A comparative study of commercial samples of ephedrae herba

A total of 22 commercial Ephedra herba samples which belonged to EPHEDRA SINICA, E. INTERMEDIA, E. EQUISETINA, and E. DISTACHYA, respectively, were collected from the Taiwan herbal market. The contents of the six ephedrine alkaloids - ephedrine (E), pseudoephedrine (PE), methylephedrine (ME), methyl-pseudoephedrine (MPE), norephedrine (NE), and nor-pseudoephedrine (NPE) - in these samples were determined by capillary electrophoresis and were found to differ greatly among the samples. Generally, E. SINICA was superior (1.594 +/- 0.467%) and E. INTERMEDIA was inferior (1.210 +/- 0.372%). Among the individual constituents, the sum of E and PE contents in each sample was about nine-tenths of the total alkaloids, and the ratios of E/PE, ME/MPE, and NE/NPE in the given species were definite and could be used to aid in identifying the origin of the herbs. The alkaloid contents were found to be higher in the internodes of the herbs and in those items with thin stems, more powdery and less fibrous characters. There were no indications for alkaloids in the roots of the plants.     

Effects of harvesting time and processing on the contents of five alkaloids in the herbaceous stems of Ephedra sinica

In the present study, we studied the changes of the contents of alkaloids in the herbaceous stems of Ephedra sinicaduring different harvest periods as well as before and after processing. The alkaloid contents of 39 batches of ephedra herb, prepared slices ephedra and honey-fried ephedra, 24 batches of ephedra herb with different harvesting periods, which were all collected from cultivation base in Inner Mongolia, and 38 batches of prepared slices ephedra purchased from the market were detected by taking norephedrine (NE), norpseudoephedrine (NPE), ephedrine (E), pseudoephedrine (PE) and methylephedrine (ME) as indicators by using HPLC method. The content of total alkaloid in prepared slices ephedra (1.71%–3.14%) was higher than that in ephedra herb (1.20%–2.53%) and honey-fried ephedra (1.52%–2.99%). Contents of different alkaloids in these three types of samples were significantly different. Prepared slices ephedra and honey-fried ephedra showed significant differences in the contents of NE, NPE and ME (P<0.05), and the contents of E were significantly different between ephedra herb and honey-fried ephedra (P<0.05). The total alkaloid content of ephedra herb was the highest in September (3.10%). Alkaloid contents of prepared slices ephedra collected in the market were uneven and 13%–91% lower than those collected from cultivation base. The results provided a basis for the quality evaluation of ephedra herb and its processed products, and had certain guiding significance for the selection of processed ephedra according to different drug purposes in clinical application. It also provided data support for the harvesting time of ephedra herb.     

Comparison of contents of five ephedrine alkaloids in three official origins of Ephedra Herb in China by high-performance liquid chromatography

In the present paper, a fast and economical HPLC method [on a Phenomenex Polar-RP column with a solution of (phosphoric acid:triethylamine:dibutylamine:water = 0.40:0.1:0.2:499.3) and methanol] is developed, and applied for the determination of norephedrine, norpseudoephedrine, ephedrine (E), pseudoephedrine (PE) and methylephedrine (ME) in 64 samples of three species from main habitats in China. Quantitation data showed that total alkaloid content in Ephedra equisetina Bge. (2.708 ± 0.642%) is higher than that in E. sinica Stapf. (1.365 ± 0.624%) and E. intermedia Schrenk et C. A. Mey. (1.537 ± 0.746%), but the range of total alkaloid content in each species is so wide that the ranges of the three species greatly overlap. The contents of E, PE and ME are different among the three species. The ratio E/total alkaloid content and ratio E/PE as well as E and ME contents can be used for identification of E. sinica, E. intermedia and E. equisetina from one another.     

