A validated high-performance liquid chromatography method for detecting geniposide, ellagic acid, piperine, costunolide and dehydrocostus lactone in Liuwei Muxiang capsules.

In this study, a sensitive high-performance liquid chromatography detector was established and validated for the simultaneous determination of geniposide, ellagic acid, piperine, costunolide and dehydrocostuslactone in Liuwei Muxiang Capsules. The analysis was achieved on CHANIN 100-5-C18-H column (5µm, 250 mm×4.6 mm) with the temperature of 30oC. Gradient elution was applied using 0.1% phosphoric acid solution-methanol-acetonitrile (50:50) as mobile phase at the flow rate of 1.0 mL/min. The determination was performed at the wavelength of 225 nm (detecting geniposide), 254 nm (detecting ellagic acid), 343 nm (detecting piperine) and 225 nm (detecting costunolide and dehydrocostuslactone) along with the sample volume of 10µL. The linear ranges of geniposide, ellagic acid, piperine, costunolide and dehydrocostuslactone demonstrated good linear relationships within their respective determination ranges. The average recoveries were 100.04%, 99.86%, 99.79%, 100.17% and 100.41%, respectively. RSD% was 1.3%, 1.2%, 1.2%, 1.2%, 1.5%, respectively. The developed method was proved to be simple, accurate and sensitive, which can provide a quantitative analysis method for the content determination of geniposide, ellagic acid, piperine, costunolide and dehydrocostuslactone in Liuwei Muxiang capsules.

Development and validation of a rapid high-performance thin-layer chromatographic method for quantification of gallic acid, cinnamic acid, piperine, eugenol, and glycyrrhizin in Divya-Swasari-Vati, an ayurvedic medicine for respiratory ailments.

Divya-Swasari-Vati is a calcium containing polyherbal ayurvedic medicine prescribed for the lung-related ailments observed in the current pandemic of Severe Acute Respiratory Syndrome Coronavirus 2 infections. The formulation is a unique quintessential blend of nine herbs cited in Ayurvedic texts for chronic cough and lung infection. Analytical standardization of herbal medicines is the pressing need of the hour to ascertain the quality compliance. This persuaded us to develop a simple, rapid, and selective high-performance thin-layer chromatographic method for Divya-Swasari-Vati quality standardization. The developed method was validated for the quantification of marker components, gallic acid, cinnamic acid, piperine, eugenol and glycyrrhizin, against reference standards in five different batches of Divya-Swasari-Vati. The analytes were identified by visualization at 254 nm, and by matching their retention factor with authentic standards. The developed method was validated as per the guidelines recommended by the International Council for Harmonization for parameters like, linearity, limit of detection, limit of quantification, accuracy, and precision. Therefore, the developed novel high-performance thin-layer chromatographic process could be employed for rapid standardization of Divya-Swasari-Vati and other related herbal formulation, which would aid in quality manufacturing and product development.

BIONOTE as an Innovative Biosensor for Measuring Endocannabinoid Levels.

In this study, a novel approach was developed to quantify endocannabinoids (eCBs), and was based on the liquid biosensor BIONOTE. This device is composed of a probe that can be immersed in a solution, and an electronic interface that can record a current related to the oxy-reductive reactions occurring in the sample. The two most representative members of eCBs have been analysed in vitro by BIONOTE: anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG). Bovine serum albumin was used to functionalize the probe and improve the sensibility of the whole analytical system. We show that BIONOTE is able to detect both AEA and 2-AG at concentrations in the low nanomolar range, and to discriminate between these eCBs and their moieties arachidonic acid, ethanolamine and glycerol. Notably, BIONOTE distinguished these five different molecules, and it was also able to quantify AEA in human plasma. Although this is just a proof-of-concept study, we suggest BIONOTE as a cheap and user-friendly prototype sensor for high throughput quantitation of eCB content in biological matrices, with an apparent diagnostic potential for tomorrow's medicine.

Identification of the metabolites of piperine via hepatocyte incubation and liquid chromatography combined with diode-array detection and high-resolution mass spectrometry.

