冬虫夏草菌丝体水提醇沉物体外抗肿瘤活性研究

目的 研究冬虫夏草菌丝体水提醇沉物的体外抗肿瘤活性及其机制。方法 采用热水浸提-醇沉法得到水提物,高效液相色谱仪测定水提醇沉物中腺苷的量;采用MTT法测定其抗肿瘤活性,利用流式细胞仪结合碘化丙锭染色法检测其对细胞周期的抑制。结果 实验表明水提醇沉物能抑制人肝癌HepG2细胞株及大细胞肺癌NCI-H460细胞株的增殖,并呈浓度相关性;半数抑制浓度（IC₅₀）值分别为（1.49±0.19）和（1.67±0.27）mg/mL。细胞周期分析表明,水提醇沉物分别阻滞HepG2及NCI-H460细胞周期于G₂/M期、S期,并可诱导上述两种细胞发生凋亡。结论 冬虫夏草水提醇沉物通过阻滞HepG2及NCI-H460细胞周期循环,诱导其凋亡,从而表现出良好的增殖抑制活性。为深入研究冬虫夏草菌丝体水提醇沉物抗肿瘤的机制提供了实验证据。     

10种常用平喘中药对磷酸二酯酶4抑制作用的研究

目的研究10种常用平喘中药桑白皮、枇杷叶、百部、马兜铃、紫菀、款冬花、苦杏仁、苏子、葶苈子、银杏叶提取物对磷酸二酯酶4（PDE4）的抑制作用。方法分别采用水煎煮、95%乙醇回流提取法提取药材,采用放射性3H标记液体闪烁计数法检测提取物对靶标PDE4的抑制活性。结果 10种平喘中药的水提物、95%乙醇提取物均显示一定的抑制PDE4活性。总体而言,平喘中药95%乙醇提取物对PDE4抑制活性高于水提取物。相同浓度下,桑白皮、枇杷叶、款冬花、马兜铃活性较高。结论平喘类中药对PDE4靶标具有一定的抑制作用,可能与其平喘作用相关。     

凌霄花提取物抗凝血活性部位研究

目的 研究凌霄花提取物抗凝血作用,确定凌霄花抗凝血活性部位。方法 通过血浆复钙实验测定小鼠血浆复钙时间（PRT）和断尾法测定小鼠出血时间（BT）实验,比较凌霄花不同提取物的药效活性。结果 凌霄花水提物、醇提物及水提醇沉上清液均具有抗凝血活性;醇提物萃取后正丁醇层药效最显著;水提醇沉上清液过大孔树脂后10%乙醇部位药效最显著。结论 凌霄花抗凝血活性主要存在于极性较大部位。     

补肝散不同分离组份抗抑郁活性筛选

目的 对补肝散醇提物不同分离组份的抗抑郁活性进行筛选。方法 采用慢性不可预知温和应激（chronicunpredictable mild stress,CUMS）对小鼠进行为期8周的造模,造模4周后,分别以盐酸帕罗西汀（paroxetine hydrochloride,PX）、补肝散醇提物、石油醚（A）、二氯甲烷（B）、乙酸乙酯（C）及正丁醇（D）4个分离组份进行为期4周的灌胃治疗,考察各组小鼠的行为学及脑内神经递质含量的变化。结果 连续给药4周（造模8周）后,PX组（0.026 g·kg^-1）、补肝散醇提物组（1.92 g·kg^-1）及其分离组份C（0.232 g·kg^-1）、D（1.04 g·kg^-1）和B（0.160 g·kg^-1）组均能明显逆转模型小鼠的抑郁表现如糖水偏好率下降、强迫游泳不动时间延长等抑郁症状。能不同程度地增加其脑内额叶皮质中5-羟色胺和多巴胺的含量,其中PX、醇提物及分离组份C的作用更为明显（与模型组比较,P<0.05或<0.01）。结论 补肝散醇提物的抗抑郁活性部位主要集中于乙酸乙酯和正丁醇分离组份,其次是二氯甲烷分离组份。     

