Validated HPLC method for simultaneous estimation of khellol glucoside, khellin and visnagin in Ammi visnaga L. fruits and pharmaceutical preparations.

Tea bags including fruits of Ammi visnaga L. are used in Egypt as remedy for the treatment of kidney stones. Our study focuses on developing simple and rapid method utilising HPLC for quantitative estimation of khellol glucoside (KG), khellin (KH) and visnagin (VS) simultaneously. Their concentrations were determined in A. visnaga L. fruits at different developmental stages and in pharmaceutical formulations together with following up them during shelf life. Separation was accomplished using HPLC. Perfect resolution between KG, KH and VS was possible through using a mobile phase consisting of water:methanol:tetrahydrofuran (50:45:5, v/v/v). Peaks were detected at 245 nm. The suggested method for the determination of KG, KH and VS was successful in determining the analytes of interest without any interference of other compounds and matrix. All validation parameters were satisfactory and the procedure was relatively easy and fast as extracts are evaluated without previous steps of purification.

Antitumor and antimicrobial activities of some hetero aromatic benzofurans derived from naturally occurring visnagin.

Bromination of visnaginone (1) yielded the dibromo derivative (2), which upon methylation with methyl iodide gave 1-(2,7-dibromo-4,6-dimethoxybenzofuran-5-yl) ethanone (3). Compound (3) reacted with dimethylformamide dimethylacetal to give (4). The reaction of (3) with aromatic aldehydes namely (vanillin, benzaldehyde and 3-anisaldehyde) in ammonium acetate, malononitrile and/or butyric cyanoanhydride gave the 2-amino substituted nicotinonitriles (5a-c) and the 2-hydroxyl substituted nicotinonitriles (7a-c), respectively, while in piperidine gave (E)-1-(2,7-dibromo-4,6-dimethoxybenzofuran-5-yl)-3-(substituted)prop-2-en-l-one (11a-c). (5a) was hydrolyzed with sulfuric acid on cold to give the nicotinic acid derivative (6a). When compound (3) reacted with hydrazines and aromatic amines, it gave the Schiff bases (8a,b) and (10a,b), respectively. (8b) reacted with thioglycolic acid to give the thiazolidin-4-one (9b). When (11a-c) reacted with thiourea, it gave the pyrimidine derivatives (12a-c). (11a,b) also reacted with butyric cyanoanhydride and hydroxylamine hydrochloride to give (13a,b) and (15a,b), respectively. When the carboxylate (13a) was treated with 2,4-dinitroaniline, it gave the carboxamide (14a). Compounds (11b,c) reacted with hydrazine derivatives (hydrazine hydrate and phenylhydrazine) yielding the substituted pyrazole derivatives (16b,c) and (17b,c), respectively. All the structures of the synthesized compounds were elucidated by elemental analyses and spectral data. The newly synthesized benzofuran compounds showed a strong to moderate cytotoxicity against liver HEPG2 cancer cell line compared to 5-fluorouracil and doxorubicin (the anticancer agents). Compounds (2, 6a, 13a, 14a, 16c and 17b) were the most active compounds in descending order. The synthesized compounds were also tested for their antimicrobial activity. Compound (10b) showed the highest activity against all the tested strains followed by 6, 10a, 5a, 8b and 7a in descending order.

Nonlinear pharmacokinetics of visnagin in rats after intravenous bolus administration.

