Pharmacology of apocynin: a natural acetophenone.

Apocynin is a naturally occurring acetophenone, found in the roots of Apocynum cannabinum and Picrorhiza kurroa. Various chemical and pharmaceutical modifications have been carried out to enhance the absorption and duration of action of apocynin, like, formulation of chitosan-based apocynin-loaded solid lipid nanoparticles, chitosan-oligosaccharide based nanoparticles, and biodegradable polyanhydride nanoparticles. Apocynin has been subjected to a wide range of experimental screening and has proved to be useful for amelioration of a variety of disorders, like diabetic complications, neurodegeneration, cardiovascular disorders, lung cancer, hepatocellular cancer, pancreatic cancer, and pheochromocytoma. Apocynin has been primarily reported as an NADPH oxidase (NOX) inhibitor and prevents translocation of its p47phox subunit to the plasma membrane, observed in neurodegeneration and hypertension. However, recent studies highlight its off-target effects that it is able to function as a scavenger of non-radical oxidant species, which is relevant for its activity against NOX 4 mediated production of hydrogen peroxide. Additionally, apocynin has shown inhibition of eNOS-dependent superoxide production in diabetic cardiomyopathy, reduction of NLRP3 activation and TGFß/Smad signaling in diabetic nephropathy, diminished VEGF expression and decreased retinal NF-κB activation in diabetic retinopathy, inhibition of P38/MAPK/Caspase3 pathway in pheochromocytoma, inhibition of AKT-GSK3ß and ERK1/2 pathways in pancreatic cancer, and decreased FAK/PI3K/Akt signaling in hepatocellular cancer. This review aims to discuss the pharmacokinetics and mechanisms of the pharmacological actions of apocynin.

Apocynin inhibits placental TLR4/NF-κB signaling pathway and ameliorates preeclampsia-like symptoms in rats.

OBJECTIVE: We aimed to investigate the potency of apocynin in ameliorating preeclampsia and explore the underlying mechanisms. METHODS: Preeclampsia model was constructed in rats by administering 200 mg/kg/day L-NAME. Apocynin was given orally in drinking water. Systolic blood pressure and proteinuria were monitored during treatment. Survival rate rate of the pups and placental weight were assessed. Serum sFlt-1, PIGF, IL-6 and placental TLR4 levels were measured using ELISA or qRT-PCR. RESULTS: Apocynin dose-dependently decreased systolic blood pressure and proteinuria during gestation. Survival rate of the pups and placental weight were improved by apocynin treatment. Apocynin ameliorated the imbalance of sFlt-1 and PIGF in serum and placenta of rats with preeclampsia. Apocynin attenuated serum inflammatory cytokine expression and placental inflammation most likely due to downregulation of the placental TLR4/NF-kB pathway in L-NAME treated rats. CONCLUSIONS: Apocynin potently ameliorates the L-NAME-induced preeclampsia, which is achieved by re-balancing the sFlt-1 and PIGF levels, attenuating inflammation, and inhibiting TLR4/NF-κB p65 signaling.

Finite Element Analysis for Predicting Skin Pharmacokinetics of Nano Transdermal Drug Delivery System Based on the Multilayer Geometry Model.

BACKGROUND: Skin pharmacokinetics is an indispensable indication for studying the drug fate after administration of transdermal drug delivery systems (TDDS). However, the heterogeneity and complex skin structured with stratum corneum, viable epidermis, dermis, and subcutaneous tissue inevitably leads the drug diffusion coefficient (Kp) to vary depending on the skin depth, which seriously limits the development of TDDS pharmacokinetics in full thickness skin. METHODS: A multilayer geometry skin model was established and the Kp of drug in SC, viable epidermis, and dermis was obtained using the technologies of molecular dynamics simulation, in vitro permeation experiments, and in vivo microdialysis, respectively. Besides, finite element analysis (FEA) based on drug Kps in different skin layers was applied to simulate the paeonol nanoemulsion (PAE-NEs) percutaneous dynamic penetration process in two and three dimensions. In addition, PAE-NEs skin pharmacokinetics profile obtained by the simulation was verified by in vivo experiment. RESULTS: Coarse-grained modeling of molecular dynamic simulation was successfully established and the Kp of PAE in SC was 2.00×10-6 cm2/h. The Kp of PAE-NE in viable epidermis and in dermis detected using penetration test and microdialysis probe technology, was 1.58×10-5 cm2/h and 3.20×10-5 cm2/h, respectively. In addition, the results of verification indicated that PAE-NEs skin pharmacokinetics profile obtained by the simulation was consistent with that by in vivo experiment. DISCUSSION: This study demonstrated that the FEA combined with the established multilayer geometry skin model could accurately predict the skin pharmacokinetics of TDDS.

