高压均质法提取云芝胞内糖肽

采用高压均质法破碎液态发酵法生产的云芝菌丝体,分析其对产品含量及得率的影响。以云芝胞内糖肽多糖含量为指标,采用L9（3^3）正交试验法,确定了提取云芝胞内糖肽最佳工艺为均质压力120MPa,料液比1：20,均质次数1次。与传统水提法比较,高压均质法能明显提高云芝胞内糖肽的得率。     

云芝胞外多糖充液胶囊的研制

从云芝培养液获得云芝胞外多糖提取物,并将其研制云芝胞外多糖充液胶囊产品;研究不同含水量的聚乙二醇400,不同的稀释剂PEG400和聚山梨酯80的适当比例和不同的封口乙醇的体积分数对明胶胶囊稳定性的影响;最后采用紫外分光光度法测定充液胶囊中云芝胞外多糖在60 min内的溶出度。结果表明:聚山梨酯80和含水量为10%的PEG400比例为1:25的云芝胞外多糖充液产品,其稳定性最好,35 min内累积云芝多糖溶出度达到90%以上。     

脱脂灵芝孢子粉多糖的提取及膜分离工艺研究

比较了热水法,超声辅助法和索氏提取法对脱脂灵芝(Ganoderma lucidum)孢子粉多糖提取的影响,确定热水法为最佳提取方法,采用正交实验对热水法提取条件进行优化,优化的提取工艺为:料液比1∶10(w/v)、提取温度100℃、提取时间3h、提取次数3次,此时多糖提取物得率9.45%,提取物多糖含量14.83%。将放大试验得到的提取液用不同孔径的膜对其进行分离,陶瓷膜(0.1μm)截留部分以及经过陶瓷膜(0.05μm)但被超滤膜(10nm)截留部分的多糖含量最高分别为23.13%和23.56%,这两部分所占提取液的比列为39.55%     

高压超临界CO_2提取灵芝三萜和甾醇成分的研究

为了研究常温高压超临界CO_2提取法提取灵芝三萜和甾醇成分的效果,采用不同高压的超临界CO_2提取法提取灵芝细粉中灵芝三萜和甾醇成分,通过高效液相色谱(HPLC)测定灵芝三萜和甾醇的含量,并与传统的乙醇回流提取法进行比较。研究表明,高压超临界CO_2提取工艺条件为:提取压力85 MPa,提取温度50℃,提取时间4 h,在此条件下灵芝三萜含量为1.35%,麦角甾醇含量为0.32%,而采用乙醇回流提取法提取的灵芝三萜含量为0.92%,麦角甾醇含量为0.2%。与传统乙醇回流提取法相比,采用高压超临界CO_2提取法,灵芝三萜含量提高了46.7%,麦角甾醇含量提高了60%。HPLC图谱表明,高压超临界CO_2提取法的提取物中三萜和甾醇不仅含量高,而且成分种类多。因此高压超临界CO_2提取法具有得率高、提取温度低、提取时间短等优点,为灵芝成分的提取提供了新技术。     

不同方法对虫草花多糖提取效果的研究

以虫草花子实体为原料,分别采用热水提取法、单一酶法(纤维素酶、果胶酶、木瓜蛋白酶)、超声波法对虫草花多糖进行提取,用苯酚-硫酸法测定虫草花多糖,并比较几种提取方法的多糖提取率和提取条件。结果表明:果胶酶法是虫草花多糖提取的较优方法,在加酶量1.00%、pH 5.5、提取温度40℃下提取90min,其多糖提取率为22.23%,而在最佳提取条件下,热水提取法、纤维素酶法、木瓜蛋白酶法和超声波法的多糖提取率分别为18.12%、15.01%、18.57%、13.34%。     

云芝胞外多糖液体发酵条件的优化

以提高云芝胞外多糖含量为主要目的,通过单因素试验,对云芝摇瓶培养条件进行优化。结果表明:云芝胞外多糖发酵培养的最适碳源为蔗糖,最佳氮源为蛋白胨,最适宜碳氮比为20,最适宜的pH为5.0;优化后的云芝发酵生物量为12.14 g/L,较优化前提高了42.49%;胞外多糖含量为3.34 g/L,较优化前提高了20.58%。     

