Induction of Biologically Active Flavonoids in Cell Cultures of Morus nigra and Testing their Hypoglycemic Efficacy

The antidiabetic activity of both leaves and MJ-treated cell cultures of Morus nigra was evaluated after their oral administration to streptozotocin-induced diabetic rats. The antidiabetic activity of extracts from leaves given to streptozotocin (STZ)-diabetic rats for 10 days increased with increasing doses of leaves extract up to 500 mg/kg/day. The administration of 500 mg/kg/day of leaves extract reduced the concentration of glucose from 370 ± 7.31 mg/dl (control) to 154 ± 6.27 mg/dl, and a significant increase in the insulin level from 11.3 ± 0.31 μU/ml (control) to 14.6 ± 0.43 μU/ml was recorded. Cell suspension cultures were established from the young leaves of Morus nigra cultivated on modified MS medium supplemented with 2.0 mg/l 1-naphthaleneacetic acid (NAA), 0.2 mg/l 6-(furfurylamino)purine (kinetin). The changes in cell weight and flavonoid content were monitored between day zero and 12. The linear increase in fresh weight was found to be parallel to flavonoids production. Cell cultures treated with 100 μM methyl jasmonate for 24 hours showed a noticeable increase in level of flavonoids and significant and more effective hypoglycemic activity than that for extract from leaves. The major flavonoids were isolated by TLC and HPLC and identified as rutin, quercetin, Morusin and cyclomorusin by co-chromatography and mass spectrometry in comparison to samples of authentic reference compounds.     

Simultaneous quantification of eight major constituents in Herba Siegesbeckiae by liquid chromatography coupled with electrospray ionization time-of-flight tandem mass spectrometry

A simple and reliable high-performance liquid chromatography coupled with electrospray ionization time-of-flight tandem mass spectrometry method was developed and validated for the determination of the major diterpenoids and flavonoids in the aerial parts of Herba Siegesbeckiae, including Kirenol, hythiemoside B, ent-16β,17,18-trihydroxy-kauran-19-oic acid, ent-17,18-dihydroxy-kauran-19-oic acid, ent-16β,17-dihydroxy-kauran-19-oic acid, 16α-hydro-ent-kauran-17,19-dioic acid, Rhamnetin, 3',4'-dimethoxy quercetin. The separation of eight compounds was performed on a Waters Symmetry Shield TM RP18 column (250mm×4.6mm i.d., 5μm) with gradient elution using a mobile phase consisting of 0.1% aqueous formic acid and acetonitrile containing 0.1% formic acid in selected ion monitoring mode. All calibration curves showed good linearity (r>0.999) within the test ranges. The precision was evaluated by intra- and inter-day tests, which revealed relative standard deviation (RSD) values less than 3.7%. The recoveries for the quantified compounds were between 97.4 and 101.2% with RSD values below 2.4%. According to the literatures, this study represents the first investigation of the simultaneous analysis of multiple components and the method can be applied to determine the amounts of the major compounds in Herba Siegesbeckiae.     

Evaluation of antimicrobial efficacy of flavonoids of withania somnifera L

In the present study antimicrobial activity of Withania somnifera L. Dunal (Solanaceae) has been evaluated against selected pathogens. Free and bound flavonoids of different parts (root, stem, leaf and fruit) of W. somnifera have been studied for their antimicrobial activity using disc diffusion assay against three Gram negative bacteria (Escherichia coli MTCC 46, Proteus mirabilis MTCC 3310 and Pseudomonas aeruginosa MTCC 1934), one Gram positive bacteria (Staphylococcus aureus MTCC 3160) and three fungi (Candida albicans MTCC 183, Aspergillus flavus MTCC 277 and Aspergillus niger MTCC 282). Minimum inhibitory concentration (MIC) of the extracts was evaluated through micro broth dilution method, while minimum bactericidal/fungicidal concentration was determined by sub culturing the relevant samples. C. albicans was found to be the most susceptible organism followed by S. aureus, P. mirabilis, E. coli and P. aeruginosa. Out of the tested organisms, A flavus and A. niger were observed to be resistant as none of the tested extracts showed activity against them. Total activity (TA) of extracts (ml/g) against each sensitive pathogens was also evaluated. Bound flavonoid extract of root showed best activity against C. albicans (IZ 30, MIC 0.039, MFC 0.039, respectively). However all the microorganisms were found to be sensitive against the extracts tested. Total activity of bound flavonoid extract of root was found to be same for E.coli, P. mirabilis, S. aureus and C. albicans (153.84 ml/g). Results of the present study reveal that extracts of W. somnifera showing great antimicrobial potential against test microorganisms may be exploited for future antimicrobial drugs.     