The risk of higenamine adverse analytical findings following oral administration of plumula nelumbinis capsules

Higenamine is a β2‐agonist that has been included in the Prohibited List of the World Anti‐Doping Agency (WADA) since 2017. Meanwhile, it exists in plumula nelumbinis, a part of the lotus seed, and is commonly used as an ingredient in cuisines, herbal medicines, and nutritional supplements in China and other countries in East Asia. Therefore, an evaluation of the risk of an adverse analytical finding (AAF) of higenamine caused by plumula nelumbinis products is necessary in doping control. In this study, 14 volunteers took plumula nelumbinis capsules orally (0.34 g/caplet, 6 caplets/day, 7 days), and another 11 volunteers ingested higenamine tablets (three 5 mg tablets/day for 7 days). Urine samples were collected over a period of 14 days. All urine samples were subjected to quantitative dilute‐and‐shoot analysis using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The analytical results showed that urinary higenamine concentrations exceeded the WADA reporting limit (10 ng/mL) during the drug period in most sample groups. The maximum higenamine concentration observed in the plumula nelumbinis capsule group was 500 ng/mL. Based on confidence interval theory, appropriate data were used to establish mathematical models. The models reflected that the higenamine concentration in urine can exceed the WADA reporting limit with a high probability after taking plumula nelumbinis capsules. In conclusion, oral administration of plumula nelumbinis capsules showed a high risk of an AAF due to higenamine.     

A rapid and simple procedure for the determination of ephedrine alkaloids in dietary supplements by gas chromatography-mass spectrometry

A simple method for the determination of ephedrine alkaloids: ephedrine (EF), pseudoephedrine (PE), norpseudoephedrine (NPE), norephedrine (NE) and methylpseudoephedrine (MPE) in dietary supplements by gas chromatography–mass spectrometry is described. After the addition of 3,4-methylenedioxypropylamphetamine as internal standard, a liquid–liquid extraction procedure in alkaline conditions with chloroform/isopropanol (9:1, v/v) was applied to the samples prior to analysis. Chromatography was performed on a fused capillary column and analytes, derivatized with pentafluoropropionic anhydride, were determined in the selected-ion-monitoring (SIM) mode. The method was validated in the range 0.3–10 μg/mg for EP, 0.06–2.5 μg/mg for PE and NPE and 0.04–1 μg/mg NE and MPE. Mean recovery ranged between 65.7 and 81.3% for the different analytes in dietary supplements. The quantification limits were 0.3 μg/mg for EP, 0.06 μg/mg for PE, 0.04 μg/mg for NPE, NE and MPE. The method was applied to analysis of various dietary supplements containing Ma-huang (Ephedra Sinica) and Sida Cordifolia plant extracts promoted for aiding weight control and boosting sports performance and energy.     

Effects of 19 herbal extracts on the sensitivity to paclitaxel or 5-fluorouracil in HeLa cells

The popularity of traditional herbal medicine (THM) being used as complementary medicines or alternative medicines is increasing. On the other hand, the development of multidrug resistance (MDR) remains a major hurdle to successful cancer chemotherapy. Some THMs capable of reversing MDR may contribute to the improvement of clinical outcomes in cancer chemotherapy. Herein, 19 kinds of herb were chosen from the ingredients of major THMs, and their effects on the sensitivity to anticancer drugs of tumor cells were investigated using the human cervical carcinoma HeLa cells. Focusing on the major mechanism for MDR, i.e., MDR1/P-glycoprotein, the effects of herbal extracts on its transport function were also examined using a MDR1 substrate Rhodamine123. Glycyrrhizae Radix, Rhei Rhizoma, Scutellariae Radix, Poria, Zizyphi Fructus, Zingiberis Rhizoma (dry), Coptidis Rhizoma, Ephedrae Herba and Asiasari Radix significantly enhanced the sensitivity to a MDR1 substrate paclitaxel, whereas none of the herbal extracts used had any effect on the sensitivity to 5-fluorouracil, which is not a substrate for MDR1. Rhodamine123 uptake was significantly increased by Rhei Rhizoma, Poria or Ephedrae Herba among nine herbal extracts sensitized to paclitaxel. This suggests that the increase in paclitaxel sensitivity by Glycyrrhizae Radix, Rhei Rhizoma, Poria or Ephedrae Herba was caused, in part, by the inhibition of MDR1 function, and the change in paclitaxel sensitivity by the other herbal extracts was not always dependent on this. Collectively, these findings indicate that the combination of anticancer drugs with some herbal extracts contributes to the enhancement of clinical outcomes in cancer chemotherapy.     

