Anti-rheumatic potential vis-à-vis aconitine and hypaconitine content analysis in different Aconitum spp. from Sikkim Himalayas (India).

Aconitum spp. are important medicinal plants mentioned in Ayurveda as Ativisa or Vatsanabha. The present study aims to evaluate anti-rheumatic potential in seven Aconitum species and correlation with aconitine and hypaconitine content. Anti-rheumatic potential was analyzed through in vitro xanthine oxidase inhibition, anti-inflammatory and ROS scavenging assays; and quantification of aconitine and hypaconitine with RP-HPLC method validated as per ICH guidelines. The findings reveal that A. palmatum possessed the most promising response (IC50 =12.68±0.15 µg/ml) followed by A. ferox (IC50 =12.912±1.87 µg/ml) for xanthin oxidase inhibition. We observed a wide variation in aconitine and hypaconitine content ranging from 0.018 %-1.37 % and 0.0051 %-0.077 % respectively on dry weight basis. Aconitine and hypaconitine showed moderate positive correlation (r=0.68 and 0.59 respectively) with anti-rheumatic potential. The study identifies potential alternative species of Aconitum that can help in sustainable availability of quality raw material.

Sensitive quantitation of ultra-trace toxic aconitines in complex matrices by perfusion nano-electrospray ionization mass spectrometry combined with gas-liquid microextraction.

The accurate analysis of ultra-trace (e.g. <10-4 ng/mL) substances in complex matrices is a burdensome but vital problem in pharmaceutical analysis, with important implications for precise quality control of drugs, discovery of innovative medicines and elucidation of pharmacological mechanisms. Herein, an innovative constant-flow perfusion nano-electrospray ionization (PnESI) technique was developed firstly features significant quantitative advantages in high-sensitivity ambient MS analysis of complex matrix sample. More importantly, double-labeled addition enrichment quantitation strategies of gas-liquid microextraction (GLME) were proposed for the first time, allowing highly selective extraction and enrichment of specific target analytes in a green and ultra-efficient (>1000-fold) manner. Using complex processed Aconitum herbs as example, PnESI-MS directly enabled the qualitative and absolute quantitative analysis of the processed Aconitum extracts and characterized the target toxic diester alkaloids with high sensitivity, high stability, wide linearity range, and strong resistance to matrix interference. Further, GLME device was applied to obtain the highly specific enrichment of the target diester alkaloids more than 1000-fold, and accurate absolute quantitation of trace aconitine, mesaconitine, and hypaconitine in the extracts of Heishunpian, Zhichuanwu and Zhicaowu was accomplished (e.g., 0.098 pg/mL and 0.143 pg/mL), with the quantitation results well below the LODs of aconitines from any analytical instruments available. This study built a systematic strategy for accurate quantitation of ultra-trace substances in complex matrix sample and expected to provide a technological revolution in many fields of pharmaceutical research.

Four New C19 -Diterpenoid Alkaloids from Aconitum nagarum Stapf.

Four new aconitine-type C19 -diterpenoid alkaloids, were isolated from the roots of Aconitum nagarum Stapf which were named as nagarutines A-D (1-4), together with eleven known compounds (5-15). The structures of the compounds were identified by IR, HR-ESI-MS, 1D and 2D NMR spectra. All compounds were tested for the inhibitory effect on LPS induced NO production in RAW 264.7 macrophages, compound 7 showed moderate anti-inflammatory activity effect and Inhibition rate is about 44.50%.

