Determination of albuterol and montelukast post-expiry drug strength by HPLC.

Background: Knowing the level of active ingredients in an expired drug is a matter of concern irrespective of its final disposition. This is also a matter of national security and defense as it has important implications on the nation's stockpile of prescription medications. Current literature has limited information about the strength of expired medications and any relevant trends. Objective: To utilize high performance liquid chromatography (HPLC) to determine the strength of selected drugs for asthma and chronic obstructive pulmonary disease (COPD) as a class of therapeutic agents commonly used in free clinics. Methods: Samples from expired lots of montelukast and albuterol pharmaceutical products were analyzed for their levels of their respective active ingredients. Two HPLC methods were developed, validated, and applied to achieve this goal. Quantitative analysis of each drug was performed using two different reversed phase C18 columns with a linear gradient of acetonitrile in 0.1% aqueous formic acid at a flow rate of 1 mL/min for both methods. Detection wavelength for montelukast and albuterol was 280 and 277 nm, respectively. Results: Expiry dates of analyzed batches ranged from 2003 to 2019. Despite the extended time range beyond expiry dates, levels of both drugs were relatively consistent and exceeded 90% of the listed strength in most analyzed lots. Conclusions: Our results introduce a new perspective towards reducing the financial burden resulting from disposal of expired medications with retained strength. They also offer supporting evidence to extend the use of out-of-date montelukast and albuterol preparations at home and in free clinics.

Stability Determination of an Extemporaneously Compounded Ambrisentan Suspension by High Performance Liquid Chromatography Analysis.

OBJECTIVE: Ambrisentan, an endothelin receptor antagonist FDA-approved for the treatment of pulmonary arterial hypertension in adult patients, lacks an acceptable pediatric dosage form. The objective of this investigation was to determine the stability of an extemporaneously compounded ambrisentan suspension. METHODS: Ambrisentan suspension was compounded to a concentration of 1 mg/mL using commercially available suspending agents. The suspension was then evenly split into 2 plastic amber prescription bottles. One bottle was stored at room temperature and under continuous fluorescent light while the other bottle was stored under refrigeration and protection from light. A fast and selective reversed-phase high-performance liquid chromatography (HPLC) method was developed and validated for the analysis of ambrisentan. HPLC analysis was performed on samples withdrawn from the stock bottles at predetermined time intervals, up to 90 days. RESULTS: The developed HPLC method enabled the elution and detection of ambrisentan peak at 4.4 minutes. HPLC analysis revealed that all samples from both storage conditions retained >90% potency throughout the study timeframe. There were no signs of any ambrisentan breakdown products on HPLC analysis. Color and odor of the final product was also consistent throughout the 90-day storage period. CONCLUSION: Ambrisentan suspension, compounded to 1 mg/mL, is stable at room temperature or under refrigeration for up to 90 days.

Phenolic compounds from nutmeg (Myristica fragrans Houtt.) inhibit the endocannabinoid-modulating enzyme fatty acid amide hydrolase.

OBJECTIVES: The study aimed to identify nutmeg compounds that indirectly interact with the endocannabinoid system through inhibition of the fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) enzymes. METHODS: Thirteen compounds were screened for FAAH and MAGL inhibition. Compounds demonstrating significant FAAH inhibition were evaluated to determine the halfmaximal inhibitory concentration (IC50 ). The most potent compound was investigated in the elevated plus maze (EPM) rodent anxiety model. KEY FINDINGS: Three compounds, licarin A (9), 5'-methoxylicarin A (8) and malabaricone C (6) were most active in inhibiting FAAH with IC50 of 7.02 µm ± 2.02, 4.57 µm ± 0.66 and 38.29 µm ± 6.18, respectively. None of the purified compounds showed significant MAGL inhibition. Because of its relative high potency and selectivity, compound 8 was further evaluated in the EPM animal model of anxiety. The compound showed significant increase in number of open arm entries (P < 0.05) when administered at 120 mg/kg dose. No effect was observed on the locomotor activity. CONCLUSIONS: Results collected introduce active nutmeg compounds as potential leads for further development. Of the three compounds, 8 possesses highest potency and FAAH selectivity as well as anxiolytic activity. Furthermore, in vivo testing in appropriate behavioural animal paradigms is warranted.

Hydroxybenzoic Acids Are Significant Contributors to the Antioxidant Effect of Borututu Bark, Cochlospermum angolensis Welw. ex Oliv.