高活性云芝α-半乳糖苷酶发酵培养基的优化

研究了云芝(Coriolus versicolor)液体发酵过程中不同诱导物及培养基组成对α-半乳糖苷酶活性的影响。采用L_9(3~4)正交设计法对云芝发酵培养基进行了优化,确定了其发酵产酶的最佳培养基。结果表明,云芝发酵生产α-半乳糖苷酶的最佳诱导物是豆粕粉,最优培养基为豆粕粉2%、半乳糖0.5%、KH_2PO_4 0.1%、MgSO_4 0.1%,发酵培养8 d,酶活性为301.6 U·mL~(-1)。     

云芝多糖含量测定及其体外抗氧化活性比较

为了解野生和人工栽培云芝多糖的含量及其抗氧化活性,采用苯酚-硫酸法对云芝多糖含量进行测定,以云芝多糖对超氧阴离子自由基(O_2~-·)和羟自由基(·OH)的清除率为考察指标,测定了野生和人工栽培云芝多糖抗氧化活性。结果表明,供试人工栽培云芝的多糖含量高于野生云芝的多糖含量,且两者之间存在极显著差异(P0.01);野生云芝对超氧阴离子自由基(O_2~-·)和羟自由基(·OH)的清除率均要高于人工栽培云芝的清除率,且存在显著性差异(P0.05)。     

超声波提取云芝发酵浸膏总三萜化合物最佳工艺研究

通过正交试验对用超声波技术提取云芝发酵浸膏中总三萜化合物进行了工艺条件的优选,确定超声波最佳提取工艺。并运用分光光度法进行含量测定,以三萜含量为指标,与传统醇提法进行比较分析。实验表明:最佳提取工艺为:超声时间30 min、提取温度40℃、提取3次、提取频率70Hkz。超声提取率为1.23%,常规提取率为0.56%。结果显示:利用超声波提取云芝发酵浸膏中总三萜化合物是有效的,最佳提取工艺简便可行,大幅度提高了提取率,并能显著减少提取时间。     

灰树花发酵液提取多糖的比较研究

以灰树花发酵液为原材料,采用酸法、碱法、盐法和中性热水法提取灰树花发酵液纯多糖,提取量分别为0.8%、1.09%、1.96%和1.34%.     

Effect of chronic hypoxia on L-Arginine/nitric oxide pathway in rat pulmonary artery

AIM: To study the effect of chronic hypoxia on L-Arginine/NO pathway in rat pulmonary artery. METHODS: Changes in pulmonary artery L-Arginine(L-Arg) transport, nitric oxide synthase (NOS) activity, plasma nitrite level and L-Arg level in HPH rats were investigated. RESULTS: (1) The mean pulmonary arterial pressure (mPAP) and weight ratio of right ventricle to left ventricle and septum (RV/LV+S) of HPH group were higher than those in control group (P＜0.01). (2) Plasma L-Arg level in HPH group was not significantly changed. (3) At low (0.2 mmol/L)or high(5.0 mmol/L)concentration of L-Arg, the velocity of L-Arg transport in HPH group was lower than that in control group (P＜0.05 or P＜0.01). (4) The activity of pulmonary artery tNOS, iNOS and cNOS in HPH group were increased by 38.0%, 32.8% and 53.0%, respectively (P＜0.01), compared with control group. (5) Plasma NO level of HPH group was decreased, which was negative correlation to mPAP and RV/LV+S (P＜0.01). CONCLUSION: The decrease of nitric oxide generation might result from L-Arg transport injury, while pulmonary artery tNOS, iNOS and cNOS activity were enhanced during chronic hypoxia.     