Analysis of the pharmacodynamic difference between Xiaojin Pills taken with Chinese Baijiu and water based on serum pharmacochemistry and pharmacokinetics.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaojin Pills (XJPs), which has the function of dissipating knots and dispersing swelling, removing blood stasis, and relieving pain, is a classic prescription for the treatment of mammary glands hyperplasia. It is also the first choice of Chinese patent medicine for the clinical treatment of mammary glands hyperplasia in contemporary traditional Chinese medicine clinics. Previous studies have shown that the efficacy of XJPs "taken orally after soaked with Chinese Baijiu" in tradition was significantly better than that of taking it orally with water in modern in terms of activating the blood, anti-inflammation, analgesia, anti-mammary gland hyperplasia, anti-breast cancer and its metastasis in vitro and in vivo, especially under low-dose conditions. However, the material basis for the difference in efficacy between XJP&B and XJP&W is still unclear. AIM OF THE STUDY: To analyze the material basis of the significant difference in efficacy between XJP&B and XJP&W from the perspective of serum pharmacochemistry and pharmacokinetics, and clarified the scientific connotation of XJPs "taken orally after soaked with Chinese Baijiu". MATERIALS AND METHODS: Ultra-high performance liquid chromatography-mass spectrometry combined with a multivariate statistical analysis method were used to screen the differential components in the Chinese Baijiu extract and the water extract of XJPs and the corresponding residues, so as to clarify the differential components between XJP&B and XJP&W in vitro. The migrating components in the blood after XJP&B and XJP&W were characterized by serum pharmacochemical methods, in order to clarify the differential components in rats. The pharmacokinetic parameters of the representative components absorbed into the blood were compared between XJP&B and XJP&W by the pharmacokinetics study method, in order to determine the dynamic changes of the representative components in rats. RESULTS: The identification results of different components in vitro showed that there were 34 and 12 different compounds between the Chinese Baijiu extract and water extract of XJPs, and the residues after Chinese Baijiu extraction and water extraction, respectively. The content of different components such as arachidonic acid, ursolic acid, 3-acetyl-11-keto-ß-boswellic acid, 2α-hydroxyursolic acid, and oleanolic acid was higher in the Chinese Baijiu extract, which was more than twice the content in the water extract. The results of the serum pharmacochemistry study indicated that 42 prototype components were identified in the serum of rats after XJP&B and XJP&W, including organic acids, alkaloids, steroids, and terpenoids. And XJP&B increased the absorption of the prototype components of organic acids in XJPs into the blood. The pharmacokinetic study results of representative components demonstrated that the mean plasma concentration-time profile and pharmacokinetic parameters of muscone, aconitine, and 3-acetyl-11-keto-ß-boswellic acid were significantly different between XJP&B and XJP&W. Compared with XJP&W, the Cmax and AUC0-t of muscone and aconitine in XJP&B were higher, and the T1/2 and MRT0-t of 3-acetyl-11-keto-ß-boswellic acid in XJP&B were relatively longer. CONCLUSION: This research proved that "taking XJPs orally after being soaked with Chinese Baijiu" can increase the dissolution and absorption of active ingredients in XJPs, increase the plasma concentration and content of representative ingredients, and prolong its action time, thus enhancing the biological activity of XJPs in vitro and in vivo. To a certain extent, this study revealed the material basis of the significantly better efficacy of XJP&B than XJP&W and clarified the scientific connotation of XJPs "taken orally after soaked with Chinese Baijiu", which can provide a theoretical basis for the optimization of XJPs' clinical administration method.

[Differences in chemical components in processing of dried ginger-steamed, sand-fried, and rice swill water-bleached Aconiti Lateralis Radix Praeparata pieces in "Jianchang" faction based on UPLC-MS/MS].

This study compared the changes in chemical components during the processing of different types of Aconiti Lateralis Radix Praeparata(ALRP) in "Jianchang" faction, i.e., dried ginger-steamed ALRP pieces(Yin-FP), sand-fried ALRP pieces(Yang-FP), and rice swill water-bleached ALRP pieces(DFP), and provided a scientific basis for the mechanism in toxicity reduction and efficacy enhancement from a compositional perspective. Samples were collected during the processing of the three types of ALRP pieces, yielding raw ALRP pieces, water-bleached Yin-FP, ginger juice-moistened Yin-FP, steamed Yin-FP, water-bleached Yang-FP, sand-fried Yang-FP, water-bleached DFP, rice swill water-bleached DFP, and roasted DFP. Aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, benzoylhypaconine, aconine, mesaconine, hypaconine, salsolinol, fuziline, and higenamine in the extracts were determined by UPLC-MS/MS, and then content analysis and cluster heatmap analysis were performed on 11 sets of samples. During the processing of the three types of ALRP pieces, bleaching significantly reduced the content of 12 alkaloids; steaming, stir-frying, and roasting significantly reduced the content of diester-type alkaloids(aconitine, mesaconitine, and hypaconitine) and significantly increased the content of monoester-type alkaloids(benzoylaconine, benzoylmesaconine, and benzoylhypaconine) and aminoalcohol-type alkaloids(aconine, mesaconine, and hypaconine). During the processing of Yin-FP, the diester-type alkaloids continuously decreased, while the monoester-type and aminoalcohol-type alkaloids showed an initial decrease followed by an increase. During the processing of Yin-FP, Yang-FP, and DFP, the diester-type alkaloids continuously decreased, while the monoester-type and aminoalcohol-type alkaloids showed an initial decrease followed by an increase. Steamed Yin-FP showed a higher increase in content than fried Yang-FP and roasted DFP. Comprehensive analysis of content differences in toxic and therapeutic components in three ALRP pieces suggests that the distinctive processing methods in "Jianchang" faction can indeed achieve detoxification and efficacy enhancement on ALRP. This study provides references for understanding the mechanisms of action of the three processing methods.

