Physicochemical quality evaluation of natural compounds isolated from crude drugs

Aconite root has high toxicity caused by diester alkaloids, thus it was necessary to define the limiting value of diester alkaloids used in medicine formulation. To give the quality of “Processed Aconite Root” and “Powdered Processed Aconite Root” in the Japanese Pharmacopoeia (14th edn, supplement II), we established the official specification and evaluation methods of standard substances. High qualitative grade diester alkaloids, aconitine, hypaconitine, jesaconitine and mesaconitine, which were useful to evaluate the purity of processed aconite root and powdered processed aconite root, were prepared and evaluated for their stability. We studied the physicochemical specification and evaluation methods of these alkaloids. In addition, an “Aconitum diester alkaloids standard solution for purity”, which was used for the purity test, was prepared, and we also studied its physicochemical specification and evaluation methods. In addition, to evaluate the quality of processed aconite root and powdered processed aconite root, a TLC identification test was established. A monoester alkaloid of benzoylmesaconine hydrochloride was used as the reference standard in the latter test, and we also investigated its physicochemical specification and evaluation methods.     

Herbal Medicines Induced Anticholinergic Poisoning in Hong Kong

In the present review, the main objective was to report the incidence and causes of herbal medicines induced anticholinergic poisoning in Hong Kong during 1989–2012 and to emphasize the importance of pharmacovigilance, investigations and preventive measures. Relevant papers, official figures and unpublished data were obtained from Medline search, the Department of Health and the Drug and Poisons Information Bureau. In the New Territories East (where ~20% of the Hong Kong population lived), the incidence of herbal medicines induced anticholinergic poisoning during 1989–1993 was 0.09 per 100,000 population. There were no confirmed cases during 1994–1996. In the whole of Hong Kong, the incidence during 2000–June 2005 was 0.03 per 100,000 population. Contamination of Rhizoma Atractylodis (50%) and erroneous substitution (42%) were the main causes. The incidence during 2008–2012 was 0.06 per 100,000 population. Contamination of non-toxic herbs (50%) and erroneous substitution (41%) were the main causes. In Hong Kong, contamination of non-toxic herbs by tropane alkaloids and substitution of Flos Campsis by toxic Flos Daturae Metelis were the predominant causes of herbal medicines induced anticholinergic poisoning. Systematic studies along the supply chain are necessary to identify the likely sources of contamination. If erroneous substitution of Flos Campsis by Flos Daturae Metelis could be prevented, 40% of herbal medicines induced anticholinergic poisoning would not have occurred. Regular inspection of the retailer, continuing education for the staff in the herbal trade and repeated publicity measures will also be required. Pharmacovigilance of herbal medicines should help determine the incidence and causes of adverse reactions and monitor the effectiveness of preventive measures.     

乌头中毒1例的救治

患者女,57岁,因患类风湿关节炎服用超剂量附子、川乌、草乌等中药方剂治疗,1d后出现全身麻木、心悸、恶心、呕吐等症状,诊断为乌头碱中毒,即予阿托品0.5mg肌内注射,后静脉滴注1mg,经对症处理后症状消失。     

Causes and prevention of herb-induced aconite poisonings in Asia

The recent reports from Hong Kong, Taiwan, China and Korea were reviewed to determine the causes and prospects for prevention of herb-induced aconite poisonings. The contributory factors included overdose (use of greater than the recommended doses), faulty processing (after harvest and during decoction), use of tincture (herbal medicinal wine), use of crude aconite roots (for preparing decoction, proprietary medicines and tincture), lack of standardisation in processing of aconite roots and preparation of tincture and proprietary medicines, unsupervised use of aconite roots and contamination or mix-up with aconite roots. As tincture (herbal medicinal wine) made from aconite roots contains a much larger amount of Aconitum alkaloids, the public should be strongly discouraged from making their own and taking it by mouth. Aconite roots should only be used after post-harvest processing and proper decoction. The public should be educated on the hazards from unsupervised use and improper decoction of processed aconite roots. There should be regular publicity measures to promote awareness among the herbalists and to publicise the risk of serious cardiotoxicity if the recommended doses of processed aconite roots are exceeded. The processing of aconite roots and their proprietary preparations should be standardised. Quality control of processed aconite roots and their proprietary preparations should be strengthened. National reporting or monitoring systems can be used to identify the causes of aconite poisonings and assess the impact of preventive measures.     

