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.

[Analysis on constituents of traditional Chinese medicine galamina].

OBJECTIVE: To analyze constituents of traditional Chinese medicine galamina, and provide basis for the quality control of galamina. METHOD: Polarization microscope and electron probe were adopted for a qualitative and quantitative analysis on microscopic appearance and micro-area constituents of galamina. Fourty-two elements contained in galamina were determined by the plasma mass-spectrometric method. Zinc oxide, the major constituent of galamina samples, was detected using the pharmacopeia method. RESULT: The micro-area analysis on galamina samples showed that galamina is mainly constituted by various mineral particulates such as hydrozincite, smithsonite, zinc oxide and dolomite in a loose texture. It is also found that there are many counterfeit galamina sold in the market, marked by a high lead content in natural galamina samples. CONCLUSION: Galamina is a mineral aggregation of multiple natural mineral substances, but those sold in the market has many quality problems. Therefore, a fast method shall be established to identify the authenticity of galamina and a standard shall be set to control harmful elements contained in galamina. It is suggested to conduct in-depth studies on excessive harmful elements contained in galamina medicinal materials.

[GC-MS analysis on volatile components of wild Trogopterus faeces from Laishui county of Hebei province].

OBJECTIVE: To analysis the volatile components of Trogopterus faeces. METHOD: The volatile components of Trogopterus faeces was extracted by steam distillation and analyzed by GC-MS. RESULT: Eighty-five volatile components were identified and the main components are cedrol (12.31%) and caryophyllene (7.5%). CONCLUSION: The volatile components of the samples are originated from the ingredients or other chemical compositions with similar basic structure of the fodders.

[Comparative research on pharmacognostic characteristics and microscopic characteristics of Radix Paeoniae Rubra from different areas].

OBJECTIVE: To compare pharmacognostic characteristics and microscopic characteristics of Radix Paeoniae Rubra (chishao) from different areas. METHOD: Pharmacognostic characteristics and microscopic characteristics of Radix Paeoniae Rubra were compared by microscope count methods. RESULT: Chishao in duolun was more straighter and longer, cortex with a set of closely spaced rill, peel off easily, pink section, etc. The wild chishao were different from the cultivated chishao on pharmacognostic characteristics and microscopic characteristics, such as appearance shape, smell, vessel arrangement, and number of crystal and starch in unit area. CONCLUSION: Chishao in duolun were different form others, appearance shape, wood fiber, difference of appearance shape, vessel arrangement, and number of crystal and starch in unit area can be used as identificatin feature of the wild chishao and the cultivated chishao.

[Shapes, properties and microstructures of gadfly].

OBJECTIVE: To search and identify the microstructure of medicinal properties of six kinds of gadfly. METHOD: The medicinal properties, different body parts and bristles microscopic characteristics were compared. RESULT: The two-spotted yellow gadfly had a smaller size. Its length no more than 14 mm, body wall debris was yellow brown or pale yellow, the seta was smaller than that of the other gadflies; while the other five varieties' body lengths all exceeded 15 mm, and their body wall debris mostly were brown black or gray and black. CONCLUSION: The authentication between the two-spotted yellow gadfly and the other five varieties can be made by the microscopic characteristics of their medicinal properties, body wall debris colors, bristle lengths, and basal diameters.

[Determination of prim-O-glucosylcimifugin and 5-O-methylvisammisoide in Saposhnikovia divaricata and HPLC fingerprint analysis].