RATIONALE: Piperine, an alkaloid isolated from Piper nigrum L., has been demonstrated to have many pharmacological effects and several health benefits. The aim of this work was to study the metabolic profiles of piperine in mouse, rat, dog and human hepatocytes. METHODS: The biotransformation was carried out by incubating piperine with hepatocytes at 37°C. After incubation for 2 h, the samples were pretreated and analyzed using liquid chromatography combined with diode-array detection and high-resolution mass spectrometry (LC/DAD-HRMS). The structures of the metabolites were assigned through a comparison of their accurate masses and product ions with those of the parent compound. RESULTS: A total of 20 metabolites were detected, and the structures were proposed. Piperine was metabolized through the following pathways: (a) oxidation to form a catechol derivative, which further underwent methylation, glucuronidation, glutathione (GSH) conjugation, and hydroxylation followed by opening of the piperidine ring; (b) hydroxylation to form a carbinolamine intermediate followed by opening of the piperidine ring and the formation of alcohol and acid derivatives; and (c) hydroxylation to form stable hydroxylated metabolites. In mouse, the formation of the catechol derivative (M12) and hydroxylation (M11) were the major metabolic pathways; in rat, the formation of the catechol derivative (M12) and glucuronidation (M9) were the main pathways; and in dog and human, the formation of the catechol derivative (M12) was the predominant pathway. No human-specific metabolite was observed. CONCLUSIONS: This study provided some new information on the metabolic profiles of piperine, which should be of great importance in the study of the pharmacology and toxicity of this compound.

Switchable-hydrophilicity solvent liquid-liquid microextraction versus dispersive liquid-liquid microextraction prior to HPLC-UV for the determination and isolation of piperine from Piper nigrum L.

Switchable-hydrophilicity solvent liquid-liquid microextraction and dispersive liquid-liquid microextraction were compared for the extraction of piperine from Piper nigrum L. prior to its analysis by using high-performance liquid chromatography with UV detection. Under optimum conditions, limits of detection and quantitation were found as 0.2-0.6 and 0.7-2.0 µg/mg with the two methods, respectively. Calibration graphs showed good linearity with coefficients of determination (R2 ) higher than 0.9962 and percentage relative standard deviations lower than 6.8%. Both methods were efficiently used for the extraction of piperine from black and white pepper samples from different origins and percentage relative recoveries ranged between 90.0 and 106.0%. The results showed that switchable-hydrophilicity solvent liquid-liquid microextraction is a better alternative to dispersive liquid-liquid microextraction for the routine analysis of piperine in food samples. A novel scaled-up dispersive liquid-liquid microextraction method was also proposed for the isolation of piperine providing a yield of 102.9 ± 4.9% and purity higher than 98.0% as revealed by NMR spectroscopy.

Selective in vivo molecular and cellular biocompatibility of black peppercorns by piperine-protein intrinsic atomic interaction with elicited oxidative stress and apoptosis in zebrafish eleuthero embryos.

Day to day consumption of black pepper raise concern about the detailed information about their medicinal, pharmaceutical values and knowledge about the biocompatibility with respect to ecosystem. This study investigates the in vivo selective molecular biocompatibility of its seed cover (SC) and seed core (SP) powder extract using embryonic zebrafish model. Gas chromatography mass spectrometry (GCMS) analysis of the extract prepared by grinding showed presence of different components with "piperine" as principle component. Biocompatibility analysis showed dose and time dependent selective effect of SC and SP with LC50 of 30.4 µg/ml and 35.6 µg/ml, respectively on survivability, hatching and heartbeat rate in embryonic zebrafish. Mechanistic investigation elucidated it as effect of accumulation and internalization of black pepper leading to their influence on structure and function of cellular proteins hatching enzyme (he1a), superoxide dismutase (sod1) and tumor protein (tp53) responsible for delayed hatching, oxidative stress induction and apoptosis. The study provided insight to selective biocompatibility of black pepper expedient to produce higher quality spices with respect to pharmaceutical, clinical and environmental aspects.