Anti‐doping analytes in serum: A comparison of SST and SST‐II Advance blood collection tubes

Serum insulin‐like growth factor‐1 (IGF‐1), procollagen type III N‐terminal peptide (PIIINP), and human growth hormone (hGH) isoforms were analyzed in identical serum samples collected into BD Vacutainer^® SST and BD Vacutainer^® SST‐II Advance serum separator tubes. Comparing the serum collected into each tube, measurement correlation was high (R² > 0.83) and percent bias was minimal (<|3.2%|) for all analytes measured using World Anti‐Doping Agency (WADA)‐approved tests. As such, it is recommended that both SST and SST‐II Advance tubes can be used interchangeably for anti‐doping purposes.     

UHPLC–MS/MS quantitation of porphyroxine in opium and application of porphyroxine‐acetylated products as signature markers for heroin

Porphyroxine, a trace alkaloid in opium, was identified in the early 1800s and isolated/characterized in the 1960s. Recently, two significant porphyroxine‐related byproducts found in the acidic and neutral extracts of illicit heroin were characterized by this laboratory as the N‐acetyl‐O¹⁴‐desmethyl‐epi‐porphyroxine ( B ) and N,O⁸‐diacetyl‐O¹⁴‐desmethyl‐epi‐porphyroxine ( C ). The prevalence of the B and C compounds has been consistent in the following order of abundance for the thousands of authentic heroin samples analyzed: Southwest Asia (SWA) > South America (SA) > Southeast Asia (SEA) > Mexico (MEX). In this research, a rapid and efficient ultra‐high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed to determine the content of porphyroxine and five primary alkaloids (morphine, codeine, thebaine, noscapine, and papaverine) in opium after extraction with methanol/water (50/50). The method was validated in terms of linearity, accuracy, recovery, and precision for porphyroxine. The limit of quantitation (LOQ) for porphyroxine was 2.5 ng/mL. The developed method was successfully applied to a total of 114 authentic opium samples from the major poppy‐growing regions. The amount of porphyroxine was determined at the level of part per thousand (‰) and the relative concentrations to morphine were in the range of 1x10^?4 and 1x10^?2 with an order of SWA > SEA, SA > MEX for its average abundance, which is consistent with the order of the average abundance of its acetylated products ( B , C ) in illicit heroin. This study reveals the significance of porphyroxine and its acylated compounds in classifying heroin and opium samples to major geographical regions of production.     

Budesonide use and misuse in sports: Elimination profiles of budesonide and metabolites after intranasal,high‐dose inhaled and oral administrations

Budesonide (BUD) is a glucocorticoid (GC) widely used in therapeutics. In sports, the World Anti‐doping Agency (WADA) controls the use of GCs, and WADA‐accredited laboratories use a reporting level of 30 ng/mL for 6β‐hydroxy‐budesonide (6βOHBUD) to detect the systemic administration of BUD. In the present work, we examined the urinary excretion profile of 6βOHBUD, BUD, and 16α‐hydroxy‐prednisolone (16αOHPRED) after intranasal (INT), inhaled (INH) (at high doses) and oral administrations in male and female volunteers. BUD was administered to healthy volunteers using INT route (256 μg/day for three days, n = 4 males and 4 females), INH route (800 μg/day for three days, n = 4 males and 4 females, and 1600 μg/day for three days, n = 4 males) or oral route (3 mg, n = 8 females). Urine samples were collected before and after administration at different time periods, and were analyzed by liquid chromatography–tandem mass spectrometry. 6βOHBUD and BUD concentrations were very low after INT treatment (0.0–7.1 and 0.0–8.1 ng/mL, respectively), and higher after INH treatments (0.0–35.4 and 0.0–48.3 ng/mL, respectively). For 16αOHPRED, elevated concentrations were detected after INT and INH treatments (2.6–66.4 and 3.4–426.5 ng/mL, respectively). Concentrations obtained following oral administration were higher than after therapeutic administrations (2.8–80.6, 1.5–36.1, and 10.4–532.2 ng/mL for 6βOHBUD, BUD, and 16αOHPRED, respectively). After all administrations, concentrations were higher in males than in females. Results demonstrated that 6βOHBUD is the best discriminatory marker and a reporting level of 40 ng/mL was found to be the best criterion to distinguish allowed from forbidden administrations of BUD.     