UHPLC-ESI-QE-Orbitrap-MS结合TRIF/p-IκBα/NF-κB/NLRP3信号通路的蛤蚧定喘丸抗炎机制与活性成分探讨

目的 探讨蛤蚧定喘丸的抗炎机制,推测蛤蚧定喘丸发挥抗炎功效的关键活性成分。方法 依次采用石油醚、无水乙醇、超纯水对蛤蚧定喘丸不同极性的化学成分进行提取;利用2、4、8、16、32、64、128 μg·mL^-1的醚提物、醇提物、水提物处理RAW264.7细胞,分别通过CellTiter-Lumi（CTL）发光法和钙黄绿素/碘化丙啶（Calcein/PI）染色法检测细胞活力,确定提取物的安全浓度;利用各提取物处理脂多糖（LPS）诱导的RAW264.7细胞炎症模型,通过格里斯试剂（Griess）测定一氧化氮（NO）释放量,通过酶联免疫吸附（ELISA）法检测白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）分泌,筛选具有抗炎活性的提取物。利用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱技术（UHPLC-ESI-QE-OrbitrapMS）对有抗炎活性的提取物进行物质分析,推测抗炎活性物质及作用机制,并利用蛋白免疫印迹法（Western blotting）进一步验证其对硫氧还蛋白互作蛋白（TXNIP）/p-核因子κB抑制蛋白α（p-IκBα）/核因子-κB（NF-κB）/NOD样受体热蛋白结构域相关蛋白3（NLRP3）信号通路相关蛋白表达的影响。结果 醚提物、醇提物、水提物在质量浓度低于64 μg·mL^-1时均未影响细胞活力。与模型组相比,醇提物在质量浓度大于8 μg·mL^-1时显著降低NO释放量（P<0.05）,醚提物和醇提物在质量浓度高达64 μg·mL^-1时仍未产生抗炎效果;醇提物显著降低了TNF-α、IL-1β的释放量（P<0.05）。醇提物中共分析出36种主要化学成分。经Western blotting实验验证,与模型组比较,蛤蚧定喘丸醇提物对RAW264.7细胞炎症模型内NLRP3炎症小体、β干扰素TIR结构域衔接蛋白（TRIF）、诱导型一氧化氮合酶（iNOS）、单核细胞趋化蛋白-1（MCP-1）、p-IκBα的表达均有显著降低作用（P<0.05）,而对TXNIP、Toll样受体4（TLR4）、丝裂原活化蛋白激酶（MAPK）信号通路蛋白的影响并不显著。结论 蛤蚧定喘丸通过抑制TRIF/p-IκBα/NF-κB/NLRP3信号通路以及iNOS、MCP-1的合成发挥抗炎功效,推测巴马汀、麻黄碱、伪麻黄碱、小檗碱、甘草素、药根碱、小檗红碱为其抗炎活性成分。     

西黄丸醇提液激活法尼酯受体的抗乳腺癌作用研究

目的 研究西黄丸醇提液抑制荷4T1小鼠乳腺癌肿瘤的生长及作用机制。方法 建立荷4T1小鼠乳腺癌模型,以低、中、高剂量（0.39、0.78、1.95 g/kg）ig给予西黄丸醇提液2周,分离肿瘤组织,称质量并计算抑瘤率;体外培养4T1乳腺癌细胞株,加入低、中、高浓度（1.25、2.50、5.00 μg/mL）的西黄丸醇提液作用48 h,CCK-8法检测细胞增殖抑制率;TUNEL染色检测细胞凋亡;实时荧光定量PCR（qRT-PCR）法检测4T1细胞中FXR mRNA表达,Western Blotting检测法尼酯受体（FXR）蛋白表达。结果 与模型组比较,西黄丸醇提液低、中、高剂量组荷瘤质量均明显下降（P<0.05）,且呈剂量相关性;CCK-8结果显示,作用48 h,4T1细胞的增殖抑制率随西黄丸醇提液浓度的升高而增加,高浓度组可达43.13%;TUNEL荧光染色结果显示,与对照组比较,西黄丸醇提液低、中、高浓度组4T1细胞凋亡数量显著增加（P<0.05）;qRTPCR、Western Blotting结果显示,4T1细胞中FXR mRNA、蛋白表达水平随西黄丸醇提液浓度的升高显著上调,且低、中、高浓度组差异均具有统计学意义（P<0.05）。结论 西黄丸可能通过激活FXR受体促进肿瘤细胞凋亡从而抑制肿瘤生长。     

