Biologically-Guided Isolation of Natural Lead Antithyroid Drug from Medicago sativa L. Sprouts and Its Toxic Profile in Comparison with Propylthiouracil

Hyperthyroidism is a common endocrine disorder associated with increased risk of cardiovascular complications and mortality. Although antithyroid drugs (ATDs) are approved as first line option for many hyperthyroidism cases, including pregnancy and childhood, they exert significant toxic profile. Medicago sativa L. (alfalfa) also called “The father of all food” was among the diet consumed by mares that gave birth to foals with congenital hypothyroidism. Since, greenfeed was accused for the development of such condition, alfalfa may possess constituents with promising antithyroid potential that could be a valuable substitute for the conventional ATDs. The current work was designed to identify the most biologically active antithyroid phytoconstituent separated from alfalfa sprouts and comparing its antithyroid mechanism, efficacy and toxic profile to the standard ATD; propylthiouracil (PTU). The most biologically active solvent fractions from alfalfa sprouts extract were identified by in vitro screening for anti-thyroid peroxidase (TPO) activity, from which different phytoconstituents were separated and identified by interpretation of spectroscopic data. These compounds were then in vitro screened for anti-TPO and virtually screened via GLIDE XP docking into the crystal structures of the enzymes; bovine lactoperoxidase, as an alternative to TPO, and mammalian selenocysteine-dependent iodothyronine deiodinase (IDI), that are both uniquely dually prohibited by PTU. The compound that showed the least TPO IC₅₀ and highest combined docking XP score was elected for comparing its antithyroid mechanism, efficacy, tendency to reverse hyperthyroidism-triggered complications and toxicity to PTU using L-thyroxine-induced hyperthyroidism model in rats. Seven compounds (1–7) were isolated from the most biologically active fraction, whilst, compounds (4–7) were reported for the first time from alfalfa sprouts. Compound 5 (apigenin) showed the least TPO IC₅₀ and highest in-silico combined score, thus, apigenin was selected for further in-vivo investigations. Apigenin was found to more effectively interfere with type 1-IDI than with TPO in vivo. Apigenin therapy resulted in nearly euthyroid state, without incidence of hypothyroidism, thyroid hypertrophy, hepatotoxity or WBCs count reduction. In addition, apigenin, but not PTU, corrected hyperthyroidism-induced left ventricular hypertyrophy. Therefore, apigenin is a natural lead antithyroid drug that represents a possible safer alternative to conventional ATDs.     

丝瓜叶化学成分的研究

从丝瓜(Luffa cylindrica Roem.)叶中分得一种新的五环三萜皂甙和芹菜素,用光谱和化学方法确定该皂甙的结构为21β-羟基齐墩果酸-28-O-β-D-吡喃葡萄糖甙,命名为丝瓜皂甙Q。     

The estrogenic effects of apigenin,phloretin and myricetin based on uterotrophic assay in immature Wistar albino rats

Chemicals that occur in vegetal food and known as phytoestrogens, because of their structures similarity to estrogen, have benefits on chronic diseases. Despite this, when they are taken at high amounts, they can cause harmful effects on endocrine system of human and animals. In this study, it has been intended to determine the estrogenic potencies of phytoestrogens apigenin, phloretin and myricetin whose affinities for estrogen receptors in vitro. The female rats divided into 17 groups, each containing six rats. There was a negative control group and there were positive control dose groups which contains ethinyl estradiol, ethinyl estradiol + tamoxifen and genistein. The other dose groups which were tested for estrogenic activity contains apigenin, myricetin and phloretin All chemicals have been given to Wistar immature female rats with oral gavage for 3 consecutive days. By using uterotrophic analysis, uterus wet and blotted weights, vaginal opening, uterus length of female rats has been recorded at the end of the experiment. For detect of cell response, luminal epithelium height, gland number and lactoferrin intensity in luminal epithelium of uterus were evaluated. Biochemical analysises in blood were performed.     

