Effects of storage conditions and heat treatment on the hesperidin concentration in Newhall navel orange (Citrus sinensis Osbeck cv. Newhall) juice

The effects of storage condition and heat treatment on the hesperidin concentration in Newhall navel orange (Citrus sinensis Osbeck cv. Newhall) juice were investigated systematically in the present study. Our results indicated that, 1) the hesperidin concentration in orange juice gradually decreased during storage, even when the juice was stored at low temperature; 2) hesperidin was stable in juice subjected to either heat or concentrating treatment; 3) the total content of hesperidin in juice remained almost unchanged, although the hesperidin would gradually settle out from the juice, possibly caused by the decomposition of vitamin C during storage, resulting in the reduction of hesperidin concentration in juice. The experimental observations in this study might help clarify some inconsistent reports on hesperidin content in the literature and provide valuable information for the measurement of hesperidin in orange juice. Meanwhile, the results of our study suggested that the hesperidin content in juice should not be considered as a marker for identification of NFC (not from concentrate) orange juice.     

金果饮的质量控制研究

目的：对金果饮的质量标准进行制定。方法：对玄参、西青果、陈皮建立薄层色谱的鉴别方法;采用高效液相色谱法测定橙皮苷的含量。用Agilent SB C18柱（4.6mm×250mm,5μm）,以甲醇-6%乙酸溶液（35∶65）为流动相;检测波长为283nm,流速1mL·min-1。结果：薄层色谱鉴别中,供试品在与对照药材色谱相应的位置上,显示相同颜色的斑点,斑点清晰,分离效果好,阴性无干扰;含量测定法中,橙皮苷在36.35～726.95ng（r=0.9999,n=5）范围内呈良好的线性关系,平均加样回收率为98.58%（RSD为1.85%）。结论：建立的方法简便、准确、专属性强、重复性好,能有效用于金果饮的质量控制。     

Effect of hesperidin and neohesperidin from bittersweet orange (Citrus aurantium var. bigaradia) peel on indomethacin-induced peptic ulcers in rats

Hesperidin and neohesperidin are the major flavanones isolated from bittersweet orange. It was recently reported that they have potent anti-inflammatory effects in various inflammatory models. In the present study, the effects of hesperidin and neohesperidin on indomethacin-induced ulcers in rats and the underlying mechanisms were investigated. Gastric ulcers were induced in rats with a single dose of indomethacin. The effects of pretreatment with hesperidin and neohesperidin were assessed in comparison with omeprazole as reference standard. Ulcer index, gene expression of gastric cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), lipid peroxidation product, malondialdhyde (MDA), and reduced glutathione (GSH) content in stomach were measured. Furthermore, gross and histopathological examination was performed. Our results indicated that both hesperidin and neohesperidin significantly aggravated gastric damage caused by indomethacin administration as evidenced by increased ulcer index and histopathological changes of stomach.     

Screening and identification of bioactive compounds from citrus against non-structural protein 3 protease of hepatitis C virus genotype 3a by fluorescence resonance energy transfer assay and mass spectrometry

BACKGROUNDHepatitis C virus genotype 3a (HCV G3a) is highly prevalent in Pakistan. Due to the elevated cost of available Food and Drug Administration-approved drugs against HCV, medicinal natural products of potent antiviral activity should be screened for the cost-effective treatment of the disease. Furthermore, from natural products, active compounds against vital HCV proteins like non-structural protein 3 (NS3) protease could be identified to prevent viral proliferation in the host.AIMTo develop cost-effective HCV genotype 3a NS3 protease inhibitors from citrus fruit extracts.METHODSFull-length NS3 without co-factor non-structural protein 4A (NS4A) and codon optimized NS3 protease in fusion with NS4A were expressed in Escherichia coli. The expressed protein was purified by metal ion affinity chromatography and gel filtration. Citrus fruit extracts were screened using fluorescence resonance energy transfer (FRET) assay against the protease and polyphenols were identified as potential inhibitors using electrospray ionization-mass spectrometry (MS)/MS technique. Among different polyphenols, highly potent compounds were screened using molecular modeling approaches and consequently the most active compound was further evaluated against HCV NS4A-NS3 protease domain using FRET assay.RESULTSNS4A fused with NS3 protease domain gene was overexpressed and the purified protein yield was high in comparison to the lower yield of the full-length NS3 protein. Furthermore, in enzyme kinetic studies, NS4A fused with NS3 protease proved to be functionally active compared to full-length NS3. So it was concluded that co-factor NS4A fusion is essential for the purification of functionally active protease. FRET assay was developed and validated by the half maximal inhibitory concentration (IC₅₀) values of commercially available inhibitors. Screening of citrus fruit extracts against the native purified fused NS4A-NS3 protease domain showed that the grapefruit mesocarp extract exhibits the highest percentage inhibition 91% of protease activity. Among the compounds identified by LCMS analysis, hesperidin showed strong binding affinity with the protease catalytic triad having S-score value of -10.98.CONCLUSIONFused NS4A-NS3 protease is functionally more active, which is effectively inhibited by hesperidin from the grapefruit mesocarp extract with an IC₅₀ value of 23.32 µmol/L.     

