Polyherbal Formulation Enhancing Cerebral Slow Waves in Sleeping Rats.

Polyherbal medicines are composed of multiple herbs and have traditionally been used in East Asian countries for the remedy of physiological symptoms. Although the effects of polyherbal formulations have been investigated at the molecular and behavioral levels, less is known about whether and how medicinal herbs affect the central nervous system in terms of neurophysiology. We introduced a novel blended herbal formulation that consisted of 35% linden, 21% mulberry, 20% lavandin, 20% butterfly pea, and 4% tulsi. After intraperitoneal administration of this formulation or saline, we simultaneously recorded epidural electrocorticograms (ECoGs) from the olfactory bulb (OB), primary somatosensory cortex (S1), and primary motor cortex (M1), along with electromyograms (EMGs) and electrocardiograms (ECGs), of rats exploring an open field arena. Using the EMGs and OB ECoGs, we segmented the behavioral states of rats into active awake, quiet awake, and sleeping states. Compared to saline, herbal medicine significantly shortened the total sleep time. Moreover, we converted the ECoG signal into a frequency domain using a fast Fourier transform (FFT) and calculated the powers at various ECoG oscillation frequencies. In the sleeping state, a slow component (0.5-3 Hz) of S1 ECoGs was significantly enhanced following the administration of the formulation, which suggests a region- and frequency-specific modulation of extracellular field oscillations by the polyherbal medicine.

Multistep Analysis of Diol-LC-ESI-HRMS Data Reveals Proanthocyanidin Composition of Complex Plant Extracts (PAComics).

Proanthocyanidins (PACs) are complex oligomeric or polymeric phenolic biopolymers composed of flavan-3-ol building blocks. PACs exert manifold functional bioactivities and are assessed as bioactive ingredients in a variety of food products, beverages, medicinal plants, and phytopharmaceuticals. Although analytical methods for PACs with low degree of polymerization (DP) are well established, a lack of methods for the detailed analysis of higher oligomers and polymers from complex plant extracts is obvious. For this, the present study investigated PAC-enriched extracts from four different plants, traditionally used for medical purpose (Lime flower, Hawthorn leaf and flower, Japanese Wisteria fruits without seeds, and Common Sorrel herb). PACs were separated on diol stationary-phase high-performance liquid chromatography according to the respective DP and detected by fluorescence and quadrupole time-of-flight mass spectrometry (qTOF-MS). The qTOF-MS contour plots [tR → m/z] provided a sufficient overview on the respective PAC distribution. Subsequently, high-resolution mass spectrometry data were used for Kendrick mass defect (KMD) analysis, with (epi)catechin, the main flavan-3-ol unit in PACs, as the reference unit. The resulting KMD plots enabled an elucidation of the general polymer chain composition with regard to DP, building blocks, and potential secondary modifications (e.g., galloylation). Subsequently, analysis of MS2 fragmentation patterns of PAC oligomers confirmed the structural features obtained from the KMD plots. While Lime flower contained oligomeric A- and B-type PACs, composed of (epi)catechin and (epi)afzelechin, Japanese Wisteria fruit contained PACs consisting of three different hydroxylated flavan-3-ols. Cinchonains, A-type PACs, and B-type PACs were detected in the Hawthorn plant material. Galloylated oligo- and polymeric PACs were detected in Common Sorrel herb. This multistep analysis reveals collective insights into the PAC composition of the extracts. The protocol offers a fast and reliable methodology to be used in a standard laboratory. On the other hand, this methodology reaches its limits for higher oligomeric PACs, and further optimization is necessary for a better detection of the polymers, as the optimal DP cluster detection depends on the resolution of diol stationary-phase chromatography and is therefore limited.

Assessment of anti-coagulant activity of Nelumbo nucifera fruit.

