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.

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.

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.

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.

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.

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.

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.

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.

Plant photosensitizers with antiviral properties.

Many antiviral compounds obtained from plants are photosensitizers, i.e., their biological properties are dependent upon or augmented by light of specific wavelengths, commonly long wave ultraviolet, UVA. Three groups of chemically distinct plant photosensitizers have been investigated in some detail in regard to antiviral properties. These are (a) thiophenes and polyacetylenes; (b) furyl compounds; (c) certain alkaloids. Some of the thiophenes and their acetylenic derivatives possess extremely potent phototoxic activities toward membrane-containing viruses. These activities are markedly affected by the chemical structures of these compounds. Inactivated virus retains its integrity, however, and penetrates cells, but does not replicate. Their mechanism of action is believed to occur via singlet-oxygen damage to the membranes, although other targets cannot be ruled out. In contrast, the antiviral activities of plant furyl compounds (such as psoralens and furanochromones) appear to depend on UVA-mediated covalent adduct formation with the viral genomes. Some of the photoactive beta-carboline alkaloids also have impressive antiviral activities, especially against viruses with single-stranded genomes. These and other types of alkaloids appear to work by mechanisms that do not require covalent bonding to nucleic acids, and may also involve other target molecules as well. Some of these compounds have potent antiviral activities at concentrations well below cytotoxic levels, and accordingly should be tested in animal models.