The influence of small doses of ethanol on the urinary testosterone to epitestosterone ratio in men and women

Endogenous steroid use can increase urinary testosterone/epitestosterone (T/E) values. In addition, ethanol in amounts >0.5 g per kg of body weight (g/kg) can also increase T/E values. However, the effect of smaller doses of ethanol on T/E values is unknown. The influence of 0.2 and 0.4 g/kg of ethanol on baseline T/E values in 20 men and 20 women with low and high baseline T/E values was investigated and correlated with ethyl glucuronide (EtG) and ethyl sulfate (EtS) concentrations. T/E values for 7 of the women were excluded from the study because of undetectable T concentrations or for other reasons. One man and 1 woman with a high T/E baseline value had a significant increase in their T/E value after ingestion of 0.2 g/kg of ethanol. One man and 2 women with a high T/E baseline, and 1 woman with a low T/E baseline had significantly increased T/E values after ingestion of 0.4 g/kg of ethanol. There was wide variability in peak EtG concentrations and a lack of correlation between ethanol dose and EtG concentrations. Interestingly, 1 man and 2 women with increased T/E values following ethanol ingestion had EtG concentrations below the World Anti‐Doping Agency (WADA) cut‐off of 5000 ng/mL. These findings demonstrate that small amounts of ethanol can elevate T/E values, with women being more susceptible. In addition, consideration should be given to the lowering of the WADA EtG cut‐off to detect samples with elevated T/E values from ingestion of low doses of ethanol.     

Simultaneous quantification of ten key Kratom alkaloids in Mitragyna speciosa leaf extracts and commercial products by ultra‐performance liquid chromatography−tandem mass spectrometry

Kratom (Mitragyna speciosa) is a psychoactive plant popular in the United States for the self‐treatment of pain and opioid addiction. For standardization and quality control of raw and commercial kratom products, an ultra‐performance liquid chromatography?tandem mass spectrometry (UPLC?MS/MS) method was developed and validated for the quantification of ten key alkaloids, namely: corynantheidine, corynoxine, corynoxine B, 7‐hydroxymitragynine, isocorynantheidine, mitragynine, mitraphylline, paynantheine, speciociliatine, and speciogynine. Chromatographic separation of diastereomers, or alkaloids sharing same ion transitions, was achieved on an Acquity BEH C18 column with a gradient elution using a mobile phase containing acetonitrile and aqueous ammonium acetate buffer (10mM, pH 3.5). The developed method was linear over a concentration range of 1–200 ng/mL for each alkaloid. The total analysis time per sample was 22.5 minutes. The analytical method was validated for accuracy, precision, robustness, and stability. After successful validation, the method was applied for the quantification of kratom alkaloids in alkaloid‐rich fractions, ethanolic extracts, lyophilized teas, and commercial products. Mitragynine (0.7%–38.7% w/w), paynantheine (0.3%–12.8% w/w), speciociliatine (0.4%–12.3% w/w), and speciogynine (0.1%–5.3% w/w) were the major alkaloids in the analyzed kratom products/extracts. Minor kratom alkaloids (corynantheidine, corynoxine, corynoxine B, 7‐hydroxymitragynine, isocorynantheidine) were also quantified (0.01%–2.8% w/w) in the analyzed products; however mitraphylline was below the lower limit of quantification in all analyses.     