Inhibitory Effects of Quercetin on Muscle-type of Nicotinic Acetylcholine Receptor-Mediated Ion Currents Expressed in Xenopus Oocytes

The flavonoid quercetin is a low molecular weight compound generally found in apple, gingko, tomato, onion and other red-colored fruits and vegetables. Like other flavonoids, quercetin has diverse pharmacological actions. However, relatively little is known about the influence of quercetin effects in the regulation of ligand-gated ion channels. Previously, we reported that quercetin regulates subsets of nicotinic acetylcholine receptors such as α3β4, α7 and α9α10. Presently, we investigated the effects of quercetin on muscle-type of nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding human fetal or adult muscle-type of nicotinic acetylcholine receptor subunits. Acetylcholine treatment elicited an inward peak current (I(ACh)) in oocytes expressing both muscle-type of nicotinic acetylcholine receptors and co-treatment of quercetin with acetylcholine inhibited I(ACh). Pre-treatment of quercetin further inhibited I(ACh) in oocytes expressing adult and fetal muscle-type nicotinic acetylcholine receptors. The inhibition of I(ACh) by quercetin was reversible and concentration-dependent. The IC(50) of quercetin was 18.9±1.2 μM in oocytes expressing adult muscle-type nicotinic acetylcholine receptor. The inhibition of I(ACh) by quercetin was voltage-independent and non-competitive. These results indicate that quercetin might regulate human muscle-type nicotinic acetylcholine receptor channel activity and that quercetin-mediated regulation of muscle-type nicotinic acetylcholine receptor might be coupled to regulation of neuromuscular junction activity.     

Wound healing potential of Sambucus ebulus L. leaves and isolation of an active component,quercetin 3-O-glucoside

Ethnopharmacological relevance

The leaves of Sambucus ebulus L. are used in Turkish folk medicine for treatment of high fever, rheumatic pains, snake bites and wounds.

Aim of the study

Four different solvent extracts by using n-hexane, diethyl ether, ethyl acetate and methanol were prepared from the leaves of Sambucus ebulus in order to investigate the claimed wound healing activity in vivo and isolation of active component(s) from the active extract through the bioassay-guided fractionation procedures.

Materials and methods

Linear incision and circular excision wound models on rats and mice were employed. The methanol extract was fractionated by successive chromatographic techniques. Wound healing activity of each fraction was investigated following the bioassay-guided fractionation procedures. Moreover, the tissue samples were examined histopathologically.

Results

Significant wound healing activity was observed for the ointment prepared with methanol extract at 1% concentration. The methanol extract treated groups of animals showed 84.3% contraction in circular excision model, which was close to contraction value of the reference drug Madecassol^® (100%). On the other hand, a significant increase (43.7%) in the wound tensile strength was determined with the same extract on incision wound model. Subfractions showed significant but reduced wound healing activity on both in vivo wound models. A flavonoid derivative “quercetin 3-O-glucoside” was isolated and determined as one of the active component of active final subfraction. The results of histopathological examination supported the outcome of linear incision and circular excision wound models.

Conclusion

The experimental data revealed that the methanolic extract of Sambucus ebulus leaves displayed remarkable wound healing activity.     

Anti-Inflammatory Activity of Parthenolide-Depleted Feverfew (Tanacetum parthenium)

Extracts of Tanacetum parthenium (L.) Sch. Bip., a plant known under the common name “Feverfew”, contains the sesquiterpene lactone parthenolide, a potent skin sensitizer. To eliminate the risk of skin sensitization from Feverfew, we developed a parthenolide-depleted extract of Feverfew (PD-Feverfew) and determined its effectiveness as an anti-inflammatory agent. We confirmed that PD-Feverfew was sufficiently depleted of parthenolide since PD-Feverfew did not inhibit TNF-α induced-NF-κB activity unlike parthenolide containing whole Feverfew. PD-Feverfew directly inhibited the activity of pro-inflammatory enzymes 5-lipoxygenase, phosphodiesterase-3 and phosphodiesterase-4. PD-Feverfew inhibited the release of pro-inflammatory mediators nitric oxide, PGE₂ and TNF-α from macrophages and TNF-α, IL-2, IFN-γ and IL-4 from human peripheral blood mononuclear cells. Additionally, PD-Feverfew inhibited TPA-induced release of PGE₂ from human skin equivalents. In vivo, PD-Feverfew inhibited oxazolone-induced dermatitis, and was more potent than whole Feverfew in reducing TPA-induced dermatitis. Finally the efficacy of PD-Feverfew was confirmed clinically by a reduction in erythema in a methyl nicotinate-induced vasodilation model. In conclusion, our results indicate that PD-Feverfew extracts have potent anti-inflammatory activity suggesting that this botanical would be efficacious in relieving inflammation without inducing immune sensitization.     

