A rapid and sensitive LC-MS/MS method for the determination of vanillic acid in rat plasma with application to pharmacokinetic study.

Vanillic acid, a phenolic compound isolated from Angelica sinensis and green tea, exhibits excellent antioxidant and anti-inflammatory activities. In this study, a rapid and sensitive ultra-high-performance liquid chromatography tandem mass spectrometry method was established and validated for the determination of vanillic acid in rat plasma. Plasma samples were prepared by protein precipitation with acetonitrile. Chromatographic separation was performed on a Zorbax RRHD Eclipse Plus C18 column (2.1 × 100 mm, 1.8 µm) with gradient elution at a flow rate of 0.3 ml/min, using mobile phase consisting of 0.1% formic acid (A) and acetonitrile (B). Vanillic acid and caffeic acid (internal standard, IS) were quantified by multiple reaction monitoring in negative ion mode. The method was fully validated according to the US Food and Drug Administration guidelines. The calibration curve was linear over the range of 2-1,000 ng/ml with a correlation coefficient of >0.99. The carryover, matrix effect, extraction recovery, dilution effect, intra- and interday precision and accuracy were within acceptable limits. The method was then applied to a pharmacokinetic study of vanillic acid in rats. After oral administration at doses of 2, 5 and 10 mg/kg, the plasma concentration reached peaks of 0.42 ± 0.09, 0.73 ± 0.21 and 0.92 ± 0.28 µg/ml at the time of 0.55-0.64 h, respectively. The oral bioavailability was calculated as 25.3-36.2% in rat plasma. The result provided pre-clinical information for further application of vanillic acid.

Bioanalytical Method Development and Validation of Veratraldehyde and Its Metabolite Veratric Acid in Rat Plasma: An Application for a Pharmacokinetic Study.

A simple, sensitive, and rapid UHPLC-MS/MS method was developed for the simultaneous determination of veratraldehyde and its metabolite veratric acid in rat plasma. Cinnamaldehyde was used as an internal standard (IS) and the one-step protein precipitation method with 0.2% formic acid in acetonitrile (mobile phase B) was used for the sample extraction. Reversed C18 column (YMC-Triart C18 column, 50 mm × 2.0 mm, 1.9 µm) was used for chromatographic separation and was maintained at 30 °C. The total run time was 4.5 min and the electrospray ionization in positive mode was used with the transition m/z 167.07 → 139.00 for veratraldehyde, m/z 183.07 → 139.00 for veratric acid, and m/z 133.00 → 55.00 for IS. The developed method exhibited good linearity (r2 ≥ 0.9977), and the lower limits of quantification ranged from 3 to 10 ng/mL for the two analytes. Intra-day precision and accuracy parameters met the criteria (within ±15%) during the validation. The bioanalytical method was applied for the determination of veratraldehyde and veratric acid in rat plasma after oral and percutaneous administration of 300 and 600 mg/kg veratraldehyde. Using the analytical methods established in this study, we can confirm the absorption and metabolism of veratraldehyde in rats for various routes.

Pharmacokinetic, Antiproliferative and Apoptotic Effects of Phenolic Acids in Human Colon Adenocarcinoma Cells Using In Vitro and In Silico Approaches.

Colon cancer is the second most common cause of cancer deaths in the USA and Europe. Despite aggressive therapies, many tumors are resistant to current treatment protocols and epidemiological data suggest that diet is a major factor in the etiology of colon cancer. This study aimed to evaluate the antioxidant activity and the influence of 3,4-dihydroxyphenylacetic (3,4-DHPAA), p-coumaric (p-CoA), vanillic (VA) and ferulic (FA) acids on cell viability, cell cycle progression, and rate of apoptosis in human colon adenocarcinoma cells (HT-29). The results showed that all compounds tested reduce cell viability in human colon cancer cells. 3,4-DHPAA promoted the highest effect antiproliferative with an increase in the percentage of cells in G0/G1 phase, accompanied by a reduction of cells in G2/M phase. Cell cycle analysis of VA and FA showed a decrease in the proportion of cells in G0/G1 phase (10.0 µM and 100.0 µM). p-CoA and FA acids increased the percentage of apoptotic cells and non-apoptotic cells. 3,4-DHPAA seems to be the substance with the greatest potential for in vivo studies, opening thus a series of perspectives on the use of these compounds in the prevention and treatment of colon cancer.

Development of nanoformulation of picroliv isolated from Picrorrhiza kurroa.

