Fertility and early embryonic development toxicity assessment of naringin in Sprague-Dawley rats.

Naringin is a dihydroflavonoid abundantly existed in grapefruit and related citrus species. The double directional adjusting function of estrogenic and anti-estrogenic activities of naringin and its aglycone naringenin has raised concern about possible risks of unwanted interference with endocrine regulation. Herein we assessed the safety of naringin on fertility and early embryonic development toxicity in Sprague-Dawley rats. Twenty-two male and 22 female rats per group were orally given naringin at 0, 50, 250, and 1250 mg/kg/day. Male rats were administered beginning 9 weeks prior to mating and continued until necropsy. Dosing to female began 2 weeks before mating and continued until gestation day 7. There were no obvious effects of naringin on physical signs, animal behavior, and survival rate, although female and male rats from 1250 mg/kg group had lower body weight and tended to have less food consumption. Importantly, no treatment-related effects of naringin were found in relation to fertility and early embryonic development. Under these experimental conditions, it was concluded that the no-observed-adverse-effect levels (NOAEL) of naringin were at least 1250 mg/kg/day for fertility and early embryonic development in rats.

Toxicological evaluation of naringin: Acute, subchronic, and chronic toxicity in Beagle dogs.

Naringin is a type of bioflavonoid widely found in a lot of dietary products. Our previous studies revealed that naringin was practically non-toxic for SD rats in an oral acute toxicity study and the no-observed-adverse-effect-level (NOAEL) in SD rats was greater than 1250 mg/kg/day when administered by oral gavage for 13 consecutive weeks or 6 consecutive months. Herein we assessed the safety of naringin in Beagle dogs. Acute oral administration of naringin was done as a single bolus dose up to 5 g/kg. Subchronic toxicity study for 3 months and chronic toxicity study for 6 months were done by oral administration at doses of 0 (control), 20, 100, and 500 mg/kg. The LD50 dosage level for dogs was determined to be > 5 g/kg body weight. In the repeated-dose 3-month and 6-month oral toxicity studies, no morbidity, mortality, and toxicologically relevant events were observed either during dosing or the post-dosing recovery period. It was concluded that the NOAEL of naringin in Beagle dogs is at least 500 mg/kg body weight per day when administered orally for 3 and 6 consecutive months. These results, combined with the previous toxicological studies in rats, demonstrate a good safety profile of naringin.

Effect of Naringenin and Its Derivatives on the Probing Behavior of Myzus persicae (Sulz.).

Substances that alter insect behavior have attracted a lot of attention as potential crop protection agents. Naringenin (5,7,4'-trihydroxyflavanone) is a naturally occurring bioactive flavanone. We evaluated the influence of naringenin on aphid activities during individual phases of probing and feeding and the effect of structural modifications of naringenin on its activity towards aphids. We monitored the probing behavior of Myzus persicae (Sulz.) (Hemiptera: Aphididae) using the Electrical Penetration Graph (EPG) technique. The chemical modifications were the substitution of hydrogen atoms with methyl, ethyl or pentyl groups and the replacement of the carbonyl group in naringenin and its derivatives with an oxime moiety. Depending on the substituents, the activity of naringenin-derived compounds varied in potency and mode of action. Naringenin was an attractant of moderate activity, which enhanced sap ingestion. The naringenin derivative with two methyl groups-7,4'-di-O-methylnaringenin-was a deterrent, which hindered aphid probing in non-phloem tissues. Naringenin oxime derivatives with methyl substituents-7,4'-di-O-methylnaringenin oxime, 7-O-methylnaringenin oxime, and 5,7,4'-tri-O-methylnaringenin oxime-and the derivative with a pentyl substituent-7-O-pentylnaringenin oxime-were strong attractants which stimulated aphid probing in non-phloem tissues and the ingestion of phloem sap.

Dibromopinocembrin and Dibromopinostrobin Are Potential Anti-Dengue Leads with Mild Animal Toxicity.

