Cytotoxic sesquiterpenoids from the fruits of Lindera communis

A new sesquiterpenoid, namely Linerenone (1), together with three known sesquiterpenoids (2–4), were isolated from the fruits of Lindera communis. Their structures were determined by extensive spectroscopic analysis including 1D, 2D-NMR and HR-MS spectra. Compound 1 showed significant cytotoxic activity against H460, ES2 and DU145 cancer cells with IC₅₀ of 2.1 μg/mL, 2.8 μg/mL and 3.0 μg/mL, respectively.     

Determination of ligustilide in rat brain after nasal administration of essential oil from Rhizoma Chuanxiong

A simple and sensitive HPLC–UV method was developed to determine ligustilide in rat brain samples after nasal administration. The lower limit of quantification (LLOQ) was 245 ng/ml for ligustilide. The calibration curve was linear over a concentration range of 245–4900 ng/ml. Brain samples were obtained at regular time intervals after nasal administration of ligustilide (45 mg/kg). Ligustilide could be detected in rat brain only after 5 min of nasal administration; which showed that ligustilide may have a rapid onset of action. This result illustrates that intranasal administration of ligustilide may act as a promising alternative to conventional routes of administration.     

Pharmacokinetic study of eight coumarins of Radix Angelicae Dahuricae in rats by gas chromatography–mass spectrometry

A sensitive, specific gas chromatography–mass spectrometry (GC–MS) method was established for the quantitative determination of eight coumarins of Radix Angelicae Dahuricae including coumarin, isopsoralen, psoralen, xanthotoxin, bergapten, osthole, imperatorin and oxypeucedanin in rat plasma. Nitrendipine was used as the internal standard (IS). The plasma samples were extracted by acetonitrile. GC separation was accomplished on a DB-5MS column with temperature programmed from 160 °C (17 min) to 190 °C (10 min) at the rate of 20 °C/min, then to 240 °C (5 min) at 20 °C/min, and finally to 280 °C (14 min) at 20 °C/min. The detection was performed on a quadrupole mass spectrometer detector operated by full-scan mode (m/z 50–500). The lower limit of quantitation was 5–10 ng/mL for eight coumarins, and the linear range was 5–1000 ng/mL for the coumarins (R² > 0.9990). All the validation data were within the required limits. After oral administration, the plasma concentration–time curves showed that the time for maximum concentration (T_max) was 1.29 for coumarin, 1.83 for isopsoralen, 1.93 for psoralen, 1.30 for xanthotoxin, 2.04 for bergapten, 0.64 for osthole, 1.41 for imperatorin and 0.51 h for oxypeucedanin. The plasma concentration of the eight coumarins was low, with a mean maximum plasma concentration (C_max) < 6.41 μg/mL. Pharmacokinetic data analysis showed that the eight coumarins had different pharmacokinetic characteristics after oral administration. The method was successfully applied to pharmacokinetic studies of eight coumarins after oral administration in rats.     

嘧啶氧磷对松材线虫的有效杀线活性

通过24孔板测定了几种农药原药和乙醇对松材线虫的杀线活性。试验结果表明嘧啶氧磷杀线活性最高,它的致死中浓度LC50为32．65μg／mL,95％的置信区间为25．08～42．51μg/mL;体积分数为5％的乙醇在20min内使94．4％的松材线虫麻痹,体积分数为10％的乙醇在90S内将96．7％的线虫杀死。另外,松材线虫浸入供试药剂的时间越长,所测定的LC50的值越小,克百威、灭线磷、嘧啶氧磷的LC50值从24h到72h分别由448．47,51．96,94．54μg／mL降低至51．47,36．94,32.65μg／mL。     

In vitro antileishmanial, antiplasmodial and cytotoxic activities of phenolics and triterpenoids from Baccharis dracunculifolia D. C. (Asteraceae)

Baccharis dracunculifolia (Asteraceae), the most important plant source of the Brazilian green propolis (GPE), displayed in vitro activity against Leishmania donovani, with an IC₅₀ value of 45 µg/mL, while GPE presented an IC₅₀ value of 49 µg/mL. Among the isolated compounds of B. dracunculifolia, ursolic acid, and hautriwaic acid lactone showed IC₅₀ values of 3.7 µg/mL and 7.0 µg/mL, respectively. Uvaol, acacetin, and ermanin displayed moderate antileishmanial activity. Regarding the antiplasmodial assay against Plasmodium falciparum, BdE and GPE gave similar IC₅₀ values (about 20 µg/mL), while Hautriwaic acid lactone led to an IC₅₀ value of 0.8 µg/mL (D6 clone).     

