Antiviral Activity of Chrysin Derivatives against Coxsackievirus B3 in vitro and in vivo

Chrysin is a 5,7-dihydroxyflavone and was recently shown to potently inhibit enterovirus 71 (EV71) by suppressing viral 3C protease (3C^pro) activity. In the current study, we investigated whether chrysin also shows antiviral activity against coxsackievirus B3 (CVB3), which belongs to the same genus (Enterovirus) as EV71, and assessed its ability to prevent the resulting acute pancreatitis and myocarditis. We found that chrysin showed antiviral activity against CVB3 at 10 μM, but exhibited mild cellular cytotoxicity at 50 μM, prompting us to synthesize derivatives of chrysin to increase the antiviral activity and reduce its cytotoxicity. Among four 4-substituted benzyl derivatives derived from C(5) benzyl-protected derivatives 7, 9–11 had significant antiviral activity and showed the most potent activity against CVB3 with low cytotoxicity in Vero cells. Intraperitoneal injection of CVB3 in BALB/c mice with 1×10⁶ TCID₅₀ (50% tissue culture infective dose) of CVB3 induced acute pancreatitis with ablation of acinar cells and increased serum CXCL1 levels, whereas the daily administration of 9 for 5 days significantly alleviated the pancreatic inflammation and reduced the elevation in serum CXCL1 levels. Collectively, we assessed the anti-CVB3 activities of chrysin and its derivatives, and found that among 4-substituted benzyl derivatives, 9 exhibited the highest activity against CVB3 in vivo, and protected mice from CVB3-induced pancreatic damage, simultaneously lowering serum CXCL1 levels.     

High-performance liquid chromatographic analysis of chrysin derivatives on a Nova-Pak C18 column

A high-performance liquid chromatographic method has been developed for the separation and quantification of chrysin and synthetic chrysin derivatives (12 chrysin alkyl and 7 chrysin acyl derivatives). The chromatography was performed using a Nova-Pak C18 column. A RP-HPLC was performed by using a binary mixture (MeOH-10 mM H3PO4) as a mobile phase, and the column temperature was maintained at room temperature. A flow rate was 1.0 ml/min, and the effluent was monitored at a wavelenth of 280 nm. The retention times for chrysin acyl and alkyl derivatives were within 10 minutes and 20 minutes, respectively. The absolute recovery of samples were all over 96%. The detection limits were 0.1-18 ng at S/N = 3 ratio.     

黄酮及其衍生物的高效合成和抗肿瘤活性

目的设计合成一系列黄酮衍生物,并考察其抗肿瘤活性,并发现1种高效合成黄酮的新方法。方法以2,46,-三羟基苯乙酮为原料,选择性甲基化、酯化、Baker-Venkataraman重排成β-二酮,进而三氟甲磺酸三甲基硅酯催化β-二酮环合合成5个黄酮;以白杨素为母体,经烷基化反应、Man-nich反应合成了一系列白杨素衍生物。采用MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide]法测试部分目标化合物对脑胶质瘤细胞(C6)和人肝癌细胞(HepG2)的抑制活性。结果与结论合成了17个化合物,其中3个为新化合物。其结构经过核磁共振氢谱、质谱确认;4个目标化合物的体外抗肿瘤活性明显强于白杨素。     

A flavonoid component from Docynia delavayi (Franch.) Schneid represses transplanted H22 hepatoma growth and exhibits low toxic effect on tumor-bearing mice

The fruit of Docynia delavayi (Franch.) Schneid is a kind of popular food in southwestern areas of China. Additionally, its rhizome has been long used as a folk medicine in the treatment of liver cancer by local people. Chrysin is a kind of flavonoid which induces cancer cell death in vitro. However, its anti-tumor activity in vivo and toxicological effects on the tumor-bearing animals still remain poorly understood. In this study, we obtained four flavonoids from this herb. Among them, chrysin showed the strongest cytotoxic effect on an array of cultured tumor cells. Further investigations revealed that it significantly repressed transplanted H22 ascitic hepatic tumor cell growth in vivo. Moreover, this compound displayed little toxic effects. Additionally, we demonstrated that in transplanted tumor tissues, chrysin not only activated caspase-3 and induced apoptosis, but also inhibited the production of vascular endothelial growth factor (VEGF) and suppressed angiogenesis. These data showed that chrysin exhibited prominent anti-tumor activities and low toxic effects in vivo.     