Dengue infection is one of the most deleterious public health concerns for two-billion world population being at risk. Plasma leakage, hemorrhage, and shock in severe cases were caused by immunological derangement from secondary heterotypic infection. Flavanone, commonly found in medicinal plants, previously showed potential as anti-dengue inhibitors for its direct antiviral effects and suppressing the pro-inflammatory cytokine from dengue immunopathogenesis. Here, we chemically modified flavanones, pinocembrin and pinostrobin, by halogenation and characterized them as potential dengue 2 inhibitors and performed toxicity tests in human-derived cells and in vivo animal model. Dibromopinocembrin and dibromopinostrobin inhibited dengue serotype 2 at the EC50s of 2.0640 ± 0.7537 and 5.8567 ± 0.5074 µM with at the CC50s of 67.2082 ± 0.9731 and >100 µM, respectively. Both of the compounds also showed minimal toxicity against adult C57BL/6 mice assessed by ALT and Cr levels in day one, three, and eight post-intravenous administration. Computational studies suggested the potential target be likely the NS5 methyltransferase at SAM-binding pocket. Taken together, these two brominated flavanones are potential leads for further drug discovery investigation.

Naringin-loaded polymeric micelles as buccal tablets: formulation, characterization, in vitro release, cytotoxicity and histopathology studies.

Naringin (NG) has been proved to have numerous notable biological effects, including anti-inflammatory effect, anti-cancer effect, and anti-ulcer effect, yet there are no clinical preparations of naringin due to its poor solubility and low dissolution rate after oral administration. In this study, in order to overcome these problems, NG was encapsulated into MPEG-PCL micelles (NGMs) by using a thin-film hydration method. NMGs were in a typical core-shell structure, with a mall particle size (23.95 ± 0.51 nm), high drug loading, and encapsulation efficiency. In vitro release of NGMs indicated that the dissolution of NG was increased after being encapsulated in the micelles. NGMs were nontoxic in the cytotoxicity and histopathology studies. Furthermore, when the freeze-dried NGMs were compressed into buccal tablets (NGBTs) by direct compression, the release speed of NG under simulated oral cavity condition from NGBTs was higher than the control tablets, with the accumulated dissolution at 93.13% in 8 hours. In conclusion, NGMs and NGBTs represent a promising drug delivery system for NG, which has the potential to improve the current treatment of oral diseases.

The Effects of Different Varieties of Aurantii Fructus Immaturus on the Potential Toxicity of Zhi-Zi-Hou-Po Decoction Based on Spectrum-Toxicity Correlation Analysis.

In accordance with the provision in China Pharmacopoeia, Citrus aurantium L. (Sour orange-SZS) and Citrus sinensis Osbeck (Sweet orange-TZS) are all in line with the requirements of Aurantii Fructus Immaturus (ZS). Both kinds of ZS are also marketed in the market. With the frequent occurrence of depression, Zhi-Zi-Hou-Po decoction (ZZHPD) has attracted wide attention. Currently, studies have shown that ZZHPD has a potential toxicity risk, but the effect of two commercial varieties of ZS on ZZHPD has not been reported. In this study, the toxicity differences of ZZHPD prepared by SZS and TZS were revealed through repeated administration experiments in rats. This indicated that different varieties of ZS could affect the toxicity of the prescription. In order to further study the chemical material basis of the toxicity difference, the fingerprints of ZZHPD prepared by different varieties of ZS were established by high-performance liquid chromatography (HPLC). Five different characteristic peaks were screened by non-target chemometrics. They were identified as geniposide, neoeriocitrin, naringin, hesperidin, and neohesperidin using an HPLC-time-of-flight mass spectrometry analyzer (TOF/MS) and an HPLC-triple stage quadrupole mass spectrometry analyzer (QqQ-MS/MS). Combined with a quantitative analysis and previous studies on promoting the intestinal absorption of geniposide, it is speculated that the synergistic effects of the components may be the main reason for the difference of toxicity among the different medicinal materials. This study provides a reference for the clinical, safe use of ZZHPD, and also provides a new perspective for the study of the potential toxic substances of traditional Chinese medicine compound preparations.