Spice oil cinnamaldehyde exhibits potent anticandidal activity against fluconazole resistant clinical isolates

Fluconazole resistance is becoming an important clinical concern. We studied the in vitro effects of cinnamaldehyde against 18 fluconazole-resistant Candida isolates. MIC₉₀ of cinnamaldehyde against different Candida isolates ranged 100–500 μg/ml. Growth and sensitivity of the organisms were significantly affected by cinnamaldehyde at different concentrations. The rapid irreversible action of this compound on fungal cells suggested membrane-located targets for its action. Insight studies to mechanism suggested that cinnamaldehyde exerts its antifungal activity by targeting sterol biosynthesis and plasma membrane ATPase activity. Inhibition of H⁺-ATPase leads to intracellular acidification and cell death. Toxicity against H9c2 rat cardiac myoblasts was studied to exclude the possibility of further associated cytotoxicity. The observed selectively fungicidal characteristics against fluconazole-resistant Candida isolates signify a promising candidature of this essential oil as an antifungal agent in treatments for candidosis.     

In vitro permeability analysis,pharmacokinetic and brain distribution study in mice of imperatorin,isoimperatorin and cnidilin in Radix Angelicae Dahuricae

Coumarins are important constituents of Radix Angelicae Dahuricae, a well-known traditional Chinese medicine possess several known bioactivities with potentials in the treatment of central nervous system diseases. By using an HPLC–MS/MS method, we analyzed the in vivo plasma and brain pharmacokinetics of three ingredients of coumarins, including imperatorin, isoimperatorin and cnidilin in mice after oral administration of Dahuricae extract at doses of 800 mg/kg. The biosamples were prepared using acetonitrile precipitation and the separation was achieved on an XDB-C18 column by gradient elution. The BBB permeability and P-gp-mediated efflux were further examined in Madin Canine kidney cells transfected with full length cDNA for human multidrug resistance gene1 (MDCKII-MDR1). Our results demonstrate that the method has excellent and satisfactory selectivity, sensitivity, linearity, precision, and accuracy for simultaneous determination of imperatorin, isoimperatorin and cnidilin. The pharmacokinetics parameters were determined by using noncompartmental analyses, including the AUC_(0 − t) in plasma (1695.22, 1326.45 and 636.98 mg*h/L), the AUC_(0 − t) in brain (1812.35, 2125.17 and 1145.83 ng*h/g) as well as the T_1/2 in plasma (0.66, 0.82, 0.97 h) and brain (0.96, 1.1, 0.99 h) for imperatorin, isoimperatorin and cnidilin, respectively, suggesting that the three coumarins could easily pass through the BBB in vivo. In the in vitro model we observed high permeability of imperatorin and isoimperatorin with the P-gp-mediated efflux ratios of 0.53 and 0.06, as well as medium permeability of cnidilin with 0.82. All data suggest that these three coumarins have high BBB permeability and have pharmacokinetic potentials for the treatment of central nervous system diseases.     

Effect of rhynchophylline on central neurotransmitter levels in amphetamine-induced conditioned place preference rat brain

The effects of rhynchophylline on expression of amphetamine reward using a conditioned place preference (CPP) paradigm and central neurotransmitter levels in rat brain was investigated. Rats were injected with amphetamine (2 mg/kg, per day for 4 consecutive days) and treated with rhynchophylline (60 mg/kg, per day for the later 3 days). Control rats were administered with rhynchophylline (60 mg/kg) instead of amphetamine to evaluate whether rhynchophylline by itself produced CPP. Glutamic acid, γ-aminobutyric acid, endorphin, acetylcholine, norepinephrine, dopamine, and 5-hydroxytryptamine contents were examined by encephalofluogram technology. Rhynchophylline reversed the expression of amphetamine-induced CPP and itself did not produce a CPP. Glutamic acid, dopamine, and norepinephrine contents in amphetamine-CPP rat brain were significantly higher; while γ-aminobutyric acid, endorphin, and acetylcholine contents were significantly lower than those of control rats. Rhynchophylline reversed those central neurotransmitter levels induced by amphetamine to control levels; rhynchophylline by itself had no effect on central neurotransmitter in control rats. These findings show that rhynchophylline reverses the expression of amphetamine-induced rewarding effect which is partly mediated by regulation of central neurotransmitter levels in the rat brain.     

