Refined assessment of heavy metal-associated health risk due to the consumption of traditional animal medicines in humans.

Little is known about the extent of heavy metal accumulation in traditional Chinese medicines (TCMs). In this study, the levels of lead (Pb), cadmium (Cd), arsenic (As), and mercury (Hg) in traditional animal medicines were monitored using inductively coupled plasma mass spectroscopy (ICP-MS). Additionally, for the first time, a heavy metal risk assessment strategy was used to evaluate the potential risks of traditional animal medicines by calculating estimated daily intake (EDI), target hazard quotient (THQ), and cancer risk (CR). To obtain a refined risk assessment, the frequency of exposure to traditional animal medicines was determined from questionnaire data, and the safe factor for TCM was applied. Based on the standard levels for leech, it was found that earthworm, hive, scorpion, and leech accumulated high levels of heavy metals. The combined THQ (cTHQ) values indicated that ingestion of most traditional animal medicines would not pose a risk to the health of either male or female human beings. However, it was indicated that attention should be paid to the potential risk associated with cicada slough, earthworm, scorpion, turtle shells, and hive. Among heavy metals, As and Hg contributed to a major extent to the risk to human health. The CR assessment for Pb and As indicated that, with the exception of earthworm, the cancer risk was less than the acceptable lifetime risk for both males and females. Owing to the higher body weight, both THQ and CR were generally lower for males than for females.

Simultaneous screening of four epidermal growth factor receptor antagonists from Curcuma longa via cell membrane chromatography online coupled with HPLC-MS.

The epidermal growth factor receptors (EGFRs) are significant targets for screening active compounds. In this work, an analytical method was established for rapid screening, separation, and identification of EGFRs antagonists from Curcuma longa. Human embryonic kidney 293 cells with a steadily high expression of EGFRs were used to prepare the cell membrane stationary phase in a cell membrane chromatography model for screening active compounds. Separation and identification of the retention chromatographic peaks was achieved by HPLC-MS. The active sites, docking extents and inhibitory effects of the active compounds were also demonstrated. The screening result found that ar-turmerone, curcumin, demethoxycurcumin, and bisdemethoxycurcumin from Curcuma longa could be active components in a similar manner to gefitinib. Biological trials showed that all of four compounds can inhibit EGFRs protein secretion and cell growth in a dose-dependent manner, and downregulate the phosphorylation of EGFRs. This analytical method demonstrated fast and effective characteristics for screening, separation and identification of the active compounds from a complex system and should be useful for drug discovery with natural medicinal herbs.

Novel bioavailability-based risk assessment of Cd in earthworms and leeches utilizing in vitro digestion/Caco-2 and MDCK cells.

In the present study, the oral bioavailability of cadmium (Cd) in earthworms and leeches was investigated through in vitro physiologically based extraction test (PBET) digestion/Caco2 and MDKC cell models. We are the first to create an innovative assessment strategy which has capacity to offer a more precise evaluation of Cd-associated health risks in traditional animal medicines (TAMs), by combinational usage of bioavailable Cd levels, the duration and frequency of the exposure to TAMs obtained by questionnaire data, as well as safety factor of TAMs. Our data showed that the percentage of bioavailability for Caco-2 cells in earthworms and leeches ranged from 3.29 to 14.17% and 4.32 to 12.61%, respectively. The percentage of bioavailability of MDCK cells in earthworms and leeches ranged from 4.83 to 15.74% and 6.53 to 15.04%, respectively. After adjusting by the bioavailability of Cd to target hazard quotient (THQ), excitingly, our findings manifested that the health risks induced by the ingestion of earthworms and leeches were acceptable in the clinic. Our key findings suggest that bioavailability characterization cannot be ruled out and health risks should be assessed on the basis of the bioavailable Cd levels rather than total levels. Our novel strategy provides insight into the bio-accumulation of Cd in organisms as well as a more realistic and accurate assessment of Cd-associated health risks in TAMs, with the main purpose of improving public health by scientifically using TAMs.

[Effect of taspine derivatives on human liver cancer SMMC7721].