Picroliv, a mixture of picroside I and kutkoside isolated from rhizome of Picrorrhiza kurroa has been reported for many pharmaceutical properties such as hepatoprotective, anticholestatic, antioxidant and immune-modulating activity. However, picroliv possessed lesser efficacy due to its poor aqueous solubility and lesser bioavailability. To find solution, picroliv was loaded into biodegradable poly lactic acid nanoparticles (PLA NPs) using solvent evaporation method. The picroliv-loaded PLA NPs were characterised by UV-vis spectroscopy, atomic force microscopy, transmission electron microscopy, Fourier transform infrared and Zeta sizer. The size of picroliv-loaded PLA NPs was 182 ± 20 nm. Zeta potential of picroliv-loaded PLA NPs was -23.5 mV, indicated their good stability. In vitro picroliv release from picroliv-loaded PLA NPs showed an initial burst release followed by slow and sustained release. The efficacy of picroliv-loaded PLA NPs was assessed against KB cell lines. Blank PLA NPs showed no cytotoxicity on KB cells. The picroliv-loaded PLA NPs showed more cytotoxic activity on KB cells as compared to the pure drug. Hence, the developed picroliv nanoformulation would find potential application in pharma-sector.

In vitro - In vivo metabolism and pharmacokinetics of picroside I and II using LC-ESI-MS method.

Picroside I and II, iridoid glycosides, are the major active markers of roots and rhizomes of Picrorhiza kurroa (family: Scrophulariaceae). The rhizomes of P. kurroa have been traditionally used to treat worms, constipation, low fever, scorpion sting, asthma and ailments affecting the liver. Various Ayurvedic and herbal preparations are available in the market which contains P. kurroa e.g. Arogyavadhini vati, Tiktadi kwath, Picrolax capsules and suspension. These preparations are used without any significant pharmacokinetics data. Previously, we have reported that oral bioavailability of picroside I and II is low. Most of the iridoid glycosides are primarily metabolized by intestinal microbial flora. So, it is necessary to determine the metabolic profile of picroside I and II and check the correlation with lower bioavailability. Therefore, this study was designed to check metabolic (in vitro and in vivo) profile along with pharmacokinetic profile of picroside I and II. For this, a sensitive and selective LC-ESI-MS method was developed and validated for simultaneous determination of picroside I and II in rat plasma. Chromatographic separations were performed on C18 column. The mobile phase consisted of acetonitrile: 10 mM ammonium acetate buffer [90:10 v/v], pH 3.5. In-vitro Metabolic study was performed on rat liver microsomes and primary hepatocytes. In-vivo pharmacokinetic and metabolic profile of picroside I and II was generated after oral administration of Kutkin (mixture of picroside I and II) to Sprague-Dawley rats. Various pharmacokinetic parameters viz. Cmax, Tmax, AUC(0-t) were determined. In metabolic study, eight metabolites of picroside I and six metabolites of picroside II were identified in vitro, out of which four metabolites for each picroside I and picroside II were identified in vivo.

Comparative Pharmacokinetics of three major bioactive components in rats after oral administration of Typhae Pollen-Trogopterus Feces drug pair before and after compatibility.

BACKGROUND: Typhae Pollen (TP) and Trogopterus Feces (TF) are well-known traditional medicine in china which widely used for thousands of years as drug pair called Shixiao San for treatment of blood stasis syndrome, specially shown great efficacy in gynecological disease. Typhaneoside, vanillic acid and p-coumaric acid are the main bioactive components of Typhae Pollen. This study was carried out for comparing the pharmacokinetic profile of these three major bioactive components in rats after oral administration of Typhae Pollen-Trogopterus Feces (TP-TF) drug pair before and after compatibility. METHODS: A sensitive and rapid UPLC-TQ/MS method has been developed for simultaneous quantification of the three main bioactive compounds in blood at different time points after oral administration of Typhae Pollen (TP) and the combination with Trogopterus Feces (TF). RESULTS: There were significant differences of C(max), T(max), T(1/2) and AUC(0~t) for three bioactive compounds among the groups, for typhaneoside with the most highest plasma concentration of 370.86 ± 315.71 ng/mL and more longer T(max) in TP-TF co-decoction group (C(M)); for vanillic acid, TP-TF co-decoction group (C(M)) had a good absorption with C(max) (3870.99 ± 2527.99 ng/mL) and T(max) (1.47 ± 3.20 h); for p-coumaric acid, it had similar pharmacokinetic characteristics with vanillic acid. CONCLUSIONS: The three bioactive components in Typhae Pollen (TP) were simultaneously determined by UPLC-TQ/MS and had a good absorption in rat plasma after the combination with Trogopterus Feces (TF).