Comparing indicators of N status of 50 beech stands (Fagus sylvatica L.) in northeastern France

We compared different potential indicators of nitrogen (N) availability across 50 beech forests growing on a wide range of soils in northeastern France. Among the 50 sites measured, high elevation acidic soils had the highest potential net N mineralization in the A horizon (PNM_0–5 cm), while low elevation neutral and calcareous soils had the lowest (PNM_0–5 cm). We found that (PNM_0–5 cm) was negatively correlated with soil pH (R² = 0.47***) and positively correlated with microbial C/N (R² = 0.34***). However, when high elevation sites were excluded from analyses, the relationship between PNM_0–5 cm and soil pH as well as microbial C/N became weaker (R² = 0.23*** for both variables). We found no relationship between PNM_0–5 cm and organic N concentration, soil C/N, or vegetation-based indices for N availability (Ellenberg N and Ecoplant C/N). Bivariate linear regression analyses showed that 69% of the variability in percent nitrification (%Nitrif) was explained by both soil pH (0–5 cm) and soil C/N. Percent nitrification was strongly correlated with vegetation-based indices for N availability. The Ellenberg N and R (pH index) values together explained 74% of the variation in %Nitrif. No relationship was found between %Nitrif and soil δ¹⁵N (natural abundance in ¹⁵N). Of the 76 plant species evaluated, the probability of presence of 61 plant species was significantly correlated with %Nitrif while the probability of presence of 27 plant species only was correlated with PNM_0–5 cm. From these results, we believe that the use of plant community composition or the combination of soil pH and C/N are robust indicators of N availability.     

The 1,4-naphthoquinone derivative from Pyrola rotundifolia activates AMPK phosphorylation in C2C12 myotubes

An aqueous ethanol extract of Pyrola rotundifolia L. induced AMP-activated protein kinase (AMPK) phosphorylation in C2C12 myotubes. The bioassay-guided fractionation of the extract led to the isolation a 2-methyl-7-hydroxymethyl-1,4-naphthoquinone, or a 7′-hydroxy-chimaphilin, which showed concentration-dependent AMPK phosphorylation activity at 2.5–20 μg/ml. At a concentration of 10 μg/ml (50 μM), an approximately four-fold increase in the AMPKα(Thr¹⁷²) phosphorylation level was observed. The stimulatory effect of naphthoquinone on AMPK activity was comparable to that of known compounds found in natural sources that activate the AMPK signaling pathway.     

Theacrine,a purine alkaloid with anti-inflammatory and analgesic activities

The anti-inflammatory and analgesic effects of theacrine (1, 3, 7, 9-tetramethyluric acid), a purine alkaloid which is abundantly present in Camellia kucha, were investigated. Xylene-induced ear edema, acetic acid-induced vascular permeability and λ-carrageenan-induced paw edema were used to investigate anti-inflammatory activity, and acetic acid-induced writhing and hot-plate tests were used to determine analgesic effect. Oral administration of theacrine (8–32 mg/kg) induced dose-related anti-inflammatory and analgesic effects. On the other hand, oral caffeine administration (8–32 mg/kg) did not show an inhibitory effect on the inhibition of inflammatory response or cause analgesia. Additionally, the result of the acute toxicity test showed that the LD₅₀ of theacrine was 810.6 mg/kg (769.5–858.0 mg/kg). The data obtained suggest theacrine possessed analgesic and anti-inflammatory activities.     