OBJECTIVE: To analyse the inhibition effect of taspine derivatives on human Liver cancer SMMC7721 cell and its mechanism. METHODS: The effects of five taspine derivatives on SMMC7721 cell growth were determined by MTT. The flow cytometry was used to determine the cell cycle. The effects of Tas-D1 on the EGF and VEGF in SMMC7721 cell were determined by ELISA. The mRNA level of EGF and VEGF in SMMC7721 cell was determined by RT-PCR. RESULTS: The MTT assay demonstrated that the taspine derivative Tas-D1 significantly inhibited the growth of SMMC7721 cell in a dose-dependent manner. Cell was stopped at S phase by Tas-D1. Tas-D1 inhibited the expression of EGF and VEGF and their mRNA in a dose-dependent manner (P<0.05). CONCLUSIONS: The taspine derivative Tas-D1 can inhibit the growth of human Liver cancer SMMC7721 cell and change cell cycle, which may be related to the inhibition of EGF and VEGF expression.

[Effects of Bai-Chuan capsule on left ventricular hypertrophy and correlative indexes].

OBJECTIVE: To investigate the effects of Bai-Chuan capsule (BC) on the left ventricular hypertrophy in spontaneously hypertensive rats (SHR). METHODS: Taking SHR and Wistar Kyoto rats (WKY) as the model group and the control group respectively, Captopril as positive drug, administered BC 0.3 g/kg, Captopril 3.75 mg/kg or 0.5% CMC-Na to each group by ig for 3 months, and measured the change of blood pressure. The left ventricular mass index was calculated after rats were sacrificed. Left ventricle was used to pathological observations, plasma angiotensin II and aldosterone were measured by radioimmunoassay. CONCLUSION: BC can inhibit left ventricular hypertrophy, plasma level of angiotensin II and aldosterone to some extent in SHR.

HPLC method for the pharmacokinetics and tissue distribution of taspine solution and taspine liposome after intravenous administrations to mice.

Taspine is a bioactive aporphine alkaloid, which has many potent pharmacological effects. A simple, rapid HPLC method to quantify taspine in mouse plasma and tissue homogenates containing either taspine solution or liposome was developed and validated. Sample preparation was achieved by liquid-liquid extraction with acetoacetate. Taspine was separated on a C(18) reversed phase HPLC column, and quantified by its absorbance at 245 nm. The pharmacokinetics and tissue distribution after intravenous administrations of taspine liposome (L-Ta) and taspine solution (Ta) to ICR mice were then compared. The area under the plasma concentration-time curve (AUC) was higher for L-Ta than for Ta. In contrast, the total body clearance (CL), apparent volume of distribution V(c) and plasma half-life for the distribution (t(1/2 alpha)) and elimination phase (t(1/2 beta)) were lower for L-Ta, in comparison to the respective parameter of Ta. The AUC values were higher in the lung than in other organs for both L-Ta and Ta. The AUC in the spleen, kidney and liver of L-Ta were higher than those of Ta. However, the heart and brain AUC of Ta was higher than that of L-Ta. It can thus be concluded that incorporation into liposomes prolonged taspine retention within the systemic circulation, increased its distribution to the spleen and liver but reduced its distribution to the heart and brain.

The use of solid lipid nanoparticles to target a lipophilic molecule to the liver after intravenous administration to mice.

Taspine solid lipid nanoparticles (Ta-SLN) and taspine solid lipid nanoparticles modified by galactoside (Ta-G2SLN) were prepared by the film evaporation-extrusion method. The nanoparticles were spherical or near-spherical particles with smooth surface, small size and high encapsulation efficiency. Ta-G2SLN and Ta-SLN showed significant inhibition on 7721 cell growth. Intravenous injection of either Ta-SLN or Ta-G2SLN resulted in a higher plasma and liver concentration and a longer retention time in mice compared with the administration of Ta. These results suggested that SLN tended to be preferentially delivered to the liver and Ta-G2SLN may further enhance liver targeting.

Down-regulatory effect of usnic acid on nuclear factor-kappaB-dependent tumor necrosis factor-alpha and inducible nitric oxide synthase expression in lipopolysaccharide-stimulated macrophages RAW 264.7.

