Tetramethylpyrazine promotes stroke recovery by inducing the restoration of neurovascular unit and transformation of A1/A2 reactive astrocytes.

2,3,5,6-Tetramethylpyrazine (TMP) as an active ingredient extracted from a traditional Chinese herbal medicine Ligusticum chuanxiong Hort. has been proved to penetrate blood-brain barrier (BBB) and show neuroprotective effects on cerebral ischemia. However, whether TMP could regulate astrocytic reactivity to facilitate neurovascular restoration in the subacute ischemic stroke needs to be urgently verified. In this research, permanent occlusion of the middle cerebral artery (MCAO) model was conducted and TMP (10, 20, 40 mg/kg) was intraperitoneally administrated to rats once daily for 2 weeks. Neurological function was evaluated by motor deficit score (MDS). Magnetic resonance imaging (MRI) was implemented to analyze tissue injury and cerebral blood flow (CBF). Magnetic resonance angiography (MRA) was applied to exhibit vascular signals. Transmission electron microscopy (TEM) was performed to detect the neurovascular unit (NVU) ultrastructure. Haematoxylin and eosin (HE) staining was utilized to evaluate cerebral histopathological lesions. The neurogenesis, angiogenesis, A1/A2 reactivity, aquaporin 4 (AQP4) and connexin 43 (Cx43) of astrocytes were observed with immunofluorescent staining. Then FGF2/PI3K/AKT signals were measured by western blot. Findings revealed TMP ameliorated neurological functional recovery, preserved NVU integrity, and enhanced endogenous neurogenesis and angiogenesis of rats with subacute ischemia. Shifting A1 to A2 reactivity, suppressing excessive AQP4 and Cx43 expression of astrocytes, and activating FGF2/PI3K/AKT pathway might be potential mechanisms of promoting neurovascular restoration with TMP after ischemic stroke.

Effect of Neurorepair for Motor Functional Recovery Enhanced by Total Saponins From Trillium tschonoskii Maxim. Treatment in a Rat Model of Focal Ischemia.

Trillium tschonoskii Maxim. (TTM), is a perennial herb from Liliaceae, that has been widely used as a traditional Chinese medicine treating cephalgia and traumatic hemorrhage. The present work was designed to investigate whether the total saponins from Trillium tschonoskii Maxim. (TSTT) would promote brain remodeling and improve gait impairment in the chronic phase of ischemic stroke. A focal ischemic model of male Sprague-Dawley (SD) rats was established by permanent middle cerebral artery occlusion (MCAO). Six hours later, rats were intragastrically treated with TSTT (120, 60, and 30 mg/kg) and once daily up to day 30. The gait changes were assessed by the CatWalk-automated gait analysis system. The brain tissues injuries, cerebral perfusion and changes of axonal microstructures were detected by multimodal magnetic resonance imaging (MRI), followed by histological examinations. The axonal regeneration related signaling pathways including phosphatidylinositol 3-kinases (PI3K)/protein kinase B (AKT)/glycogen synthase kinase-3 (GSK-3)/collapsin response mediator protein-2 (CRMP-2) were measured by western blotting. TSTT treatment significantly improved gait impairment of rats. MRI analysis revealed that TSTT alleviated tissues injuries, significantly improved cerebral blood flow (CBF), enhanced microstructural integrity of axon and myelin sheath in the ipsilesional sensorimotor cortex and internal capsule. In parallel to MRI findings, TSTT preserved myelinated axons and promoted oligodendrogenesis. Specifically, TSTT interventions markedly up-regulated expression of phosphorylated GSK-3, accompanied by increased expression of phosphorylated PI3K, AKT, but reduced phosphorylated CRMP-2 expression. Taken together, our results suggested that TSTT facilitated brain remodeling. This correlated with improving CBF, encouraging reorganization of axonal microstructure, promoting oligodendrogenesis and activating PI3K/AKT/GSK-3/CRMP-2 signaling, thereby improving poststroke gait impairments.

