Exploring the multifaceted effects of Ammi visnaga: subchronic toxicity, antioxidant capacity, immunomodulatory, and anti-inflammatory activities.

Ammi visnaga (A. visnaga) is an annual herb that has been used in traditional medicine to treat various ailments attributed to the presence of its bioactive compounds. The purpose of this study was to identify and examine the phytochemical properties of the hydroalcoholic extract of A. visnaga using in vitro and in vivo models. Our findings demonstrated that the extract contained a variety of beneficial components, including phenols, flavonoids, tannins, coumarins, saponins, khellin, and visnagin. The total polyphenolic content and total flavonoid content were 23.26 mg/GAE/g dry weight and 13.26 mg/GAE/g dry weight, respectively. In vitro tests demonstrated that the extract possessed antioxidant properties as evidenced by the ability to scavenge free radicals, including DPPH, ABTS, nitric oxide (NO), phosphomolybdate, and ferric-reducing antioxidant power (FRAP). Further, the extract was found to inhibit hydrogen peroxide (H2O2)-induced hemolysis. In a 90-d in vivo study, female Wistar rats were administered 1 g/kg of A. visnaga extract orally resulting in a significant increase in total white blood cell count. Although morphological changes were observed in the liver, no marked alterations were noted in kidneys and spleen. In a female Swiss albino mice model of acetic acid-induced vascular permeability, A. visnaga significantly inhibited extravasations of Evans blue at doses of 0.5 or 1 g/kg with inhibition percentages of 51 and 65%, respectively, blocking tissue necrosis. The extract also demonstrated potential immunomodulatory properties in mice by enhancing antibody production in response to antigens. In silico molecular docking studies demonstrated a strong affinity between khellin or visnagin and immunomodulatory proteins, NF-κB, p52, and TNF-α. These findings suggest that A. visnaga may be considered a beneficial antioxidant with immunomodulatory properties and might serve as a therapeutic agent to combat certain diseases.

Comparative study on the impacts of visnagin and its methoxy derivative khellin on human lymphocyte proliferation and Th1/Th2 balance.

BACKGROUND: Visnagin is a phenolic and natural compound in turmeric and fenugreek, and its anti-inflammatory effect has been indicated. Therefore, this study aimed to investigate and compare the anti-inflammatory properties of visnagin and its methoxy derivative khellin on human lymphocytes. METHODS: Human lymphocytes were treated with khellin, visnagin (10, 30, and 100 µM), and dexamethasone (0.1 mM) in the presence of phytohemagglutinin (PHA). The levels of cell proliferation, nitric oxide (NO), glutathione (GSH), malondialdehyde (MDA), and MDA/GSH ratio were measured using biochemistry methods. Furthermore, the mRNA levels of interferon-γ (IFN-γ), interleukin (IL)-4, and IL-10 were assessed using real-time PCR, while IFN-γ/IL-4(Th1/Th2), IFN-γ/IL-10(Th1/Treg), and IL-4/IL-10(Th2/Treg) ratios were made by dividing their exact values. RESULTS: In the PHA-stimulated group, GSH and IFN-γ/IL-4 levels were markedly diminished, but other variables were significantly elevated compared to the control group. Khellin and visnagin significantly declined the levels of cell proliferation, MDA, MDA/GSH ratio, and NO production. Khellin and visnagin concentration-dependently diminished IFN-γ and IL-4 levels and increased IL-10 levels compared to the PHA-stimulated group. Two higher concentrations of khellin and visnagin (30 and 100 µM) considerably diminished the IFN-γ, IFN-γ/IL-10, and IL-4/IL-10 values compared to the PHA-stimulated group. However, 100 µM of khellin and visnagin significantly increased GSH level compared to the PHA-stimulated group. CONCLUSIONS: In PHA-stimulated lymphocytes, representing Th2 dominant allergic diseases, khellin and visnagin provides more specific anti-oxidant, anti-inflammatory, and immunomodulatory functions than dexamethasone. In addition, the effects of khellin were more prominent than visnagin.

Nanostructured Lipid Carriers Can Enhance Oral Absorption of Khellin, a Natural Pleiotropic Molecule.

