Diosmin ameliorates LPS-induced depression-like behaviors in mice: Inhibition of inflammation and oxidative stress in the prefrontal cortex.

Diosmin is a flavone glycoside with a confirmed therapeutic effectiveness on the chronic venous disorders. In this paper, the classical mouse depression model induced by LPS was established to explore the effect of Diosmin on depression. Firstly, we found that Diosmin could inhibit the inflammation and neuronal damage in the prefrontal cortex (PFC) of mice, and thus alleviating the LPS-induced depressive-like behaviors. Specifically, Diosmin treatment significantly suppressed the secretion of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1ß), reduced the activation of microglia, and inhibited the expression of NLRP3 inflammasome and its downstream effector caspase-1 in both PFC of mice and BV2 microglial cells exposed to LPS. Then, we demonstrated that pretreatment with Diosmin dramatically suppressed the LPS-induced oxidative stress in the PFC of mice, manifested in the decrease of reactive oxygen species and malondialdehyde while increase of catalase activity. Consistently, Diosmin also alleviated the oxidative stress in BV2 cells exposed to LPS. Finally, we confirmed that Diosmin effectively suppressed the activation of NF-κB signaling pathway in the PFC of LPS-treated mice. Further in vitro experiments also verified that Diosmin could prevent the p65 transposition to nucleus in LPS-treated BV2 cells, suggesting that the antidepressant effects of Diosmin are partially mediated by blocking of NF-κB signaling. Taken together, this study proposes the potential antidepressant effect of Diosmin, which provides useful support to the development of new therapies for depression.

Nephroprotective effect of diosmin against sodium arsenite-induced renal toxicity is mediated via attenuation of oxidative stress and inflammation in mice.

Arsenic compounds, which are used in different industries like pesticide manufacturing, cause severe toxic effects in almost all organs, including the kidneys. Since the primary route of exposure to arsenic is through drinking water, and millions of people worldwide are exposed to unsafe levels of arsenic that can pose a threat to their health, this research was performed to investigate the nephroprotective effects of Diosmin (Dios), a flavonoid found in citrus fruits, against nephrotoxicity induced by sodium arsenite (SA). To induce nephrotoxicity, SA (10 mg/kg, oral gavage) was administered to mice for 30 days. Dios (25, 50, and 100 mg/kg, oral gavage) was given to mice for 30 days prior to SA administration. After the study was completed, animals were euthanized and blood and kidney samples were taken for biochemical and histopathological assessments. Results showed that SA-treated mice significantly increased the blood urea nitrogen and creatinine levels in the serum. This increase was associated with significant kidney tissue damage in SA-treated mice, which was confirmed by histopathological studies. Furthermore, SA enhanced the amounts of renal thiobarbituric acid reactive substances and decreased total thiol reserves, as well as the activity of antioxidant enzymes such as catalase, superoxide dismutase, and glutathione peroxidase. Also, in the SA-exposed group, an increase in the levels of kidney inflammatory biomarkers, including nitric oxide and tumor necrosis factor-alpha was observed. The western blot analysis indicated an elevation in the protein expression of kidney injury molecule-1 and nuclear factor-kappa B in SA-treated mice. However, pretreatment with Dios ameliorated the SA-related renal damage in mice. Our findings suggest that Dios can protect the kidneys against the nephrotoxic effects of SA by its antioxidant and anti-inflammatory characteristics.

Diosmin Promotes Myogenesis via Activating the Akt/FOXO1 Pathway to Facilitate the Proliferation of C2C12 Myoblasts.

Our previous study with artificial intelligence (AI)-assisted screening found that diosmin, a natural flavonoid extracted from citrus, may affect myoblast proliferation and differentiation. At present, few studies have been conducted regarding the biological function of diosmin in muscle cells. Here, using molecular biological techniques, we found that diosmin elevated the proliferation ability of C2C12 myoblasts via activating the Akt/FOXO1 pathway to promote FOXO1 nuclear export, thus repressing p27 protein expression, increasing CDK2, CDK4, and cyclin D1 and cyclin E1 protein expression and accelerating cell cycle transformation, which contributed to myogenesis. Moreover, diosmin suppressed differentiation of C2C12 myoblasts by delaying the terminal exit of the cell cycle in early differentiated myoblasts and inhibiting autophagic flux in mature myotubes. Furthermore, diosmin promoted myogenesis by activating the Akt/FOXO1 pathway to facilitate myoblast proliferation, which had a positive biological effect on the repair of muscle injury. This study revealed the effect and mechanism of diosmin on skeletal muscle cells and simultaneously provided a new candidate drug for the treatment of myopathy.