Expression and distribution of 5-HT1B receptors in lung tissue in rats with hypoxic pulmonary hypertension

AIM: To investigate the changes of 5-hydroxytryptamine(5-HT)levels and to observe the expression and distribution of 5-HT_1B receptors in the lung tissues of hypoxic pulmonary hypertension(HPH) rats for exploring the mechanisms of hypoxic pulmonary hypertension.METHODS: Forty male Sprague-Dawley rats were randomly divided into 4 groups: normoxia control(control group), 3 weeks hypoxia group, 4 weeks hypoxia group and 5 weeks hypoxia group. The rats in normoxia control group stayed in normal environment. The rats in 3 weeks hypoxia group, 4 weeks hypoxia group and 5 weeks hypoxia group were kept respectively in hypoxia chamber for 3 weeks, 4 weeks and 5 weeks respectively to establish the HPH animal model. After HPH was established, the mean pulmonary pressure(mPAP) and the right ventricular systolic pressure(RVSP) were recorded by a micro-catheter. RV/(LV+S) ratio was calculated to assess the right ventricular hypertrophy. 5-HT levels in plasma and lung tissues of HPH rats were measured by ELISA. The expression and distribution of 5-HT_1B receptors in the lung tissues were measured by the methods of immunohistochemistry and Western blotting. RESULTS: Compared to the normoxia controls, mPAP, RVSP and RV/(LV+S)% in 3 weeks hypoxic rats increased significantly(P<0.05), and continued to increase following prolonged hypoxia. The results of ELISA showed that 5-HT levels in plasma and lung tissues of HPH rats continued to increase following prolonged hypoxic exposure(P<0.05). The 5-HT_1B receptors were localized mainly in the intima of the pulmonary arteries in normal rats. Exposed to hypoxia, the immuno-reactivity for 5-HT_1B receptors increased in the media of pulmonary arteries in 3 weeks hypoxic rats, particularly those bordering the adventitia. The increase in the expression of 5-HT_1B receptor was observed following prolonged hypoxic exposure. The results of Western blotting showed the same changes of 5-HT_1B receptor expression in the lung tissues as that of 5-HT_1B immuno-reactivity in pulmonary arteries.CONCLUSION: Hypoxia induces the high 5-HT levels and the over-expression of 5-HT_1B receptors in the pulmonary arteries of HPH rats, indicating the underlying mechanism of 5-HT in the development of HPH.     

Nitric oxide opens the second window of protection in ischemic preconditioning via induction of heat shock protein 72

AIM:To examine the inhibition of nitric oxide (NO) synthesis during ischemic preconditioning (IP) on the induction of heat shock protein 72 (HSP72) and infarct size-limiting effect of the second window of protection. METHODS:Rabbits were subjected to 4 cycles of 5 min of coronary artery occlusion separated by 10 min reperfusion, or received a sham operation. During this procedure, N^G-nitro-L-arginine methyl ester (L-NAME, an inhibitor of NO synthase) was injected intravenously 5 min before IP followed by its continuous infusion. Twenty-four hours later, the hearts were rapidly excised for assaying HSP72 expression or were subjected to 30 min coronary artery occlusion followed by 120 min reperfusion and then measured infarct size (IS). RESULTS:Twenty-four hours later, immunoblotting revealed an increase in HSP72 protein levels in the IP group, and this was blocked by L-NAME. IS of the IP rabbits was reduced as compared with the control (29.8%±3.7% vs 50.8%±4.3%, P＜0.01). IS in the IP rabbits was elevtated as a result of L-NAME treatment (46.0%±5.1%). Administration of L-arginine reversed the effects of L-NAME on the induction of HSP72 and IS (33.5%±4.0%). The intravenous administration of S-nitroso-N-acetylpenicillamine (SNAP, a NO donor) increased the induction of HSP72 and reduced IS (31.3%±5.7%, P＜0.01vs control) 24 h later. CONCLUSION:These findings suggest that NO may be involved in the induction of HSP72 and the opening of the second window of protection of IP.     