Characteristics of nobiletin-induced effects on jejunal contractility

Nobiletin, a citrus polymethoxylated flavone, exhibits multiple biological properties including anti-inflammatory, anti-carcinogenic, and anti-insulin resistance effects. The present study found that nobiletin exerted significant stimulatory effects on the contractility of isolated rat jejunal segments in all 6 different low contractile states, and meanwhile significant inhibitory effects in all 6 different high contractile states, showing characteristics of bidirectional regulation (BR). Nobiletin-exerted BR on jejunal contractility was abolished in the presence of c-kit receptor tyrosine kinase inhibitor imatinib or Ca^2 + channel blocker verapamil. In the presence of neuroxin tetrodotoxin, nobiletin only exerted stimulatory effects on jejunal contractility in both low and high contractile states. Hemicholinium-3 and atropine partially blocked nobiletin-exerted stimulatory effects on jejunal contractility in low-Ca^2 +-induced low contractile state. Phentolamine or propranolol or l-NG-nitro-arginine significantly blocked nobiletin-exerted inhibitory effects on jejunal contractility in high-Ca^2 +-induced high contractile state respectively. The effects of nobiletin on myosin light chain kinase (MLCK) mRNA expression, MLCK protein content, and myosin light chain phosphorylation extent were also bidirectional. In summary, nobiletin-exerted BR depends on the contractile states of rat jejunal segments. Nobiletin-exerted BR requires the enteric nervous system, interstitial cell of Cajal, Ca^2 +, and myosin phosphorylation-related mechanisms.     

Cerebral nuclei distribution study of dehydrodiisoeugenol as an anxiogenic agent determined by RP-HPLC

A sensitive RP-HPLC–DAD method was established to quantify dehydrodiisoeugenol (DDIE) in rat cerebral nuclei. The assay procedure involved one-step extraction of DDIE and daidzein, as an internal standard, from rat plasma and various cerebral nuclei with ethyl acetate. Chromatographic separation was performed on a Diamonsil™ ODS C₁₈ column with methanol–water (81:19, v/v) as a mobile phase. The UV absorbance of the samples was measured at the wavelength of 270 nm. The analysis method was proved to be precise and accurate at linearity ranges in plasma and each cerebral nucleus with correlation coefficients of ≥ 0.9971. The results indicated that the method established was successfully applied to cerebral nuclei distribution study of DDIE after intravenous administration at a single dose of 40 mg/kg to rat. DDIE showed high concentration in all of cerebral nuclei at 8 min, which indicated that DDIE could cross the blood–brain barrier rapidly and might be one of the main bioactive substances of nutmeg. The results provide fundamental data for evaluating the effects of DDIE on the central nervous system and to be developed into an effective anxiogenic agent.     

Pharmacokinetics and tissue distribution study of scoparone in rats by ultraperformance liquid-chromatography with tandem high-definition mass spectrometry

Scoparone is an important constituent of Yinchenhao (Artemisia annua L.), a famous Chinese medicinal plant, and has several known bioactivities, and displayed bright prospects in prevention and therapy of jaundice and liver disorders. The aim of this study was to investigate the in vivo plasma pharmacokinetic and tissue distribution characteristics of scoparone after oral administration. The levels of scoparone in plasma, and tissues were measured by a rapid and sensitive UPLC-MS/MS method. The biosamples were prepared using methanolic precipitation and the separation of scoparone was achieved on a UPLC HSS T3 column by linear gradient elution using water (containing 0.1% formicacid) and acetonitrile (containing 0.1% formic acid) as the mobile phase at a flow rate of 0.5mL/min The total run time was only 3.9min. Our results successfully demonstrate that this method has excellent and satisfactory selectivity, sensitivity, linearity, precision, accuracy and recovery. The estimated pharmacokinetic parameters (i.e., C(max), AUC and CL), were C(max)=14.67mg/L, AUC=81.15mg*h/L, CL=1.23L/h for scoparone. The pharmacokinetic study found that scoparone was distributed and eliminated rapidly in rats. Tissue distribution showed the highest level was observed in liver, followed by the kidney and spleen; the lower level appeared in the muscle, thyroid, and adrenal. It was not detected in the brain which indicated that scoparone does not cross the blood-brain barrier after oral administration. Our developed method was suitable for the study on pharmacokinetics and tissue distribution of scoparone after oral administration.     

