UV spectrometric and DC polarographic studies on apigenin and luteolin

Remarks on polyphenolic compounds has been arisen since past few years. The flavonoids appears to be the important groups of compounds with their capability to inhibit DNA damage, lipid peroxidation, to quench free radicals and, at least, anticarcinogenic and antiproliferative effects. On the other hand, their mechanism of action is still unexplained. Apigenin and luteolin are the most wide-spread flavones and they exhibited to be useful in chemoprevention. UV spectrometric and DC polarographic studies on these two compounds have been carried out with regard to changing pH. The most significant changes were observed at basic pH. These results could aid to elucidation of their mechanism of action as pH is one of the important factors for bioprocesses passing in living organisms.     

芹菜素抑制人卵巢癌CAOV3细胞增殖的研究

目的探讨芹菜素对人卵巢癌CAOV3细胞增殖的抑制作用。方法2006年10月至12月在中国医科大学附属第一医院肿瘤研究所应用四甲基偶氮唑蓝（MTT）比色法检测芹菜素对CAOV3细胞增殖的抑制作用，流式细胞仪技术分析芹菜素对CAOV3细胞增殖周期的影响。结果20—160μmol／L芹菜素均能抑制CAOV3细胞的生长，且呈明显的时间、剂量依赖性关系。细胞周期分析显示芹菜素组G2／M期细胞比例明显增多，呈量效关系。结论芹菜素抑制CAOV3细胞增殖，可能通过使CAOV3细胞停滞在G2／M期而诱导其凋亡。     

In vitro antiproliferative characteristics of flavonoids and diazepam on SNU-C4 colorectal adenocarcinoma cells

The need for beneficial use of sedatives in oncologic patients is increasing. Therefore, in this study, antiproliferative characteristics of herbal and synthetic sedatives were examined in vitro in SNU-C4 human colorectal adenocarcinoma cells. Apigenin (50% inhibition concentration, IC₅₀ = 1.8 ± 0.5 μM) and diazepam (IC₅₀ = 7.0 ± 0.5 μM) showed concentration-dependent inhibition of SNU-C4 cancer cell survival. Efficacy of cancer cell survival inhibition by apigenin and diazepam was much lower than that of 5-fluorouracil (5-FU), a known chemotherapeutic drug. However, 10⁻⁶ M concentration of apigenin and diazepam potentiated 5-FU-induced cytotoxicity. In SNU-C4 cells, 10⁻⁶ M concentrations of diazepam, flumazenil (Ro15-1788), Ro5-4864, or PK11195, all ligands for central- or peripheral-type benzodiazepine (BZD) receptors, inhibited cell survival like the flavonoid apigenin (4′,5,7-trihydroxyflavone) and fisetin (3,7,3′,4′-tetrahydroxyflavone). Also like the plant flavonoids, treatment with 10⁻⁶ M concentration of diazepam for 3 days hardly affect the peripheral-type BZD receptor (PBR) messenger RNA (mRNA) expression and inhibited glucose utilization of SNU-C4 cells. Treatment with flavonoids or diazepam for 6 days upregulated PBR mRNA expression and cell cytotoxicity of SNU-C4 cells. Furthermore, treatment with 10⁻⁶ M concentration of apigenin, a natural sedative material originating from traditional herbs, positively modulated BZD-induced antiproliferative cytotoxicity in SNU-C4 cells. Overall, the in vitro antiproliferative activity on SNU-C4 cancer cells of herbal sedatives, such as apigenin, plus additive enhancement of synthetic BZD- and 5-FU-induced antiproliferative activities, were shown. In conclusion, this study provides experimental basis for advanced trial in the future.     

反相高效液相色谱法测定亳菊提取物中木犀草素及芹菜素的含量

目的 建立同时测定亳菊提取物中木犀草素及芹菜素的反相高效液相色谱检测方法.方法 采用Restek Pinnacle Ⅱ C18(250 mm×4.6 mm,5 μm)色谱柱,配制流动相为甲醇∶四氢呋喃∶0.3%H3PO4水溶液=42∶13∶56,在流速为1.0 ml/min,检测波长为350 nm,柱温为室温,进样量为10 μl的情况下建立测定方法.结果 木犀草素线性范围为3.99～99.75 μg/ml(r=0.999 6),平均加样回收率为105.9%,RSD=1.5%;芹菜素线性范围为4.095～102.375 μg/ml(r=0.999 6),平均加样回收率为103.7%,RSD=2.5%.结论 用该方法测定亳菊提取物中木犀草素及芹菜素的含量,具有分析时间较短,分离度高的特点.     

Expression pattern of NMDA receptors reveals antiepileptic potential of apigenin 8-C-glucoside and chlorogenic acid in pilocarpine induced epileptic mice

The present study was aimed to evaluate the effect of apigenin 8-C-glucoside (Vitexin) and chlorogenic acid on epileptic mice induced by pilocarpine and explored its possible mechanisms. Intraperitonial administration of pilocarpine (85 mg/kg) induced seizure in mice was assessed by behavior observations, which is significantly (p > 0.05) reduced by apigenin 8-C-glucoside (AP8CG) (10 mg/kg) and chlorogenic acid (CA) (5 mg/kg), similar to diazepam. Seizure was accompanied by an imbalance in the levels of Gamma-aminobutyric acid (GABA) and glutamate in the pilocarpine administered group. Moreover, convulsion along with reduced acetylcholinesterase, increased monoamine oxidase and oxidative stress was observed in epileptic mice brain. AP8CG and CA significantly restored back to normal levels even at lower doses. Further, increased lipid peroxidation and nitrite content was also significantly attenuated by AP8CG and CA. However, CA was found to be more effective when compared to AP8CG. In addition, the mRNA expression of N-methyl-d-aspartate receptor (NMDAR), mGluR1 and mGlu5 was significantly (P ≤ 0.05) inhibited by AP8CG and CA in a lower dose. The mRNA expression of GRIK1 did not differ significantly in any of the group and showed a similar pattern of expression. Our result shows that AP8CG and CA selectively inhibit NMDAR, mGluR1 and mGlu5 expression. Modification in the provoked NMDAR calcium response coupled with neuronal death. Hence, these findings underline that the polyphenolics, AP8CG and CA have exerted antiepileptic and neuroprotective activity by suppressing glutamate receptors.     