Selective PPARγ modulator diosmin improves insulin sensitivity and promotes browning of white fat.

Peroxisome proliferator-activated receptor γ (PPARγ) is a master regulator of adipocyte differentiation, glucolipid metabolism, and inflammation. Thiazolidinediones are PPARγ full agonists with potent insulin-sensitizing effects, whereas their oral usage is restricted because of unwanted side effects, including obesity and cardiovascular risks. Here, via virtual screening, microscale thermophoresis analysis, and molecular confirmation, we demonstrate that diosmin, a natural compound of wide and long-term clinical use, is a selective PPARγ modulator that binds to PPARγ and blocks PPARγ phosphorylation with weak transcriptional activity. Local diosmin administration in subcutaneous fat (inguinal white adipose tissue [iWAT]) improved insulin sensitivity and attenuated obesity via enhancing browning of white fat and energy expenditure. Besides, diosmin ameliorated inflammation in WAT and liver and reduced hepatic steatosis. Of note, we determined that iWAT local administration of diosmin did not exhibit obvious side effects. Taken together, the present study demonstrated that iWAT local delivery of diosmin protected mice from diet-induced insulin resistance, obesity, and fatty liver by blocking PPARγ phosphorylation, without apparent side effects, making it a potential therapeutic agent for the treatment of metabolic diseases.

Depigmenting topical therapy based on a synergistic combination of compounds targeting the key pathways involved in melasma pathophysiology.

Melasma has a complex pathophysiology with different cell types and signalling pathways involved. Paracrine factors secreted by keratinocytes, fibroblasts and endothelial cells act on melanocytes and stimulate melanogenesis. These paracrine factors are involved in the oxidative stress, inflammatory, vascular and hormonal pathways, among others. Damage of the dermoepidermal barrier also occurs and facilitates melanin deposition in the dermis, also known as dermal or mixed melasma. We used artificial intelligence tools to define the best combination of compounds for skin pigmentation inhibition. Mathematical models suggested the combination of retinol, diosmin and ferulic acid to be the most effective one. In vitro cellular tyrosinase activity assay proved that this combination had a synergistic depigmenting effect. Further assays proved that the combination could inhibit key pathways involved in melasma by downregulating ET-1 and COX-2 gene expression and IBMX-induced dendricity in human melanocytes, and upregulated the gene expression of IL-1b, TIMP3 and several endogenous antioxidant enzymes. The combination also reduced melanin levels in a phototype VI 3D epidermis model. These results indicate that the combination of retinol, diosmin and ferulic acid is an effective synergistic complex for the treatment of melasma by regulating the key molecular pathways involved in skin hyperpigmentation pathophysiology.

Diosmin and its glycocalyx restorative and anti-inflammatory effects on injured blood vessels.

