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OBJECTIVE： To study the effects of ethanol extract of Sanguis Draconis on the survival of perforating flap model in rats and PI3K/Akt/eNOS pathway. METHODS： Perforating flap model was established by cutting off surrounding vessels and keeping one perforator. After modeling， the rats were divided into model group （external use， normal saline） and ethanol extract of Sanguis Draconis （EESD， the content of dracorhodin was 75.08 mg/g） group （external use， 0.21 g/cm2）， with 10 rats in each group. They were given relevant medicine for consecutive 7 days， once a day. The flap survival rate and flap microvessel density were determined after given relevant medicine 7 days. Human umbilical vein endothelial cells （HUVECs） were reoxygenated and glycoconjugated 16 h after hypoxia and hypoglycemia to establish oxygen-glucose deprivation/oxygen-glucose recovery model of HUVECs. After modeling， model cells were divided into normal group， model group， dracorhodin high-concentration， medium- concentration and high-concentration groups （2.5， 1.0， 0.5 μg/mL）. After reoxygenated and glycoconjugated for 24 h， cells morphology was observed by microscope； cell viability and the content of NO were detected by MTT assay and colorimetry. mRNA expression of Akt， PI3K and eNOS， PI3K protein expression， the phosphorylation of Akt and eNOS protein were determined by RT-PCR and Western blot assay. RESULTS： In rat experiment， compared with model group， flap survival rate and microvessel density of rats were increased significantly in EESD group （P＜0.01）. In cell experiment， compared with normal group， the survival rate of HUVEC， NO content， mRNA expression of PI3K， Akt， eNOS，PI3K protein expression， the phosphorylation of Akt and eNOS protein were decreased significantly （P＜0.05 or P＜0.01）. Compared with model group， dracorhodin high-concentration， medium-concentration and high-concentration groups survival rate of HUVEC cells， NO content， mRNA expression of PI3K， Akt and eNOS， PI3K protein expression， the phosphorylation of Akt and eNOS protein were increased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS： The survival rate of perforating flap model in rat can be increased by treating with EESD， the mechanism of which may be associated with the activation of PI3K/Akt/eNOS pathway to protect endothelial cells.
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OBJECTIVE： To establish a method for simultaneous determination of 4 triglyceride anti-tumor components in Coix lacryma seed oil. METHODS： HPLC-ELSD was adopted. The determination was performed on Inertsil ODS-3 C18 column with mobile phase consisted of acetonitrile-isopropanol （57 ∶ 43， V/V） at the flow rate of 1.0 mL/min. The column temperature was 30 ℃， and the sample size was 10 μL. Evaporative light scattering detector was used， the drift tube temperature was 70 ℃， and the gas flow rate was 2 L/min. Using glycerol trioleateas internal standard， relative correction factors （RCF） of linolein trilinolein， 1，2-linoleic acid-3-palmitic acid glyceride and 1-palmitic acid-2-oleic acid-3-linoleic acid glyceride were calculated respectively. The contents of above 3 components in C. lacryma seed oil were calculated by RCF. The contents of 4 components in C. lacryma seed oil were determined by external standard. The results of content determination by quatitative analysis of multi-components by single marker （QAMS） were compared with external standard method. RESULTS： The linear ranges were 0.15-4.50 μg for linolein trilinolein， 0.15-4.50 μg for 1，2-linoleic acid-3-palmitate， 0.35-10.50 μg for 1-palmitic acid-2-oleic acid-3-linoleic acid glyceride， 0.35-10.50 μg for glycerol trioleate （r≥0.999 5）. The limits of quantification were 0.13， 0.06， 0.07， 0.12 μg. The limits of detection were 0.04， 0.02， 0.02， 0.03 μg， respectively. RSDs of precision， stability， and repeatability tests were less than 2.0％（n＝6）. The average recoveries were 95.43％-102.67％（RSD＜2.0％， n＝6）. Average RCFs of linolein trilinolein， 1，2- linoleic acid-3-palmitic acid glyceride and 1-palmitic acid-2- oleic acid-3-linoleic acid glyceride were 0.31， 0.88， and 1.21， respectively. RCFs reproducibility was perfect under different experiment conditions. There was no significant difference in results of content determination between QAMS and external standard method （P＞0.05）. CONCLUSIONS： The method is simple， rapid， accurate and reliable. It is used for simultaneous determination of linolein trilinolein， 1， 2-linoleic acid-3-palmitate， 1-palmitic acid-2-oleic acid-3-linoleic acid glyceride and glycerol trioleateas in C. lacryma seed oil.