Ammi visnaga L. (syn. Khella, Apiaceae) preparations have traditionally been used in the Middle East for the treatment of kidney stone disease. Visnagin, a furanocoumarin derivative, is one of the main compounds of Ammi visnaga with potential effects on kidney stone prevention. To date, no information is available about the pharmacokinetic (PK) properties of visnagin. It was the aim of the study to characterize the PK properties of visnagin after intravenous (i.v.) bolus administration in rats and to develop an adequate model for the description of the observed data, including model parameter estimates. Therefore, three doses of visnagin (1.25, 2.5, and 5mg/kg) solubilized in 25% Captisol® were administered by i.v. bolus injection to male Sprague-Dawley rats. Plasma samples were extracted and subsequently analyzed using a validated LC-MS/MS method. Both non-compartmental and compartmental PK analyses were performed. A stepwise model building approach was applied including nonlinear mixed effect modeling for final model selection and to obtain final model estimates in NONMEM VI. The average areas under the curve (AUC(0-last)) after doses of 1.25, 2.5, and 5mg/kg were 1.03, 3.61, and 12.6 mg *h/l, respectively. The shape of the plasma concentration-time profiles and the observed disproportionate increase in AUC(0-last) with increasing dose suggested nonlinearity in the elimination of visnagin. A two-compartment Michaelis-Menten model provided the best fit with following typical values of the parameter estimates: 2.09 mg/(l*h) (V(max)), 0.08 mg/l (K(M)), 0.175 l (V(C)), 1.0 h⁻¹ (k12), and 1.22 h⁻¹ (k21). Associated inter-subject variability estimates (% CV) for V(max), K(M) and V(C) were 21.8, 70.9, and 9.2, respectively. Intra-subject variability (constant CV error model) was estimated to be 7.0%. The results suggest the involvement of a saturable process in the elimination of visnagin, possibly an enzyme or transporter system.

Synthesis of new visnagen and khellin furochromone pyrimidine derivatives and their anti-inflammatory and analgesic activity.

6-[(4-Methoxy/4,9-dimethoxy)-7-methylfurochromen-5-ylideneamino]-2-thioxo-2,3-dihydropyrimidin-4-ones 1a,b were prepared by reaction of 6-amino-2-thiouracil with visnagen or khellin, respectively. Reaction of 1a,b with methyl iodide afforded furochromenylideneaminomethylsulfanylpyrimidin-4-ones 2a,b. Compounds 2a,b were reacted with secondary aliphatic amines to give the corresponding furochromen-ylideneamino-2-substituted pyrimidin-4-ones 3a-d. Reaction of 3a-d with phosphorus oxychloride yielded 6-chlorofurochromenylidenepyrimidinamines 4a-d, which were reacted with secondary amines to afford furochromenylideneamino-2,6-disubstituted pyrimidin-4-ones 5a-d. In addition, reaction of 5a-d with 3-chloropentane-2,4-dione gave 3-chloro-furochromenylpyrimidopyrimidines 6a-d. The latter were reacted with piperazine and morpholine to give 1-(furochromenyl)-pyrimidopyrimidine-3,6,8-triylpiperazines or -3,6,8-triylmorpholines 7a-d. The chemical structures of the newly synthesized compound ware characterized by IR, ¹H-NMR, ¹³C-NMR and mass spectral analysis. These compounds were also screened for their analgesic and anti-inflammatory activities. Some of them, particularly 3-7, exhibited promising activities.

Prevention of renal crystal deposition by an extract of Ammi visnaga L. and its constituents khellin and visnagin in hyperoxaluric rats.

In Egypt, teas prepared from the fruits of Ammi visnaga L. (syn. "Khella") are traditionally used by patients with urolithiasis. The aim of this study was to evaluate whether oral administration of an aqueous extract prepared from the fruits of A. visnaga as well as two major constituents khellin and visnagin could prevent crystal deposition in stone-forming rats. Hyperoxaluria was induced in male Sprague-Dawley rats by giving 0.75% ethylene glycol and 1% ammonium chloride via the drinking water. The Khella extract (KE; 125, 250 or 500 mg/kg) was orally administered for 14 days. The histopathological examination of the kidneys revealed that KE significantly reduced the incidence of calcium oxalate (CaOx) crystal deposition. In addition, KE significantly increased urinary excretion of citrate along with a decrease of oxalate excretion. Comparable to the extract, khellin and visnagin significantly reduced the incidence of CaOx deposition in the kidneys. However, both compounds did not affect urinary citrate or oxalate excretion indicating a mechanism of action that differs from that of the extract. For KE, a reasonably good correlation was observed between the incidence of crystal deposition, the increase in citrate excretion and urine pH suggesting a mechanisms that may interfere with citrate reabsorption. In conclusion, our data suggest that KE and its compounds, khellin and visnagin, may be beneficial in the management of kidney stone disease caused by hyperoxaluria but that it is likely that different mechanism of action are involved in mediating these effects.

