Paronychia argentea Lam. protects renal endothelial cells against oxidative injury Publication date: 10 February 2020 Source: Journal of Ethnopharmacology, Volume 248 Author(s): L. Arkoub-Hamitouche, V. González-del-Campo, M.E. López-Oliva, Fatiha Bedjou, O.M. Palomino AbstractEthnopharmacological relevanceParonychia argentea Lam. (Arabic tea), a species spontaneously growing in the Mediterranean area, has been used in folk medicine for renal diseases.Aim of the studyTo assess the antioxidant and protective potentials of different extracts from P. argentea in the renal endothelial NRK-52E cell line by several in vitro models, including a H2O2-induced oxidative stress model.Material and methodsAerial parts of P. argentea were collected in Algeria and ethanolic, chloroform and aqueous-chloroform extracts were obtained from dried plant. The antioxidant capacity was first evaluated by the Oxygen Radical Absorbance Capacity (ORAC) and the free radical scavenging activity (DPPH) methods. Cellular viability was assessed by MTT method assay after 24 h pretreatment with each extract concentration in order to measure protection from H2O2 in NRK-52E cells. Furthermore, the intracellular ROS formation (DCFH-DA method), was determined.ResultsP. argentea showed in vitro antioxidant activity as evidenced by the ORAC and DPPH assays. No cell toxicity was observed for concentrations ranging from 0.1 to 100 μg/mL of each extract. These extracts also exerted a protective effect on renal endothelial cells simultaneously treated with 1 mM H2O2. Chemical composition for the aqueous-chloroform extract was assessed by HPLC, as it showed the strongest antioxidant ability, revealing three quercetin derivatives as the main phenolic compounds.ConclusionP. argentea is endorsed with antioxidant activity and protects renal endothelial cells against oxidative damage which indicate this plant constitutes a potential treatment for renal diseases.Graphical abstract |
Natural products against acute respiratory infections: Strategies and lessons learned Publication date: 10 February 2020 Source: Journal of Ethnopharmacology, Volume 248 Author(s): Julia Langeder, Ulrike Grienke, Ya Chen, Johannes Kirchmair, Michaela Schmidtke, Judith M. Rollinger AbstractEthnopharmacological relevanceA wide variety of traditional herbal remedies have been used throughout history for the treatment of symptoms related to acute respiratory infections (ARIs).Aim of the reviewThe present work provides a timely overview of natural products affecting the most common pathogens involved in ARIs, in particular influenza viruses and rhinoviruses as well as bacteria involved in co-infections, their molecular targets, their role in drug discovery, and the current portfolio of available naturally derived anti-ARI drugs.Materials and methodsLiterature of the last ten years was evaluated for natural products active against influenza viruses and rhinoviruses. The collected bioactive agents were further investigated for reported activities against ARI-relevant bacteria, and analysed for the chemical space they cover in relation to currently known natural products and approved drugs.ResultsAn overview of (i) natural compounds active in target-based and/or phenotypic assays relevant to ARIs, (ii) extracts, and (iii) in vivo data are provided, offering not only a starting point for further in-depth phytochemical and antimicrobial studies, but also revealing insights into the most relevant anti-ARI scaffolds and compound classes. Investigations of the chemical space of bioactive natural products based on principal component analysis show that many of these compounds are drug-like. However, some bioactive natural products are substantially larger and have more polar groups than most approved drugs. A workflow with various strategies for the discovery of novel antiviral agents is suggested, thereby evaluating the merit of in silico techniques, the use of complementary assays, and the relevance of ethnopharmacological knowledge on the exploration of the therapeutic potential of natural products.ConclusionsThe longstanding ethnopharmacological tradition of natural remedies against ARIs highlights their therapeutic impact and remains a highly valuable selection criterion for natural materials to be investigated in the search for novel anti-ARI acting concepts. We observe a tendency towards assaying for broad-spectrum antivirals and antibacterials mainly discovered in interdisciplinary academic settings, and ascertain a clear demand for more translational studies to strengthen efforts for the development of effective and safe therapeutic agents for patients suffering from ARIs.Graphical abstract |