附子心脏毒性及配伍减毒增效机制研究进展

附子来源于毛茛科植物乌头Aconitum carmichaeli Debx.,最主要的毒性反应表现为心脏毒性和神经毒性,其中心脏是主要的靶器官之一。目前普遍认为附子所致心脏毒性是由双酯型二萜生物碱直接或间接作用于心脏的结果,其毒性机制与Ca²⁺超载、脂质过氧化反应、细胞凋亡以及迷走神经兴奋性增高有关。研究发现附子与其他传统中草药配伍能通过影响体内代谢过程、抗Ca²⁺超载、抗氧化应激、抗凋亡等机制发挥减毒增效作用。就近年来国内外对附子心脏毒性及配伍减毒增效相关机制的研究进展进行综述,以期为附子的深入研究和临床安全用药提供参考。     

Removal of Toxin (Tetrodotoxin) from Puffer Ovary by Traditional Fermentation

The amounts of puffer toxin (tetrodotoxin, TTX) extracted from the fresh and the traditional Japanese salted and fermented “Nukazuke” and “Kasuzuke” ovaries of Takifugu stictonotus (T. stictonotus) were quantitatively analyzed in the voltage-dependent sodium current (I_Na) recorded from mechanically dissociated single rat hippocampal CA1 neurons. The amount of TTX contained in “Nukazuke” and “Kasuzuke” ovaries decreased to 1/50–1/90 times of that of fresh ovary during a salted and successive fermented period over a few years. The final toxin concentration after fermentation was almost close to the TTX level extracted from T. Rubripes” fresh muscle that is normally eaten. It was concluded that the fermented “Nukazuke” and “Kasuzuke” ovaries of puffer fish T. Stictonotus are safe and harmless as food.     

附子抗心律失常作用机制研究进展

心律失常是一类较常见的心血管系统疾病,种类多、病因复杂,严重可致死。临床药物治疗多以β受体阻滞剂和离子通道阻滞剂为主,但也伴有一定毒副作用。附子是毛茛科植物乌头子根的加工品,味辛、甘,性大热,归心、肾、脾经,有抗心律失常的功能。目前,关于附子及配伍方剂拮抗心律失常的研究日趋增加。本文通过查阅文献,在阐述附子及配伍方剂对心律失常的拮抗作用上,进一步分析两者抗心律失常的机制,为后续附子的安全使用及研究提供参考。     

Rapid investigation of α-glucosidase inhibitory activity of Phaleria macrocarpa extracts using FTIR-ATR based fingerprinting

Phaleria macrocarpa, known as “Mahkota Dewa”, is a widely used medicinal plant in Malaysia. This study focused on the characterization of α-glucosidase inhibitory activity of P. macrocarpa extracts using Fourier transform infrared spectroscopy (FTIR)-based metabolomics. P. macrocarpa and its extracts contain thousands of compounds having synergistic effect. Generally, their variability exists, and there are many active components in meager amounts. Thus, the conventional measurement methods of a single component for the quality control are time consuming, laborious, expensive, and unreliable. It is of great interest to develop a rapid prediction method for herbal quality control to investigate the α-glucosidase inhibitory activity of P. macrocarpa by multicomponent analyses. In this study, a rapid and simple analytical method was developed using FTIR spectroscopy-based fingerprinting. A total of 36 extracts of different ethanol concentrations were prepared and tested on inhibitory potential and fingerprinted using FTIR spectroscopy, coupled with chemometrics of orthogonal partial least square (OPLS) at the 4000–400 cm⁻¹ frequency region and resolution of 4 cm⁻¹. The OPLS model generated the highest regression coefficient with R²Y = 0.98 and Q²Y = 0.70, lowest root mean square error estimation = 17.17, and root mean square error of cross validation = 57.29. A five-component (1+4+0) predictive model was build up to correlate FTIR spectra with activity, and the responsible functional groups, such as −CH, −NH, −COOH, and −OH, were identified for the bioactivity. A successful multivariate model was constructed using FTIR-attenuated total reflection as a simple and rapid technique to predict the inhibitory activity.     