Development and Validation of an LC-MS/MS Assay to Quantitate 2',4',6'-Trihydroxyacetophenone in Rat and Dog Plasma and its Application to a Pharmacokinetic Study.

In the present study, a simple, rapid, and reliable bioanalytical method was developed using liquid chromatography with tandem-mass spectrometry (LC-MS/MS) to quantify 2',4',6'-trihydroxyacetophenone (THAP) in rat and dog plasma with 2',4',6'-trihydroxybenzaldehyde as an internal standard (IS). The LC-MS/MS instrument was operated in the multiple reaction monitoring (MRM) mode to detect THAP at m/z transition 166.89 > 82.8 and IS at 152.89 > 82.8, respectively. A simple, one-step protein precipitation (PP) method was employed with acetonitrile for sample preparation. Utilizing a Gemini C18 column, THAP and IS were separated with an isocratic mobile phase consisting of 10 mM ammonium acetate and methanol (10:90, v/v) at a flow rate of 0.2 mL/min. Total chromatographic run time was 2.5 min per sample injection. The standard calibration curve for THAP was linear (r2 ≥ 0.9987) over the concentration range of 0.1 to 100 µg/mL with the lower limit of quantitation (LLOQ) of 0.1 µg/mL (S/N ratio > 10). According to the regulatory guidelines from the U.S. Food and Drug Administration (FDA) and the Korea Ministry of Food and Drug Safety (MFDS), our newly developed biomedical analytical method was fully and adequately validated in terms of selectivity, sensitivity, linearity, intra- and inter-day precision and accuracy, recovery, matrix effect, stability, and dilution integrity. Our validated assay was successfully utilized in a nonclinical pharmacokinetic study of THAP in rats and dogs.

In vivo and in silico characterization of apocynin in reducing organ oxidative stress: A pharmacokinetic and pharmacodynamic study.

Apocynin has been widely used in vivo as a Nox2-contaninig nicotinamide adenine dinucleotide phosphate oxidase inhibitor. However, its time-dependent tissue distribution and inhibition on organ reactive oxygen species (ROS) production remained unclear. In this study, we examined apocynin pharmacokinetics and pharmacodynamics (PKPD) after intravenous (iv) injection (bolus, 5 mg/kg) of mice (CD1, 12-week). Apocynin was detected using a HPLC coupled to a linear ion-trap tandem mass spectrometer. Apocynin peak concentrations were detected in plasma at 1 minute (5494 ± 400 ng/mL) (t1/2  = 0.05 hours, clearance = 7.76 L/h/kg), in urine at 15 minutes (14 942 ± 5977 ng/mL), in liver at 5 minutes (2853 ± 35 ng/g), in heart at 5 minutes (3161 ± 309 ng/g) and in brain at 1 minute (4603 ± 208 ng/g) after iv injection. These were accompanied with reduction of ROS production in the liver, heart and brain homogenates. Diapocynin was not detected in these samples. Therapeutic effect of apocynin was examined using a mouse model (C57BL/6J) of high-fat diet (HFD, 16 weeks)-induced obesity and accelerated aging. Apocynin (5 mmol/L) was supplied in drinking water during the HFD period and was detected at the end of treatment in the brain (5369 ± 1612 ng/g), liver (4818 ± 1340 ng/g) and heart (1795 ± 1487 ng/g) along with significant reductions of ROS production in these organs. In conclusion, apocynin PKPD is characterized by a short half-life, rapid clearance, good distribution and inhibition of ROS production in major organs. Diapocynin is not a metabolite of apocynin in vivo. Apocynin crosses easily the blood-brain barrier and reduces brain oxidative stress associated with metabolic disorders and aging.