HPLC法同时测定旱莲草中木犀草素和芹菜素的含量

目的:建立同时测定旱莲草提取物中木犀草素和芹菜素含量的方法。方法:采用高效液相色谱法。色谱柱为Nucleodur100-5 C18(250 mm×4.6 mm,5μm),流动相为甲醇-0.2%磷酸水溶液(50∶50,V/V),流速为1 ml/min,检测波长为350 nm,柱温为40△。结果:木犀草素和芹菜素的质量浓度均在0.02⁵0.00μg/ml范围内与各自峰面积积分值呈良好的线性关系(r分别为0.999 5和0.999 3);精密度、稳定性、重复性试验的RSD<2%;平均加样回收率分别为97.2%和98.9%,RSD分别为2.41%和2.91%(n均为6)。结论:该方法快速、简便,结果准确,可用于不同产地旱莲草药材的质量评价。     

同等剂量槲皮素、芹菜素对高尿酸血症大鼠的影响及其机制研究

目的研究同等剂量槲皮素(Quercetin,Que)和芹菜素(Apigenin,Api)对腺嘌呤+盐酸乙胺丁醇诱导大鼠高尿酸血症的影响及其机制。方法利用腺嘌呤+盐酸乙胺丁醇片灌胃法诱导SD大鼠制备高尿酸血症模型,槲皮素、芹菜素预防治疗3周,测定血清尿酸(SerumUricAcid,SUA)、血清以及肝脏中的黄嘌呤氧化酶(Xanthine Oxidase,XOD)、腺苷脱氨酶(adenosine deaminase,ADA)的活性。结果槲皮素和芹菜素均可显著降低SUA水平,抑制血清和肝脏中的ADA水平,槲皮素能够显著抑制血清和肝脏中的XOD的活性,但是芹菜素对肝脏XOD活性影响不大。结论槲皮素、芹菜素均有干预腺嘌呤+盐酸乙胺丁醇诱导高尿酸血症的作用。可能的机制是槲皮素通过抑制机体XOD和ADA的活性,减少血尿酸的生成量,从而降低血清尿酸水平;而芹菜素可能通过抑制体内ADA的活性,或许还有别的途径来降低SUA水平,但是具体机制有待于进一步研究。     

芹菜素对肺癌细胞的抗增殖作用和抗肿瘤药物的增敏作用

目的:研究芹菜素(apigenin)对肺癌细胞的抗肿瘤作用以及增敏作用.方法:通过细胞台盼蓝染色计数和MTS法分别检测芹菜素在肺癌细胞中的抗增殖作用以及增敏作用,研究芹菜素在肺癌细胞中与其他抗癌药物联用的效果.结果:芹菜素可以显著抑制4种肺癌细胞的增殖( A549:P =0.041,H460:P=0.050,LTEP-a2:P=0.039,H292:P =0.016);MTS实验结果表明,芹菜素的过夜预处理可以大大提高抗癌药物奥沙利铂( oxaliplatin)和博来霉素(bleomycin)在4种肺癌细胞中的IC50值,并且与抗癌药物存在协同作用(芹菜素与抗癌药在每个浓度处的联合作用指数CI值绝大多数＜1).结论:芹菜素可以抑制多种肺癌细胞的增殖以及增强肺癌细胞对抗癌药物奥沙利铂和博来霉素的敏感性.     

Strategic formulation of apigenin-loaded PLGA nanoparticles for intracellular trafficking,DNA targeting and improved therapeutic effects in skin melanoma in vitro

The aim of the present study was the evaluation of anti-proliferative potentials of apigenin (Ap), (a dietary flavonoid) loaded in poly (lactic-co-glycolide) nanoparticles (NAp) in A375 cells in vitro. NAp was characterized for particle size, morphology, zeta potential, drug release and encapsulation. Cellular entry and intracellular localization of NAp were assessed by transmission electron and confocal microscopies. Circular dichroic spectral analysis and stability curve for Gibb's free energy of dsDNA of A375 cells were also analyzed. DNA fragmentation, intracellular ROS accumulation, superoxide-dismutase activity, intracellular glutathione-reductase content and mitochondrial functioning through relevant markers like mitochondrial transmembrane potential, ATPase activity, ATP/ADP ratio, volume changes/swelling, cytochrome-c release, expressions of Apaf-1, bax, bcl-2, caspase-9, 3, and PARP cleavage were analyzed. NAp produced better effects due to their smaller size, faster mobility and site-specific action. Photostability studies revealed that PLGA encapsulations were efficient at preserving apigenin ultraviolet-light mediated photodegradation. NAp readily entered cancer cells, could intercalate with dsDNA, inducing conformational change. Corresponding increase in ROS accumulation and depletion of the antioxidant enzyme activities exacerbated DNA damage, mediating apoptosis through mitochondrial dysfunction. Overall results indicate that therapeutic efficacy of NAp may be enhanced by PLGA nanoparticle formulations to have better ameliorative potentials in combating skin melanoma.     