The Protective Effect of Naringenin on Airway Remodeling after Mycoplasma Pneumoniae Infection by Inhibiting Autophagy-Mediated Lung Inflammation and Fibrosis.

Our previous study has shown that Chinese medicine, Qingfei Tongluo formula (QTF), has a significantly therapeutic effect to Mycoplasma pneumoniae (MP) pneumonia (MPP). The aim of this study was to investigate the therapeutic effect and mechanism of naringenin (NRG) on MPP which was an important component of QTF. Here, we studied 124 children with or without MPP and compared inflammatory cytokines and fibrinogen-related protein expression with enzyme-linked immunosorbent assay. We also employed a BALB/c mouse model of MPP and divided the mice into three groups: ctrl (normal control mice), MPP (MP-infected mice), and MPP + NRG (MP-infected mice treated with NRG). BEAS-2B cells were used to confirm the relationship between autophagy, inflammation, and fibrosis. The results show proinflammatory cytokines (interleukin- [IL-] 6, IL-1ß, and tumor necrosis factor-α), and transforming growth factor beta (TGF-ß) expression was significantly increased after MP infection from both clinical and animal experiment. In vivo experimental confirmation showed that NRG treatment decreased MPP-induced lung injury in mice by inhibiting autophagy-mediated inflammatory cytokine expression and pulmonary fibrosis. In vitro experiments confirmed it. These results indicate that NRG treatment suppressed the inflammatory response and pulmonary fibrosis by inhibition of autophagy after MP infection.

Naringenin; a bioflavonoid, impairs the reproductive potential of male mice.

Present study analyzed the effect of naringenin, a bioflavonoid, on male reproductive function in adult mouse, after intraperitoneal treatment with varying concentrations of naringenin (2, 8 and 20 mg/kg b.wt.) for two weeks. Naringenin increased the generation of reactive oxygen species and lipid peroxidation in the testis with concomitant decrease in sperm count and motility in a dose-dependent manner. Activities of antioxidant enzymes catalase, superoxide dismutase, glutathione peroxidase and levels of reduced glutathione were found to be decreased in a dose-dependent manner. Also, the levels of oxidized glutathione were increased leading to a shift in redox ratio. Naringenin treatment also led to a dose-dependent increase in the mRNA expression of c-jun, c-fos and NF-κB. The testicular histomorphology was also altered dose dependently. Additionally, the number of apoptotic germ cells increased with increasing doses of naringenin as evident from acridine orange/ethidium bromide costaining and JC-1 staining. In conclusion, our study reveals that naringenin despite being a potent antioxidant with numerous important biological functions may also act as pro-oxidant, thus causing damaging effects in the testicular tissue.

Phytotoxic potential of Onopordum acanthium L. (Asteraceae).

Onopordum acanthium L. (Asteraceae) is a plant native to southern Europe and southwestern Asia, but it is invasive in disturbed areas and agricultural fields around the world, causing many agronomic problems by interfering with crops or preventing animals from grazing on pastures. Allelopathy could be one of the reasons that this plant has spread over different continents. The aim of the present study was to bioprospect O. acanthium leaf extracts through the isolation and purification of allelopathic secondary metabolites with phytotoxicity to explain their invasive behavior. Phytotoxic activity was tested using etiolated wheat coleoptiles. The most active extract was selected to perform a bioassay-guided isolation of two flavonoids, pectolarigenin (1) and scutellarein 4'-methyl ether (2), and two sesquiterpene lactones, elemanolide 11(13)-dehydromelitensin ß-hydroxyisobutyrate (3) and acanthiolide (4). All compounds were isolated for the first time from O. acanthium, and acanthiolide (4) is described for the first time. Compound 3 strongly inhibited the growth of wheat coleoptiles and 1 showed an intermediate effect. The results indicate that these compounds could contribute to the invasion of O. acanthium in ecological systems and agricultural fields.

Design, synthesis and inhibitory activities of naringenin derivatives on human colon cancer cells.