The endothelium, a crucial homeostatic organ, regulates vascular permeability and tone. Under physiological conditions, endothelial stimulation induces vasodilator endothelial nitric oxide (eNO) release and prevents adhesion molecule accessibility and leukocyte adhesion and migration into vessel walls. Endothelium dysfunction is a principal event in cardiovascular disorders, including atherosclerosis. Minimal attention is given to an important endothelial cell structure, the endothelial glycocalyx (GCX), a negatively charged heterogeneous polysaccharide that serves as a protective covering for endothelial cells and enables endothelial cells to transduce mechanical stimuli into various biological and chemical activities. Endothelial GCX shedding thus plays a role in endothelial dysfunction, for example by increasing vascular permeability and decreasing vessel tone. Consequently, there is increasing interest in developing therapies that focus on GCX repair to limit downstream endothelium dysfunction and prevent further downstream cardiovascular events. Here, we present diosmin (3',5,7-trihydroxy-4'-methoxyflavone-7-rhamnoglucoside), a flavone glycoside of diosmetin, which downregulates adhesive molecule expression, decreases inflammation and capillary permeability, and upregulates eNO expression. Due to these pleiotropic effects of diosmin on the vasculature, a possible unidentified mechanism of action is through GCX restoration. We hypothesize that diosmin positively affects GCX integrity along with GCX-related endothelial functions. Our hypothesis was tested in a partial ligation left carotid artery (LCA) mouse model, where the right carotid artery was the control for each mouse. Diosmin (50 mg/kg) was administered daily for 7 days, 72 h after ligation. Within the ligated mice LCAs, diosmin treatment elevated the activated eNO synthase level, inhibited inflammatory cell uptake, decreased vessel wall thickness, increased vessel diameter, and increased GCX coverage of the vessel wall. ELISA showed a decrease in hyaluronan concentration in plasma samples of diosmin-treated mice, signifying reduced GCX shedding. In summary, diosmin supported endothelial GCX integrity, to which we attribute diosmin's preservation of endothelial function as indicated by attenuated expression of inflammatory factors and restored vascular tone.

Chemopreventive potential of Diosmin against benzo[a]pyrene induced lung carcinogenesis in Swiss Albino mice.

Lung cancer, one of the most common cancer is a cause of concern associated with cancer-related mortality. Benzo[a]pyrene [B(a)P], a potent carcinogen as well as an environmental contaminant is reported to be found in cigarette smoke among various sources. The present study focuses on the chemopreventive potential of Diosmin against B[a]P-induced lung carcinogenesis and its possible mechanism in male Swiss Albino mice (SAM). SAM were treated orally with Diosmin (200 mg/kg b.w.) for 16 weeks and/or B[a]P (50 mg/kg b.w) for a period of 4 weeks. B[a]P treated cancerous mice showed increased peroxidation of membrane lipid as well as a decrease in the level/activity of antioxidant proteins. Cancerous mice also showed an increased level of carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE). Diosmin treatment, however, leads to decreased peroxidation of lipids, increased antioxidant proteins as well decrease in the level of CEA and NSE. B[a]P-induced cancerous animals also exhibited increased expression of cyclic AMP response element-binding protein (CREB), COX2 as well as prostaglandin-E2 (PGE2) while Diosmin-treated mice were found to have an ameliorative effect. Histopathological results further confirm the protective effect of Diosmin in averting B[a]P-induced pathological alterations of lung tissue. Overall, our results suggest Diosmin exerts its chemopreventive potential possibly via targeting the CREB/cyclooxygenase-2 (COX-2)/PGE2 pathway thereby repressing inflammation.

Repurposing of FDA-approved drugs against active site and potential allosteric drug-binding sites of COVID-19 main protease.

The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still has serious negative effects on health, social life, and economics. Recently, vaccines from various companies have been urgently approved to control SARS-CoV-2 infections. However, any specific antiviral drug has not been confirmed so far for regular treatment. An important target is the main protease (Mpro ), which plays a major role in replication of the virus. In this study, Gaussian and residue network models are employed to reveal two distinct potential allosteric sites on Mpro that can be evaluated as drug targets besides the active site. Then, Food and Drug Administration (FDA)-approved drugs are docked to three distinct sites with flexible docking using AutoDock Vina to identify potential drug candidates. Fourteen best molecule hits for the active site of Mpro are determined. Six of these also exhibit high docking scores for the potential allosteric regions. Full-atom molecular dynamics simulations with MM-GBSA method indicate that compounds docked to active and potential allosteric sites form stable interactions with high binding free energy (∆Gbind ) values. ∆Gbind values reach -52.06 kcal/mol for the active site, -51.08 kcal/mol for the potential allosteric site 1, and - 42.93 kcal/mol for the potential allosteric site 2. Energy decomposition calculations per residue elucidate key binding residues stabilizing the ligands that can further serve to design pharmacophores. This systematic and efficient computational analysis successfully determines ivermectine, diosmin, and selinexor currently subjected to clinical trials, and further proposes bromocriptine, elbasvir as Mpro inhibitor candidates to be evaluated against SARS-CoV-2 infections.