Borututu (Cochlospermum angolensis) is an African tree whose bark has recently emerged as a herbal dietary supplement with claims for antioxidant activity. In order to substantiate the claimed activity of borututu supplements, we performed an activity-guided fractionation of the total extract utilizing a 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay. Subsequent flash and centrifugal chromatography resulted in the isolation of gallic acid (1) and protocatechuic acid (2) as the main antioxidant constituents. Two apocarotenoids and one flavonoid were also isolated from the chloroform fraction and were identified as cochloxanthin (3), dihydrocochloxanthin (4), and 7,4'-dimethyltaxifolin (5), respectively. A High-performance liquid chromatography (HPLC) method was also developed for fingerprinting borututu samples, with Compounds 1-4 suggested as chemical markers for quality control purposes.

HPLC-Guided Isolation, Purification and Characterization of Phenylpropanoid and Phenolic Constituents of Nutmeg Kernel (Myristica fragrans).

Many studies on the biological activities of nutmeg continue to appear in the literature. The most common targets include GIT, CNS, oxidative stress and inflammation. However, results obtained from most studies are often inconsistent due to the variability of utilized samples, lack of standardized nutmeg products or insufficient amounts of pure compounds for comprehensive follow-up investigation. To address the consistency and supply issue we utilized available technology to develop a reproducible procedure for preparation of specific extracts and isolation of the major phenolic constituents present in nutmeg kemel. A well-defined and reproducible sequence of extraction, fractionation and chromatographic purification was adopted and was guided by HPLC fingerprinting. Spectroscopic methods, mainly NMR, and mass spectrometry were utilized to identify each compound. Thirteen compounds were isolated in a pure form and identified as: elemicin (1), isoelemicin (2), myristicin (4), surinamensin (5), malabaricone C (6), 2-(3'-allyl-2',6'-dimethoxy-phenyloxy)-l- acetoxy-(3,4-dimethoxyphenyl)-propyl ester (7), methoxylicarin A (8), licarin A (9), malabaricone B (10), licarin C (11), 5'-methoxylicarin B (12), licarin B (13), and 2-(3'-allyl-2',6'-dimethoxy-phenyloxy)-l-methyl-5-methoxy-1,2-dihydrobenzofuran (3, a new compound). With repeated isolation runs, these pure compounds can be prepared in quantities sufficient for biological evaluation as needed. The availability of purified compounds will also allow the development of specific, accurate, and sensitive analytical procedures for pharmacokinetic studies and for quality control of nutmeg products. Both aspects are essential for nutmeg-focused drug discovery. The same approach can also be adapted to other medicinal plants of potential interest.

Indirect modulation of the endocannabinoid system by specific fractions of nutmeg total extract.

CONTEXT: Nutmeg [Myristica fragrans Houtt. (Myristicaceae)] has a long-standing reputation of psychoactivity. Anecdotal reports of nutmeg use as a cheap marijuana substitute, coupled to previous studies reporting a cannabimimetic-like action, suggest that nutmeg may interact with the endocannabinoid system. OBJECTIVE: The study evaluates nutmeg fractions for binding capacity with various CNS receptors and their potential interaction with the endocannabinoid system. MATERIALS AND METHODS: Dichloromethane (DF) and ethyl acetate (EF) fractions were prepared from the methanol extract of powdered whole nutmeg. The HPLC-profiled fractions were assayed by the NIMH Psychoactive Drug Screening Program (PDSP) in a panel of CNS targets at a 10 µg/mL concentration. The fractions were also screened for fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) inhibition, initially at a concentration of 500 µg/mL, then by concentration-dependent inhibition studies. RESULTS: None of the tested fractions showed significant binding to CNS receptors included in the PDSP panel. However, both fractions exerted significant inhibition of the FAAH and MAGL enzymes. The DF fraction inhibited FAAH and MAGL enzymes at IC50 values of 21.06 ± 3.16 and 15.34 ± 1.61 µg/mL, respectively. Similarly, the EF fraction demonstrated FAAH and MAGL inhibition with IC50 values of 15.42 ± 3.09 and 11.37 ± 6.15 µg/mL, respectively. DISCUSSION AND CONCLUSION: The study provides the first piece of evidence that nutmeg interacts with the endocannabinoid system via inhibition of the endocannabinoid catabolizing enzymes. This mechanism provides insight into reported cannabis-like action as well as expands the potential therapeutic utility of nutmeg.