蜜炙对甘草饮片免疫活性影响的实验研究

目的 观察甘草饮片蜜炙前后对小鼠离体脾淋巴细胞转化增殖能力的影响。方法 无菌操作分离小鼠脾脏,制备脾淋巴细胞。加入刺激剂刀豆蛋白和不同浓度的甘草、蜜炙甘草饮片及其配伍的炙甘草汤水提物、醇提物以及多糖与甘草苷样品溶液培养48 h,以噻唑蓝（MTT）法检测细胞增殖情况。结果 甘草、蜜甘草饮片和炙甘草汤水提物组以及多糖组与对照组相比均能显著增强经ConA诱导的小鼠脾淋巴细胞增殖能力（P＜0.01）,同时上述各样品蜜炙品水提物组明显高于相应的生品组（P＜0.05）。各样品醇提物、甘草苷、蜂蜜组与对照组比较无明显差别（P＞0.05）。结论 甘草生炙饮片免疫活性存在明显差别,蜜炙后免疫活性增强,其中多糖成分是甘草的主要免疫活性成分。甘草饮片生品和蜜炙品在临床应用中应区分应用,保证临床疗效。     

白子草总黄酮及总多糖降血糖有效部位的提取工艺研究

目的 研究建立白子草总黄酮及总多糖降血糖有效部位的提取工艺。方法 在单因素试验的基础上,考察提取次数、药材粒径、浸泡时间、料/液比、乙醇浓度、提取时间对白子草总黄酮提取率的影响,并通过L₉（3）⁴正交试验优化总黄酮最佳提取工艺;以前期研究为基础,结合L₉（3）⁴正交试验,优化总多糖最佳提取工艺。结果 白子草总黄酮最佳提取工艺为：药材最粗粉加20倍80%乙醇,浸泡0.5 h,回流提取2次,每次1.0 h;总多糖最佳提取工艺为：醇提后药渣加20倍纯净水,80℃回流提取2次,每次1.0 h。该条件下白子草总黄酮及总多糖提取率均大于80%。结论 本研究建立的白子草总黄酮及总多糖降血糖有效部位提取工艺稳定、重复性好,为白子草降血糖活性研究奠定了实验基础。     

泽泻水提物的化学成分研究

目的 研究泽泻水提物中的化学成分,并筛选激活核因子E2相关因子2(Nrf2)的活性成分。方法 综合应用多种色谱技术分离纯化泽泻水提物,采用凝胶色谱或制备液相色谱法,获得单体,通过¹H-NMR、¹³C-NMR、MS等波谱技术进行结构鉴定,采用荧光素酶报告基因法筛选泽泻水提物中激活Nrf2的活性成分。结果 从泽泻水提物中分离鉴定15个化合物,分别为β-D-呋喃果糖（1）,β-D-乙基呋喃果糖苷（2）,5-羟甲基糠醛（3）,蔗糖（4）,棉子糖（5）,水苏糖（6）,毛蕊花糖（7）和甘露三糖（8）以及环氧泽泻烯（9）,泽泻烯醇（10）,泽泻醇C（11）,泽泻醇L（12）,16-羰基泽泻醇A（13）,16-羰基-24-乙酰泽泻醇A（14）,16-羰基-11-去氧泽泻醇A（15）,其中化合物11至化合物15具有激活Nrf2的活性。结论 从泽泻水提物中共分离15个成分,包括8个糖类（化合物1至8）,2个倍半萜类（化合物9和10）和5个16位羰基取代的三萜类（化合物11至15）,化合物2、3、5、6、7、8为首次从泽泻水提物中分离,15个化合物经活性评价,5个三萜类成分具有激活Nrf2的作用,16-羰基取代的泽泻三萜可能是泽泻激活Nrf2信号系统的药效物质基础之一。     