Differences in chemical components in processing of dried ginger-steamed, sand-fried, and rice swill water-bleached Aconiti Lateralis Radix Praeparata pieces in "Jianchang" faction based on UPLC-MS/MS / 中国中药杂志

This study compared the changes in chemical components during the processing of different types of Aconiti Lateralis Radix Praeparata(ALRP) in "Jianchang" faction, i.e., dried ginger-steamed ALRP pieces(Yin-FP), sand-fried ALRP pieces(Yang-FP), and rice swill water-bleached ALRP pieces(DFP), and provided a scientific basis for the mechanism in toxicity reduction and efficacy enhancement from a compositional perspective. Samples were collected during the processing of the three types of ALRP pieces, yielding raw ALRP pieces, water-bleached Yin-FP, ginger juice-moistened Yin-FP, steamed Yin-FP, water-bleached Yang-FP, sand-fried Yang-FP, water-bleached DFP, rice swill water-bleached DFP, and roasted DFP. Aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, benzoylhypaconine, aconine, mesaconine, hypaconine, salsolinol, fuziline, and higenamine in the extracts were determined by UPLC-MS/MS, and then content analysis and cluster heatmap analysis were performed on 11 sets of samples. During the processing of the three types of ALRP pieces, bleaching significantly reduced the content of 12 alkaloids; steaming, stir-frying, and roasting significantly reduced the content of diester-type alkaloids(aconitine, mesaconitine, and hypaconitine) and significantly increased the content of monoester-type alkaloids(benzoylaconine, benzoylmesaconine, and benzoylhypaconine) and aminoalcohol-type alkaloids(aconine, mesaconine, and hypaconine). During the processing of Yin-FP, the diester-type alkaloids continuously decreased, while the monoester-type and aminoalcohol-type alkaloids showed an initial decrease followed by an increase. During the processing of Yin-FP, Yang-FP, and DFP, the diester-type alkaloids continuously decreased, while the monoester-type and aminoalcohol-type alkaloids showed an initial decrease followed by an increase. Steamed Yin-FP showed a higher increase in content than fried Yang-FP and roasted DFP. Comprehensive analysis of content differences in toxic and therapeutic components in three ALRP pieces suggests that the distinctive processing methods in "Jianchang" faction can indeed achieve detoxification and efficacy enhancement on ALRP. This study provides references for understanding the mechanisms of action of the three processing methods.

A simple and rapid method for quantifying aconitines and their metabolites in whole blood by modified QuEChERS and liquid chromatography/tandem mass spectrometry (LC/MS/MS).

Aconitum contains highly toxic alkaloids such as aconitine, hypaconitine, jesaconitine, and mesaconitine. Since Aconitum ingestion causes fatal intoxication, it is important to analyze aconitines and their metabolites in the blood. In forensic toxicology, postmortem drug redistribution is known as one factor that would hamper accurate evaluation of concentrations. Therefore, it is recommended to collect multiple blood samples from various sites and compare the results to avoid miss identification of causative compounds for intoxication. In this study, we evaluated aconitines and their metabolites in postmortem blood specimens from ten sites by QuEChERS extraction and liquid chromatography-tandem mass spectrometry (LC/MS/MS). The recovery rates and matrix effects of analytes were approximately 74-80% and 94-100%, respectively. The correlation coefficients were over 0.99. The validation studies revealed that accuracies and precisions were around 97-2% (intraday) and 100-4% (interday), respectively. Finally, the concentrations of aconitine and jesaconitine were from 2.72 to 7.20 ng/mL and from 14.9 to 26.3 ng/mL, respectively. The concentrations of mesaconitine were from 0.32 to 0.88 ng/mL in four samples and detected in two. The concentrations were highest in the right atrium and lowest in the femoral vein. Our results suggest that aconitine and jesaconitne are accumulated in right atrium blood after death, and that right atrium specimen is suitable for measuring aconitine compounds in fatal intoxication cases.

