淫羊藿苷元在大鼠体内的血浆动力学和组织分布研究

目的研究淫羊藿中的主要活性成分淫羊藿苷元在大鼠体内的血浆动力学和组织分布特征,为黄酮类天然药物的开发提供依据。方法应用高效液相色谱-串联质谱(HPLC-MS/MS)测定大鼠血浆及组织中游离淫羊藿苷元(淫羊藿苷元原型)和总淫羊藿苷元(淫羊藿苷元原型及其二相代谢产物的总和)的浓度,并应用Win Nonlin7.0软件非房室模型计算药动学参数。结果大鼠iv淫羊藿苷元(5 mg/kg)后,血浆中游离淫羊藿苷元和总淫羊藿苷元的血药峰浓度(Cmax)分别为(978±107)、(2390±295)ng/m L,血药浓度-时间曲线下面积(AUC0-t)分别为(422±38)、(3000±932)ng·h/m L,消除半衰期(t1/2)分别为(1.640±0.767)、(3.82±2.16)h。大鼠ig淫羊藿苷元(10mg/kg)后,血浆中游离型淫羊藿苷元及总淫羊藿苷元的达峰时间(Tmax)为0.292h,Cmax分别为(12.90±5.77)、(3800±1110)ng/m L,AUC0-t分别为(12.00±8.01)、(5760±2200)ng·h/m L,t1/2分别为(0.812±0.263)、(2.73±1.28)h。大鼠ig淫羊藿苷元(10mg/kg)后,游离淫羊藿苷元在组织中的分布程度由高到低排序依次为胃、肠、肺(110～220倍血浆)、肝、子宫(20～65倍血浆)、心、卵巢、脾(4～15倍血浆)、骨髓、肾、肌肉(1～2倍血浆)、血浆、脑、脂肪(20%～50%血浆)、睾丸(未检测到);总淫羊藿苷元在组织中的分布由高到低排序依次为肠、胃、肺(1～2倍血浆)、血浆、肝、肾、子宫、卵巢(20%～60%血浆)、肌肉、心、脾、睾丸、骨髓、脂肪、脑(1%～7%血浆)。结论大鼠ig给药后,血浆中游离淫羊藿苷元及总淫羊藿苷元吸收迅速,消除较快,与iv给药相比,游离淫羊藿苷元的绝对生物利用度仅为1.42%,而总淫羊藿苷元绝对生物利用度达到96.0%。血浆中游离淫羊藿苷元的暴露量明显低于总淫羊藿苷元,仅占总淫羊藿苷元暴露量的0.208%,提示淫羊藿苷元ig给药后在大鼠血液循环系统中主要以II相结合型产物暴露为主。但在多数组织中游离淫羊藿苷元的暴露量、暴露持续时间均明显高于血浆,而且游离淫羊藿苷元与总淫羊藿苷元的比例也明显大于血浆。     

Metabolic profiling of isomeric aglycones central‐icaritin (c‐IT) and icaritin (IT) in osteoporotic rats by UPLC‐QTOF‐MS

The isomers, although of similarly chemical structures, have different pharmacological activities due to their metabolic processes in vivo. Central‐icaritin (c‐IT) and icaritin (IT) are isomers and major bioactive aglycones of the Herba Epimedii. In this study, we found that the anti‐osteoporotic effect of c‐IT was stronger than IT on bone structural changes in osteoporotic rats evaluated by Micro‐μCT with the parameters of bone mineral density (BMD), bone mineral content (BMC), tissue mineral content (TMC), and tissue mineral density (TMD). c‐IT treatment significantly increased the bone microarchitecture, compared with IT (p < 0.05). In order to explain their differences in anti‐osteoporosis, the metabolic profiling and pathways of c‐IT and IT in the plasma, bile, urine, and faeces of ovariectomized (OVX) rats were investigated by ultra‐performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC‐QTOF‐MS) after oral administration of c‐IT or IT (80 mg/kg). Finally, 59 metabolites of c‐IT and 43 metabolites of IT were identified by elucidating their corresponding quasimolecular ions and fragment ions. IT could be quickly absorbed into blood and reached a maximum plasma concentration, and then be rapidly conversed to its glucuronidation metabolites, most of which were excreted out by urine. Interestingly, the absorbed and conjugated speeds of c‐IT were slower than IT. The metabolic processes of c‐IT existed enterohepatic circulation, which decreased the metabolism and excretion rate of c‐IT, and prolonged the anti‐osteoporosis effect. Our findings provided evidence on the difference on metabolic profiles of c‐IT and IT in osteoporotic rats, which might shed new lights on improving anti‐osteoporotic effects of IT and c‐IT. Copyright © 2014 John Wiley & Sons, Ltd.     