Pharmacokinetics and tissue distributions of veratric acid after intravenous administration in rats.

The present study was designed to investigate the pharmacokinetics and tissue distributions of veratric acid following intravenous administration in rats. The concentrations of veratric acid in rat plasma at various times after administrated at doses of 2.5, 5, and 10 mg·kg(-1) were quantified by HPLC. The tissue distributions of veratric acid at various times after a single intravenous dose of 2.5 mg·kg(-1) were also analyzed. The plasma pharmacokinetic parameters at the three doses were as follows: t(1/2), (86.23 ± 6.83), (72.66 ± 4.10) and (71.20 ± 2.90) min; C0, (11.10 ± 1.47), (23.67 ± 1.24) and (39.17 ± 3.90) µg·mL(-1); and AUC(0→∞), (1 240.90 ± 129.14), (2 273.84 ± 132.47) and (3 516.4 ± 403.37) min·µg·mL(-1), respectively. The compound was distributed into tissues rapidly and extensively after intravenous administration and was mainly distributed into the liver, heart and kidneys.

UHPLC-MS simultaneous determination and pharmacokinetic study of three aromatic acids and one monoterpene in rat plasma after oral administration of Shaofu Zhuyu decoction.

We developed a sensitive and rapid method for determination of ferulic acid, caffeic acid, vanillic acid, and paeoniflorin in rat plasma based on ultra high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). The separation of the four compounds was carried out on an AcQuity UHPLC™ BEH C18 column using a mobile phase consisting of acetonitrile and water (containing 0.1% formic acid). Electrospray ionization in positive and negative ion mode and multiple reaction monitoring was used to identify and quantify active components. All calibration curves gave good linearity (r > 0.991) over the concentration range from 4.24-2875 ngmL(-1) for all components. The precision of the in vivo study was evaluated by intraday and interday assays and the percentages of RSD were all within 10.6%. The recovery ranged from 60.2 to 77.9%. The method was successfully applied to pharmacokinetic study of all three aromatic acids and one monoterpene in rat plasma. Furthermore, we compared the pharmacokinetics profile of the four compounds in normal and primary dysmenorrhea rats' plasma following oral administration of Shaofu Zhuyu decoction (SFZYD) and its ethanol supernatant extract (SFE).

Comparative pharmacokinetic profiles of picrosides I and II from kutkin, Picrorhiza kurroa extract and its formulation in rats.

Picrosides I and II are the active chemical constituents, present in the roots and rhizomes of Picrorhiza kurroa Royle (family: Scrophulariaceae). The plant is ethnomedically claimed for the treatment of liver and upper respiratory tract infection, fever, dyspepsia and scorpion sting. This study attempts to determine the in vivo pharmacokinetic profile of picrosides I and II in rats after oral administration of three different preparations namely, kutkin (a mixture of picrosides I and II), P. kurroa extract and Picrolax® capsule (marketed formulation). A simple, precise, specific and sensitive method was developed for simultaneous quantification of picrosides I and II in rat plasma and was applied for the pharmacokinetic study. Pharmacokinetic parameters were calculated from the observed plasma concentration of picrosides I and II. The results showed a significant difference (p≤0.05) in oral bioavailability of picrosides I and II from different preparations. Both the compounds were found to be more bioavailable from P. kurroa extract followed by Picrolax® capsule and kutkin. Moreover, we also developed a novel method for isolation of kutkin from roots of P. kurroa with a high yield of 2.4% w/w. The information gained from this study provides a meaningful basis for clinical application and mechanistic study of such phytochemicals.

Bioprocessing of wheat bran in whole wheat bread increases the bioavailability of phenolic acids in men and exerts antiinflammatory effects ex vivo.

