Saikosaponins (SS) are the main active components of Bupleuri Radix. In this study, the yields of SS a, b1, b2, c, d, e, and f were simultaneously determined using the HPLC-DAD dual wavelength method, and the ultrasound-assisted extraction process of saikosaponins was optimized using the response surface methodology. The antioxidant effect of saikosaponins was investigated using the scavenging rate of 1, 1-diphenyl-2-picrylhydrazyl (DPPH), 2, 2-diazo-bis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS), and hydroxyl (-OH) groups, and the mechanism was clarified via network pharmacological analysis. The results showed that the optimal extraction process of SS was a 5% ammonia–methanol solution as an extraction solvent, a material–liquid ratio of 1:40, a temperature of 46.66 °C, an extraction time of 65.07 min, and an ultrasonic power of 345.56 W. The total content of the seven saikosaponins under this condition was up to 6.32%, which was close to the model’s predicted value of 6.56%, where the yields of the seven saikosaponins a, b1, b2, c, d, e, and f were 1.18%, 0.11%, 0.26%, 1.02%, 3.02%, 0.38%, and 0.44%, respectively. The saikosaponins have an obvious scavenging ability for DPPH, ABTS, and -OH radicals. The interactions of seven saikosaponins with antioxidant targets were studied, and a database was used to collate the core of saikosaponins and antioxidants through network pharmacology. The mechanisms of the antioxidant effects of the saikosaponins were derived via GO enrichment analysis and KEGG pathway analysis. Finally, the binding energy of the saikosaponins to the antioxidant targets was found to be less than −5.0 kcal·mol−1 via molecular docking, indicating that the antioxidant capacity of the saikosaponins are good. Therefore, this study developed a rapid and efficient method for the extraction of saikosaponins, which provides a theoretical basis for an in-depth understanding of the rational utilization of saikosaponins and the development of their medicinal value.

This study focused on extracting coumarins from Angelicae Pubescentis Radix (APR) using deep eutectic solvents (DES). The process parameters were optimized using response surface methodology and characterized by projected scanning electron microscopy analysis. Methods: Eighteen types of DESs based on Choline chloride, Betaine, Sodium acetate, and Ammonium acetate were synthesized. The ultrasonic-assisted effect of these DESs on the yield of coumarins was investigated using high-performance liquid chromatography (HPLC). We studied and optimized the technological conditions for extracting coumarins from APR using a one-factor-at-a-time method and response surface methodology. Additionally, scanning electron microscopy was employed to observe the degree of fragmentation of APR powder before and after extraction with methanol solvent and DESs. The biological activity of DESs extract was evaluated using the free radical scavenging model of 1,1-diphenyl-2picrylhydrazide (DPPH), while the molecular docking approach was used to explore the binding potential between the coumarins and superoxide dismutase (SOD) enzymes. The results indicated that the natural deep eutectic system with a molar ratio of betaine to ethylene glycol of 1:4 exhibited the most effective extraction. The optimum extraction conditions included a water content of 16.11 % in the DESs, a temperature of 43.52 degrees C, an extraction time of 59.61 min, and an extraction yield of 3.37 %. Furthermore, scanning electron microscopy revealed that the cells of APR powder obtained through ultrasonically assisted treatment with DESs exhibited the most severe breakage. The experiment demonstrated that the DESs extract exhibited a high scavenging capacity for DPPH free radicals. Conclusion: DESs present as an environmentally friendly solvent option for effectively extracting coumarins from APR, indicating promising potential for substitution of organic solvents.