Chemical diversity and pharmacological significance of the secondary metabolites of nutmeg (Myristica fragrans Houtt.).

Nutmeg is a valued kitchen spice that has been used for centuries all over the world. In addition to its use in flavoring foods and beverages, nutmeg has been used in traditional remedies for stomach and kidney disorders. The antioxidant, antimicrobial and central nervous system effects of nutmeg have also been reported in literature. Nutmeg is a rich source of fixed and essential oil, triterpenes, and various types of phenolic compounds. Many of the secondary metabolites of nutmeg exhibit biological activities that may support its use in traditional medicine. This article provides an overview of the chemistry of secondary metabolites isolated from nutmeg kernel and mace including common methods for analysis of extracts and pure compounds as well as recent approaches towards total synthesis of some of the major constituents. A summary of the most significant pharmacological investigations of potential drug leads isolated from nutmeg and reported in the last decade is also included.

Content Variation of Catechin Markers, Total Phenolics and Caffeine in Green Tea Dietary Supplements.

Green tea (Camellia sinensis) preparations are among the top selling products in the United States dietary supplements market. Numerous manufacturers claim different levels of specific catechin markers in their products while many others use total phenolic concentration instead, or not at all. Limited quality control results have been published for green tea dietary supplements over the past seven years. Thus, the goal of this work was to correlate determined levels of phenolics, catechins, and caffeine with manufacturer label claims for selected dietary supplement products (26 total) purchased in the United States. The Folin-Ciocalteu (FC) method was used to determine the total phenolic content while reversed-phase (RP) HPLC was used to quantify the major catechins: epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG). The developed HPLC method was validated for accuracy and precision. It utilized a C8 column with gradient elution of acetonitrile in 0.1% aqueous formic acid over 11 min total run time. Peak detection was performed at 280 nm. Caffeine was also included in the HPLC method as another non-phenolic alkaloid marker commonly found in green tea. Both methods showed a good correlation between the content of catechins and polyphenolic compounds in the selected products. The ranges of total catechins and polyphenol concentrations were 3.8-70.2% and 3.6-95.8%, respectively, while that of caffeine was 0.8-11.2%. The selected products displayed a wide range of marker levels. A lack of conformity in disclosing the actual levels of marker compounds was also noticed in the labeling of many products.

Potential of Horse Apple Isoflavones in Targeting Inflammation and Tau Protein Fibrillization.

In our ongoing search for anti-inflammatory and neuroprotective agents of natural origin, the total methanolic extract (MPE) of horse apple (Maclura pomifera) and its two major prenylated isoflavones, osajin (OSA) and pomiferin (POM), were evaluated in vitro for their ability to affect four mediators of inflammation and to inhibit tau protein fibrillization. The two isoflavones were effective in enhancing the activity of NSAID activated gene (NAG-1) at 2.5 pg/mL (1.5-1.8 fold increase) and inhibiting iNOS and NF-κB activity with IC50 values in the range of 6-13 µg/mL. Pomiferin also inhibited intracellular oxidative stress with IC50 of 3.3 µg/mL, while osajin did not show any effect. The extract activated NAG-1 and inhibited iNOS and oxidative stress without affecting NF-κB. As observed by transmission electron microscopy (TEM), MPE, OSA and POM also inhibited arachidonic acid-induced tau fibrillization in a concentration-dependent manner.

Recently disclosed chemical entities as potential candidates for management of tuberculosis.

Tuberculosis (TB) is one of the deadliest infectious diseases worldwide. The drug discovery process of novel, safe and effective agents to combat TB involves identification of new molecular targets and novel chemical scaffolds. The current anti-TB drug pipeline includes several small molecules with more to follow as new candidates are disclosed. This review highlights the most significant findings described in 78 international, European and US patents for chemically diverse compounds as prospective anti-TB medications. Main points of emphasis include chemical classification, in vitro and in vivo activity, ADME/Tox profile and mycobacterial target as described in each patent. The collective mass of compounds disclosed in the reviewed patents introduces new candidates as potential therapeutic agents for TB infections.

Evaluation of cost versus antioxidant determinants in green tea dietary supplements.