Comparative Phytochemical Investigation of the Sources of Ayurvedic Drug Patha: A Chromatographic Fingerprinting Analysis

Standardization of herbal drugs based on their chemical and biological activity profile is an important prerequisite for acquiring the herbal market. The main problem encountered in standardization of Ayurvedic drugs is proper identification of the source plant. The present study was aimed to establish identification characters, quality control parameters, chemical and biological parameters for roots of three plants Cissampelos pareira, Cyclea peltata and Stephania japonica (Fam. Menispermaceae) which are being used as source of Patha, in the market. All the three plant were subjected for evaluation of quality control parameters as per WHO guidelines and root extracts and total alkaloidal fractions were subjected for HPTLC and HPLC fingerprinting analysis using a marker compound Bebeerine isolated from roots of Cissampelos pareira. The parameters studied clearly indicated the significant differences among the three plant materials. The roots of Cissampelos pareira can be distinguished from other two plants by presence of high concentration of alkaloids especially the presence of high concentration of pharmacologically active alkaloid bebeerine, which was found to be present in very low concentration in Stephania japonica and absent in roots of Cyclea peltata. The roots of Cyclea peltata were found to contain high concentration of saponins and comparatively in low concentration in Cissampelos pareira where as it was found to be absent in roots of Stephania japonica.     

乌头中主要生物碱的高效液相色谱测定

用高效液相色谱分离、测定乌头及附子中一些主要生物碱。生药的乙醚提取物在十八烷基键合相柱上,用甲醇—水—氯仿—三乙胺(70:30:2:0.1)作流动相,中乌头碱、乌头碱及次乌头碱与其它杂质能很好分离。以β-甲基萘作内标化合物,用峰高比测定各生物碱含量。曾测定了不同品种、不同产地及不同加工炮制方法的一些样品,其生物碱组成及比例相差较大。     

Phormidium autumnale Growth and Anatoxin-a Production under Iron and Copper Stress

Studies on planktonic cyanobacteria have shown variability in cyanotoxin production, in response to changes in growth phase and environmental factors. Few studies have investigated cyanotoxin regulation in benthic mat-forming species, despite increasing reports on poisoning events caused by ingestion of these organisms. In this study, a method was developed to investigate changes in cyanotoxin quota in liquid cultures of benthic mat-forming cyanobacteria. Iron and copper are important in cellular processes and are well known to affect growth and selected metabolite production in cyanobacteria and algae. The effect of iron (40–4000 μg L⁻¹) and copper (2.5–250 μg L⁻¹) on growth and anatoxin-a quota in Phormidium autumnale was investigated in batch culture. These concentrations were chosen to span those found in freshwater, as well as those previously reported to be toxic to cyanobacteria. Anatoxin-a concentrations varied throughout the growth curve, with a maximum quota of between 0.49 and 0.55 pg cell⁻¹ measured within the first two weeks of growth. Growth rates were significantly affected by copper and iron concentrations (P < 0.0001); however, no statistically significant difference between anatoxin-a quota maxima was observed. When the iron concentrations were 800 and 4000 μg L⁻¹, the P. autumnale cultures did not firmly attach to the substratum. At 250 μg L⁻¹ copper or either 40 or 4000 μg L⁻¹ iron, growth was suppressed.     

