Free and Glycosidically Bound Volatiles of Mentha longifolia Growing in Croatia

The glycosides of volatile compounds and the essential oil were isolated from wild Mentha longifolia. After the enzymatic hydrolysis of glycosides, fourteen volatile aglycones were identified by GC/MS. The main aglycones were eugenol, 2-phenylethanol, benzyl alcohol, lavandulol, trans- and cis-carveol, 3-octanol, and 3-hexen-1-ol. The content of aglycones was 40.85 mg kg^-1 of dried plant material. The main components of the essential oil (yield 0.93 w/w) were carvone, piperitenone oxide, limonene, and -caryophyllene. Eugenol, carveol, 3-octanol, and -terpineol were identified in the aglycones and in the essential oil.     

Developing a validated liquid chromatography-mass spectrometric method for the simultaneous analysis of five bioactive quassinoid markers for the standardization of manufactured batches of Eurycoma longifolia Jack extract as antimalarial medicaments

An extensive comparative study on the electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) mass spectrometry using automated flow injection analysis (FIA), was performed on eurycomanone (1), 13α(21)-epoxyeurycomanone (2), eurycomanol (3), eurycomanol-2-O-β-d-glucopyranoside (4), and 13,21-dihydroeurycomanone (5), the bioactive markers isolated from Eurycoma longifolia. The effects of eluent mixture (methanol or acetonitrile in water) and acidic modifiers (acetic acid, formic acid and trifluoroacetic acid) on the ionization efficiency of the markers were also investigated. The ESI in the positive ion mode with methanol containing 0.1% (v/v) acetic acid was selected for the subsequent optimization of nebulizer pressure, dry gas flow, dry gas temperature and capillary voltage to improve the sensitivity of the total ion chromatogram (TIC). Fragmentation of the analytes was further investigated by varying the capillary exit offset voltage and fragmentation amplitude in positive mode of ESI. The detection limits (LODs) were determined in isolation mode (selected ion monitoring, SIM). Their limits of detection (LODs) ranged between 0.03 and 0.1μgmL(-1) while the intra-day and inter-day precisions were less than 5.72% and 4.82%, respectively. The method was next applied for the simultaneous analysis of the markers to standardize various batches of manufactured extracts of E. longifolia for potential use as antimalarial products. Multiple Reaction Monitoring (MRM) mode was used for the quantification of analytes which gave protonated molecular ion, [M+H](+). For those without pseudo-molecular ions, SIM mode was used to quantify the analytes. The batches contained 5.65-9.95% of eurycomanone (1), 5.21-19.75% of eurycomanol (3) and 7.59-19.95% of eurycomanol-2-O-β-d-glucopyranoside (4) as major quassinoids whereas, 13α(21)-epoxyeurycomanone (2), and 13,21-dihydroeurycomanone (5) were much lower in concentrations of 0.78-3.90% and 0.47-1.76%, respectively.     

A new anti-inflammatory β-carboline alkaloid from the hairy-root cultures of Eurycoma longifolia

One new β-carboline alkaloid 7-methoxy-(9H-β-carbolin-1-il)-(E)-1-propenoic acid (1) together with 9-methoxycanthin-6-one (2) and 9-hydroxycanthin-6-one (3) were isolated from the hairy-root cultures of Eurycoma longifolia. The effects of these compounds on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells were investigated. Compound 1 strongly inhibited the production of NO while 2 and 3 having weak or inactive effect. Consistently, compound 1 decreased the expression of cyclooxygenase-2 and inducible nitric oxide synthase.     

Quassinoids from the Roots of Eurycoma longifolia and Their Anti-Proliferation Activities

A phytochemical investigation on the roots of medicinal plant Eurycoma longifolia resulted in the isolation of 10 new highly oxygenated C₂₀ quassinoids longifolactones G‒P (1–10), along with four known ones (11–14). Their chemical structures and absolute configurations were unambiguously elucidated on the basis of comprehensive spectroscopic analysis and X-ray crystallographic data. Notably, compound 1 is a rare pentacyclic C₂₀ quassinoid featuring a densely functionalized 2,5-dioxatricyclo[5.2.2.0^4,8]undecane core. Compound 4 represents the first example of quassinoids containing a 14,15-epoxy functionality, and 7 features an unusual α-oriented hydroxyl group at C-14. All isolated compounds were evaluated for their anti-proliferation activities on human leukemia cells. Among the isolates, compounds 5, 12, 13, and 14 potently inhibited the in vitro proliferation of K562 and HL-60 cells with IC₅₀ values ranging from 2.90 to 8.20 μM.     

