An unusual lanostane-type triterpenoid,spiroinonotsuoxodiol, and other triterpenoids from Inonotus obliquus

An unusual lanostane-type triterpenoid, spiroinonotsuoxodiol (1), and two lanostane-type triterpenoids, inonotsudiol A (2) and inonotsuoxodiol A (3), were isolated from the sclerotia of Inonotus obliquus. Their structures were determined to be (3S,7S,9R)-3,7-dihydroxy-7(8 → 9)abeo-lanost-24-en-8-one (1), lanosta-8,24-dien-3β,11β-diol (2), and (22R)-3β,22-dihydroxylanosta-8,24-dien-11-one (3) on the basis of NMR spectroscopy, including 1D and 2D (¹H–¹H COSY, NOESY, HMQC, HMBC) NMR, and FABMS. Compounds 1–3 showed moderate activity against cultured P388, L1210, HL-60 and KB cells.     

桦褐孔菌三萜化合物提取工艺的优选

为了提高桦褐孔菌三萜化合物的产量,对其三萜化合物的提取工艺进行了初步优选。试验以桦褐孔菌发酵菌丝体为材料,齐墩果酸为标准品,用香草醛比色法测桦褐孔菌三萜化合物含量。通过单因素实验考察了不同提取剂、提取剂用量、提取温度、提取时间对桦褐孔菌三萜化合物提取的影响。结果表明,提取的最佳工艺为：以异丙醇为提取溶剂,用量为7倍,最佳提取温度为80℃,提取时间2h,在此条件下三萜化合物占菌丝体干质量的9．03％。该工艺的三萜桦褐物的提取率高,可用于桦褐孔菌中三萜化合物的提取。     

双水相超声法提取桦褐孔菌三萜

通过单因素实验和正交实验对影响桦褐孔菌三萜提取的异丙醇体积浓度、硫酸铵质量浓度、料液比和超声时间等因素进行探讨,旨在建立桦褐孔菌Inonotus obliquus菌丝体三萜类化合物的提取工艺。结果表明,提取桦褐孔菌三萜的最佳条件为：异丙醇体积浓度40%,硫酸铵质量浓度0.125g/mL,料液比（W/V）0.9:1,超声时间为35min,三萜提取率为6.18%,提取效率明显高于常规超声波提取法（5.38%）。利用该方法获得的三萜具有较强清除自由基的能力。通过正交实验获得的桦褐孔菌三萜的提取工艺是一种经济、有效的方法。     

Antioxidant activities of five polysaccharides from Inonotus obliquus

Five polysaccharides (IOP1b, IOP2a, IOP2c, IOP3a and IOP4) were isolated and purified from Inonotus obliquus by DEAE-Sepharose fast flow and SepharoseCL-6B column chromatography. Their chemical and physical characteristics were determined and antioxidant activities were investigated on the basis of hydroxyl radical assay, superoxide radical assay and ferric-reducing antioxidant power assay. The results showed that five polysaccharides exhibited antioxidant activities, and the higher content of uronic acid and proteinous substances, the stronger antioxidant activities of polysaccharides. Besides, molecular weights of polysaccharides also influence their antioxidant activities. IOP3a and IOP4 showed higher antioxidant properties than IOP1b, IOP2a and IOP2c.     

桦褐孔菌子实体多糖提取研究

目的:为了获得桦褐孔菌多糖最大量的多糖收率,研究了影响多糖提取的最佳条件.方法:对影响桦褐孔菌胞内水溶性多糖提取效果的6个因素进行了单一因素影响实验,并对多糖提取影响因子的3个因素进行正交试验,对多糖提取工艺参数进行了优化.结果:正交试验确定桦褐孔菌子实体多糖的最佳提取条件是:料水比为1∶40,温度80℃,提取1.5h,多糖收率达2.53％.结论:确立了桦褐孔菌子实体多糖提取工艺,建立了一套简便、高效的桦褐孔菌胞内多糖的提取方法.     