Thermodynamics of the Interaction between the Spike Protein of Severe Acute Respiratory Syndrome Coronavirus-2 and the Receptor of Human Angiotensin-Converting Enzyme 2. Effects of Possible Ligands.

Since the end of 2019, the coronavirus SARS-CoV-2 has caused more than 1000000 deaths all over the world and still lacks a medical treatment despite the attention of the whole scientific community. Human angiotensin-converting enzyme 2 (ACE2) was recently recognized as the transmembrane protein that serves as the point of entry of SARS-CoV-2 into cells, thus constituting the first biomolecular event leading to COVID-19 disease. Here, by means of a state-of-the-art computational approach, we propose a rational evaluation of the molecular mechanisms behind the formation of the protein complex. Moreover, the free energy of binding between ACE2 and the active receptor binding domain of the SARS-CoV-2 spike protein is evaluated quantitatively, providing for the first time the thermodynamics of virus-receptor recognition. Furthermore, the action of different ACE2 ligands is also examined in particular in their capacity to disrupt SARS-CoV-2 recognition, also providing via a free energy profile the quantification of the ligand-induced decreased affinity. These results improve our knowledge on molecular grounds of the SARS-CoV-2 infection and allow us to suggest rationales that could be useful for the subsequent wise molecular design for the treatment of COVID-19 cases.

Metabolism and pharmacological activities of the natural health-benefiting compound diosmin.

Diosmin is a famous natural flavonoid for treating chronic venous insufficiency and varicose veins. Recently, extensive study has indicated that diosmin possesses diverse pharmacological activities, including anti-inflammation, anti-oxidation, anti-diabetes, anti-cancer, anti-microorganism, liver protection, neuro-protection, cardiovascular protection, renoprotection, and retinal protection activities. Due to its low water solubility, diosmin is dramatically limited in clinical application. Expectedly, many potential strategies have been developed for improving its pharmacokinetic values and bioavailability. This health-benefiting compound has been explored as the major component of Daflon and micronized purified flavonoid fraction (MPFF), which have been used in clinics to improve micro-circulation. However, no specific drug targets for diosmin are reported, although some potential factors have been involved in screening, such as P-glycoprotein (P-gp), IKKß, acetylcholinesterase (AChE), and aldose reductase (AR). More investigations on the underlying mechanisms of diosmin in mediating cellular processes with high specificity is still needed.

An In Vitro elucidation of the antiaggregatory potential of Diosminover thermally induced unfolding of hen egg white lysozyme; A preventive quest for lysozyme amyloidosis.

Protein misfolding diseases are associated with human pathologies. These neurodegenerative diseases remain challenging task for researchers because of their adverse effect on vital organs system. Lysozyme amyloidosis is also associated with multi-organ dysfunction. Hence elucidation of its folding pathway is of great importance, for which hen egg white lysozyme (HEWL) being homological to its human counterpart was taken into consideration. Here in this study we have investigated the effect of diosmin (DSN), a flavonoid over thermally aggregated HEWL. Decrease in ANS, ThT and Rayleigh scattering fluorescence intensity suggests the transition between ß to α conformations. Further decrease in absorbance at 360 nm and of congo red with slight blue shift also indicated the disappearance of ß sheeted structure under the under the influence of increasing concentration of DSN. These results were also supported by circular dichroism in which gradual appearance α helical structure was observed. Finally visualization under transmission electron microscopy (TEM) authenticated the maximum structural alteration in the previously formed aggregates of HEWL at 250 µM DSN. Molecular docking followed by 100 ns MD simulations help to understand the interaction mechanism of HEWL with DSN. Results suggest DSN could be a useful in the treatment of amyloid related disorders.

Influence of diosmin on the metabolism and disposition of carbamazepine in healthy subjects.

