蛹虫草及其培养残基中腺苷和虫草素含量的快速测定

建立了以氰基正相色谱柱反相条件下快速分析腺苷和虫草素的方法。以微波辅助提取蛹虫草及其培养残基样品,每次提取1.5 min,共提取2次。采用Eclipse XDB-CN色谱柱测定提取液中腺苷和虫草素的含量,以甲醇-水(7:93, v/v)为流动相进行等度洗脱,检测波长260 nm,并考察和分析了影响分离性能的流动相组成和pH值。结果腺苷和虫草素在4.5 min内实现完全分离且无基质干扰。腺苷和虫草素在线性范围内线性关系良好,线性相关系数r2分别为0.9998和0.9995,定量限(以10倍信噪比计)分别为0.21 mg/L(腺苷)和0.083 mg/L(虫草素)。该方法日内和日间精密度的相对标准偏差(RSD)小于2%;腺苷和虫草素的平均加标回收率为93.8%～102.9%, RSD值不大于3.62%(n=5)。本方法简便、快速、准确、成本低,可用于冬虫夏草、蛹虫草子实体、虫草培养残基及虫草制剂中腺苷和虫草素含量的快速测定。     

虫草素与羟丙基-β-环糊精的包合行为及性能研究

通过饱和溶液方法制备了虫草素(COR)与羟丙基-β-环糊精(HPβCD)形成的包合物.采用紫外-可见光谱法对水溶液中HPβCD与虫草素(COR)的包合行为进行研究,利用Job曲线法确定COR/HPβCD包合物的包合比,通过1 H NMR和2D NMR、DSC、TG、XRD、FTIR和SEM对COR/HPβCD包合物进行表征和性能测定.结果表明,COR/HPβCD包合物的包合比为1:1,虫草素与HPβCD形成包合物后,其水溶性、热稳定性及生物环境稳定性都得到明显提高.COR/HPβCD包合物在医药领域具有潜在的应用前景.     

毛细管区带电泳检测九州虫草核苷类成分

1　引　　言核苷是参与遗传信息储存、传递与表达的核酸“构件”分子 ,亦是生物氧化、能量代谢中的能源物质和抗病毒、抗肿瘤药物的中间体。虫草素和腺苷作为虫草属重要的核苷活性成分 ,具有显著的药理作用。高效毛细管电泳应用于中药活性组分的分离、鉴定具有极大的优越性 ,是中药现代化的有益探索。本文采用毛细管区带电泳检测虫草素 ,首次报道了九州虫草 (Ｃｏｒｄｙｃｅｐｓｋｙｕｓｈｕｅｎｓｉｓ)子座中虫草素的存在 ,测定了虫草素、腺苷的含量 ,并初步确定了部分核苷及相关碱基的特征峰。2　实验部分2 .1　仪器与试剂　Ｐ ＡＣＥＴＭ…     

毛细管电泳高频电导法测定虫草中的有效成份

建立了毛细管电泳高频电导法同时测定腺苷和虫草素的方法。实验对电泳介质的种类、浓度以及操作电压和进样时间等因素进行了优化,在4mmol／L乳酸＋10％异丙醇＋80μg／mL羟甲基纤维素钠（pH=4．0）,分离电压20．OkV的条下测定了天然虫草和人工虫草菌丝制品中的腺苷和虫草素的含量,线性范围分别为2．0μg／mL～120μg／mL和3．0μg／mL～110μg／mL,检出限分别为0．5μg／mL和1．0μg／mL。     

虫草素的全合成研究

虫草素具有广谱的生物活性和药理学作用,有着重要的开发价值.为了大量制备高纯度虫草素,分别以D-葡萄糖和D-木糖为原料,以Barton-McCombie反应脱去葡糖糖和木糖3’-羟基合成得到3’-脱氧核糖为关键步骤,分别经过8步和7步化学反应以37%和40%的总产率完成了虫草素的全合成,产物纯度>98.5%.     

高效液相色谱法同时测定虫草素和腺苷

本文用微波法提取蛹虫草培养基和子实体中的虫草素及腺苷,并利用高效液相色谱法同时测定虫草素和腺苷.选择Inertsil ODS-SP柱(150×4.6 mm,5 μm)为色谱柱,乙腈-水为流动相,流速1.0 mL/min,柱温35℃,二极管阵列检测器(DAD)检测,检测波长260 nm,进样量10 μL.在2～10 μg...     