Antifungal and anti-aflatoxigenic activity of Heliopsis longipes roots and affinin/spilanthol against Aspergillus parasiticus by downregulating the expression of alfD and aflR genes of the aflatoxins biosynthetic pathway.

In the present study, ethanolic extract from Heliopsis longipes roots and affinin/spilanthol against Aspergillus parasiticus growth and aflatoxins production were studied in relation to the expression of aflD and aflR, two key genes of aflatoxins biosynthetic pathway. Phytochemical analysis of the ethanolic extract by GC-EIMS identified affinin/spilanthol (7.84 ± 0.27 mg g-1) as the most abundant compounds in H. longipes roots. The antifungal and anti-aflatoxigenic assays showed that affinin/spilanthol at 300 µg mL-1 produced the higher inhibition of radial growth (95%), as well as, the higher aflatoxins production inhibition (61%) in comparison to H. longipes roots (87% and 48%, respectively). qRT-PCR revealed that the expression of aflD and aflR genes showed a higher downregulation in affinin/spilanthol at 300 µg mL-1. The expression ratio of alfD was suppressed by affinin/spilanthol in 79% and aflR in 84%, while, a lower expression ratio suppressed by H. longipes was obtained, alfD (55%) and aflR (59%). Affinin/spilanthol possesses higher antifungal and anti-aflatoxigenic activity against A. parasiticus rather than H. longipes roots, and this anti-aflaxotigenic activity occurring via downregulation of the aflD and aflR genes. Thus, H. longipes roots and affinin/spilanthol can be considered potent antifungal agents against aflatoxigenic fungus, especially, affinin/spilanthol.

Simultaneous determination of six components in commercial Roukou Wuwei pills using ultra-high-performance liquid chromatography with diode-array detector.

An efficient ultra-performance liquid chromatography with diode-array detector method was established for simultaneous determination of six active components in Roukou Wuwei pills, namely gallic acid, piperine, costundide, dehydrocostus lactone, isoalantolactone and alantolactone. Chromatographic separation of six components was successfully achieved on an Waters BEH C18 column (50 × 2.1 mm, 1.7 µm) with a mobile phase composed of acetonitrile and water using a gradient elution. Gallic acid and piperine were detected at 270 nm and 343 nm, respectively; while costundide, dehydrocostus lactone, isoalantolactone and alantolactone were simultaneously measured at 225 nm. All six calibration curves showed good linearity (R2 ≥ 0.9994) between the peak area of each component and corresponding concentration. Relative standard deviations for inter- and intra-day precisions were <0.45 and 0.77%, respectively. The mean recovery rates ranged from 96.72 to 102.2% with relative standard deviations <2.07%. The developed method was validated in terms of linearity, precision and accuracy and then successfully applied for the quality control of commercial Roukou Wuwei samples.

[Quantitative Analysis of Piperine and the Derivatives in Long Pepper Extract by HPLC Using Relative Molar Sensitivity].

A novel method was developed for quantification of five major piperine derivatives (piperanine, piperine, chavicine, isopiperine, and isochavicine) in a hot water extract of long pepper fruit (LPE) using the relative molar sensitivity (RMS) based on the combination of HPLC/UV and 1H- quantitative NMR (1H-qNMR). The RMSs of piperanine, chavicine, isopiperine, and isochavicine to piperine of which the absolute purity was determined by 1H-qNMR were calculated to be 0.3693, 1.138, 0.9164, and 1.277, respectively. The total amount of piperine derivatives in LPE was quantified by both 1H-qNMR and HPLC/UV based on the RMS using piperine as a single-reference material (RMS method). The relative difference in quantitation values of 1H-qNMR and calibration curve method from the RMS method was 2.01% or less. The relative difference of the total cis-trans piperine isomers content between before and after photoirradiation in piperine solution was quantified to be 2.84% by the RMS method. In addition, the interlaboratory difference of the RMS method was confirmed in the range of 0.600 to 4.00 µg/g when analysis was performed on piperine derivatives in LPE containing tablets, while the total amount of piperine derivatives in the tablets was quantified at 606 µg/g. Our proposed method is a reliable tool for determining the contents of piperine and the derivatives in LPE and processed foods containing LPE.