Pharmacokinetics and Metabolism of Liposome-Encapsulated 2,4,6-Trihydroxygeranylacetophenone in Rats Using High-Resolution Orbitrap Liquid Chromatography Mass Spectrometry.

2,4,6-trihydroxy-3-geranylacetophenone (tHGA) is a bioactive compound that shows excellent anti-inflammatory properties. However, its pharmacokinetics and metabolism have yet to be evaluated. In this study, a sensitive LC-HRMS method was developed and validated to quantify tHGA in rat plasma. The method showed good linearity (0.5-80 ng/mL). The accuracy and precision were within 10%. Pharmacokinetic investigations were performed on three groups of six rats. The first two groups were given oral administrations of unformulated and liposome-encapsulated tHGA, respectively, while the third group received intraperitoneal administration of liposome-encapsulated tHGA. The maximum concentration (Cmax), the time required to reach Cmax (tmax), elimination half-life (t1/2) and area under curve (AUC0-24) values for intraperitoneal administration were 54.6 ng/mL, 1.5 h, 6.7 h, and 193.9 ng/mL·h, respectively. For the oral administration of unformulated and formulated tHGA, Cmax values were 5.4 and 14.5 ng/mL, tmax values were 0.25 h for both, t1/2 values were 6.9 and 6.6 h, and AUC0-24 values were 17.6 and 40.7 ng/mL·h, respectively. The liposomal formulation improved the relative oral bioavailability of tHGA from 9.1% to 21.0% which was a 2.3-fold increment. Further, a total of 12 metabolites were detected and structurally characterized. The metabolites were mainly products of oxidation and glucuronide conjugation.

Chemoreactive-Inspired Discovery of Influenza A Virus Dual Inhibitor to Block Hemagglutinin-Mediated Adsorption and Membrane Fusion.

Owing to the emergence of drug resistance and high morbidity and mortality, the need for novel anti-influenza A virus (IAV) drugs with divergent targets is highly sought after. Herein, we reveal the discovery of an anti-IAV agent as a dual inhibitor to block hemagglutinin-mediated adsorption and membrane fusion using a chemoreactive ortho-quinone methide (o-QM) equivalent. Based on the o-QM equivalent nonenzymatically multipotent behavior, we created a series of clavatol-derived pseudo-natural products and found that penindolone (PND), a new diclavatol indole adduct, exhibited potent and broad-spectrum anti-IAV activities with low risk of inducing drug resistance. Distinct from current anti-IAV drugs, PND possesses a novel scaffold and is the first IAV inhibitor targeting both HA1 and HA2 subunits of virus hemagglutinin to dually block the IAV adsorption and membrane fusion process. More importantly, intranasal and oral administration of PND can protect mice against IAV-induced death and weight loss, superior to the effects of the clinical drug oseltamivir. Thus, the use of chemoreactive intermediates could expand our understanding of chemical diversity and aid in the development of anti-IAV drugs with novel targets.

Apocynin and catalase prevent hypertension and kidney injury in Cyclosporine A-induced nephrotoxicity in rats.