芹菜素诱导胰腺癌PANC- 1 细胞凋亡与GSH 耗竭的关系

目的研究芹菜素（apigenin）诱导体外培养人胰腺癌PANC-1细胞凋亡作用是否涉及细胞内谷胱甘肽（glutathione,GSH）耗竭。方法体外培养PANc-1细胞,分光光度法测量细胞内GSH水平;流式细胞术检测细胞凋亡率;ELISA法测定细胞DNA断裂。结果apigenin以浓度依赖方式诱导PANC-1细胞凋亡率增高俨〈0．05）,并导致细胞组蛋白／DNA碎片增加。apigenin显著降低PANC-1细胞内GSH水平（P〈0．05）。呈浓度依赖性。500μmol．L^-1GSH预孵育1h,能有效阻断apigenin诱导细胞内GSH耗竭,同时,拮抗apigenin触发细胞凋亡作用（P〈0．05）。结论apigenin诱导人胰腺癌PANC-1细胞凋亡作用与其耗竭细胞内GSH相关。     

芹菜素调控lncRNA MEG8对高糖诱导的人肾小管上皮细胞损伤的影响

目的 探讨芹菜素对高糖诱导的人肾小管上皮细胞损伤的影响及分子机制是否与ln-cRNA MEG8有关.方法 肾小管上皮细胞HK-2细胞分为正常(NG)组、高糖(HG)组、芹菜素低、中、高剂量组、HG+si-NC组、HG+si-MEG8组、HG+芹菜素+pcDNA组、HG+芹菜素+pcDNA-MEG8组.实时荧光定量PCR...     

芹菜叶对自发性高血压大鼠内皮型一氧化氮合酶及长链非编码RNA-sONE的影响研究

背景　高血压是心血管疾病和全因死亡的重要危险因素。研究表明,长链非编码RNA（LncRNA）的异常表达与心血管疾病的发生密切相关。目的　探讨芹菜叶对自发性高血压大鼠内皮型一氧化氮合酶（eNOS）、LncRNA-sONE的影响。方法　2015年9月,将购买的48只（雌雄各24只）10周龄、SPF级、自发性高血压大鼠正常饮食（0.25%的NaCl饲料）饲养2周后,随机分为高盐组、正常盐组和高盐芹菜叶组,每组16只（雌雄各半）。高盐组给予5.00%的NaCl饲料饲养12周;正常盐组给予0.25%的NaCl饲料饲养12周;高盐芹菜叶组先予5.00%的NaCl饲料饲养8周后,再予5.00%的NaCl饲料+10.00%芹菜叶饲养4周;此外,各组予以足量其他饮食。比较3组大鼠干预前及干预4周、8周、12周体质量、收缩压。采用Western blotting法检测eNOS蛋白表达水平,qPCR法检测LncRNA-sONE、eNOS mRNA表达水平。结果　高盐组、高盐芹菜叶组大鼠干预8周、12周体质量均低于正常盐组（P<0.05）。高盐组、高盐芹菜叶组大鼠干预4周、8周、12周收缩压高于正常盐组（P<0.05）;高盐芹菜叶组大鼠干预12周收缩压低于高盐组（P<0.05）。高盐组大鼠干预12周eNOS蛋白表达水平低于正常盐组,LncRNA-sONE、eNOS mRNA表达水平高于正常盐组（P<0.05）;高盐芹菜叶组大鼠干预12周eNOS蛋白、eNOS mRNA表达水平高于正常盐组（P<0.05）;高盐芹菜叶组大鼠干预12周eNOS蛋白、eNOS mRNA表达水平高于高盐组,LncRNA-sONE表达水平低于高盐组（P<0.05）。结论　芹菜叶能升高自发性高血压大鼠eNOS蛋白及其mRNA表达水平,降低LncRNA-sONE表达水平;同时eNOS、LncRNA-sONE可能参与了芹菜叶的降压机制。