香菇子实体多糖分步酶解法提取研究

首先采用正交试验优化纤维素酶﹑果胶酶和木瓜蛋白酶对香菇(Lentinula edodes)子实体多糖酶解提取的工艺参数,然后在优化酶解条件下,依次采用纤维素酶﹑果胶酶和木瓜蛋白酶分步处理香菇子实体以提取香菇多糖,并与单一酶解提取法和传统热水浸提法进行对比.结果表明,纤维素酶﹑果胶酶﹑木瓜蛋白酶的最佳提取工艺参数依次为加酶量0.8%、温度45 ℃、pH 4.5、提取时间1 h,加酶量1.0%、温度45 ℃、pH 3.5、提取时间2.0 h和加酶量1.0%、温度45 ℃、pH 4.0、提取时间1.5 h;在优化提取条件下,分步酶解法提取香菇粗多糖的提取率可达14.17%,比传统热水浸提法提高128.2%,比单独采用纤维素酶﹑果胶酶﹑木瓜蛋白酶酶解提取分别提高了43.71%、46.99%和23.11%.紫外光谱分析表明,分步酶解法提取的香菇多糖纯度明显高于热水浸提法提取的香菇多糖.-     

Effect of ischemic preconditioning on myoelectricity and the structure of myocardial gap junction during heart valve replacement surgery

AIM: To investigate the cardio-protective mechanism of ischemic preconditioning (IP) during heart valve replacement of the perspective of architectural changes of myocardial gap junction. METHODS: Fifty-four patients were prospectively randomized to receive or not ischemic preconditioning before cold cardioplegic arrest. The IP protocol in IP patients (n=22) consisted of a single 2-minute ischemia followed by 3-minute reperfusion just before aortic clamping and cold crystalloid cardioplegia for myocardial protection. The control group (n=32) received no ischemic preconditioning prior to cold cardioplegic arrest. The parameters including arrhythmias occurrence, Cx43 expression (immunohistochemistry SABC method) and myocardial structure and intercalated discs observed under electronic microscope were recorded before and after surgery in each group. RESULTS: In IP group, one case (4.55%) of ventricular arrhythmia (sporadic ventricular premature beat), 11 cases (50.00%) of supraventricular arrhythmia (atrial fibrillation, atrial flutter, supraventricular tachycardia, atrioventricular block) and 10 cases (45.50%) of ischemic ST-T changes were observed. In control group, there were 14 cases (43.75%) of ventricular arrhythmia (ventricular premature beat, tachycardia), 18 cases (56.25%) of supraventricular tachycardia and 28 cases (87.50%) of ischemic ST-T changes. No statistical difference in preoperative positive unit of Cx43 expression between the two groups was found (P>0.05). Postoperatively, the positive unit of Cx43 expression in IP group was 16.15±4.40, but the difference was not significant compared to the preoperative value (P>0.05). In control group, Cx43 expression was 11.92±1.26, significantly lower than that of the preoperative value (P<0.05). Cx43 expression between the two postoperative groups showed a significant difference (P<0.05). In control group, electronic microscopic observation revealed disrupted intercalated discs, with some partially or even totally ruptured and disintegrated. Enormous necrotic structural changes of myocardial fibers were also observed, including swelling, dissolution and disorganization of myofilaments and fibers, widening of the Z striae and disorganization. However in IP group, the intercalated discs appeared intact, continuous with normal myocardial structure. CONCLUSION: IP maintains normal expression of the myocardial junctional gap protein Cx43, which preserves a seamless intercellular gap junction and a normal myocardial electric conduction activity.     

Hyaluronan preserves the membrane transport properties in chronic peritoneal dialysis animal model of rats