枸骨茎抗炎活性成分研究

目的 研究枸骨（Ilex cornuta Lindl.et Paxt）茎的抗炎活性成分。方法 运用80%乙醇提取、大孔吸附树脂、正反相硅胶、Sephadex-LH20色谱柱等多种方法进行分离和纯化,根据理化性质和波谱数据进行结构鉴定。采用脂多糖（LPS）诱导的小鼠巨噬细胞RAW264.7的NO生成模型对部分化合物的抗炎活性进行测试。结果 共分离得到10个化合物,经结构鉴定分别为α-香树脂醇（1）、齐墩果酸（2）、熊果酸（3）、羽扇豆醇（4）、menisdaurin（5）、lithospermoside（6）、槲皮素（7）、芦丁（8）、汉黄芩苷（9）、5,2''-二羟基-6,7,8,3''-四甲氧基黄酮（10）。测试了化合物2~8对LPS诱导的小鼠巨噬细胞RAW264.7的NO生成抑制活性。结论 化合物5、6、9、10为首次从该植物中分离得到。化合物3和7能较明显地抑制LPS诱导的小鼠巨噬细胞RAW264.7的NO生成。     

cAMP-specific phosphodiesterase inhibitors: promising drugs for inflammatory and neurological diseases

Introduction: PDEs are key enzymes in the adenosine and guanosine cyclic nucleotides (cAMP and cGMP) signaling cascade. Their inhibition increases cyclic nucleotide levels inside the cell. Thus, pharmacological modulation of PDE activity can have profound effects on the function of cells and organ systems throughout the body.

Areas covered: Among the large PDE families, only PDE4, PDE7 and PDE8 are cAMP-specific hydrolyzing enzymes. cAMP is an important second messenger not only by its involvement in a vast number of physiological processes but also by activation of protein kinase A, exchange protein activated by cAMP (Epac) and cAMP response element-binding (CREB) or cyclic nucleotide-gated channels. Clearly, such enzymes represent ideal drug targets for the pharmacological treatment of many pathologies. The discovery and development of small molecules targeting cAMP-specific PDEs reported in the last 5 years is the focus of the present review.

Expert opinion: The first PDE4 inhibitors recently reached the market, having avoided, by different strategies, their dose-limiting side effects (after more than two decades of drug development). Meanwhile, new cAMP-specific PDE7 and PDE8 inhibitors emerged as effective and safe drugs for severe unmet diseases. The therapeutic potential of these inhibitors will be tested in the near future, as many of these drug candidates are ready to start clinical trials.     

苯二醛缩氨基硫脲的合成及其酪氨酸酶抑制活性研究

目的 合成苯二醛单缩和二缩氨基硫脲类化合物,并初步研究其抑制酪氨酸酶的活性和作用机制。方法 以5种苯二醛和氨基硫脲为原料,通过缩合反应合成9个目标化合物;采用蘑菇酪氨酸酶多巴速率氧化法和酶抑制动力学实验,测定目标化合物对酪氨酸酶的抑制活性和作用机制;选择化合物3a和4a进行抑制机制和抑制动力学研究。结果 目标化合物的结构经¹H-NMR、¹³C-NMR及MS确证;所有化合物抑制酪氨酸酶的活性均优于对照药物曲酸;苯二醛二缩氨基硫脲3a~3d的活性明显强于相应的单缩氨基硫脲4a~4d;化合物3a和4a对酪氨酸酶的抑制作用均表现为混合型可逆抑制作用。结论 苯二醛二缩氨基硫脲类化合物具有优异的抑制酪氨酸酶的活性,值得进一步深入研究。     

Design and synthesis of 1,2,4‐triazolo[1,5‐a]pyrimidine derivatives as PDE 4B inhibitors endowed with bronchodilator activity

A series of 1,2,4‐triazolo[1,5‐a]pyrimidine derivatives was designed, synthesized, and screened for their phosphodiesterase (PDE 4B) inhibitory activity and bronchodilation ability. Compound 7e showed 41.80% PDE 4B inhibition at 10 µM. Eight compounds were screened for their bronchodilator activity, where compounds 7f and 7e elicited promising bronchodilator activity with EC₅₀ values of 18.6 and 57.1 µM, respectively, compared to theophylline (EC₅₀ = 425 µM). Molecular docking at the PDE 4B active site revealed a binding mode and docking scores comparable to those of a reference ligand, consistent with their PDE 4B inhibition activity.     