The herbal agents rich in flavonoids are progressively becoming popular these days as they are considered to have anti-oxidant effects and also lack bothersome effects. That's why existing research was encircled around the anti-coagulant effect of Nelumbo nucifera fruit (NNF) as it is also a rich source of flavonoids and cultivated in abundance especially in tropical regions of Asia but its usefulness as anti-coagulant agent was never determined pharmacologically. Anticoagulant assessment was done in thirty five male Wister rats which were separated equally in 5 groups. Results of the current study revealed that NNF 200mg/kg significantly prolonged prothrombin time and thrombin time, whereas fibrinogen level was highly significantly reduced as compared to control. Fibrinogen level was also reduced highly significantly with NNF 100 mg/kg as compared to control without affecting other parameters of coagulation i.e. activated partial thromboplastin time, prothrombin time and thrombin time. NNF exhibited strong anti-coagulant activity which may be due to the inhibitory effects on platelet activation, adhesion and aggregation along with inhibitory effects on thromboxane A2 formation. Presence of alkaloid i.e. neferine and flavonoids in it may be a reason of its anti-coagulant activity but more pre-clinical and clinical evaluation needs to be conducted to establish these findings.

Phytochemical analysis of non-polar solvent extracts of the Wisteria sinensis leaves.

A comparative study on the phytochemical composition of the n-hexane and chloroform extracts from Wisteria sinensis leaves collected in June and October is described. Continuous extraction in Soxhlet apparatus, as well as ultrasound-assisted technique, was used for the preparation of the extracts. All the extracts were analysed by GC/MS method. As a result, α-tocopherol was identified as the main component (56%) of the extracts from October leaves, whereas, ß-sitosterol was identified as the main compound (47%) in the extracts from the June leaves. Additionally, pure α-tocopherol was isolated from n-hexane extract of the October leaves using column chromatography. A total of 6.25 mg of α-tocopherol was isolated from 1 g of dried leaves. The presence of the vitamin E in extracts from W. sinensis leaves is described here for the first time.

Quantitative flavonoid variation accompanied by change of flower colors in Edgeworthia chrysantha, Pittosporum tobira and Wisteria floribunda.

The flavonoids in the flowers of Edgeworthia chrysantha, Pittosporum tobira and Wisteria floribunda were isolated and identified. Quercetin and kaempferol 3-O-glucosides and 3-O-rutinosides were found in E. chrysantha, and quercetin 3-O-rutinoside, 3-O-glucoside and 3-O-pentosylrhamnosylglucoside, kaempferol 3-O-robinobioside, 3-O-rutinoside, 3-O-glucoside and 3-O-pentosylrhamnosylglucoside, and isorhamnetin 3-O-rutinoside were isolated from P. tobira. Ten flavonoids, quercetin 3-O-sophoroside, 3-O-rutinoside, 3-O-glucoside, kaempferol 3-O-sophoroside and 3-O-glucoside, luteolin 5-O-glucoside, 7- O-glucoside and 7-O-hexoside, and apigenin 7-O-glucoside and 4'-O-hexoside were isolated from W floribunda. The major pigments of E. chrysantha were carotenoids. Their content decreased with the change in flower color to white from yellow via cream, and total flavonoid content also slightly decreased by ca. 0.8 in cream and ca. 0.9 fold in white flowers. In contrast with E. chrysantha, white flowers of P. tobira turn to cream and then yellow in which the major pigments are also carotenoids. In this species, both carotenoid and flavonoid contents are gradually increased from white to yellow flowers. Though the petal color of Wisteria floribunda is mauve, due to anthocyanin pigments, the yellow areas are due to carotenoids; these turn to white in the late flowering stage. However, their flavonoid contents were essentially the same among the yellow, cream and white spots of flags. Thus, it was shown by HPLC analysis of the flower flavonoids of E. chrysantha, P. tobira and W. floribunda, although the visible pigments such as carotenoids and anthocyanins are quantitatively varied, the quantitative variation in UV-absorbing substances, such as flavones and flavonols, differs with plant species.

Starmerella caucasica sp. nov., a novel anamorphic yeast species isolated from flowers in the Caucasus.

