Simultaneous determination of glaucocalyxin A and glaucocalyxin B in rat plasma by LC-MS/MS and its application to a pharmacokinetic study after oral administration of Rabdosia japonica extract.

A specific and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for the analysis of glaucocalyxin A and glaucocalyxin B in rat plasma using praeruptorin A as an internal standard. Separation was performed on a Hypurity C18 column (2.1 × 50 mm, 5 µm) with isocratic elution using 0.2% formic acid in water-acetonitrile (20:80, v/v). Mass spectrometric detection was conducted using selected reaction monitoring via an electrospray ionization source. Both analytes exhibited good linearity within their concentration ranges (r2 > 0.9932). The lower limit of quantitation of glaucocalyxin A and glaucocalyxin B was 1.10 ng/mL. Intra- and inter-day precision exhibited an RSD within 14.5%, and the accuracy (RE) ranged from -12.1 to 15.0% at the lower limit of quantitation and three quality control levels. The developed assay was successfully applied to a pharmacokinetic study of glaucocalyxin A and glaucocalyxin B in rats after oral administration of Rabdosia japonica extract.

GE11 peptide conjugated selenium nanoparticles for EGFR targeted oridonin delivery to achieve enhanced anticancer efficacy by inhibiting EGFR-mediated PI3K/AKT and Ras/Raf/MEK/ERK pathways.

Selenium nanoparticles (Se NPs) have attracted increasing interest in recent decades because of their anticancer, immunoregulation, and drug carrier functions. In this study, GE11 peptide-conjugated Se NPs (GE11-Se NPs), a nanosystem targeting EGFR over-expressed cancer cells, were synthesized for oridonin delivery to achieve enhanced anticancer efficacy. Oridonin loaded and GE11 peptide conjugated Se NPs (GE11-Ori-Se NPs) were found to show enhanced cellular uptake in cancer cells, which resulted in enhanced cancer inhibition against cancer cells and reduced toxicity against normal cells. After accumulation into the lysosomes of cancer cells and increase of oridonin release under acid condition, GE11-Ori-Se NPs were further transported into cytoplasm after the damage of lysosomal membrane integrity. GE11-Ori-Se NPs were found to induce cancer cell apoptosis by inducting reactive oxygen species (ROS) production, activating mitochondria-dependent pathway, inhibiting EGFR-mediated PI3K/AKT and inhibiting Ras/Raf/MEK/ERK pathways. GE11-Se NPs were also found to show active targeting effects against the tumor tissue in esophageal cancer bearing mice. And in nude mice xenograft model, GE11-Ori-Se NPs significantly inhibited the tumor growth via inhibition of tumor angiogenesis by reducing the angiogenesis-marker CD31 and activation of the immune system by enhancing IL-2 and TNF-α production. The selenium contents in mice were found to accumulate into liver, tumor, and kidney, but showed no significant toxicity against liver and kidney. This cancer-targeted design of Se NPs provides a new strategy for synergistic treating of cancer with higher efficacy and reduced side effects, introducing GE11-Ori-Se NPs as a candidate for further evaluation as a chemotherapeutic agent for EGFR over-expressed esophageal cancers.

[Intestinal Absorption Characteristics and Mechanism of Polygala tenuifolia Hydrolysate in Rats].

OBJECTIVE: To evaluate the absorption feature and mechanism of tenuifolin(TF) and polygalaxanthone III (PT) in different intestinal parts of rats and the impact of MRP2 and P-glycoprotein (P-gp) on it. METHODS: In situ unidirectional perfusion was used to detect the concentration of TF and PT through HPLC-DAD with gravimetric method. Furthermore, impact of different parts, cosolvents and inhibitors to TF and PT was also explored with data of Ka and Papp. RESULTS: Tween as cosolvent, Ka and Papp of TF was significantly higher in colon than in other intestinal parts(P <0. 05 or P <0. 01). Whereas, Ka of PT was in sequence of colon, duodenum, jejunum, ileum,but with no significant difference among them(P >0. 05). SDS as cosolvent, Papp of TF was higher in colon than in duodenum(P <0. 05). K. of TF was significantly higher compared with control when added with VH, an inhibitor of P-gp(P <0. 05). In addition, Papp of PT in different concentration of VH increased(P <0. 05, P <0. 01). Papp of TF significantly increased with IT at the concentration of 0. 02 and 0. 04 mmol/L, an inhibitor of MRP2(P <0. 05, P <0. 01). Meanwhile, Ka of PT,with IT at the concentration of 0. 04 and 0. 08 mmol/L, was significantly higher(P <0. 05, P <0. 01). CONCLUSION: TF is mainly absorbed in colon, whereas PT is in duodenum. P-gp but not MRP2 influences the intestinal absorption of TF, indicating TF as substrate of P-gp. However, both of P-gp and MRP2 impact the absorption of PT, illustrating PT as substrate of P-gp and MRP2. It also indicates that inhibitors of P-gp and/or MRP2 in combined application may improve the absorption of PT and TF.