A novel formulation based on nanostructured lipid carriers (NLCs) was developed to increase solubility and intestinal absorption of khellin. K-NLCs were prepared with stearic acid, hempseed oil, Brij S20, and Labrafil M 1944 CS, using the emulsification-ultrasonication method. Developed nanoparticles were chemically and physically characterized by liquid chromatography, light scattering techniques, and electron microscopy. The size, about 200 nm, was optimal for oral delivery, and the polydispersity index (around 0.26), indicated high sample homogeneity. Additionally, K-NLCs showed a spherical morphology without aggregation by microscopic analysis. The encapsulation efficiency of khellin was about 55%. In vitro release studies were carried out in media with different pH to mimic physiological conditions. K-NLCs were found to be physically stable in the simulated gastric and intestinal fluids, and they preserved about 70% of khellin after 6 h incubation. K-NLCs were also successfully lyophilized testing different lyoprotectants, and obtained freeze-dried K-NLCs demonstrated good shelf life over a month. Lastly, permeability studies on Caco-2 cells were performed to predict khellin passive diffusion across the intestinal epithelium, demonstrating that nanoparticles increased khellin permeability by more than two orders of magnitude. Accordingly, developed NLCs loaded with khellin represent a versatile formulation with good biopharmaceutical properties for oral administration, possibly enhancing khellin's bioavailability and therapeutic effects.

Visnagin prevents isoproterenol-induced myocardial injury by attenuating oxidative stress and inflammation and upregulating Nrf2 signaling in rats.

Oxidative tissue injury and inflammatory responses play major roles in cardiovascular diseases and heart failure. Visnagin (VIS) is a natural bioactive component of Ammi visnaga, with promising radical scavenging and anti-inflammatory activities. This study explored the protective effect of VIS against isoproterenol (ISO)-induced acute myocardial injury and oxidative stress in rats. VIS was supplemented for 14 days, and the rats received ISO (100 mg/kg) twice at an interval of 24 h. ISO-induced myocardial injury was characterized by elevated serum CK-MB, LDH, and troponin-I associated with increased heart weight and several histopathological changes. ISO increased reactive oxygen species (ROS), malondialdehyde (MDA), NF-κB p65, TNF-α, IL-6, and decreased glutathione and antioxidant enzymes in rats' hearts. VIS prevented myocardial injury and ameliorated the cardiac function markers, ROS, MDA, NF-κB p65, and pro-inflammatory cytokines in ISO-intoxicated rats. In addition, VIS decreased Bax mRNA and caspases, and upregulated Nrf2, HO-1, Bcl-2, and PPARγ. Molecular docking simulations revealed the binding method of VIS to NF-κB, Keap1, and PPARγ. In conclusion, VIS protects against ISO-induced acute myocardial injury by attenuating oxidative tissue injury and reducing key inflammatory and apoptosis markers. In vivo and in silico results showed that activation of Nrf2/HO-1 signaling and PPARγ mediates the cardioprotective effect of VIS.

Assessment of Conventional Solvent Extraction vs. Supercritical Fluid Extraction of Khella (Ammi visnaga L.) Furanochromones and Their Cytotoxicity.

BACKGROUND: Khella (Ammi visnaga Lam.) fruits (Apiaceae) are rich in furanochromones, mainly khellin and visnagin, and are thus incorporated in several pharmaceutical products used mainly for treatment of renal stones. METHODS: The objective of this study was to compare the yield of khellin and visnagin obtained using different conventional solvents and supercritical fluid extraction (SCFE) with carbon dioxide (containing 5% methanol as co-solvent). Water, acetone and ethanol (30% and 95%) were selected as conventional solvents. RESULTS: Highest extract yield was obtained from 30% ethanol (15.44%), while SCFE gave the lowest yield (4.50%). However, the percentage of furanochromones were highest in SCFE (30.1%), and lowest in boiling water extract (5.95%). HPLC analysis of conventional solvent extracts showed other coumarins that did not appear in supercritical fluid extraction chromatogram due to non-selectivity of solvent extraction. Ammi visnaga extracts as well as standard khellin and visnagin were tested for their cytotoxic activity using sulforhodamine B assay on breast cancer (MCF-7) and hepatocellular carcinoma (Hep G2) cell lines. Results revealed a strong cytotoxic activity (IC50 < 20 µg/mL) for the SCFE and standard compounds (khellin and visnagin) (IC50 ranging between 12.54 ± 0.57 and 17.53 ± 1.03 µg/mL). However, ethanol and acetone extracts had moderate cytotoxic activity (IC50 20-90 µg/mL) and aqueous extract had a weak activity (IC50 > 90 µg/mL). CONCLUSIONS: Thus, supercritical fluid extraction is an efficient, relatively safe, and cheap technique that yielded a more selective purified extract with better cytotoxic activity.