Identification and quantification of eight alkaloids in Aconitum heterophyllum using UHPLC-DAD-QTOF-IMS: A valuable tool for quality control.

INTRODUCTION: Aconitum spp. are prime medicinal plants rich in alkaloids and have been used as the main constituents of traditional medicine in India and China. The whole plant can be toxic and creates pathophysiological conditions inside the human body. Therefore, simultaneous quantification of alkaloids within plant parts and herbal medicines associated with this genus is essential for quality control. OBJECTIVE: We aimed to develop and validate methods using ultra-high-performance liquid chromatography-diode array detector-quadrupole time-of-flight ion mobility mass spectrometry (UHPLC-DAD-QTOF-IMS) and to develop an analytical strategy for the identification and quantification of alkaloid compounds (aconitine, hypaconitine, mesaconitine, aconine, benzoylmesaconitine, benzoylaconine, bulleyaconitine A, and deoxyaconitine) from Aconitum heterophyllum. METHODOLOGY: We developed a simultaneous identification and quantification method for eight alkaloids using UHPLC-DAD-QTOF-IMS. The method was validated as per International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines and also in IMS mode. RESULTS: The developed method has good linearity (r2 = 0.997-0.999), LOD (0.63-8.31 µg/mL), LOQ (0.63-2.80 µg/mL), recovery (86.01-104.33%), reproducibility, intra- and inter-day variability (<3.25%), and stability. Significant qualitative and quantitative variations were found among different plant parts (flower, leaf, stem, root, and tuber) and five market products of A. heterophyllum. Furthermore, a total of 21 metabolites were also profiled based on the fragmentation pattern of MS2 using the validated method. CONCLUSION: An appropriate mobile phase using acetonitrile and water in a gradient elution gave a satisfactory chromatographic separation of eight Aconitum alkaloids with their adjacent peaks. Therefore, this method could provide a scientific and technical platform for quality control assurance.

Pharmacokinetics-based chronoefficacy of Fuzi against chronic kidney disease.

OBJECTIVES: Identifying drugs with time-varying efficacy or toxicity, and understanding the underlying mechanisms would help to improve treatment efficacy and reduce adverse effects. In this study, we uncovered that the therapeutic effect of Fuzi (the lateral root of Aconitum carmichaelii Debeaux) depended on the dosing time in mice with adenine-induced chronic kidney disease (CKD). METHODS: The Fuzi efficacy was determined by biomarker measurements [i.e. plasma creatinine (CRE), blood urea nitrogen (BUN) and urinary N-acetyl-ß-D-glucosaminidase (NAG)], as well as inflammation, fibrosis and histological analyses. Circadian regulation of Fuzi pharmacokinetics and efficacy was evaluated using brain and muscle Arnt-like protein-1 (Bmal1)-deficient (Bmal1-/-) mice. KEY FINDINGS: The Fuzi efficacy was higher when the drug was dosed at ZT10 and was lower when the drug was dosed at other times (ZT2, ZT6, ZT14, ZT18 and ZT22) according to measurements of plasma CRE, BUN and urinary NAG. Consistently, ZT10 (5 PM) dosing showed a stronger protective effect on the kidney (i.e. less extensive tubular injury) as compared to ZT22 (5 AM) dosing. This was supported by lower levels of inflammatory and fibrotic factors (IL-1ß, IL-6, Tnf-α, Ccl2, Tgfb1 and Col1a1) at ZT10 than at ZT22. Pharmacokinetic analyses showed that the area under the curve (AUC) values (reflective of systemic exposure) and renal distribution of aconitine, hypaconitine and mesaconitine (three putative active constituents) for Fuzi dosing at ZT10 were significantly higher than those for herb dosing at ZT22, suggesting a role of circadian pharmacokinetics in Fuzi chronoefficacy. Drug efficacy studies confirmed that aconitine, hypaconitine and mesaconitine possessed a kidney-protecting effect. In addition, genetic knockout of Bmal1 in mice abolished the time-dependency of Fuzi pharmacokinetics and efficacy. This reinforced the existence of chronoefficacy for Fuzi and supported the role of circadian pharmacokinetics in Fuzi chronoefficacy. CONCLUSIONS: The efficacy of Fuzi against CKD depends on the dosing time in mice, which is associated with circadian pharmacokinetics of the three main active constituents (i.e. aconitine, hypaconitine and mesaconitine). These findings highlight the relevance of dosing time in the therapeutic outcomes of herbal medicines.