Astragaloside IV alleviates ischemia reperfusion-induced apoptosis by inhibiting the activation of key factors in death receptor pathway and mitochondrial pathway Publication date: 10 February 2020 Source: Journal of Ethnopharmacology, Volume 248 Author(s): Fei Yin, Huifen Zhou, Yuchen Fang, Chang Li, Yu He, Li Yu, Haitong Wan, Jiehong Yang AbstractEthnopharmacological relevanceApoptosis plays an important role in cerebral ischemia-reperfusion injury and triggers a series of pathological changes which may even be life-threatening. Astragaloside-IV (AS-IV), a natural compound extracted from Astragalus (Astragalus membranaceus (Fisch.) Bunge., Leguminosae, Huangqi in Chinese), showed neuroprotective effects in the study of cerebral ischemia-reperfusion injury. In this study we investigate the effects of AS-IV on apoptosis induced by transient cerebral ischemia and reperfusion in rats, as well as the associated regulatory factors.MethodsAS-IV was administrated to male Sprague-Dawley (SD) rats after transient cerebral ischemia and reperfusion surgery (12.5, 25, and 50 mg/kg, once per day, continued for 7 days after surgey). After seven days of continuous administration, neurological function, cerebral infarction volume, and pathological changes of brain tissue were detected. Fas, FasL, Caspase-8, Bax, and Bcl-2 mRNA levels were determined by real-time PCR. Caspase-8, Bid, Cytochrome C (Cyto C), cleaved Caspase-3 proteins were determined by western blot and immunohistochemistry was used to quantify Cyto C.ResultsAS-IV significantly attenuated the neurological deficit in rats with ischemica-reperfusion injury, and reduced cerebral infarction and neuronal apoptosis. AS-IV inhibited the mRNA upregulation of Fas, FasL, Caspase-8, and Bax/Bcl-2. Furthermore, the protein level of apoptosis cytokines Caspase-8, Bid, cleaved Caspase-3 and Cyto C were also inhibited after ischemia reperfusion, suggesting that AS-IV might alleviate ischemia reperfusion-induced apoptosis by inhibiting the activation of key factors in death receptor pathway and mitochondrial pathway.Graphical abstract |
Polysaccharides derived from Morinda citrifolia Linn reduce inflammatory markers during experimental colitis Publication date: 10 February 2020 Source: Journal of Ethnopharmacology, Volume 248 Author(s): Jalles Arruda Batista, Diva de Aguiar Magalhães, Stefany Guimarães Sousa, Jayro dos Santos Ferreira, Cynthia Maria Carvalho Pereira, José Victor do Nascimento Lima, Ieda Figueira de Albuquerque, Nayonara Lanara Sousa Dutra Bezerra, Tarcisio Vieira de Brito, Carlos Eduardo da Silva Monteiro, Alvaro Xavier Franco, David Di Lenardo, Lorena Almeida Oliveira, Judith Pessoa de Andrade Feitosa, Regina Célia Monteiro de Paula, Francisco Clarck Nogueira Barros, Ana Lúcia Ponte Freitas, Jefferson Soares de Oliveira, Daniel Fernando Pereira Vasconcelos, Pedro Marcos Gomes Soares AbstractEthnopharmacological relevanceThere are many reports of pharmacological activities of extracts and fractions of different vegetable-derived products in the scientific literature and in folk medicine. Ethnopharmacological use of these products by various communities continues to be extensively explored, and they account for more than half of all medications used worldwide. Polysaccharides (PLS) extracted from plants such as Morinda Citrifolia Linn present therapeutic potential in treatment of inflammatory bowel diseases (IBD) such as ulcerative colitis (UC).Aim of the studyTo