Simultaneous determination of ginsenoside Rb1, naringin, ginsenoside Rb2 and oridonin in rat plasma by LC-MS/MS and its application to a pharmacokinetic study after oral administration of Weifuchun tablet.

A sensitive, specific and rapid liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for analysis of ginsenoside Rb1, naringin, ginsenoside Rb2 and oridonin in rat plasma using sulfamethoxazole as an internal standard (IS). Separation was conducted out on an Agilent Eclipse XDB C18 column with liner gradient elution using acetonitrile (A) and 0.1% aqueous acetic acid (B). A tandem mass spectrometric detection was conducted using multiple reaction monitoring (MRM) via an electrospray ionization (ESI) source. A novel multi-determination-periods program was executed to achieve a higher sensitivity by setting three scanning periods. All analytes exhibited good linearity within the concentration range (r>0.9973). The lower limits of quantitation (LLOQ) of ginsenoside Rb1, naringin, ginsenoside Rb2 and oridonin were 2.64, 4.32, 2.32 and 1.56ng/mL, respectively. Intra-day and inter-day precisions of the investigated components exhibited an RSD within 8.3%, and the accuracy (RE) ranged from -8.6% to 6.0% at all quality control levels. The developed method was successfully applied to a pharmacokinetic study of ginsenoside Rb1, naringin, ginsenoside Rb2 and oridonin in rats after oral administration of a Weifuchun tablet.

Comparative pharmacokinetics study of a kaurane diterpenoid after oral administration of monomer and Siegesbeckiae pubescens Makino extract to rats.

In this paper, a sensitive, rapid and reproducible high-performance liquid chromatography-tandem mass spectrometry method was developed to analyze 16α-hydro-ent-kauran-17,19-dioic acid in rat plasma. First, this study compared the pharmacokinetics of 16α-hydro-ent-kauran-17,19-dioic acid after oral administration of monomer and Siegesbeckiae pubescens Makino extract in rat plasma with approximately the same dosage of 6.0 mg/kg. Second, chromatographic separation was performed on a Waters Symmetry C18 column (2.1 × 100 mm, 3.5 µm) with isocratic elution using methanol-water containing 5 mmol/L ammonium acetate (70:30, v/v) as mobile phase at a flow rate of 0.2 mL/min. The calibration curves were linear over the range of 30-12000 ng/mL for monomer. At different time points (0, 0.083, 0.25, 0.75, 1, 2, 4, 6, 8, 12, 18, 24, 36, 48, 60 and 72 h) after administration, the concentrations of monomer in rat plasma were determined and main pharmacokinetic parameters were estimated. The double absorption presented in this study indicates that the pharmacokinetics of monomer in rat plasma have significant differences between different groups.

Simultaneous quantification of Kirenol and ent-16ß,17-dihydroxy-kauran-19-oic acid from Herba Siegesbeckiae in rat plasma by liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic studies.

A rapid and specific liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method was developed for the simultaneous determination of two active diterpenoids: Kirenol and ent-16ß,17-dihydroxy-kauran-19-oic acid (DHKA) from Herba Siegesbeckiae in rat plasma using osthole as an internal standard (IS). Plasma sample pretreatment involved a one-step liquid-liquid extraction with ethyl acetate. Chromatographic separation was performed on a Waters Symmetry C18 column (2.1mm×100mm, 3.5µm) with isocratic elution using methanol-5mmol/L aqueous ammonium acetate (80:20, v/v) as the mobile phase at a flow rate of 0.2mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode under positive and negative electrospray ionization. The calibration curves were linear over the range of 50.0-25,000ng/mL for Kirenol, and 25.0-12,500ng/mL for DHKA. The extraction recoveries of the two analytes and the IS were all over 85%. The intra- and inter-day precision (relative standard deviation) values were less than 16.8% and the accuracy (relative error) ranged from -10.7 to 10.6% at four quality control levels. The validated method was successfully applied to a comparative pharmacokinetic study of the two diterpenoids in rat plasma after intragastric administration of Kirenol, DHKA and Herba Siegesbeckiae extract. The results showed that there were obvious differences between the pharmacokinetic behaviors after oral administration of Herba Siegesbeckiae extract compared with each of the substances alone.

