Ethno-medicinal study of Artemisia ordosica Krasch. (traditional Chinese/Mongolian medicine) extracts for the treatment of allergic rhinitis and nasosinusitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia ordosica Krasch. (AOK) has been used for rheumatic arthritis, cold headache, sore throat, etc. in traditional Chinese/Mongolian medicine and is used for nasosinusitis by local Mongolian "barefoot" doctors. Up to now, their mechanisms are still unclear. AIM: To evaluate the in vivo anti-inflammatory and allergic rhinitis (AR) alleviating effect as well as in vitro antimicrobial activities of AOK extracts to verify its ethno-medicinal claims. MATERIALS AND METHODS: Crude extracts (methanol/95%-ethanol/ethyl acetate) of AOK root/stem/leaf and fractions (petroleum ether/ethyl acetate/n-butanol/aqueous) of AOK root extract were prepared. Xylene-induced ear swelling model in mouse and ovalbumin (OVA)-induced AR model in guinea pig were established. Ear swelling degrees of mice were measured. The numbers of rubbing movement and sneezes of guinea pigs were counted to evaluate the symptoms of AR. The serum levels of histamine, INF-γ, IL-2/4/10, and VCAM-1 were measured by ELISA assay. The histological changes of nasal mucosa were investigated by light microscope after H&E staining. Antimicrobial activities of AOK extracts were also tested. LC-MS/MS analysis was performed to characterize the constituents of active extract and molecular docking was conducted to predict the biological mechanism. RESULTS: In ear-swelling model, extract (100.00 mg/kg) from the ethyl acetate layer of 95% ethanol (100.00 mg/kg) showed better swelling inhibition in mice than positive control (dexamethasone, 191.91 mg/kg). In AR model, extract from the ethyl acetate layer of 95% ethanol significantly alleviated the AR symptoms in guinea pigs, decreased the serum levels of histamine, INF-γ, IL-2/4/10, and VCAM-1, and reduced the infiltration of eosinophil in nasal mucosa. For Staphylococcus aureus, the ethyl acetate extract of AOK stem showed the highest inhibition (MIC=1.25 mg/mL), for Escherichia coli, n-butanol layer of 95% ethanol extract of AOK root showed the highest inhibition (MIC=15.00 mg/mL), for Candida glabrata, 95%-ethyl acetate extract of AOK leaf showed the best inhibition (MIC=0.064 mg/mL), while ethyl acetate and n-butanol layers showed similar inhibition on MRSA (MIC=7.50 mg/mL). LC-MS/MS characterization showed that dicaffeoylquinic acids account for more than 30% of ethyl acetate layer of AOK extract. Dicaffeoylquinic acids bind with histamine-1 receptor with high affinities and interesting modes. CONCLUSIONS: Extracts from AOK had interesting anti-inflammatory activity in mice, alleviating effect against OVA-induced AR in guinea pigs, and antimicrobial activities in vitro, which support the ethno-medicinal use of it. The main constituents in ethyl acetate layer of AOK root extract are dicaffeoylquinic acids and could bind with histamine-1 receptor well. These findings highlighted the importance of natural product chemistry study of AOK.

[Influence of Four Kinds of PPCPs on Micronucleus Rate of the Root-Tip Cells of Vicia-faba and Garlic].

In order to determine the degree of biological genetic injury induced by PPCPs, the genotoxic effects of the doxycycline (DOX), ciprofloxacin (CIP), triclocarban (TCC) and carbamazepine (CBZ) in the concentration range of 12.5-100 mg · L⁻¹ were studied using micronucleus rate and micronucleus index of Vicia-fabe and garlic. The results showed that: (1) When the Vicia-faba root- tip cells were exposed to DOX, CIP, TCC and CBZ, micronucleus rates were higher than 1.67 ‰ (CK1), it was significantly different from that of the control group (P < 0.05), and the micronucleus index was even greater than 3.5; With the increasing concentrations of the PPCPs, the micronucleus rates first increased and then decreased. (2) When the garlic root tip cells were exposed to DOX, CIP, TCC and CBZ respectively, the micronucleus rates were less than those of the Vicia-faba, while in most treatments significantly higher than that of the control group (0.67‰). The micronucleus index was higher than 3.5 in the groups exposed to CIP with concentrations of 25, 50, 100 mg · L⁻¹ and TCC and CBZ with concentrations of 25 mg · L⁻¹; With the increase of exposure concentrations, the micronucleus rate showed a trend of first increasing and then decreasing as well. (3) Under the same experimental conditions, the cells micronucleus rates of the garlic cells caused by the four tested compounds were significantly lower than those of Vicia-faba. (4) The micronucleus index of the root tip cells of Vicia-faba and garlic treated with the four kinds of compounds followed the order of CIP > CBZ > TCC > DOX. These results demonstrated that the four compounds caused biological genetic injury to root-tip cells of Vicia-faba and garlic, and the genetic damage caused to garlic was significantly lower than that to Vicia-faba. The damages caused by the four kinds of different compounds were also different.