A green , efficient extraction method for Radix Bupleuri polysaccharide (RBP) by natural deep eutectic solvents (NADES) combined with the ultrasonic-assisted enzymolytic extraction (UAEE) was established. Using UAEE, the NADES with different ion combinations were selected to extract RBP. The best technical conditions of NADES combined with UAEE were determined by a single factor experiment and response surface optimization experiment. The results showed that the best extraction process of RBP with NADES was as follows: the molar ratio of urea to choline chlo-ride was 1:3, the water content was 20%, the ratio of material to liquid was 1:30, the ultrasonic power was 400 W, the temperature was 57.34 degrees C, pH was 4.89 , the amount of enzyme was 2.63%. The extraction rate of RBP was 10.1042%, and the relative error compared with the pre-dicted value was 2.03%. The reuse of NADES not only has little effect on the extraction rate of RBP but also has the highest extraction rate when it is reused for the third time, indicating that NADES has certain recyclability. The in vitro antioxidant activity of polysaccharides extracted from Radix Bupleuri by NADES showed that the extract had a specific ability to DPPH, ABTS, and hydroxyl radicals. Therefore, this study proposed a new, green, and recyclable extraction method for extracting RBP from Radix Bupleuri, which provided a theoretical basis for the in-depth under-standing of the physical and chemical properties of RBP and the development and utilization of its biological activities.

Objectives. Our previous study has used RNA-seq technology to show that apoptotic molecules were involved in the myocardial protection of electroacupuncture pretreatment (EAP) on the ischemia/reperfusion (I/R) animal model. Therefore, this study was designed to investigate how EAP protects myocardium against myocardial I/R injury through antiapoptotic mechanism. Methods. By using rats with myocardial I/R, we ligated the left anterior descending artery (LAD) for 30 minutes followed by 4 hr of reperfusion after EAP at the Neiguan (PC6) acupoint for 12 days; we employed arrhythmia scores, serum myocardial enzymes, and cardiac troponin T (cTnT) to evaluate the cardioprotective effect. Heart tissues were harvested for western blot analyses for the expressions of pro-and antiapoptotic signaling molecules. Results. Our preliminary findings showed that EAP increased the survival of the animals along with declined arrhythmia scores and decreased CK, LDH, CK-Mb, and cTnT levels. Further analyses with the heart tissues detected reduced myocardial fiber damage, decreased number of apoptotic cells and the protein expressions of Cyt c and cleaved caspase 3, and the elevated level of Endo G and AIF after EAP intervention. At the same time, the protein expressions of antiapoptotic molecules, including Xiap, BclxL, and Bcl2, were obviously increased. Conclusions. The present study suggested that EAP protected the myocardium from I/R injury at least partially through the activation of endogenous antiapoptotic signaling.

BACKGROUND:Angina pectoris (Angina) is a medical condition related to myocardial ischemia. Although acupuncture has been widely accepted as a clinical approach for angina, there is no sufficient evidence of its effectiveness against this syndrome, and its mechanisms have not yet been well elucidated. We develop this protocol to confirm the clinical efficacy of electro-acupuncture on stable angina pectoris by needling on acupoint Neiguan (PC6). Furthermore, we employ high-throughput sequencing technology to investigate the gene expression profiling and determine involvement of histone modifications in the regulation of genes after electro-acupuncture treatment.METHODS/DESIGN:A randomized, controlled, double-blinded (assessor and patients) trial will be carried out. Sixty participants will be randomly assigned to two acupuncture treatment groups and one control group in a 1:1:1 ratio. Participants in acupuncture groups will receive 12 sessions of electro-acupuncture treatment across 4 weeks, followed by a 12-week randomization period. The acupuncture groups are divided into Neiguan (PC6) on Pericardium Meridian of Hand-jueyin or a non-acupoint. The primary clinical measure of effect is the frequency of angina attacks between these groups for four weeks after randomization. RNAs are extracted from peripheral neutrophils collected from all participants on day 0, day 30, and week 16, and are processed to RNA-Seq. We then investigate profiles of histone modifications by ChIP-Seq, for H3 Lysine 4 (H3K4me) and acetylation of H3 Lysine 27 (H3K27ac), in the presence or absence of acupuncture treatment.DISCUSSION:This study determines the efficacy and mechanisms of electro-acupuncture on stable angina pectoris. We focus on effectiveness of acupuncture on alleviating symptoms of myocardial ischemia and the gene regulation and the chromatin remodeling marks, including H3K4me1, H3K4me2, and H3K27ac, which could be key factors for regulating gene expressions caused by electro-acupuncture treatment at Neiguan. This is the first genome-wide study of electro-acupuncture treatment in angina patients, and will provide valuable information for future studies in the fields of acupuncture and its underlying mechanisms. Fourteen patients have been recruited since recruitment opened in November of 2012. This study is scheduled to end in November of 2014.TRIALS REGISTRATION:ChiCTR-TRC-12002668.

