Structural characterization, in vivo toxicity and biological activity of two new pyro-type diterpenoid alkaloids derived from 3-acetylaconitine.

OBJECTIVE: The transformations that occur in diterpenoid alkaloids during the process of sand frying for Chinese herbal medicine preparation have yet to be clarified. This study investigated the structural changes that take place in 3-acetylaconitine during a simulation of heat-processing and evaluated the toxicity and biological activity of the pyrolysis products. METHODS: The diterpenoid alkaloid 3-acetylaconitine was heated at 180 °C for 15 min to simulate the process of sand frying. The pyrolysis products were separated using column chromatography, and their structures were investigated using high-resolution electrospray ionization mass spectroscopy and nuclear magnetic resonance spectroscopy. Further, in vivo cardiotoxicity and acute toxicity of 3-acetylaconitine and its pyrolysis products were compared, and the aconitine-induced arrhythmia model was employed to evaluate the antiarrhythmic effect of the pyrolysis products. RESULTS: Two new diterpenoid alkaloids, pyroacetylaconitine and 16-epi-pyroacetylaconitine, a pair of epimers at C-16, were isolated. After comparing the structures of these compounds, possible transformation pathways were proposed. Compared with the prototype compound, 3-acetylaconitine, the cardiotoxicity and acute toxicity of the heat-transformed products were significantly decreased. In the biological activity assay, the two pyrolysis products exhibited an effective increase in ventricular premature beat latency, a reduction in the occurrence of ventricular tachycardia, as well as an increase in the rate of arrhythmia inhibition, implying strong antiarrhythmic activity. CONCLUSION: Compared with 3-acetylaconitine, its pyrolysis products displayed lower toxicity and good antiarrhythmic effects; thus, they have potential for being developed into antiarrhythmic medicines. Please cite this article as: Wang YJ, Wang Y, Tao P. Structural characterization, in vivo toxicity and biological activity of two new pyro-type diterpenoid alkaloids derived from 3-acetylaconitine. J Integr Med. 2023; 21(3): 302-314.

Investigating the molecular mechanism of traditional Chinese medicine for the treatment of placental syndromes by influencing inflammatory cytokines using the Mendelian randomization and molecular docking technology.

Introduction: Placental syndromes, which include pregnancy loss, preterm birth, gestational diabetes mellitus (GDM), and hypertensive disorders in pregnancy (HDP), have a strong association with disorder inflammatory reactions. Nonetheless, the exact causal relationship has not been established. This study aims to investigate the causal relationship between placental syndromes and inflammatory cytokines utilizing Mendelian randomization (MR). Additionally, we examined the interaction between small molecular compounds derived from traditional Chinese medicine and inflammatory cytokines using molecular docking method. Methods: After obtaining the data of inflammatory cytokines and placental syndromes, as well as establishing single nucleotide polymorphisms (SNPs), we employed the inverse variance weighted (IVW) method to assess the causal relationship. We also accessed the heterogeneity and the horizontal pleiotropy of these data. The "ClusterProfiler" R package was utilized for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) term analyses. The protein-protein interaction (PPI) network was constructed using STRING database. AutoDock Vina software was used for molecular docking, and Discovery Studio 2019 was used for visualization purposes. Results: We found that the growth regulated oncogene A (GROA) and interleukin-9 (IL-9) were associated with the development of pregnancy hypertension, whereas interleukin-10 (IL-10) and hepatocyte growth factor (HGF) were linked to the occurrence of preeclampsia. Moreover, there were correlations observed between interleukin-18 (IL-18), IL-10, macrophage colony-stimulating factor (MCSF), and platelet-derived growth factor BB (PDGFbb) in cases of chronic hypertension combined with pregnancy (CHP). Additionally, macrophage migration inhibitory factor (MIF) exhibited a connection with GDM, and TNF related apoptosis inducing ligand (TRAIL) demonstrated a causal relationship with preterm birth. It is plausible to suggest that interleukin-1ß (IL-1ß) might contribute to the promotion of pregnancy loss. All of the binding free energy values of small molecular compounds with inflammatory cytokines were below -5.0 kcal/mol. Furthermore, all of the RMSD values were less than 2. Conclusions: GROA, IL-1ß, IL-9, IL-10, IL-18, MIF, MCSF, HGF, PDGFbb and TRAIL were found to be causally associated with placental syndromes. Molecular docking analysis revealed that small molecular compounds, such as puerarin, magnolol, atractylenolide I, paeoniflorin, tumulosic acid and wogonin, are closely bound to these inflammatory cytokines.

Light-Decomposable Polymeric Micelles with Hypoxia-Enhanced Phototherapeutic Efficacy for Combating Metastatic Breast Cancer.