Oxidative stress is involved in the pathogenesis of a number of diseases including hypertension and renal failure. There is enhanced expression of nicotinamide adenine dinucleotide (NADPH oxidase) and therefore production of hydrogen peroxide (H2O2) during renal disease progression. This study investigated the effect of apocynin, an NADPH oxidase inhibitor and catalase, an H2O2 scavenger on Cyclosporine A (CsA) nephrotoxicity in Wistar-Kyoto rats. Rats received CsA (25mg/kg/day via gavage) and were assigned to vehicle, apocynin (2.5mmol/L p.o.), catalase (10,000U/kg/day i.p.) or apocynin plus catalase for 14 days. Renal functional and hemodynamic parameters were measured every week, and kidneys were harvested at the end of the study for histological and NADPH oxidase 4 (NOX4) assessment. Oxidative stress markers and blood urea nitrogen (BUN) were measured. CsA rats had higher plasma malondialdehyde (by 340%) and BUN (by 125%), but lower superoxide dismutase and total antioxidant capacity (by 40%, all P<0.05) compared to control. CsA increased blood pressure (by 46mmHg) and decreased creatinine clearance (by 49%, all P<0.05). Treatment of CsA rats with apocynin, catalase, and their combination decreased blood pressure to near control values (all P<0.05). NOX4 mRNA activity was higher in the renal tissue of CsA rats by approximately 63% (P<0.05) compared to controls but was reduced in apocynin (by 64%), catalase (by 33%) and combined treatment with apocynin and catalase (by 84%) compared to untreated CsA rats. Treatment of CsA rats with apocynin, catalase, and their combination prevented hypertension and restored renal functional parameters and tissue Nox4 expression in this model. NADPH inhibition and H2O2 scavenging is an important therapeutic strategy during CsA nephrotoxicity and hypertension.

Chemistry, pharmacokinetics, pharmacology and recent novel drug delivery systems of paeonol.

Paeonol is a bioactive phenol present in Dioscorea japonica, Paeonia suffruticosa and Paeonia lactiflora. It is reported for various pharmacological activities. AIM: To review chemistry, pharmacokinetics, pharmacological activities as well as various formulations of paeonol. MATERIALS AND METHODS: A literature search was done using different search terms for paeonol by using different scientific databases like PubMed, Scopus and ProQuest. Scientific papers published during the period 1969 to 2019 were comprehensively reviewed. KEY FINDINGS: Researchers have synthesized methoxy, ethoxy, piperazine, chromonylthiazolidine, phenol-phenylsulfonyl, alkyl ether, aminothiazole, tryptamine hybrids and paeononlsilatie derivatives to enhance the stability of paeonol. These derivatives were synthesized and evaluated for in vitro series of biological activities like anti-inflammatory, tyrosinase inhibitory, neuroprotective, anticancer and antiviral activity. Regardless of valuable therapeutic potential, the clinical use of paeonol is restricted due to poor water solubility, low oral bioavailability, low stability and high volatility at room temperature. To enhance the bioavailability of paeonol various formulations are prepared and evaluated for its activity. Paeonol formulations can be categorized as conventional-tablets, topical gel and hydrogel; polymeric delivery system-microparticles, microsponges, dendrimers, nanocapsules, polymeric nanoparticles, nanospheres; lipid-based delivery systems-microemulsion, self-micro-emulsifying drug delivery, liposome, transethosomes, ethosomes, niosomes, proniosomes, lipid-based nanoparticles and nanoemulsion of paeonol. SIGNIFICANCE: Paeonol has a potential to be developed as a techno-commercial product with respect to its multi-faceted pharmacological properties. Even though in vitro and in vivo studies have been reported the important activities of paeonol, its commercial utilization requires extensive safety and efficacy data.

Lipopolysaccharide-induced depressive-like, anxiogenic-like and hyperalgesic behavior is attenuated by acute administration of α-(phenylselanyl) acetophenone in mice.