OBJECTIVE: To investigate the association between cost and (a) chemical constituents and (b) antioxidant activity as quality determinants of select green tea supplements available in the United States. DESIGN/SETTING: Laboratory analysis of green tea using HPLC and antioxidant assay methods. MAIN OUTCOME MEASURES: Correlation between selected quality parameters and daily cost based on the serving size as stated in the label. Quality was defined in terms of (a) catechin levels (validated high-performance liquid chromatography method), (b) total phenolic content (Folin-Ciocalteu method), and (c) antioxidant activity (total antioxidant capacity and diphenylpicryl hydrazyl free-radical scavenging). RESULTS: A wide range of variation in marker levels and antioxidant activity was observed in the evaluated products. Catechin levels correlated well with the total phenolic content in each product while antioxidant activities were not as consistent when correlated with catechin/polyphenol levels. There was also a low correlation between product cost and quality. CONCLUSION: Our results indicate that product cost does not always reflect quality, at least within the selected range of products. Thus, for a pharmacist to be able to recommend quality green tea dietary supplements, factors other than cost should be considered.

HPLC determination of isoflavone levels in osage orange from the Midwest and southern United States.

The fruit of the Maclura pomifera tree is a sustainable source for the pharmacologically interesting isoflavones, osajin and pomiferin. A reversed-phase HPLC method was developed to identify osage orange samples with high isoflavone content and to determine the optimum conditions for sample preparation. Analytical run time was 8 min at a flow rate of 1 mL/min using a gradient of acetonitrile in H2O (0.1% formic acid) and UV peak detection at 274 nm. The method was validated for specificity, accuracy, precision, and limits of detection and quantitation (LOD/LOQ). The method was applied to determine the levels of osajin and pomiferin in extracts prepared from different samples of osage orange growing in the Midwest and southern United States. Results demonstrated the effect of different variables, such as sample preparation, geographical location, and growth stage, on the levels of osajin and pomiferin in analyzed samples.

Bioavailability of Plant-Derived Antioxidants.

Natural products with antioxidant properties have been extensively utilized in the pharmaceutical and food industry and have also been very popular as health-promoting herbal products. This review provides a summary of the literature published around the first decade of the 21st century regarding the oral bioavailability of carotenoids, polyphenols and sulfur compounds as the three major classes of plant-derived antioxidants. The reviewed original research includes more than 40 compounds belonging to the above mentioned classes of natural antioxidants. In addition, related reviews published during the same period have been cited. A brief introduction to general bioavailability-related definitions, procedures and considerations is also included.

Bioconversion of silybin to phase I and II microbial metabolites with retained antioxidant activity.

Microbial transformation of silybin (1), the major flavonolignan of milk thistle (Silybum marianum, Asteraceae), resulted in the isolation of four metabolites. The structures of the isolated metabolites were determined by spectroscopic methods. One phase I metabolite was produced by Beauveria bassiana and was characterized as 8-hydroxysilybin (2). Three phase II metabolites were produced by two Cunninghamella species and were identified as 2,3-dehydrosilybin-3-O-ß-d-glucoside (3), obtained from Cunninghamella species; and silybin-7-sulfate (4) and 2,3-dehydrosilybin-7-sulfate (5), obtained from Cunninghamella blakesleana. Compared to 1 (IC(50) 284 µg/mL), the generated metabolites displayed varying levels of antioxidant activities in the DPPH free-radical scavenging assay.

Antimycobacterial activity of ferutinin alone and in combination with antitubercular drugs against a rapidly growing surrogate of Mycobacterium tuberculosis.

The aim of this study was to investigate the antimycobacterial activity of the major daucane constituent, ferutinin (jaeschkeandiol p-hydroxybenzoate, 1), four of its natural analogues, its hydrolysis products, as well as methyl p-hydroxybenzoate (methylparaben) against Mycobacterium smegmatis, a rapidly growing surrogate of Mycobacterium tuberculosis. The agar dilution assay was utilised for an antimycobacterial evaluation of single compounds. A modified agar dilution assay, the checkerboard method, was utilised for evaluating the potentiating effect of 1 on different antitubercular drugs, namely isoniazid, ethionamide, rifampin and streptomycin. In the agar dilution assay, 1 exhibited higher potency (minimum inhibitory concentration [MIC] 10 µg mL⁻¹) than streptomycin and rifampin (MIC 20 µg mL⁻¹ for each). Of the natural analogues, 8,9-epoxyjaeschkeandiol p-hydroxybenzoate and 8,9-epoxyjaeschkeandiol benzoate exhibited marginal activity (MIC ≥ 40 and 80 µg mL⁻¹, respectively). The checkerboard method showed that the combination of 1 with each antitubercular drug led to mutual enhancement of the antimycobacterial activity with isoniazid and ethionamide, while no such effect was observed with rifampin or streptomycin. Based on this study and earlier studies with Staphylococcus aureus, the major constituent 1 may be responsible for the major part of the antimicrobial activity of the root of Ferula hermonis.