RGD-modified poly(D,L-lactic acid) nanoparticles enhance tumor targeting of oridonin.

OBJECTIVE: The purpose of this study was to develop an active targeting strategy to improve the therapeutic antitumor efficacy of oridonin (ORI), the main active ingredient in the medicinal herb Rabdosia rubescens. METHODS: A modified spontaneous emulsification solvent diffusion method was used to prepare the ORI-loaded atactic poly(D,L-lactic acid) nanoparticles (ORI-PLA-NPs). Surface cross-linking with the peptide Arg-Gly-Asp (RGD) further modified the ORI-PLA-NPs, generating ORI-PLA-RGD-NPs. The NPs were characterized and release experiments were performed in vitro. The pharmacokinetics, tissue distribution, and antitumor activity of the NPs were studied in mice bearing hepatocarcinoma 22 (H22)-derived tumors. RESULTS: The ORI-PLA-NPs and ORI-PLA-RGD-NPs were smooth, sphere-like, and relatively uniform in size. The RGD surface modification slightly increased the mean particle size (95.8 nm for ORI-PLA-NPs versus 105.2 nm for ORI-PLA-RGD-NPs) and considerably altered the surface electrical property (-10.19 mV for ORI-PLA-NPs versus -21.95 mV for ORI-PLA-RGD-NPs), but it had no obvious influence on ORI loading (8.23% ± 0.35% for ORI-PLA-NPs versus 8.02% ± 0.38% for ORI-PLA-RGD-NPs), entrapment efficiency (28.86% ± 0.93% for ORI-PLA-NPs versus 28.24% ± 0.81% for ORI-PLA-RGD-NPs), or the release of ORI. The pharmacokinetic properties of free ORI were improved by encapsulation in NPs, as shown by increased area under the concentration-time curve (11.89 ± 0.35 µg·mL(-1) · h for ORI solution versus 22.03 ± 0.01 µg · mL(-1) · h for ORI-PLA-RGD-NPs) and prolonged mean retention time (2.03 ± 0.09 hours for ORI solution versus 8.68 ± 0.66 hours for ORI-PLA-RGD-NPs). In the tissue distribution study, more ORI targeted tumor tissue in the mice treated with ORI-PLA-RGD-NPs than with ORI-PLA-NPs or ORI solution. Consistent with these observations, ORI-PLA-RGD-NPs showed greater antitumor efficacy than ORI-PLA-RGD-NPs or ORI solution, as reflected by the decreased tumor growth and the prolonged survival time of mice bearing H22 tumors. CONCLUSION: The tumor-targeting efficiency and subsequent antitumor efficacy of ORI is increased by incorporation into ORI-PLA-RGD-NPs.

[Study on pharmacokinetics of oridonin in mice].

OBJECTIVE: To develop a HPLC method for the determination of plasma concentration of oridonin (ORI) and study the pharmacokinetics of ORI in mice. METHOD: Blood was sampled from mice which were injected ORI by 10 mg x kg(-1) at different time intervals, and the concentration of ORI was determined by HPLC. The pharmacokinetic parameters were accessed by 3P97. RESULT: The calibration curve was linear (r = 0.998 7) within the range of 0.202-20.0 mg x L(-1) for ORI in plasma. The average recoveries were more than 93%. The within-day and between-day precisions were no more than 9%. After i.v. oridonin in mice, the plasma concentration-time course fitted well to two-compartment model. The pharmacokinetic equation was C = 16.192 5e(-0.554 6t) + 5.475 7e(-0.016 3t). The pharmacokinetic parameters were below: t1/2alpha 1.249 9 min, t1/2beta 42.638 4 min, K21 0.152 3 min(-1), K12 0.359 3 min(-1), K10 0.0592 min(-1), AUC 366.035 0 microg x min x mL(-1), CL 0.0273 L x min(-1) x kg(-1), V(c)0.461 5 L x kg(-1). CONCLUSION: The method can be used to determine the concentration and to investigate the pharmacokinetics of ORI in mice. ORI was absorbed and distributed very fast in mice. The effect of ORI was rapid. The elimination was the main process.

Stevioside and related compounds: therapeutic benefits beyond sweetness.

