灵芝子实体中三萜类物质抗肿瘤活性的谱效关系

通过HPLC指纹图谱结合多元线性回归分析对不同产地灵芝子实体的功效性特征进行评价,为寻找灵芝中活性三萜提供理论依据。利用高效液相分析方法,结合样品对肿瘤细胞L1210的增殖抑制率,运用“中药色谱指纹图谱相似度评价系统2004A版”软件和多元线性回归分析11批不同品种灵芝子实体中的三萜活性成分。样品与标准指纹图谱的相似度均在0.9以上,共标定了12个共有物质峰,其中与抗L1210肿瘤细胞活性关系密切的三萜物质有灵芝酸C₂、灵芝酸G、灵芝烯酸B、灵芝烯酸A、灵芝酸K、灵芝酸A、灵芝酸F和灵芝醛A。     

HPLC法测定灵芝孢子粉中三萜含量

为准确测定灵芝孢子粉中三萜的含量,运用高效液相建立适合孢子粉的分析测定方法。通过对前处理条件的优化,确定40%乙醇为孢子粉中等极性三萜酸类的最佳提取溶剂,浓缩倍数是子实体提取条件的50倍。通过色谱柱和洗脱条件的优化,建立了包括灵芝酸I、灵芝烯酸C、灵芝酸C2等13种标准品测定方法,方法学考察显示该分析方法精密度、重复性、稳定性的RSD值均小于5%,可以用于灵芝孢子粉中三萜类成分的定量检测。通过5组样品的分析发现,灵芝酸C6、灵芝酸G、灵芝酸A、灵芝酸D、灵芝酸F是灵芝孢子粉中的主要三萜类成分,其中灵芝酸A含量最高,平均占样品三萜总量的比例达19.71%;三萜类成分的溶出量与是否破壁没有相关性。三萜类成分在灵芝孢子粉和灵芝孢子油产品中的含量非常低,孢子粉的三萜含量为14.24-99.70μg/g,仅为子实体的1/100,灵芝孢子油中三萜含量也均低于50μg/g,因此三萜类成分不适合作为灵芝孢子粉及其相关产品的定量检测指标。     

栽培条件对灵芝子实体的三萜组成及其活性的影响

以不同灵芝品种、不同栽培基质、不同管理方式、不同生长时期获得的灵芝子实体为原料,用95%乙醇超声提取,对提取物进行了含量测定、三萜组成分析和体外抗肿瘤实验。结果表明,不同子实体醇提物中三萜和甾醇类物质的含量在4.60-6.20mg/g之间;高效液相分析发现10种三萜化合物的种类和含量在样品间存在明显差异。所有灵芝子实体醇提物对肿瘤细胞L1210的增殖均有一定的抑制作用,开伞期的灵芝子实体醇提物抑制肿瘤增殖的活性优于其他生长时期。     

灵芝孢子粉中核苷类成分分析

本文利用高效液相色谱方法（HPLC）同时对灵芝孢子粉中的15种核苷类成分的含量进行测定。采用Ultimate AQ-C₁₈（4.6mm×250mm,5μm）色谱柱,以甲醇和水为流动相进行梯度洗脱,流速1.0mL/min,检测波长259nm,柱温30℃,进样量10μL。方法学考察结果表明,该方法准确度高,稳定性、精密度、重现性好,适用于灵芝孢子粉中核苷类成分的测定分析。运用建立的方法对不同破壁时间、不同采收时期龙泉、奉化、大别山、黄山4个产区的灵芝孢子粉中的15种核苷类成分的含量进行测定。结果表明破壁处理对灵芝孢子粉中核苷类成分提取率的影响不大,不同产地的灵芝孢子粉中核苷类成分的组成和含量具有显著差异,且孢子粉中的核苷含量随着产粉时间的延长有所增加。各待测样品中均含有胞嘧啶、尿苷、腺嘌呤、鸟苷、腺苷等成分,其中尿苷、鸟苷、腺苷3种核苷的含量占总量的比例在待测样品中均达到70%以上,为灵芝孢子粉中的主要核苷类成分。     