Reduced levels of the endocannabinoid arachidonylethanolamide (AEA) in hair in patients with borderline personality disorder - a pilot study.

Endocannabinoids are involved in depressive and anxious symptoms and might play a role in stress-associated psychiatric disorders. While alterations in the endogenous cannabinoid system have been repeatedly found in patients with posttraumatic stress disorder (PTSD), this system has been mostly neglected in borderline personality disorder (BPD). However, there is first evidence for elevated serum levels of the endocannabinoids arachidonylethanolamide (AEA) and 2-arachidonyl-sn-glycerol (2-AG) in BPD patients compared to healthy controls and PTSD patients. In this study, hair endocannabinoids were analyzed, reflecting long-term endocannabinoid concentrations. We assessed AEA concentrations as well as 2-AG and the 2-AG main isomer 1-AG (1-AG/2-AG) in hair in women with BPD (n = 15) and age- and education-matched healthy women (n = 16). We found significantly reduced log AEA in BPD patients compared to healthy women (p = .03) but no differences in log 1-AG/2-AG concentrations. In addition, there was no association between 1-AG/2-AG and hair cortisol, but we found a non-significant correlation between hair concentrations of AEA and cortisol (p = .06). Our data indicate altered long-term release of endogenous cannabinoids in women with BPD depending on type of endocannabinoid. AEA has been suggested to modulate the basal activity of the endocannabinoid system and seems to attenuate depressive and anxious symptoms. Thus, chronically reduced AEA might contribute to psychiatric symptoms in BPD.

Characterization of piperine metabolites in rats by ultra-high-performance liquid chromatography with electrospray ionization quadruple time-of-flight tandem mass spectrometry.

RATIONALE: Piperine is a major constituent of Piper nigrum L. and is a naturally bioactive alkaloid. Structural changes in piperine have been shown to result in different biological effects. The present study aims to investigate piperine metabolites in rat plasma, bile, urine, and feces after oral administration. METHODS: The metabolic pathway of piperine in vivo was investigated using ultra-high-performance liquid chromatography (UHLPC) combined with electrospray ionization quadruple time-of-flight tandem mass spectrometry (QTOF-MS). Piperine metabolites were found and identified by fragmentation patterns and accurate mass measurements. RESULTS: The 12 metabolites detected and identified were divided into three groups: methylenedioxycyclic ring-opening metabolites (M01-M08), methylenedioxycyclic ring-oxidizing metabolites (M09-M11), and piperidine ring-cleavage metabolites (M12). Seven piperine metabolites, including M02, M03, M04, M05, M09, M10 and M11, were reported for the first time in the literature. CONCLUSIONS: Results showed that the principal metabolism pathways of piperine in rat were reduction and demethylation after ring-opening, and that UHPLC/QTOF-MS can serve as an important analytical platform to gather the piperine metabolism profile. Copyright © 2017 John Wiley & Sons, Ltd.

Characterization of endocannabinoids and related acylethanolamides in the synovial fluid of dogs with osteoarthritis: a pilot study.