The lipopolysaccharide (LPS) is an endotoxin derived from gram-negative bacteria, which induces inflammation. The aims of this study were to evaluate the possible α-(phenylselanyl) acetophenone (PSAP) activity in reducing comorbid hyperalgesia, depressive-like and anxiogenic-like symptoms induced by LPS in mice. In additional, investigated physical chemical properties of PSAP through in silico analysis by ADMET predictor software. The LPS (100 µg/kg, intraperitoneally) or saline were administered and after 4 h the treatment with PSAP (0.001-10 mg/kg, intragastric route [i.g.]) or FLX (10 mg/kg, i.g.) was performed, and after 30 min, the behavioral tests were carried out. LPS reduced the latency time for the first episode of immobility and increased the immobility time in the FST as well as decreased the grooming time in the splash test. PSAP reversed these alterations demonstrating an antidepressive-like effect. LPS also enhances the anxiogenic behavior in the elevated plus maze test (EPM). PSAP reversed these parameters, showing anxiolytic-like effect. LPS also decreased the latency time (s) on the hot plate and the treatment with PSAP at all doses significantly reversed the hyperalgesic effect of LPS. LPS increased the activation of p38MAPK and p-p65NF-κB pathways as well as the COX-2 levels in the cerebral cortex, which are indicative of an inflammatory response. Besides, it also reduced the levels of mBDNF, involved in neuroplasticity. Treatment with PSAP restored all these neurochemical alterations induced by LPS. The results demonstrated that PSAP presents antidepressive-like, anxiolytic-like and anti-hyperalgesic effects related to reduction in neuroinflammation.

Novel chitosan-based solid-lipid nanoparticles to enhance the bio-residence of the miraculous phytochemical "Apocynin".

Apocynin (APO), a specific NADPH oxidase inhibitor, is a bioactive phytochemical that exhibits versatile pharmacological activities. However, its rapid elimination and poor bioavailability represent great challenges to pharmaceutical scientists. Accordingly, novel chitosan-based APO-loaded solid lipid nanoparticles (CS,APO - loaded SLNS) were developed to address such obstacles. A full 24 factorial design of experiment approach was employed to evaluate the individual and combined effect of critical process parameters namely; the amount of glycerol tristearate (GTS, XA) and sucrose mono palmitate (SMP, XB) as well as the concentration of chitosan (CS, XC) and polyvinyl alcohol (PVA, XD), on different critical quality attributes. Full characterization and extensive in vitro-in vivo evaluations of the optimized SLNs formula were conducted. The optimized formula, with core (CS,APO) and shell (PVA), has enhanced oral and intravenous bioavailability in rats as clearly verified when compared with APO solution. Probably, PVA hindered opsonization intravenously and SLNs reduced pre-systemic effect. In conclusion, the novel chitosan-based SLNs system would open new vistas in potentiating the bioavailability and sustaining the effect of APO and other bioactive phytochemicals with comparable properties.

Paeonol-Loaded Ethosomes as Transdermal Delivery Carriers: Design, Preparation and Evaluation.

Paeonol exhibits a wide range of pharmacological activities, such as anti-inflammatory, antidiabetic as well as pain-relieving activities. However, its intrinsic properties, such as low water solubility, poor stability and low oral bioavailability, restrict its clinical application. The current study aimed to optimize paeonol-loaded ethosomal formulation and characterize it in terms of encapsulation efficiency (EE), vesicle size (VS), zeta potential (ZP) and polydispersity index (PDI), in addition to differential scanning calorimetry (DSC), X-ray diffraction (XRD) and Fourier-transform infrared spectroscopy (FT-IR) studies. Here, paeonol-loaded ethosomes were prepared by the injection method and optimized by the single-factor test and central composite design-response surface methodology. The optimized paeonol-loaded ethosomes had an EE of 84.33 ± 1.34%, VS of 120.2 ± 1.3 nm, negative charge of -16.8 ± 0.36 mV, and PDI of 0.131 ± 0.006. Ethosomes showed a spherical morphology under the transmission electron microscope (TEM). DSC, XRD and FT-IR results indicated that paeonol was successfully incorporated into the ethosomes. In-vitro transdermal absorption and skin retention of paeonol from paeonol-loaded ethosomes were 138.58 ± 9.60 µg/cm² and 52.60 ± 7.90 µg/cm², respectively. With reasonable skin tolerance, ethosomes could be a promising vehicle for transdermal delivery of paeonol.

Pharmacokinetic and Pharmacodynamics of Self-Assembled Cubic Liquid Crystalline Nanoparticle Gel After Transdermal Administration.

