Potential hepatic and renal toxicity induced by the biflavonoids from Ginkgo biloba.

Evidence continues to grow on potential health risks associated with Ginkgo biloba and its constituents. While biflavonoid is a subclass of the flavonoid family in Ginkgo biloba with a plenty of pharmacological properties, the potential toxicological effects of biflavonoids remains largely unknown. Thus, the aim of this study was to investigate the in vitro and in vivo toxicological effects of the biflavonoids from Ginkgo biloba (i.e., amentoflavone, sciadopitysin, ginkgetin, isoginkgetin, and bilobetin). In the in vitro cytotoxicity test, the five biflavonoids all reduced cell viability in a dose-dependent manner in human renal tubular epithelial cells (HK-2) and human normal hepatocytes (L-02), indicating they might have potential liver and kidney toxicity. In the in vivo experiments, after intragastrical administration of these biflavonoids at 20 mg·kg-1·d-1 for 7 days, serum biochemical analysis and histopathological examinations were performed. The activity of alkaline phosphatase was significantly increased after all the biflavonoid administrations and widespread hydropic degeneration of hepatocytes was observed in ginkgetin or bilobetin-treated mice. Moreover, the five biflavonoids all induced acute kidney injury in treated mice and the main pathological lesions were confirmed to the tubule, glomeruli, and interstitium injuries. As the in vitro and in vivo results suggested that these biflavonoids may be more toxic to the kidney than the liver, we further detected the mechanism of biflavonoids-induced nephrotoxicity. The increased TUNEL-positive cells were detected in kidney tissues of biflavonoids-treated mice, accompanied by elevated expression of proapoptotic protein BAX and unchanged levels of antiapoptotic protein BCL-2, indicating apoptosis was involved in biflavonoids-induced nephrotoxicity. Taken together, our results suggested that the five biflavonoids from Ginkgo biloba may have potential hepatic and renal toxicity and more attentions should be paid to ensure Ginkgo biloba preparations safety.

Toxicological evaluation of the biflavonoid, agathisflavone in albino Swiss mice.

Agathisflavone (AGF) is a biflavonoid with a number of important biological and pharmacological activities, such as antioxidant, antimicrobial, and neuroprotective effects. However, its toxicological effects have not been fully investigated. Accordingly, the aim of this study was to investigate the toxicological effects of AGF in mice. For this purpose, the median lethal dose 50% (LD50) was determined along with the anatomic and histopathological parameters (weight, alimentation, excretion, biochemical, and hematological) in fertile untouched female Swiss mice. Results suggest that during the treatment, no deaths were reported at 300 and 2000 mg/kg (n = 03/group, p.o.). Moreover, AGF did not cause significant change in the above mentioned parameters in test animals when compared with the control group (0.05% Tween 80 dissolved in 0.9% saline). Taken all together, this non-clinical toxicological study revealed that AGF has an LD50 larger than 2000 mg/kg and did not change significantly the hematological, biochemical, histopathological, behavioral, as well as physiological parameters in the female mice.

Anti-inflammatory and analgesic activities of cupressuflavone from Cupressus macrocarpa: Impact on pro-inflammatory mediators.

Hit, Lead & Candidate Discovery Inflammation is a complex biological process that is generally occurs in response to pathological triggers. Both neurodegenerative diseases and cancer have been linked to inflammation. The analgesic and anti-inflammatory effects of cupressuflavone (CUF) isolated from Cupressus macrocarpa were examined. The analgesic effects of CUF (40, 80 and 160 mg/kg po) were assessed in the acetic acid-induced writhing and hot plate models in mice with diclofenac sodium as the reference standard (100 mg/kg). CUF dose-dependently inhibited the writhing response in mice by 25, 48, and 62%, at the three CUF doses with 160 mg/kg being equivalent to the diclofenac control. CUF dose-dependently increased the hot plate model reaction time with a maximal effect after 120 min. In the carrageenan-induced paw edema model of inflammation, CUF demonstrated anti-inflammatory activity by inhibiting paw edema by 55, 60, and 64% at doses of 40, 80, and 160 mg/kg po, respectively. CUF also reduced the plasma pro-inflammatory mediators PGE2 (44, 54, and 58%), TNF-α (26, 37, and 53%), IL-1ß (19, 33, and 41%), and IL-6 (32, 44, and 55%) at the three doses tested with the highest dose having similar effects to diclofenac sodium (100 mg/kg). This finding from this study indicates that CUF has both analgesic and anti-inflammatory effects.

