[Systematic comparison of two kinds of Bufonis Venenum derived from different Bufo gargarizans subspecies based on metabolomics and antitumor activity].

This paper compared the differences between two kinds of Bufonis Venenum produced by Bufo gargarizans gargarizans and B. gararizans andrewsi, and verified the rationality of the market value orientation of Bufonis Venenum based on the zebrafish mo-del. Twenty batches of Bufonis Venenum from Jiangsu province, Hebei province, Liaoning province, Jilin province, and Liangshan, Sichuan province, including B. gargarizans gargarizans and B. gararizans andrewsi, were collected. The UHPLC-LTQ-Orbitrap-MS combined with principal component analysis was used to compare the differences between two kinds of Bufonis Venenum. According to the limiting conditions of VIP>1, FC<0.5 or FC>2.0, and peak total area ratio>1%, 9 differential markers were determined, which were cinobufagin, cinobufotalin, arenobufagin, resibufogenin, scillaredin A, resibufagin, 3-(N-suberoylargininyl)-arenobufagin, 3-(N-suberoylargininyl)-marinobufagin, and 3-(N-suberoylargininyl)-resibufogenin. The content of 20 batches of Bufonis Venenum was determined according to the Chinese Pharmacopoeia(2020 edition) by high-performance liquid chromatography, and the 2 batches of Bufonis Venenum, CS7(8.99% of total content) and CS9(5.03% of total content), with the largest difference in the total content of the three quality control indexes of the Chinese Pharmacopoeia(bufalin, cinobufagin, and resibufogenin) were selected to evaluate their anti-liver tumor activity based on the zebrafish model. The tumor inhibition rates of the 2 batches were 38.06% and 45.29%, respectively, proving that only using the quality control indexes of the Chinese Pharmacopoeia as the value orientation of Bufonis Venenum market circulation was unreasonable. This research provides data support for the effective utilization of Bufonis Venenum resources and the establishment of a rational quality evaluation system of Bufonis Venenum.

The grading quality markers identification of Panax notoginseng under the guidance of traditional experience using untargeted metabolomics and anti-myocardial ischemia evaluation of zebrafish.

BACKGROUND: Panax notoginseng (PN) was an edible Chinese herbal medicine. PN's current quality control standard cannot precisely match the traditional grading experience. PURPOSE: In this study, under the guidance of the traditional grading experience, the combined metabolomics and biological effect evaluation were used to reveal the distinct chemical quality of PN. METHODS: The quality of PN was evaluated by traditional experience and characterized by the electronic tongue. A zebrafish myocardial ischemia model was developed to verify the grading experience. The untargeted metabolomics method was used to identify and validate the grading markers of PN. RESULTS: The taste was the critical indicator for classifying the quality. Based on the experience sensory scores (ranged from 47.0 to 87.8), PNs could be divided into two grades. The experience scores were significantly associated with umami and richness of the electronic tongue(p<0.01). Besides, superior PN showed substantially stronger anti-myocardial ischemia activity(p<0.001). Thirty-nine differential components were found using UHPLC-LTQ-Orbitrap MS, of which 22 were identified. A new kind of grading quality markers alkynols in PN-associated efficacy was identified, which revealed stronger anti-myocardial ischemia activities than saponin. CONCLUSION: This study evaluated PN through untargeted metabolomics and anti-myocardial ischemia evaluation of zebrafish and proposed the critical role of alkynols in PN's quality classification.

A Zebrafish Heart Failure Model for Assessing Therapeutic Agents.

Heart failure is a leading cause of death and the development of effective and safe therapeutic agents for heart failure has been proven challenging. In this study, taking advantage of larval zebrafish, we developed a zebrafish heart failure model for drug screening and efficacy assessment. Zebrafish at 2 dpf (days postfertilization) were treated with verapamil at a concentration of 200 µM for 30 min, which were determined as optimum conditions for model development. Tested drugs were administered into zebrafish either by direct soaking or circulation microinjection. After treatment, zebrafish were randomly selected and subjected to either visual observation and image acquisition or record videos under a Zebralab Blood Flow System. The therapeutic effects of drugs on zebrafish heart failure were quantified by calculating the efficiency of heart dilatation, venous congestion, cardiac output, and blood flow dynamics. All 8 human heart failure therapeutic drugs (LCZ696, digoxin, irbesartan, metoprolol, qiliqiangxin capsule, enalapril, shenmai injection, and hydrochlorothiazide) showed significant preventive and therapeutic effects on zebrafish heart failure (p < 0.05, p < 0.01, and p < 0.001) in the zebrafish model. The larval zebrafish heart failure model developed and validated in this study could be used for in vivo heart failure studies and for rapid screening and efficacy assessment of preventive and therapeutic drugs.