Whole grain consumption has been linked to a lower risk of metabolic syndrome, which is normally associated with a low-grade chronic inflammation. The benefits of whole grain are in part related to the inclusion of the bran, rich in phenolic acids and fiber. However, the phenols are poorly bioaccessible from the cereal matrix. The aim of the present study was to investigate the effect of bioprocessing of the bran in whole wheat bread on the bioavailability of phenolic acids, the postprandial plasma antioxidant capacity, and ex vivo antiinflammatory properties. After consumption of a low phenolic acid diet for 3 d and overnight fasting, 8 healthy men consumed 300 g of whole wheat bread containing native bran (control bread) or bioprocessed bran (bioprocessed bread) in a cross-over design. Urine and blood samples were collected for 24 h to analyze the phenolic acids and metabolites. Trolox equivalent antioxidant capacity was measured in plasma. Cytokines were measured in blood after ex vivo stimulation with LPS. The bioavailabilities of ferulic acid, vanillic acid, sinapic acid, and 3,4-dimethoxybenzoic acid from the bioprocessed bread were 2- to 3-fold those from the control bread. Phenylpropionic acid and 3-hydroxyphenylpropionic acid were the main colonic metabolites of the nonbioaccessible phenols. The ratios of pro-:antiinflammatory cytokines were significantly lower in LPS-stimulated blood after the consumption of the bioprocessed bread. In conclusion, bioprocessing can remarkably increase the bioavailability of phenolic acids and their circulating metabolites, compounds which have immunomodulatory effects ex vivo.

Drug development for liver diseases: focus on picroliv, ellagic acid and curcumin.

The use of herbal drugs for the treatment of liver diseases has a long tradition in many eastern countries. The easy accessibility without the need for laborious pharmaceutical synthesis has drawn increased attention towards herbal medicines. Few herbal preparations exist as standardized extracts with major known ingredients or even as pure compounds. Some of the herbals, which show promising activity, are ellagic acid for antifibrotic treatment, phyllanthin for treating chronic hepatitis B, glycyrrhizin to treat chronic viral hepatitis and picroliv for liver regeneration. These compounds, which have proven antioxidant, antiviral or anticarcinogenic properties, can serve as primary compounds for further development as hepatoprotective drugs. This review provides the chemistry, pharmacology and future aspects of picroliv, ellagic acid and curcumin with focus on hepatoprotective properties. These phytochemicals may prove to be very useful in the treatment of hepatotoxicity induced by viral agents, toxic drugs and plant poisons. The high safety profile may be an added advantage. However, poor bioavailability and temperature and light sensitivity can reduce the efficacy of drugs like curcumin. In future, the derivatives or new combinations of these drugs may prove to be useful.

Evaluation of the antioxidant properties and bioavailability of free and bound phenolic acids from Trichilia emetica Vahl.

Trichilia emetica Vahl. is commonly used in folk medicine of Mali for the treatment of various diseases. In this study, the content and the antioxidant activity of phenolic acids from Trichilia emetica root were evaluated. Free phenolic acids were extracted with a mixture of methanol and 10% acetic acid. Bound phenolic acids were released using first alkaline and then acid hydrolysis. All fractions were quantified separately by HPLC. After alkaline hydrolysis, a remarkable increase in caffeic acid, ferulic acid, p-coumaric acid, syringic acid, vanillic acid, protocathecuic acid and gallic acid content was observed, showing that most of phenolic acids in the drug are present as bound forms. Moreover, the extracts submitted to alkaline hydrolysis showed high antioxidant properties in two in vitro assays: autooxidation of methyl linoleate (MeLo) and ascorbate/Fe(2+)-mediated lipid peroxidation in rat microsomes. An in vivo study was also performed to investigate the intestinal absorption of phenolic acids after oral administration of Trichilia emetica extracts. Results showed high levels of phenolic acids, free or conjugated to glucuronide, in the plasma of rats treated with the hydrolyzed extract. Due to the absence of chlorogenic acid in plasma samples, the presence of caffeic acid seems to be derived from hydrolysis of chlorogenic acid in the gastrointestinal tract.

Kinetics of finite dose absorption through skin 1. Vanillylnonanamide.

Despite the considerable success in predicting the steady-state dermal absorption rates of chemical compounds from large reservoirs applied to skin, correspondingly little progress has been made in predicting the absorption rate and extent for small doses of topically applied compounds. In the latter case, steady-state absorption rates are generally not obtained, and rapid evaporation or penetration of the dose solvent makes application of permeability coefficient models problematic. This report presents a new analysis of the finite dose problem in terms of a diffusion model with three parameters-a characteristic time for diffusion, h2/D; a skin solubility factor, S(m)h; and a capacity factor for absorption of the dose during the dry down period, M*. These parameters can be related to the molecular weight and oil and water solubilities of the permeant in a manner similar to models describing steady-state absorption from saturated solutions. Some variation of the parameter values based on the chemical nature and volume of the dose solvent is anticipated. The applicability of the model is demonstrated by analyzing the in vitro absorption rates of varying doses of vanillylnonamide (VN, synthetic capsaicin) applied to excised human skin from propylene glycol. The analysis shows that a three-parameter model that assigns all of the resistance to transport to diffusion through the stratum corneum is able to explain most of the significant features of VN absorption through skin.