Evaluation of Antiaris africana methanol extract and compounds for antioxidant and antitumor activities

A methanol extract from the stem bark of Antiaris africana Engler (Moraceae), as well as compounds isolated and identified as betulinic acid (1), 3β-acetoxy-1β,11α-dihydroxy-olean-12-ene (2), ursolic acid (3), oleanolic acid (4), strophanthidol (5), periplogenin (6), convallatoxin (7), strophanthidinic acid (8), methyl strophanthinate (9), and 3,3′-dimethoxy-4′-O-β-d-xylopyronosylellagic acid (10), were tested for their antioxidant and anticancer activities. The DPPH radical scavenging assay was used for the antioxidant test while the potato disk tumor induction and XTT assays were used to detect antitumor activities. The antioxidant test showed that the methanol extract and compounds 1, 9, and 10, as well as vitamin C, used as reference antioxidant drug, were able to interact with more than 50% DPPH. The results of the potato disk tumor induction assay also indicated a pronounced tumor reducing activity of the methanol extract (83.12%) and compound 10 (96.64%). Samples showing more than 20% inhibition of Crown gall tumors were then tested against human DU-145 and hepatocarcinoma Hep G2 cells. The results showed inhibitory activities of 64.12% and 73.62%, respectively, on DU-145 and Hep G2 cells for the methanol extract. Compound 10 showed the highest inhibition potency on both cell lines with more than 70% inhibition at 50?μg/mL. The methanol extract showed an IC₅₀ value lower than this at 30?μg/mL, a threshold value for potential antineoplastic extracts. The results of the present study provide supportive data for the traditional anticancer use of A. africana and indicate that the methanol extract as well as compound 10 represent a potential source of medicine for the treatment of cancer, having also interesting antioxidant properties.     

A new selaginellin derivative and a new triarylbenzophenone analog from the whole plant of Selaginella pulvinata

Abstract

Five selaginellin derivatives (1 and 3–6) including a new one, selaginellin T (1), and a new triarylbenzophenone analog, selagibenzophenone A (2), were isolated from the whole plants of Selaginella pulvinata. Their structures were determined by 1D- and 2D-NMR and HR-ESI-MS data. Selagibenzophenone A (2) is the first example of naturally occurring triarylbenzophenone. The results of the phosphodiesterase-4 (PDE4) inhibitory screening assays showed that compounds 1?6 exhibited potent activities with the IC₅₀ values in the range of 1.04?9.35 μM.     

Design of Novel β‐Carboline Derivatives with Pendant 5‐Bromothienyl and Their Evaluation as Phosphodiesterase‐5 Inhibitors

New derivatives with the tetrahydro‐β‐carboline‐imidazolidinedione and tetrahydro‐β‐carboline‐piperazinedione scaffolds and a pendant bromothienyl moiety at C‐5/C‐6 were synthesized and tested for their ability to inhibit PDE5 in vitro. The following SAR can be concluded: The tetracyclic scaffold is essential for PDE5 inhibition; the ethyl group is the most suitable among the adopted N‐substituents on the terminal ring (hydantoin/piperazinedione); the appropriate stereochemistry of C‐5/C‐6 derived from the aldehyde rather than C‐11a/C‐12a derived from tryptophan appears crucial for inhibition of PDE5; surprisingly, derivatives with the hydantoin terminal ring are more active than their analogs with the piperazinedione ring; the selectivity versus PDE5 relative to PDE11 with cGMP as a substrate is mainly a function of the substitution and stereochemistry pattern of the external ring, in other words of the interaction with the H‐loop residues of the isozymes. Thirteen derivatives showed PDE5 inhibitory activity with IC₅₀ values in the range of 0.16–5.4 µm. Compound 8 was the most potent PDE5 inhibitor and showed selectivity towards PDE5 versus other PDEs, with a selectivity index of 49 towards PDE5 rather than PDE11 with cGMP as the substrate.     

Real-world incidence of inflammatory bowel disease among patients with other chronic inflammatory diseases treated with interleukin-17a or phosphodiesterase 4 inhibitors

Objectives: (1) To assess the real-world incidence of inflammatory bowel disease (IBD) in patients with or without other chronic inflammatory diseases (CIDs), and (2) to understand whether IBD incidence differs in CID patients receiving interleukin-17a signaling antagonists (anti-IL-17a) or phosphodiesterase 4 inhibitors (PDE4i) versus patients using a biologic not indicated for IBD or biologic-naïve patients.