The purpose of this study was to investigate the molecular mechanisms that are responsible for the antiinflammatory effect of usnic acid (UA). UA is one of the most common and abundant lichen metabolites. The present study examined the effects of UA on the tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) production induced by lipopolysaccharide (LPS) in RAW264.7 macrophages and the underlying molecular mechanisms. UA decreased the TNF-alpha level in LPS-stimulated RAW264.7 macrophages in dose-dependent manner, the IC(50) value was 12.8 microM. RT-PCR analysis indicated that it inhibited TNF-alpha mRNA expression. Furthermore, it inhibited NO production in LPS-activated RAW264.7 macrophages, the IC(50) value was 4.7 microM. Western blot analysis showed that UA attenuated LPS-induced synthesis of iNOS protein and nuclear translocation of NF-kappaB p65 in the macrophages, in parallel. UA also inhibited LPS-mediated I-kappaBalpha degradation. Taken together, this suggests that UA has an antiinflammatory effect by inhibiting TNF-alpha and iNOS expression, possibly through suppression of nuclear translocation of NF-kappaB p65 and I-kappaBalpha degradation.

Interfacial Engineering of Supported Liquid Membranes by Vapor Cross-Linking for Enhanced Separation of Carbon Dioxide.

Supported liquid membranes (SLMs) based on ionic liquids (ILs) with not only high gas permeability and selectivity, but also high stability under high pressure, are highly desired for gas separation applications. In this work, permeable and selective polyamide network (PN) layers are deposited on the surface of SLMs by utilizing the cross-linking reaction of trimesoyl chloride, which was pre-dispersed in the SLMs, and vapor of amine linkers. The vapor cross-linking method makes it easy to control the growth and aggregation of PN layers, owing to the significantly reduced reaction rate, and thereby ensuring the good distribution of PN layers on the surface of SLMs. With rational choice of amine linkers and optimization of vapor cross-linking conditions, the prepared sandwich-like PN@SLMs with ILs embedded homogeneously within polymeric matrices displayed much-improved CO2 permeability and CO2 /N2 selectivity in relation to the pristine SLMs. Moreover, those SLMs with ILs impregnated into porous supports physically displayed improved stability under high pressure after vapor cross-linking, because the PN layers formed on the surface of SLMs help prevent the ILs from being squeezed out. This interfacial engineering strategy represents a significant advance in the surface modification of SLMs to endow them with promising applications in CO2 capture.

Imperatorin induces vasodilatation possibly via inhibiting voltage dependent calcium channel and receptor-mediated Ca2+ influx and release.

The purpose of the present study was to investigate the effect of imperatorin on vasodilatation and its possible mechanisms. Isometric tension of rat mesenteric arterial rings was recorded by a myograph system in vitro. The results showed that imperatorin at more than 10 muM concentration-dependently relaxed rat mesenteric arteries pre-contracted by potassium chloride (KCl) and endothelin-1, and human omental arteries pre-contracted by noradrenaline and U46619. Removal of the endothelium did not affect imperatorin-induced relaxant responses, suggesting that the vasodilatation effect is independent of the endothelium. Co-incubation with imperatorin resulted in rightward shift of concentration-response curves of KCl, calcium chloride (CaCl(2)) and noradrenaline in a non-parallel manner; 5-hydroxytryptamine (5-HT) concentration-response curves were shifted towards right in a parallel manner by imperatorin 10 and 30 muM, but markedly suppressed by imperatorin 100 muM. These results suggest that the inhibitory effect of imperatorin is mainly via voltage dependent calcium channel and possibly receptor operated calcium channel. beta-adrenoceptor, ATP-sensitive potassium channel and inwardly rectifying potassium channel were not involved in the vasodilatation, whereas blockage of calcium-activated potassium channel with tetraethylammonium had effect. Furthermore, in Ca(2+)-free medium, imperatorin concentration-dependently depressed the vasoconstrictions derived from noradrenaline and CaCl(2), and resulted in a decreased contractile response induced by caffeine, indicating a role of inhibiting extracellular Ca(2+) influx and intracellular Ca(2+) release from Ca(2+) store. Taken together, our results suggest that imperatorin induces vasodilatation by possible mechanisms inhibiting voltage dependent calcium channel and receptor-mediated Ca(2+)influx and Ca(2+)release. Opening calcium-activated potassium channel and competitive antagonism of 5-HT receptors may also contribute to this vasodilatation effect.