Method Development and Quantitative Elemental Analysis of Mentha Longifolia L. Leaves from Saudi Arabia by Total Reflection X-Ray Fluorescence

A method has been developed for the quantitative elemental analysis of Mentha longifolia L. leaves collected from different cities in Saudi Arabia using total reflection X-ray fluorescence. Using a microwave digestion system, 100?mg of each sample was completely digested using 3?mL of nitric acid and 2?mL of hydrogen peroxide. The stabilization of the digested samples on the silicon reflectors was studied using a silicone solution and polyvinyl alcohol. 5?µL of either silicone solution or polyvinyl alcohol (1% m/v) was pipetted and dried on the silicon reflector prior to the deposition of the digested samples. It was recognized that there is some enhancement on the intensity of the peak area with the silicone solution at photon energies less than 11?keV. However, the obtained results confirm the ability of using silicone solution or polyvinyl alcohol (1% m/v) as the stabilizer prior to the deposition of the sample droplet on the quartz reflector. However, the silicone solution was more applicable. Based on the developed method, 15 elements were quantified, namely, P, S, Cl, K, Ca, Ti, Cr, Mn, Fe, Cu, Zn, Br, Rb, Sr, and Ba. Based on the present quantitative analysis results, M. longifolia samples collected from Al-Madina city had the highest concentration of P, Cl, K, Ti, Fe, Ni, and Cu. The lowest concentrations of P, S, Cl, K, Mn, Ni, and Br were found in Taif-Shafa.     

Simultaneous estimation of 16α-hydroxycleroda-3,13(14) Z-dien-15,16-olide from Polyalthia longifolia and its metabolite in hamster plasma: application to pharmacokinetic study

A selective and sensitive LC–MS‐MS method was developed and validated for simultaneous estimation and pharmacokinetic studies of 16α‐hydroxycleroda‐3,13(14) Z‐dien‐15,16‐olide (K‐09) obtained from Polyalthia longifolia and its metabolite (K‐9T), a novel antidyslipidemic agent. Sample clean‐up involved liquid–liquid extraction of both the analytes and internal standard (rosuvastatin) from 200 μL of hamster plasma. The analytes were chromatographically separated on a Symmetry‐Shield C₁₈ (5 µm, 4.6 × 150 mm) column, using acetonitrile–0.1% aqueous formic acid (92:08, v/v) as the mobile phase. Detection was performed using negative ion electrospray ionization in multiple reaction monitoring mode. The MS/MS response was linear over the concentration range 1.56–200 ng/mL, with a correlation coefficient (r²) of 0.998 or better. The within‐ and between‐batch precisions (relative standard deviation, %RSD) and the accuracy (percentage bias) were within acceptable limits as per FDA guidelines. The validated method was successfully applied to reveal the pharmacokinetic parameters of K‐09 and metabolite after oral administration. This method will therefore be highly useful for future studies of K‐09 and metabolite K‐9T pharmacokinetics in preclinical and clinical studies. Copyright © 2011 John Wiley & Sons, Ltd.     

The Modulation of PCSK9 and LDLR by Supercritical CO2 Extracts of Mentha longifolia and Isolated Piperitone Oxide,an In Vitro Study

In the present study the ability of supercritical carbon dioxide (SCO₂) extracts of M. longifolia L. leaves to modulate low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) expression was evaluated in cultured human hepatoma cell lines Huh7 and HepG2. Two SCO₂ extracts, one oil (ML-SCO₂) and a semisolid (MW-SCO₂), were subjected to detailed chemical characterization by mono- and bidimensional nuclear magnetic resonance (1D, 2D-NMR), gas chromatography coupled with mass spectrometry (GC-MS) and liquid chromatography coupled with mass spectrometry (LC-MS). Chemical analysis revealed significant amounts of fatty acids, phytosterols and terpenoids. ML-SCO₂ was able to induce LDLR expression at a dose of 60 µg/mL in HuH7 and HepG2 cell lines. Furthermore, ML-SCO₂ reduced PCSK9 secretion in a concentration-dependent manner in both cell lines. Piperitone oxide, the most abundant compound of the volatile constituent of ML-SCO₂ (27% w/w), was isolated and tested for the same targets, showing a very effective reduction of PCSK9 expression. The overall results revealed the opportunity to obtain a new nutraceutical ingredient with a high amount of phytosterols and terpenoids using the SCO₂ extraction of M. longifolia L., a very well-known botanical species used as food. Furthermore, for the first time we report the high activity of piperitone oxide in the reduction of PCSK9 expression.     

The In Vitro Anti-Cancer Activities and Mechanisms of Action of 9-Methoxycanthin-6-one from Eurycoma longifolia in Selected Cancer Cell Lines