Oxygen dependence and anabatic hypoxia are the major factors responsible for the poor outcome of photodynamic therapy (PDT) against cancer. Combining of PDT and hypoxia-activatable bioreductive therapy has achieved remarkably improved antitumor efficacy compared to single PDT modality. However, controllable release and activation of prodrug and safety profiles of nanocarrier are still challenging in the combined PDT/hypoxia-triggered bioreductive therapy. Herein, we developed a near infrared (NIR) light-decomposable nanomicelle, consisting of PEGylated cypate (pCy) and mPEG-polylactic acid (mPEG2k-PLA2k) for controllable delivery of hypoxia-activated bioreductive prodrug (tirapazamine, TPZ) (designated TPZ@pCy), for combating metastatic breast cancer via hypoxia-enhanced phototherapies. TPZ@pCy was prepared by facile nanoprecipitation method, with good colloidal stability, excellent photodynamic and photothermal potency, favorable light-decomposability and subsequent release and activation of TPZ under irradiation. In vitro experiments demonstrated that TPZ@pCy could be quickly internalized by breast cancer cells, leading to remarkable synergistic tumor cell-killing potential. Additionally, metastatic breast tumor-xenografted mice with systematic administration of TPZ@pCy showed notable tumor accumulation, promoting tumor ablation and lung metastasis inhibition with negligible toxicity upon NIR light illumination. Collectively, our study demonstrates that this versatile light-decomposable polymeric micelle with simultaneous delivery of photosensitizer and bioreductive agent could inhibit tumor growth as well as lung metastasis, representing a promising strategy for potent hypoxia-enhanced phototherapies for combating metastatic breast cancer.

Attenuated Structural Transformation of Indaconitine during Sand Frying Process and Anti-Arrhythmic Effects of Its Transformed Products.

The transformation pathways of diterpenoid alkaloids have been clarified clearly in the boiling and steaming process, but remain to be determined in the sand frying process. The aims of the study were to investigate the transformation pathways of indaconitine in the sand frying process, as well as examine the cardiotoxicity and anti-arrhythmic activity of indaconitine and its transformed products. The transformed product was separated by column chromatography, and the structure was identified by 1H NMR, 13C NMR, and HR-ESI-MS. The cardiotoxicity of indaconitine and its transformed products was clarified by observing the electrocardiogram (ECG) changes at the same dose. Furthermore, the anti-arrhythmic activity of the transformed products was investigated using an aconitine-induced rat arrhythmia model. Consequently, Δ15(16)-16-demethoxyindaconitine, a new diterpenoid alkaloid, was isolated from processed indaconitine. Intravenous injection of 0.06 mg/kg indaconitine induced arrhythmias in SD rats, while Δ15(16)-16-demethoxyindaconitine did not exhibit arrhythmias at the same dose. In the anti-arrhythmic assay, mithaconitine, obtained in the previous research, together with Δ15(16)-16-demethoxyindaconitine, could dose-dependently delay the onset time of ventricular premature beat (VPB) and reduce the incidence of ventricular tachycardia (VT), combined with the increasing arrhythmia inhibition rate, exhibiting strong anti-arrhythmic activities. These results indicated that two or more pathways exist in the sand frying process, and the transformed products exhibited lower cardiotoxicity and strong anti-arrhythmic activities, which had the possibility of being developed into anti-arrhythmic drugs.

Attenuated Structural Transformation of Aconitine during Sand Frying Process and Antiarrhythmic Effect of Its Converted Products.

The transformation pathways of diterpenoid alkaloids have been clarified in the boiling and steaming process. Aconitine, a famous diterpenoid alkaloid, is successively transformed into benzoylaconine and aconine during the processes of boiling and steaming, but the transformation pathway remains to be determined in the sand frying process. The present study aims at investigating the transformation pathways of aconitine in the process of sand frying, as well as assessing the cardiotoxicity and antiarrhythmic activity of aconitine and its converted products. The parameters of temperature and time for the structural transformation of aconitine were confirmed by HPLC. The converted products were further separated and identified by column chromatography, NMR, and HR-ESI-MS. Furthermore, by observing the lead II electrocardiogram (ECG) changes in rats under an equivalent dose, the cardiotoxicity of aconitine and its converted products were compared. Ultimately, the antiarrhythmic effect of the converted products was investigated by employing the model of aconitine-induced arrhythmia. Consequently, the structure of aconitine was converted when processed at 120°C-200°C for 1-40 min. Two diterpenoid alkaloids, a pair of epimers, namely, pyroaconitine and 16-epi-pyroaconitine, were further isolated from processed aconitine. 0.03 mg/kg aconitine induced arrhythmias in normal rats, while the converted products did not exhibit arrhythmias under an equal dose. In the antiarrhythmic assay, 16-epi-pyroaconitine could dose-dependently delay the onset time of VPB, reduce the incidence of VT, and increase the arrhythmia inhibition rate, demonstrating comparatively strong antiarrhythmic activity. Conclusively, compared with the prototype compound aconitine, the converted products exhibited lower cardiotoxicity. Further investigations on the cardiotoxicity indicated that pyroaconitine with ß configuration had a stronger cardiotoxicity than 16-epi-pyroaconitine with α configuration. Furthermore, 16-epi-pyroaconitine could antagonize the arrhythmogenic effect caused by the prototype compound aconitine; the antiarrhythmic effect of 16-epi-pyroaconitine was stronger than lidocaine and propafenone, which had the potential to be developed as antiarrhythmic drugs.