Quality Assessment of Psoralea fructus by HPLC Fingerprint Coupled with Multi-components Analysis

Psoralea Fructus, the dried and ripe fruit of Psoralea corylifolia L., have been used as traditional medicine. There is substantial evidence that multiple constituents are responsible for the beneficial effects of this medicine. To effectively control the quality of this herbal medicine, HPLC fingerprint analysis was performed on a SinoChrom ODS-BP column with mobile phase of a gradient prepared from H₂O and CH₃CN, which the conditions used for gradient elution were: 0–10 min, 5–45% CH₃CN; 10–45 min, 45–70% CH₃CN; 45–50 min, 70–100% CH₃CN; 50–60 min, 100–100% CH₃CN, and the flow rate was 1.0 ml/min. It was obtained on the basis of the chromatographic data from 28 batches of samples, which contained 26 common peaks and 13 peaks were identified by the electrospray ionization-mass spectrometry as psoralen, isopsoralen, isobavachin, neobavaisoflavone, bavachin, corylin, broussochalcone B, psoralidin, isobavachalcone, bavachinin, corylifol A, bavachalcone and backuchiol. The contents of these 13 compounds were also simultaneously examined. By using principal component analysis, 28 batches of samples collected from 6 producing locations with different collecting time were evaluated and differentiated. In summary, the data as described in this study offer valuable information for quality control and proper use of Psoralea Fructus.     

Meltlets? of Soy Isoflavones: Process Optimization and the Effect of Extrusion Spheronization Process Parameters on Antioxidant Activity

In the current research work an attempt was made to develop “Melt in mouth pellets” (Meltlets^®) containing 40% herbal extract of soy isoflavones that served to provide antioxidants activity in menopausal women. The process of extrusion–spheronization was optimized for extruder speed, extruder screen size, spheronization speed, and time. While doing so the herbal extract incorporated in the pellet matrix was subjected to various processing conditions such as the effect of the presence of other excipients, mixing or kneading to prepare wet mass, heat generated during the process of extrusion, spheronization, and drying. Thus, the work further investigates the effect of these processing parameters on the antioxidant activity of the soy isoflavone herbal extract incorporated in the formula. Thereby, the antioxidant activity of the soya bean herbal extract, Meltlets^® and of the placebo pellets was evaluated using DPPH free radical scavenging assay and total reduction capacity.     

Use,history, and liquid chromatography/mass spectrometry chemical analysis of Aconitum

Aconitum and its products have been used in Asia for centuries to treat various ailments, including arthritis, gout, cancer, and inflammation. In general, their preparations and dispensing have been restricted to qualified folk medicine healers due to their low safety index and reported toxicity. In the past few decades, official guidelines have been introduced in Asian pharmacopeias to control Aconitum herbal products. However, these guidelines were based on primitive analytical techniques for the determination of the whole Aconitum alkaloids and were unable to distinguish between toxic and nontoxic components. Recent advances in analytical techniques, especially high performance liquid chromatography (HPLC) and electrophoresis coupled with highly sensitive detectors, allowed rapid and accurate determination of Aconitum secondary metabolites. Reports focusing on liquid chromatography/mass spectrometry analysis of Aconitum and its herbal products are discussed in the current review. This review can be used by the health liquid chromatography/mass spectrometry regulatory authorities for updating pharmacopeial guidelines of Aconitum and its herbal products.     

A phase I study on pharmacokinetics and pharmacodynamics of higenamine in healthy Chinese subjects

Aim:

To investigate the pharmacokinetics, pharmacodynamics, and safety of higenamine, an active ingredient of Aconite root, in healthy Chinese volunteers.

Methods:

Ten subjects received continuous, intravenous infusion of higenamine at gradually escalating doses from 0.5 to 4.0 μg·kg⁻¹·min⁻¹, each dose was given for 3 min. Blood and urine samples were collected at designated time points to measure the concentrations of higenamine. Pharmacodynamics was assessed by measuring the subject''s heart rate. A nonlinear mixed-effect modeling approach, using the software Phoenix NLME, was used to model the plasma concentration-time profiles and heart rate.