BACKGROUND: Cannabis-based drugs have been shown to be effective in inflammatory diseases. A number of endocannabinoids including N- arachidonoylethanolamide (anandamide, AEA) and 2-arachidonyl glycerol (2-AG) with activity at the cannabinoid receptors (CBR) CBR1 and CBR2, have been identified. Other structurally related endogenous fatty acid compounds such as oleoylethanolamide (OEA) and palmitoyl ethanolamide (PEA) have been identified in biological tissues. These compounds do not bind to CBR but might be involved in facilitating the actions of directly acting endocannabinoids and thus are commonly termed "entourage" compounds due to their ability to modulate the endocannabinoid system. The aim of this study was to evaluate the presence of endocannabinoids and entourage compounds in the synovial fluid of dogs with osteoarthritis subjected to arthrotomy of the knee joint. Cytokines and cytology were studied as well. RESULTS: AEA, 2-AG, OEA and PEA were all present in the synovial fluid of arthritic knees and in the contralateral joints; in addition, a significant increase of OEA and 2AG levels were noted in SF from OA knees when compared to the contralateral joints. CONCLUSION: The identification and quantification of endocannabinoids and entourage compounds levels in synovial fluids from dogs with OA of the knee is reported for the first time. Our data are instrumental for future studies involving a greater number of dogs. Cannabinoids represent an emerging and innovative pharmacological tool for the treatment of OA and further studies are warranted to evaluate the effectiveness of cannabinoids in veterinary medicine.

Characteristic fingerprinting based on macamides for discrimination of maca (Lepidium meyenii) by LC/MS/MS and multivariate statistical analysis.

BACKGROUND: Macamides with a benzylalkylamide nucleus are characteristic and major bioactive compounds in the functional food maca (Lepidium meyenii Walp). The aim of this study was to explore variations in macamide content among maca from China and Peru. Twenty-seven batches of maca hypocotyls with different phenotypes, sampled from different geographical origins, were extracted and profiled by liquid chromatography with ultraviolet detection/tandem mass spectrometry (LC-UV/MS/MS). RESULTS: Twelve macamides were identified by MS operated in multiple scanning modes. Similarity analysis showed that maca samples differed significantly in their macamide fingerprinting. Partial least squares discriminant analysis (PLS-DA) was used to differentiate samples according to their geographical origin and to identify the most relevant variables in the classification model. The prediction accuracy for raw maca was 91% and five macamides were selected and considered as chemical markers for sample classification. CONCLUSION: When combined with a PLS-DA model, characteristic fingerprinting based on macamides could be recommended for labelling for the authentication of maca from different geographical origins. The results provided potential evidence for the relationships between environmental or other factors and distribution of macamides. © 2016 Society of Chemical Industry.

[Study on the Extraction, Geometry Structure and Spectral Characterization of Piperine Alkaloid].

Using pepper fruit of Hainan as raw material and 95% ethanol as solvent, the alkaloid in pepper is extracted with reflux method in this paper. The piperonylic acid is removed by adjusting the pH; the fat-soluble substance being removed by adding ethyl ether; the piperine alkaloid being purified with acetone by recrystallization anddetected with HPLC, as well as characterized with IR. The characterizations of piperine are discussed. Meanwhile, B3LYP/6-31G (d,p) method of DFT is applied to optimize the structure, calculate frequency and energy of pepper alkaloid, then obtain four kinds of configurations (configuration Ⅰ as Piperine, configuration Ⅱ as Iso Piperine, configuration Ⅲ as Iso Chavicine, configuration Ⅳ as Chavicine) with 64 kinds of stability conformational structure. The distribution of the thermodynamic equilibrium of stable conformations of four kinds of configurations of the molecular is calculated with Gibbs free energy at room temperature (298.15 K). And IR spectra of the experimental were compared with the IR spectra of the theoretical. The results show that the alkaloid extracted from pepper is mainly conformer 1 in configuration Ⅰ, that is, Piperine; after purifying, the content of piperine is 7% with the purity of 99%. With analysis, the methods of extraction, separation and purification of piperine in this paper achieve good results. Established models are in good agreement with the experimental results. This research is of great significance in guiding extracting process, building structural model and the characterization and application of piperine.

A robust capillary liquid chromatography/tandem mass spectrometry method for quantitation of neuromodulatory endocannabinoids.