Based on the previous result, several naringenin derivatives modified at position 7 with bulky substituents were designed and synthesized, and their inhibitory effects on HCT116 human colon cancer cells were tested using a clonogenic assay. The half maximal inhibitory concentrations (IC(50)) of five naringenin derivatives ranged between 1.20 µM and 20.01 µM which are much better than naringenin used as a control. In addition, new structural modification at C-4 of flavanone results in improving both the anti-cancer effect and anti-oxidative effect. In vitro cyclin dependent kinase 2 (CDK2) binding assay was carried out based on the previous results. To elucidate the possible interaction between naringenin derivatives and CDK2, in silico docking study was performed. This result demonstrates the rationale for the different inhibitory activities of the naringenin derivatives. These findings could be used for designing cancer therapeutic or preventive flavanone-derived agents.

Structure-cytotoxic activity relationship of 3-arylideneflavanone and chromanone (E,Z isomers) and 3-arylflavones.

The E,Z-isomers of 3-arylidene substituted flavanone, chromanone and 3-aryl substituted flavone derivatives were tested in vitro for their cytotoxic activity against three cancer cell lines (HL-60, NALM-6, WM-115) and normal cell line (HUVEC). It was observed that substitution at C3 position led to significant enhance in cytotoxicity. Isomeric configuration of 3-arylideneflavanones had an influence on the cytotoxic potential. Multiple regression analysis combined with variable selection by genetic algorithm was used to model relationships between molecular descriptors and the cytotoxic activity. The most accurate QSAR models were based on a combination between energy of LUMO, experimental value of logP and partial charge on carbonyl oxygen (δO2).

Phytotoxicity of secondary metabolites isolated from Flourensia oolepis S.F.Blake.

The aim of this study was to isolate the active principles of Flourensia oolepis S.F.Blake (Asteraceae), which completely inhibited the germination of Raphanus sativus seeds at 10 mg/ml. Flavanone pinocembrin and sesquiterpene ilicol, were isolated by bioassay-guided fractionation. They were active both against monocot and dicot seeds. Pinocembrin was the most active compound, with an IC50 (germination) value of 0.24, 3.40, 3.28, and 3.55 mM against Panicum miliaceum, Avena sativa, Lactuca sativa, and R. sativus, respectively; ilicol, however, exhibited IC50 (germination) values of 0.67, 2.73, 5.25, and 9.66 mM for the same species, respectively. Pinocembrin and ilicol inhibited root growth and showed IC50 (root growth) values of 0.199, 14.68, 8.05, 7.69 mM, and 1.22, 2.90, 7.35, 8.07 mM, against P. miliaceum, A. sativa, L. sativa, and R. sativus, respectively. Pinocembrin and ilicol reduced Allium cepa cell division without chromosome aberrations.

Antimicrobial effect of sophoraflavanone G isolated from Sophora flavescens against mutans streptococci.

In this study, the antibacterial properties of sophoraflavanone G isolated from the methanol extract of Sophora flavescens were tested against 16 strains of mutans streptococci to screen and determine the optimal concentration of anti-caries natural extract. The antimicrobial activity was evaluated by measuring minimum bactericidal concentration (MBC). The cell viability of normal human gingival fibroblast (NHGF) cells was tested using the methyl thiazolyl tetrazolium assay after exposure to sophoraflavanone G. The data showed that sophoraflavanone G had a remarkable antimicrobial effect on the bacteria tested with an MBC ranging from 0.5 µg/ml to 4 µg/ml. Sophoraflavanone G had no cytotoxic effect on NHGF cells at concentrations where it produced an antimicrobial effect. These findings demonstrate that sophoraflavanone G has strong antimicrobial activity against mutans streptococci and could be useful in the development of novel oral hygiene products, such as a gargle solution or dentifrice.

Lavandulyl flavanones from the stems of Hypericum calycinum L.