高效液相色谱法测定蛹虫草中腺苷和虫草素

在蛹虫草中腺苷和虫草素含量的反相高效液相色谱分析中,所用色谱柱为WatersNOVA-PAK C18(3.9 mm×300 mm,4μm),流动相为0.01 mol.L-1KH2PO4-K2HPO4缓冲溶液(pH 6.86)+1%(体积分数)四氢呋喃,流速为1.0 mL.min-1。紫外检测的波长为260 nm,峰面积与腺苷浓度和虫草素浓度之间的线性范围依次为1.27~50.5 mg.L-1和3.23~129.0 mg.L-1,其检出限依次为0.18 ng和0.25 ng。在腺苷及虫草素的浓度水平依次为5.05 mg.L-1和12.9 mg.L-1时测试方法的精密度,求算得RSD(n=7)值依次为0.094%和0.792%。用标准加入法试验方法的回收率,求得两者的平均回收率依次为103.2%和98.5%。     

人工蚕蛹虫草和冬虫夏草水溶性成分的相对归属分析

为对相近中药的化学指纹图谱进行比较研究,充分利用联用色谱所得的光谱、色谱信息与光谱相关色谱方法,分析了蚕蛹虫草和冬虫夏草的水溶性成分的异同,实现了蚕蛹虫草和冬虫夏草水溶性成分化学指纹图谱中相应化学组分的归属分析。其中,蚕蛹虫草和冬虫夏草有5个共有组分,分别有4个和6个相异组分。实验表明：该方法可对复杂中药成分及其指纹图谱进行快速比较分析。     

La(NO3)3对蛹虫草活性物质含量的影响EI北大核心CSCD

蛹虫草是一种著名的药用和食用真菌,研究者们常在培养基中添加亚硒酸钠、Fe SO4等无机物来提高蛹虫草活性物质的含量。本文采用高效液相色谱法和紫外分光光度法测定了La(NO3)3处理后蛹虫草活性物质的含量,探究La(NO3)3对蛹虫草活性物质含量的影响。结果表明,在实验浓度范围内,10,50和200 mg·L-1这3个浓度对蛹虫草的活性物质含量影响较大。La(NO3)3浓度为10 mg·L-1时,对喷司他丁、虫草酸和多糖合成具有促进作用,其含量分别为空白组的6.2倍、2.4倍和1.3倍,La(NO3)3对蛹虫草的腺苷和N6-2-羟乙基腺苷合成具有抑制作用,其含量仅为空白组的24.29%和16.87%。La(NO3)3浓度为50 mg·L-1时,对虫草素合成具有显著促进作用,其含量为空白组的8.2倍,La(NO3)3对多糖合成具有抑制作用,其含量仅为空白组的73.14%。La(NO3)3浓度为200 mg·L-1时,对腺苷和N6-2-羟乙基腺苷合成具有促进作用,其含量分别为空白组的1.5倍和2.6倍。La(NO3)3对麦角甾醇和蛋白质的含量未产生显著影响(P>0.05)。     

蛹虫草中硒的赋存形态及蛋白硒分析

为研究蛹虫草中硒的赋存形态,并进一步探究蛹虫草硒蛋白组分中硒含量的分布,以蛹虫草子实体为研究对象,用DAN荧光法测定了硒含量,用连续提取法进行了蛋白硒分析。结果表明,蛹虫草最高硒含量为89.486mg·kg-1,有机硒占85.96%;蛹虫草中蛋白硒、多糖硒和核酸硒分别占有机硒的71.4%~77.1%、19.7%~32.3%和0.138%~2.727%;在硒蛋白组分中虫草盐溶性蛋白、水溶性蛋白、醇溶性蛋白和碱溶性蛋白结合硒分别占总蛋白硒的55.13%~56.80%、20.47%~24.60%、15.30%~16.49%和2.99%~3.35%。蛹虫草具有很强的富硒能力;在蛹虫草子实体中,有机硒主要与蛋白质、多糖、核酸等生物大分子结合,其中蛋白质结合硒是硒的主要赋存形态;在蛋白质组分中,又以盐溶性蛋白质结合硒量最多。     