Studies on cultivated ephedra plants in inner mongolia autonomous region and ningxia hui autonomous region

Progression of the desertification in northern China has been causing damage to wild Ephedra plants on which we depend for most of supply of the traditional herbal medicine, "Ma huang." The Chinese government encourages the cultivation of Ephedra plants, and Ephedra fields have been reclaimed in the original Ephedra habitats in recent years. We surveyed 7 Ephedra fields that have been recently developed in the Inner Mongolia Autonomous Region and Ningxia Hui Autonomous Region to collect information on Ephedra plant cultivation, especially pertaining to crop species. Specimens taken from those Ephedra fields were genetically and morphologically analyzed, and their ephedrine alkaloid content was examined. DNA analyses of Ephedra specimens, including DNA sequencing of ITS (internal transcribing sequence of nuclear ribosomal DNA) and trn L/F (intron of trnL and intergenic spacer between the trnL and trnF of chloroplast DNA) region and species-specific amplification of trn L/F were conducted to identify Ephedra species. Based on the results of DNA sequencing and morphological determination, the crops grown in 6 fields ware identified as Ephedra sinica, while co-planting of E. sinica and E. intermedia was found in one field where a higher appearance rate of plants with varied morphology from wild Ephedra plants was observed. Furthermore, direct sequencing of the PCR product of the trn L/F region of some specimens from the field and their species-specific PCR showed ambivalent result. Cloning and sequencing of the PCR product of the trn L/F region of those specimens DNA suggested their heteroplasmy, containing both E. sinica- and E. intermedia-type chloroplasts. On the other hand, the profile of the ephedrine alkaloid content was clearly correlated with the result of direct sequencing of the trn L/F region; the specimens showing the E. sinica-type sequence contained more ephedrine than pseudoephedrine, and the specimens of the E. intermedia-type more pseudoephedrine.     

Presence of higenamine in beetroot containing ‘foodstuffs’ and the implication for WADA-relevant anti-doping testing

Higenamine is an alkaloid found within plant species including some that are used in traditional Asian and Chinese herbal medicines. Identified as having mixed mode adrenergic receptor activity, higenamine is present within some nutritional supplements marketed for stimulant and/or weight loss. Its inclusion within nutritional supplements can be via its natural presence within botanical ingredients or as a synthetic additive, often added in mg amounts. The World Anti-doping Agency (WADA) prohibited list has contained higenamine since 2017 as banned at all times in the beta-2 agonist (S3) category, with a reporting level of 10 ng/ml for the free parent form in urine. In this study, an investigation into the content of beetroot or beetroot-containing foodstuffs and supplement products was conducted. Higenamine was confirmed as present within the majority of foodstuffs and supplements, with experimental evidence that higenamine can arise within beetroot extracts through heating. The results in this paper demonstrate the first reported evidence of a link between beetroot and this WADA prohibited substance. To investigate the link between intake and excretion, concentrated beetroot drinks were consumed by six individuals and higenamine quantified in their urine. Free higenamine was detected in the urine of all individuals, with maximum measured concentration in samples of less than 1% of the current WADA reporting limit. Although the risk of an inadvertent doping violation by consumption of the foodstuffs and products investigated in this study is low, beetroot as a source of higenamine should be considered by athletes.     

Genetic diversity of Ephedra plants in mongolia inferred from internal transcribed spacer sequence of nuclear ribosomal DNA

Ephedrae herba has been used for treating colds, relieving coughs and asthma from ancient times. We previously reported the distribution of Ephedra sinica, E. equisetina, E. przewalskii, E. regeliana, E. monosperma and Ephedra sp. in Mongolia, and among them E. sinica and E. equisetina were potential new resources of Ephedrae herba of Japanese pharmacopoeia grade, based on our field survey and subsequent molecular and chemical assessments. However, the Ephedra population in southwestern areas showed a high possibility of having hybrid origins. Further field surveys in southwestern areas, and sequence analysis of the partial nuclear internal transcribed spacer 1 (ITS1) region, besides trnK and 18S ribosomal RNA (rRNA) gene regions, were conducted in order to obtain detailed evidence of hybridization status. As a result, the distribution of E. glauca in western area and E. lomatolepis in western-most area was confirmed. The ITS sequences from all 8 Ephedra species collected in Mongolia were roughly divided into 5 types (types I-V). Type II sequence, having several additive nucleotides, was found in Ephedra sp., E. glauca, E. regeliana and E. sinica, which provided useful information for tracing hybrid origins. Morphological, genetic and distribution evidence suggested that the hybridization of Ephedra species occurred widely in southwestern Mongolia, and several Ephedra species including E. przewalkskii and E. intermedia were involved in these events. Integrated with our previous report, trnK-, 18S- and ITS-types from pure lines of each species are proposed. In addition, we propose a practicable method for detecting additive peaks on a direct sequencing electropherogram.     