Screening for the anti-inflammatory activity of fractions and compounds from Atractylodes macrocephala koidz.

The aim of this study was to screen for the anti-inflammatory activity of fractions and compounds from Atractylodes macrocephala Koidz. The rhizomes of Atractylodes macrocephala were treated with supercritical CO(2) fluid and the extract was separated by normal-phase and reverse-phase column chromatography. The separated samples were screened with white blood cell membrane (WBCM) chromatography (WBCM-C). The anti-inflammatory effects of these fractions and components were tested pharmacologically in vivo. The results indicated that the retention characteristics of the petrol-ether (1:1, v/v) fraction (BZC-2) of the supercritical CO(2) extract, the atractylenolide I and 14-acetoxy-12-senecioyloxytetradeca-2E,8E,10E-trien-4, 6-diyn-1-ol isolated from BZC-2 as active fractions and components were similar to that of dexamethasone in WBCM-C. Therefore, they may act on WBCM and its receptors. BZC-2 has shown anti-inflammatory effects in acute and chronic inflammation models in rats and mice. Oral administration of atractylenolide I and 14-acetoxy-12-senecioyloxytetradeca-2E,8E,10E-trien-4,6-diyn-1-ol produced significant anti-inflammatory effects in acute and chronic inflammation models in mice. The screening results with WBCM-C were correlated significantly with pharmacological effects in vivo. Atractylenolide I and 14-acetoxy-12-senecioyloxytetradeca-2E,8E,10E-trien-4,6-diyn-1-ol were the main components of Atractylodes macrocephala that were effective as anti-inflammatory agents.

[Analysis of supercritical fluid extracts of Radix caulophylli with gas chromatography-mass spectrometry].

To analyze the constituents in supercritical fluid CO2 extraction (SFE-CO2) of Radix caulophylli, the Radix caulophylli was extracted with SFE-CO2, and analyzed by gas chromatography-mass spectrometry (GC-MS). The GC-MS analysis with a DB-5MS capillary column (30 mm x 0.32 mm ID, 0.25 microm film thickness) was used. The inlet temperature was maintained at 280 degrees C. The column oven was held at 80 degrees C for 2 min, then programmed from 80 to 280 degrees C at 5 degrees C x min(-1) and, finally, held for 4 min. Helium at a constant flow rate of 2.0 mL x min(-1) was used as the carrier gas. The mass spectrometry conditions were as follows: ionization energy, 70 eV; ion source temperature, 200 degrees C. The mass selective detector was operated in the TIC mode (m/z was from 40 - 500). For the first time 49 peaks were separated and identified, the compounds were quantitatively determined by normalization method, and the identified compounds represent 97.44% of total GC peak areas. Viz, n-hexadecanoic acid (31.4%), (E, E) -9, 12-octadecadienoic acid (26.54%), (Z)-7-tetradecenal (9.4%), hexadecenoic acid (3.23%), 10-undecenal (3.22%), octadecanoic acid (2.25%), and caulophylline (1.76%) etc. The results will provide important foundation for understanding the constituents and further exploitation of Radix caulophylli.

[Study on thaspine in inducing apoptosis of A549 cell].

OBJECTIVE: To investigate the effect of thaspine on the cellular proliferation, apoptosis and cell cycle in A549 cell line. METHODS: A549 cell was cultured with different concentrations of thaspine. Cellular proliferation was detected with MTT, apoptosis and cell cycle were checked with Flow Cytometer, and change of microstructure was observed by transmission electron microscope. RESULTS: Thaspine could inhibit the proliferation and induce apoptosis of A549 cell in a time-dose dependent manner. Cell cycle was significantly stopped at the S phase by thaspine with FCM technology. Under electronic microscope, the morphology of A549 cell showed nuclear karyopycnosis, chromatin agglutination and typical apoptotic body when the cell was treated with thaspine. CONCLUSION: Thaspine has the effects of anti-tumor and inducing apoptosis.

[Inhibitory effect of taspine on mouse S180 sarcoma and its mechanism].