Aconitum Diterpenoid Alkaloid Profiling to Distinguish between the Official Traditional Chinese Medicine (TCM) Fuzi and Adulterant Species Using LC-qToF-MS with Chemometrics.

The lateral roots of Aconitum carmichaelii, known in Chinese as fuzi, are officially recognized as a materia medica in the Chinese Pharmacopoeia and used culinarily to prepare herbal soups. A strategy combining UPLC-qToF-MS analysis of A. carmichaelii and its intraspecies and interspecies chemometrics study was developed to examine the distribution of Aconitum marker metabolites. Four diterpenoid alkaloids were recognized to be important markers in fuzi, and another 15 markers were identified to differentiate A. carmichaelii from adulterant species. The detected fuzi markers, mesaconitine (47) and hypaconitine (51), are known to be the principal toxins in this herb, while fuziline (6) and benzoylmesaconine (25) are associated with its medicinal properties. Additional marker compounds have been detected in other Aconitum species that are useful for identifying adulteration. This study provides a useful resource for detecting traditional Chinese medicine (TCM) adulterants and assisting in the quality control of botanical products in TCM and beyond.

Supramolecular structure, in vivo biological activities and molecular-docking-based potential cardiotoxic exploration of aconine hydrochloride monohydrate as a novel salt form.

Despite the high profile of aconine in WuTou injection, there has been no preparative technology or structural studies of its salt as the pharmaceutical product. The lack of any halide salt forms is surprising as aconine contains a tertiary nitrogen atom. In this work, aconine was prepared from the degradation of aconitine in Aconiti kusnezoffii radix (CaoWu). A green chemistry technique was applied to enrich the lipophilic-poor aconine. Reaction of aconine with hydrochloride acid resulted in protonation of the nitrogen atom and gave a novel salt form (C25H42NO9+·Cl-·H2O; aconine hydrochloride monohydrate, AHM), whose cation in the crystal structure was elucidated based on extensive spectroscopic and X-ray crystallographic analyses. The AHM crystal had a Z' = 3 structure with three independent cation-anion pairs, with profound conformational differences among the aconine cations. The central framework of each aconine cation was compared with that of previously reported aconitine, proving that protonation of the nitrogen atom induced the structure rearrangement. In the crystal of AHM, aconine cations, chloride anions and water molecules interacted through inter-species O-H...Cl and O-H...O hydrogen bonds; this complex hydrogen-bonding network stabilizes the supramolecular structure. The seriously disordered solvent molecules were treated using the PLATON SQUEEZE procedure [Spek (2015). Acta Cryst. C71, 9-18] and their atoms were therefore omitted from the refinement. Bioactivity studies indicated that AHM promoted in vitro proliferative activities of RAW264.7 cells. Molecular docking suggested AHM could target cardiotoxic protein through the hydrogen-bonding interactions. The structural confirmation of AHM offers a rational approach for improving the pharmaceutical technology of WuTou injection.

["Attenuation-preservation effects" of different processed products of Aconiti Kusnezoffii Radix in industrialized production].

To study the correlation between toxicity and efficacy of different processed Aconiti Kusnezoffii Radix productsin industrial production, in order to define the optimal processing method for "attenuation-preservation effects". The HPLC method was used to determine the content of six aconitine alkaloids in Aconiti Kusnezoffii Radix and its different processed products. The Bliss method was used to determine the half-lethal dose(LD_(50)) or the maximum dose of Aconiti Kusnezoffii Radix and its different processed products in mice. The toluene-induced ear swelling method and the acetic acid twist method were used to investigate the anti-inflammatory and analgesic effects of different processed products. The results showed that: ① the total amount of diester alkaloids incrude Aconiti Kusnezoffii Radix was 0.358 8%; the total amount of diester alkaloids in Aconiti Kusnezoffii Radix prepared by pharmacopoeia-based boiling method was 0.002 2%, and the total amount of monoester alkaloids was 0.036 2%; the total amount of diester alkaloids in Aconiti Kusnezoffii Radix produced by atmospheric steaming method was 0.006 0%, and the total amount of monoester alkaloids was 0.056 7%; ② the LD_(50) of Aconiti Kusnezoffii Radix was 5.4 g·kg~(-1),and the maximum dose of processed products by two methods were 133.34 g·kg~(-1); pathological observation showed that compared with the normal group, the two kinds of processed products of Aconiti Kusnezoffii Radix had certain damage to the heart, liver and kidney; ③products processed by pharmacopoeia-based boiling method and atmospheric steaming method had anti-inflammatory and analgesic effects(P<0.01 or P<0.05). The anti-inflammatory and analgesic effects were as follows: the atmospheric steaming method was superior to the pharmacopoeia-based boiling method. The above results suggest that the crude Aconiti Kusnezoffii Radixis more toxic. The atmospheric steaming method can significantly reduce the toxicity, while retaining its good anti-inflammatory and analgesic effects, which is significantly better than the pharmacopoeia-based boiling method. The atmospheric steaming process is simple and easy to operate, and suitable for industrial production.