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OBJECTIVE:To optimize the soxhlet extraction technology of Atractylodis macrocephalae,and to provide evi-dence for research and preparation of its formula granules. METHODS:Using the contents of atractylenolide Ⅰ,Ⅱ,Ⅲ as index, based on single factor test,the Soxhlet extraction technology of A. macrocephalae formula granules was optimized and verified by L9(34)orthogonal test with extraction time,solid-liquid ratio,extraction times as factors,and then compared with other technolo-gies (normal temperature extraction method,ultrasonic extraction method,reflux extraction method). RESULTS:The optimal ex-traction technology was as follows as 6-fold ethanol,extracting for 3 times,lasting for 8 h. Results of validation test showed that the extraction amounts of atractylenolide were 0.769,0.752,0.781 mg/g (RSD=1.99%,n=3) for 3 times,which were higher than the extraction amounts of other 3 methods(0.683,0.489,0.693 mg/g). CONCLUSIONS:The optimized extraction technolo-gy possesses high extraction rates of atractylenolide Ⅰ,Ⅱ,Ⅲ,and can be used for the extraction of internal ether from A. macro-cephalae formula granules.
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Objective To discuss the clinical effect of the phase-one treatment scheme for traumatic osteomyelitis in tibia by combining flap,vancomycin-loaded calcium sulfate and autogenous iliac bone.Methods From January,2009 to July,2014,49 patients which had traumatic osteomyelitis in tibia and met the inclusive criteria were investigated and treated.By taking these patients as treatment group A(34 cases),they were treated by adopting the phase-one treatment scheme of combing tissue flap,vancomycin-loaded calcium sulfate and autogenous iliac bone.Fifteen patients who were treated by using the phase-one treatment scheme,namely,removing the lesion,implanting vancomycin-loaded calcium sulfate and repairing the defect by means of tissue flap,were chosen as control group B.Concerning treatment group A,drainage fluid was collected after operation every day to measure the concentration of vancomycin until drainage tube was removed.All the patients were followed up to study the following indexes:the standing time of drainage tube,the healing time of fracture,infection control rate,bone nonunion rate and other complications.Results All cases were followed up during 17 to 40 months after operation and no amputation was conducted for the affected limb.To repair soft tissue defect,flap and direct suture were adopted for 25 and 9 cases respectively in group A;The results indicated that all flaps survived,the poor healing of flap defect was observed for 2 cases which were healed after dressing change.However,to repair soft tissue defect,all group B cases used flaps;results revealed that distal flap necrosis was found in 2 cases applying neurocutaneous flap,with defect exudation and infection while the 2 cases were cured after debridement and dressing change without performing a second flap operation.In group A,3 cases recurred during 5 months to 2 years after operation;in group B,it was 1;other complications included pintract infection,nonunion,numbness of anterolateral thigh,hematocele in iliac 1 region.In group B,refracture occurred for 2 cases at the original lesion location 18 and 25 months after healing and was cured after plate refixation and the graft of autogenous iliac bone;intraoperative pathology validated no recurrence of osteomyelitis.The standing time of drainage tube was (12.53±4.56) days on average for group A while (17.07±3.87) days for group B;The difference was statistically significant (P＜0.05).The healing time of fracture was (6.20±2.16) months on average for group A while(8.36±2.84) months for group B.The difference was statistically significant(P＜0.05).Conclusion In one stage treatment of localized and diffused traumatic osteomyelitis,the scheme of combining tissue flap,vancomycin-loaded calcium sulfate and autogenous iliac bone effectively shortened the healing time of fracture,increased the healing strength,and reduced the exudation after operation,without increasing infection recurrence rate.The scheme was superior to merely implanting vancomycin-loaded calcium sulfate.