Cordycepin Inhibits Virus Replication in Dengue Virus-Infected Vero Cells

Dengue virus (DENV) infection causes mild to severe illness in humans that can lead to fatality in severe cases. Currently, no specific drug is available for the treatment of DENV infection. Thus, the development of an anti-DENV drug is urgently required. Cordycepin (3′-deoxyadenosine), which is a major bioactive compound in Cordyceps (ascomycete) fungus that has been used for centuries in Chinese traditional medicine, was reported to exhibit antiviral activity. However, the anti-DENV activity of cordycepin is unknown. We hypothesized that cordycepin exerts anti-DENV activity and that, as an adenosine derivative, it inhibits DENV replication. To test this hypothesis, we investigated the anti-DENV activity of cordycepin in DENV-infected Vero cells. Cordycepin treatment significantly decreased DENV protein at a half-maximal effective concentration (EC50) of 26.94 μM. Moreover, DENV RNA was dramatically decreased in cordycepin-treated Vero cells, indicating its effectiveness in inhibiting viral RNA replication. Via in silico molecular docking, the binding of cordycepin to DENV non-structural protein 5 (NS5), which is an important enzyme for RNA synthesis, at both the methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, was predicted. The results of this study demonstrate that cordycepin is able to inhibit DENV replication, which portends its potential as an anti-dengue therapy.     

A rapid assay to screen adenosine deaminase inhibitors from Ligustri Lucidi Fructus against metabolism of cordycepin utilizing ultra-high-performance liquid chromatography–tandem mass spectrometry

Cordycepin has recently received increased attention owing to its extensive pharmacological activity. Adenosine deaminase (ADA) is widely distributed in mammalian blood and tissues; as a result, cordycepin is quickly metabolized upon entering into the body and converted into the inactive metabolite 3′-deoxyinosine, thus limiting its activity when administered alone. We herein present a novel ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method for screening ADA inhibitors against the metabolism of cordycepin. Cordycepin and 3′-deoxyinosine were chosen as substrate and product, respectively. A proper separation was achieved for all analytes within 3 min. 3′-Deoxyinosine was quantified in the presence or absence of potential ADA inhibitors to evaluate ADA activity. The assay can simultaneously determine substrate and product, with the endogenous substance and ADA inhibitors added not interfering in its activity. After optimizing the enzymatic incubation and UHPLC–MS/MS conditions, K_m and V_max values for ADA deamination of cordycepin were 95.18 ± 7.85 μm and 363.90 ± 12.16 μmol/min/unit, respectively. Oleanolic acid and ursolic acid from Ligustri Lucidi Fructus were chosen as ADA inhibitors with half maximal inhibitory concentration values of 21.82 ± 0.39 and 18.41 ± 0.14 μm , respectively. A non-competitive inhibition model was constructed and this assay can be used to screen other potential ADA inhibitors quickly and accurately.     

Voltammetric determination of boron by using Alizarin Red S

Cordycepin is the main active metabolite of Cordyceps militaris extracts; according to recent studies it has interesting therapeutic activities. A new capillary electrophoresis (CE) procedure with UV detection at 254 nm for determination of cordycepin was developed and optimized. Optimal conditions found were 20 mM sodium borate buffer with 28.6% methanol, pH 9.5, separation voltage 20 kV, hydrodynamic injection time 10 s and temperature 25 °C. Linearity was found over the 20-100 μg/mL concentration ranges of cordycepin. The developed method has been applied for determination of cordycepin in various pharmaceutical products. A comparison was made between CE and a high performance liquid chromatography (HPLC) method. Both of these methods gave comparable results. The shorter analysis time and low running cost are the main advantages of CE method.     