Methods: The MarketScan Research Databases (January 2010–July 2017) were used. A CID population was created from patients with ankylosing spondylitis, psoriatic arthritis, psoriasis or rheumatoid arthritis (RA). The CID population was stratified into different cohorts based on the baseline treatments received: (1) anti-IL-17a, (2) PDE4i, (3) biologic-naïve, and (4) non-IBD-indicated biologic (i.e. biologics not indicated for the treatment of IBD and excluding anti-IL-17a and PDE4i); a non-CID cohort was also created. The 1?year incidence rate (IR) of IBD was compared between cohorts using a logistic regression model adjusting for baseline characteristics.

Results: CID cohorts included older patients than the non-CID cohort (mean age range: 48.4–54.4 versus 46.3?years). The 1?year IR of IBD was 1.41% in the anti-IL-17a cohort (N?=?355), 0.68% in the PDE4i cohort (N?=?2195), 0.47% in the biologic-naïve cohort (N?=?424,767), 0.51% in the non-IBD-indicated biologic cohort (N?=?56,317) cohort and 0.25% in the non-CID cohort (N?=?1,008,436). After 1?year of follow-up, the odds of having IBD were 2.85 (p?=?.0213) and 1.42 (p?=?.1891) times higher in the anti-IL-17a and PDE4i cohorts, respectively, compared to the biologic-naïve cohort, and 2.86 (p?=?.0253) and 1.21 (p?=?.4978) times higher compared to the non-IBD-indicated biologic cohort. Similar results were observed in sensitivity analyses where patients with RA only were excluded (since anti-IL-17a and PDE4i agents are not indicated for RA).

Conclusions: Anti-IL-17a treatment was associated with a nearly three-fold higher risk of IBD in CID patients. Treatment decisions for patients with CIDs should take into account the risk of developing of IBD.     

Two pairs of rare naturally occurring 4-hydroxy-4-methyl-2,5-heptanedione derivatives from the red alga Chondria crassicaulis

Two pairs of rare naturally occurring racemic lipids, (±)-4,7-dihydroxy-4-methyl-2,5-heptanedione (1), and (±)-7-butoxy-4-hydroxy-4-methyl-2,5-heptanedione (2) were isolated from the red alga Chondria crassicaulis Harv. The structures of the racemic mixtures of 1 and 2 were elucidated by detailed spectroscopic techniques, including 1D and 2D NMR (¹H and ¹³C NMR, ¹H–¹H COSY, HSQC, and HMBC) as well as mass spectrometry and optical rotation experiments, and by comparison with data for related known analogs. This is the first report of naturally occurring 4-hydroxy-4-methyl-2,5-heptanedione derivatives. Antifungal, PTP1B inhibitory, and receptor tyrosine kinase inhibitory activities of these two compounds were investigated. The results showed that compounds 1 and 2 exhibited good selective inhibition against RET tyrosine kinase activity with IC₅₀ values of 9.56 and 8.93 μM, respectively. Compound 1 also displayed moderate antifungal activity against Cryptococcus neoformans (32609), showing a MIC₈₀ value of 32 μg/ml.     

具有酪氨酸酶抑制活性的丹参有效成分筛选研究

目的 筛选丹参中对酪氨酸酶活性具有抑制作用的有效成分。方法 基于受试物抑制酪氨酸酶催化底物L-多巴的反应原理,将丹参经80%乙醇提取后依次经石油醚、氯仿、醋酸乙酯、饱和正丁醇和水5种不同极性溶剂萃取,分别检测各提取部位对酪氨酸酶活性的抑制作用;并通过UPLC-MS/MS技术鉴定活性最强提取部位化学成分,结合分子对接技术预测各成分潜在活性,并通过酶学检测验证。结果 丹参氯仿萃取层具有较强的酪氨酸酶抑制活性。其生物活性与丹参素、原儿茶醛等6种成分有关,酶学检测验证结果与筛选结果一致。结论 丹参中丹参素和原儿茶醛等成分对酪氨酸酶活性具有显著的抑制作用。