Anti-inflammatory effect of visnagin in lipopolysaccharide-stimulated BV-2 microglial cells.

Visnagin, which is found in Ammi visnaga, has biological activity as a vasodilator and reduces blood pressure by inhibiting calcium influx into the cell. The present study demonstrates the anti-inflammatory effect of visnagin on lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. When cells were treated with visnagin prior to LPS stimulation, production of nitric oxide and expression of iNOS were attenuated in a dose-dependent manner. Visnagin also caused a significant decrease of mRNA expression and release of TNF-α, IL-1ß and IFNγ. In addition, visnagin reduced LPS-induced IL-6 and MCP-1 mRNA level. We further found that visnagin dose-dependently inhibited LPS-induced AP-1 and NF-κB luciferase activities. Taken together, our results for the first time suggest that the anti-inflammatory effect of visnagin might result from the inhibition of transcription factors, such as AP-1 and NF-κB.

An aqueous extract of Ammi visnaga fruits and its constituents khellin and visnagin prevent cell damage caused by oxalate in renal epithelial cells.

Teas prepared from the fruits of Ammi visnaga L. (syn. "Khella") have been traditionally used in Egypt as a remedy to treat kidney stones. It was the aim of our study to evaluate the effect of a Khella extract (KE) as well as the two major constituents khellin and visnagin on renal epithelial injury using LLC-PK1 and Madin-Darby-canine kidney (MDCK) cells. Both cell lines provide suitable model systems to study cellular processes that are possibly involved in the development of a renal stone. LLC-PK1 and MDCK cell lines were exposed to 300 microM oxalate (Ox) or 133 microg/cm(2) calcium oxalate monohydrate (COM) in presence or absence of 10, 50, 100 or 200 microg/mL KE. To evaluate cell damage, cell viability was assessed by determining the release of lactate dehydrogenase (LDH). KE (e.g. 100 microg/ml) significantly decreased LDH release from LLC-PK1 (Ox: 8.46+0.76%; Ox + 100 microg/ml KE: 5.41+0.94%, p<0.001) as well as MDCK cells (Ox: 30.9+6.58%; Ox+100 microg/ml KE: 17.5+2.50%, p<0.001), which indicated a prevention of cell damage. Similar effects for KE were observed in both cell lines when COM crystals were added. In LLC-PK1 cells khellin and visnagin both decreased the % LDH release significantly in cells that were pretreated with Ox or COM crystals. However, khellin and visnagin exhibited different responses in MDCK cells. Whereas khellin slightly reduced the % LDH release after exposure of the cells to Ox and COM crystals, visnagin significantly decreased % LDH release only after COM crystal exposure. Overall both compounds were more active in LLC-PK1 than in MDCK cells. In summary, exposure of renal epithelial cells to Ox or COM crystals was associated with a significant release of LDH indicating cell injury. Our data demonstrate that KE as well as khellin and visnagin could prevent renal epithelial cell damage caused by Ox and COM and could therefore play a potential role in the prevention of stone formation associated with hyperoxaluria.

Determination of visnagin in rat plasma by liquid chromatography with tandem mass spectrometry and its application to in vivo pharmacokinetic studies.

A rapid and sensitive method for the determination of visnagin in rat plasma was developed using liquid chromatography tandem mass spectrometry (LC-MS/MS). The plasma samples were processed by protein precipitation with methanol:acetonitrile (25:75) and warfarin was used as the internal standard. After vortex mixing and centrifugation, the supernatants were diluted with water (1:5) and then directly injected onto a Phenomenex Synergi Max RP column (75 mm x 2.0 mm ID, 4 microm) with isocratic elution at a flow rate of 0.2 mL/min. The mobile phase consisted of water and methanol (15:85, v/v) containing 0.1% formic acid and 5mM ammonium acetate. The total run time (injection to injection) was less than 4 min. Detection of the analytes was achieved using positive ion electrospray in the selected reaction monitoring (SRM) mode. The linear standard curve ranged from 1.0 to 5000 ng/mL and the precision and accuracy (inter- and intra-run) were within 4.5% and 4.3%, respectively. The method, which is rapid, simple, and precise, was successfully used to support a visnagin pharmacokinetics study.