evaluate the anti-inflammatory action of Noni-PLS against the intestinal damage in UC induced by acetic acid in mice.Materials and methodsIn acetic acid-induced colitis, the mice were treated intraperitoneally (ip) with Noni-PLS (0.1, 0.3, and 3.0 mg/kg) or subcutaneously (sc) with dexamethasone (2.0 mg/kg) 30 min before euthanasia to determine the best dose of Noni-PLS with an anti-inflammatory effect in the course of UC. The colonic tissue samples were collected for macroscopic, wet weight, microscopic and biochemical (myeloperoxidase (MPO), glutathione (GSH), malondialdehyde (MDA), nitrate/nitrite (NO3/NO2), cytokines, cyclooxygenase (COX-2) and inducible nitric oxide (iNOS)) analyses.ResultsTreatment with Noni-PLS reduced the intestinal damage induced by acetic acid as it reduced macroscopic and microscopic scores and the wet weight of the colon. In addition, MPO activity and levels of GSH, MDA, NO3/NO2, pro-inflammatory cytokines, and COX-2 expression reduced.ConclusionsThis study suggests that Noni-PLS exhibits anti-inflammatory action against intestinal damage by reducing inflammatory cell infiltration, oxidative stress, pro-inflammatory action of cytokines, COX-2 and iNOS expression in the inflamed colon. Noni-PLS shows therapeutic potential against inflammatory disorders like UC.Graphical abstract |
Yin Yang Gong Ji pill is an ancient formula with antitumor activity against hepatoma cells Publication date: 10 February 2020 Source: Journal of Ethnopharmacology, Volume 248 Author(s): Yongwei Li, Yujie Li, Zengcheng Zou, Yue Li, Heping Xie, Hongzhi Yang AbstractEthnopharmacological relevanceYin Yang Gong Ji pill (YYGJ) is a formula that was used in the Ming Dynasty. This study investigated the effects of YYGJ on HepG2 and MHCC97H hepatoma cells.Material and methodsThe effects of YYGJ drug-containing rat serum (YYGJ serum) on cell proliferation and the cell cycle were investigated by a tetrazolium dye-based MTS assay and flow cytometry. Apoptosis was assayed by TUNEL and flow cytometry. E-cadherin, vimentin, c-Myc, Smad4, and MMP2 expression were assayed by real-time quantitative PCR and Western blot assays. The effects on cell invasiveness and migration were evaluated by wound healing and transwell assays. The antitumor activity of 10% YYGJ serum was compared to that of blank control, 10% rat serum control and 5-fluorouracil(FU).ResultsHepG2 and MHCC97H cell proliferation was inhibited by YYGJ serum in a time- and concentration-dependent manner. Cells accumulated in G0/G1 and apoptosis was increased in both cell lines by 10% YYGJ serum. The effects of apoptosis in 10% YYGJ serum were weaker than those in response to 5-FU. E-cadherin and Smad4 expression were upregulated by 10% YYGJ serum, but c-Myc, vimentin and MMP2 expression were downregulated in both hepatoma cell lines. The protein expression of Smad4 in HepG2, and mRNA expression of MMP2 and E-cadherin in both cell lines had no difference between 10% YYGJ serum and 5-FU treated groups. Cell invasion and migration were decreased by 10%YYGJ serum while cell cytotoxicity was shown in 5-FU treated group.ConclusionsYYGJ drug-containing serum inhibited HepG2 and MHCC97H cell proliferation, induced apoptosis, and regulated the expression of tumor-related genes and proteins. It reduced tumor cell invasion and migration. Further study to investigate the antitumor activity of YYGJ is warranted.Graphical abstract |
Stachydrine hydrochloride alleviates pressure overload-induced heart failure and calcium mishandling on mice Publication date: 10 February 2020 Source: Journal of Ethnopharmacology, Volume 248 Author(s): Hui-Hua Chen, Si-Ning Wang, Tong-Tong Cao, Jia-Li Zheng, Jing Tian, Xiao-Li Shan, Pei Zhao, Wei Guo, Ming Xu, Chen Zhang, Rong Lu AbstractEthnopharmacological relevanceTraditional Chinese medicine Leonurus japonicus Houtt. has a long history in the treatment of cardiovascular diseases. Stachydrine hydrochloride, the main bioactive ingredient extracted from Leonurus japonicus Houtt., has been shown to have cardioprotective effects. However, the underlying