AIM: We have shown that intraperitoneal (IP) addition of hyaluronan could increase peritoneal fluid removal by decreasing peritoneal fluid absorption rate. In this study, we investigated the impact of chronic use of hyaluronan on peritoneal membrane transport characteristics. METHODS: Twenty male SD rats received daily IP injection of 25 mL 4.25% glucose dialysis solution without (HP, n=8) or with 0.025% hyaluronan (HA, n=6) for one week, another six rats did not receive any peritoneal injection. Twenty-four hours after the last injection, a 4h dwell study using 25 mL 4.25% glucose dialysis solution with IP volume marker and frequent dialysate and blood samplings were performed in each rat. RESULTS:IPV was significantly higher in the HA group as compared to the HP group (ANOVA repeated measurement, P＜0.01). The peritoneal fluid absorption rate, K_E, was significantly increased in the HP group as compared to the other groups. The direct lymphatic fluid absorption rate, K_EB, was significantly higher in the two daily infusion groups (HP and HA) as compared to the control group. However, the tissue absorption rate, K_ET, was significantly higher in HP group as compared with HA group and control group. CONCLUSION: The present study suggests that repeated intraperitoneal addition of hyaluronan may prevent, at least in part, the membrane damage caused by currently used hypertonic dialysis solution.     

Changes of nuclear factor-κB and inducible nitric oxide synthase in hypoxic pulmonary hypertension

AIM: To observe the changes of nuclear factor-κB (NF-κB) activity and inducible nitric oxide synthase (iNOS) expression in hypoxic pulmonary hypertension (HPH). METHODS :The rat model of HPH was used. The NF-κB activity and iNOS expression in lung tissue were determined by immunohistochemistry (IHC), in situ hybridization (ISH), RT-PCR and Western blot. RESULTS: ISH showed that iNOS mRNA expression in intraacinar pulmonary arteriole (IAPA) in H28d group (hypoxic treatment for 28 days) was stronger than that in normal group, H5d group and H14d group. RT-PCR showed that the iNOS mRNA in H28 group was 2.1 times, 1.9 times and 1.8 times higher than that in normal group, H5d group and H14d group, respectively. The nucleic anti-NF-κB stain was observed in H28d group, which was significantly stronger, but the I-κB amount was 2.8 times, 2.7 times and 2.5 times lower than that in normal group, H5d group and H14d group, respectively. CONCLUSION: The activity of NF-κB was correlated with the hypoxic pulmonary vessel structural remodeling and iNOS expression.     

Genistein downregulates survivin gene expression and induces apoptosis in hepatocellular carcinoma HepG2 cells

AIM: To probe into the role of 1, 4, 5-trisphosphate inositol (IP3) and survivin protein in apoptosis of HepG2 cells induced by genistein. METHODS: HepG2 cells were treated with 60 μmol/L genistein for 12 h, 24 h, 48 h and 72 h. IP3, survivin and apoptosis rate were assayed by IP3-［3H］ Birtrak assay, Western blotting and flow cytometry, respectively. RESULTS: IP3 in groups incubated for 12 h, 24 h, 48 h and 72 h with 60 μmol/L genistein were significantly lower than that in control (P<0.01) ［(12.0±1.4) pmol/106cells, (7.5±0.8) pmol/106 cells, (5.6±0.5) pmol/106cells, (3.3±0.6) pmol/106 cells， vs (29.2±0.6) pmol/106 cells］. V-survivin/ V-β-actin, which was the gray degree multiply area of survivin/the gray degree multiply area of β-actin in groups incubated for 12 h, 24 h, 48 h and 72 h with 60 μmol/L genistein, were significantly lower than that in control (P<0.01) ［（0.36±0.13, 0.33±0.03, 0.23±0.04, 0.18±0.04）， vs 0.63±0.06］. The apoptosis rate in groups incubated with 60 μmol/L genistein for 24 h, 48 h and 72 h was significantly higher than that in control （P<0.01） ［(7.4%±0.5%, 20.5%±2.0%, 30.7%±1.6%) vs 2.6%±0.1%］. CONCLUSION: Genistein induces apoptosis in HepG2 cells by reducing IP3 production and survivin protein expression.     