Synthesis of 6- and 9-alkylaminomethyl furoflavones as gastroprotective agents.

The synthesis of 9- and 6-alkylaminomethyl furoflavones 5a, b, 9a-c, 13a, b, 15a-g and 18 from the naturally occurring chromones visnagin and khellin. Gastroprotective potency of these compounds in the ethanol damage model was determined. The results indicate that, through appropriate substitution, furoflavones can be obtained that are gastroprotective.

Ultraviolet photoionization of the photosensitizers khellin and visnagin in aqueous solution and in micelles: one-photon ionization is a minor process.

One-photon ionization, leading to formation of hydrated electrons and radical cations, has been proposed as a possible mechanism of action of some sensitizers in photobiology. In this contribution, we have investigated this proposal for the compounds khellin and visnagin, used in photomedical applications. Nanosecond transient absorption spectroscopy covering a wide range of laser pulse energies was employed to measure the formation of radical cations and hydrated electrons in aqueous solution and in cationic (CTAB) as well as anionic (SDS) micellar solutions. A model allowing for simultaneous one- and two-photon processes and fully accounting for the nonlinearity of the pulse energy dependence was used to simulate the data. The results did not support the hypothesis of a significant role of one-photon ionization, the upper limits of the quantum yields of radical cation formation being phi < 0.01 for visnagin and phi < 0.004 for khellin.

Nanosecond time-resolved infrared studies of visnagin and khellin triplets and radical ions.

Time-resolved infrared spectroscopy (TRIR) and density functional theory (DFT) calculations were used to directly observe and assign the vibrational spectra of the triplet states of visnagin and khellin, and to investigate their electron-transfer chemistry. The TRIR spectra of triplet visnagin and triplet khellin, and of their radical cations and anions, were obtained upon 266 nm laser flash photolysis in acetonitrile and in deuterated acetonitrile. The radical cations were observed in the presence of chloranil, and the radical anions were formed in the presence of NaI and KSCN. The TRIR spectra are in good agreement with the calculated vibrational spectra. We did not observe the related neutral radicals by TRIR spectroscopy upon laser flash photolysis (LFP) of khellin in the presence of hydroquinone, but we found evidence for the formation of semiquinone and neutral visnagin radicals upon LFP of visnagin and hydroquinone.

Determination of khellin and visnagin in Ammi visnaga fruits by capillary electrophoresis.

A new, simple and rapid capillary electrophoresis method was developed for the identification and quantitative determination of two medically active constituents-khellin and visnagin-in the extracts of Ammi visnaga fruits. Micellar electrochromatographic separation of khellin and visnagin was carried out using 10 mmol/l borate, 50 mmol/l sodium dodecylsulfate, 25% (v/v) acetonitrile at pH 9 as running buffer. Ammi visnaga fruits were extracted with methanol and the extracts were directly injected without any purification and pre-separation processes. Coumarin was used as internal standard for quantitation and the limits of detection for khellin and visnagin were 2.36 and 1.97 microg/ml, respectively using UV detection at 245 nm.

Photosynthesis of furocoumarin- and furochromone-types potential intercalative alkylating and oxidizing agents of DNA through photooxidations using gamma-ray.

The photooxygenation of imperatorin (1a) under gamma-ray irradiation afforded the hydroperoxides 2a and 3a. Similarly, the photooxygenation of alloimperatorin (1b) gave the hydroperoxide (2b). Visnagin (1c) was also photooxygenated to give the hydroperoxide (2c) as sole product. On the other hand, the photooxygenation of khellin (1d) gave the endoperoxide (2d) as a sole product. The epoxidation of imperatorin (1a) using hydrogen peroxide under gamma-ray irradiation afforded the epoxide 5a. Similarly visnagin (1c) and khellin (1d) were epoxidized to give the epoxides 5c and 5d.