层迭灵芝子实体的体外抗肿瘤及免疫活性

【背景】层迭灵芝Ganoderma lobatum是灵芝属中的一个种,在民间有药用历史,但缺乏对其化学成分和药理活性的科学研究。【目的】以赤芝Ganoderma lingzhi子实体为参照,研究对比层迭灵芝子实体的抗肿瘤及免疫活性的强弱,探讨层迭灵芝的药用价值。【方法】采用化学分析及仪器分析的方法,比较2种灵芝子实体中三萜及多糖含量差异,并进行体外抗肿瘤及免疫活性研究。【结果】层迭灵芝和赤芝的子实体中三萜含量差异不大,分别为1.14%和1.21%,但2种灵芝中三萜化合物的种类差异较大。层迭灵芝子实体中的多糖含量较赤芝稍高,分别为3.60%和2.67%,2种子实体中多糖的重均分子量分布特征有所差别。2种灵芝醇提物对肿瘤细胞K562及SW620的增殖均具有一定的抑制活性,其中,层迭灵芝对SW620细胞具有较强的抑制活性,其IC50值达到了52.5μg/mL。2种灵芝水提物可以促进RAW 264.7细胞释放NO,说明两者均具有一定的免疫活性。【结论】层迭灵芝具有较好的抗肿瘤及免疫活性,可以作为药用开发的原料来源。     

中药非药用部位栽培灵芝的活性成分及药效变化

【目的】以丹参(Salvia miltiorrhiza Bge.)、菊花(Chrysanthemum morifolium Ramat.)、桔梗(Ptatycodongrandiflorum A.DC.)3种中药材的非药用部位作为灵芝袋料栽培的原料,研究灵芝子实体活性成分及药效变化。【方法】试验比较测定了非药用部位配方组与常规组(常规组作为实验对照组,配方由玉米芯、棉籽壳等常规基质组成)的灵芝农艺性状,子实体多糖和三萜含量,并对各组灵芝进行了小鼠急性毒性试验及药效试验。【结果】结果表明,非药用部位栽培灵芝生物转化率接近或者高于常规组,生长周期有所延长;活性成分上,除了丹参组(SM.G)的三萜含量有所降低外,其余各组的活性成分较常规配方组(OF.G)均有提高,菊花组(CM.G)灵芝的多糖和三萜含量最高,分别为2.47%和0.79%。最大耐受量试验表明,非药用部位栽培的灵芝子实体的小鼠最大耐受量均为100 g/kg。溶血素试验和促睡眠试验中菊花组效果优于常规组灵芝;抗疲劳上,只有常规组灵芝表现出一定的抗疲劳功效,而非药用部位栽培的灵芝没有该药效。【结论】中药材非药用部位栽培灵芝是可行的,且子实体活性成分含量和部分药效发生了改变。     

灵芝子实体中的羊毛脂烷型灵芝酸酯类成分研究

目的系统研究灵芝子实体中的羊毛脂烷型灵芝酸酯类成分。方法通过氯仿对盐酸酸化的灵芝子实体提取物中的羊毛脂烷型三萜酸类成分进行萃取,结合硅胶柱层析、中压柱层析(RP C18色谱柱)、制备型HPLC(RP C18色谱柱)对提取物中的三萜酸酯类化学成分进行系统的分离、纯化。通过UV、ESIMS、HRESI-MS、1 H-NMR、13C-NMR、HSQC、HMBC、NOESY等波谱学手段结合文献数据对制备得到的三萜类单体成分进行准确的结构测定。结果从灵芝子实体的氯仿提取物中共分离鉴定了10个羊毛脂烷型灵芝酸酯类成分,分别为:(1)Ganoderic acid AP,(2)Methyl Ganoderate G,(3)Ganoderic acid H,(4)12β-Hydroxy-3,7,11,15,23-pentaoxo-5α-lanost-8-en-26-oic acid,(5)Ganoderic acid K,(6)Ganoderenic acid B,(7)Lucienic acid A,(8)Lucidenic acid B,(9)20(21)-Dehydrolucidenic acid A,(10)Lucidone D。结论灵芝子实体中极性较小的化学成分主要为羊毛脂烷型三萜及其降碳衍生物,该类成分多具有羧基或羧酸酯官能团,既而也称灵芝酸类,本文共分离鉴定了10个该系列成分,其中化合物1为新化合物。     