Antiviral activity of theaflavin digallate against herpes simplex virus type 1.

Tea is the second most consumed drink in the world. The beneficial effects of tea have been mostly attributed to its catechin content. Black tea is derived from the leaves of Camellia sinensis plant, and it is rich in theaflavin polyphenols, in particular theaflavin (TF1), theaflavin-3-monogallate (TF2A), theaflavin-3'-monogallate (TF2B), and theaflavin-3,3'-digallate (TF3). Vero and A549 cells were used to evaluate the effect of purified individual black tea theaflavins as anti-herpes simplex virus 1 agents. With the rise of HSV resistant strains, there is a critical need to develop novel antiherpesviral treatments. Results of the cytotoxicity assay tested by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium] showed that TF1, TF2, and TF3 are not toxic to Vero and A549 cells at a concentration up to 75 µM. The antiviral activity of the individual theaflavins was tested by plaque reduction assay, MTS assay, flow cytometric analysis and confocal microscopy observations. The results showed that TF1, TF2, and TF3 exhibit potent, dose-dependent anti-HSV-1 effect, with TF3 being the most efficient in both Vero and A549 cells. A concentration of 50 µM TF3 and above was sufficient to inhibit >99% of the production of HSV-1 viral particles. The anti-HSV-1 effect of TF3 is due to a direct effect on the virions, and treating Vero or A549 cells with TF3 for 1h prior to infection, or treating the cells at different times post infection does not inhibit HSV-1 production. TF3 is stable at vaginal pH, indicating its potential to be a promising natural and affordable remedy against herpes simplex viral infections.

Altered profile of gut microbiota after subchronic exposure to neochamaejasmin A in rats.

Neochamaejasmin A, isolated from Stellera chamaejasme L., has been widely used in China. Gut microbiota represent the first barrier against xenobiotics. This study aimed to evaluate the effects of subchronic exposure to neochamaejasmin A on the composition of gut microbiota. We found that neochamaejasmin A altered 21 OTUs in female rats and 46 OTUs in male rats. Among these OTUs, OTU86, OTU338 and OTU482 were shared in neochamaejasmin A-fed groups in both genders, implying that neochamaejasmin A might promote the growth of these three genera. In contrast, little or no effect on 226 OTUs was observed at all doses in both genders, suggesting their resistance to neochamaejasmin A. These findings could help improve our understanding of the health effects of neochamaejasmin A and provide an example of the risk assessment of pharmaceuticals or food contaminants on the gut microbiota composition.

Anti-Mycobacterium tuberculosis activity and cytotoxicity of Calophyllum brasiliense Cambess (Clusiaceae)

We evaluated the in vitro anti-Mycobacterium tuberculosis activity and the cytotoxicity of dichloromethane extract and pure compounds from the leaves of Calophyllum brasiliense. Purification of the dichloromethane extract yielded the pure compounds (-) mammea A/BB (1), (-) mammea B/BB (2) and amentoflavone (3). The compound structures were elucidated on the basis of spectroscopic and spectrometric data. The contents of bioactive compounds in the extracts were quantified using high performance liquid chromatography coupled to an ultraviolet detector. The anti-M. tuberculosis activity of the extracts and the pure compounds was evaluated using a resazurin microtitre assay plate. The cytotoxicity assay was performed in J774G.8 macrophages using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide colourimetric method. The quantification of the dichloromethane extract showed (1) and (2) at concentrations of 31.86 ± 2.6 and 8.24 ± 1.1 µg/mg of extract, respectively. The dichloromethane and aqueous extracts showed anti-M. tuberculosis H37Rv activity of 62.5 and 125 µg/mL, respectively. Coumarins (1) and (2) showed minimal inhibitory concentration ranges of 31.2 and 62.5 µg/mL against M. tuberculosis H37Rv and clinical isolates. Compound (3) showed no activity against M. tuberculosis H37Rv. The selectivity index ranged from 0.59-1.06. We report the activity of the extracts and coumarins from the leaves of C. brasiliense against M. tuberculosis.

Anti-Mycobacterium tuberculosis activity and cytotoxicity of Calophyllum brasiliense Cambess (Clusiaceae).