Effect of Visnagin on Altered Steroidogenesis and Spermatogenesis, and Testicular Injury Induced by the Heavy Metal Lead.

BACKGROUND: Lead (Pb) is an environmental pollutant causing serious health problems, including impairment of reproduction. Visnagin (VIS) is a furanochromone with promising antioxidant and anti-inflammatory effects; however, its protective efficacy against Pb toxicity has not been investigated. OBJECTIVE: This study evaluated the protective effect of VIS on Pb reproductive toxicity, impaired steroidogenesis and spermatogenesis, oxidative stress and inflammation. METHODS: Rats received VIS (30 or 60 mg/kg) and 50 mg/kg lead acetate for 3 weeks and blood and testes samples were collected. RESULTS: Pb intoxication impaired the pituitary-testicular axis (PTA) manifested by the decreased serum levels of gonadotropins and testosterone. Pb decreased sperm count, motility and viability, increased sperm abnormalities, and downregulated the steroidogenesis markers StAR, CYP17A1, 3ß-HSD and 17ß-HSD in the testis of rats. VIS significantly increased serum gonadotropins and testosterone, alleviated sperm parameters and upregulated steroidogenesis. In addition, VIS decreased pro-inflammatory cytokines, testicular lipid peroxidation and DNA fragmentation, downregulated Bax, and enhanced antioxidants and Bcl-2. CONCLUSION: These results demonstrate the protective effect of VIS against Pb reproductive toxicity in rats. VIS improved serum gonadotropins and testosterone, enhanced steroidogenesis and spermatogenesis, and attenuated oxidative injury, inflammation and apoptosis. Therefore, VIS is a promising candidate for the protection against Pb-induced reproduction impairment.

Visnagin and benzofuran scaffold-based molecules as selective cyclooxygenase-2 inhibitors with anti-inflammatory and analgesic properties: design, synthesis and molecular docking.

A series of new visnagin and benzofuran scaffold-based molecules was designed and synthesized as anti-inflammatory and analgesic agents. Biological screening of these compounds showed that they exhibit potent anti-inflammatory/analgesic activity with a safer side effect profile in in vivo mouse models. In vitro cyclooxygenase (COX) inhibition assay showed that the compounds elicit their function through selective COX-2 inhibition. Molecular docking study also revealed the ability of the compounds to correctly recognize the active site and achieve noncovalent binding interactions with key residues therein. The best combined profile of anti-inflammatory, analgesic and COX-2 selective inhibition properties in association with low gastrotoxicity was displayed by the analogs 8, 11b and 19d, which can be considered as promising leads for further future optimization.

The anticancer activity of visnagin, isolated from Ammi visnaga L., against the human malignant melanoma cell lines, HT 144.

Melanoma is a cancer of melanocyte cells and has the highest global incidence. There is a need to develop new drugs for the treatment of this deadly cancer, which is resistant to currently used treatment modalities. We investigated the anticancer activity of visnagin, a natural furanochromone derivative, isolated from Ammi visnaga L., against malignant melanoma (HT 144) cell lines. The singlet oxygen production capacity of visnagin was determined by the RNO bleaching method while cytotoxic activity by the MTT assay. Further, HT 144 cells treated with visnagin were also exposed to visible light (λ ≥ 400 nm) for 25 min to examine the illumination cytotoxic activity. The apoptosis was measured by flow cytometry with annexin V/PI dual staining technique. The effect of TNF-α secretion on apoptosis was also investigated. In standard MTT assay, visnagin (100 µg/mL) exhibited 80.93% inhibitory activity against HT 144 cancer cell lines, while in illuminated MTT assay at same concentration it showed lesser inhibitory activity (63.19%). Visnagin was induced apoptosis due to the intracellular generation of reactive oxygen species (ROS) and showed an apoptotic effect against HT 144 cell lines by 25.88%. However, it has no effect on TNF-α secretion. Our study indicates that visnagin can inhibit the proliferation of malignant melanoma, apparently by inducing the intracellular oxidative stress.