Taxonomic analysis of budding yeast strains isolated from flowers of Wisteria sinensis (Fabales, Fabaceae) abundantly visited by flying insects, mainly bees in city parks of Baku is described. The isolates forming slightly pink colonies and propagating by budding represent a hitherto unknown yeast species for which the name Starmerella caucasica is proposed. The sequences of the D1/D2 domains of the large subunit rRNA genes and the ITS1-5.8S-ITS2 regions were highly similar in the isolates and indicated a close relationship with Candida kuoi and Starmerella bombicola in the phylogenetic analysis. S. caucasica can be separated from these species by its growth on glucosamine and D-tryptophan, in vitamin-free medium and at 37°C, and its inability to grow on citrate, ethylamine, cadaverine and in media supplemented with 0.01% of cycloheximide. The type strain is 11-1071.1(T). It has been deposited in Centralbureau voor Schimmelcultures (Utrecht, the Netherlands) as CBS 12650(T), the National Collection of Agricultural and Industrial Microorganisms (Budapest, Hungary) as NCAIM Y.02030(T) and the Culture Collection of Yeasts (Bratislava, Slovakia) as CCY 90-1-1(T). The GenBank accession numbers for nucleotide sequences of S. caucasica are JX112043 (D1/D2 domain of the 26S rRNA gene) and JX112044 (ITS1-5.8S-ITS2). Mycobank: MB 800536.

Diversity of rhizobia associated with leguminous trees growing in South Korea.

This study was carried out to examine the diversity of 34 isolates collected from 11 species of leguminous trees growing in South Korea. Phylogenetic relationships between these 34 isolates and reference strains of the Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium and Ensifer/Sinorhizobium were analysed by using 16S rRNA gene sequences. Twenty-one isolates were related to Mesorhizobium, four isolates to Rhizobium, and nine isolates to Bradyrhizobium. But none of isolates were related to Sinorhizobium/Ensifer and Azorhizobium. Robinia pseudoacacia and Amorpha fruticosa were nodulated by various genotypes of rhizobia out of them, most of the isolates belonged to the genus Mesorhizobium. The isolates from Lespedeza bicolar belonged to diverse genera of Mesorhizobium, Rhizobium, and Bradyrhizobium. The isolates from Maackia amurensis and Lespedeza maximowiezii var. tomentella were phylogenetically related to the genera of Bradyrhizobium. PCR-based RAPD method and phylogenetic analysis of the 16S rRNA results revealed a high phylogenetic diversity of rhizobial strains nodulating leguminous trees in South Korea. Also, the relationships between host and bacterial phylogenies showed that only Robinia pseudoacacia, and Wisteria floribunda have significantly unique branch length than expected by chance based on phylogenetic tree.

Fever, sickness behavior, and expression of inflammatory genes in the hypothalamus after systemic and localized subcutaneous stimulation of rats with the Toll-like receptor 7 agonist imiquimod.

The Toll-like receptor 7 (TLR7) agonist imiquimod is used for topical treatment of skin cancers. We studied the consequences of injections of imiquimod into a subcutaneous (s.c.) air pouch or of intraperitoneal (i.p.) injections on the manifestation of fever, sickness behavior, and the peripheral and brain-intrinsic induction of a variety of inflammatory molecules. Rats were given imiqimod s.c. or i.p. (1 or 5 mg/kg). Body temperature, motor activity, and food and water intake were recorded by telemetric devices. Peripheral and brain-intrinsic induction of inflammatory mediators was analyzed by real-time polymerase chain reaction (RT-PCR), bioassays, enzyme-linked immunosorbent assays (ELISAs), and immunohistochemistry. Imiquimod is the first TLR-agonist to produce more potent effects with s.c. than i.p. administration. Peripheral induction of interferons (IFNs) and putative circulating pyrogens corresponded to the magnitude of the illness responses. In the brain, an expression of cytokines (TNFα, IL-1ß, and IL-6) and inducible forms of enzymes for prostaglandin E2 synthesis (COX-2 and mPGES) occurred, which was accompanied by a moderate activation of the transcription factors NFκB and STAT3, and a strong activation of the transcription factor NF-IL6, in cells of specific areas with an open blood-brain barrier. These inflammatory responses noted within the brain were more marked after s.c. administration, than i.p. administration of imiquimod. At a dose of 5 mg/kg, imiquimod causes rather moderate brain-inflammatory responses, which are related to peripheral IFN-expression and possibly mediated by brain-intrinsic activation of NF-IL6 and induction of a proinflammatory cocktail. The lack of a septic-like state in imiquimod-treated rats reinforces the therapeutic use of this drug.