OBJECTIVE: To study the inhibition effect of taspine on mouse S180 sarcoma and its mechanism. METHOD: The mouse S180 sarcoma model was established and used to observe the antitumor activity of taspine. The microvessel density and protein expressing of the VEGF, bFGF, Bcl-2 and Bax in the tumor were measured by immunohistochemistry. RESULT: Taspine showed antitumor activity on the mouse S180 sarcoma in a good dose-dependent manner. The inhibition rates on tumor of taspine at low, middle and high concentrations were 39.08% , 43.99% and 48.60%, respectively. The microvessel density and protein expressing of the VEGF, bFGF, Bcl-2 and Bax in the tumor were decreased compared with the negative control. The ratio of Bax to Bcl-2 was increased. CONCLUSION: Taspine has antitumor effect on the S180 sarcoma, and the mechanism may be through the way of decreasing the expressing of the VEGF, bFGF, Bcl-2 and Bax and inducing the vascular endothelial cell apoptosis.

[Study on the extraction of the total alkaloids from Caulopyhllum robustum].

OBJECTIVE: To study the technological parameters of the extraction process of the total alkaloids from Caulopyhllum robstum. METHODS: Taspine, whiVh is main component of the total alkaloids from Caulopyhllum robustum, was selected as an evaluating marker and determined by HPLC. The orthogonal test was used to optimize extracting conditions in the process of acid water extraction. Then the optimized conditions for purification using cation exchange resin were investigated. RESULTS: The optimized conditions in the process of acid water extraction were 1% hydrochloric acid as much as seven times of the medicine amount for 24hs and three times. Then the extraction of acid water was purified with a column of macroporous cation exchange resin LSD001 at 2 ml/min of flow rate, then eluted with 10BV of 4% aqueous ammonia ethanol. The extraction ratio of the total alkaloids was 1. 35% and the content of taspine of the total alkaloids was 6. 80%. CONCLUSION: This technology is simply, cheap effective and feasible for manufacture in great scale.

Determination of three bile acids in artificial Calculus Bovis and its medicinal preparations by micellar electrokinetic capillary electrophoresis.

A micellar electrokinetic capillary electrophoresis (MEKCE) method for the determination of cholic acid (CA), hyodeoxycholic acid (HDCA) and chenodeoxycholic acid (CDCA) in artificial Calculus Bovis and its four medicinal preparations is described. The buffer solution consisted of 40 mM disodic phosphate and 40 mM sodium dodecylsulfate (SDS) adjusted to pH 9.0. UV detection was set to 200 nm. Under optimum conditions, the analytes were baseline separated within 11min. The linear calibration range was 12.1-970 microgml(-1) for CA and 18.8-950 microgml(-1) for HDCA and CDCA, respectively. It was found that overall recoveries were within the range of 98-102%, and R.S.D.s were less than 5% for the analytes. This method, due to its convenience, high accuracy and good reproducibility can be employed in quality control of artificial Calculus Bovis and its medicinal preparations.

Ligustilide induces vasodilatation via inhibiting voltage dependent calcium channel and receptor-mediated Ca2+ influx and release.

The purpose of the present study was to investigate the effect of ligustilide on vasodilatation in rat mesenteric artery and the mechanisms responsible for it. Isometric tension of rat mesenteric artery rings was recorded by a sensitive myograph system in vitro. The results showed that ligustilide at concentrations more than 10 microM relaxed potassium chloride (KCl)-preconstricted rat mesenteric artery in a concentration-dependent manner. The vasodilatation effect of ligustilide was not dependent on endothelium. Ligustilide rightwards shifted concentration-response curves induced by KCl, calcium chloride (CaCl(2)), noradrenaline (NA) or 5-hydroxytryptamine (5-HT) in a non-parallel manner. This suggests that the vasodilatation effects were most likely via voltage-dependent calcium channel (VDCC) and receptor-operated calcium channel (ROCC). Propranolol, glibenclamide, tetraethylammonium and barium chloride did not affect the vasodilation induced by ligustilide, showing that beta-adrenoceptor, ATP sensitive potassium channel, calcium-activated potassium channel and inwardly rectifying potassium channel were not involved in the vasodilatation. Ligustilide concentration-dependently inhibited the vasoconstriction induced by NA or CaCl(2) in Ca(2+)-free medium, indicating that the vasodilatation relates to inhibition of extracellular Ca(2+) influx through VDCC and ROCC, and intracellular Ca(2+) release from Ca(2+) store. Since caffeine-induced contraction was inhibited by ligustilide, inhibition of intracellular Ca(2+) released by ligustilide occurred via the ryanodine receptors. Our results suggest that ligustilide induces vasodilatation in rat mesenteric artery by inhibiting the VDCC and ROCC, and receptor-mediated Ca(2+) influx and release.