Cordycepin inhibits UVB-induced matrix metalloproteinase expression by suppressing the NF-��B pathway in human dermal fibroblasts

Cordycepin (3''-deoxyadenosine) has been shown to exhibit many pharmacological activities, including anti-cancer, anti-inflammatory, and anti-infection activities. However, the anti-skin photoaging effects of cordycepin have not yet been reported. In the present study, we investigated the inhibitory effects of cordycepin on matrix metalloproteinase-1 (MMP-1) and -3 expressions of the human dermal fibroblast cells. Western blot analysis and real-time PCR revealed cordycepin inhibited UVB-induced MMP-1 and -3 expressions in a dose-dependent manner. UVB strongly activated NF-κB activity, which was determined by IκBα degradation, nuclear localization of p50 and p65 subunit, and NF-κB binding activity. However, UVB-induced NF-κB activation and MMP expression were completely blocked by cordycepin pretreatment. These findings suggest that cordycepin could prevent UVB-induced MMPs expressions through inhibition of NF-κB activation. In conclusion, cordycepin might be used as a potential agent for the prevention and treatment of skin photoaging.     

The Role of Autophagy in Anti-Cancer and Health Promoting Effects of Cordycepin

Cordycepin is an adenosine derivative isolated from Cordyceps sinensis, which has been used as an herbal complementary and alternative medicine with various biological activities. The general anti-cancer mechanisms of cordycepin are regulated by the adenosine A3 receptor, epidermal growth factor receptor (EGFR), mitogen-activated protein kinases (MAPKs), and glycogen synthase kinase (GSK)-3β, leading to cell cycle arrest or apoptosis. Notably, cordycepin also induces autophagy to trigger cell death, inhibits tumor metastasis, and modulates the immune system. Since the dysregulation of autophagy is associated with cancers and neuron, immune, and kidney diseases, cordycepin is considered an alternative treatment because of the involvement of cordycepin in autophagic signaling. However, the profound mechanism of autophagy induction by cordycepin has never been reviewed in detail. Therefore, in this article, we reviewed the anti-cancer and health-promoting effects of cordycepin in the neurons, kidneys, and the immune system through diverse mechanisms, including autophagy induction. We also suggest that formulation changes for cordycepin could enhance its bioactivity and bioavailability and lower its toxicity for future applications. A comprehensive understanding of the autophagy mechanism would provide novel mechanistic insight into the anti-cancer and health-promoting effects of cordycepin.     

毛细管区带电泳法测定冬虫夏草及鹿茸制品中核苷和碱基成分

应用毛细管区带电泳法测定分别以冬虫夏草菌丝体粉和鹿茸血为主要原料制品中的多种核苷和碱基成分。对实验条件进行了优化,结果表明,以20mmol/L硼砂-15mmol/Lβ-环糊精为缓冲溶液(pH=9.4),分离电压22kV,检测波长254nm,电动进样为10kV、5s时,在10min内同时分离测定了虫草素、腺嘌呤、鸟嘌呤、尿嘧啶、腺苷、次黄嘌呤、尿苷、鸟苷和肌苷。各组分在0.2～200μg/mL范围内呈线性关系,检出限的范围是0.07～1.67μg/mL。5个批次的冬虫夏草菌丝粉保健品中腺嘌呤、尿嘧啶、腺苷、鸟苷、尿苷5组分的定量结果分别在0.15～0.19mg/g、0.72～0.92mg/g、1.44～1.59mg/g、1.51～2.32mg/g和1.77～2.56mg/g范围内,加标回收率的范围是82.83%～109.21%;2个批次的鹿茸血保健品中次黄嘌呤、尿苷的定量结果在36.55～49.97μg/mL和86.08～108.97μg/mL范围内,加标回收率的范围分别是89.68%～96.79%和99.05%～102.81%。     

Cordycepin Sensitizes Cholangiocarcinoma Cells to Be Killed by Natural Killer-92 (NK-92) Cells