One novel lavandulyl flavanone (=2,3-dihydro-2-phenyl-4H-1-benzopyran-4-one) with an unusual 5,2',4',6'-tetrahydroxy substitution, calycinigin A (1), was isolated from the stems of Hypericum calycinum L. (Hypericaceae). The structure was elucidated on the basis of 1D- and 2D-NMR analysis, as well as mass spectrometry (LR-EI- and HR-EI-MS) and circular dichroism. Three known lavandulyl flavanones with 5,7,2',4',6'-pentahydroxy substitution, i.e., 2-4, were also isolated. Chemosystematically, this is the first report on the occurrence of prenylated flavanones in the family Hypericaceae. Reduction of cell viability by all compounds was evaluated in a MTT (=3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay using HeLa cells. Compound 1 showed moderate activity with an IC50 value of 9.7±1.8 µM, whereas compounds 2-4 were less active exhibiting IC50 values of 11.6±0.9, 19.3±1.5, and 40.7±2.4 µM, respectively. The antioxidant activity was evaluated by an ORAC (Oxygen Radical Absorbance Capacity) assay, and calycinigin A (1) was again the most active compound with a Trolox equivalent of 2.3±0.2. None of the compounds was able to reduce the TNF-α induced ICAM-1 expression in vitro using human microvascular endothelial cells (HMEC-1).

Evaluation of the cytotoxicity of Satureja spicigera and its main compounds.

Satureja spicigera (Lamiaceae) grows wildly in Northwest of Iran. In this study, bioassay-guided isolation and identification of the main compounds has been reported using various chromatographic methods and comparison of their spectral data with those reported in the literature. Brine shrimp lethality and four cancerous cell lines HT29/219, Caco(2), NIH-3T3, and T47D were used for cytotoxicity evaluations. From the aerial parts of S. spicigera, nine known compounds including two flavanones, 5,7,3',5'-tetrahydroxy flavanone (8) and 5,4'-dihydroxy-3'-methoxyflavanone-7-(6''-O-α-L-rhamnopyranosyl)-ß-D-glucopyranoside (9), one dihydrochalcone, nubigenol (7), together with thymoquinone (1), thymol (2), carvacrol (3), ß-sitosterol (4), ursolic acid (5) and oleanolic acid (6) were identified. Among the isolated chalcone and flavanones, compound 8 was effective against Artemia salina larva (LC(50)= 2 µg/mL) and only the compound 9 demonstrated IC(50) value of 98.7 µg/mL on the T47D (human, breast, ductal carcinoma). Other compounds did not show significant inhibition of the cell growth.

A new prenylated flavanone from Derris trifoliata Lour.

A new flavanone, 4',5,7-trihydroxy-6,8-di-(2-hydroxy-3-methylbut-3-enyl)- flavanone, was isolated from the aerial parts of Derris trifoliate, together with eleven known compounds: rotenone, tephrosin, 12a-hydroxyrotenone, deguelin, 6a,12a-dehydro-rotenone, dehydrodeguelin, 7a-O-methyldeguelol, 7a-O-methylelliptonol, 5,7,3',4'-tetra-hydroxy-6,8-diprenylisoflavone, daidzein and 4'-hydroxy-7-methoxyflavanone. 7a-O-Methylelliptonol was isolated for the first time from the genus Derris. Their structures were characterized on the basis of spectral data. Eight of the isolated compounds were found to be significantly toxic to brine shrimp (LC(50) range 0.06-9.95 µg/mL). The new compound showed weak toxicity (LC(50) = 211.31 µg/mL).

