidemic pharmacodynamic material basis and action mechanism of JZN.

Uses, chemical compositions, pharmacological activities and toxicology of Clematidis Radix et Rhizome- a Review.

ETHNOPHARMACOLOGICAL RELEVANCE: Clematis chinensis Osbeck (C. chinensis), Clematis hexapetala Pall (C. hexapetala) and Clematis terniflora var. mandshurica Rupr (C. mandshurica) are collectively referred to as Clematidis Radix et Rhizome (CRR) in China. CRR is widely distributed in China, which is used as a traditional Chinese medicine to treat rheumatic arthralgia, limb numbness, tendon constriction and inconvenience in flexion and extension. AIMS OF THIS REVIEW: This review systematically summarized the research progress on uses, chemical components, pharmacological activities and toxicology of CRR, listed the chemical structures of main compounds for clarifying the differences in chemical compositions. Meanwhile, the review will provide a theoretical and practical basis for the further research and development of CRR. MATERIALS AND METHODS: The available information on CRR was collected using published materials and electronic databases, including ancient and modern books, Chinese Pharmacopoeia, Ph.D. and M. Sc. dissertations, CNKI, SciFinder, WanFang data, PubMed, ScienceDirect and Web of Science. The starting and ending years of references is 1965-2020, the search strategy was conducted by key words such as uses, chemical components, pharmacology and toxicology of CRR. RESULTS: Up to now, CRR has been used to treat various diseases/disorders, such as relieving rheumatism pain, treating cervical spondylopathy and scapulohumeral periarthritis, treating hepatic carcinoma and gastrointestinal, etc. In addition, more than 200 compounds have been isolated from the three plant species of Clematidis. Moreover, the crude extracts and isolated compounds of CRR have been reported to have a wide range of pharmacological activities, such as anti-inflammatory, anti-tumor, antimicrobial and antioxidant activities, etc. Toxicity studies have shown that CRR can cause oral burning, swelling, abdominal pain or severe diarrhea, difficulty breathing, dilated pupils, renal tissue structural changes, and severe death. CONCLUSIONS: Researches in recent years mainly focused on C. chinensis and C. mandshurica, while there are a few reports on the pharmacological studies of C. hexapetala. Therefore, it is necessary to conduct further research on C. hexapetala. Meanwhile, it is important to pay attention to pursue research on the similarities and differences between the three plant species of Clematidis to find their respective advantages and make rational use of CRR. In addition, there is no report on the mechanism of toxicity research, which needs more attention.

Screening out the anti-insomnia components from Prunella vulgaris L. based on plasma pharmacochemistry combined with pharmacodynamic experiments and UPLC-MS/MS analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Prunella vulgaris L. (P. vulgaris) is a medicinal plant belonging to the Labiatae family, and its dried spikes is called as Xiakucao in China, which is a common traditional Chinese medicine with the activities of clearing the liver and expelling fire, improving eyesight, dispersing nodules and detumescence. Modern pharmacological studies have proved that P. vulgaris has various pharmacological activities such as immunomodulatory, antiviral, antibacterial and anti-insomnia activities. AIMS OF THIS REVIEW: P. vulgaris have been reported to have anti-insomnia effects. Nevertheless, the pharmacodynamic substance basis of this anti-insomnia effect is still unclear. The aim of this study was to identify the active components responsible for evoking the anti-insomnia effect of P. vulgaris and to evaluate its anti-insomnia effect. MATERIALS AND METHODS: In this study, we proposed a method combined with pharmacodynamic experiments, extraction and enrichment of chemical components, and the plasma pharmacochemistry to screen out the anti-insomnia components of P. vulgaris. Firstly, the active eluted fraction of the ethanol extract was screened out based on pharmacodynamic tracing method, and then the chemical composition was analyzed systematically by UPLC-MS/MS. Thirdly, pharmacodynamic tracing method and silica gel column chromatography were employed to screen out the active fraction of 70% ethanol eluted fraction, and its bioactive components in vitro and in vivo were identified by UPLC-MS/MS. Finally, screening out the anti-insomnia components of P. vulgaris by comparing the difference between in vivo and in vitro components, and three potentially bioactive ingredients were validated experimentally. RESULTS: It was confirmed that the fraction eluted with 70% ethanol from macroporous adsorption resin column was responsible for the anti-insomnia efficacy, and 55 compounds were identified or preliminarily identified. Then totally 9 compounds in vitro and 12 compounds in vivo from the active fraction of 70% ethanol eluted fraction were tentatively identified. Among them, mangiferin, rosmarinic acid and salviaflaside were the prototype components of P. vulgaris, which indicated that the three compounds might play the key role in the anti-insomnia activities. In vivo, compared to blank control group, the three compounds significantly shortened the sleeping latency and prolonged the sleeping time produced by pentobarbital sodium. CONCLUSIONS: This study clarified that mangiferin, rosmarinic acid and salviaflaside were considered as the anti-insomnia components of P. vulgaris. This is the first study on screening out the active ingredients responsible for evoking the anti-insomnia effect of P. vulgaris. The three compounds of P. vulgaris may help develop one or more drugs to prevent or treat insomnia. Further investigations are recommended to define the mechanism of the anti-insomnia activity of P. vulgaris.