Induction of apoptosis in human hepatocarcinoma SMMC-7721 cells in vitro by flavonoids from Astragalus complanatus

Aim of the study

Flavonoids extracted from the seeds of Astragalus complanatus R.Br. reduce the proliferation of many cancer cells. The present study was carried out to evaluate the effects of these flavonoids from Astragalus complanatus (FAC) on human hepatocarcinoma cell viability and apoptosis and to investigate its mechanisms of action in SMMC-7721 cells.

Materials and methods

Cell viability was measured using the MTT assay. To detect apoptotic cells, SMMC-7721 cells treated with FAC were stained with Hoechst 33258 and subjected to agarose gel electrophoresis. Quantitative detection of apoptotic cells was performed by flow cytometry. The effects of FAC on apoptosis and cell cycle regulatory genes and proteins in SMMC-7721 cells were examined using an S series apoptosis and cell cycle gene array and Western blot analysis.

Results

The growth of SMMC-7721 and HepG2 cells was inhibited by treatment with FAC. Cell death induced by FAC was characterized by nuclear condensation and DNA fragmentation. Moreover, the cell cycle was arrested in the G0/G1 and S phases in FAC-treated SMMC-7721 cells. A sub-G1 peak with reduced DNA content was also formed. The activity of caspase-3 was significantly increased following FAC treatment. Microarray data indicated that the expression levels of 76 genes were changed in SMMC-7721 cells treated with FAC: 35 genes were up-regulated and 41 were down-regulated. Western blot analysis showed that caspase-3, caspase-8, Bax, P21, and P27 protein levels in SMMC-7721 cells were increased after 48 h of FAC treatment, while cyclinB1, cyclinD1, CDK1, and CDK4 protein levels were decreased.

Conclusions

These results suggest that FAC may play an important role in tumor growth suppression by inducing apoptosis in human hepatocarcinoma cells via mitochondria-dependent and death receptor-dependent apoptotic pathways.     

Water-soluble derivative of propolis mitigates scopolamine-induced learning and memory impairment in mice

The water-soluble derivative of propolis (WSDP) was prepared from fresh Chinese propolis. Its major constituents were identified by high performance liquid chromatography (HPLC) analysis. It has been reported that propolis possessed a broad spectrum of biological activities but including few studies on learning and memory by now. Thus, this study was aimed to investigate the effect of WSDP on scopolamine-induced learning and memory impairment in mice. WSDP (50 mg/kg, 100 mg/kg) was given by intragastric administration (i.g.) 40 min prior to the intraperitoneal (i.p.) injection of scopolamine (1 mg/kg).The effect on amnesia was investigated with both hidden-platform acquisition training and probe trial testing in Morris water maze test. The results from 100 mg/kg WSDP group showed significant mitigation scopolamine-induced amnesia in mice. Furthermore, WSDP's effect on the acetylcholinesterase (AChE) activity in the cerebral cortex and hippocampus was also assayed. As a result, WSDP (100 mg/kg) significantly inhibited AChE activity in the hippocampus of scopolamine-treated mice. These results indicated that WSDP may mitigate amnesia in vivo through inhibition of AChE activity in the hippocampus, which suggested propolis may have potential as a pharmaceutical of brain protection with elderly population for preventing Alzheimer's disease (AD) and other neurodegenerative diseases.     

Bioassay-guided isolation of anti-inflammatory and antinociceptive principles from a folk remedy, Rhododendron ponticum L. leaves

Ethnopharmacological relevance: Rhododendron ponticum L. (Ericaceae) is used for the treatment of inflammatory diseases and to alleviate rheumatic pain and against toothache in Turkish traditional medicine.     

Galangin increases the cytotoxic activity of imatinib mesylate in imatinib-sensitive and imatinib-resistant Bcr-Abl expressing leukemia cells

Resistance to imatinib mesylate is an emergent problem in the treatment of Bcr-Abl expressing myelogenous leukemias and additional therapeutic strategies are required. We observed that galangin, a non-toxic, naturally occurring flavonoid was effective as anti-proliferative, and apoptotic agent in Bcr-Abl expressing K562 and KCL22 cells and in imatinib mesylate resistant K562-R and KCL22-R cells. Galangin induced an arrest of cells in G0-G1phase of cell cycle and a decrease in pRb, cdk4, cdk1, cycline B levels; moreover, it was able to induce a monocytic differentiation of leukemic Bcr-Abl+ cells. Of note, galangin caused a decrease in Bcl-2 levels and markedly increased the apoptotic activity of imatinib both in sensitive or imatinib-resistant Bcr-Abl+ cell lines. In contrast, flavonoids unable to modify the Bcl-2 intracellular levels, such as fisetin and chrysin, did not increase the apoptotic effect of imatinib. These data suggest that galangin is an interesting candidate for a combination therapy in the treatment of imatinib-resistant leukemias.