Dispersive solid-phase extraction cleanup combined with accelerated solvent extraction for the determination of carbamate pesticide residues in Radix Glycyrrhizae samples by UPLC-MS-MS.

Dispersive solid-phase extraction (DSPE) cleanup combined with accelerated solvent extraction (ASE) is described here as a new approach for the extraction of carbamate pesticides in Radix Glycyrrhizae samples prior to UPLC-MS-MS. In the DSPE-ASE method, 15 carbamate pesticides were extracted from Radix Glycyrrhizae samples with acetonitrile by the ASE method at 60 °C with a 5 min heating time and two static cycles. Cleanup of a 1 mL aliquot of the extract by the DSPE method used 20 mg PSA (primary secondary amine), 50 mg Al(2)O(3)-N, and 20 mg GCB (graphitized carbon black) (as cleanup sorbents) under the determined optimum conditions. The linearity of the method was in the range of 10 to 200 ng/mL with correlation coefficients (r(2)) of more than 0.996. The limits of detection were approximately 0.2 to 5.0 µg/kg. The method was successfully used for the analysis of target pesticides in Radix Glycyrrhizae samples. The recoveries of the carbamate pesticides at the spiking levels of 50, 100, and 200 µg/kg ranged from 79.7% to 99.3% with relative standard deviations lower than 10%. This multi-residue analytical method allows for a rapid, efficient, sensitive and reliable determination of target pesticides in Radix Glycyrrhizae and other medicinal herbs.

[Effects of nitrogen and phosphorus fertilizer on atrazine degradation and detoxification by degrading strain HB-5].

An atrazine-degrading strain HB-5 was used as a bacteria for biodegradation. Treatments of soil with nitrogen single, phosphate single and nitrogen phosphate together with HB-5 were carried out for degradation and eco-toxicity test; then, relationship between atrazine degradation rate and soil available nitrogen, available phosphorus were discussed. Atrazine residues were determined by HPLC; available nitrogen was determined with alkaline hydrolysis diffusion method; available phosphorus was determined with 0.5 mol/L-NaHCO3 extraction and molybdenum stibium anti-color method, and toxicity test was carried out with micronucleus test of Vicia faba root tip cells. The results showed that: After separately or together application, nitrogenous and phosphorous fertilizers could significantly accelerate atrazine degradation than soil with HB-5 only. On day 5, the order of atrazine degradation was ANP > AP > AN > A; 7 days later, no statistically significant differences were found between treatments. The available nitrogen and phosphorus level in soil reduced as the degradation rate increased in the soil. The soil of eco-toxicity test results indicated that the eco-toxicity significantly reduced with the degradation of atrazine by HB-5, and the eco-toxicity on treatments of soil with fertilizer were all below the treatments without fertilizer. On day 5, the order of eco-toxicity was ANP < AP < AN < A; 7 days later, all treatments were decreased in control levels. So, adjusting soil nutrient content could not only promote atrazine degradation in soil but also could reduce the soil eco-toxicity effects that atrazine caused. All these results could be keystone of atrazine pollution remediation in contaminated soil in the future.

[Study of liquorice processing fructus].

OBJECTIVE: To establish the processing method of fructus evodiae and its standard for quality control, toxicity aspects and pharmacodynamics were carried out at the same time. METHOD: In the studies of processing techniques, the optimized technical parameters were determined by the contents of evodiamine and evodine. And the acute toxicity and pharmacodynamics were studied by rats. RESULT: The process was that the liquorice-processed fructus evodiae was wetted by liquorice decoction by sixth of raw fructus evodiae (V/W) and fried below 230 degrees C. The method of detecting the contents of evodiamine and evodine was that Alltima ODS C18 (4.6 mm x 250 mm, 5 microm); mobile phase acetonitrile-water-tetrahydrofuran-phosphoric acid (51:48: 1: 0.05); column temperature 25 degrees C; mobile rate 0.8 mL x min(-1); wave length 225 nm. The toxicity experimentation show that rats didnt show any notable changes after affused the raw material and the processed fructus evodiae's decotion 40 g x kg(-1) b. w. at one time seven days constantly. The analgesic effect was observed after 0.6 g (material) x kg(-1) (weight) b. w. CONCLUSION: The toxicity of the raw material and the processed one were low and the liquorice-processed fructus evodia analgesic effect was good.