桦褐孔菌野生分离菌株的比较与栽培选育

通过菌丝形态比较、ISSR分析鉴定及人工驯化栽培选育,对1株野生分离菌株和4株保藏菌株进行比较。结果显示:5个菌株在菌丝形态和长势上差异显著,1号菌株(野生分离)菌丝洁白,长速最快;ISSR扩增图谱谱带差异明显,1号菌株与5号菌株在遗传相似水平85%时聚为1类,其他菌株各自聚为独立1类。驯化栽培试验结果表明:1号菌株出核较快,菌核大,产量高,较其他菌株产量差异显著。被试菌株为各自独立菌株,1号菌株经驯化适宜高产栽培,是重要珍稀野生种质资源。本研究可为野生桦褐孔菌菌株种质资源收集和为实现大规模栽培育种提供亲本资源。     

New antioxidant polyphenols from the medicinal mushroom Inonotus obliquus

The fruiting body of Inonotus obliquus, a medicinal mushroom called chaga, has been used as a traditional medicine for cancer treatment. Although this mushroom has been known to exhibit potent antioxidant activity, the mechanisms responsible for this activity remain unknown. In our investigation for free radical scavengers from the methanolic extract of this mushroom, inonoblins A (1), B (2), and C (3) were isolated along with the known compounds, phelligridins D (4), E (5), and G (6). Their structures were established by extensive spectroscopic analyses. These compounds exhibited significant scavenging activity against the ABTS radical cation and DPPH radical, and showed moderate activity against the superoxide radical anion.     

桦褐孔菌酚类化合物及其免疫促进作用

Phenolic compounds from field-grown Inonotus obliquus sclerotia (Chaga) consist mainly of hispidin analogs and melanins, and are thought to be the active constituents to treat several human diseases. In submerged cultures of the fungus, however, no information is currently available on the production of phenolic compounds and their corresponding pharmacological functions. In this study, phenolic compounds from Chaga and submerged cultures of the fungus were assayed for their composition and immune-stimulating effects. Phenolic compounds produced by I. obliquus in submerged cultures mostly consist of flavonoids, together with small amounts of hispidin analogs and melanins. This is quite contrary to the situation in Chaga, where flavonoids are determined as trace elements. Furthermore, phenolic compounds from Chaga show capacity about two-fold higher than those produced in submerged cultures in inhibiting cyclophosphamide-induced reduction of bodyweight, spleen index and viability of peripheral lymphocytes in test mice. Thus less production of hispidin analogs and melanins is likely to be responsible for less immune-stimulating effects in phenolic compounds from submerged cultures, and additional factors should be imposed during submerged cultures of I. obliquus to regulate biosynthesis of phenolic compounds directed to the composition similar to Chaga.     

Antitumor and immunomodulatory activity of water-soluble polysaccharide from Inonotus obliquus

The medicinal mushroom Inonotus obliquus has been used as a folk remedy for a long time in Russia and East-European countries to treat gastrointestinal cancer, cardiovascular disease and diabetes. In our study, a water-soluble polysaccharide (ISP2a) was successfully purified from I. obliquus by DEAE-Sepharose CL-6B and Sepharose CL-6B column chromatography. In vivo ISP2a had not only shown antitumor activity, but also could significantly enhance the immune response of tumor-bearing mice. In addition, ISP2a significantly enhanced the lymphocyte proliferation and increased the production of TNF-α. Results of these studies demonstrated that ISP2a had a potential application as natural antitumor agent with immunomodulatory activity.     

Synthetic lanostane-type triterpenoids as inhibitors of DNA topoisomerase II

DNA topoisomerase (Topo) II is one of the target enzymes for chemotherapeutic drug development. Lanostane-type triterpenoids with various functional groups (-Cl, -Br, -OMe, -CHO, -CN, -COOH, and -COOMe) at C-2 were synthesized from 3-oxolanost-9(11)-en-24S,25-diol (9) isolated from Pinus luchuensis and their inhibitory effects on Topo II activity and cytotoxic activities against A549 cells were examined. All the derivatives showed Topo II inhibitory effects with IC50 values ranging from 1.86 to 149.97 microM and cytotoxic activities with ED50 values ranging from 3.96 to 38.15 microM.     

Melanin complex of the fungus Inonotus obliquus

The fungus Inonotus obliquus (Pers.) Pil. synthesised high-molecular-weight phenolic pigments that were assigned to melanins according to their physicochemical properties. It was showed that copper ions (0.008%), pyrocatechol (1.0 mM), and tyrosine (20.0 mM) stimulated the melanogenesis. The production of melanin correlated with the synthesis of o- and p-diphenoloxidases. The fungal melanin had strong antioxidant and genoprotective effects.     