Stevioside, an abundant component of Stevia rebaudiana leaf, has become well-known for its intense sweetness (250-300 times sweeter than sucrose) and is used as a non-caloric sweetener in several countries. A number of studies have suggested that, beside sweetness, stevioside along with related compounds, which include rebaudioside A (second most abundant component of S. rebaudiana leaf), steviol and isosteviol (metabolic components of stevioside) may also offer therapeutic benefits, as they have anti-hyperglycemic, anti-hypertensive, anti-inflammatory, anti-tumor, anti-diarrheal, diuretic, and immunomodulatory actions. It is of interest to note that their effects on plasma glucose level and blood pressure are only observed when these parameters are higher than normal. As steviol can interact with drug transporters, its role as a drug modulator is proposed. This review summarizes the current knowledge of the pharmacological actions, therapeutic applications, pharmacokinetics and safety of stevioside and related compounds. Although much progress has been made concerning their biological and pharmacological effects, questions regarding chemical purity and safety remain unsolved. These issues are discussed to help guide future research directions.

Studies on pharmacokinetics and tissue distribution of oridonin nanosuspensions.

The purpose of the present study was to investigate the effects of particle size on the pharmacokinetics and tissue distribution of oridonin nanosuspensions after intravenous administration. Two oridonin nanosuspensions with markedly different size were prepared by high pressure homogenization method. The particle size of nanosuspension A is 103.3+/-1.5nm, while B is 897.2+/-14.2nm. Dissolution studies showed that complete dissolution could be obtained within 10min for nanosuspension A, however, nanosuspension B showed a slower dissolution, only 85.2% dissolved by 2h. The pharmacokinetics and tissue distribution of oridonin nanosuspensions A and B were studied after intravenous administration using New Zealand rabbits and Kunming mice as experimental animals, respectively. An Oridonin control solution was studied parallelly. The results showed that oridonin nanosuspension A exhibited pharmacokinetic and biodistribution properties similar to solution due to its rapid dissolution in blood circulation. Oridonin nanosuspension B, however, showed a high uptake in RES organs, meanwhile exhibited a markedly different pharmacokinetic property compared to nanosuspension A. These differences could be attributed to the different particle size of the two nanosuspensions considering their zeta potential had no significant difference. In conclusion, particle size showed obvious effects on pharmacokinetics and tissue distribution of nanosuspensions.

Farmacología de Terpenoides de Helianthus annuus L / Pharmacology of Terpenoids from Helianthus annuus L

Desde los albores de la humanidad, las plantas han sido fuente de interés porsus características nutricionales y medicinales. Por ello, se realizó un estudio farmacológicode terpenoides de Helianthus annuus L., optimizando un método deextracción, aislamiento y purificación de dichos metabolitos, para evaluar unaposible actividad farmacológica sobre modelos de inflamación. También se evaluóuna potencial actividad antiinflamatoria utilizando extractos (metanólico y acuoso)de la planta. Se aislaron tres diterpenos, dos kauranos y un traquilobano, mediantetécnicas cromatográficas, identificándose sus estructuras por RMN (1H, 13C) y evaluando su pureza por HPLC. Paralelamente se evaluó la variación estacional delácido grandiflorólico y de la mezcla de ácido kaurenoico y traquilobánico obtenida,en diferentes sumidades de la planta durante su desarrollo, con el fin de determinardónde y cuándo se produce una máxima acumulación del metabolito estudiado,para así obtener una extracción con un mayor rendimiento. Se encontrarondiferencias significativas útiles para el control de calidad de preparados a base deplantas medicinales que contengan algunos de estos metabolitos secundarios. Parael estudio de la actividad farmacológica de los terpenoides y de los extractos, setrabajó sobre dos modelos de inflamación (in vivo e in vitro). En el modelo in vivose evaluó la acción de los compuestos sobre el edema producido por aplicacióntópica de TPA en oreja de ratón, evaluando en paralelo la infiltración leucocitariaen términos de actividad mieloperoxidasa. En este modelo los ácidos kaurenoicoy traquilobánico demostraron actividad antiinflamatoria. En el modelo in vitro seutilizaron cultivos celulares de una línea de macrófagos, evaluando la actividad delos compuestos sobre la liberación de mediadores inflamatorios (NO, PGE2 y TNFa),y sobre la enzima COX, donde se observó que el ácido grandiflorólico y elextracto metanólico mostraron actividad inhibidora sobre la liberación de estosmediadores, además el ácido grandiflorólico mostró actividad inhibidora de laenzima COX. Sin embargo, no se descarta que en las actividades antiinflamatoriasde dichos compuestos estén implicados otros mediadores