Modeling internal temperature changes of timber poles during ACA treatment

Summary The temperature-time-location relationships during steam conditioning and pressure treatment of timber poles using ammoniacal copper arsenate (ACA) have been studied and a new mathematical model that incorporates both the thermal properties of the poles and the parameters of the treatment process is discussed. Prediction equations and charts are presented that show the minimum required steaming time to satisfy the 1982 Rural Electrification Authority (REA) purchase specification, i.e. a center temperature above 150 °F (65.5 °C) for 2 hours. A six hour steaming time, commonly used for ACA treatment, has been found to be too short to bring poles with diameters larger than about 40 cm to the required sterilization conditions. Therefore longer steaming times, predicted using the methods given here, are recommended. The temperature of the preservative used does not appear to be a major factor in determining the maximum temperature achieved at the center of a pole, but it can influence the length of time the pole is above 65.5 °C.Symbols D pole diameter, cm - k₁ rate of surface temperature change during vacuuming, °C/h - K₂ rate of surface temperature change early in the pressure period, °C/h - J₀ Bessel function of the first kind of order zero - J₁ Bessel function of the first kind of order one - r radial location in a pole measured from the center, cm - R pole radius, cm - T interior temperature of a pole during preservative treatment, °C - T_f final preservative temperature, °C - T_mid average of the steam and the final preservative temperatures, °C - t₀ initial temperature of a pole, °C - T_s temperature of the steam used for conditioning the poles, °C - T₁ surface temperature at the end of the vacuum period, °C - t time measured from the opening of the steam valve for steaming, h - t_above time the center of a pole remained above 65.5°C during preservatives treatment, h - t_c1 total time required for the surface temperature to reach T_f, °C - t_pr time elapsed at which initial vacuuming stops and pressure is applied, h - t_stm minimum required steaming time used, h - t_vc time elapsed at which initial vacuuming starts, h - thermal diffusivity, cm²/h - _n Roots of a particular characteristic equation - _d the time delay period between initial flow of steam and surface temperature response, h     

Study on Cytoplasmic polyhedrosis virus onDendrolimus superans

A strain of Cytoplasmic polyhedrosis virus (CPV) was separated from the infected larva during the research of integrated pest management ofDendrolimus superans. The morphology, bioassay, histopathology and field-test for this CPV were studied. The size of CPV is 0.16 μm×1.57 μm and the virion is 16.0 nm×58.1 nm. The L_C50 to the 3rd and 5th instar larva ofDendrolimus superans were 2.81×10⁴ PIB/mL and 7.17×10⁴ PIB/mL respectively. The polyhedrosis were formed after midgut of larva were infected for 72 h. A large amount of polyhedrosis was formed after 144 h. The mortality was more than 82% and average mortality was 84.62% when using 1.17×10⁸ PIB/mL virus suspension to control the pest in field test. The project was supported by the Natural Sciences Foundation of Heilongjiang Province. (Responsible Editor: Chai Ruihai)     

Two new cytotoxic isomeric indole alkaloids from the roots of Nauclea orientalis

A pair of new isomeric indole alkaloids, naucleaorals A (1) and B (2) were isolated from the roots of Nauclea orientalis. The structures of compounds 1 and 2 were fully characterized using spectroscopic data, and were tested for their cytotoxicity (HeLa and KB cells) and antimalarial activity. Compound 1 showed significant cytotoxicity to HeLa cells with an IC₅₀ value of 4.0 µg/mL, while compound 2 exhibited very modest cytotoxicity to both cell lines with IC₅₀ values of 7.8 and 9.5 µg/mL, respectively. Both compounds proved to be inactive in antimalarial assays (IC₅₀ > 10.00 µg/mL).     