Arjunic acid, a strong free radical scavenger from Terminalia arjuna.

This study was designed to investigate the antioxidant and free radical scavenging capacities of arjunic acid, an aglycone obtained from the fruit of medicine Terminalia Fruit. Liver microsomes, mitochondria, and red blood cells (RBCs) were prepared from Wistar rats. The antioxidant capacity was determined by the inhibitory effect on lipid peroxidation, hydrogen peroxide induced RBCs hemolysis, and RBCs autoxidative hemolysis. The free radical scavenging activity was tested by DPPH method and 2',7'-dichlorodihydrofluoresc in diacetate (DCFH(2)-DA) assay. Ascorbic acid was chosen as the positive controls. Results showed that arjunic acid was a strong antioxidant and a free radical scavenger, more potent than ascorbic acid, in microsomes lipid peroxidation, DPPH, hydrogen peroxide induced RBCs hemolysis, and (DCFH(2)-DA) assay (p < 0.05). However, no significant difference was observed in the RBCs autoxidative hemolysis assay (p > 0.05).

[Development of virulent heat-evil-induced thrombosis animal model].

OBJECTIVE: To develop a virulent heat-evil-induced thrombosis animal model, and provide a rational animal model for pathogeny and pathogenesis research of thrombosis-related diseases, anti-thrombosis activity screening and pre-clinical studies of CAHT formula. METHOD: SD rats were pretreated with carrageenin (Ca) intraperitoneal injection, followed by intravenous injection of endotoxin (LPS from E. coli O111:B4) 50 microg x kg(-1) 16 h later. Thrombosis in rat tails were observed during 12-24 h after injection of LPS. The inflammatory mechanism of this model were investigated by analyzing serum level of TNF-alpha, IL-6, TXB2 and 6-keto-PGF 1alpha, CD11b/CD18 expression of white blood cells (WBC) and P-selectin expression of vessel walls. RESULT: In LPS/Ca model group, thrombosis can be clearly observed in the distal part of rat tails after 12-24 h of LPS/Ca treatment. High level of TNF-alpha and IL-6 can be measured in serum. The expression of CD11b/CD18 in WBC and P-selectin in vessel endothelium significantly increased and the number of WBC in peripheral blood markedly decreased shortly after LPS/Ca treatment. The adherence of white blood cells to vessel endothelium which can be seen by microscope mainly contributed to the decrease of WBC. The results indicated that there was obvious inflammation after treatment with LPS/Ca, suggesting that inflammation was the key mechanism for this model. CONCLUSION: This model was developed through treatment of LPS in combination with Ca, of which LPS is considered to be an exotic virulent heat-evil in TCM, while the inflammatory molecules produced in this model, such as TNF-alpha, IL-6, CD11b/CD18 and P-selectin belong to internal virulent heat-evils, so this animal model consists of pathogeny and pathogenesis of virulent heat-evils. virulent heat-evil.

[Pharmacodynamic and toxicologic comparative study of crude and processed radix aristolochice].

OBJECTIVE: To study the accumulated toxic action to bandicoot of aqueous extract of crude and processed Radix Aristolochice and the pharmacodynamic action of aqueous and alcoholic extract of crude and processed Radix Aristolochice. METHOD: The LD50 of acute toxicity to mice and chronic accumulated toxicity to bandicoots of crude and processed Radix Aristolochice were observated. Intestinal and myokinetic influence of normal and revulsive hyperactive gastrointestinal motility of mice induced by neostigmine were observated by giving aqueous extract and alcoholic extract of crude and processed Radix Aristolochice. Relieving pain and eliminating inflammation to mice also were observated. RESULT: The LD50 of aqueous extract of crude and processed Radix Aristolochice were 146. 45, 846.06 g X kg(-1) (equivalently to crude drug) respectively by intragastric administration. Bandicoot' general condition, peripheral blood, serum, organic coefficient, histopathologic examination weren't obvious changes after 1 month administrating aqueous extract of crude and processed drug in three dose. Serum indicators-urea nitrogen, cholesterol total, alkaline phosphatase manifestly were heightened and some animals'hepatic cells, nephric tubules and mucosa emerged differently damage at histomorphology by giving crude high dose after 2 months. Above organs emerged different damage in crude middle and high dose and processed high dose after 3 months and serum indicators- creatinine, urea nitrogen manifestly were increased, the coefficients of liver, kidney and gaster manifestly were heightened. However, the toxicity of identical dose processed product was lower than that of crude one. Aqueous extract and alcoholic extract of crude and processed Radix Aristolochice could obviously inhibite normal and revulsive hyperactive gastrointestinal motility by neostigmine of mice, relieve pain in mouse, stretching and heat stimulation models and inhibite dimethyl benzene-induc mouse, auricle inflammation. Pharmacodynamic action wasnt obvious difference in same dose of crude product and processed one. CONCLUSION: Acute toxicity and chronic accumulated toxicity are stepped down after giving processed Radix Aristolochice, but pharmacodynamic effect wasn t lower. In pharmacodynamic effect, aqueous extract can't compare with alcoholic extract in same dose.