Chrysin abrogates early hepatocarcinogenesis and induces apoptosis in N-nitrosodiethylamine-induced preneoplastic nodules in rats

Flavonoids possess strong anti-oxidant and cancer chemopreventive activities. Chrysin (5,7-dihydroxyflavone) occurs naturally in many plants, honey, and propolis. In vitro, chrysin acts as a general anti-oxidant, causes cell cycle arrest and promotes cell death. However, the mechanism by which chrysin inhibits cancer cell growth and the subcellular pathways activated remains poorly understood. Effect of dietary supplementation with chrysin on proliferation and apoptosis during diethylnitrosamine (DEN)-induced early hepatocarcinogenesis was investigated in male Wistar rats. To induce hepatocarcinogenesis, rats were given DEN injections (i.p., 200 mg/kg) three times at a 15 day interval. An oral dose of chrysin (250 mg/kg bodyweight) was given three times weekly for 3 weeks, commencing 1 week after the last dose of DEN. Changes in the mRNA expression of COX-2, NFkB p65, p53, Bcl-xL and β-arrestin-2 were assessed by quantitative real-time PCR. Changes in the protein levels were measured by western blotting. Chrysin administration significantly (P < 0.001) reduced the number and size of nodules formed. Also, a significant (P < 0.01) reduction in serum activities of AST, ALT, ALP, LDH and γGT was noticed. Expression of COX-2 and NFkB p65 was significantly reduced whereas that of p53, Bax and caspase 3 increased at the mRNA and protein levels. Likewise, a decrease in levels of β-arrestin and the anti-apoptotic marker Bcl-xL was also noted. These findings suggest that chrysin exerts global hepato-protective effect and its chemopreventive activity is associated with p53-mediated apoptosis during early hepatocarcinogenesis.     

Chrysin promotes tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induced apoptosis in human cancer cell lines

Chrysin exists widely in plants, honey and propolis. The anti-cancer property of chrysin has been demonstrated though the molecular mechanism is not clear. In this study, we found that pre-treatment with chrysin could promote the cell death induced by TRAIL according to the morphological changes and appearance of sub-G1 peak in four human cancer cell lines. In HCT-116 cells, the results of flow cytometry analysis showed that the percentage of sub-G1 reached (38.89 ± 3.78) % when pre-treatment of chrysin was used at 40 μM, but that was only (2.53 ± 0.10) % in the untreated group and (13.22 ± 0.20) % in TRAIL alone group. The differences between the combination and the untreated or TRAIL alone group were all significant (P < 0.05) and dose-dependent effect was obvious. Similar results were obtained in CNE1 cells. In the search of molecular mechanisms, we found that pre-treatment with chrysin could increase TRAIL-induced degradation of caspase 3, caspase 8, PARP proteins. Z-VAD-fmk, which is a pan-caspase inhibitor, could inhibit the apoptosis enhanced by the combination of chrysin and TRAIL. All data indicate that chrysin can enhance the apoptosis induced by TRAIL, and the apoptosis is caspase-dependent and related to the activation of caspase 8.     

Modulation of the activity of pro-inflammatory enzymes, COX-2 and iNOS, by chrysin derivatives.

Chrysin, a natural flavone compound found in plants, has anti-inflammatory activity that has been previously explained in part by the suppression of promoter activities of pro-inflammatory enzymes, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Here we present evidence that several chrysin derivatives modulate the activities, as well as the expression, of COX-2 and iNOS enzymes. Nitrate production triggered by lipopolysaccharide (LPS) was suppressed by treatment of cultured Raw264.7 cells (mice macrophage/monocyte) with chrysin, 5-hydroxy-7-methoxyflavone (Ch-2), and 5,7-diacetylflavone (Ch-4). Interestingly, COX-2 enzyme was strongly inhibited by Ch-4 (IC(50)=2.7 microM) but not by other derivatives. Furthermore, the inhibition of COX enzyme by Ch-4 was selective for COX-2 over COX-1. Three-dimensional modeling showed that Ch-4 fits well into the binding pocket of COX-2. The modeling suggested that a hydrogen bond exists between the oxygen of the ketone group at the 7-position of Ch-4 and the hydroxyl group of Tyr355. Docking Ch-4 into the V523I mutant of COX-2 indicated that Ile523 of COX-1 might contribute to the selectivity of COX-2 over COX-1. Ch-4 showed no effect on iNOS activity. Chrysin and Ch-2 weakly inhibited iNOS enzyme activity in the hemoglobin assay, but the underlying mechanisms of inhibition of iNOS by chrysin are not understood.     