BACKGROUND The aim of this study was to assess the pharmacokinetics after transdermal administration by a novel skin microdialysis technology in rats. The guinea pig model was established by investigating the pharmacodynamics. MATERIAL AND METHODS Three different agents were given after hair removal, and the samples were extracted by microdialysis and detected by HPLC. Subcutaneous/plasma concentration-time curves of the 3 different agents were analyzed and the pharmacokinetic parameters were calculated. The SS-04B UV light therapy instrument was used in the modeling. Changes in melanin index and histopathology were observed with HE staining. RESULTS The increment and decrement results showed that the concentration had no significant effect on drug recovery both in vivo and in vitro. After the paeonol cubic liquid crystalline nanoparticles gel (PAE-LCNPs) was administered, the maximum peak time (tmax) of paeonol skin concentration appeared at 2.42±0.20 h, the maximum skin concentration Cmax was (926±105) ng/ml, and the area under the curve AUC0-8 was (8056±954) ng/h/ml. The tmax was shortened much more than in the other groups, and the performance of PAE-LCNPs targeting was good. Pharmacodynamic results showed that PAE-LCNPs can reduce melanocytes and reduce the melanin index, proving its utility in the treatment of melanin deposition. CONCLUSIONS The skin microdialysis study indicated PAE-LCNPs have good transdermal permeability and efficacy. Pharmacological experiments based on the study found that the topical pigmentation model of guinea pigs showed a better therapeutic effect.

Kinetics and metabolism of apocynin in the mouse brain assessed with positron-emission tomography.

BACKGROUND: Apocynin is a constituent of the medicinal herb Picrorhiza kurroa. It is an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase. This compound shows potential anti-inflammatory and antioxidant effects and has been tested as a neuroprotectant in many animal models of brain disease. In such studies, understanding the brain kinetics of apocynin would be important for interpreting its in vivo efficacy; however, little has been reported on the kinetics of apocynin in the brain. PURPOSE: The purpose of this study is to investigate the kinetics and metabolism of apocynin in the brain of mice. STUDY DESIGN: The kinetics and metabolism of apocynin were examined using [11C]apocynin and positron-emission tomography (PET). METHODS: In vivo PET scanning was performed in mice for 20min after intraperitoneal administration of an apocynin solution containing [11C]apocynin. Metabolites in the brain were analyzed using high-performance liquid chromatography. The doses of apocynin used ranged from <1.5 µg/kg (tracer dose) to 100mg/kg. RESULTS: Brain radioactivity during the period of 0 to 20min after administration was negligible at the tracer dose and extremely low at the dose of 10mg/kg. Moderate radioactivity was observed in the brain a few minutes after administration at the doses of 25 and 50mg/kg and rapidly decreased thereafter. At a dose of 100mg/kg, [11C]apocynin resulted in a high uptake of radioactivity followed by a gradual washout. In contrast to the brain, a clear dose-dependent increase in radioactivity was not observed in the blood. The fraction of the unchanged form in the brain decreased with time, and the degree of the reduction depended on apocynin doses: apocynin was rapidly metabolized in the brain at lower doses, whereas it was slowly decomposed at higher doses. On the basis of these data, the maximum apocynin concentrations in the brain were calculated to be 10 µM (10mg/kg), 49 µM (25mg/kg), 150 µM (50mg/kg), and 380 µM (100mg/kg). A metabolite observed in the brain was found to be apocynin glucuronide but not diapocynin, an active metabolite. CONCLUSION: These results would be useful for an evaluation of the potential efficacy of apocynin as a neuroprotective agent.

Simultaneous determination of saikosaponin a, paeonol, and imperatorin, components of DA-9805, in rat plasma by LC-MS/MS and application to a pharmacokinetic study.