Quantitative analysis of aconitine in body fluids in a case of aconitine poisoning.

Aconitine belongs to the Aconitum alkaloids and is a natural toxic substance. Aconitine has been used as a traditional medicine in East Asian culture. Today, aconitine is still in use with or without a prescription, in the Republic of Korea. Here we present a case report of accidental death due to acute aconitine poisoning. An 81-year-old woman ingested liquid that had been heat extracted from the root of the Aconitum plant; she presented to the emergency room 1 h after ingestion. Her electrocardiogram showed irregular ventricular arrhythmias including ventricular tachycardia; she progressed to cardiac arrest. Cardiopulmonary resuscitation and anti-arrhythmic drugs were administered, but the patient did not survive. An autopsy was performed 2 days postmortem. Toxicological analysis was performed, and aconitine was detected by liquid chromatography tandem mass spectrometry. The antemortem blood concentration of aconitine was 39.1 ng/ml and the concentrations of aconitine in the postmortem cardiac blood, peripheral blood, cerebrospinal fluid (CSF), pericardial fluid, and urine were 21.1 ng/ml, 28.6 ng/ml, 6.8 ng/ml, 24.1 ng/ml, and 67.4 ng/ml, respectively. This is the first forensic case report of an aconitine poisoning death in the Republic of Korea with quantitative measurement of aconitine in the antemortem blood and various postmortem body fluids. To the best of our knowledge, this is the first report of the detection of aconitine in the CSF. These data about the distribution of aconitine in the antemortem blood and various postmortem body fluids is helpful for future aconitine poisoning death cases.

Optimization of the traditional processing method for precision detoxification of CaoWu through biomimetic linking kinetics and human toxicokinetics of aconitine as toxic target marker.

ETHNOPHARMACOLOGICAL RELEVANCE: CaoWu (Aconiti Kusnezoffii Radix), well known for its high toxicity leading to fatal ventricular arrhythmias, is detoxified by HeZi (Terminalia Chebula Retz) decoction to prepare ZhiCaoWu (Aconiti Kusnezoffii Radix Preparata) as one part of ingredients of NaRu-3 pill which is used for the treatment of rheumatoid arthritis (RA). Aconitine (AC) is a highly toxic alkaloid of CaoWu and it is used as toxic target marker for the quality control (QC) of ZhiCaoWu. In the traditional processing method, the vanish of astringent or spicy feeling in tongue is the important detoxification indicator of ZhiCaoWu. However, how CaoWu is detoxified to ZhiCaoWu and whether the appropriate content of AC in ZhiCaoWu can be efficiently perceived after the empirical detoxification still lack factual basis. AIM OF THE STUDY: The present study aimed to optimize the traditional processing method for precision detoxification of CaoWu through biomimetic linking kinetics and human toxicokinetics (TK) of AC, with a view of providing insights into the changes of toxic target marker. MATERIALS AND METHODS: CaoWu medicinal slices (Mes) and coarse powder (Cop) were processed by blank HeZi decoction through the soaking method for 7 days. High-performance liquid chromatography (HPLC) was used for the analysis of the samples. The acidity of blank HeZi decoction and HeZi processing decoction was directly determined by pH meter. The non-compartment analysis (NCA) was used to have an intuitive appreciation for AC and pH changes in HeZi processing decoction while the compartment model method was used to build the biomimetic linking kinetics model with the covariate. The inter-species scaling of animal TK parameters was conducted to predict human AC TK profiles. The possible uptake ways of AC (rapid-release or extended-release) for humans were attempted to assess the poisoning risk of AC in NaRu-3 pill. Based on the target content of AC in ZhiCaoWu, the biomimetic linking kinetics model was explored to optimize the traditional processing detoxification method of CaoWu. The assays of determining inflammatory cytokines in lipopolysaccharides (LPS)-induced RAW264.7 cells were performed to investigate the inflammatory modulation effects of AC in vitro. RESULTS: ZhiCaoWu was prepared by eliminating redundant AC in CaoWu through the repeatable replacement of HeZi processing decoction in which its acidity (pH) was affected. AC-pH changes in HeZi processing decoction were adequately depicted by a biomimetic linking kinetics model whose predictive power was determined by comparing the predictions of AC in ZhiCaoWu with the reported data. Rapid-release AC at the converted dose of 111.1 and 417.6 µg (0.011 and 0.042% of AC in NaRu-3 pill) reached maximum blood concentrations of 26.1 and 98.1 ng/mL at 0.3 h, in comparison with minimum human lethal concentration (100 ng/mL). Achieving the target content of AC (0.04%) in ZhiCaoWu or AC (0.011%) in NaRu-3 pill to precisely control the poisoning risk, the potential optimized protocols were that the processing time at 0.2-0.8% of AC in CaoWu was 2.0-4.4 days for Cop and 2.7-6.2 days for Mes. Correspondingly, pH values in HeZi processing decoction were 3.95 and 3.77 for Cop and Mes, respectively. Meanwhile, Lipopolysaccharides (LPS)-induced RAW264.7 cells were exposed to 0, 20, and 200 µM of AC for 12 h and AC at 20 µM enhanced the levels of IL-6, IL-10 and TNF-α. CONCLUSIONS: Thus, for the first time, a biomimetic linking kinetics model was built to optimize the traditional detoxification method. Moreover, pH changes could be developed as surrogate endpoint for guiding the processing detoxification of CaoWu. Notably, setting the content limit of AC (0.011%) was very rational to control the poisoning risk of NaRu-3 pill. In addition, it was possible that there existed the more complex mechanisms of AC for inflammatory modulation in vitro.