半夏糖浆含量测定研究

目的建立高效液相色谱(HPLC)法测定半夏糖浆中甘草酸和橙皮苷的含量测定方法。方法采用高效液相色谱法,以Hypersil BDS C18柱为固定相,橙皮苷以甲醇-水(32∶68)为流动相,流速1 ml/min,检测波长284 nm,柱温为室温;甘草酸以甲醇-0.2 mol/L醋酸铵溶液-冰醋酸(67∶33∶1),流速1 ml/min,检测波长250 nm,柱温为室温。结果橙皮苷的量在0.204～1.530μg范围内与峰面积积分值呈良好的线性关系,r=0.999 9,RSD=0.68%。甘草酸的量在0.394 6～2.959 5μg范围内与峰面积积分值呈良好的线性关系r=0.999 9,RSD=0.96%。结论所建立的方法能准确、快速地进行定量检测,可用于半夏糖浆的质量控制。     

单面针茎中氯化两面针碱和橙皮苷含量测定

目的利用HPLC测定单面针茎中氯化两面针碱和橙皮苷的含量。方法采用AgilentZorbaxSB—C18柱（250mm×4．6mm,5μm）,分别以0．02mol／ml磷酸二氢钾：乙腈（74：26）、甲醇：水（40：60）为流动相,流速均为1．0ml／min,柱温均为30℃,检测波长分别为328nm和283nm。结果氯化两面针碱,橙皮苷分别在0．072～0．36μg和0．1472～0．736μg范围内线性关系良好;平均回收率（n=5）分别为100．56％、100．86％。结论该方法为单面针药材的质量控制提供实验依据,该方法具有简便、快速、准确、重现性好的特点。     

橙皮苷对慢性支气管炎大鼠炎性因子表达的影响

目的:观察橙皮苷对慢性支气管炎大鼠模型的炎性因子表达变化的影响。方法:将大鼠随机分为3组:对照组、模型组和橙皮苷给药组。采用熏烟法建立慢性支气管炎大鼠模型,采用HE染色检测支气管肺组织的病理学情况;ELISA法检测支气管肺泡灌洗液中肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)和白细胞介素10(IL-10)含量;Western blot法检测肺组织中ICAM-1和VCAM-1的蛋白表达水平。结果:与模型组相比,橙皮苷给药组的支气管肺组织的炎性浸润减少,肺泡结构的完整性增加,支气管肺泡灌洗液中TNF-α和IL-6的含量显著下降(P0.05),IL-10含量无显著变化;肺组织中ICAM-1和VCAM-1的蛋白表达水平显著下降(P0.05)。结论:橙皮苷具有降低慢性支气管炎中炎性因子TNF-α和IL-6含量的作用,可能与调节ICAM-1和VCAM-1的蛋白表达有关。     

藿香正气水中橙皮苷的超声提取工艺

目的:设计正交试验,对藿香正气水中橙皮苷的超声提取条件进行优化。方法:超声提取橙皮苷,用HPLC测定其含量。结果:在提取液为40%甲醇、温度为50℃、超声15min条件下,提取率高。橙皮苷在5～100μg·ml~(-1)(r=0.999 6,n=6)浓度范围具有良好线性关系,平均回收率为99.78%,RSD为1.38%。结论:方法简单、易操作,稳定可行,为藿香正气水的质量控制提供了方法。     

HPLC法测定保和咀嚼片中橙皮苷的含量

目的:高效液相色谱法测定保和咀嚼片中橙皮苷的含量.方法:采用Luna C18色谱柱,以乙腈-水-冰醋酸(20:80:1)为流动相,检测波长280 nm.结果:橙皮苷在0.06μg～0.18μg之间,线性关系良好,r=0.999 8(n=7),平均回收率为100.45%.RSD为1.20%(n=5).结论:方法简便可行,重现性好,能有效控制产品质量.