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Objective To investigate the effect of lateral ventricle transplantation of neurotrophic factor-transfected cells derived from Glia cell line on vascular dementia in rats and gene expression of Drebrin in hippocampal region.Methods By using gene clone technique,the GDNF gene was transfected into SH-SY5Y cell lines.104 adult male Sprague-Dawley rats weighing (200± 20) gram were divided into groups:transplanted group,injected group,control group,all of which accepted operation by permanent ligation of left common carotid artery and clipping right common carotid artery repeatedly to build up model of vascular dementia,and sham operation group which accepted no ligation or clipping.6 rats from each group were decapitated on the third day,seventh day and tenth day after transplanting treatment were for fluorescence detection.The rest 20 rats in each group were used to detect learning and memory functions by Morris water maze on the third day and decapitated on the fourth day after transplanting treatment.Then GDNF level in temporal lobe were detected by enzyme-linked immunosorbent assay (ELISA),while Drebrin mRNA and protein levels in hippocampal region were detected by real time-PCR and Westernblot respectively.Results There was strong fluorescent light detected around lateral ventricle of rats in transplanted group on the third day after transplantation,which faded on the seventh day and disappeared on the tenth day.The learning and memory functions of rats in transplanted group were improved significantly.The escape latency was shorter in transplanted group than in injected group and control group [(34.89±4.15) s vs.(43.86±6.95) s,(50.89±3.66) s,both P＜0.05],while shuttle times through the third quadrant were more often in transplanted group than in injected group and control group [(11.00±1.49) vs.(9.26 ±1.38),(8.04 ± 1.12),both P＜0.05].GDNF level and Drebrin mRNA and protein levels were higher in transplanted group than in injected group and control group [GDNF:(315.71±27.43) vs.(256.26±19.90),(141.95±21.33),Drebrin mRNA:(5.54±0.35) vs.(3.10±0.33),(1.32±0.23),Drebrinprotein:(0.55±0.05) vs.(0.43±0.06),(0.26±0.06),all P＜0.05].Conclusions GDNF-transfected cells could survive in the lateral cerebral ventricle of rats for about seven days.The method for treating vascular dementia through the technique of transplanting GDNF-transfected cells is certain feasible,which has a better therapeutic effect than GDNF-injection directly into lateral cerebral ventricle.The therapeutic effect of GDNF on vascular dementia may be related to its action of regulating neural plasticity.
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Objective:To establish the fingerprints of Rhizoma Atractylodis macrocephalae from different origins with high performance liquid chromatography(HPLC)technology,which was furthermore applied in the quality comparison of plant resource.Methods:In this method,C1 8column(4.6mm?250 mm)was used with the mobile phase containing acetonitrile-water for gradient elution,acetonitrile(A):0-15min,75%;15-20min,75%-95%;20-35min,95%;35-40min,95%-75%;40-50min,75%,flow rate 1 mL/min and wave length 220nm.Results:The HPLC fingerprints of Rhizoma Atractylodis macrocephalae from different origins were established and the correlated coefficients of each were calculated.9 common peaks were determined in HPLC chromatogram,three of them were identified as atractylenolideⅢ,Ⅰand atractylon respectively.Conclusion:The method was simple,reproducible and can be used as plant resource selection and quality control of Rhizoma Atractylodis macrocephalae.
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OBJECTIVE:To study the change in grain size and in vitro dissolution ratio of Atractylodes macrocephala after ultramicronization. METHODS: The particle size before and after ultramicronization was analyzed using particle size analyzer. The content of the sample was determined by HPLC using atractylenolide Ⅲ and atractylenolide Ⅰ as indexes to reflect the dissolution ratio. RESULTS: After ultramicronization, the particle size of the sample became thinner obviously, about 30% that of the common fine powder, and the content increased by 27% as compared with the common fine powder. CONCLUSION: The ultramicronization can significantly decrease the particle size, increase specific surface area and contribute to the dissolution of atractylenolide Ⅲ and atractylenolide I from Atractylodes macrocephala.
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Object To research the effect of laurocapram upon the percutaneous absorption of Huayu Babu Sticking Plaster (HBSP) in vitro. Methods The improved Franz diffusing cells were chosen to study percutaneous absorption of piperine from HBSP. The content of permeated piperine was determined by HPLC; while the content of augenol was determined by GC to inspect the stability of HBSP. Results The result showed that the process of penetrating of piperine in HBSP through skin could be in accordance with zero-grade releasing equation and HBSP was stable during the course of experiment. Conclusion Laurocapram could promote percutaneous absorption of piperine.
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Object To study specific TLC identification of the root bark of Morus alba L. (RBMA)Methods The ethanol extracts of RBMA and other confusable species were subjected to TLC in different development system. The TLC plats were examined under shortwavelength UV light or colored by FeCl 3 solution.Results A mixture of chloroform and methanol (5∶1) is used for development and ferric chloride solution is used to color, a specific purple spot of the certified RBMA can be found.Conclusion The specific spot showed with TLC can be regarded as the basis of identification of RMBA and it was separated and identified as sanggenon C.
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Objective: To study the best gathering period and processing method of Cortex Mori.Methods: A HPLC method was used to determine the content of scopoletin. Results: The scopoletin contents in Cortex Mori picked in Jan., Feb. Apr., Jul. and Aug. were higher than that picked in the other months. The scopoletin content in Cortex mori of which rough barks were unremoved was higher than that of which rough barks were removed.Conclusion: Spring and Summer are the best gathering periods, but the processing method shouldn't be stricthy limited.