We evaluated the in vitro anti-Mycobacterium tuberculosis activity and the cytotoxicity of dichloromethane extract and pure compounds from the leaves of Calophyllum brasiliense. Purification of the dichloromethane extract yielded the pure compounds (-) mammea A/BB (1), (-) mammea B/BB (2) and amentoflavone (3). The compound structures were elucidated on the basis of spectroscopic and spectrometric data. The contents of bioactive compounds in the extracts were quantified using high performance liquid chromatography coupled to an ultraviolet detector. The anti-M. tuberculosis activity of the extracts and the pure compounds was evaluated using a resazurin microtitre assay plate. The cytotoxicity assay was performed in J774G.8 macrophages using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide colourimetric method. The quantification of the dichloromethane extract showed (1) and (2) at concentrations of 31.86 ± 2.6 and 8.24 ± 1.1 µg/mg of extract, respectively. The dichloromethane and aqueous extracts showed anti-M. tuberculosis H37Rv activity of 62.5 and 125 µg/mL, respectively. Coumarins (1) and (2) showed minimal inhibitory concentration ranges of 31.2 and 62.5 µg/mL against M. tuberculosis H37Rv and clinical isolates. Compound (3) showed no activity against M. tuberculosis H37Rv. The selectivity index ranged from 0.59-1.06. We report the activity of the extracts and coumarins from the leaves of C. brasiliense against M. tuberculosis.

Antiproliferative and apoptotic effects triggered by Grape Seed Extract (GSE) versus epigallocatechin and procyanidins on colon cancer cell lines.

Grape seed extract has been proven to exert anticancer effects on different tumors. These effects are mainly ascribed to catechin and procyanidin content. Analytical studies demonstrated that grape seed extract composition is complex and it is likely other components could exert biological activities. Using cell count and flow cytometry assays, we evaluated the cytostatic and apoptotic effects produced by three different grape seed extracts from Italia, Palieri and Red Globe cultivars, on Caco2 and HCT-8 colon cancer cells. These effects were compared to those induced by epigallocatechin and procyanidins, alone or in association, on the same cell lines. All the extracts induced growth inhibition and apoptosis in Caco2 and HCT-8 cells, along the intrinsic apoptotic pathway. On both cell lines, growth inhibition induced by Italia and Palieri grape seed extracts was significantly higher than that it has been recorded with epigallocatechin, procyanidins and their association. In Caco2 cells, the extract from Red Globe cultivar was less effective in inducing growth inhibition than procyanidins alone and in association with epigallocatechin, whereas, in HCT-8 cells, only the association of epigallocatechin and procyanidins triggers a significant proliferation decrease. On both cell lines, apoptosis induced by Italia, Palieri and Red Globe grape seed extracts was considerably higher than has been recorded with epigallocatechin, procyanidins and their association. These data support the hypothesis by which other compounds, present in the grape seed extracts, are likely to enhance the anticancer effects.

High-throughput screening for anti-influenza A virus drugs and study of the mechanism of procyanidin on influenza A virus-induced autophagy.

In this research, we have established a high-throughput screening (HTS) platform based on the influenza A virus (IAV) vRNA promoter. Using this HTS platform, we selected 35 medicinal plants out of 83 examples of traditional Chinese medicine and found that 7 examples had not been reported. After examining many previous reports, we found that Vaccinium angustifolium Ait., Vitis vinifera L, and Cinnamomum cassia Presl had a common active compound, procyanidin, and then determined the anti-IAV effect of procyanidin and explored its mechanism of action. With a plaque inhibition assay and a time-of-addition experiment, we found that procyanidin could inhibit the IAV replication at several stages of the life cycle. In the Western blot and EGFP-LC3 localization assays, we found that procyanidin could inhibit the accumulation of LC3II and the dot-like aggregation of EGFP-LC3. In the RT-PCR and Western blot assays, we found procyanidin could inhibit the expression of Atg7, Atg5, and Atg12. Finally, by the bimolecular fluorescence complementation-fluorescence resonance energy transfer and co-immunoprecipitation assays, we found that procyanidin could inhibit the formation of the Atg5-Atg12/Atg16 heterotrimer and the dissociation of the beclin1/bcl2 heterodimer. In conclusion, we have established an HTS platform and identified procyanidin as a novel and promising anti-IAV agent.

