芒果叶提取物芒果苷安全性评价Ⅲ:长期毒性试验

目的:通过Beagle犬长期灌胃给药,考察芒果苷的长期毒性,为临床安全用药的剂量设计提供参考。方法:芒果苷以0.2,1.0,0 g/kg剂量灌服给予Beagle犬,对照组给予0.5%CMC-Na。给药1次/d,连续1个月。测体重、体温1次/周,并根据体重调整给药量。于给药前、给药结束和恢复结束时进行眼科检查、心电图分析、尿液分析,并采血进行血液、血液生化学、血液电解质、血凝等检测,给药结束时各组取4只犬麻醉后取骨髓涂片检查,然后放血处死,进行系统尸解和组织病理学检查。给药结束后各组剩余2只犬停药,常规喂2周,进行可逆性和延迟毒性观察。结果:在给药期间,给药犬粪便颜色加深,但粪便形状、排便次数、排便量均正常;其余外观体征、行为活动、腺体分泌、摄食情况等无明显异常。芒果苷连续给予Beagle 1犬1个月:犬体重增长正常,各给药组在各时点对对照组比较,差异均无统计学意义(P0.05);犬心率G波、PR间期、QRS间期、QT间期、ST段偏移量均正常,各给药组在各时点与对照组比较,心率差异无统计学意义(P0.05);血液学各指标均正常;血液生化学各指标均正常。无论给药前,还是给药后,各犬左右两眼曲光介质透明度、视网膜、血管、视乳头均基本正常,未见有供试品引起的明显改变。芒果苷连续给予Beagle犬1个月,与对照组比较,给药犬骨髓增生活跃,粒系细胞与红系有核细胞比例正常,粒系及红系各阶段细胞比例未见异常,淋巴系和巨核细胞系亦无特殊,属正常骨髓象;各给药组在各时点与对照组比较,骨髓各类细胞均无明显异常。芒果苷连续给予Beagle犬1个月及停药恢复2周,与对照组比较,给药犬脏器重量及系数基本正常,各给药组在各时点与对照组比较,高剂量组在给药结束时睾丸和附睾重量和系数均明显高于对照组,差异有统计学意义(P0.05)。结论:芒果苷连续给药1个月和停药恢复2周,各给药组均有一些散在病变发生,但病变特征没有共性,也未见有剂量-毒性关系,且部分改变也见于对照组,未发现在供试品剂量范围,因供试品导致的Beagle犬的特征病变和毒性靶器官,给药剂量与药时曲线下面积呈正相关,长期经口给予芒果苷于Beagle犬体内显露无蓄积倾向。Beagle犬安全剂量上限范围为4 g/kg,为人临床拟推荐剂量的127倍。因此,按临床拟推荐剂量用药应是比较安全的。     

芒果苷对骨关节炎的潜在治疗机制：通过抑制巨噬细胞NF-κB调节巨噬细胞M2极化

目的芒果苷通过抑制巨噬细胞NF-κB调节巨噬细胞极化为M2,探索芒果苷治疗骨关节炎的潜在机制。 方法RAW264.7小鼠巨噬细胞系进行体外培养,IL-4诱导巨噬细胞M₂极化,用芒果苷对巨噬细胞进行干预。实验分为空白组、对照组和实验组。CCK-8检测芒果苷的细胞毒性。钙黄绿素（Calcein-AM）和碘化丙啶（PI）双荧光染色法检测细胞活性,流式细胞仪检测细胞凋亡的情况。免疫荧光染色评估甘露糖受体（CD206抗体）在巨噬细胞的分泌情况,实时荧光定量PCR（qRT-PCR）法检测NF-κB通路相关基因RelA（p65）及M2巨噬细胞相关基因CD206、IL-10的表达情况。 结果浓度为20 μM的芒果苷对巨噬细胞无明显毒性作用,芒果苷在该浓度下能显著下调RelA的表达和上调IL-10、CD206的表达,从而抑制NF-κB通路激活巨噬细胞M2极化。 结论芒果苷能够抑制巨噬细胞NF-κB调节巨噬细胞极化为M2,M2巨噬细胞具有抗炎功能,促进软骨修复功能,为芒果苷在骨关节炎的研究中提供可能的机制理论。     

Mangiferin and hesperidin metabolites are absorbed from the gastrointestinal tract of pigs after oral ingestion of a Cyclopia genistoides (honeybush tea) extract

