天然茯苓和液体发酵茯苓粉末的研究

液体发酵茯苓为一种新型的发酵中药。本文在粉末显微特征、多糖、灰分和氨基酸含量等方面,对液体发酵茯苓粉末和天然茯苓粉末展开了研究。研究结果表明二者在显微特征和化学成分等方面均存在较大的差异,探讨了上述差异产生的原因。     

16种茯苓菌株液体发酵产胞内多糖与三萜的比较研究

筛选茯苓高产胞内多糖和胞内三萜的优良液体发酵出发菌株。采用PDA富集固体平板培养与液体发酵培养测定菌丝体生长速率;采用液体发酵策略分析16种茯苓菌株产胞内多糖与胞内三萜的潜能。实验结果表明菌株生长于固体培养基与种子培养基的生长速率之间没有关联性;降低一级种子培养基初始pH值到4.0时能有效缓解茯苓菌株培养物褐化现象;AS5.137胞内多糖含量最高,达377.60±0.10 mg/g,而DB菌株显示出最高的胞内多糖产量,达1.01±0.13 g/L;Y1菌株胞内三萜含量最高,达83.89±4.28 mg/g,而Jingzhou28菌株胞内三萜产量最高,达136.63±26.66 mg/L。就生产茯苓胞内多糖与胞内三萜而言,AS5.137与DB菌株适合作为液体发酵产胞内多糖的出发菌株;Y1,Jingzhou28,Z(z)与Xingpinzhong菌株均较适合作为液体发酵产胞内三萜的出发菌株。     

茯苓健脾作用活性部位的研究

通过建立饮食失节、过食肥甘所致的小鼠脾虚模型,以体重、饮食量、外部表现特征及脾指数为指标,从茯苓总三萜类、茯苓总多糖及茯苓水煎剂中确定具有健脾作用的有效活性部位。结果表明:茯苓总三萜类对脾虚小鼠有明显的恢复作用,茯苓水煎剂也有一定作用,而茯苓总多糖作用不明显。说明茯苓总三萜类是茯苓治疗过食肥甘所致小鼠脾虚证的活性部位。     

茯苓菌丝发酵液粗多糖对肠道益生菌的影响

目的探究茯苓菌丝发酵液中提取的粗多糖对肠道益生菌的影响。方法运用发酵工程相关技术获得茯苓菌丝发酵液,提纯分离以获取茯苓菌丝中分泌的粗多糖。将提取出的茯苓粗多糖以10.0%、5.0%、2.5%和空白对照(0.0%)的浓度对4种肠道益生菌——嗜酸乳杆菌、干酪乳杆菌、乳杆菌MZ900、青春双歧杆菌的生长进行观察。结果在茯苓粗多糖含量10.0%以下的范围内,10.0%、5.0%、2.5%浓度下肠道益生菌数量均高于对照组,并且随着茯苓发酵液粗多糖浓度的含量增加,4种肠道益生菌数量呈正相关增长。结论茯苓菌丝发酵液粗多糖对以上4种肠道益生菌有增菌效果。实验结果为茯苓菌丝发酵液粗多糖有保护人类肠道的作用提供理论依据,并为后续的生化研究与动物实验提供理论基础。     

茯苓菌液体培养条件的优化及其多糖的提取

研究了茯苓菌株F2.10的液体培养条件,并对其中的多糖进行了提取和分析.结果表明,最佳培养条件为初始pH 5.5、摇床转速150r/min、培养温度26℃、装液量80 mL/250mL三角瓶、培养时间5 d;最适碳、氮源分别为4 %蔗糖和3 %豆饼粉.在此条件下100 mL培养液可获得2.21 g干重生物量.用水煮和稀碱浸提法可从30 g干菌丝体中分别获得1.38 g水溶性多糖和19.75 g碱溶性多糖.IR分析结果表明这两种多糖的主要成分为β-D-葡聚糖.     