Multiple incidence of the prescription diuretic hydrochlorothiazide in compounded nutritional supplements

Diuretic agents are prohibited in sports in‐ and out‐of‐competition according to the regulations of the World Anti‐Doping Agency (WADA) because of their possible masking effects on other doping agents in urine samples, and their ability to produce fast acute weight losses. Despite previous studies reported adverse analytical findings (AAFs) resulting from contaminations at ppm level (μg/g) of medicinal products, and recommended to introduce reporting limits for diuretics in doping controls, these are not adopted in analyses performed by WADA‐accredited laboratories. We report the case of an athlete with two AAFs for hydrochlorothiazide (HCTZ) at low urinary concentrations (<10 ng/mL), who declared the use of nutritional supplements prepared in a compounding pharmacy. His nutritional supplements were analyzed revealing HCTZ presence in different concentrations, at the ppm level (μg/g and ng/mL). With the aim of testing the plausibility of the observed urinary HCTZ concentrations with the nutritional supplement ingestion, a urinary excretion study with three healthy volunteers was performed. HCTZ‐contaminated powder (6.4 μg/g of HCTZ) was administered to each subject in different dosages, reproducing the possible ingestion pattern occurred. Urine specimens were collected before and after ingestion of the powder, up to 24 hours, and underwent liquid–liquid extraction and liquid chromatography–tandem mass spectrometry determination. Post‐administration specimens were found to contain HCTZ at concentrations of 5–230 ng/mL, which supported the accidental inadvertent intake of the prohibited substance by the athlete. This study makes the argument that the introduction of reporting limits for diuretics are warranted in doping control samples, in order to protect against inadvertent AAFs due to contaminated products.     

Alpha‐PVP as an active component of herbal highs in Poland between 2013 and 2015

Alpha‐PVP (alpha‐pyrrolidinovalerophenone, α‐PVP) is a synthetic derivative of cathinone. It has been one of the most frequently detected new psychoactive substances (NPS) available on the drug market in recent years in Poland. The usual routes of administration of the drug include oral, insufflation, and injection. Unexpectedly, we dealt with a great number of herbal samples that turned out to contain α‐PVP. A total number of 352 herbal samples from 19 cases in which we detected synthetic cathinones, were investigated in the Institute of Forensic Research (IFR) from 2013 to 2015. The seized products that were received by our laboratory were first screened by gas chromatography coupled to mass spectrometry (GC–MS). Quantification of α‐PVP and other cathinones was performed by ultra‐performance liquid chromatography with photodiode array detection (UPLC‐PDA). Of the samples, 84% contained only α‐PVP. Other groups of products were those containing only α‐PVT, α‐PVP and α‐PVT, α‐PVP and synthetic cannabinoid A‐834, 735, and α‐PVP and cannabis. In one herbal sample, α‐PVP was detected along with caffeine and tadalafil. The herbal products present on the market containing only α‐PVP usually had a mass of 0.3 to 0.6 g, and concentration range in this group of samples was 3.0–44.0% (content: 13.0–222.0 mg per package). The amount of α‐PVP in samples below 0.30 g was in a range 9–18 mg whiles in samples above 0.60 g it was in the range 30–716 mg. There were also products containing a mixture of α‐PVP and α‐PVT. In those samples, α‐PVP concentrations were: 3.0–6.0% (amount: 15.0–34.0 mg). Copyright © 2016 John Wiley & Sons, Ltd.     