Synthetic flavanones augment the anticancer effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as the most promising anticancer agent in the TNF superfamily because of its selective cytotoxicity against tumor cells versus normal primary cells. However, as more tumor cells are reported to be resistant to TRAIL-mediated death, it is important to develop new therapeutic strategies to overcome this resistance. Flavonoids have been shown to sensitize cancer cells to TRAIL-induced apoptosis. The aim of this study was to examine the cytotoxic and apoptotic activities of TRAIL on HeLa cancer cells in combination with two synthetic compounds: 6-hydroxyflavanone (6-HF) and its derivative 6-propionoxy-flavanone (6-PF) and to determine the mechanism by which the flavanones overcome the TRAIL-resistance. The cytotoxicity was measured by MTT and LDH assays. The apoptosis was detected by annexin V-FITC fluorescence staining in flow cytometry and microscopy. Death receptor (TRAIL-R1/DR4 and TRAIL-R2/DR5) expression were analysed using flow cytometry. Mitochondrial membrane potential was evaluated using DePsipher staining by fluorescence microscopy. The synthetic flavanones enhanced TRAIL-induced apoptosis in HeLa cells through increased expression of TRAIL-R2 death receptor and reduction of mitochondrial membrane potential. Our study indicates that the 6-HF and 6-PF augmented the anticancer effects of TRAIL and confirm a potential use of flavanones in TRAIL-based anticancer therapy and prevention.

Developmental toxicity and genotoxicity studies of wogonin.

We studied the developmental toxicities and genotoxic potency of a widely bioactive plant medicine-wogonin in vivo and in vitro. In the in vivo developmental experiments, high dose of wogonin (40mg/kg, intravenous injection) significantly induced the maternal weight gains and affected fetus including bodyweight, resorptions, live birth index and fetal skeletal alterations. In Ames test, no concentration-dependently increased TA98, TA100, and TA102 revertants were detected in wogonin groups whether in presence of metabolic activating enzymes or not. In the chromosome aberration test, wogonin dose-dependently increased structural chromosomal aberrations in CHL cells both with and without S9, even the effect was all judged (-). In micronucleus assay, no significant changes of MNPCE/PCE and PCE/NCE were found on mouse bone marrow micronucleus in wogonin groups. We concluded that wogonin induced developmental toxicities on pregnant mice and fetus, and the genotoxicities were positive. However no significant malformation was observed and only in vitro potency of chromosome aberration was weak, which suggested us wogonin could be a relatively safe drug in clinic.

Two new cytotoxic phenylallylflavanones from Mexican propolis.

Two new phenylallylflavanones, (2R,3R)-6-[1-(4'-hydroxy-3'-methoxyphenyl)prop-2-en-1-yl]pinobanksin (1) and (2R,3R)-6-[1-(4'-hydroxy-3'-methoxyphenyl)prop-2-en-1-yl]pinobanksin 3-acetate (2) were isolated from a methanolic extract of Mexican propolis. Their structures were elucidated with spectroscopic analysis. Both compounds (1, 2) exhibited preferential cytotoxic activity against PANC-1 human pancreatic cancer cells in a nutrient-deprived medium with the concentration at which 50% cells died preferentially in NDM (PC50) values of 17.9 µM and 9.1 µM, respectively.

Antiviral activity of silymarin and baicalein against dengue virus.

Dengue is an arthropod-borne viral disease that has become endemic and a global threat in many countries with no effective antiviral drug available currently. This study showed that flavonoids: silymarin and baicalein could inhibit the dengue virus in vitro and were well tolerated in Vero cells with a half-maximum cytotoxic concentration (CC50) of 749.70 µg/mL and 271.03 µg/mL, respectively. Silymarin and baicalein exerted virucidal effects against DENV-3, with a selective index (SI) of 10.87 and 21.34, respectively. Baicalein showed a better inhibition of intracellular DENV-3 progeny with a SI of 7.82 compared to silymarin. Baicalein effectively blocked DENV-3 attachment (95.59%) to the Vero cells, while silymarin prevented the viral entry (72.46%) into the cells, thus reducing viral infectivity. Both flavonoids showed promising antiviral activity against all four dengue serotypes. The in silico molecular docking showed that silymarin could bind to the viral envelope (E) protein with a binding affinity of - 8.5 kcal/mol and form hydrogen bonds with the amino acids GLN120, TRP229, ASN89, and THR223 of the E protein. Overall, this study showed that silymarin and baicalein exhibited potential anti-DENV activity and could serve as promising antiviral agents for further development against dengue infection.