C-Isoprenylation of flavonoids enhances binding affinity toward P-glycoprotein and modulation of cancer cell chemoresistance

Previous studies have shown that flavones bind to P-glycoprotein (Pgp) with higher affinity than isoflavones, flavanones, and glycosylated derivatives. In the present work, a series of C- or O-substituted hydrophobic derivatives of chrysin were synthesized to further investigate structural requirements of the A ring toward Pgp modulation. Increasing hydrophobicity at either position 6, 8, or 7 increased the affinity of in vitro binding to a purified cytosolic domain of Pgp, but only benzyl and 3,3-dimethylallyl C-substitution produced a high maximal quenching of the protein intrinsic fluorescence. Inhibition of membrane Pgp within leukemic cells, characterized by intracellular drug accumulation, was specifically produced by isoprenylated derivatives, with 8-(3,3-dimethylallyl)chrysin being even more efficient than the commonly used cyclosporin A.     

Nephroprotective efficacy of chrysin against cisplatin-induced toxicity via attenuation of oxidative stress

Objectives Cisplatin‐induced nephrotoxicity is the main cause for its dose‐limited use in the treatment of various cancers and results in acute renal cell injury through generation of reactive oxygen species. Chrysin possess antioxidant, anti‐inflammatory and anti‐cancer properties. The aim of this study was to investigate the protective efficacy of chrysin against cisplatin‐induced nephrotoxicity. Methods Thirty male Wistar rats were divided into five groups with six rats in each group. Group I served as control and received corn oil (vehicle of chrysin) for 14 days and 0.9% saline (vehicle of cisplatin) on day 14 only. Group II received a single intraperitoneal injection of cisplatin on day 14. Group III and IV were pretreated with two different doses of chrysin in addition to cisplatin and group V received chrysin only. Rats were examined for the effect of chrysin on cisplatin induced depletion of antioxidant enzymes, induction of lipid peroxidation and DNA damage in the kidney, utilizing a well‐established model of cisplatin‐induced nephropathy. Key findings Pretreatment with chrysin significantly attenuated cisplatin‐induced renal oxidative damage by diminishing the DNA damage and toxicity markers, such as creatinine and blood urea nitrogen, lipid peroxidation and xanthine oxidase activity, accompanied by increase in enzymatic (catalase, glutathione peroxidase, glutathione reductase and glutathione‐S‐transferase) and non‐enzymatic (reduced glutathione) antioxidant status. Histological findings further substantiated the protective efficacy of chrysin, which reduced cisplatin‐induced renal damage. Conclusions The data of the present study suggest that chrysin effectively suppress cisplatin‐induced renal injury by ameliorating oxidative stress.     

The effect of chrysin‐loaded nanofiber on wound healing process in male rat

Wound healing is an inflammatory process. Chrysin, a natural flavonoid found in honey, has been recently investigated to have anti‐inflammatory and antioxidant effects. In this work, the effects of chrysin‐loaded nanofiber on the expressions of genes that are related to wound healing process such as P53, TIMPs, MMPs, iNOS, and IL‐6 in an animal model study were evaluated. The electrospinning method was used for preparation the different concentrations of chrysin‐loaded PCL‐PEG nanofiber (5%, 10%, and 20% [w/w]) and characterized by FTIR and SEM. The wound healing effects of chrysin‐loaded PCL‐PEG nanofiber were in vivo investigated in rats, and the expressions of genes related to wound healing process were evaluated by real‐time PCR. The study results showed chrysin‐loaded PLC‐PEG compared to chrysin ointment and control groups significantly increase IL‐6, MMP‐2, MMP‐8, MMP‐9, TIMP‐1, and TIMP‐2 (p < .05). On the other hand, nanofibers containing chrysin significantly decreased p53 and iNOS expression compared to chrysin ointment and control groups (p < .05). According to the results, chrysin‐loaded PCL‐PEG‐PCL nanofibers have positive effects on the expression of the genes that have pivotal role in wound healing.     