Furanochromone radical cations: generation, characterization and interaction with DNA.

The radical cations of naturally occurring furanochromones visnagin (VI) and khellin (KH) have been generated and identified for the first time by use of laser flash photolysis and pulse radiolysis techniques. The lifetimes of VI(.+) and KH(.+) are determined as approximately 6 and approximately 35 micros under these conditions, respectively. Direct 308-nm excitation of VI in aqueous buffer at physiological pH results in monophotonic photoionization to generate VI(.+), with a quantum yield of 0.075, which is much higher than that of 8-methoxypsoralen and KH under identical conditions. Though VI(.+) is a more powerful oxidant than KH(.+), both of them react with guanosine mononucleotide (k=1.2x10(9) and 3.8x10(7) dm(3) mol(-1) s(-1), respectively) via electron transfer to give the guanine radical cation. Furthermore, selective oxidation of guanine in single and double strand DNA by VI(.+) was also observed. These novel findings suggest that electron transfer reactions involving furanochromone radical cations may be of considerable importance in furanochromone photochemotherapy.

Cardiovascular effects of visnagin on rats.

The present article describes the effects of visnagin on systolic blood pressure and heart rate in the anaesthetized rat. Intravenous administration of visnagin (0.3-5 mg kg-1) produced dose-related decreases in blood pressure with no significative changes in heart rate. Under nitric oxide synthase inhibition (L-NAME, 50 mg kg-1) the hypotensive effects of visnagin (5 mg kg-1) were not affected. Visnagin (5 x 10(-6) M-10(-4) M) produced a weak decrease in the rate and amplitude of spontaneous contractions in right atria. Visnagin also caused a weak decrease in peak contractile force and the df/dtmax with no significant changes in the time to peak tension or the time for total contraction in left atria driven at a basal rate of 1 Hz. Visnagin (10(-5) M, 5 x 10(-5) M and 10(-4) M) concentration-dependently decreased pressor response to KCl (IC50 = 5.1 +/- 2.5 x 10(-5) M) and noradrenaline (IC50 = 2.6 +/- 0.9 x 10(-5) M) in rat isolated mesenteric beds. Visnagin (3 x 10(-7) M-10(-4) M) induced a concentration-dependent relaxation of isolated mesenteric arteries contracted by noradrenaline (IC50 = 1.7 +/- 0.8 x 10(-5) M). The relaxant effects in the absence of functional endothelium were not significantly different (IC50 = 1.5 +/- 0.3 x 10(-5) M, P > 0.05) from those observed in segments with intact endothelium. In conclusion, the main mechanism responsible for the acute hypotensive effect of visnagin is the vasorelaxant response induced by this drug in resistance arteries.

Effects of visnagin on cyclic nucleotide phosphodiesterases and their role in its inhibitory effects on vascular smooth muscle contraction.

1. Visnagin relaxed aortae previously contracted by noradrenaline. This effect was unalterated by endothelium removal and potentiated, at high concentrations, by the previous incubation with sodium nitroprusside. 2. Visnagin weakly inhibited the hydrolytic activity of the cyclic nucleotide phosphodiesterase (PDE) isozymes (PDE5, PDE4, PDE3, cyclic GMP activated PDE2 and PDE1). 3. The present results indicate an involvement of PDE inhibition in the relaxant effect of visnagin at high concentration (>5x10(-5) M).

Inhibition of metabolic activation of the promutagens, benzo[a]pyrene, 2-aminofluorene and 2-aminoanthracene by furanochromones in Salmonella typhimurium.

Khellin, a naturally occurring furanochromone (Ammi visnaga fruits), inhibited the mutagenicity of the promutagens benzo[a]pyrene, 2-aminofluorene and 2-aminoanthracene in Salmonella typhimurium TA98. The effect varied greatly and depended on the S9 fraction used. Cytosolic activation of 2-aminoanthracene was also inhibited. Khellin produced no effect or only weak activity against the direct acting mutagens 2-nitrofluorene, 4-nitro-o-phenylenediamine, 1-nitropyrene and ethylmethane sulfonate (in TA100). Daunomycin mutagenicity was inhibited to a greater extent. Visnagin was more toxic, but showed similar effects. Khellol and its glucoside were inactive against all the mutagens tested. We conclude that khellin acts as an inhibitor or the microsomal cytochrome P450 sub-enzymes analogous to the related furanocoumarins and is also capable of inhibiting cytosolic enzymes. The extract from Ammi visnaga fruits showed a higher inhibition potency than khellin alone against 2-aminoanthracene, 1-nitropyrene and daunomycin. This might be due to additional inhibitors, e.g. coumarins, or to the synergistic effects of accompanying compounds.