Since the dawn of the humanity plants have been a source of interest becauseof their nutritional and medicinal characteristics. For that reason, a pharmacologicstudy of the terpenoids of Helianthus annuus L. was done, optimizing a method ofextraction, isolation and purification of these metabolites in order to evaluate apossible pharmacologic activity in inflammation models. Also, potentialantiinflammatory activity was evaluated using extracts (methanolic and watery) ofthe plant. Three diterpenes, two kauranes and a trachylobane were isolated, bymeans of chromatographic techniques; their structures were identified by RMN(1H, 13C) and their purity was evaluated by HPLC. Parallelly, the seasonal variationof grandiflorolic acid and kaurenoic and the trachylobanic acid mixture obtainedwas evaluated, in different above-ground parts from the plant during itsdevelopment, with the purpose of determining where and when the highestaccumulation of the studied metabolite occurs thus to obtain an extraction withthe greatest yield. Useful significant differences were found for the quality controlof preparations with medicinal plants that contain some of these secondarymetabolites. For the study of the pharmacologic activity of the terpenoids and theextracts, two models of inflammation (in vivo and in vitro) were worked on. In thein vivo model, the action of compounds was evaluated on edema produced by topical application of TPA in mouse ear, evaluating in parallel the leucocitaryinfiltration in terms of mieloperoxidase activity.In this model the kaurenoic and trachylobanic acids demonstratedantiinflammatory activity. In the in vitro model cellular cultures of a line ofmacrophages were used, the activity of compounds on the liberation ofinflammatory mediators (NO, PGE2 and TNF-a), and the COX enzyme wereevaluated, where it was observed that the grandiflorolic acid and the methanolicextract showed inhibiting activity on the liberation of these mediators. Also, thegrandiflorolic acid showed inhibiting activity on the COX enzyme. However, onedoes not rule out other mediators being implicated in the antiinflammatory activities of these said compounds

Pharmacokinetic behaviors and oral bioavailability of oridonin in rat plasma.

AIM: To study the intravenous and oral pharmacokinetic behavior of oridonin and its extent of absolute oral bioavailability in rats. METHODS: Oridonin was administered to rats via iv (5, 10 and 15 mg/kg), po (20, 40 and 80 mg/kg) or ip administration (10 mg/kg). The concentrations of oridonin in rat plasma were determined by a high performance liquid chromatography with electrospray ionization mass spectrometric detection (HPLC/ESI-MS) method and the pharmacokinetic parameters were determined by non-compartmental analysis. RESULTS: The plasma concentration of oridonin after intravenous administration decreased polyexponentially, and the pharmacokinetic parameters of oridonin were dose-independent within the examined range. Oridonin was absorbed rapidly after oral gavage with a t(max) of less than 15 min; the extent of absolute bioavailability of oridonin following oral administration was 4.32%, 4.58% and 10.8%. The extent of absolute bioavailability of oridonin following intraperitoneal administration was 12.6%. CONCLUSION: First order rate pharmacokinetics were observed for oridonin within the range of iv doses, while the extent of absolute oral bioavailability was rather low and dose- dependent. The low and dose-dependent extent of oral bioavailability may be due to the saturation of first-pass effects.

[The tissue distribution in mice and pharmacokinetics in rabbits of oridonin-solid lipid nanoparticles].

AIM: To investigate the tissue distribution and pharmacokinetics of oridonin-solid lipid nanoparticles in animals. METHODS: HPLC method was established to determine the concentration of oridonin in serum of rabbits and in different tissues of mice. The results after tail iv administration of oridonin and oridonin solid lipid nanoparticles were compared. RESULTS: The relative tissue content of oridonin of solid lipid nanoparticles in the liver, spleen, lung, heart and kidney were 4.25%, 3.44%, 1.19%, 0.52% and 0.60%, respectively. The concentration-time curves of oridonin and oridonin solid lipid nanoparticles were both fitted to the three-compartment model. T(1/2)pi = 0.087 h, T(1/2)alpha = 1.65 h, T(1/2)beta = 32.36 h, V(C) = 0.66 mL.kg(-1). CONCLUSION: Solid lipid nanoparticles could increase the hepatic and lienic targeting efficiency of oridonin in mice and improve its bioavailability. Solid lipid nanoparticles were helpful for oridonin to reach a long circulation time and were hopeful to be its novel drug carrier.