Trillium tschonoskii rhizomes' saponins induces oligodendrogenesis and axonal reorganization for ischemic stroke recovery in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Trillium tschonoskii Maxim. is one of traditional Chinese medical herbs that has been utilized to treat brain damages and cephalalgia. The neuroprotective effect of total saponins from Trillium tschonoskii rhizome (TSTT) has been demonstrated efficacy in rats following ischemia. However, the axonal remodeling effect of TSTT and the detailed mechanisms after ischemic stroke have not been investigated. AIM OF THE STUDY: We aimed to estimate therapeutic role of TSTT in axonal remodeling using magnetic resonance imaging (MRI) technique, and explored possible mechanisms underlying this process followed by histological assays in ischemic rats. METHODS: Male Sprague-Dawley (SD) rats underwent permanently focal cerebral ischemia induced by occluding right permanent middle cerebral artery. TSTT was intragastrically administrated 6 h after surgery and once daily for consecutive 15 days. Neurological function was assessed by the motor deficit score and beam walking test. T2 relaxation mapping and diffusion tensor imaging (DTI) were applied for detecting cerebral tissues damages and microstructural integrity of axons. Luxol fast blue (LFB) and transmission electron microscope (TEM) were performed to evaluate histopathology in myelinated axons. Double immunofluorescent staining was conducted to assess oligodendrogenesis. Furthermore, the protein expressions regarding to axonal remodeling related signaling pathways were detected by Western blot assays. RESULTS: TSTT treatment (65, 33 mg/kg) markedly improved motor function after ischemic stroke. T2 mapping MRI demonstrated that TSTT decreased lesion volumes, and DTI further confirmed that TSTT preserved axonal microstructure of the sensorimotor cortex and internal capsule. Meanwhile, diffusion tensor tractography (DTT) showed that TSTT elevated correspondent density and length of fiber in the internal capsule. These MRI measurements were confirmed by histological examinations. Notably, TSTT significantly increased Ki67/NG2, Ki67/CNPase double-labeled cells along the boundary zone of ischemic cortex and striatum. Meanwhile, TSTT treatment up-regulated the phosphorylation level of Ser 9 in GSK-3ß, and down-regulated phosphorylated ß-catenin and CRMP-2 expression. CONCLUSION: Taken together, our findings indicated that TSTT (65, 33 mg/kg) enhanced post-stroke functional recovery, amplified endogenous oligodendrogenesis and promoted axonal regeneration. The beneficial role of TSTT might be correlated with GSK-3/ß-catenin/CRMP-2 modulating axonal reorganization after ischemic stroke.

[Morphological and microscopical identification of Genkwa Ramulus and its adulterants].

The purpose of this article is to identify Daphne genkwa and its adulterants, Wikstroemia chamaedaphne, according to the morphological and microstructure characteristics of their stem and foliage. The root of D.genkwa was studied simultaneously. The results indicated that the crude drug and processed pieces of Genkwa Ramulus were mainly composed of stems and branches where obvious opposite petiole scars and branch marks were able to be seen on their nodes. Otherwise, foliage or peduncles generally couldn't be found. Moreover, the fine silver flocculent fibers could be observed in the bark of fracture surface. The adulterants were the plant segments which were composed of stems, foliage and peduncles with spikelet-pedicel scars. There existed microstructures differences between Genkwa Ramulus and its adulterants. In the former, single thick lignified phloem fibers were interspersed in the stem phloem of the transverse section with very thick wall and unicellular non-glandular hairs could be observed on the lower epidermis of foliage. Nevertheless, in the latter, there was no thick lignified phloem fibers in cross section of stem phloem, the outer wall of epidermal cells of foliage hadthick cuticles and no non-glandular hairs in lower epidermis of foliage. The results can be used for the identification and the quality standard of the crude drug and processed pieces of D.genkwa.The characteristics of the microstructures and the transverse section can be used to identify the radix D.genkwa.

[Research on quality standards of Euonymus fortunei].

To establish quality standards of Euonymus fortunei, and supply scientific evidence for the quality control of Euonymus fortunei. Empirical and microscopic identification methods were adopted to observe morphological and histological characters. The contents of water, total ash, acid-insoluble ash and alcohol-soluble extractive were analysed according to the methods of Chinese Pharmaco- poeia (2010). Dulcitol and reference herbs were used to identify materia medica of Euonymus fortunei by TLC method. The total flavonol glycosides contents were analysed by HPLC method, using quercetin and kaempferol as reference substances. Quercetin and kaempferol were separated on a C18 column (4.6 mm x 250 mm, 5 µm) with methanol-0.1% formic acid(51:49) as the mobile phase and detected at 366 nm. The flavonoid aglycones content was then multiplied by a conversion coefficient, and the result was the total flavonol glycosides content. The macroscopical identification, microscopic features and TLC methods were proper. The average contents of water, total ash, acid-insoluble ash, alcohol-soluble extractive and total flavonol glycosides were 8.76%, 6.48%, 0.31%, 17.48% and 0.211% , respectively. The quality standards established on the basis of the research results were suitable for the quality evaluation of Euonymus fortunei.