[Comparative research of thrombosis animal model induced byendotoxin in combination with carrageenan in different animals].

OBJECTIVE: To compare the development of thrombosis animal model induced by endotoxin(LPS) in combination with carrageenan (Ca) in different animals. METHOD: Two species of rats (SD and Wistar) and three species of mice (Kunming, ICR and Balb/c mice) were employed in the study. The animals of each species were randomly divided into control group and model group (LPS/Ca treatment). The animals in the model group were pretreated with Ca ip at the doses of 25 mg x kg(-1) for rats and 150 mg x kg(-1) for mice, and then treated by LPS iv sixteen hours later, while in the control group were given normal saline (NS). Thrombosis in tails was observed at 24 h after LPS iv. Hematologic parameters were tested for all the animals from each species, and the blood concentration of TNFalpha and IL-6 at different time in SD and Wistar rats were measured. RESULT: LPS/Ca combinatory treatment could induce thrombosis animal model in all five animal species, and the thrombus could be clearly observed on the tails. All species had the similar change in hematologic parameters characterized as the significant decrease of white blood cells and platlets. Inflammatory factors TNFalpha and IL-6 could be largely induced in blood of both SD and Wistar rats at 2 h after LPS iv, but both inflammatory factors only transitorily exist in blood at the early stage of thrombosis model formation. CONCLUSION: LPS/Ca combinatory treatment can successfully induce thrombosis animal model in all tested animal species, and thus this model has extensive animal candidates. The secretion of a large amount of inflammatory factors plays a crucial role in the formation of thrombosis animal model.

[Nephrotoxicity of Aristolochia manshuriensis and aristolochic acids in mice].

OBJECTIVE: The acute toxic effects of Aristolochia manshuriensis (GMT) and the total aristolochic acids (TA) were compared in mice with aristolochic acid A (AA) as the dose standard. The dose relationship of the renal toxicity induced by Aristolochia manshuriensis was determined. METHOD: A single dose of GMT extract or TA was given intragastrically to mice at different doses. LD50 values, the blood levels of BUN, Cr and ALT were measured. A histomorphological study was also performed in livers and kidneys of mice. RESULT: LD50 value of GMT extract was 4.4 g x kg(-1) which was equivalent to 40 mg x kg(-1) as calculated by the content of AA in GMT extract, and this value was comparable with LD50 obtained from TA given intragastrically in mice (equivalent to 33 mg x kg(-1) of AA for male and 37 mg x kg(-1) for female). GMT extract caused a significant increase in blood BUN and Cr and an obvious morphological change in kidney in a dose-dependent manner at doses of AA 4.5 mg x kg(-1) and above. Liver damage, characterized by both an increase in blood level of AST and histomorphological change, was observed at doses of AA 25 mg x kg(-1) and above. All changes were in proportion to the doses of AA. CONCLUSION: GMT causes both renal and liver toxicity. The dose leading to nephrotoxicity is much lower than that inducing hepatatoxicity. Aristolochic acids existed in GMT are the main toxic components to cause renal toxicity which is a crucial cause to result in death. The lethality and nephrotoxicity of GMT is in proportion to the doses of AA.

[Development of an animal model of blood stasis syndrome and thrombosis].

OBJECTIVE: To develop an animal model of thrombosis and blood stasis syndrome in rats by using lipopolysaccharide (LPS) in combination with carrageenan (Ca). METHOD: SD rats in control group were randomly divided into control group and model group (LPS/Ca treatment). The rats in model group were firstly treated with Ca ip, and followed by LPS iv sixteen hours later. The rats in control group were given normal saline (NS). The moment of LPS iv was served as 0 h for the observation. The ear microcirculation, blood rheology parameters (whole blood viscosity etab, plasma viscosity etap and platelet aggregation PA), cruor parameters (thrombin time TT, prothrombin time PT, and partial thromboplastin time APIT) and inflammation factors (TNFalpha, IL-6) were observed at different time after treatment. RESULT: LPS/Ca combinatory treatment can induce a stable and repeatable thrombosis animal model. The thrombus can be observed on the tails of rats by naked eyes, and can be quantitatively measured without necessary of autopsy. Obstacle in microcirculation, increase in whole blood viscosity (etab) and a change of platelets aggregation (PA) rate were observed after LPS/Ca treatment. Cruor parameters were significantly prolonged due to large consumption of cruor factors and platelets. The concentration of inflammation factors TNFalpha and IL-6 in blood was obviously increased at the early stage of the model. The results indicate that this animal model has the characteristics of blood stasis syndrome caused by pyrogen and toxin accompanied by thrombosis. CONCLUSION: LPS/Ca combinatory treatment can induce a easily practicable and repeatable animal model characterized as thrombosis and blood stasis syndrome