Toxicology evaluation of a procyanidin-rich extract from grape skins and seeds.

The procyanidin-rich extract from grape seeds and skins (GSSE) has antioxidant properties which may have cardioprotective effects. Since it might be interesting to incorporate this extract into a functional food, toxicological tests need to be made to determine how safe it is. In this study we carried out a limit test to determine the acute oral toxicity and the lethal dose 50 (LD50) and some genotoxicity tests of the extract in rats. The LD50 was higher than 5000 mg/kg. Doses of up to 2000 mg/kg showed no increase in micronucleated erythrocytes 72 h after treatment. The bacterial reverse mutation test showed that the extract was weakly mutagenic to the dose of 5 mg/plate and 19.5 and 9.7 µg/ml of GSSE did not show significant differences in the frequency of aberrant metaphases in relation to negative controls. Our results indicated slight mutagenicity under the study conditions, so further studies should be conducted at lower doses to demonstrate that this extract is not toxic.

Mutagenic activity promoted by amentoflavone and methanolic extract of Byrsonima crassa Niedenzu.

Byrsonima crassa is a plant pertaining to the Brazilian central savannah-like belt of vegetation and popularly used for the treatment of gastric dysfunctions and diarrhoea. The methanol extract contains catechin, tannins, terpenes and flavonoids; both mutagenic potential and antioxidant properties have been ascribed to flavonoids. The mutagenicity of some flavonoids is believed to be associated with the formation of reactive oxygen species and seems to depend on the number and position of hydroxyl groups. In the present study the mutagenic activity of the methanol, chloroform and 80% aqueous methanol extracts, as well as acetate and aqueous sub-fractions, of this medicinal plant were evaluated by Salmonella typhimurium assay, using strains TA100, TA98, TA102 and TA97a, and in mouse reticulocytes. The results showed mutagenic activity of the methanolic extract in the TA98 strain without S9, but no mutagenicity to mouse cells in any of the extracts. The acetate fraction showed strong signs of mutagenicity without S9, suggesting that in this enriched fraction were concentrated the compounds that induced mutagenic activity. The aqueous fraction showed no mutagenic activity. The TLC and HSCCC analyses of the acetate fraction with some standard compounds permitted the isolation of the quercetin-3-O-beta-D-galactopyranoside, quercetin-3-O-alpha-L-arabinopyranoside, amentoflavone, methyl gallate and (+)-catechin, of which only the amentoflavone exhibited positive mutagenicity to TA98 (+S9, -S9).

Comparative antiplasmodial, leishmanicidal and antitrypanosomal activities of several biflavonoids.

The antiplasmodial, leishmanicidal and antitrypanosomal activities of eight natural biflavonoids were estimated in vitro on a chloroquine-resistant strain of Plasmodium falciparum, axenically grown Leishmania donovani amastigotes and Trypanosoma cruzi trypomastigotes and Trypanosoma brucei rhodesiense bloodstream forms. Lanaroflavone showed the highest antiplasmodial activity (IC(50) = 0.48 microM), isoginkgetin was the most active leishmanicidal compound (IC(50) = 1.9 microM), whereas ginkgetin (IC(50) = 11 microM) and isoginkgetin (IC(50) = 13 microM) showed the best antitrypanosomal activity in our assays. The cytotoxicity and the selectivity indices for the most active compounds were also estimated. Lanaroflavone exhibited a high selectivity index value (SI = 159), indicating selective antiplasmodial activity.

The biflavonoid, amentoflavone degrades DNA in the presence of copper ions.

Previous reports from this laboratory have shown that flavonoids including apigenin are capable of inducing oxidative DNA cleavage in the presence of copper ions. In the present report, we have examined the ability of amentoflavone, a biflavonoid which is a dimer of apigenin, to catalyze the degradation of DNA. Amentoflavone was found to degrade calf thymus DNA in the presence of Cu(II) at a rate almost twice that of apigenin. Amentoflavone was also shown to reduce Cu(II) to Cu(I) and to generate hydroxyl radicals in the presence of copper ions. In the presence of Cu(II), the absorption spectrum of amentoflavone undergoes a shift and a quenching effect indicating that the biflavonoid is capable of binding to copper ions. Amentoflavone and apigenin were isolated from Cycas rumphii and Trifolium alexandrinum, respectively. The results are discussed in relation to the putative chemopreventive mechanism of amentoflavone.