DA-9805 is a new botanical antiparkinson drug candidate formulated using an ethanolic extract of the root of Bupleurum falcatum, the root cortex of Paeonia suffruticosa, and the root of Angelica dahurica. In this study, a sensitive and rapid LC-MS/MS method was developed to simultaneously determine, saikosaponin a, paeonol, and imperatorin, three active/representative ingredients of DA-9805, in rat plasma. Plasma was extracted by mixture of ethyl acetate and methyl tertiary butyl ether. Chromatographic separation was carried out using a C18 column and a gradient elution of mobile phases consisting of 5mM formic acid in water and acetonitrile. Total chromatographic run time was 10.5min. Multiple reaction monitoring mode was used for mass spectrometry; the transitions were m/z 779.5→617.2 for saikosaponin a in negative-ion mode, m/z 167→149 for paeonol and m/z 271.1→203 for imperatorin in positive-ion mode. Calibration curves were constructed in the range of 0.5-1000ng/mL for saikosaponin a, 20-10000ng/mL for paeonol, and 0.2-1000ng/mL for imperatorin. All the validation data, including the selectivity, linearity, precision, accuracy, recovery, matrix effect, and stability satisfied the acceptance requirements. The method was successfully applied in a pharmacokinetic study of saikosaponin a, paeonol, and imperatorin following oral administration of DA-9805.

[Transdermal permeation of effective components in Huoxue Zhitong patch].

To establish a determination method for the contents of paeonol, eugenol and piperine in receptor liquid and to research the transdermal permeability of Huoxue Zhitong patch. The contents of paeonol, eugenol and piperine in receptor liquid were determined by high pressure liquid chromatography(HPLC); and the receptor liquid was optimized by taking accumulative amount penetrated within 24 hours, percutaneous permeation rate and skin irritation as indexes. In vitro Franz diffusion experiment was applied to assess the percutaneous penetration characteristics and regularity of Huoxue Zhitong patch. The results showed that the accumulative penetration amount and penetration rate by using PEG 400-ethanol-normal saline 3∶3∶4 as receptor liquid were higher than those by using propylene glycol∶ethanol∶normal saline 3∶3∶4 and ethanol-normal saline 3∶7, the and skin irritation of PEG 400-ethanol-normal saline 3∶3∶4 was smaller than propylene glycol:ethanol: normal saline 3∶3∶4. Results of percutaneous permeability experiments displayed that the accumulative amount penetrated of paeonol, eugenol and piperine within 24 hours was 2.84, 19.9, and 0.753 µg•cm⁻² respectively in Huoxue Zhitong patch and the penetration rate was 0.18, 1.22, and 0.02 µg•cm⁻²â€¢h⁻¹ respectively. Thus, the permeation of paeonol, eugenol and piperine through the skin was a diffusion process, which was irrelevant to their content in patch.

PEGylated niosomes-mediated drug delivery systems for Paeonol: preparation, pharmacokinetics studies and synergistic anti-tumor effects with 5-FU.

This work describes the preparation of a PEGylated niosomes-mediated drug delivery systems for Paeonol, thereby improving the bioavailability and chemical stability of Paeonol, prolonging its cellular uptake and enhancing its synergistic anti-cancer effects with 5-Fu. PEGylated niosomes, which are prepared from biocompatible nonionic surfactant of Spans 60 and cholesterol, and modified with PEG-SA. Pae-PEG-NISVs were evaluated in vitro and in vivo. The cytotoxicity of Pae-PEG-NISVs was investigated against HepG2 cells. Fluorescence microscope was used to detect the apoptotic morphological changes. Growth inhibition assays were carried out to investigate whether Pae-PEG-NISVs could enhance the antiproliferative effects of Pae co-treated with 5-FU on HepG2 cells. The optimized Pae-PEG-NISVs had mean diameters of approximately 166 nm and entrapment efficiency (EE) of 61.8%. Furthermore, the in vitro release study of Paeonol from PEGylated niosomes exhibited a relatively prolonged release profile for 12 h. Pharmacokinetic studies in rats after i.v. injection showed that Pae-PEG-NISVs had increased elimination half-lives (t1/2, 87.5 versus 17.0 min) and increased area under the concentration-time curve (AUC0-t, 38.0 versus 19.48 µg/ml*min) compared to Paeonol solution. Formulated Paeonol had superior cytotoxicity versus the free drug with IC50 values of 22.47 and 85.16 µg/mL at 24 h on HepG2 cells, respectively, and we found that low concentration of Pae-PEG-NISVs and 5-Fu in conjunction had obviously synergistic effect. Our results indicate that the PEG-NISVs system has the potential to serve as an efficient carrier for Paeonol by effectively solubilizing, stabilizing and delivering the drug to the cancer cells.