Multi Criteria Assessment of Zero Residue Apple Production

During the 2008–2010 growing seasons, an alternative ‘zero residue apple production system’ was compared with integrated apple production with cvs ‘Idared’, ‘Golden Delicious’, ‘Jonagold’ and ‘Gala’ in commercial orchards at several locations throughout Slovenia, using data envelopment analysis (DEA), and multi criteria assessment by an analytical hierarchical process (AHP). The principle of the ‘zero residue apple production’ is a combination of integrated (IP) and organic apple production. During the first 3 months of the growing season (1 April–30 June), pesticides used in IP with rapid degradation (8–10 applications) were used to control pests and diseases. During the second part of the season from 1 July to harvest, organic products (6–12 applications) were employed compared with 19–25 applications overall in IP. The goal of the alternative system was to reduce the amount of applied conventional pesticides by 40 % and to minimize pesticide residues in fruits to below the limit of 0.5 % of the legal maximum residue level (MRL) or below the residue concentrations of 0.005–0.01 mg/kg and to retain the high long-term level of yield, fruit quality, and net income per hectare. The number of pesticide residues was reduced from 4.2–5.5 in IP to 1.8–3.4 in zero residue cultivation, while 3 year average yields (class 1 fruit) were 4–9 % lower than in IP. The break even prices ranged from € 0.31 for Idared in IP, € 0.34 for ‘Elstar’ of both production systems to € 0.35/kg for zero residue cultivated ‘Golden Delicious’. Overall, a price increase of just € 0.02/kg for residue free apples would make this new ‘zero residue apple production’ profitable then representing a realistic alternative to the standard integrated apple production system.     

Expression of NO and H2S in hypoxic pulmonary hypertension and intervention of sodium nitrite in rats

AIM: To observe the change of nitric oxide(NO)and hydrogen sulfide(H2S)in blood and lung homogenate of hypoxic pulmonary hypertension(HPH)rat model, and to discuss the meaning of inhalation sodium nitrite and these factors in the treatment of HPH. METHODS: Fifty healthy male Wistar rats were assigned randomly into 5 groups(10 rats each): normoxia control group(NC), normoxia sodium nitrite group(NNI), hypoxic control group(HC), hypoxic normal saline group(HNS)and hypoxic sodium nitrite group(HNI). The mean pulmonary arterial pressure(mPAP), weight of right ventricle, weight of left ventricle plus septum, and the ratio of the weight of right ventricle to that of left ventricle plus septum(right ventricle hypertrophy index, RVHI)were also determined. The serum level of NO and plasma level of H2S were measured, and at the same time the levels of NO in the lung homogenate were detected. The structures in pulmonary arteries were examined using optical microscope. RESULTS: After model established, compared to that in the normoxia groups, the body weight decreased significantly in hypoxia groups(P<0.05), although no difference of body weight in five groups before producing model was observed. Compared to that in normoxia groups, the levels of mPAP and RVHI increased significantly in hypoxia groups, and compared to that in hypoxia control groups and hypoxia normal saline group, mPAP and RVHI levels decreased significantly in hypoxia sodium nitrite group(P<0.05). Compared to that in normoxia groups, the serum level of NO decreased significantly in hypoxia groups(P<0.05). NO level in lung homogenate decreased significantly in hypoxia control group and hypoxia normal saline group as compared to that in normoxia groups(P<0.05), and no obvious difference between hypoxic sodium nitrite group and normoxia groups was found. The plasma level of H2S was decreased significantly in hypoxia groups(P<0.05)as compared to that in normoxia groups. H2S level increased significantly in hypoxia sodium nitrite group as compared to that in hypoxia control groups and hypoxia normal saline group(P<0.05). Observation under optical microscope, the lumen structure of lung in normoxia control group was normal. No significant change in normoxia sodium nitrite group was found. The proliferation of smooth muscle cells(SMCs), the collagen fiber deposition in the vessel wall and every caliber thickening was observed in hypoxic control group. The same changes were also observed in hypoxic normal saline group. The thickened caliber was relieved significantly in hypoxic nitrite group. CONCLUSION: Pulmonary hypertension and right ventricle reconstitution can be relieved by inhalation of sodium nitrite, and can be regulated by the level of NO and H2S in rats. Above all, inhalation of sodium nitrite may degrade HPH directly or by affecting the externalization and synthesizing of gas signaling molecule indirectly.