Health-promoting properties such as antioxidative, anticarcinogenic, and cholesterol-lowering effects are described for mangiferin and hesperidin, the major phenolic compounds present in Cyclopia genistoides (honeybush). However, knowledge of their metabolic fate and their absorption from the gastrointestinal tract is very limited. The aim of this study was to determine the concentrations of mangiferin, hesperidin, and their metabolites in plasma, urine, and feces samples from pigs consuming an extract of Cyclopia genistoides. Pigs were administered up to 74 mg mangiferin per kilogram of body weight and 1 mg hesperidin per kilogram of body weight per day for 11 days. Plasma samples were collected at various time points on days 9 and 11 of the study and days 1 and 2 after termination of extract administration. Urine and feces were collected in fractions for 24 hours. In the plasma samples, the aglycone of mangiferin (norathyriol) was detected. Mean plasma concentrations ranged from 7.8 to 11.8 μmol/L. Six metabolites of mangiferin and hesperidin were detected in the urine, including methyl mangiferin, norathyriol, its monoglucuronide, hesperetin, hesperetin monoglucuronide, and eriodictyol monoglucuronide. Between 26.0% and 30.8% of the administered dose of hesperidin and only between 1.4% and 1.6% of mangiferin could be detected in the urine on days 9 and 11 of the study. Approximately 8.2% of the administered dose of mangiferin was determined in the feces. The main metabolite was norathyriol. Neither hesperidin nor metabolites ascribed to hesperidin intake were detected. The results suggest that formation of norathyriol from mangiferin occurs in vivo, and specific metabolites were identified in blood and excretion products in urine and feces. This study will aid in investigating the physiological functions of the parent compounds in vivo.     

Combination treatment with oxaliplatin and mangiferin causes increased apoptosis and downregulation of NFκB in cancer cell lines

Mangiferin-mediated down-regulation of NFκB showed potential for chemotherapeutic agent-mediated cell death, suggesting a role in combination therapy for cancer. In this study the combined mechanism of the anticancer action of oxaliplatin and mangiferin was investigated. MTT dose response curves, trypan blue staining, caspase 3 assays as well as DNA cell cycle analyses were performed on HeLa, HT29 and MCF7 cancer cell lines, with and without the addition of 10 μg/ml mangiferin. Mitochondrial membrane potential, DNA fragmentation, resistance induction studies and NFκB assays were performed on HT29 cells only. Addition of 10 μg/ml mangiferin reduced oxaliplatin IC(50) values in HT29 (3.4 fold) and HeLa (1.7 fold) cells in the MTT assay while reducing trypan blue staining. This was accompanied by increased caspase 3 activation and DNA fragmentation and a delay in the S-phase of the cell cycle. Mitochondrial membrane permeabilization was not enhanced in the combination treatment. Mangiferin was shown to cause a reduction of NF-κB activation in HT29 cells rendered resistant to oxaliplatin. The present study indicates that mangiferin in combination with oxaliplatin favours apoptotic cell death and thereby improves the efficacy of oxaliplatin in vitro. In addition, combination therapy with mangiferin may also counteract the development of resistance in cancer cell lines.     

HPLC法测定小儿咳宁糖浆中芒果苷的含量

目的建立以高效液相色谱法测定小儿咳宁糖浆中芒果苷含量的方法。方法采用Phenomenex Gemini C18(250 mm×4.6 mm,5μm)色谱柱,以甲醇-水-甲酸(30:70:3)为流动相,流速为0.8mL.min-1,检测波长为275 nm,采用外标法定量测定。结果芒果苷在8.160~102.0μg.mL-1(r=0.999 8)浓度范围内线性关系良好;平均加样回收率(n=6)为98.4%,RSD=1.75%。结论该方法简便、准确、重复性好,可用于测定小儿咳宁糖浆中芒果苷的含量。     

知母总黄酮提取工艺的研究

目的建立知母中总黄酮提取工艺，为知母总黄酮开发和利用提供依据。方法对提取方法、提取溶剂、提取时间进行研究，采用紫外分光光度法测定知母中总黄酮含量。结果知母中总黄酮较佳提取工艺为：70％乙醇超声提取30min。结论该方法简便、快速、准确，可作为知母中总黄酮提取及含量测定方法。     

UPLC-MS/MS法测定大补阴丸中小檗碱等4种成分含量

目的：建立同时测定大补阴丸中小檗碱、黄柏碱、毛蕊花糖苷、芒果苷的含量方法。方法：采用液质联用技术,以Agilent ZORBAX SB-C18为色谱柱,以甲醇-甲酸铵溶液为流动相,等度洗脱。结果：小檗碱、黄柏碱、毛蕊花糖苷、芒果苷的线性范围分别为5～500 ng·mL-1（r=0.9991）、0.5～50 ng·mL-1（r=0.9994）、1～100 ng·mL-1（r=0.9987）、1.5～150 ng·mL-1（r=0.9990）;加样回收率为98.35%～102.55%,RSD均＜1.60%;平均含量分别为3.55 、0.322、0.511、0.886 mg·g-1。结论：该法专属性强、快速灵敏,可用于大补阴丸中上述4种成分的含量测定。     