RATIONALE: Methods for quantifying anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) are needed to support programs investigating molecular mechanisms of the central nervous system. Existing methods, while useful, are not well adapted to efficiently process large numbers of very small tissue samples. A unique challenge involves the disparity in endogenous levels of AEA (pmol/g tissue) and 2-AG (nmol/g tissue). METHODS: A simplified one-step solvent extraction procedure was developed for recovering endocannabinoids from rat brain tissues, and combined with capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS). Various multiple reaction monitoring (MRM)-based methods were evaluated for limit of detection (LOD) and robustness. RESULTS: The optimized simultaneous quantitation method achieves an LOQ of 50 amol for AEA and 25 fmol for 2-AG, both with a linearity over 3 orders of magnitude, and elution times under 3 min. Accuracy, expressed as relative error (RE), is less than 12% for AEA and less than 6% for 2-AG. Precision, expressed as relative standard deviation (RSD), is less than 6% for AEA and less than 3% for 2-AG. Sample handling routines are sufficiently robust to support the automated analysis of thousands of samples from a range of tissue types. CONCLUSIONS: The microscale method is a sensitive, economical and robust alternative to the larger scale LC/MS methods currently implemented for quantitation of AEA and 2-AG.

Isolation, characterization and identification of pericarp-degrading bacteria for the production of off-odour-free white pepper from fresh berries of Piper nigrum L.

AIM: To isolate, fermentatively evaluate and identify black pepper (Piper nigrum L.)-associated bacteria for the microbial decortication of fresh ripened berries and dried black pepper for preparation of off-odour-free white pepper. METHODS AND RESULTS: Among 45 bacterial isolates obtained from black pepper, seven of them were found to decorticate black pepper (>60%) and fresh pepper berries (98-100%) into white pepper within 5 days of immersion in bacterial suspension. The 16S rRNA genes (1500-bp amplicon) of these bacteria were sequenced, and species identity was established by closest match in GenBank. Superior-quality white pepper was obtained with Bacillus subtilis (IISR WP 33, 34, 38), Bacillus licheniformis (IISR WP 43), Acinetobacter baumanii (IISR WP 35), Klebsiella pneumoniae (IISR WP 19) and Microbacterium barkeri (IISR WP25). The bacterial isolates were found to secrete multiple hydrolytic enzymes such as cellulase, pectinase, amylase, protease and xylanase. Bacterial cultures were deposited with International Depository Authority at Microbial Type Culture Collection, India, as patent deposits as prescribed in Budapest Treaty for microbial deposits. The white pepper, thus obtained from bacterial decortication process, was free from off-odour compound, especially skatole. Other biochemical constituents such as oleoresin, piperine and essential oils were found in the acceptable range. The bacterial decortication did not affect inherent constituents of pepper such as essential oil constituents, oleoresin and piperine content. CONCLUSION: One of the most significant findings of the work is identification of specific bacterial species for decortication of fresh berries or black pepper berries into value-added white pepper. SIGNIFICANCE AND IMPACT OF THE STUDY: This work paved way for developing a technological process for microbial decortication of fresh/black pepper for the production of superior-quality white pepper.

HPTLC method for simultaneous determination of piperine, embeline, and carvone in the ayurvedic formulation Catpusphadhya churna.

Churnas are an important group of formulations used by traditional physicians to treat various types of diseases. The principle of using a churna is based on the fact that the therapeutic value of most substances greatly increases when they are reduced to a very fine state of subdivision. Catpusphadhya churna, as per the Ayurvedic system of Indian medicine, is used for acute rheumatoid arthritis. In the present study, an attempt was made to develop an HPTLC method for the quantitative determination of piperine, embeline, and carvone in a laboratory-prepared formulation. Raw materials used in formulations were obtained from two different suppliers and were subjected to methanol extractions by using a Soxhlet apparatus. Piperine, embeline, and carvone were quantified in the extracts by using HPTLC. The detection and quantification were performed at 254 nm. The formulation contained 2.35% (w/w) of piperine, 4.86% (w/w) of embeline, and 1.48% (v/w) of carvone. Linearity studies indicated that piperine, embeline, and carvone were in the linear ranges, while the recovery studies revealed a recovery of 99.32% (w/w) of piperine, 101.82% (w/w) of embeline, and 100.09% (v/w) of carvone, thus proving the accuracy of the analysis. The developed HPTLC method resolved and quantified piperine, embeline, and carvone effectively, so it could be an important method for the QC of polyherbal formulations.