桦褐孔菌的研究进展

概述了国内外桦褐孔菌的分类学、生态学、人工栽培及药用研究动态的最新进展和应用前景。     

桦褐孔菌膜二肽酶在巴斯德毕赤酵母中的分泌表达

本研究利用巴斯德毕赤酵母Pichia pastoris蛋白表达体系表达了药用担子菌桦褐孔菌的一个二肽酶基因。该二肽酶基因编码区全长1814bp,包含6个内含子,编码465个氨基酸。生物信息学分析发现,二肽酶基因编码的蛋白中不含信号肽序列,但在第55–77位氨基酸之间存在一个跨膜结构。将含跨膜结构和去跨膜结构蛋白的cDNA序列分别克隆到酵母分泌型表达载体pPICZαA上,电转化至巴斯德毕赤酵母X-33中,用1%(V/V)甲醇诱导重组菌株表达目标蛋白,采用SDS-PAGE和Western-blot检测表达蛋白。结果显示,巴斯德毕赤酵母可表达含跨膜结构的完整基因,但目标蛋白不能分泌到胞外,存在于破碎细胞的沉淀中,且没有催化活性;而去跨膜结构的蛋白则可分泌表达到胞外,并具有催化活性。Ni-NTA纯化去跨膜结构的桦褐孔菌二肽酶浓度可达0.12mg/mL,并发现其在pH 7.3、反应温度50℃、反应时间2h的条件下,以Gly-Gly为底物时,其比活为433U/mg。同时检测到其对Ile-Leu、Trp-Trp和Phe-Phe具有较高的水解活性。     

Folin-Ciocalteu比色法测定桦褐孔菌多酚的条件优化

以没食子酸为标准品,通过正交试验和单因素实验研究了Folin-Ciocalteu 比色法测定桦褐孔菌中多酚含量的适宜条件。结果表明,优化后的显色条件为Folin-Ciocalteu试剂用量0.3mL、10% Na2CO3溶液0.6mL,25℃时避光反应30min,于750nm处测定其吸光值。多酚质量浓度在0.0032－0.0256mg/mL范围内与吸光值有良好的线性关系。根据拟合的线性回归方程对桦褐孔菌多酚进行定量测定,野生桦褐孔菌和人工培养桦褐孔菌中多酚含量最高分别为（72.05±0.08）mg/g、（52.76±0.06）mg/g（n=6）。不同加标水平的加标回收率测定实验获得的平均回收率为98.95%,RSD为1.27%。该法测定桦褐孔菌多酚具有快速、准确,稳定性强,重现性好,精密度高的特点,可应用于实际样品的测定。     

Cloning, expression and characterization of squalene synthase from Inonotus obliquus

The chaga mushroom Inonotus obliquus has been widely used as a folk medicine in Russia, Poland and most of the Baltic countries. The total triterpene saponins of I. obliquus have significant pharmacological activity. Though the triterpene component has been well characterized in terms of its pharmaceutical activity, there is little information on the genes responsible for the biosynthesis of these compounds in I. obliquus. Squalene synthase represents a potential branching point and the first committed step to diverge the carbon flux from the main isoprenoid pathway towards sterol biosynthesis. In this study, we cloned and characterized squalene synthase from I. obliquus. A 1476-bp full-length cDNA consisting of the entire coding region of squalene synthase (GenBank accession number is KC182754) was cloned by RT-PCR. The DNA sequence showed as much as 76 % similarity with the sequence of Fomitiporia mediterranea squalene synthase, and phylogenetic analysis indicated that it is most closely related to F. mediterranea squalene synthase at both DNA and protein levels. I. obliquus squalene synthase was actively expressed in the yeast Pichia pastoris as a secreted form and purified by gel filtration using Superdex G-75 column. The purified recombinant squalene synthase was able to convert farnesyl diphosphate (FPP) to squalene in an NADPH-dependent reaction. The result of this study could serve as an important step toward the manipulation of triterpenoids biosynthesis in I. obliquus at the level of squalene through engineering better SQS for reintroduction into the mushroom.     

Two perenniporiol derivatives,lanostane-type triterpenoids,from the cultured mycelia of Perenniporia ochroleuca

Two new perenniporiol derivatives, lanostane-type triterpenoids, were isolated from the benzene extract of cultured mycelia of Perenniporia ochroleuca. The structures of the two new compounds were elucidated by spectral data to be 3-O-acetyl-7,11-dihydroperenniporiol and 12β-acetoxyperenniporiol.     