1. Carbamazepine (CBZ) is an antiepileptic drug with narrow therapeutic window and administration in humans receiving long-term therapy with diosmin (DSN) may occur, which leads to CYP3A4-mediated drug interactions. The purpose of the present study was to assess the influence of DSN on the metabolism and pharmacokinetics of CBZ in healthy volunteers. 2. An open-label, sequential, two-period study was conducted in 12 healthy male volunteers. A single dose of DSN 500 mg was administered once daily for 10 days during treatment phase. A single dose of CBZ 200 mg was administered during control and after treatment phases under fasting conditions. The blood samples were collected after CBZ dosing at predetermined time intervals and analyzed by LC-MS/MS. 3. Treatment with DSN significantly enhanced the maximum plasma concentration (Cmax), area under the curve (AUC), half-life (t1/2) and significantly decreased the apparent oral clearance (CL/F) and elimination rate constant (Kel) of CBZ. On the other hand, treatment with DSN significantly decreased the Cmax and AUC of carbamazepine 10, 11-epoxide (CBZE). Furthermore, treatment with DSN significantly decreased the metabolite to parent ratios of Cmax and AUC, indicating the reduced metabolism of CBZ to CBZE. 4. The results suggest that the altered CYP3A4 enzyme activity and pharmacokinetics of CBZ might be attributed to DSN-mediated inhibition of CYP3A4 enzyme, which indicates pharmacokinetic interaction present between DSN and CBZ. Therefore, we conclude that DSN has an inhibiting effect on the metabolism and disposition of CBZ.

The effect of diosmin on the pharmacokinetics of fexofenadine in healthy human volunteers.

1. Diosmin (DSN) has been found to possess P-glycoprotein (P-gp) inhibition activity in vitro and in vivo in rats, which may have potential to cause P-gp-mediated interactions in humans. The purpose of the present study was to investigate the effect of DSN on pharmacokinetics of fexofenadine (FEX) in healthy human volunteers. 2. An open-label, two-period, sequential study was conducted in 12 healthy male volunteers. A single dose of FEX 120 mg was administered to volunteers during control and treatment phases. A single dose of DSN 500 mg was administered to volunteers daily for period of 10 days. The blood and urine samples were collected at predetermined time intervals after FEX dosing and analyzed by LC-MS/MS. 3. Treatment with DSN significantly increased the peak maximum plasma concentration (Cmax) and area under the plasma concentration-time curve (AUC) of FEX by 49.2% (523.28 versus 780.63 ng/mL) and 64.4% (3459.48 versus 5687.74 ng h/mL), respectively as compared to control phase. On the other hand, apparent oral clearance (CL/F) of FEX was significantly decreased by 41.3% (37.03 versus 21.75 L/h) and there was no significant change was observed in Tmax, T1/2, and CLr (renal clearance) upon treatment with DSN when compared to control. 4. The results suggest that altered pharmacokinetics of FEX might be attributed to DSN-mediated inhibition of P-gp-mediated efflux in humans. Therefore, we conclude that intake of DSN or dietary supplements containing DSN may potentially increase the absorption or bioavailability of FEX, a P-gp substrate in humans.

Diosmin downregulates the expression of T cell receptors, pro-inflammatory cytokines and NF-κB activation against LPS-induced acute lung injury in mice.

Diosmin, a natural flavonoid glycoside present abundantly in the pericarp of various citrus fruits. Because of its anti-inflammatory and antioxidant properties, it can be used in many diseases. In this study, we investigated the possible protective mechanisms of the diosmin on LPS-induced lung injury through inhibition of T cell receptors, pro-inflammatory cytokines and NF-κB activation. Animals were pretreated with diosmin (50 and 100mg/kg, p.o.) for seven days prior to lipopolysaccharides (LPS) treatment. LPS administration increased neutrophils, monocytes, lymphocytes, total leukocyte count (TLC) and platelets which were decreased by diosmin. We observed that mice exposed to LPS showed increased malondialdehyde level and MPO activity whereas marked decrease in glutathione content. These changes were significantly reversed by treatment with diosmin in a dose dependent manner. Diosmin treatment showed a substantial reduction in T cell (CD4(+) and CD8(+)) receptors and pro-inflammatory (IL-2(+) and IL-17(+)) cytokines in whole blood. In addition, RT-PCR analysis revealed increased mRNA expression of IL-6, IL-17, TNF-α, and NF-κB in the LPS group, while reduced by treatment with diosmin. Western blot analysis confirmed the increased protein expression of IL-1ß, TNF-α and NF-κB p65 in the LPS group and treatment of animals with diosmin reversed these effects. The levels of cytoplasmic p-IκB-α and p-NF-κB p65 expression also were mitigated by diosmin. The histological examinations revealed protective effect of diosmin while LPS group aggravated lung injury. These results support the potential for diosmin to be investigated as a potential agent for the treatment of lung injury and inflammatory diseases.