An alkaloid compound from the hairy root culture of Eurycoma longifolia has been isolated and characterised as 9-methoxycanthin-6-one. The aims of these studies were to investigate the in vitro anti-cancer activities of 9-methoxycanthin-6-one against ovarian cancer (A2780, SKOV-3), breast cancer (MCF-7), colorectal cancer (HT29), skin cancer (A375) and cervical cancer (HeLa) cell lines by using a Sulphorhodamine B assay, and to evaluate the mechanisms of action of 9-methoxycanthin-6-one via the Hoechst 33342 assay and proteomics approach. The results had shown that 9-methoxycanthin-6-one gave IC₅₀ values of 4.04 ± 0.36 µM, 5.80 ± 0.40 µM, 15.09 ± 0.99 µM, 3.79 ± 0.069 µM, 5.71 ± 0.20 µM and 4.30 ± 0.27 µM when tested in A2780, SKOV-3, MCF-7, HT-29, A375 and HeLa cell lines, respectively. It was found that 9-methoxycanthin-6-one induced apoptosis in a concentration dependent manner when analysed via the Hoechst 33342 assay. 9-methoxycanthine-6-one were found to affect the expressions of apoptotic-related proteins, that were proteins pyruvate kinase (PKM), annexin A2 (ANXA2), galectin 3 (LGAL3), heterogeneous nuclear ribonucleoprotein A1 (HNRNP1A1), peroxiredoxin 3 (PRDX3), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the differential analysis of 2-DE profiles between treated and non-treated 9-methoxycanthine-6-one. Proteins such as acetyl-CoA acyltransferase 2 (ACAA2), aldehyde dehydrogenase 1 (ALDH1A1), capping protein (CAPG), eukaryotic translation elongation factor 1 (EEF1A1), malate dehydrogenase 2 (MDH2), purine nucleoside phosphorylase (PNP), and triosephosphate isomerase 1 (TPI1) were also identified to be associated with A2780 cell death induced by 9-methoxycanthine-6-one. These findings may provide a new insight on the mechanisms of action of 9-methoxycanthin-6-one in exerting its anti-cancer effects in vitro.     

Determination of eurycomanone in rat plasma using hydrophilic interaction liquid chromatography–tandem mass spectrometry for pharmacokinetic study

In this study, a rapid, sensitive, and reliable hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC‐MS/MS) method for the determination of eurycomanone in rat plasma was developed and validated. Plasma samples were pretreated with a protein precipitation method and quercitrin was used as an internal standard (IS). A HILIC silica column (2.1 × 100 mm, 3 μm) was used for hydrophilic‐based chromatographic separation, using the mobile phase of 0.1% formic acid with acetonitrile in gradient elution at a flow rate of 0.25 mL/min. Precursor–product ion pairs for multiple‐reaction monitoring were m /z 409.1 → 391.0 for eurycomanone and m /z 449.1 → 303.0 for IS. The linear range was 2–120 ng/mL. The intra‐ and inter‐day accuracies were between 95.5 and 103.4% with a precision of <4.2%. The developed method was successfully applied to the pharmacokinetic analysis of eurycomanone in rat plasma after oral dosing with pure compound and E. longifolia extract. The C _max and AUC_0–t , respectively, were 40.43 ± 16.08 ng/mL and 161.09 ± 37.63 ng h/mL for 10 mg/kg eurycomanone, and 9.90 ± 3.97 ng/mL and 37.15 ± 6.80 ng h/mL for E. longifolia extract (2 mg/kg as eurycomanone). The pharmacokinetic results were comparable with each other, based on the dose as eurycomanone.     

Analysis of Secondary Metabolite Production in Somatic Embryo of Pasak Bumi (Eurycoma Longifolia Jack.)

Pasak bumi (Eurycoma longifolia Jack.) has been known as a plants that can produce secondary metabolites for medicinal purposes such as: aphrosidiac, antimalaria, dysentri, antitumor, etc. Poor seed germination of pasak bumi will affect the avaibility of plant material for drug extraction. Over exploitation of this plant will also reduce plant population in its natural habitat. In vitro culture, i.e. through somatic embryogenesis, therefore, can be used as one of an alternative method for plant regeneration as well as for in vitro metabolite production. Based on this reason, the research has been done with an objective to analyze the presence of secondary metabolite in somatic embryo of pasak bumi. Seed-derived callus was used as an explant. This callus was maintained to proliferate in MS (Murashige&Skoog, 1962) medium supplemented with 2.25 mg/L 2,4-D and 2.0 mg/L kinetin. A half gram of callus from proliferation medium was transferred into the MS liquid medium containing 1.0 or 2.25 mg/L 2,4-D, and 2.0 mg/L BAP or 2.0 mg/L kinetin. Histochemical examination using Jeffrey's reagen and neutral red showed that alkaloid and terpenoid substances were presence in somatic embryo of pasakbumi. In accordance with histochemical test, GC-MS analyses showed that secondary metabolites was also synthesized by non embryogenic callus and the mixture ofembryogenic callus and somatic embryo, although the concentration in the mixture of embryogenic callus and somatic embryo was lower than those in non-embryogenic callus. Secondary metabolites, including 3-[(cyclohexyl-methyl-amino)-methyl]-3H-benzooxazole-2-one (0.06%) and 2-furancarboxaldehyde, 5-(hydroxymethyl) (43.024%) were found in embryogenic callus and somatic embryo. In addition, the mixture of embryogenic callus and somatic embryo also synthesized fatty acid and lipids (52.751%) which was higher than non-embryogenic callus (24.789%). Based on the result, the mixture of embryogenic callus and somatic embryo could produce secondary metabolites, such as alkaloid, terpenoid subtances, and phenol. The concentration of metabolites in the mixture of embryogenic callus and somatic embryo, however, was lower compare to non-embryogenic callus.