[Comparative studies on pharmacological effects of angong niuhuang pill with its simplified prescicription].

OBJECTIVE: Based on the therapeutic claims of Angong Niuhuang pill, a series of pharmacodynamic experiments were designed, where pharmacological effects were investigated comparatively with its simplified prescription(realgar and cinnabar are removed from the original pill) as a parallel control in order to explore possible contribution of cinnabar and realgar to pharmacodynamic activities of the pill as a whole. METHOD: Anti-pyretic, sedative, anti-convulsive, and mice-protected effects of the pill and its simplified prescription as a control were observed, respectively, in rabbits with fever induced by typhoid bacillus, in pentobarbital sodium-induced sleeping mice, in mice with convulsion induced by strychnine, or pentylenetetrazole, and in mice with anoxia induced by NaNO2. RESULT: Both the pill and its simplified prescription were found to have Anti-pyretic action and protective effect against the mouse death induced by anoxia, and synergistic interaction with pentobarbital sodium in sedative activity, although neither of them was found to have any effects on the convulsion of mice. CONCLUSION: No significant difference between Angong Niuhuang pill and its simplified prescription was found in the above pharmacodynamic experiments.

[Absorption and distribution of mercury and arsenic from realgar and cinnabar of angong niuhuang pill in normal rats and rats with cerebral ischemia].

OBJECTIVE: To study comparatively the characteristics of absorption and distribution of mercury and arsenic from realgar and cinnabar of Angong Niuhuang Pill in normal rats and the rats with cerebral ischemia after oral administration. METHOD: The blood samples and homogenates of liver, kidney and brain were prepared at various intervals after the animals were treated with Angong Niuhuang pill ig. The levels of total mercury and total arsenic in the blood and the organ homogenates were measured with Microwava Accelerated Reaction System and AAs, respectively. RESULT: The blood concentrations of mercury and arseic reached the highest point in normal rats at one hour following single oral dosing of Angong Niuhuang pill. In normal rats, the mercury distribution was characterized by its higher level in blood and kidneys than in other organs, while a higher distribution of arsenic was found in blood than in organs. No difference in the distribution of mercury or arsenic was found between normal rats and rats with cerebral ischemia after the treatment with the pill. CONCLUSION: The highest level of mercury or arsenic in blood occurs at one hour after oral administration of the pill in normal rats. There is a higher distribution of mercury in blood and kidneys, while a higher distribution of the arsenic only in blood. There is no significant difference in the distribution of mercury or arsenic between the normal rats and the ischemic rats.

[Reversal of anti-apoptotic action by tetrandrine in human breast carcinoma multidrug-resistant MCF-7 cells].

OBJECTIVE: To study whether the anti-apoptotic action is reversed by tetrandrine in a combination with vincristine in human breast carcinoma MCF-7 multidrug-resistant cells. METHOD: Chromatin condensation was observed by co-staining of fluorescent dyes Hoechst 33342 and propidium iodide; and G1 sub-peak was detected by flow cytometry. Apoptotic cells were detected with TUNEL method. Cellular free ca2+ was determined with Fluo-3 staining method. RESULT: Two types of chromatin condensation were observed after the sensitive and drug-resistant MCF-7 cells were treated with an antitumor drug vincristine 5 mumol.L-1 for 24 h. The number of cell with chromatin condensation was obviously reduced in the drug-resistant cells treated with the same concentration of vincristine, as compared with the sensitive MCF-7 cells. The number of the apoptotic cells was increased by a combination of non-cytotoxic tetrandrine 20 mumol.L-1 and vincristine in both the sensitive and drug-resistant cells, which was confirmed with fluorescent indication and TUNEL method. The increment of introcellular free Ca2+ level in the cells treated with tetrandrine in a combination of vincristine was detected with Fluo-3 staining method. CONCLUSION: The anti-apoptotic action of human breast carcinoma MCF-7 cells can be effectively reversed by tetrandrine.