Inhibitory effects of ligustilide and butylidenephthalide on bFGF-stimulated proliferation of rat smooth muscle cells.

AIM: To investigate the bio-affinities of ligustilide and butylidenephthalide to rat aortic smooth muscle cells and the inhibitory effects of them on bFGF-stimulated proliferation of rat vascular smooth muscle cell (VSMC). METHODS: VSMCs were cultured from rat aorta pectoralis and identified by an immunohistochemical method. The bio-affinities between solute (ligustilide or butylidenephthalide) and cell membrane were measured by rat aortic cell membrane chromatography (CMC). The inhibitory effects of ligustilide and butylidenephthalide on bFGF-stimulated VSMC proliferation were evaluated by MIT colorimetric method. RESULTS: Both ligustilide and butylidenephthalide had selective affinities to rat aortic smooth muscle cell as the same as verapamil, one of the calcium ion antagonists. They could potently inhibit the bFGF-stimulated VSMC proliferation at the concentrations of 5.5 and 11.1 micromol x L(-1), separately (P < 0.05), but had no effects on the normal VSMC growth. CONCLUSION: Both ligustilide and butylidenephthalide can inhibit the abnormal proliferation of VSMC induced by bFGF.

[Pharmacokinetics and tissue distribution of atractylenolide III in rats].

OBJECTIVE: [corrected] To establish an HPLC method for the analysis of pharmacokinetics and tissue distribution of atractylenolide III in rats. METHODS: The biological samples were extracted with ether. The chromatographic conditions were as follows: Hypersil ODS column (150 mm x 4.6 mm, 5 microm) was used. The mobile phase was methnol/warter (67 : 33) with a flow rate of 1.0 ml/min under the column temperature of 25 degrees C, and the detection wavelength was set at 220 nm. RESULTS: The recovery of the method was 85.12% (RSD = 5.57%). The linear range was 0.2 microg/ml - 18.5 microg/ml (r = 0.9996) in rat plasma. The Lowest Limit of detection was 0.10 microg/ ml (S/N > 3). The within-day and between-day precision were from 0.98% to 6.19% and 12.95% to 15.48%, respectively. After oral administration of atractylenolide III (100 mg/kg), the concentration-time profiles of atractylenonlide III fit a two compartment model. In main effect tissues, the atractylenolide III concentration was followed as in order C(lung) > C(cerebellum) > C(heart) > C(cerebrum), and that was C(spleen) > C(liver) > C(kidney) in eliminated tissues. CONCLUSION: The method is accurate, stable and reliable, and can be used for the investigation of atractylenolide III in plasma and tissues of rats.

[Protecting effect of brevifolin and 8,9-single-epoxy brevifolin of Phyllanthus simplex on rat liver injury].

OBJECTIVE: To investigate the effect of protecting liver of brevifolin and 8,9-single-epoxy brevifolin of Phyllanthus simplex. METHOD: Rats were administered with CCl4 (ip) or alcohol (ig) to establish acute or chronic liver injured model, respectively. ALT, AST and TBIL in serum were measured using colorimetric analysis to evaluate liver function. MDA content or SOD activity in serum and liver tissue was measured by thiobarbituric acid chromatometry and xanthine oxidase methods, respectively. The hemorheological parameters were observed. RESULT: Brevifolin and 8,9-single-epoxy brevifolin reduced the increase of ALT induced by CCl4, but they did not influence the increase of AST. And it could inhibit the pathologic increase of serum TBIL induced by alcohol. They could ameliorate the MDA increase or SOD decrease in serum and liver tissue in rats with liver injury, and decrease abnormal changed hemorheological parameters. CONCLUSION: Brevifolin and 8,9-single-epoxy brevifolin show protective effective against acute and chronic liver injuries, and the mechanism is relevant to antagonizing the lipid peroxidation of free radical and improving the blood circulation.