[The dose-dependent protective effect of curcumin on hepatocyte of rats with sepsis].

Objective: To observe the protective effect of different doses of curcumin on hepatocytes of rats with sepsis. Methods: 100 healthy male Sprague-Dawley (SD) rats were randomly divided into sham operation group, sepsis group, and low, medium, high dose curcumin intervention groups (L-cur, M-cur, H-cur groups), with 20 rats in each group. The animal model of sepsis was reproduced by cecal ligation and puncture (CLP) method, and in the sham operation group the cecum was just taken out and returned. In the L-cur, M-cur, H-cur groups curcumin was immediately injected after CLP with a dose of 50, 100, 150 mg/kg, respectively, and the rats in sham operation group and sepsis group were given the same amount of normal saline. Five rats in each group were sacrificed at 2, 6, 12, 24 hours after operation, and the hepatic tissues and blood samples were obtained. The pathological changes in hepatic tissues were observed under a microscope, and hepatocytes apoptosis and apoptosis index (AI) of hepatocytes were determined with transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) method, and the levels of serum procalcitonin (PCT), tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were determined with enzyme linked immunosorbent assay (ELISA) method. Results: Microscopic examination showed that the damage degree of hepatic tissues was significantly increased in sepsis group; the number of apoptotic cells and damage degree of hepatic tissues were increased gradually over time. The damage degree of hepatic tissues in curcumin groups was lessened as compared with sepsis group, especially in M-cur group. There were no significant changes in AI and serum PCT, TNF-α, and IL-1ß levels at any of the time points tested in the sham operation group. The AI, serum PCT, TNF-α, and IL-1ß levels in the sepsis group were significantly higher than those in the sham operation group from 2 hours after operation on [AI: (23.59±2.00)% vs. (2.02±0.13)%, PCT (µg/L): 2.41±0.21 vs. 0.81±0.01, TNF-α (ng/L): 217.28±14.24 vs. 80.02±2.26, IL-1ß (ng/L): 61.84±3.21 vs. 25.78±1.29, all P < 0.05], and they showed a gradually increasing tendency. AI reached peak value at 24 hours after operation [(52.05±1.31)%]; PCT, TNF-α and IL-1ß reached the peak values at 12 hours after operation [(8.68±0.58) µg/L, (314.13±14.39) ng/L, (132.24±2.58) ng/L, respectively]. Curcumin intervention significantly reduced the levels of AI, TNF-α, PCT and IL-1ß in hepatocytes of septic rats, especially in M-cur group [AI: (11.56±0.96)% vs. (23.59±2.00)% at 2 hours, (30.35±1.20)% vs. (52.05±1.31)% at 24 hours; PCT (µg/L): 1.13±0.19 vs. 2.41±0.21 at 2 hours, 5.09±0.42 vs. 8.68±0.58 at 12 hours; TNF-α (ng/L): 124.73±7.47 vs. 217.28±14.24 at 2 hours, 168.68±6.95 vs. 314.13±14.39 at 12 hours; IL-1ß (ng/L): 35.05±1.00 vs. 61.84±3.21 at 2 hours, 84.06±3.42 vs. 132.24±2.58 at 12 hours; all P < 0.05]. Conclusions: Curcumin can inhibit the inflammatory reaction of hepatocytes of rats, prevent apoptosis, and protect the hepatocytes of rats with sepsis. The concentration of curcumin with the most significant effect is 100 mg/kg, which is the medium dosage.