Transport of the flavonoid chrysin and its conjugated metabolites by the human intestinal cell line Caco-2.

Chrysin (5,7-dihydroxyflavone), a natural product present in our daily diet, is a potent inhibitor of drug-metabolizing enzymes. However, its oral bioavailability is not known. This study examined the intestinal epithelial transport of chrysin (20 microM), using the human colonic cell line Caco-2 as a model of human intestinal absorption. The apical to basolateral flux of chrysin, with an apparent permeability coefficient (P(app)) during the first hour of 6.9 +/- 1.6 x 10(-6) cm x sec(-1) (mean +/- SEM), was more than 10-fold higher than for the paracellular transport marker mannitol, 0.42 +/- 0.12 x 10(-6) cm x sec(-1). Interestingly, the reverse, basolateral to apical flux of chrysin, P(app) = 14.1 +/- 1.6 x 10(-6) cm x sec(-1), was about 2-fold higher than the apical to basolateral flux (P < 0.01). In transport studies beyond 1 hr, there was a rapid decline in P(app). This correlated with the appearance of two metabolites, M1 (chrysin glucuronide) and M2 (chrysin sulfate), identified by enzymatic hydrolysis procedures and HPLC. Following apical loading of chrysin, as much as 90% of M1 + M2 appeared on the apical side, thus indicating clear efflux of the chrysin metabolites. The addition of the anion transport inhibitor MK-571 (50 microM) on the apical side produced a 71% (P < 0.0001) and 20% (P < 0.05) inhibition of the efflux of M1 and M2, respectively, suggesting the involvement of the multidrug resistance protein MRP2 pump. Indeed, using specific antibodies, MRP2 was in fact detected by western blotting in Caco-2 plasma membranes, whereas MRP1 was not. These observations suggest that chrysin has favorable membrane transport properties but that its intestinal absorption may be seriously limited by surprisingly efficient glucuronidation and sulfation by the enterocytes and almost quantitative efflux by MRP2 of the metabolites formed.     

白杨素Mannich碱衍生物的合成及其抗癌活性

目的设计合成新型白杨素Mannich碱衍生物,并寻求具有抗癌活性的新化合物。方法利用Baker-Venkataraman重排法完成白杨素的全合成,再与甲醛、胺类进行Mannich缩合反应得到目标化合物。采用MTT法,以5-氟尿嘧啶为阳性对照,评价目标化合物对人宫颈癌细胞(Hela)、人肺腺癌细胞(A549)、人胃癌细胞(SGC-901)、人结肠癌细胞(HCT-116)、人白血病细胞(K562)5种肿瘤细胞的抗癌活性。结果与结论合成了10个未见文献报道的新化合物,其结构经1H-NMR、IR和MS确证。体外抗癌活性实验表明,部分化合物显示出较好的抗癌活性。     

Chrysin inhibits metastatic potential of human triple‐negative breast cancer cells by modulating matrix metalloproteinase‐10, epithelial to mesenchymal transition,and PI3K/Akt signaling pathway

Chrysin, a naturally occurring flavone, has been shown to inhibit cell proliferation and induce cell apoptosis in various cancers. However, the effect and mechanisms of chrysin on cancer metastasis are still enigmatic. In this study, metastatic triple‐negative breast cancer (TNBC) cell lines were used to evaluate the antimetastatic activity of chrysin. The results showed that chrysin (5, 10 and 20 μM) significantly suppressed TNBC cell migration and invasion in a dose‐dependent manner. Human matrix metalloproteinase (MMP) antibody array demonstrated that MMP‐10 was downregulated by chrysin, which was further verified by Western blotting and ELISA. Moreover, it was shown that chrysin induced increased E‐cadherin expression and decreased expression of vimentin, snail and slug in TNBC cells, suggesting that chrysin had a reversal effect on epithelial–mesenchymal transition. More importantly, it was demonstrated that inhibiting the Akt signal pathway might play a central role in chrysin‐induced antimetastatic activity by regulating MMP‐10 and epithelial–mesenchymal transition. In conclusion, our study indicates that chrysin exerts antimetastatic activities in TNBC cells, which suggests that chrysin might be a potential therapeutic candidate for the treatment of advanced or metastatic breast cancer. Copyright © 2013 John Wiley & Sons, Ltd.     