Towards a better understanding of the psychopharmacology of nutmeg: Activities in the mouse tetrad assay.

ETHNOPHARMACOLOGICAL RELEVANCE: Nutmeg, the seeds of Myritica fragrans (family Myristicaceae), is a well known kitchen spice with a long-standing reputation as a psychoactive herb. Nutmeg at high doses is considered a cheap substitute to several drugs of abuse. Earlier reports have attributed amphetamine-like activities to nutmeg. AIM OF THE STUDY: To characterize the neuropharmacological effects of different nutmeg extracts, administered orally and intraperitoneally, in comparison to Delta(9)-terahydrocannabinol, amphetamine, and morphine. MATERIALS AND METHODS: Methanolic (ME), dichloromethane (DE), and hexane (HE) extracts were obtained from a chromatographically fingerprinted batch of nutmeg. Biological evaluation was conducted in sets of 6-8 mice in the tetrad assay at doses ranging from 100 to 500 and 500 to 1000 mg/kg for i.p. and oral administration, respectively. RESULTS: While oral administration of all the nutmeg extracts at 500 mg/kg caused a significant increase in locomotor activity, the i.p. administration of DE showed significant reduction in rectal temperature along with a significant increase in tail flick latency at 300 mg/kg. A significant decrease in core body temperature was observed with HE at 100 mg/kg, while higher doses caused significant increases in hot plate latency. CONCLUSION: Different behavioral effects were observed that varied by the type of extract as well as by the route of administration.

Phytochemical and biological studies on Saudi Commiphora opobalsamum L.

The aerial part of Commiphora opobalsamum L. (Burseraceae) growing in Saudi Arabia was subjected to a phytopharmacological investigation in order to identify its major chemical constituents and to evaluate its extracts and isolated compounds in preliminary in vitro assays for antimicrobial, antimalarial, antitumor, anti-inflammatory (COX-2 inhibition), antioxidant and estrogenic activity. Six compounds were isolated and identified as the triterpenes friedelin, canophyllal, and oleanonic acid; the flavonols mearnsetin and quercetin; and syringic acid. The ethyl acetate extract was moderately active against Staphylococcus aureus, Pseudomonas aeruginosa, and Plasmodium falciparum; while the petroleum ether and chloroform extracts inhibited COX-2 at 5 and 10 microg mL(-1), respectively. Of the isolated compounds, syringic acid showed moderate antimalarial, anticandidal, and antimycobacterial activity; while mearnsetin and quercetin exhibited antioxidant activity comparable to ascorbic acid and trolox. This is the first detailed phytochemical investigation of C. opobalsamum L. growing in Saudi Arabia and elsewhere. The isolated compounds are reported from this plant for the first time and their full (1)H and (13)C NMR assignments are included.

HPTLC determination of caffeine in stimulant herbal products and power drinks.

The caffeine content of selected herbal products and energy drinks available in the Saudi market was determined by HPTLC-UV densitometric analysis. Pre-coated HPTLC silica gel plates (20cm x 10cm) were used for the analysis. The solvent system consisted of ethyl acetate-methanol (85:15, v/v), and caffeine was detected at 275nm. The developed method was validated for specificity, repeatability (C.V. < 5%), recovery (98.90 +/- 3.46), and accuracy (99.84 +/- 2.87). The levels of caffeine were 4.76-13.29% (w/w) and 0.011-0.032% (w/v), for the herbal products and the energy drinks, respectively.

Lotaustralin from Rhodiola rosea roots.

Lotaustralin was isolated as a mixture of two diastereoisomeric forms from the methanol extract of Rhodiola rosea roots, together with the known compounds rosavin, rosarin, rosin, rosiridin, salidroside, and beta-sitosterol. The structure of lotaustralin was established by 1D and 2D-NMR spectroscopy, including 1H-1H COSY, NOESY, HMQC, and HMBC, and FAB and HR MS.