Ultra-performance liquid-chromatography with tandem mass spectrometry performing pharmacokinetic and biodistribution studies of croomine,neotuberostemonine and tuberostemonine alkaloids absorbed in the rat plasma after oral administration of Stemonae Radix

Stemonae Radix (Stemona tuberosa Lour, Bai Bu) is an important traditional Chinese medicinal (TCM) plant known for its antitussive activity. Croomine, neotuberostemonine and tuberostemonine alkaloids of Stemonae Radix are major components responsible for antitussive action. In this work, plasma pharmacokinetic and biodistribution characteristics of the three alkaloids after oral administration of Stemonae Radix are investigated using a rapid and sensitive UPLC-Q-TOF–HDMS method. Mass spectrometry (MS) was performed on a Waters Micromass high-definition technology with an electrospray ionization source in positive ion mode, with excellent MS mass accuracy and enhanced MS data acquisition. Separation of main alkaloids was achieved on a Waters BEH C₁₈ column by linear gradient elution. Data were analyzed and estimated by compartmental methods and pharmacokinetic parameters calculated using WinNonlin Professional version 5.1. It was found that croomine, neotuberostemonine and tuberostemonine had faster absorbed into the bloodstream, maintain the high plasma concentration, and pose a large AUC value. The biodistribution of neotuberostemonine and tuberostemonine showed that the higher levels were in liver, and lung. Croomine was discovered in brain and showed that it could cross the blood–brain barrier, indicating that croomine plays an antitussive effect as acting on the central nervous system. Neotuberostemonine and tuberostemonine were not discovered in brain, demonstrating that they play an antitussive effect as peripheral antitussive. This work suggests that the pharmacokinetics and biodistribution based-UPLC-Q-TOF–HDMS can provide a reliable tools for screening bioactive components contributing to pharmacological effects of medicinal herbs.     

Pharmacokinetics of brucine after intravenous and oral administration to rats

The toxicity depending on both dose and administration route is the major obstacle to the development of brucine, a bioactive alkaloid from Semen Strychni. In this study, the apparent partition coefficient and plasma protein binding extent of brucine were determined. In addition, the dose-dependency of the pharmacokinetics of brucine was investigated. Three intravenous (2.5, 5 and 10 mg/kg) and three oral (10, 20 and 40 mg/kg) doses were administered to rats. After intravenous administration, the systemic clearance was reduced and AUC was nonlinearly increased as a function of dose. Upon oral administration, brucine was rapidly absorbed (T_max < 0.5 h), which was consistent with previously reported high Caco-2 P_app values. The increase in AUC was proportional to the increase in dose. The oral bioavailability (F) did not vary with the dose (F = 40.31%, 47.15% and 43.02% for 10, 20, 40 mg/kg doses, respectively). However, the dose-proportionality was not observed with C_max. The values of C_max/Dose were calculated to be 92.92 ± 45.83, 55.73 ± 24.01 and 36.29 ± 22.44 μg/L for 10, 20 and 40 mg/kg, respectively. The results of dose-dependent pharmacokinetic behavior under different administration routes may account for the significantly different toxicities of brucine between intravenous and oral administration.     

Different pharmacokinetics of the two structurally similar dammarane sapogenins,protopanaxatriol and protopanaxadiol,in rats

Dammarane Sapogenins (DS), with main ingredients of protopanaxatriol (PPT, 33%) and protopanaxadiol (PPD, 16%), is an alkaline hydrolyzed product of ginsenosides and had significant activities in improving learning and memory and decreasing chemotherapy-induced myelosuppression. In the present study, the pharmacokinetics and oral bioavailabilities of PPT and PPD were investigated when a single dose of DS was administrated orally (75 mg/kg) and intravenously (i.v., 30 mg/kg) to rats. Their in vitro stabilities in the GI tract were also investigated. PPT and PPD concentrations were measured by LC–MS. The results showed that PPT was eliminated rapidly from the body with an average t_{1/2, λz} value of 0.80 h and CL of 4.27 l/h/kg after i.v. administration, while PPD was eliminated relatively slowly with a t_{1/2, λz} of 6.25 h and CL of 0.98 l/h/kg. After oral administration, both PPD and PPT could be absorbed into the body, but their systemic exposures were quite different. PPT was absorbed into the body quickly, with a T_max of 0.58 h and a C_max of 0.13 μg/ml, while PPD was absorbed relatively slowly with a T_max of 1.82 h and a C_max of 1.04 μg/ml. The absolute bioavailabilities of PPT and PPD were estimated as 3.69% and 48.12%, respectively. The stability test found that PPT was instable in the stomach with 40% degradation after 4 h incubation at 37 °C, both in pH 1.2 buffer and in the stomach content solution. The instability in the stomach might be one of the reasons for PPT's poor bioavailability.