7-羟乙基白杨素对PC12细胞的抗氧化作用及机制研究

目的 研究7-羟乙基白杨素对PC12细胞的抗氧化作用，并对其保护机制进行探讨。方法 使用CCK-8试剂盒检测PC12细胞的存活率，筛选出7-羟乙基白杨素作用的最佳浓度进行实验。之后将PC12细胞随机分为4组，分别为对照组、缺氧组、白杨素组和7-羟乙基白杨素组。使用微量酶标法测定细胞培养基中LDH活性，DCFH-DA染色观察细胞内ROS含量，同时使用试剂盒对细胞内MDA、SOD和CAT水平进行测定，提取细胞总蛋白通过Western blotting检测评价Nrf2及其下游蛋白表达量。结果 缺氧后PC12细胞存活率显著下降，经7-羟乙基白杨素预处理后可获得改善；白杨素组和7-羟乙基白杨素组上清液中的LDH和细胞内ROS、MDA含量与缺氧组相比显著降低，SOD和CAT含量与缺氧组相比显著升高，同时发现7-羟乙基白杨素在各个方面的保护作用都明显强于白杨素。缺氧条件会使Nrf2、Keap1、HO-1、NQO1蛋白表达增加，7-羟乙基白杨素干预后可以进一步提高它们的蛋白表达。结论 7-羟乙基白杨素具有比白杨素更好的保护效果，它可以通过激活Nrf2/ARE通路减轻PC12细胞由于缺氧造成的损伤。     

Isolation,characterization, development and evaluation of phytoconstituent based formulation for diabetic neuropathy

BackgroundMorus alba Linn, referred to as white mulberry, is a potential traditional medicine for diabetes and neuroprotection.AimIsolation, characterization, development and evaluation of phytoconstituent based formulation for diabetic neuropathy.Material and methodsThe stem Bark of M. alba was peeled and subjected to extraction. A phytoconstituent was then isolated by column chromatography and characterized using Mass spectroscopy, FTIR, and NMR. The isolated phytoconstituent was used to formulate a nanoemulsion. Nanoemulsion was also characterized for viscosity, surface tension, refractive index, pH, and particle size. Selected nanoemulsion formulations were then tested for acute oral toxicity and diabetic neuropathy, including behavioral, hematological, histopathological, and biomarker examinations.ResultsThe spectral analysis affirmed that the isolated compound was found to be chrysin. A nanoemulsion formulation was made using the chrysin and was characterized and found to be stable during the stability testing and fulfilled all other testing parameters. Then acute oral toxicity study of the formulations was found to be safe. Formulations were found to possess significant results against diabetic neuropathy in rats. Biomarkers were analyzed for their mechanistic involvement in reducing neuropathy in rats, and it was found that the oxidative pathway was considerably restored, suggesting that chrysin causes these effects via this pathway.ConclusionsResults suggests that isolated phytoconstituent (chrysin) from the bark of Morus alba derived nanoemulsion has protective and beneficial effects by diminishing the oxidative damage against alloxan-induced diabetic neuropathy in rats.     