Rapid identification of herbal toxins using electrospray laser desorption ionization mass spectrometry for emergency care.

The unintentional ingestion of toxic compounds in herbs is not uncommon in many parts of the world. To provide timely and life-saving care in the emergency department, it is essential to develop a point-of-care analytical method that can rapidly identify these toxins in herbs. Since electrospray laser desorption ionization mass spectrometry (ELDI/MS) has been successfully used to characterize non-volatile chemical compounds without sample preparation, it was used to identify toxic herbal compounds in this study. The herbal toxins were collected either by sweeping a metallic probe across the surface of a freshly cut herb section or by directly sampling extracts of ground herbal powder. The analytes on the probe were then desorbed, ionized and detected using ELDI/MS, wherein analysis of the herbal toxins was completed within 30 s. This approach allows for the rapid morphological recognition of herbs and early point-of-care identification of herbal toxins for emergency management and is promising in providing important toxicological information to ensure appropriate medical treatment.

Evaluation of noninvasive specimens to diagnose livestock exposure to toxic larkspur (Delphinium spp.).

Larkspurs (Delphinium spp.) are native perennial plants that have a serious toxic potential to cattle on foothill and mountain rangelands in the western United States. Livestock death due to larkspur toxicity is attributed to norditerpenoid alkaloids. Diagnosing plant poisonings in livestock is often challenging. The objective of this study was to evaluate the use of three matrices; earwax, oral fluid, and nasal mucus, as noninvasive specimens to determine livestock exposure to larkspurs. Reversed phase high performance liquid chromatography - high resolution mass spectrometry was used to analyze for norditerpene alkaloids, in all three matrices, in cattle administered a single dose of larkspur. Earwax, oral fluid, and nasal mucus were collected over 6 days post-dosing. Methyllycaconitine (MLA) and deltaline concentrations in earwax ranged from 0.4 ±â€¯0.1 to 0.2 ±â€¯0.06 and 0.6 ±â€¯0.5 to 0.11 ±â€¯0.08 ng/mg, respectively. MLA and deltaline concentrations in oral fluid ranged from 0.08 ±â€¯0.03 to 0.01 ±â€¯0.002 ng/mg and 0.07 ±â€¯0.03 ng/mg to not detected (ND), respectively. MLA and deltaline concentrations in nasal mucus ranged from 0.2 ±â€¯0.13 to 0.03 ±â€¯0.01 ng/mg and 0.2 ±â€¯0.12 ng/mg to ND, respectively. The ability to detect differing norditerpene alkaloid chemotypes from two different Delphinium spp. was also possible in the three matrices. This study demonstrates the potential of earwax, oral fluid, and nasal mucus as noninvasive specimens for chemical analyses to aid in the diagnosis of livestock that may have been exposed to and poisoned by larkspur plants.