蝉拟青霉菌丝体与天然蝉花中化学成分的比较分析

以天然蝉花Cordyceps cicadae做对比,对一株蝉拟青霉Paecilomyces cicadae菌株发酵菌丝体的化学成分进行了分析,包括粗成分、生物活性成分、无机元素、氨基酸、脂肪酸等。结果表明:蝉拟青霉菌丝体中虫草多糖的含量为33.2mg/g,甘露醇的含量为78.9mg/g,麦角甾醇的含量为0.6256mg/g,腺苷的含量为1.0620mg/g,钙、铁、锌、硒和锰微量元素的含量分别为5187.59μg/g、207.23μg/g、41.93μg/g、0.087μg/g和28.24μg/g,18种氨基酸齐全,不饱和脂肪酸是优势脂肪酸,以亚麻油酸、油酸为主,其相对含量分别为53.91%和20.63%,与天然蝉花相比,发酵菌丝体中虫草多糖、甘露醇、麦角甾醇、腺苷、氨基酸总量、必需氨基酸总量和不饱和脂肪酸的含量明显高于后者,其它化学成分和天然蝉花中相应成分基本一致。     

蝉拟青霉菌丝体与天然蝉花中化学成分的比较分析

以天然蝉花Cordyceps cicadae做对比,对一株蝉拟青霉Paecilomyces cicadae菌株发酵菌丝体的化学成分进行了分析,包括粗成分、生物活性成分、无机元素、氨基酸、脂肪酸等。结果表明:蝉拟青霉菌丝体中虫草多糖的含量为33.2mg/g,甘露醇的含量为78.9mg/g,麦角甾醇的含量为0.6256mg/g,腺苷的含量为1.0620mg/g,钙、铁、锌、硒和锰微量元素的含量分别为5187.59μg/g、207.23μg/g、41.93μg/g、0.087μg/g和28.24μg/g,18种氨基酸齐全,不饱和脂肪酸是优势脂肪酸,以亚麻油酸、油酸为主,其相对含量分别为53.91%和20.63%,与天然蝉花相比,发酵菌丝体中虫草多糖、甘露醇、麦角甾醇、腺苷、氨基酸总量、必需氨基酸总量和不饱和脂肪酸的含量明显高于后者,其它化学成分和天然蝉花中相应成分基本一致。     

羧甲基茯苓多糖体外抗氧化活性研究

目的：比较不同浓度洗脱液洗脱得到的羧甲基茯苓多糖的抗氧化活性。方法：以茯苓为原料提取茯苓多糖,进行羧甲基取代反应,分离和纯化得到了均一性羧甲基茯苓多糖CMP-1、CMP-2、CMP-3、CMP-4,通过测定还原能力、DPPH自由基清除率、羟基自由基清除率、超氧阴离子自由基清除率比较其体外抗氧化活性。结果：羧甲基茯苓多糖均表现出与浓度正相关的体外抗氧化活性,其中CMP-4具有相对更强的体外抗氧化活性。结论：所得羧甲基茯苓多糖样品具有不同的体外抗氧化能力,随着洗脱液浓度增加,抗氧化活性增强。     

硫酸化茯苓多糖急性毒性实验研究

按10、8、6.4、5.12、4.1g/kg体重剂量的硫酸化茯苓多糖(SP)分别灌胃给药,通过观察小鼠的活动和急性毒性反应,记录小鼠的死亡数,用综合计算法计算半数致死量(LD50)等相关数据.结果表明硫酸化茯苓多糖灌胃给药的LD50为7.358/kg,LD50的95%平均可信限为(7.358±0.894)g/kg,属低毒性物质.该研究为开发应用硫酸化茯苓多糖新药提供科学的药理学依据.     

羧甲基茯苓多糖的制备及体内抗肿瘤作用的实验研究

目的 :探讨羧甲基茯苓多糖的制备方法及体内抗肿瘤作用。方法 :选择性氧化茯苓菌发酵液得羧甲基茯苓多糖 ,检测三个剂量的羧甲基茯苓多糖 (2 mg/ ml、4mg/ ml、6mg/ ml)对 H2 2 荷瘤小鼠淋巴细胞转化率和 NK细胞杀伤活性以及血清中 TNF-α含量的影响。结果 :羧甲基茯苓多糖三个剂量组均可提高荷瘤小鼠的淋巴细胞转化率和 NK细胞杀伤活性 ,与生理盐水组相比差异有显著性 (P<0 .0 5) ,中剂量组抗肿瘤的效果显著 ,与其他两组相比差异有显著性 (P<0 .0 5) ;羧甲基茯苓多糖三个剂量组均可提高荷瘤小鼠血清中 TNF-α的含量 ,与生理盐水组相比差异有显著性 (P<0 .0 5) ,但三个剂量组之间比较差异无显著性 (P<0 .0 5)。结论 :羧甲基茯苓多糖可改善荷瘤小鼠的免疫功能 ,具有抗肿瘤作用 ,且功效与作用剂量间有一定的关系 ,在最佳剂量时活性最高     

茯苓多糖抗氧化性研究

采用分光光度法研究了茯苓多糖提取物的体外抗氧化作用,并与Vc进行比较.结果表明其具有较强的抗氧化能力,在清除DPPH·的体系中和还原能力体系中,样品的清除能力均超过Vc,但在羟基自由基·OH和超氧阴离子O-2体系中,其清除能力低于对照样Vc.     