Highly potent visnagin derivatives inhibit Cyp1 and prevent doxorubicin cardiotoxicity.

Anthracyclines such as doxorubicin are highly effective chemotherapy agents used to treat many common malignancies. However, their use is limited by cardiotoxicity. We previously identified visnagin as protecting against doxorubicin toxicity in cardiac but not tumor cells. In this study, we sought to develop more potent visnagin analogs in order to use these analogs as tools to clarify the mechanisms of visnagin-mediated cardioprotection. Structure-activity relationship studies were performed in a zebrafish model of doxorubicin cardiomyopathy. Movement of the 5-carbonyl to the 7 position and addition of short ester side chains led to development of visnagin analogs with 1,000-fold increased potency in zebrafish and 250-fold increased potency in mice. Using proteomics, we discovered that doxorubicin caused robust induction of Cytochrome P450 family 1 (CYP1) that was mitigated by visnagin and its potent analog 23. Treatment with structurally divergent CYP1 inhibitors, as well as knockdown of CYP1A, prevented doxorubicin cardiomyopathy in zebrafish. The identification of potent cardioprotective agents may facilitate the development of new therapeutic strategies for patients receiving cardiotoxic chemotherapy. Moreover, these studies support the idea that CYP1 is an important contributor to doxorubicin cardiotoxicity and suggest that modulation of this pathway could be beneficial in the clinical setting.

Larvicidal activity of extracts from Ammi visnaga Linn. (Apiaceae) seeds against Culex quinquefasciatus Say. (Diptera: Culicidae).

Efficacies of the Ammi visnaga seeds extract and a majority of substances on larval Culex quinquefasciatus mortality in various development stages including pupae were studied. The effect of exposure time on larval mortality was also studied. The effect of sublethal concentrations or short exposure times on further larval development and subsequent fecundity in adults were studied as well. Lethal doses of the extract were estimated for the 2nd, 3rd and 4th instar of C. quinquefasciatus (LC50 for 18, 23 and 180 mg L(-1), respectively). The majority of furanochromenes, khellin and visnagin, were identified by analysing the extract. Khellin was significantly more effective compared to visnagin, whose LC50 was estimated at 8, 10 and 41 mg L(-1) for the 2nd, 3rd and 4th instar larvae. Khellin showed very fast efficacy on mortality for the 3rd instar larvae in a concentration of 100 mg L(-1). Fifty percent mortality was determined 30 min after application, a time which was considerably shorter compared to the extract (113 min) or visnagin (169 min). The effect of the application of lethal concentrations on C. quinquefasciatus larval mortality was studied. The least number of adults were hatched after application of the extract and khellin (41.8% and 37.9%, respectively), less than after visnagin application (46.7%) or in the control (94.2%). LC50 application caused lower fecundity in the hatched adults, lower hatchability of the eggs, and also very low natality, more than 77% lower for khellin compared to the control. A short exposure, corresponding to our estimated LT30, caused no significant acute toxicity in the larvae (until 24 h) for the extract or visnagin (4.3% and 11.5%, respectively); however, 18 min of action from khellin caused a 54.3% mortality rate of the larvae within 24 h.

Visnagin protects against doxorubicin-induced cardiomyopathy through modulation of mitochondrial malate dehydrogenase.