Immunotherapy harnessing immune functions is a promising strategy for cancer treatment. Tumor sensitization is one approach to enhance tumor cell susceptibility to immune cell cytotoxicity that can be used in combination with immunotherapy to achieve therapeutic efficiency. Cordycepin, a bioactive compound that can be extracted from some Cordyceps spp. has been reported to effectively inhibit tumor growth, however, the mechanism of its tumor sensitization activity that enhances immune cell cytotoxicity is unknown. In the present study, we investigated the potency of cordycepin to sensitize a lethal cancer, cholangiocarcinoma (CCA), to natural killer (NK) cells. Treatment with cordycepin prior to and during co-culturing with NK-92 cells significantly increased cell death of KKU-213A as compared to solitary cordycepin or NK treatment. Moreover, sensitization activity was also observed in the combination of NK-92 cells and Cordyceps militaris extract that contained cordycepin as a major component. The cordycepin treatment remarkably caused an increase in TRAIL receptor (DR4 and DR5) expression in KKU-213A, suggesting the possible involvement of TRAIL signaling in KKU-213A sensitization to NK-92 cells. In conclusion, this is the first report on the sensitization activity of cordycepin on CCA cells to NK cytotoxicity, which supports that cordycepin can be further developed as an alternate immunomodulating agent.     

SE-HPLC-ICP-MS联用技术在富硒蛹虫草硒蛋白形态分析中的应用研究

从富硒培养的虫草中提取可溶性蛋白质, 经过(NH4)2SO4分级盐析, 采用ICP-MS对不同盐析组分中蛋白及硒分布进行测定, 结果表明, 富硒蛹虫草的可溶性硒含量占总硒含量的69.3%以上, 在所得的各级蛋白组分中, 除A组分(0~30%饱和度的盐析)外, 其它各组分均存在含硒蛋白, 其硒含量高低顺序为B组分(30%~50%饱和度的盐析)>C组分(50%~75%饱和度的盐析)>D组分(75%~100%饱和度的盐析)>A组分. 采用SE-HPLC-ICP-MS联机技术对样品中的硒蛋白进行分离检测, 结果表明, 其分子量分布在114000和1000之间， 且含量各异. 进一步研究结果表明, 硒在虫草体内参与了蛋白的合成, 这对于以蛹虫草为载体将无机硒有机化具有重要意义.     

An effective and convenient synthesis of cordycepin from adenosine

Cordycepin is a purine nucleoside analog with potent and diverse biological activities. Herein, we designed two methods to synthesize cordycepin. One method mainly converted the 3′-OH group into an iodide group and further dehalogenation to yield the final product. Although this method presented a short synthetic procedure, the synthesis had a low overall yield, resulting in only 13.5% overall yield. To improve the overall yield of cordycepin, another synthetic route was studied, which consisted of four individual steps: (1) 5′-OH protection (2) esterification (3) -O-tosyl (-OTs) group removal (4) deprotection. The key step in the synthetic method involved the conversion of 5′-O-triphenylmethyladenosine to 3′-O-tosyl-5′-O-triphenylmethyladenosine, using LiAlH₄ as reducing agent. The main advantages of this route were an acceptable total product yield and the commercial availability of all starting materials. The optimal reaction conditions for each step of the route were identified. The overall yield of cordycepin obtained from adenosine as the starting material was 36%.     

Simultaneous determination of cordycepin and its metabolite 3′-deoxyinosine in rat whole blood by ultra-high-performance liquid chromatography coupled with Q Exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometry and its application to accurate pharmacokinetic studies

Cordycepin from Cordyceps possesses excellent pharmacological properties, including anti-inflammation and anti-tumor effects, therefore representing a potential alternative medicine. However, doubts about the pharmacokinetic results of cordycepin had been raised in the previous study due to its rapid deamination. The organic solvent methanol was immediately added to terminate the degradation of cordycepin in anticoagulated blood samples and enable the accurate evaluation of pharmacokinetics in vivo. A sensitive and selective ultra-high-performance liquid chromatography coupled with Q Exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometry method was developed and validated to simultaneously determine cordycepin and its deamination metabolite 3′-deoxyinosine using 2-chloroadenosine as an internal standard in rat whole blood. The calibration curves of cordycepin and 3′-deoxyinosine showed excellent linearity within the concentration range of 1.05–10 000.00 ng/ml with acceptable accuracy, precision, selectivity, recovery, matrix effect, and stability. This method was successfully applied to the pharmacokinetic study of cordycepin and its metabolite in rat blood. The effect of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride on the pharmacokinetics of cordycepin was investigated. In summary, the reliable pharmacokinetic parameters of cordycepin and its deamination metabolite 3′-deoxyinosine in rat blood were successfully elucidated. Erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride considerably prolonged the half-life of cordycepin in vivo.