[Comparative study on identification of Clematidis Radix et Rhizoma from different origins based on microstructure and NIRS method].

Microscopic identification and NIRS methods were applied to identify Clematidis Radix et Rhizoma of two different origins. The results showed that both methods could identify the samples. NIRS could identify the two samples nondestructively, and provides a basis for establishment of a standard herbs radix clematidis NIRS fingerprint in the future.

[Comparative study on chemcial composition between cultivated and wild Scutellariae radix].

OBJECTIVE: To do some comparative study on chemical composition between the cultivated and wild samples of Radix Scutellariae. METHOD: Thirty three samples of Radix Scutellariae were collected from different habitats. An HPLC method for fingerprint identification and simultaneous determination of baicalin, baicalein and wogonin was established, and the content of ethanolic extractive was measured according to the method in Chinese Pharmacopoeia. RESULT: The number of chromatographic peaks in fingerprint of wild sample was more than that of cultivated sample in Hebei and Inner Mongolia. In 95% confidence intervals, the contents of baicalin, baicalein and wogonin were (15.89 +/- 1.52)%, (1.04 +/- 0.26)% and (0.27 +/- 0.07)%, respectively in the cultivated samples and (11.93 +/- 1.62)%, (1.03 +/- 0.26)% and (0.27 +/- 0.06)%, respectively in wild samples. The content of ethanolic extractive was 52.07 +/- 3.05% in the cultivated and (41.21 +/- 2.86)% in the wild within 95% confidence interval. CONCLUSION: Slight difference between the fingerprint of the cultivated and wild samples was found, and the contents of baicalin and ethanolic extraction in the cultivated were higher than those in the wild.

[Textual research on origin and development of genuine medicinal herbs of Shanyao].

Making textual research on Bencao and documents, this article inquires to the origin and development of genuine medicinal herbs of Shanyao (Rhizoma Dioscoreae) on the points of change of the growing areas and the development of cultivating and processing techniques and clinical uses. The study indicates that the medicinal use of Dioscorea oposita went through several periods: the period of the use of wild D. oposita before Tang dynasty, the period of the mixed use of wild and cultivating D. oposita from Song to the middle of Qing dynasty, and the period of the main use of cultivating D. oposita after the latter stage of Qing dynasty (18th century). It considers that the growing area of genuine medicinal herbs of Shanyao appeared in Ming dynasty and finally formed "Huaishanyao" on the early of 20th century. The acknowledgement of Huaishanyao as genuine medicinal herbs is related closely to its cultivating and processing techniques and clinical uses. The development of cultivating techniques provided the resource of Shanyao, the invention of processing techniques improved its appearance and quality, and the clinical uses and practices by modern and contemporary famous medical men played an important role to the social approval and development of Huaishanyao.

[Dynamic change of paeoniflorin content in root of Paeonia lactiflora growing at Duolun County, Inner Mongolia].

UNLABELLED: To explore the dynamic change of paeoniflorin content in root of Paeonia lactiflora. METHOD: The samples were collected and paeoniflorin content was determined by HPLC. RESULT: The results showed that the average content of paeoniflorin was the lowest at 3.05% in the roots collected in May, and during the fruit time it was rather high in August at 4.72%, and in September at 4.58%. The average content showed no significant differences during June to October. CONCLUSION: Wild plant of P. lactiflora might be collected for medical use during blooming stage until wilting stage.

[Determination of carvacrol and thymol in Mosla chinensis by HPLC].

OBJECTIVE: To establish a quantitative method of determination of carvacrol and thymol in Mosla chinensis. METHOD: The sample was extracted with 95% ethanol, ODS column was used with methanol-water-acetic acid (60:40:2) as mobile phase. The detection wavelength was set at 274 nm. RESULT: The linearities of carvacrol and thymol were respectively in the range of 0.23-2.15 microg (r = 0.9999) and 0.39-2.36 microg (r = 0.9999); the average recoveries were 99.9% (RSD 1.4%) and 98.6% (RSD 1.3%); the RSD of repeatability were 1.1% and 1.6%. CONCLUSION: The method is reliable, and can be used for quality control of M. chinensis.

[Quick identification of scullcaps in different geographical origins using clustering analysis method and infrared fingerprint spectra].