Doxorubicin is a highly effective anticancer chemotherapy agent, but its use is limited by its cardiotoxicity. To develop a drug that prevents this toxicity, we established a doxorubicin-induced cardiomyopathy model in zebrafish that recapitulates the cardiomyocyte apoptosis and contractility decline observed in patients. Using this model, we screened 3000 compounds and found that visnagin (VIS) and diphenylurea (DPU) rescue the cardiac performance and circulatory defects caused by doxorubicin in zebrafish. VIS and DPU reduced doxorubicin-induced apoptosis in cultured cardiomyocytes and in vivo in zebrafish and mouse hearts. VIS treatment improved cardiac contractility in doxorubicin-treated mice. Further, VIS and DPU did not reduce the chemotherapeutic efficacy of doxorubicin in several cultured tumor lines or in zebrafish and mouse xenograft models. Using affinity chromatography, we found that VIS binds to mitochondrial malate dehydrogenase (MDH2), a key enzyme in the tricarboxylic acid cycle. As with VIS, treatment with the MDH2 inhibitors mebendazole, thyroxine, and iodine prevented doxorubicin cardiotoxicity, as did treatment with malate itself, suggesting that modulation of MDH2 activity is responsible for VIS' cardioprotective effects. Thus, VIS and DPU are potent cardioprotective compounds, and MDH2 is a previously undescribed, druggable target for doxorubicin-induced cardiomyopathy.

Khellin and visnagin differentially modulate AHR signaling and downstream CYP1A activity in human liver cells.

Khellin and visnagin are two furanochromones that can be frequently found in ethnomedical formulations in Asia and the Middle East. Both compounds possess anti-inflammatory and analgesic properties, therefore modern medicine uses these compounds or structurally related derivatives for treatment of vitiligo, bronchial asthma and renal colics. Despite their frequent usage, the potential toxic properties of visnagin and khellin are not well characterized up-to-now. Many natural compounds modulate the expression and activity of cytochrome P450 1A1 (CYP1A1), which is well-known to bioactivate pro-carcinogens. The expression of this enzyme is controlled by the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor and regulator of drug metabolism. Here, we investigated the influence of both furanochromones on AHR signaling in human HepG2 hepatocarcinoma cells and primary human hepatocytes. Both compounds transactivated xenobiotic response element (XRE)-driven reporter gene activity in a dose-dependent manner and induced CYP1A1 transcription in HepG2 cells and primary hepatocytes. The latter was abolished in presence of a specific AHR antagonist. CYP1A enzyme activity assays done in HepG2 cells and primary hepatocytes revealed an inhibition of enzyme activity by both furanochromones, which may become relevant regarding the metabolism of xenobiotics and co-administered therapeutic drugs. The observed induction of several other members of the AHR gene battery, whose gene products are involved in regulation of cell growth, differentiation and migration, indicates that a further toxicological characterization of visnagin and khelllin is urgently required in order to minimize potential drug-drug interactions and other toxic side-effects that may occur during therapeutic usage of these furanochromones.

Molecular modeling study bioactive natural product of khellin analogues as a novel potential pharmacophore of EGFR inhibitors.

Khelline is naturally occurring furochromone exhibited significant Epidermal Growth Factor Receptor (EGFR) inhibitory activity. The newly synthesized compounds 2-5 displayed the most potent EGFR inhibitory activity on MCF-7 and HeLa. In vitro study against 59 different human tumour cell lines derived from nine cancer type in NCI (USA), which was presented and documented. Molecular docking simulation was performed to position compounds 1-5 into the EGFR active site to determine the probable binding mode.

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.

DNA binding, antiviral activities and cytotoxicity of new furochromone and benzofuran derivatives.