[Possibilities of pharmacotherapy for chronic venous insufficiency with diosmin preparations from the position of the endothelial functional state].

Despite a high level of the development of modern angiology and vascular surgery, the problem of chronic venous insufficiency (CVI) complicating the course of various venous diseases seems to have no tendency towards being solved, thus calling forth permanent search for optimization of methods of treatment and rehabilitation of patients presenting with the above-mentioned syndrome. The article presents a review of contemporary studies dedicated to the problem of correcting CVI. Special attention is paid to the endothelial state in CVI and possibilities of correcting endothelial dysfunction with the use of bioflavonoids, in particular, diosmin. Also presented herein are the results of an original experimental study dedicated to peculiarities of the endothelial functional state, endothelial dysfunction, and correction thereof on the background of the existing CVI.

Development of novel polymer-stabilized diosmin nanosuspensions: in vitro appraisal and ex vivo permeation.

Scanty solubility and permeability of diosmin (DSN) are perpetrators for its poor oral absorption and high inter-subject variation. This article investigated the potential of novel DSN nanosuspensions to improve drug delivery characteristics. Bottom-up nanoprecipitation technique has been employed for nanosuspension development. Variables optimized encompassed polymeric stabilizer type, DSN: stabilizer ratio, excess stabilizer removal, spray drying, and mannitol incorporation. In vitro characterization included particle size (PS), infrared spectroscopy (IR), differential scanning calorimetry (DSC), X-ray powder diffraction (XRD), transmission electron microscope (TEM), scanning electron microscopy (SEM), and dissolution profile. Ex vivo permeation was assessed in rats using non-everted sac technique and HPLC. Optimal DSN nanosuspension (DSN:hydroxypropylmethyl cellulose HPMC 2:1) was prepared with acid base neutralization technique. The formula exhibited the lowest PS (336 nm) with 99.9% drug loading and enhanced reconstitution properties after mannitol incorporation. SEM and TEM revealed discrete, oval drug nanocrystals with higher surface coverage with HPMC compared to MC. DSN nanosuspension demonstrated a significant enhancement in DSN dissolution (100% dissolved) compared to crude drug (51%). Permeation studies revealed 89% DSN permeated from the nanosuspension after 120 min compared to non-detected amounts from drug suspension. Conclusively, novel DSN nanosuspension could successful improve its dissolution and permeation characteristics with promising consequences of better drug delivery.

Diosmin binding to human serum albumin and its preventive action against degradation due to oxidative injuries.

Diosmin is a glycosylated polyphenolic compound, commonly found in fruits and vegetables, which is utilized for the pharmacological formulation of some drugs. The interactions of diosmin to human serum albumin have been investigated by fluorescence, UV-visible, FTIR spectroscopy, native electrophoresis and protein-ligand docking studies. The fluorescence studies indicate that the binding site of the additive involves modifications of environment around Trp214 at the level of subdomain IIA. Combining the curve-fitting results of infrared Amide I' band, the modifications of protein secondary structure have been estimated, indicating a decrease in α-helix structure following flavonoid binding. Data obtained by fluorescence and UV-visible spectroscopy, FTIR experiments and molecular modeling afforded a clear picture of the association mode of diosmin to HSA, suggesting that the primary binding site of diosmin is located in Sudlow's site I. Computational mapping confirms this observation suggesting that the possible binding site of diosmin is located in the hydrophobic cavity of subdomain IIA, whose microenvironment is able to help and stabilize the binding of the ligand in non-planar conformation. Moreover the binding of diosmin to HSA significantly contributes to protect the protein against degradation due to HCLO and Fenton reaction.