Effect of spleen-invigorating, Qi-replenishing and blood-arresting formula on zebrafish models with simvastatin-induced hemorrhage caused by spleen failing to control blood, in terms of theory of Traditional Chinese Medicine.

OBJECTIVE: To investigate the hemostasis effect of spleen-invigorating, Qi-replenishing and blood-arresting formula, on a zebrafish models with simvastatin-induced hemorrhage, and with symptom pattern caused by spleen failing to control blood, in terms of theory of Traditional Chinese Medicine (TCM). METHODS: In the first experiment, 60 AB strain wild type zebrafishes were randomly assigned into two groups: normal group and model group. The model group was treated with 50 µM simvastatin for 24 h. The second experiment: The melanin allele mutated Albino strain zebrafishes were divided into normal, model, A group and B group. The observational parameters were as follows: blood flow, velocity of movement, hemorrhage ratio and improvement ratio of hemorrhage. RESULTS: Hemorrhage ratio: in the first experiment, brain hemorrhage ratio was 75%. In the second experiment, heart hemorrhage ratio was 65%. Blood flow: compared with the normal group, there was a significantly decrease in the model group (P < 0.001). Velocity of movement: in the first experimental, compared with the normal group, there was a significantly decrease in the model group (P < 0.001). Improvement ratio of hemorrhage: agents A had little effect in heart hemorrhage of the zebrafish; agents B could reduce heart hemorrhage ratio of the zebrafish, and increase the improvement ratio of hemorrhage. CONCLUSION: The manifestation of zebrafish model with simvastatin-induced hemorrhage is basically similar to that of the clinical symptom pattern caused by spleen's failure to control blood. The Spleen-invigorating, Qi-replenishing and Blood-arresting Formula can reduce the heart hemorrhage ratio of zebrafish induced by simvastatin, and increase the Improvement ratio of hemorrhage.

Bioactive C21 Steroidal Glycosides from the Roots of Cynanchum otophyllum That Suppress the Seizure-like Locomotor Activity of Zebrafish Caused by Pentylenetetrazole.

Six new C21 steroidal glycosides, cynotophyllosides A-F (1-6), together with 16 known compounds, were isolated from the roots of Cynanchum otophyllum. The structures of the new compounds were elucidated by spectroscopic analysis and chemical methods. The three major components, otophylloside F (15), otophylloside B (17), and rostratamine 3-O-ß-D-oleandropyranosyl-(1→4)-ß-D-cymaropyranosyl-(1→4)-ß-D-cymaropyranoside (18), suppressed the seizure-like locomotor activity caused by pentylenetetrazole in zebrafish. Preliminary structure-activity relation studies revealed that a pregnene skeleton with a C-12 ester group (ikemaoyl > cinnamoyl > hydroxy > p-hydroxybenzoyl) and a C-3 sugar chain consisting of three 2,6-dideoxysaccharide units is essential for this suppressive activity.

Rapid analysis of hypolipidemic drugs in a live zebrafish assay.

INTRODUCTION: Hyperlipidemia is the most common form of dyslipidemia, which is the key risk factor for cardiovascular disease and stroke. The development of effective and safe drug treatments for hyperlipidemia has been proven challenging. METHODS: In this study, taking advantage of the transparency of larval zebrafish, we developed a zebrafish hyperlipidemia model for drug screening and efficacy assessment. Zebrafish at 5 d.p.f (days post fertilization) were fed with 0.1% egg yolk for 48 h (hours), followed by drug treatment for 24h or 48 h. Tested drugs were administered into the zebrafish by direct soaking. Drug effect was evaluated based on quantitative analysis of Oil Red O (ORO) in zebrafish vena caudalis. RESULTS: All 5 human hypolipidemic drugs (simvastatin, lovastatin, ezetimibe, bezafibrate and hyodesoxycholic acid) showed significant hypolipidemic effects (p<0.01) in a dose-dependent manner in the zebrafish hyperlipidemia model. 'We also found a well-known Chinese tea Pu-erh tea significantly reduced lipids in this model (p<0.001 and p<0.01). DISCUSSION: Our results demonstrate that the zebrafish hyperlipidemia model developed and validated in this study could be used for in vivo hyperlipidemia studies and drug screening and for assessing hypolipidemic drugs with different mechanisms.