Results:

Peak concentrations (C_max) of higenamine ranged from 15.1 to 44.0 ng/mL. The half-life of higenamine was 0.133 h (range, 0.107–0.166 h), while the area under concentration-time curve (AUC), extrapolated to infinity, was 5.39 ng·h·mL⁻¹ (range, 3.2-6.8 ng·h·mL⁻¹). The volume of distribution (V) was 48 L (range, 30.8–80.6 L). The total clearance (CL) was 249 L/h (range, 199-336 L/h). Within 8 h, 9.3% (range, 4.6%–12.4%) of higenamine was recovered in the urine. The pharmacokinetics of higenamine was successfully described using a two-compartment model with nonlinear clearance. In the pharmacodynamic model, heart rates were related to the plasma drug concentrations using a simple direct effect model with baseline. The E₀, E_max, and EC₅₀ were 68 bpm, 73 bpm and 8.1 μg/L, respectively.

Conclusion:

Higenamine has desirable pharmacokinetic and pharmacodynamic characteristics. The results provide important information for future clinical studies on higenamine.     

Incidence of herb-induced aconitine poisoning in Hong Kong: impact of publicity measures to promote awareness among the herbalists and the public.

BACKGROUND: In Hong Kong 'chuanwu' (the main root of Aconitum carmichaeli) and 'caowu' (the root of Aconitum kusnezoffii) are used by herbalists to treat patients with various musculoskeletal disorders. These aconite roots contain aconitine, mesaconitine and hypaconitine, which are neurotoxins and cardiotoxins. During 1989 to 1991, 31 patients were treated in public hospitals because of poisoning by aconite roots and there were two deaths from ventricular arrhythmias. In late 1991, healthcare officials together with cardiologists held a press conference to warn the public, healthcare professionals and herbalists of the potential toxicity of aconite roots. The risk of ventricular arrhythmias and the need for urgent medical attention were highlighted. An information leaflet was also sent to hospital doctors. Since 1992, the topic was covered periodically in the local newspapers, medical journals and continuing medical education programmes. OBJECTIVE: The objective of this study was to assess the impact of these publicity measures on the incidence of herb-induced aconitine poisoning in the New Territories East, based on the number of admissions to the Prince of Wales Hospital. METHODS: During 1989 to 1993 and 1996 to 1998, all patients admitted to our medical wards because of herb-induced aconitine poisoning were identified by on-going surveillance of medical patients, searching our computerised medical record system and reviewing reports received by the 24-hour Drug and Poisons Information Bureau. RESULTS: The number of hospitalisations due to aconitine poisoning markedly decreased from four to six per year in 1989 to 1991 to one to two per year in 1992 to 1993. The annual incidence of aconitine poisoning showed a marked decrease from 0.49 to 0.69 [overall 0.60, 95% confidence intervals (CI) 0.34 to 0.99] to 0.10 to 0.22 (overall 0.16, 95% CI 0.03 to 0.46) per 100,000 population (p = 0.024). During 1996 to 1998, herb-induced aconitine poisoning remained uncommon, with zero to two hospital admissions per year or an annual incidence of zero to 0.33 (overall 0.17, 95% CI 0.05 to 0.43) per 100,000 population (p = 0.016). DISCUSSION: It is possible that the herbalists could have used smaller doses of 'chuanwu' and 'caowu' than before. Patients could be more compliant with the instructions on how to prepare the herbal decoction at home. However, our experience suggested that publicity measures to promote awareness, among the herbalists and the public, may reduce the incidence of poisoning due to toxic herbs such as aconite roots.     

Alteration in brain-derived neurotrophic factor (BDNF) after treatment of mice with herbal mixture containing Euphoria longana,Houttuynia cordata and Dioscorea japonica

Background

Literature data indicate that brain-derived neurotrophic factor (BDNF), cyclic-AMP response element-binding protein (CREB) and phospho-CREB (pCREB) may have a place in depression. BDNF belongs to the neurotrophin family that plays an important role in proliferation, survival and differentiation of different cell populations in the mammalian nervous system. The herbal mixture used in the present study consists of Euphoria longana, Houttuynia cordata and Dioscorea japonica. The purpose of the present study was to determine the neuroprotective effect of herbal mixture. We also tested the hypothesis that administration of herbs reverses memory deficits and promotes the protein expression of BDNF in the mouse brain.