OBJECTIVE: To develop HPLC methods for the determination of prim-O-glucosylcimifugin and 5-O-methylvisammisoide in Saposhnicovia divaricata and of HPLC fingerprint to compare the wild and culture varieties. METHOD: Conditions of determination: Shimadzu C18 column (4.6 mm x 150 mm, 5 microm), methanol-water (40:60) as mobile phase with the flow rate of 1 mL x min(-1). The detection wavelength was 254 nm. Conditions of HPLC fingerprint: MG II C18 column (4.6 mm x 250 mm, 5 microm), the mobile phase was acetonitrile-water with the flow rate of 1 mL x min(-1), using linear gradient elution, the column temperature was 30 degrees C. RESULT: The average recovery of prim-O-glucosylcimifugin was 99.6% (RSD 0.72%, n=6). The average recovery of 5-O-methylvisammisoide was 102.6% (RSD 0.88%, n=6). The contents of prim-o-glucosylcimifugin in wild and culture varieties were (4.96 +/- 2.59) and (3.61 +/- 1.82) mg x g(-1) respectively. The contents of 5-O-methylvisammisoide were (3.91 +/- 2.09) and (4.37 +/- 2.02) mg x g(-1) respectively. The compositions of S. divaricata were effective separated under the conditions of HPLC fingerprint. CONCLUSION: The HPLC determination method of prim-O-glucosylcimifugin and 5-O-methylvisammisoide is convenient and accurate. The HPLC fingerprint analysis method could be a basis for quality control and classification evaluate of S. divaricata.

[Differences in shapes and properties and microscopic frameworks of wild and cultivated Radix Salviae Miltiorrhizae from different regions].

OBJECTIVE: To compare the differences in shapes and properties and the microscopic frameworks of the wild and cultivated Radix Salviae Miltiorrhizae from different regions. METHOD: The differences in the shapes and properties, the characters of transverse sections, the powder and disintegrated tissue of roots were compared using microscopic measurement and statistics analysis. RESULT: The wild Radix Salviae Miltiorrhizae had several long cylinder roots with rough flaky squama skin and brown red or wine culour, the cultivated had root of many branch with cling skin and brick-red or chestnut culour. The difference of microscopic histological structure was that the xylem vessel of wild Radix Salviae Miltiorrhizae had bunched vessel with the rank form of big diameter alternating with small diameter, and had stone cell on samples from some producing region, the xylem vessel of the cultivated had no bunched vessel and no stone cell with the rank form of tangential radial. Radix Salviae Miltiorrhizae cultivated in Sichuan Province is called original-region medicinal materials and named Chuandanshen. Chuandanshen had the differences with the Salviae Miltiorrhizae Radix cultivated in other region. The root of Chuandanshen had 1.2 cm diameter, and was bulky and fat with solid fabric and the fracture with brownish yellow color and cutin-alikeness, its xylem vessel of transverse section of root was thin with the rank form of tangential radial, and 19-24 vascular bundle and a few wood fiber. CONCLUSION: Salviae Miltiorrhizae Radix of the wild and the cultivated, of the original-region (Chuandanshen) and the other-region, have the differences in the shapes and properties, and the microscopic frameworks. The character can be identified by the differences in the shapes of medicinal materials, and the rank form of vascular bundle of transverse section of root.

[Study on quality standard of Rhizoma Diosoreae and Rhizoma Dioscorea stir-baked with bran].

OBJECTIVE: To perfect the current standard of Rhizoma Diosoreae and Rhizoma Diosoreae stir-baked with bran by improving quality standards of the two processed pieces. METHOD: The quality standards were established according to 9 batches of processed pieces, separately. The standards contains items of identification, water, total ash, acid-insoluble ash, extractives, heavy metals limit, organochlorine limit, microbial limit and assay. RESULT: The TLC of the two pieces was characteristed. The contents of acid-insoluble ash in the two pieces were increased, not more than 0.5%, 0.3%, respectively. The content limits of five kinds of heavy metals and harmful elements, two kinds of residual organochlorine pesticides and three microbial limits were increased. There were no more than 2 x 10(-7) of lead, 2 x 10(-7) of cadmium, 1 x 10(-5) of copper, 3 x 10(-7) of arsenic, 1 x 10(-7) of mercury, 1 x 10(-7) of hexachlorocyclohexane (BHC) and 1 x 10(-7) of chlorophenothane (DDT) in the two processed pieces, respectively. There were no more than 2 000 and 600 cfu x mL(-1) in the two pieces, respectively and no more than 30 MPN x 100 g(-1) and fungi can not be tested in the two pieces. The contents of allantoin in the two pieces were no more than 0.15%. CONCLUSION: This method is simple and suitable for the quality control of the two processed pieces.