Bromination of visnagin (1) afforded 9-bromovisnagin (2) which on its alkaline hydrolysis afforded the 3-acetyl benzofuran derivative (3). The condensation of (3) with hydrazine hydrate, phenylhydrazine and/or hydroxylamine hydrochloride afforded the corresponding pyrazole derivatives (4a, b) and isoxazole derivative (4c). On the other hand, when compound 3 was condensed with some aromatic aldehydes, this yielded corresponding α, ß-unsaturated keto derivatives (5a-e). Furthermore, when 1 was subjected to chlorosulfonation, the visnaginsulfonylchloride derivative 6 was afforded, which on amidation using morpholine, a sulonamido derivative (7) was obtained. Alkaline hydrolysis of the latter compound yielded 7-N-morpholinosulsamidobenzofuran (8) which was condensed with some aromatic aldehydes to yield the corresponding chalcone compounds (9a-e). Demethylation of visnagin afforded norvisnagin (10). The reaction of 10 with ethylbromoacetate in dry acetone yielded the ester benzopyran derivative (11) which reacted with hydrazine hydrate to afford the corresponding hydrazide derivative (12) and this was condensed with 3,4,5-trimethoxybenzaldehyde to give the corresponding hydrazone (13). A thaizolidinone derivative (14) was obtained by condensation of (13) with thioglycolic acid. Chloromethylation of norvisnagin afforded a 4-chloromethyl derivative (15) which reacted with different primary and secondary amines to yield the corresponding ethylamino derivative (16a, b). Moreover, mannich bases (16a, b) and (17a-c) were obtained by reacting norvisnagin with different primary and secondary amines in the presence of formalin but benzoylation of (16a, b) and (17a-c) afforded 4-oxybenzoyl derivative (18a-e). The prepared compounds were tested for their interaction with DNA; bromovisnagin 2 showed the highest affinity and compounds 6, 15, 8a, > 14, > 16b, 17a, and 16a showed moderate activity in decreasing potency. Moreover, compound 2 also was the most active as antiviral agent toward HS-I virus and compounds 6, 7, 15, 14, 16a, and 18a were found to be moderately active. CD(50) of the active compounds were also measured.

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.

Computational study of khellin excited states and photobinding to DNA.

A theoretical investigation of the formation and spectroscopic properties of the furan and pyrone monoadducts between the photosensitizer khellin and DNA base thymine is reported. The thermal reaction pathways involve very high barriers, whereas the excited state surfaces display low barriers in regions leading to the ground state TS structures and potential wells at the ground state TS geometries. Computed UV absorption spectra are interpreted with the support of molecular orbital calculations, and the role of solvent effects on the spectra is discussed. The red-shift in the khellin spectra upon intercalation in DNA is excellently reproduced by the computational methodology, as is the solvent induced spectral shift. The data also provides an explanation to why khellin predominantly forms furan monoadducts in DNA, as opposed to the closely related psoralen compounds.

KUVA (khellin plus ultraviolet A) stimulates proliferation and melanogenesis in normal human melanocytes and melanoma cells in vitro.

BACKGROUND: Khellin is a naturally occurring furochromone which, when combined with artificial ultraviolet (UV) A or solar irradiation (KUVA), is reported to repigment vitiligo skin as effectively as PUVA photochemotherapy. The exact mechanism of KUVA-induced repigmentation is unknown. OBJECTIVES: The aim of this study was to test the effect of khellin and KUVA on proliferation and melanogenesis of normal human melanocytes and Mel-1 melanoma cells in vitro. METHODS: Cultured normal human melanocytes, Mel-1 melanoma cells and fibroblasts were treated with khellin, UVA and KUVA and the effect on proliferation determined by cell counting. The effect on melanogenesis was determined by a radiometric melanin formation assay. Changes in gene expression and protein synthesis were determined by Northern blot, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analyses. RESULTS: Khellin stimulated proliferation of Mel-1 melanoma cells and melanocytes at concentrations between 1 nmol L-1 and 0.5 mmol L-1 with a peak effect at 0.01 mmol L-1 khellin. In contrast, khellin inhibited proliferation of fibroblasts over the entire concentration range tested. At concentrations above 0.5 mmol L-1, khellin was cytotoxic to both melanocytic cells and fibroblasts. Exposure of khellin-treated cells to single doses of UVA between 150 and 280 mJ cm-2 resulted in an enhanced proliferative effect. Khellin and KUVA also stimulated the melanogenic enzyme activity of pigmented cells, with the most effective treatment being 0.01 mmol L-1 khellin with 250 mJ cm-2 UVA. Western blot, Northern blot and RT-PCR analysis revealed that these increases in melanogenic enzyme activity were not due to increases in gene expression or protein synthesis. UVA treatment resulted in an increase in enzyme glycosylation and this correlated with the increase in melanogenesis. CONCLUSIONS: We conclude that khellin activated by UVA stimulates melanocyte proliferation and melanogenesis. Our results point to the possibility that current treatment regimens might be improved if reduced khellin doses are applied and suggest that improved delivery vehicles be tested.