南方水稻黑条矮缩病毒诱导的水稻挥发物及白背飞虱成虫对其组分的行为反应

【目的】南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)是白背飞虱Sogatella furcifera在稻苗间传播的一种持久性病毒。本研究旨在调查SRBSDV是否诱导水稻挥发物的含量及成分发生变化,并探究变化的组分是否对白背飞虱具引诱或驱避作用。【方法】采用动态顶空法收集3个水稻品种病/健植株的挥发物,利用气相色谱-质谱联用仪(GC-MS)分析各品种不同生育期病/健株挥发物的组分,使用嗅觉测定仪测定了白背飞虱对病株特有挥发物的嗅觉行为反应。【结果】从健株检测到11大类化合物共36种;从病株检测到12大类化合物共37种。病株特有挥发物12种,即正十六烷、癸醚、butyl octyl phthalate、正十八烷、己二酸二异丁酯、正十五碳醛、十六烷酸甲酯、2-methylhexacosane、二苯甲酮、雪松醇、正二十烷和叶绿醇;病/健株中共有但含量差异显著的挥发物6种,即1-tricosene、octadecanal、萘、植酮、正十二烷醇和2,6-二叔丁基对甲酚。嗅觉选择实验表明,在50μL/L浓度下,仅叶绿醇对白背飞虱成虫(健康与饲毒)表现出显著的驱避活性。【结论】SRBSDV的侵染改变了水稻挥发物的组分,且不同品种、不同生育期挥发物组分均具差异。叶绿醇为孕穗期荣优华占病株特有挥发物,对健康与饲毒白背飞虱均有驱避活性,是否有助于促进该病毒传播,尚需进一步验证。     

灵芝子实体中不同极性的三萜体外抗肿瘤及抗炎活性比较

为了解灵芝中不同极性三萜活性的差异,运用大孔树脂将灵芝总三萜根据极性大小进行分段,采用高效液相法分析各极性部分的HPLC指纹图谱和三萜含量,并比较其对不同肿瘤细胞增殖的抑制作用和抗炎活性。结果显示D-101大孔树脂可以有效地将灵芝总三萜分为中等极性和低极性两部分,两部分的三萜含量分别为(279.00±2.90)mg/g、(94.52±2.03)mg/g。其中,低极性三萜的体外抗肿瘤活性明显强于中等极性三萜,其对K562、SW620、L1210细胞增殖抑制的IC₅₀值分别为(74.12±1.94)μg/mL、(121.45±2.13)μg/mL、(13.52±1.13)μg/mL。另外,低极性三萜对RAW264.7细胞呼吸爆发的抑制作用也明显强于中等极性三萜。进一步对低极性三萜和中等极性三萜单体的抗炎活性进行比较,发现低极性三萜灵芝烯酸F、灵芝酸DM、灵芝醇B对RAW264.7细胞呼吸爆发的抑制作用明显强于极性较高的灵芝酸C₂、灵芝酸A。该研究阐明了灵芝子实体中不同极性三萜部位的抗肿瘤及抗炎活性并不相同,为将来新药开发和灵芝质量标准的改进建立了基础。     

人工栽培灵芝中多糖的部分理化性质及免疫调节作用

【背景】灵芝是一种广受关注的药食两用真菌,在中国作为传统药材和健康食品已有几千年历史,近年来中国人工栽培灵芝规模扩展迅速,而多糖被认为是灵芝中最主要的活性成分。【目的】分离和纯化人工栽培灵芝子实体多糖,并对其结构和免疫调节活性进行研究。【方法】采用水提醇沉法提取灵芝子实体多糖(GLP),采用DE-52纤维素柱和Sephadex G-100进行多糖的分离纯化,高效凝胶渗透色谱法测定多糖分子量,1-苯基-3-甲基-5-吡啶啉酮(PMP)柱前衍生法测定单糖组成,红外光谱进行结构分析,噻唑蓝(MTT)比色法测定多糖对小鼠脾细胞增殖的影响,同时评价其对RAW 264.7细胞吞噬能力和细胞因子分泌能力的促进作用。【结果】GLP平均分子量为1.93×10~4 Da,单糖组成包含葡萄糖(Glc)、半乳糖(Gal)及甘露糖(Man),占比为Glc:Gal:Man=3.3:1.3:1.0。红外光谱显示,GLP的异头碳为β构型。GLP能直接促进脾细胞的增殖,且能显著增强刀豆蛋白A(Con A)诱导的T淋巴细胞和脂多糖(LPS)诱导的B淋巴细胞的增殖。此外,对于RAW 264.7细胞的吞噬能力及细胞因子分泌具有一定的促进作用。【结论】灵芝多糖可作为一个潜在的免疫调节药物而开发利用。     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.