Research on Q-markers of Qiliqiangxin capsule for chronic heart failure treatment based on pharmacokinetics and pharmacodynamics association.

BACKGROUND: Qiliqiangxin capsule (QLQX), composed of 11 herbs, is an effective traditional Chinese medicine (TCM) that has been widely used for treatment of chronic heart failure (CHF) in China. In the Chinese pharmacopoeia (Ch.P.) only astragaloside was described as the marker component to control the quality of QLQX, which could not reflect the overall effectiveness. PURPOSE: The aim of this work was to investigate the quality markers (Q-markers) of QLQX based on the association of the pharmacodynamics (PD) of inhibitory effect on activated renin-angiotensin-aldosterone system (RAAS) and the pharmacokinetics (PK) of bioactive compounds according to the Q-marker theory. METHODS: The contents of astragaloside, calycosin-7-glucoside, sinapine, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rg1, salvianolic acid A, salvianolic acid B, danshensu, rosmarinic acid, formononetin, aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine and benzoylhypacoitine were determined by an HPLC-MS/MS method both in QLQX preparation and in the plasma of CHF rats administered intragastrically with QLQX. The effect of lowering angiotensin II (Ang II) production by QLQX was assayed by ELISA. The association between PK and PD was explored and the bioactive compounds with higher content in vitro and better exposure in vivo, which were closely related to the inhibitory effect on the activated RAAS, were identified as Q-markers of QLQX for CHF treatment. RESULTS: The contents of 17 constituents were in the order of salvianolic acid B > danshensu > ginsenoside Rb1 > sinapine > benzoylmesaconine > astragaloside > benzoylhypacoitine > ginsenoside Rb2 > salvianolic acid A > ginsenoside Rg1 > calycosin-7-glucoside > rosmarinic acid > formononetin > benzoylaconine > hypaconitine > aconitine > mesaconitine in QLQX preparation. PK and PD association study of 14 bioactive compounds of QLQX showed the maximum effect (Emax) of astragaloside, calycosin-7-glucoside, sinapine and ginsenoside Rg1 and their peak concentration (Cmax) appeared at the same time; while the time of Emax of ginsenoside Rb1, ginsenoside Rb2, salvianolic acid A, salvianolic acid B, danshensu, rosmarinic acid, formononetin, benzoylaconine, benzoylmesaconine and benzoylhypacoitine was delayed from the time of their Cmax. CONCLUSIONS: Astragaloside, calycosin-7-glucoside, sinapine and ginsenoside Rg1 are suitable as the Q-markers of QLQX for CHF treatment, which have higher content in vitro, finer exposure in vivo and a direct correlation with the inhibitory effect on activated RAAS.

[Content determination of four diester diterpenoid alkaloids in leaves of Aconitum kusnezoffii by HPLC].

This present study is to develop an HPLC method for simultaneous determination of four diester diterpenoid alkaloids, beiwutine, mesaconitine, hypaconitine and aconitine in the leaves of Aconitum kusnezoffii, so as to provide evidence of the quality control of this herb. The four constituents were measured on a Waters XBridge CC18 column(4.6 mmχ250 mm, 5 µm). The mobile phase was acetonitrile-40 mmol·L⁻¹ ammonium acetate solution(adjusted pH to 10.5 with ammonia solution)(33:67) with isocratic elution at a flow rate of 1.0 mL·min⁻¹; the detection wavelength was 235 nm; the column temperature was 30 °C, and the injection volume was 10 µL. Next, this contents of the four diester diterpenoid alkaloids in 12 samples were 0.025 5-0.088 5, 0.039 1-0.071 5, 0.026 6-0.081 0 and 0.008 12-0.031 2 mg·g⁻¹, respectively. Next, this method has been successfully applied to the analysis of A. kusnezoffii folium in different harvest periods. The contents of the four alkaloids decreased primarily, and then increased with the postponing of harvest. The established method is proved to be accurate and sensitive for the determination of alkaloids in A. kusnezoffii folium, and may be useful for the quality improvement of this herbal medicine. Moreover, these results indicated the scientific significance for the toxicity and the suitable harvest time of this herb.