Development and evaluation of transdermal patches of nonivamide and sodium nonivamide acetate.

Transdermal patches were developed and evaluated for two synthetic derivatives of capsaicin, nonivamide (NVA) and sodium nonivamide acetate (SNA), by in vitro and in vivo skin permeation. The designed system was essentially based on natural rubber or polymeric Gelva dispersion. The characterization of physicochemical properties of the patches showed adequate adhesive force and low thickness which suggested these patch formulations possessed good quality. In the study of in vitro percutaneous experiment, the predicted therapeutic activities of patches were all higher than that of a commercially available plaster after the calculation of antinociceptive index. The highest in vitro penetration flux was found in the Gelva patch for NVA. The result of in vivo experiments revealed a high obverse correlation with that of in vitro experiments. The information of this present study is helpful for the further development of NVA and SNA transdermal drug delivery system.

Percutaneous absorption of vanilloids: in vivo and in vitro studies.

The percutaneous absorption of three highly lipophilic analogs of capsaicin--vanillylnonanamide (VN), olvanil, and NE-21610--was measured in vivo in the CD:VAF rat, and in vitro through excised CD: VAF and SkH:Fz rat skin and human cadaver skin. Absorption and skin metabolism were monitored by radiolabel techniques. The rank order of penetration in all species was VN > olvanil > NE-21610, in accordance with that expected from their physical properties. Rat skin was more permeable than human skin by factors ranging from 4 to 8 for VN, 10 to 20 for olvanil, and approximately 10 to 100 for NE-21610. All three compounds were extensively metabolized during passage through fresh SkH:Fz rat skin, with the primary route of degradation for at least two of the compounds involving hydrolysis of the amide bond (the metabolites of NE-21610 were not identified). For the in vitro studies a range of receptor solutions was employed to determine a set of conditions that best mimicked in vivo absorption. The results with phosphate-buffered saline containing a preservative and 1-6% polyoxyethylene-20 oleyl ether (Oleth-20) were in good agreement with in vivo results for all three compounds for periods up to 24 h post-dose; after this time, in vivo absorption rates declined but in vitro rates remained relatively constant. Buffered saline or saline containing 0.5% bovine serum albumin led to marked underestimates of in vivo penetration for olvanil and NE-21610, whereas a 1:1 ethanol: water solution led to gross overestimates of the in vivo absorption rates for all three compounds.

Facile hydrolysis of mebeverine in vitro and in vivo: negligible circulating concentrations of the drug after oral administration.

The HPLC methods for the determination of plasma concentrations of the antispasmodic agent mebeverine (0.01-10 micrograms/mL) and its hydrolysis product veratric acid (0.1-50 micrograms/mL) are presented. Mebeverine was demonstrated to hydrolyze readily in fresh unbuffered human and rat plasma samples ex vivo. Hydrolysis in human plasma was completely inhibited in the presence of the esterase inhibitor physostigmine sulfate, at a concentration of 130 micrograms/mL. However, the inhibitor was only partially effective in blocking mebeverine hydrolysis in rat plasma. After oral administration of mebeverine.HCl (270 mg) to fasted human volunteers, measurable concentrations of the drug were not found in plasma. By contrast, the metabolite veratric acid achieved considerable concentrations (mean peak plasma concentration of 13.5 micrograms/mL at 40-80 min). After iv administration of mebeverine.HCl (2 mg) to rats, the drug was rapidly eliminated from plasma (mean half-life of 29 min) with simultaneous appearance of veratric acid (mean peak plasma concentration of 1.80 micrograms/mL at 15-30 min). However, after oral administration of the same dose, only traces of mebeverine were found in plasma, with the exception of one rat. Veratric acid again achieved considerable concentrations (mean peak plasma concentration of 0.90 micrograms/mL at 15 min-4 h). The results show that mebeverine undergoes rapid and extensive first-pass metabolism involving hydrolysis of the ester function, and that negligible circulating concentrations of the parent drug are found in humans.