基于UPLC-Q/TOF-MS分析淫羊藿炮制前后黄酮组分的变化规律

目的基于UPLC-Q/TOF-MS技术建立淫羊藿炮制前后指纹图谱,对其全成分进行分析并找出标志性化学成分,以明确淫羊藿炮制前后黄酮组分的变化规律。方法采用UPLC-Q/TOF-MS技术,在正离子模式下采集淫羊藿生品及炮制品样品数据,并在此基础上根据正交-偏最小二乘判别分析(OPLS-DA)整体探究9个不同产地、批次的淫羊藿炮制前后化学成分的差异。结果从淫羊藿生品及炮制品中寻找并鉴定出9个标志性化学成分,即8-乙烯-山柰酚、淫羊藿素、淫羊藿次苷I、淫羊藿素-3-O-葡萄糖苷、异戊醇基箭藿苷B、1,3-异戊二烯基朝藿定C、1,3-异戊二烯基-箭藿苷B-7-O-葡萄糖醛酸、3-O-(4-乙酰氧基)鼠李糖-2-O-(间二乙酰氧基)葡萄糖-淫羊藿苷及其同分异构体。结论淫羊藿炮制后黄酮组分结构发生变化,次级糖苷增加,多级糖苷减少,淫羊藿黄酮组分总体向低糖苷组分转化,进一步阐明了淫羊藿加热炮制后黄酮组分的变化规律。     

Biofabrication of a PLGA–TCP‐based porous bioactive bone substitute with sustained release of icaritin

A phytomolecule, icaritin, has been identified and shown to be osteopromotive for the prevention of osteoporosis and osteonecrosis. This study aimed to produce a bioactive poly (l ‐lactide‐co‐glycolide)–tricalcium phosphate (PLGA–TCP)‐based porous scaffold incorporating the osteopromotive phytomolecule icaritin, using a fine spinning technology. Both the structure and the composition of icaritin‐releasing PLGA–TCP‐based scaffolds were evaluated by scanning electron microscopy (SEM). The porosity was quantified by both water absorption and micro‐computed tomography (micro‐CT). The mechanical properties were evaluated using a compression test. In vitro release of icaritin from the PLGA–TCP scaffold was quantified by high‐performance liquid chromatography (HPLC). The attachment, proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) on the composite scaffold were evaluated. Both an in vitro cytotoxicity test and an in vivo test via muscular implantation were conducted to confirm the scaffold's biocompatibility. The results showed that the PLGA–TCP–icaritin composite scaffold was porous, with interconnected macro‐ (about 480 µm) and micropores (2–15 µm). The mechanical properties of the PLGA–TCP–icaritin scaffold were comparable with those of the pure PLGA–TCP scaffold, yet was spinning direction‐dependent. Icaritin content was detected in the medium and increased with time. The PLGA–TCP–icaritin scaffold facilitated the attachment, proliferation and osteogenic differentiation of BMSCs. In vitro cytotoxicity test and in vivo intramuscular implantation showed that the composite scaffold had no toxicity with good biocompatibility. In conclusion, an osteopromotive phytomolecule, icaritin, was successfully incorporated into PLGA–TCP to form an innovative porous composite scaffold with sustained release of osteopromotive icaritin, and this scaffold had good biocompatibility and osteopromotion, suggesting its potential for orthopaedic applications. Copyright © 2012 John Wiley & Sons, Ltd.     

淫羊藿素及相关衍生物的抗肿瘤机制研究进展

综述淫羊藿素及相关衍生物的抗肿瘤机制研究进展。淫羊藿素及相关衍生物对不同类型肿瘤有抑制作用,其作用机制主要包括诱导细胞凋亡、调控细胞周期、抗血管生成、抑制肿瘤转移和免疫调节等。     

Protective effect of icaritin on focal cerebral ischemic–reperfusion mice

Objective: To investigate the protective effects of icaritin(ICT),one of the active ingredients in Epimedii Folium,on mouse model of cerebral ischemia-reperfusion(I/R)in vivo.Methods: ICR mice were subjected to an 1 h transient middle cerebral artery occlusion(MCAO)and followed by 24 h of reperfusion.Neurological deficits,infarct volume,brain edema and survive rate were measured,respectively.The levels of brain IL-1β,TNF-α,ROS and DNA-binding activity of NF-κB p65 were measured by ELISA kits.The levels of malondialdehyde(MDA)and activities of superoxide dismutase(SOD)were detected by spectrophotometry,and the release of nitric oxide(NO)were detected by Griess kit.Results: ICT markedly reduced the neurological deficit scores,brain edema,infarct volume and increased the survival rate of the cerebral I/R mice.The expression of IL-1 β,TNF-α,NO,MDA and DNA-binding activity of NF-κB p65 were significantly inhibited by ICT,while the activity of SOD were up-regulated at the same time.Conclusion: ICT possessed significant neuroprotective effects in cerebral I/R mice,which might be related to prevent neuroinflammatory and oxidative damage.     