Protective effects of acupuncture on myocardial ischemia in each stage have been confirmed increasingly, while there was no in-depth research in terms of histone modifications. Histone modification is an important part of epigenetics. It is also the focus of molecular biology and basic medical research. In this paper, the relationship of histone modifications and gene expression of heart was first explained. Then, the recent research progress of modified histones, which was the direct alteration of gene expression induced by histone modifications on the degree and prognosis of myocardial ischemic injury, was reviewed. This investigation introduced epigenetic regulation in the protection mechanism of myocardial ischemia by acupuncture. It provided new ideas and directions for the study of molecular mechanism of acupuncture efficacy.

This study investigated genome-wide gene expressions and the cardioprotective effects of electro-acupuncture pretreatment at the PC6 Neiguan acupoint on myocardial ischemia reperfusion (I/R) injury. Male SD rats were randomly divided into four groups: sham operation (SO), I/R, electro-acupuncture at the PC6 Neiguan acupoint pretreatment (EA) and electro-acupuncture at non-acupoint pretreatment (NA). Compared with the I/R group, the survival rate of the EA group was significantly increased, the arrhythmia score, infarction area, serum concentrations of CK, LDH and CK-Mb and plasma level of cTnT were significantly decreased. RNA-seq results showed that 725 genes were up-regulated and 861 genes were down-regulated under I/R conditions compared to the SO group; both EA and NA reversed some of these gene expression levels (592 in EA and 238 in NA group). KEGG pathway analysis indicated that these genes were involved in multiple pathways, including ECM, MAPK signaling, apoptosis, cytokine and leukocyte pathways. In addition, some pathways were uniquely regulated by EA, but not NA pretreatment, such as oxidative stress, cardiac muscle contraction, gap junction, vascular smooth muscle contraction, hypertrophic, NOD-like receptor, and P53 and B-cell receptor pathways. This study was first to reveal the gene expression signatures of acute myocardial I/R injury and electro-acupuncture pretreatment in rats.

Background Acupuncture exerts cardioprotective effects on several types of cardiac injuries, especially myocardial ischemia (MI), but the mechanisms have not yet been well elucidated. Angiogenesis mediated by VEGF gene expression and its modification through histone acetylation has been considered a target in treating myocardial ischemia. This study aims to exam whether modulation of angiogenesis through H3K9 acetylation regulation at VEGF gene is one possible cardioprotective mechanism of acupuncture. Results We generated rat MI models by ligating the left anterior descending coronary artery and applied electroacupuncture (EA) treatment at the Neiguan (PC6) acupoint. Our results showed that acupuncture reversed the S-T segment change, reduced Q-wave area, decreased CK, CK-MB, LDH levels, mitigated myocardial remodeling, and promoted microvessel formation in the MI heart. RNA-seq analysis showed that VEGF-induced angiogenesis signaling was involved in the modulation of EA. Western blot results verified that the protein expressions of VEGF, Ras, phospho-p44/42 MAPK, phospho-p38 MAPK, phospho-SAPK/JNK and Akt, were all elevated significantly by EA treatment in the MI heart. Furthermore, increased H3K9 acetylation was also observed according with the VEGF. ChIP assay confirmed that EA treatment could notably stimulate the recruitment of H3K9ace at the VEGF promoter. Conclusions Our study demonstrates for the first time that acupuncture can effectively up-regulate VEGF expression through H3K9 acetylation modification directly at the VEGF promoter and hence activate VEGF-induced angiogenesis in rat MI models. We employed high throughput sequencing in this study and, for the first time, generated genome-wide gene expression profiles both in the rat MI model and in acupuncture treatment.