桦褐孔菌的化学成分及抗炎活性

本实验研究了桦褐孔菌的化学成分及其抗炎活性。方法是采用溶剂法和柱色谱法提取分离桦褐孔菌的化学成分,根据化合物的理化性质及核磁共振氢谱、碳谱、质谱对其化学结构进行鉴定,然后采用二甲苯致小鼠耳肿胀和二甲苯致小鼠腹膜炎模型对所得化合物的抗炎活性进行考察。最终从桦褐孔菌的乙醇提取物中分离得到3个羊毛脂烷型三萜类化合物,分别鉴定为羊毛甾醇(1)、inotodiol(2)和trametenolic acid(3)。化合物1、2、3在10 mg/kg均显著抑制二甲苯致小鼠耳肿胀,化合物1和2在10 mg/kg时显著降低血管通透性。由此得出化合物1～3均有抗炎活性,其中trametenolic acid(3)的抗炎活性最强。     

New lanostane-type triterpenoids, inonotsutriols D, and E, from Inonotus obliquus

Two new lanostane-type triterpenoids, inonotsutriols D (1) and E (2), were isolated from the sclerotia of Inonotus obliquus (Pers.: Fr.) Pil. (Japanese name: kabanoanatake; Russian name: chaga). Their structures were determined to be lanost-8-ene-3β,22R,24R-triol (1) and lanost-8-ene-3β,22R,24S-triol (2) on the basis of spectral data, including 2D NMR analysis. In addition, major compounds, inotodiol (3), trametenolic acid (4), 3β-hydroxylanosta-8,24-dien-21-al (5), 21-hydroxylanosterol (6), inonotsuoxide A (7) and inonotsuoxide B (8) were identified, and all compounds, except 2, were evaluated for their cancer cell growth inhibitory activity against P388, HL-60, L1210 and KB cell lines.     

Anti-cancer effect and structural characterization of endo-polysaccharide from cultivated mycelia of Inonotus obliquus

The endo-polysaccharide extracted from mycelia of Inonotus obliquus (Pers.:Fr.) Pil. (Hymenochaetaceae) is a specific activator of B cells and macrophages. However, the in vivo anti-cancer effects and the chemical structure of the endo-polysaccharide are unknown. We purified the endo-polysaccharide, investigated its anti-cancer effects via in vitro and in vivo assays, and performed a structural characterization. The endo-polysaccharide was extracted from I. obliquus mycelia cultivated in a 300-l pilot fermenter, followed by hot water extraction and ethanol precipitation. Purification was achieved by DEAE-cellulose ion-exchange chromatography and gel-permeation chromatography. Chemical analysis revealed that the purified endo-polysaccharide is an alpha-linked fucoglucomannan with a molecular weight of approximately 1,000 kDa. The anti-cancer activities of the endo-polysaccharide against various types of tumor cells were determined. No direct toxicity against either cancer or normal cells was observed. Intraperitoneal administration of the endo-polysaccharide significantly prolonged the survival rate of B16F10-implanted mice, resulting in a 4.07-fold increase in the survival rate at a dose of 30 mg/kg/day. After 60 days of feeding, approximately 67% of the initial number of mice survived with no tumor incidence based on macroscopic examination. These results indicate that the anti-cancer effect of endo-polysaccharide is not directly tumorcidal but rather is immuno-stimulating.     

桦褐孔菌菌质活性组分的提取及其体外抗氧化作用

为提取桦褐孔菌菌质各活性组分并研究其体外抗氧化活性,将桦褐孔菌菌质醇提后分别梯度萃取获得各极性组分,将残渣沸水浸提醇沉得粗多糖;将获取的各活性组分别进行DPPH自由基清除率、羟基自由基清除率和总抗氧化能力的测定,并测定各组分中总多酚、总三萜的含量;同时与桦褐孔菌菌核进行对比。研究结果表明菌质和菌核乙酸乙酯、正丁醇组分对DPPH自由基、OH自由基清除能力及其总抗氧化能力明显优于相应的其他组分(P0.01);菌质的乙酸乙酯、正丁醇组分对DPPH自由基的清除能力与菌核相应组分比较无显著性差异(P0.05),菌质和菌核的乙酸乙酯、正丁醇部位的总三萜和总多酚含量较高。结果显示桦褐孔菌菌质的乙酸乙酯、正丁醇组分的体外抗氧化活性较好,总多酚和总三萜含量较高,抗氧化活性的强弱可能与该二者活性成分的含量相关。通过双向固体发酵技术制得的桦褐孔菌菌质体外抗氧化作用较好。