In vitro estrogenicity of ambient particulate matter: contribution of hydroxylated polycyclic aromatic hydrocarbons

Atmospheric particulate matter (PM1) was collected at an urban and a rural site in Switzerland during a hibernal high air pollution episode and was investigated for estrogenicity using an estrogen‐sensitive reporter gene assay (ER‐CALUX). All samples that were tested induced estrogen receptor‐mediated gene expression in T47D human breast adenocarcinoma cells. Observed estrogenic activities corresponded to 17β‐estradiol (E2) CALUX equivalent concentrations ranging from 2 to 23 ng E2‐CEQ per gram of PM1 (particulate matter of ≤ 1 µm aerodynamic diameter) and from 0.07 to 1.25 pg E2‐CEQ per m³ of sampled air. There was a strong correlation between the PM1 estrogenicity of the urban and rural sites (r = 0.92). Five hydroxylated polycyclic aromatic hydrocarbons (hydroxy‐PAHs), which show structural similarities to E2, were assessed for their estrogenic activity. The following order of estrogenic potency was found: 2‐hydroxychrysene > 2‐hydroxyphenanthrene > 1‐hydroxypyrene > 2‐hydroxynaphthalene > 1‐hydroxynaphthalene. Three of these hydroxy‐PAHs, namely 2‐hydroxyphenanthrene, 2‐hydroxynaphthalene and 1‐hydroxynaphthalene, were detected in all PM1 extracts. However, they contributed only 0.01–0.2% to the overall estrogenic activity. Hence, mainly other estrogenic compounds not yet identified by chemical analysis must be responsible for the observed activity. The temporal trend of PM1 estrogenicity at the urban and rural site, respectively, was compared with the time course of several air pollutants (NO₂, NO, SO₂, O₃, CO) and meteorological parameters (temperature, humidity, air pressure, solar irradiation, wind velocity). However, specific emission sources and formation processes of atmospheric xenoestrogens could not be elucidated. This study showed that ambient particulate matter contains compounds that are able to interact with estrogen receptors in vitro and potentially also interfere with estrogen‐regulated pathways in vivo. Copyright © 2008 John Wiley & Sons, Ltd.     

Cytotoxic and trypanocidal activities of cinchona alkaloid derivatives

A series of 27 cinchona alkaloid derivatives ( 1f–w , 2a–e and 3a–d ) were investigated for their cytotoxic and trypanocidal activities using seven different cancer cell lines (KB, HeLa, MCF‐7, A‐549, Hep‐G2, U‐87 and HL‐60), two normal cell lines (HDF and CHO) and bloodstream forms of Trypanosoma brucei brucei, respectively. Four compounds ( 1u , 1w , 2e and 3d ) were identified with promising cytotoxic activity with 50% growth inhibition (GI₅₀) values below 10 μM. Two ( 2e and 3d ) of the four compounds also exhibited potent anti‐trypanosomal activity with GI₅₀ values of 0.3–0.4 μM. All four active compounds represented derivatives modified at their C‐9 hydroxy group. With respect to anti‐proliferative activity and selectivity, 2e (epi‐N‐quinidyl‐N′‐bis(3,5‐trifluoromethyl)phenylthiourea) proved to be the most promising derivative for both cancer cells and bloodstream forms of T. b. brucei. The cytotoxic activity of compounds 1u , 1w , 2e and 3d was attributed to their ability to induce apoptosis in cancer cells. The results demonstrate the potential of cinchona alkaloid derivatives as novel anti‐cancer and anti‐trypanosome drug candidates.     

Studies of <Emphasis Type="Italic">Ephedra</Emphasis> plants in Asia. Part 6: Geographical changes of anatomical features and alkaloids content of <Emphasis Type="Italic">Ephedra sinica</Emphasis>

Ephedra sinica Stapf is the main botanical origin of the Chinese herbal drug Mahuang, Ephedra Herb. Eighty-five samples of E. sinica, collected across eastern China, Mongolia, and Buryatia (Russia), were studied anatomically and chemically to elucidate the local variations and the relation between environmental factors and the variations. The results showed that samples grown in more arid conditions tended to have a more sinuous epidermis, more cuticular tubers, and more subepidermal fiber bundles anatomically, and contained more total ephedrine alkaloids. These samples also had a high pseudoephedrine content. These results imply that Ephedra Herb with good quality should be collected from arid fields, and the chemical quality can be estimated by observing the anatomical characteristics.