Antitumor activities of heteropolysaccharides of Poria cocos mycelia from different strains and culture media

Ten water-soluble heteropolysaccharide fractions were isolated from Poria cocos mycelia cultured from one wild and one cultivated strain in two identical culture media differing only in one component: either corn steep liquor or bran extract. The chemical compositions, including monosaccharide profile, protein content, and molecular mass M(w) of the mycelial polysaccharides were determined. Both the in vitro and in vivo antitumor activities of the heteropolysaccharides were evaluated and compared. The heteropolysaccharides from Poria cocos mycelia cultured with the wild strain in a medium containing corn steep liquor exhibited the highest antitumor activities against Sarcoma 180 in vivo.     

Nematicidal activity and chemical component of Poria cocos

Poria cocos, a famous traditional Chinese medicine, was found to have nematicidal activity in experiments searching for nematicidal fungi. The experiment showed it could kill 94.9% of the saprophytic nematode, Panagrellus redivivue, 92.6% of the root-knot nematode, Meloidogyne arenaria, and 93.5% of the pine nematode, Bursaphelenchus xylophilus, on PDA plate within 12 hours. According to the nematicidal activity, three new compounds, 2, 4, 6-triacetylenic octane diacid, 2, 4, 5, 6-tetrahydroxyhexanoic acid and 3, 4-dihydroxy-2-keto-n-butyl 2,4,5,6-tetrahydroxyhexanate, were isolated from submerged cultures of Poria cocos. Of these, 2, 4, 6-triacetylenic octane diacid could kill 83.9% Meloidogyne arenaria and 73.4% Panagrellus redivivus at 500 ppm within 12 hours. Here, it is reported for the first time that Poria cocos has nematicidal activity.     

Solution properties of (1-->3)-alpha-D-glucan and its sulfated derivative from Poria cocos mycelia via fermentation tank

From Poria cocos mycelia yielded via a pilot scale facility-fermentation tank, a water-insoluble (1-->3)-alpha-D-glucan coded as Pi-PCM3-I was isolated by extraction with 0.5 M NaOH/0.01 M NaBH(4) aqueous solution. Nine fractions from F1 to F9 with a weight-average molecular mass (M(w)) range from 7.75 x 10(4) to 57.3 x 10(4) were prepared from the Pi-PCM3-I sample by a nonsolvent addition method. The fractions were reacted with chlorosulfonic acid-pyridine complex to product water-soluble sulfated derivatives coded as S1 to S8 with M(w) from 2.36 x 10(4) to 14.5 x 10(4) and degree of substitution (DS) of 0.86-1.38. M(w), z-average radius of gyration (s(2) (z) (1/2)), the second virial coefficient (A(2)), and the intrinsic viscosity ([eta]) of the native and sulfated Pi-PCM3-I were measured by laser light scattering (LLS), size-exclusion chromatography combined with LLS (SEC-LLS), and viscometry at 25 degrees C. The Mark-Houwink equations for Pi-PCM3-I in 0.25 M LiCl/dimethylsulfoxide (DMSO) (Me(2)SO) and for its sulfated derivative in 0.15 M NaCl aqueous solution at 25 degrees C were established to be [eta] = 1.33 x 10(-2) M(w) (0.75+/-0.01) (mL g(-1)) and [eta] = 1.46 x 10(-4) M(w) (1.13+/-0.01) (mL g(-1)), respectively. On the basis of theories for a wormlike cylinder model, the conformational parameters of the native and sulfated Pi-PCM3-I were calculated to be 760 +/- 50 and 1060 +/- 30 nm(-1) for the molar mass per unit contour length (M(L)), 6.3 +/- 0.5 and 13.1 +/- 1 nm for the persistence length (q), and 14.9 +/- 0.2 and 31.8 +/- 1 for the characteristic ratio (C( proportional, variant)), respectively. The results revealed that Pi-PCM3-I existed as an extended flexible chain in 0.25 M LiCl/Me(2)SO, and its sulfated derivative existed as a semistiff chain in 0.15 M NaCl aqueous solution. Furthermore, Pi-PCM3-I possessed similar structure and molecular parameters to wc-PCM3-I from a rotary shaker; this suggests promising industrialization of Poria cocos polysaccharides.     