Microemulsions based on paeonol-menthol eutectic mixture for enhanced transdermal delivery: formulation development and in vitro evaluation.

In this work, microemulsion-based gels were prepared for transdermal delivery of paeonol. Microemulsions containing eutectic mixtures of paeonol and menthol were developed. The obtained microemulsions were evaluated for particle size, viscosity and physical stability. The selected microemulsions were incorporated into Carbopol gels. Drug crystallization behavior during a short-term storage was compared and in vitro permeation and deposition study were conducted on mouse skin. Results showed that the eutectic liquids of paeonol and menthol at all ratio (6:4, 5:5 and 4:6) could form microemulsions but with significantly different physical characteristics. As the ratio of paeonol increased, the prepared microemulsions exhibited larger droplet size, higher viscosity and quicker crystal growth. Microemulsion containing paeonol and menthol at a ratio of 4:6 possessed the smallest size of 27 nm. Accordingly, the related gel showed better physical stability during 10 days of storage, as well as the highest percent of drug deposition (111.8 µg/cm2) and steady-state flux (0.3 µg/cm2 h). These results suggested that the microemulsion formulation is a preferable approach for enhanced skin permeation, and the microemulsion based on drug-menthol eutectic mixture might be used as a potential transdermal delivery system for better therapeutic efficacy.

Integrated identification, qualification and quantification strategy for pharmacokinetic profile study of Guizhi Fuling capsule in healthy volunteers.

Guizhi Fuling capsule (GZFL), a traditional Chinese medicine formulation, is widely used in China to relieve pain from dysmenorrhea and is now in a Phase II clinical trial in the USA. Due to the low exposure of the five main medicative ingredients (amygdalin, cinnamic acid, gallic acid, paeoniflorin and paeonol) of GZFL in human, a strategy was built to qualitatively and quantitatively identify the possible metabolites of GZFL and to describe the pharmacokinetic profiles of GZFL in human. In this strategy, LC-Q-TOF/MS was used to identify and structurally elucidate the possible metabolites of GZFL in vivo; and a time-based metabolite-confirming step (TBMCs) was used to confirm uncertain metabolites. The simultaneously quantitation results by LC-MS/MS showed low exposure of the five medicative ingredients. According to the strategy we built, a total of 36 metabolites were found and structurally elucidated. The simultaneously semi-quantitative analysis by LC-MS/MS showed that obvious time-concentration curves could be established for 12 of the metabolites, and most of them showed a relatively higher exposure. This study provides a better understanding of the metabolic processes of GZFL in human.

In vitro Penetration and in vivo Distribution of Honokiol into the Intervertebral Disc in Rat.

Honokiol is a potential candidate for the treatment of intervertebral disc (IVD) degeneration. In this study, we develop in vitro and in vivo methods to detect the distribution of honokiol in intervertebral discs using high-performance liquid chromatography. A rat tail disc was used for both experimental models. For the in vivo animal experiment, blood samples and tail discs were collected at 15, 30, 60, 120 and 240 min after honokiol administration (30 mg/kg, i.v.). The analyte was separated by a mobile phase of methanol and 10 mM NaH2PO4 buffer at pH 2.8 (78:22, v/v) and pumped through a reversed-phase analytical column (250 × 4.6 mm, particle size 5 µm) at room temperature. The in vitro experimental results demonstrated that honokiol diffused into the intervertebral disc and was concentration-dependent. The active concentration is obtained for the therapeutic level at 15 and 30 min after honokiol administration in the in vivo model.