Methods

Mice were randomized into four different treatment groups (n = 10/group). Normal and stress groups received regular lab chow without stress and under stress conditions, respectively, for 3 weeks. The animals in the stress group were immobilized for 4 hours a day for 2 weeks. Different doses of herbal mixture (206 and 618 mg/kg) were administered for 3 weeks to those mice under stress conditions. Mice were analyzed by behavioral tests and immunoblotting examination in the hippocampus and cortex. An additional in vitro investigation was performed to examine whether herbs induce neurotoxicity in a human neuroblastoma cell line, SH-SY5Y cells.

Results

No significant toxicity of herbs on human neuroblastoma cells was observed. These herbs demonstrated an inductive effect on the expression of BDNF, pCREB and pAkt. For spatial working memory test, herbal mixture fed mice exhibited an increased level of spontaneous alternation (p < 0.01) compared to those in stress conditions. Moreover, herbal mixture produced highly significant (p < 0.01) reduction in the immobility time in the tail suspension test. Mice in the herbal mixture groups demonstrated lower serum corticosterone concentration than mice in the stress group (p < 0.05). Effects of the oral administration of herbal mixture on protein levels of BDNF in the hippocampi and cortices were significant.

Conclusions

Our study showed that herbal mixture administration has antidepressant effects in mice. It is proposed that adverse events such as stress and depression can modulate the expression of molecular players of cellular plasticity in the brain.     

Open Field Study of Some Zea mays Hybrids,Lipid Compounds and Fumonisins Accumulation

Lipid molecules are increasingly recognized as signals exchanged by organisms interacting in pathogenic and/or symbiotic ways. Some classes of lipids actively determine the fate of the interactions. Host cuticle/cell wall/membrane components such as sphingolipids and oxylipins may contribute to determining the fate of host–pathogen interactions. In the present field study, we considered the relationship between specific sphingolipids and oxylipins of different hybrids of Zea mays and fumonisin by F. verticillioides, sampling ears at different growth stages from early dough to fully ripe. The amount of total and free fumonisin differed significantly between hybrids and increased significantly with maize ripening. Oxylipins and phytoceramides changed significantly within the hybrids and decreased with kernel maturation, starting from physiological maturity. Although the correlation between fumonisin accumulation and plant lipid profile is certain, the data collected so far cannot define a cause-effect relationship but open up new perspectives. Therefore, the question—“Does fumonisin alter plant lipidome or does plant lipidome modulate fumonisin accumulation?”—is still open.     

Quantitative Analysis of Cereulide Toxin from Bacillus cereus in Rice and Pasta Using Synthetic Cereulide Standard and 13C6-Cereulide Standard—A Short Validation Study

A single laboratory validation study of a rapid and sensitive quantitative method for the analysis of cereulide toxin produced by Bacillus cereus using ultra high performance liquid chromatography-electrospray-tandem mass spectrometry is presented. The analysis of this cyclic peptide toxin was validated for pasta and rice samples using a newly presented synthetic cereulide peptide standard, together with ¹³C₆-cereulide that previously have not been commercially available. The use of cereulide standard was also compared to the most frequently used surrogate standard, the antibiotic valinomycin. The performance of the method was evaluated by analyzing spiked sample pools from different types of rice and pasta, as well as 21 individual rice and pasta samples from differently prepared meals. Inoculation of samples with three cereulide toxin-producing strains of Bacillus cereus was finally used to mimic naturally contaminated foods. The quantification range of the method was 1–500 ng/g (R² = 0.999) and the limits of detection and quantification were 0.1 and 1 ng/g, respectively. The precision varied from 3% to 7% relative standard deviation and the trueness from −2% to +6% relative bias at different concentration levels in cooked rice and pasta.