Application of Aidi injection (艾迪注射液) in the bronchial artery infused neo-adjuvant chemotherapy for stage III A non-small cell lung cancer before surgical operation / 中国结合医学杂志

<p><b>OBJECTIVE</b>To study the effect of Aidi Injection (艾迪注射液,ADI) applied in the bronchial artery, applied in the bronchial artery infused (BAI) neo-adjuvant chemotherapy for stage III A non-small cell lung cancer (NSCLC) before surgical operation.</p><p><b>METHODS</b>The 60 patients with NSCLC stage III A underwent two courses BAI chemotherapy before tumor incision were assigned to two groups, the treatment and the control groups, using a random number table, 30 in each group. ADI (100 mL) was given to the patients in the treatment group by adding into 500 mL of 5% glucose injection for intravenous dripping once daily, starting from 3 days before each course of chemotherapy, and it lasted for 14 successive days, so a total of 28 days of administration was completed. The therapeutic effectiveness and the adverse reaction that occurred were observed, and the levels of T-lymphocyte subsets, natural killer cell activity, and interleukin-2 in peripheral blood were measured before and after the treatment.</p><p><b>RESULTS</b>The effective rate in the treatment group was higher than that in the control group (70.0% vs. 56.7%, P<0.05). Moreover, as compared with the control group, the adverse reaction that occurred in the treatment group was less and mild, especially in terms of bone marrow suppression and liver function damage (P<0.05). Cellular immune function was suppressed in NSCLC patients, but after treatment, it ameliorated significantly in the treatment group, showing significant difference as compared with that in the control group (P<0.05).</p><p><b>CONCLUSION</b>ADI was an ideal auxiliary drug for the patients in stage III A NSCLC received BAI neo-chemotherapy before surgical operation; it could enhance the effectiveness of chemotherapy, ameliorate the adverse reaction and elevate patients' cellular immune function; therefore, it is worthy for spreading in clinical practice.</p>

Effects of electroacupuncture on the expression of nestin in endogenous neural stem cell in hippocampus in rats with focal cerebral ischemia-reperfusion / 中国针灸

<p><b>OBJECTIVE</b>To explore the mechanism of electroacupuncture for treating focal cerebral ischemia-reperfusion injury.</p><p><b>METHODS</b>Seventy-five Wistar rats were randomly divided into a control group, a model group and an electroacupuncture group, 25 cases in each group. The model of focal cerebral ischemia-reperfusion was established by inserting nylon thread into the internal carotid artery except the control group which was only separated of the carotid artery without occlusion. Electroacupuncture group was treated with electroacupuncture at "Baihui (GV 20)" and "Dazhui (GV 14)" and the other groups without electroacupuncture treatment. The number of nestin positive cells expression at 1st, 3rd, 7th, 14th and 21st days after focal cerebral ischemia reperfusion was observed by use of immunohistochemistry method.</p><p><b>RESULTS</b>The number of nestin positive cells in electroacupuncture group at ischemia side was significantly more than that in the model group at 3rd, 7th, 14th and 21st days (P < 0.05, P < 0.01), and at contralateral ischemia side, the number of nestin positive cells in the electroacupuncture group was significantly more than that in the model group only at 7th day (P < 0.05).</p><p><b>CONCLUSION</b>Electroacupuncture at "Baihui (GV 20)" and "Dazhui (GV 14)" in rats can increase the number of nestin positive cells in hippocampus after focal cerebral ischemia-reperfusion, which may be one of the important mechanisms of electroacupuncture in treating acute cerebral ischemic diseases.</p>

Methane and carbon dioxide emission from two pig finishing barns.

Agricultural activities are an important source of greenhouse gases. However, comprehensive, long-term, and high-quality measurement data of these gases are lacking. This article presents a field study of CH(4) and CO(2) emission from two 1100-head mechanically ventilated pig (Sus scrofa) finishing barns (B1 and B2) with shallow manure flushing systems and propane space heaters from August 2002 to July 2003 in northern Missouri. Barn 2 was treated with soybean oil sprinkling, misting essential oils, and misting essential oils with water to reduce air pollutant emissions. Only days with CDFB (complete-data-full-barn), defined as >80% of valid data during a day with >80% pigs in the barns, were used. The CH(4) average daily mean (ADM) emission rates were 36.2 +/- 2.0 g/d AU (ADM +/- 95% confidence interval; animal unit = 500 kg live mass) from B1 (CDFB days = 134) and 28.8 +/- 1.8 g/d AU from B2 (CDFB days = 131). The CO(2) ADM emission rates were 17.5 +/- 0.8 kg/d AU from B1 (CDFB days = 146) and 14.2 +/- 0.6 kg/d AU from B2 (CDFB days = 137). The treated barn reduced CH(4) emission by 20% (P < 0.01) and CO(2) emission by 19% (P < 0.01). The CH(4) and CO(2) released from the flushing lagoon effluent were equivalent to 9.8 and 4.1% of the CDFB CH(4) and CO(2) emissions, respectively. The emission data were compared with the literature, and the characteristics of CH(4) and CO(2) concentrations and emissions were discussed.