[Processing technics of Rhizoma Dioscoreae stired by wheat bran].

OBJECTIVE: To study the processing technics of prepared slice of Rhizoma Dioscoreae for its industrial production. METHOD: The effect factors, such as stiring temperature (60, 80, 100 degrees C), stiring time (10, 20, 30 min) and the proportion of pieces and wheat bran (100: 5, 100: 10, 100: 15) were evaluated by orthogonal method. The content of allantoin in Rhizoma Dioscoreae was determine by HPLC method. RESULT: The amount of wheat bran could affect the content of allation significantly, and stiring temperature and stiring time almost had no effect on content of allantoin. The processing technics was tested by industrial produce, according to 11 batches from 4 growing regions. The result showed that the quality of industrial product was stable. CONCLUSION: That the amount of wheat bran can affect the quality of Rhizoma Dioscoreae greatly. The determined technics is simple and suitable for prepared slice of Rhizoma Dioscoreae industrival manufacture.

[Difference of shapes and propertiesand microscopic frameworks between wild and cultivated Radix Saposhnikovia].

OBJECTIVE: To find the difference of the shapes and properties and the microscopic frameworks between wild and cultivated Radix Saposhnikovia. METHOD: The shapes and properties, the characters of transverse section, the powder and disintegrated tissue of roots of medical materials were compared by microscopic measuring. RESULT: Wild Radix Saposhnikovia had a long conical or cylindrical root, and fewer root branches. It showed a close annulus grain on top root, cortical section of root in light brown colour, many brown oil spots and possessed typical odor, While cultivated Radix Saposhnikovia had many root branches, and showed less annulus grain on top root, cortical section of root in light yellow brown colour, less brown oil spots and possessed light odor. The difference of microscopic histological structure was that wild Radix Saposhnikovia had phloem transverse section of root with many rotundity oil tube lining up 10-22 rings, xylem vessel with radiate rank, and indistinct annual ring. While cultivated Radix Saposhnikovia had phloem transverse section of root with oil tube lining up 10-11 rings and xylem vessel with distinct annual ring. CONCLUSION: There exists several differences between wild and cultivated Radix Saposhnikovia in shapes and properties and differences of microscopic frameworks. The main characteristics are the differences of shapes and numbers of oil tube of phloem transverse section of root. The cultivated Radix Saposhnikovia of 1-4 years can be recognized by annual rings of xylem vessel.

[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.

Rapid determination of aristolochic acid I and II in Aristolochia plants from different regions by beta-cyclodextrin-modified capillary zone electrophoresis.

Chinese herbs nephropathy (CHN) is a kind of severe kidney disease caused by excessively taking aristolochic acid (AA). Hence, it is essential for health security and quality control of related herbal medicines to develop an efficient method for separation and determination of these two important components in Traditional Chinese Medicines. In this study, a rapid capillary zone electrophoresis (CZE) method using 120 mM sodium borate buffer containing 10 mM beta-cyclodextrin (beta-CD) as modifier was firstly developed for the analysis of AA-I and AA-II within 4min in some medicinal plant samples. The separation conditions including pH of running buffer, CD content in the buffer system, applied voltage and capillary temperature were systematically optimized, and two kinds of aristolochic acids in 37 herbal samples of Aristolochia plants were successfully determined with high separation efficiency, satisfactory sensitivity, repeatability and recovery. The result indicated high variability in the contents of aristolochic acids due to different species and regions. The comparison of CZE method with high performance liquid chromatography (HPLC) was also discussed.

[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.