6,8-二-三氟甲基-5-羟基-7-乙酰基白杨素的抗肿瘤作用

目的　研究 6 ,8 二 三氟甲基 5 羟基 7 乙酰基白杨素的抗肿瘤作用及机制。方法　采用MTT比色法检测白杨素及白杨素乙酰化、卤化、三氟甲基化衍生物体外抗肿瘤活性 ;小鼠Lewis肺癌移植瘤模型观察目标化合物 (6 ,8 二 三氟甲基 5 羟基 7 乙酰基白杨素 )体内抗肿瘤作用 ;PI染色流式细胞术分析 6 ,8 二 三氟甲基 5 羟基 7 乙酰基白杨素对人胃癌(SGC 790 1 )细胞周期的影响。结果　白杨素及白杨素乙酰化、卤化、三氟甲基化衍生物对体外培养人胃癌 (SGC 790 1 )细胞 ,人急性粒细胞性白血病 (HL 6 0 )细胞和人结肠癌 (HT 2 9)细胞具有抑制增殖作用 ;以 6 ,8 二 三氟甲基 5 羟基 7 乙酰基白杨素作用最强 ,其对SGC 790 1细胞 ,HT 2 9细胞和HL 6 0细胞的IC50 分别是 2 5 1 ,1 96和 0 1 8μmol·L-1 。 6 ,8 二 三氟甲基 5 羟基 7 乙酰基白杨素对小鼠Lewis肺癌皮下移植原发瘤及自发性肺转移具有抑制作用 ,呈剂量依赖关系。6 ,8 二 三氟甲基 5 羟基 7 乙酰基白杨素 (1～ 2 μmol·L-1 )能使SGC 790 1细胞周期阻滞于G1 期。结论　 6 ,8 二 三氟甲基 5 羟基 7 乙酰白杨具有抗肿瘤作用 ,其机制可能与使细胞周期G1 期阻滞相关     

Chrysin serves as a novel inhibitor of DGKα/FAK interaction to suppress the malignancy of esophageal squamous cell carcinoma (ESCC)

Among current novel druggable targets, protein–protein interactions (PPIs) are of considerable and growing interest. Diacylglycerol kinase α (DGKα) interacts with focal adhesion kinase (FAK) band 4.1-ezrin-radixin-moesin (FERM) domain to induce the phosphorylation of FAK Tyr397 site and promotes the malignant progression of esophageal squamous cell carcinoma (ESCC) cells. Chrysin is a multi-functional bioactive flavonoid, and possesses potential anticancer activity, whereas little is known about the anticancer activity and exact molecular mechanisms of chrysin in ESCC treatment. In this study, we found that chrysin significantly disrupted the DGKα/FAK signalosome to inhibit FAK-controlled signaling pathways and the malignant progression of ESCC cells both in vitro and in vivo, whereas produced no toxicity to the normal cells. Molecular validation specifically demonstrated that Asp435 site in the catalytic domain of DGKα contributed to chrysin-mediated inhibition of the assembly of DGKα/FAK complex. This study has illustrated DGKα/FAK complex as a target of chrysin for the first time, and provided a direction for the development of natural products-derived PPIs inhibitors in tumor treatment.Key Words: Chrysin, Esophageal squamous cell carcinoma, DGKα, FAK, Protein–protein interactions     

Alleviation of hepatic injury by chrysin in cisplatin administered rats: Probable role of oxidative and inflammatory markers

BackgroundCisplatin is an effective and extensively used chemotherapeutic agent to treat range of malignancies, but its therapeutic use is limited because of dose-dependent nephrotoxicity and hepatotoxicity. Several published reports advocate that supplementation with antioxidant can influence cisplatin induced hepatic damage.MethodIn the present study the Wistar rats were subjected to concurrent prophylactic oral treatment of chrysin (25 and 50 mg/kg b.wt.) against the hepatotoxicity induced by intraperitoneal administration of cisplatin (7.5 mg/kg b.wt.). Efficacy of chrysin against the hepatotoxicity was evaluated in terms of biochemical estimation of antioxidant enzyme activities, histopathological changes and expression levels of molecular markers of inflammation.ResultsChrysin ameliorated cisplatin-induced lipid peroxidation, xanthine oxidase activity, glutathione depletion, decrease in antioxidant (catalase, glutathione reductase, superoxide dismutase, glutathione peroxidase and glucose-6 phosphate dehydrogenase) and phase-II detoxifying (glutathione-S-transferase and quinone reductase) enzyme activities. Chrysin also attenuated expression of COX-2, iNOS and levels of NFκB and TNF-α, and hepatic tissue damage which were induced by cisplatin. Histological findings further supported the protective effects of chrysin against cisplatin-induced hepatic damage.ConclusionThe results of the present study demonstrate that oxidative stress and inflammation are closely associated with cisplatin-induced toxicity and chrysin shows the protective efficacy against cisplatin-induced hepatotoxicity possibly via attenuating the oxidative stress and inflammatory response.