Determination of furanochromones and pyranocoumarins in drugs and Ammi visnaga fruits by combined solid-phase extraction-high-performance liquid chromatography and thin-layer chromatography-high-performance liquid chromatography.

A new, simple and rapid solid-phase extraction method for the determination of furanochromones and pyranocoumarins in Ammi visnaga L. fruits and pharmaceuticals by reversed-phase high-performance liquid chromatography (RP-HPLC) was developed. The isolation of compounds examined was carried out on octadecyl BakerBond SPE columns using various concentrations of methanol, acetonitrile and tetrahydrofuran in water. High and reproducible recoveries were obtained. To compare the results of quantitative analysis a preparative TLC procedure was also elaborated and carried out.

Photophysical properties and photobiological activity of the furanochromones visnagin and khellin.

The larger photobiological activity of visnagin (VI) versus khellin (KH) toward several living organisms, including fungi, viruses, yeasts and bacteria, induced a detailed investigation of the photophysical properties of these naturally occurring furanochromones, using laser-flash-photolysis, photoacoustic calorimetry and fluorescence (steady-state and time-resolved) techniques in solvents with different polarity and content of water, including micelles and vesicles. The results have shown that the magnitude of all the three rate constants out of S1 (radiative, kf; internal conversion, kic and intersystem crossing, kisc) for VI and KH strongly depend on the solvent, namely on its hydrogen bonding ability and polarity. The changes of kf and kisc are due to the solvent-assisted mixing and/or inversion of the two first singlet excited states (1n, pi and 1 pi, pi), while kic increases with a decrease of the S0-S1 energy gap. As a consequence, the quantum yield of triplet formation (phi T) strongly decreases from values of approximately 0.8 in dioxane to < 0.05 in water for both compounds. The magnitude of solvent polarity/hydrogen bonding ability required, at which the state order is inverted and phi T starts to decrease, is greater for VI than for KH and consequently phi T (VI) >> phi T (KH) over a broad range of water content including that appropriate to the environment of the compounds in a living system. These facts account for the larger photobiological activity of VI with respect to KH, regarding both the fungus Fusarium culmorum L. and the wild strain of Escherichia coli, studied by us.

Studies on the photobiological activity of two naturally occurring furochromones, visnagin and khellin, in Chlamydomonas reinhardtii.

Irradiation of arg-1 cells of the green alga Chlamydomonas reinhardtii with UV-A in the presence of visnagin (10 microg/ml) produced weak mutagenic effects when a fluence rate of 5.1 W/m2 and fluences of 1.5-36.7 kJ/m2 were applied. A maximum number of revertants was obtained at approximately 9.2 kJ/m2. When a fluence rate of 20.4 W/m2 was used the photomutagenicity of visnagin was markedly enhanced with fluences of > or = 36.7 kJ/m2. In survival experiments with a fluence rate of 5.1 W/m2 the surviving fraction decreased continuously to approximately 4%. In experiments with a fluence rate of 20.4 W/m2, however, higher survival rates were observed compared at equal UV-A doses. Visnagin was much less phototoxic and photomutagenic than bergapten when compared at equimolar concentrations and equal UV-A doses. Re-irradiation with UV-A in the absence of unbound visnagin did not alter survival and mutagenicity which had been induced by the first treatment. The mutation frequency plotted versus the UV-A fluence exhibited second-order kinetics. Khellin showed only marginal photosensitizing capacity and no significant mutagenicity up to a concentration of 100 microg/ml and a total UV-A fluence of 73.4 kJ/m2.