淫羊藿素在大鼠体内的药动学研究

 目的 研究淫羊藿素灌胃或静脉注射给药后原型药物及结合型药物在大鼠体内的血浆药动学和排泄特征，为新药开发提供参考依据。方法 采用高效液相色谱-串联质谱法测定生物样品经酶水解前后淫羊藿素浓度。结果 大鼠灌胃给予不同剂量淫羊藿素（20、40和80 mg·kg-1）后，ρ_max和AUC_0-∞与给药剂量呈正相关，生物利用度分别为17.29%、13.80%和10.70%。灌胃给予80 mg·kg-1淫羊藿素后，大鼠血浆中游离淫羊藿素的ρmax和AUC_0-∞分别为13.26 ng·mL-1，495.67 ng·h·mL-1；游离与结合形式总和的药动学参数分别为597.50 ng·mL-1，12 038 ng·h·mL-1。静脉给药20 mg·kg-1后，大鼠血浆中游离淫羊藿素的ρmax和AUC_0-∞分别为5 896 ng·mL-1，2 470 ng·h·mL-1；游离与结合形式总和的药动学参数分别为11 598 ng·mL-1，23 303 ng·h·mL-1。大鼠灌胃给予80 mg·kg-1淫羊藿素72 h后，尿、粪样品中游离淫羊藿素的排泄量分别占给药量的0.04%和25.09%，游离与结合形式总和的排泄量分别占给药量的0.14%和32.46%；大鼠静脉注射给予20 mg·kg-1淫羊藿素72 h后，尿、粪样品中游离淫羊藿素的排泄量分别占给药量的0.58%和8.76%，游离与结合形式总和的排泄量分别占给药量的2.48%和10.82%。结论 淫羊藿素给药后在体内主要以结合形式存在，生物利用度较低，主要通过粪便排泄。     

Anticancer effect of icaritin on human lung cancer cells through inducing s phase cell cycle arrest and apoptosis

Icaritin, a prenylflavonoid derivative from Epimedium Genus, has been shown to exhibit many pharmacological and biological activities. However, the function and the underlying mechanisms of icaritin in human non-small cell lung cancer have not been fully elucidated. The purpose of this study was to investigate the anticancer effects of icaritin on A549 cells and explore the underlying molecular mechanism. The cell viability after icaritin treatment was tested by MTT assay. The cell cycle distribution, apoptosis and reactive oxygen species（ROS） levels were analyzed by flow cytometry. The mRNA and protein expression levels of the genes involved in proliferation and apoptosis were respectively detected by RT-PCR and Western blotting. The results demonstrated that icaritin induced cell cycle arrest at S phase, and down-regulated the expression levels of S regulatory proteins such as Cyclin A and CDK2. Icaritin also induced cell apoptosis characterized by positive Hoechst 33258 staining, accumulation of the Annexin V-positive cells, increased ROS level and alteration in Bcl-2 family proteins expression. Moreover, icaritin induced sustained phosphorylation of ERK and p38 MAPK. These findings suggested that icaritin might be a new potent inhibitor by inducing S phase arrest and apoptosis in human lung carcinoma A549 cells.     

淫羊藿素通过Akt/mTOR调控糖酵解抑制肝内胆管癌细胞增殖的作用机制研究

目的 探究淫羊藿素对人肝内胆管癌HuCCT1细胞增殖的影响及其作用机制。方法 CCK-8法检测淫羊藿素对HuCCT1细胞增殖活性的影响;平板克隆法检测淫羊藿素对HuCCT1细胞集落形成能力的影响;流式细胞术检测淫羊藿素对HuCCT1细胞周期的影响;分光光度法检测淫羊藿素对Hu CCT1细胞葡萄糖摄取量、乳酸生成量、三磷酸腺苷(adenosine triphosphate,ATP)生成量以及己糖激酶(hexokinase,HK)和丙酮酸激酶(pyruvate kinase,PK)活性的影响;Western blotting法检测淫羊藿素对HuCCT1细胞中增殖相关蛋白和蛋白激酶B(protein kinase B,Akt)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,m TOR)通路及糖酵解相关蛋白表达的影响;Western blotting检测淫羊藿素对瞬时转染Akt基因质粒的HuCCT1细胞中Akt/mTOR及糖酵解相关蛋白表达的影响。结果 淫羊藿素显著抑制HuCCT1细胞的活力,并呈时间和剂量相关性;淫羊藿素呈剂量相关性地抑制HuCCT1细胞...