Antiprotozoal activity of Achillea ptarmica (Asteraceae) and its main alkamide constituents.

In the course of our ongoing screening of plants of the family Asteraceae for antiprotozoal activity, a CH2Cl2-extract from the flowering aerial parts of Achillea ptarmica L. (sneezewort yarrow) was found to be active in vitro against Trypanosoma brucei rhodesiense (IC50 = 0.67 µg/mL) and Plasmodium falciparum (IC50 = 6.6 µg/mL). Bioassay guided fractionation led to the isolation and identification of five alkamides from the most active fractions. Pellitorine and 8,9-Z-dehyropellitorine are the main components of the extract. Beside these olefinic acid amides, four alkamides with diene-diyne structures were isolated. All alkamides were tested for antiprotozoal activity in vitro. Pellitorine was the most active compound so far within this study against P. falciparum (IC50 = 3.3 µg/mL), while 8,9-Z-dehydropellitorine was most active against T. b. rhodesiense (IC50 = 2.0 µg/mL). The activity of pure pellitorine against Plasmodium is higher than that of the crude extract and thus explains the activity of the latter. None of the isolated alkamides, however, was as active against T. b. rhodesiense as the crude extract whose antitrypanosomal activity must therfore be due to a synergistic effect of the isolated compounds or to more active yet to be identified constituents.

Development and validation of simultaneous estimation method for curcumin and piperine by RP-UFLC.

Curcumin and piperine are proven for their potent medicinal benefits to treat various diseases and they are most commonly used combination in various Indian systems of medicine such as Ayurveda, Siddha and Unani. The objective of the present work is to develop a simultaneous estimation of curcumin and piperine by reverse phase Ultra-fast liquid chromatographic (RP-UFLC) method. The chromatographic separation was performed on a C8 column (250 x 4.6 mm, 5µ i.d.) stationary phase using a mobile phase of 25mM potassium dihydrogen ortho phosphate buffer (pH 3.5) and acetonitrile (30: 70 v/v) at a flow rate of lml/min at detection wave length of 280nm. The calibration curve was plotted in the concentration range of 0-2200ng/ml and found to be linear for both curcumin (r(2)=0.996) and piperine (r(2)=0.999). The method was validated for parameters such as accuracy, sensitivity, precision, linearity, specificity, ruggedness and robustness as per ICH guidelines. The developed simple, precise and specific method can be used as a quality control tool for qualitative and quantitative estimation of curcumin and piperine in various food products, herbal medicines and nutraceuticals.

Quantitative LC-MS/MS analysis of arachidonoyl amino acids in mouse brain with treatment of FAAH inhibitor.

An additional class of endogenous lipid amides, N-arachidonoyl amino acids (Ara-AAs), is growing in significance in the field of endocannabinoids. The development, validation, and application of a sensitive and selective method to simultaneously monitor and quantify the level of Ara-AAs along with anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) in mouse brain has been established. The linearity of the method over the concentration ranges of 0.2-120 pg/µl for the standards of N-arachidonoyl amino acids, N-arachidonoyl alanine (NAAla), serine (NASer), γ-aminobutyric acid (NAGABA), and glycine (NAGly); 0.7-90 pg/µl for AEA-d(0)/d(8); and 7.5-950 pg/µl for 2-AG was determined with R(2) values of 0.99. Also the effects of the FAAH inhibitor URB 597 on the endogenous levels of these analytes were investigated. AEA and NASer brain levels exhibit a dose-dependent increase after systemic administration of URB 597, whereas NAGly and NAGABA were significantly decreased after treatment. NAAla and 2-AG were not altered after URB 597 treatment. The potential benefit of establishing this assay extends beyond the quantification of the Ara-AAs along with AEA and 2-AG in mouse brain, to reveal a variety of pharmacological effects and physiological roles of these analytes.