The phytochemical analysis and antioxidant activity assessment of orange peel (Citrus sinensis) cultivated in Greece-Crete indicates a new commercial source of hesperidin.

The flavonoid content of several methanolic extract fractions of Navel orange peel (flavedo and albedo of Citrus sinensis) cultivated in Crete (Greece) was first analysed phytochemically and then assessed for its antioxidant activity in vitro. The chemical structures of the constituents fractionated were originally determined by comparing their retention times and the obtained UV spectral data with the available bibliographic data and further verified by detailed LC-DAD-MS (ESI+) analysis. The main flavonoid groups found within the fractions examined were polymethoxylated flavones, O-glycosylated flavones, C-glycosylated flavones, O-glycosylated flavonols, O-glycosylated flavanones and phenolic acids along with their ester derivatives. In addition, the quantitative HPLC analysis confirmed that hesperidin is the major flavonoid glycoside found in the orange peel. Interestingly enough, its quantity at 48 mg/g of dry peel permits the commercial use of orange peel as a source for the production of hesperidin. The antioxidant activity of the orange peel methanolic extract fractions was evaluated by applying two complementary methodologies, DPPH(*) assay and the Co(II)/EDTA-induced luminol chemiluminescence approach. Overall, the results have shown that orange peel methanolic extracts possess moderate antioxidant activity as compared with the activity seen in tests where the corresponding aglycones, diosmetin and hesperetin were assessed in different ratios.

Effects of citrus phytochemicals on liver and lung cytochrome P450 activity and on the in vitro metabolism of the tobacco-specific nitrosamine NNK.

NNK is a potent environmental carcinogen to which both smokers and nonsmokers are exposed. The response to NNK may be affected by factors including nutrition. We investigated the effects of five citrus phytochemicals on the in vitro metabolism of the tobacco-specific nitrosamine NNK and on the dealkylation of methoxyresorufin (MROD) and pentoxyresorufin (PROD) in liver and lung microsomes of the Syrian golden hamster. In the NNK metabolism experiments in vitro incubations contained 3 microCi [5-H3] NNK, 0.5 mg microsomal protein and 0.5 mumole of the citrus phytochemical diosmin, naringin, naringenin, quercetin or rutin. In the dealkylation studies incubations contained 0.5 microM methoxyresorufin or pentoxyresorufin, 0.5 mg microsomal protein and 0.5 mumole of citrus phytochemical. The major NNK metabolism pathway in hamster liver microsomes was NNK-reduction while in lung microsomes it was alpha-hydroxylation. The alpha-hydroxylation pathway produces metabolic products that methylate and pyridyloxobutylate DNA. Naringenin, a metabolite of naringin, and quercetin were the most potent inhibitors of alpha-hydroxylation of NNK in both liver and lung microsomes. This inhibition correlated with a potent inhibition of MROD and PROD activity in liver but not in lung microsomes. The metabolic activation of NNK is associated with cytochrome P450 isoforms 1A1, 1A2, 2B1, 2D6 and 2E1. Our results suggest that naringenin and quercetin from citrus fruits inhibit the activity of cytochrome P450 (CYP) isoforms that activate NNK and may afford protection against NNK-induced carcinogenesis.

Pharmacokinetic studies of diosmin and diosmetin in perfused rat liver.

1. The kinetics and metabolism of diosmin and diosmetin were investigated in the isolated perfused rat liver in order to assess the role of the liver. 2. Diosmetin and especially diosmin disappeared quickly from the perfusion medium. 3. Both flavonoids were rapidly metabolized and diosmetin was partly excreted in bile as the glucuronide and sulphate and diosmin was partly excreted in bile as such and as the glucuronide conjugate. 4. Diosmin and diosmetin did not appear to affect various parameters of liver function.