Production and characterization of antioxidant apigenin nanocrystals as a novel UV skin protective formulation

Flavonoids have many positive effects on various cell layers of the skin (e.g. anti-oxidant, anti-allergic and anti-inflammatory effects). However, the limiting factor of the use of flavonoids is their low water solubility. To overcome the poor solubility, apigenin nanosuspensions were prepared using the combination technology (CT), i.e. bead milling and subsequently high pressure homogenization. Distinct reduction in particle size was observed after each bead milling passage resulting in z-average (PCS) of 413 nm and a polydispersity index of 0.202. The LD data showed a similar pattern of particle size reduction reaching a diameter 99% (d(v)99%) of 0.515 μm. The antioxidant capacity of apigenin nanocrystals were almost doubled compared to the original coarse suspension. The developed smartCrystals can be easily incorporated into gels, which makes apigenin nanocrystals available for dermal application as efficient antioxidant.     

HPLC法测定紫花地丁中槲皮素、芹菜素、木犀草素含量

目的:建立高效液相色谱法测定紫花地丁中槲皮素、芹菜素和木犀草素的含量测定方法。方法:采用HPLC法对紫花地丁中的槲皮素、芹菜素、木犀草素进行含量测定。色谱条件为:C18柱(5μm,4.6 mm×250 mm),柱温30℃,流动相为乙腈-甲醇-0.2%磷酸水溶液梯度洗脱,流速0.8 mL.min-1,检测波长350 nm。结果:槲皮素、芹菜素和木犀草素色谱分离良好,浓度与峰面积呈良好线性关系,线性范围分别为槲皮素7.5~90.0 mg.L-1,r=0.999 9(n=6);芹菜素7.5~180.0 mg.L-1,r=0.999 8(n=6),木犀草素2.5~55.0 mg.L-1,r=0.999 9(n=6)。平均加样回收率分别为95.1%(n=9,RSD=1.0%)、95.7%(n=9,RSD=1.2%)、97.8%(n=9,RSD=0.7%)。结论:本方法测定了5批紫花地丁的槲皮素、芹菜素和木犀草素的含量,该方法分离度好、快速、简便、重现性好。     

高压液相色谱法测定中药米口袋中木犀草素和芹菜素的含量

目的建立一种可用于评价米口袋药材质量的含量的高压液相色谱(HPLC)测定方法。方法采用Agilent ZORBAX SB-C18色谱柱,以0.2%醋酸水溶液和甲醇为流动相,流速:1.0ml/min;检测波长:254nm;柱温:30℃。用梯度洗脱法同时测定米口袋药材甲醇提取物中木犀草素和芹菜素的含量。结果测得木犀草素和芹菜素的线性范围分别为0.01044～0.35g/L(r2=0.9997)和0.00391～0.125g/L(r2=0.9994);加样回收率分别为95.96%和98.41%。结论该法能快速、准确地测定米口袋中2个黄酮类成分的含量,为米口袋的质量控制提供了1种较好的检测方法。     

Combination of N-(4-hydroxyphenyl) retinamide and apigenin suppressed starvation-induced autophagy and promoted apoptosis in malignant neuroblastoma cells

Autophagy is a catabolic process for recycling of cellular contents in response to metabolic stress in malignant tumors. We explored efficacy of the synthetic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) and the isoflavonoid apigenin (APG) in the serum-starved human malignant neuroblastoma cells. Combination of 0.5 μM 4-HPR and 50 μM APG synergistically decreased cell viability in the serum-starved neuroblastoma SH-SY5Y, SK-N-BE2, and IMR-32 cells. Acridine orange (AO) staining and LC3 II upregulation showed that serum-starvation for 12 and 24 h progressively increased the formation of acidic vesicular organelles (AVO) and autophagy in SH-SY5Y cells. Further, AO staining and flow cytometry showed blockage of formation of AVO and accumulation of auophagic population, respectively, following the treatment of the serum-starved SH-SY5Y cells with combination of 0.5 μM 4-HPR and 50 μM APG. Combination therapy downregulated autophagy inducing proteins such as Beclin 1, LC3 II, TLR-4, and Myd88 while upregulated autophagy inhibitory p-Akt/mTOR singaling pathway. Consistent with the hypothesis that inhibition of autophagy could induce apoptosis, we noticed inhibition of autophagy and induction of apoptosis in the serum-starved SH-SY5Y cells with the suppression of the survival factor NF-κB, upregulation of pro-apoptotic Bax, downregulation of anti-apoptotic Bcl-2, activation of caspase-3, and degradation of poly(ADP-ribose) polymerase (PARP) after combination therapy. Collectively, combination of 4-HPR and APG worked synergistically to suppress autophagy and promote apoptosis in human malignant neuroblastoma cells.