Based on the infrared fingerprint spectra, scullcap samples from 15 geographical origins were clustered into 6 classes with principal component analysis. The classification was related to their geographical origins and weather. In the same class, chemical components of scullcaps are similar to each other, which can be considered as the criterion of evaluating the quality of scullcaps. 43 geographical origins were predicted with radial basis function, which was demonstrated to be a powerful ANN method in discrimination. The classes of scullcaps can be distinguished by the method. The method can be used to class and identify geographical origins of scullcaps. It is one of the methods in the quality control of traditional Chinese medicines.

[Study of stinging crystals in tian nanxing].

OBJECTIVE: To test whether the raphides in Tian Nanxing (Pinellia pedatisecta Schott) caused irritation. METHOD: Scan Electron Microscope and Microscope Oberservation; Animal experimental study. RESULT: Through the comparision of unprocessed Tian Nanxing to processed ones which included 36 h, 72 h, 120 h processed samples, the great modifications in the structure of raphides, especially for the ones with barbs in the processed samples was observed with time course study. A further animal experimental study went to show that the rate of change for raphides with barbs existed a dose-reponse relationship to irritation. CONCLUSION: Investigations of the causes of these reactions showed that raphides of calcium oxalate are, at least in part, responsible for the Tian Nanxing's irritation.

[Study on the physicochemical properties of cultivated soil of genuine crude and no-enuine crude Chinese Angelica].

OBJECTIVE: To provide basis for environmental factors of genuine crude Chinese angelica. METHOD: On-the-spot investigation and indoor chemical analysis were made to study the physicochemical properties of cultivated soil of Chinese angelica. RESULT AND CONCLUSION: The physical properties and the organic matte and mineral nutrition of cultivated soil of Chinese angelica are best in Min County, Gansu Province. The ecological environment is the leading factor in forming genuine crude Chinese angelica.

[Determination of flavone for Scutellaria baicalensis from different areas by HPLC].

OBJECTIVE: To describe the difference between native and nonative herbs by determining contents of seven kinds of flavone for twenty-five samples from seventeen areas. METHODS: HPLC. Fluid phase: MEOH-H2O-CH3COOH(ICE) (41:59:0.2) and (50:50:0.2). Detection wavelength: 275. RESULTS: The contents of baicalin are 6%-9%, wogenin are 2%-8%, baicalein are 0.1%-1.6%, neobaicalein are 0.01%-0.2%, wogonin are 0.01%-0.3%, visidulin and oroxylin are trace amounts or undetected. CONCLUSION: The native and nonative herbs have no distinct differce in absolute component ratio. The ratio of baicalin and wogenin is under three. The ratio of baicalin and baicalein, baicalin and wogonin is between twenty and fifty.

[A study on genetic variation between wild and cultivated populations of Paeonia lactiflora Pall].

AIM: To discuss the genetic differentiation between wild and cultivated populations of Paeonia lactiflora Pall., and to find the reason for forming the genuineness of Radix Paeoniae rubra (Chishao) and Radix Paeoniae alba (Baishao). METHODS: Forty three representative samples of P. lactiflora from 11 localities were analyzed by RAPD method with 21 random primers. According to RAPD results, the genetic diversity and genetic differentiation of P. lactiflora were detected by the percentage of polymorphic sites (PPB), the coefficient of gene differentiation (GST) and analysis of molecular variance (AMOVA). RESULTS: (1) At species level, the PPB of P. lactiflora was 85.26%, the Nei's gene diversity (Ht) was 0.166. The PPB in wild population (WP) was 77.61%, which was more than that (54.96%) in cultivated population for medicine (MP), and that (61.76%) in cultivated population for ornament (OP). (2) AMOVA showed that 29.50% of the genetic diversity resulted from differentiation among populations. Pairwise Phist distance (0.3632) between WP and MP was furthest, while that (0.0973) between MP and OP was closest, indicating population differentiation was significant (P < 0.001). (3) In general, cluster analysis revealed that the samples of P. lactiflora were divided in wild and cultivated groups (except for 39). In WP, individuals of Duolun were separated from those of other localities. In MP, the clusters of samples corresponded well with their own habitats. CONCLUSION: In addition to environmental factor, genetic differentiation should be the main cause for the genuineness of "Chishao" and "Baishao". Because of over collection and worse habitat, P. lactiflora in Duolun, whose root is the famous Chinese Geo-herbal--"Duolun Chishao", is progresively rare. So, it should become the endangered germplasm resource to protect.