茯苓中多糖的提取及含量测定

采用水煎煮、稀碱浸提茯苓中的多糖,通过正交试验优选工艺条件。结果表明,稀碱浸提茯苓中的多糖的工艺较为合理,其操作简单,提取时间短,收率较高．为测定提取液中的多糖含量,以苯酚—硫酸法,制得有色糖醛衍生物,用分光光度法,在490nm波长处测定吸光度,回归方程线性关系好。方法简单易行、稳定、快速。     

Triterpenes from the fungus Poria cocos and their inhibitory activity on nitric oxide production in mouse macrophages via blockade of activating protein-1 pathway

Two new triterpenes, 29-hydroxydehydrotumulosic acid (1) and 29-hydroxydehydropachymic acid (2), together with six known compounds, dehydropachymic acid (3), dehydrotumulosic acid (4), 29-hydroxypolyporenic acid C (5), polyporenic acid C (6), tumulosic acid (7), and pachymic acid (8), were isolated from the dried sclerotia of Poria cocos. In the in vitro bioassays, these isolated compounds reduced, in a dose-dependent manner, nitric oxide (NO) production from lipopolysaccharide (LPS)-induced RAW 264.7 cells, with compounds 5 and 6, the IC(50) values of which were 16.8±2.7 and 18.2±3.3 μM, respectively, exhibiting the greatest inhibition activity. Further Western blot analysis conducted on cells pre-treated with compounds 5 and 6, and luciferase assays on activator protein 1-dependent gene expression revealed that the inhibited NO release was attributed to the reduced expression of iNOs (=inducible NO synthase) enzymes, which might be regulated via the blockade of activator protein-1 signaling pathway.     

茯苓原生质体制备与再生条件的研究

研究了酶、酶解时间、菌龄、稳渗剂等对茯苓原生质体制备与再生的影响。茯苓原生质体制备的最佳条件为:纤维素酶(1．5%)和蜗牛酶(1．5%)的等量混合酶解系统,酶解时间3h,7d菌龄菌丝,产量可达1．77×10~7个/mL。以甘露醇为稳渗剂,采用CYM再生培养基,酶解时间3h,7d菌龄菌丝,其原生质体再生率最高,为0．164%。这一结果为茯苓通过原生质体技术进行菌种改良提供了重要技术参数。     

Tetranorlanostane and Lanostane Triterpenoids with Cytotoxic Activity from the Epidermis of Poria cocos

Two unprecedented tetranorlanostane triterpenoids, poricolides A ( 1 ) and B ( 2 ), and two new lanostane triterpenoids, 3β-acetoxy-24-methyllanosta-8,16,24(31)-trien-21-oic acid ( 3 ) and 3β-acetoxylanosta-7,9(11),16,23-tetraen-21-oic acid ( 4 ), were isolated from the epidermis of Poria cocos. The structures of 1–4 were determined via analysis of ¹H-, ¹³C-, and 2D-NMR, and HR-ESI-MS data, and the absolute configurations of 1 and 3 were established by single-crystal X-ray diffraction analysis. Compounds 1 and 2 were the first report of tetranorlanostane triterpenoid having a δ-lactone ring at C(17). Compounds 3 and 4 were rare lanostane triterpenoids having a double bond between C(16) and C(17). Compounds 1–4 exhibited potent antiproliferative effects against A549, SMMC-7721, MCF-7, and SW480 cancer cell lines with IC₅₀ values from 16.19±0.38 to 27.74±1.12 μM.     

Evaluating the basidiomycetes Poria medula-panis and Wolfiporia cocos for xylanase production

Xylanase, oxidative enzymes and iron-binding compounds were detected in the filtrates of Wolfiporia cocos and Poria medula-panis grown in wheat bran liquid medium. Xylanase and iron-binding compounds were produced at high levels by the brown-rot fungus (BR) W. cocos and at low levels by the white-rot fungus (WR) P. medula-panis. Phenoloxidase was produced only by P. medula-panis. Polyacrylamide gel electrophoresis (PAGE) (SDS-PAGE) showed a wide variety of bands for extracellular proteins produced by W.cocos, with low molecular weight (<30 kDa) and minor bands with molecular weight above 45 kDa. Two bands with xylanase activity derived from W. cocos extracts were detected in the gels, whereas many different bands with xylanase activity were found in the extracts from P. medula-panis. P. medula-panis is a selective lignin degrader, whereas W. cocos preferentially removes cellulose from wood.