RP-HPLC法测定芒果叶中芒果苷的含量

目的 :建立芒果叶中芒果苷的含量测定方法 ;测定不同产地和月份芒果叶中芒果苷的含量。方法 :采用RP HPLC法 ,内标法定量 ,ShimpackCLC ODS柱分离 ,流动相 :甲醇 0.05mol·L^-1H₃PO₄(三乙胺调pH为 3.5)65∶134 ,检测波长 2 58nm ,柱温为室温 ,流速 1mL·min^-1。从广西南宁市、钦州市和田阳县分别采集芒果叶供试品 ;测定南宁市 1～12月份同一株芒果树的芒果叶中芒果苷的含量。结果 :所建立的RP-HPLC含测方法平均回收率 99.2 % ,RSD =1.05% (n =5)。不同产地和月份的芒果叶中芒果苷的含量存在差异。结论 :所建立的RP-HPLC含测方法灵敏度高 ,准确 ,快速 ,专属性强 ,重现性好。南宁市和田阳县芒果叶中芒果苷的含量相差不大 ,均明显比钦州的高。 7～10月份的芒果苷含量较高 ,最高的为 9月 ,最低的为 2月份。     

芒果苷抑制中波紫外线诱导人成纤维细胞提前衰老的相关研究

【摘要】 目的 研究芒果苷是否对反复亚毒性剂量中波紫外线（UVB）诱导的人二倍体成纤维细胞的早衰起抑制作用及其可能的机制。 方法 实验分成空白对照组（不做处理）,UVB-应激诱导的提前衰老（SIPS）组（单纯照光）,芒果苷4.0 mg/L组（单纯加药）,UVB + 芒果苷1.0 mg/L组、UVB + 芒果苷2.0 mg/L组、UVB + 芒果苷4.0 mg/L组（UVB照射联合药物处理）。成纤维细胞经5次UVB 10 mJ/cm2照射后,用细胞计数法（CCK8法）检测细胞增殖活性,β半乳糖苷酶染色（SA-β-Ga1）计算衰老细胞百分比,流式细胞仪测定细胞周期,蛋白印迹检测衰老相关蛋白p53、p21、p16含量,实时荧光定量PCR（RT-PCR）检测基质金属蛋白酶1（MMP1）、基质金属蛋白酶3（MMP3）、Ⅰ型胶原、Ⅲ型胶原mRNA和衰老相关基因p53、p21、p16 mRNA表达。采用单因素方差分析进行数据统计分析。 结果 UVB + 芒果苷1.0 mg/L组、UVB + 芒果苷2.0 mg/L组、UVB + 芒果苷4.0 mg/L组以及UVB-SIPS组细胞增殖活性（A450）依次为0.322 9 ± 0.011 3、0.336 1 ± 0.016 3、0.342 6 ± 0.014 4、0.288 2 ± 0.020 7（F = 110.08,P < 0.05）,β半乳糖苷酶染色阳性率依次为（88.83 ± 4.54）%、（46.33 ± 5.51）%、（32.17 ± 6.05）%、（93.67 ± 3.75）%（F = 283.54,P < 0.05;与UVB-SIPS组比较,除UVB + 芒果苷1.0 mg/L组外,其他两组均P < 0.05）;G1期细胞阻滞率依次为（72.19 ± 3.42）%、（60.99 ± 2.70）%、（49.80 ± 2.10）%、（82.09 ± 0.89）%（F = 156.01,P < 0.05）。与UVB-SIPS组相比,UVB + 各浓度芒果苷组细胞MMP1和MMP3 mRNA表达降低（F = 69.41、106.41,均P < 0.05）,Ⅰ型胶原和Ⅲ型胶原mRNA表达增加（F = 66.41、46.81,除UVB + 芒果苷1.0 mg/L组P > 0.05外,其他各组均P < 0.05）,衰老相关基因p53、p21、p16 mRNA表达降低（F = 265.60、151.82、329.85,均P < 0.05）,衰老相关蛋白p53、p21和p16的合成减少（F = 160.51、158.53、75.38,均P < 0.05）,各指标检测值的变化与芒果苷浓度之间呈一定依赖性。 结论 芒果苷可能通过下调p53、p21、p16基因的表达来抑制UVB诱导的人二倍体成纤维细胞的早衰。     

芒果苷对大鼠心肌缺血再灌注损伤的保护作用

目的探讨芒果苷对大鼠心肌缺血再灌注损伤的保护作用及机制。方法48只SD大鼠随机分为6组:假手术组、缺血再灌注模型组、芒果苷10mg/kg、20mg/kg和40mg/kg剂量组、地奥心血康组,分别给予相应剂量芒果苷、地奥心血康或等容量的生理盐水灌服21d,末次给药后1h采用左冠状动脉前降支结扎40min再灌120min建立在体大鼠心肌缺血再灌注模型。假手术组完成模型全部操作只穿线不结扎。电镜观察心肌超微结构改变,测定心肌组织髓过氧化物酶,观察中性粒细胞浸润;检测血清乳酸脱氢酶活性;分别检测心肌组织超氧化物歧化酶和丙二醛含量。结果芒果苷可以改善心肌缺血再灌注大鼠心肌超微结构,芒果苷小剂量组就能使超氧化物歧化酶活力升高,心肌组织髓过氧化物酶、丙二醛、乳酸脱氢酶含量降低。结论芒果苷对缺血再灌注损伤的心肌具有保护作用,其机制可能与提高超氧化物歧化酶活性,增强心肌抗氧化能力,减轻氧自由基损伤,稳定细胞膜,减轻心肌组织中性粒细胞浸润有关。