Wisteria floribunda gall extract inhibits cell migration in mouse B16F1 melanoma cells by regulating CD44 expression and GTP-RhoA activity.

Extracts from galls grown on Wisteria floribunda are used as an anti-tumoral preparation in oriental traditional medicine. Here, we investigated the molecular mechanism of this anti-tumoral effect by first examining whether the extract inhibited cell migration in a B16 cell-based wound healing assay. The gall extract delayed wound healing in a dose- and time-dependent manner, indicating that one or more components of the fraction inhibited cell migration. Examination of two molecules known to be involved in metastasis, CD44, and RhoA-GTP, revealed that the gall extract decreased CD44 expression in a concentration-dependent manner, and also increased RhoA-GTP activity in comparison to untreated controls. Taken together, these results suggest that the Wisteria gall extract may inhibit cancer cell migration via inhibition of CD44 mRNA expression and activation of the GTP-RhoA protein.

Potential cytoplasmic inheritance in Wisteria sinensis and Robinia pseudoacacia (Leguminosae).

We examined pollen cells of Wisteria sinensis and Robinia pseudoacacia (Leguminosae) to determine a possible mode for cytoplasmic inheritance in these species. Epifluorescence microscopy revealed distinct mature generative cells. Mature generative cells of W. sinensis were associated with large numbers of punctuated fluorescent signals corresponding to cytoplasmic DNA aggregates, but no fluorescent signals were observed in the generative cells of R. pseudoacacia. Closer examination showed that the punctate fluorescent signals corresponded to plastid but not mitochondrial DNA. These results suggest a strong potential for paternal transmission of the plastid genome in W. sinensis. Electron microscopy confirmed the presence of plastids in the generative cells of W. sinensis and the absence of plastids in R. pseudoacacia cells due to an unequal distribution of plastids during the first pollen mitosis. Mitochondria were present and intact in the mature generative cells of both species. The lack of fluoresced mitochondrial DNA suggests a very low level of mitochondrial DNA in the cells. Immunoelectron microscopy demonstrated that the labeling of mitochondrial DNA in these cells was reduced by nearly 90% during pollen development. Such a dramatic reduction suggests an active degradation of paternal mitochondrial DNA, which may contribute greatly to the maternal inheritance of mitochondria. In short, we found that W. sinensis exhibits a strong potential for paternal transmission of plastids and that both W. sinensis and R. pseudoacacia appear to share the same mechanism for maternal mitochondrial inheritance.

[Relationships between light and physiological characters of five climbing plants].

Studies on the photosynthetic and respiratory rates, light utilization efficiencies and light compensations of five climbing plants showed that the diurnal variations of photosynthetic rates presented double peak, the first peak was between 10:00 to 12:00, and the second was between 14:00 to 16:00. The phenomenon of "noon break" was obvious. The diurnal variations of respiration rates also presented double peak, the first peak was between 11:00 to 13:00, and the second was between 14:00 to 17:00. The light compensation point of Hedera nepatensis, H. helix, Parthenocissus tricuspidata, P. quinuefolia and Wisteria sinensis was 5.73, 5.07, 9.96, 6.40 and 18.93 micromol x m(-2) x s(-1), respectively, and the light utilization efficiency of W. sinensis was higher under strong light, P. quinuefolia was the second, but that of H. helix was higher under weak light. The results showed that Wisteria sinensis was a typical heliophytic plant, Parthenocissus tricuspidata and P. quinuefolia were neuter plants, and Hedera nepalensis and H. helix were typical sciophytic plants.