mechanisms of stachydrine hydrochloride haven't been comprehensively studied so far.Aim of the studyThe aim of this study was to investigate the protective role of stachydrine hydrochloride in heart failure and elucidate its possible mechanisms of action.Materials and methodsIn vivo, transverse aorta constriction was carried out in C57BL/6J mice, and thereafter, 7.2 mg/kg telmisartan (a selective AT1R antagonist as positive control) and 12 mg/kg stachydrine hydrochloride was administered daily intragastrically for 4 weeks. Cardiac function was evaluated by assessing morphological changes as well as echocardiographic and haemodynamic parameters. In vitro, neonatal rat cardiomyocytes or adult mice cardiomyocytes were treated with stachydrine hydrochloride and challenged with phenylephrine (α-AR agonist). Ventricular myocytes were isolated from the hearts of C57BL/6J mice by Langendorff crossflow perfusion system. Intracellular calcium was measured by an ion imaging system. The length and movement of sarcomere were traced to evaluate the systolic and diastolic function of single myocardial cells.ResultsStachydrine hydrochloride improved the cardiac function and calcium transient amplitudes, and inhibited the SR leakage and the amount of sparks in cardiac myocytes isolated from TAC mice. We also demonstrated that stachydrine hydrochloride could ameliorated phenylephrine-induced enhance in sarcomere contraction, calcium transients and calcium sparks. Moreover, our data shown that stachydrine hydrochloride blocked the hyper-phosphorylation of CaMKII, RyR2, PLN, and prevented the disassociation of FKBP12.6 from RyR2.ConclusionOur results suggest that stachydrine hydrochloride exerts beneficial therapeutic effects against heart failure. These cardioprotective effects may be associated with the regulation of calcium handling by stachydrine hydrochloride through inhibiting the hyper-phosphorylation of CaMKII.Graphical abstract |
Repeated-dose 26-week oral toxicity study of ginsenoside compound K in Beagle dogs Publication date: 10 February 2020 Source: Journal of Ethnopharmacology, Volume 248 Author(s): Chunmei Li, Zhezhe Wang, Tong Wang, Guangfei Wang, Guisheng Li, Chengfeng Sun, Jian Lin, Liqin Sun, Xilin Sun, Susan Cho, Hongbo Wang, Yonglin Gao, Jingwei Tian AbstractEthnopharmacological relevanceGinsenoside compound K (CK), a product produced by the intestinal bacteria-mediated breakdown of ginsenoside, exhibits a wide array of pharmacological activities against diverse targets. However, few of preclinical safety evaluation of CK is reported.Aims of the study: The present study therefore sought to assess the toxicity of oral CK in Beagle dogs over a 26-week period. Material and methodsAll dogs received 4, 12, or 36 mg/kg oral CK doses for 26 weeks with regular monitoring, followed by a 4-week recovery period. Animals were monitored through measurements of temperature, weight, food intake, blood chemistry and hematological findings, electrocardiogram (ECG) measurements, urinalysis, gross necropsy and organ weight and tissue histopathology.ResultsAnimals in the 36 mg/kg group exhibited an apparent reduction in body weight over the study period, in addition to the presence of focal liver necrosis and increased plasma enzyme levels (alanine aminotransferase, ALT; alkaline phosphatase, ALP) consistent with hepatotoxicity, although there was some evidence suggesting this toxicity was reversible. Animals in the 4 and 12 mg/kg groups did not exhibit any apparent toxicity for any measured parameters.ConclusionThese results thus indicate that the no observed adverse effect level (NOAEL) in dogs is 12 mg/kg.Graphical abstract |
Sorocea guilleminiana Gaudich.: Wound healing activity, action mechanisms, and chemical characterization of the leaf infusion Publication date: 10 February 2020 Source: Journal of Ethnopharmacology, Volume 248 Author(s): Fabiana de Freitas Figueiredo, Valdir Cechinel Filho, Amilcar Sabino Damazo, Karuppusamy Arunachalam, Edson Moleta Colodel, Marlon Ribeiro, Claudio Luis Venturini, Darley Maria Oliveira, Marco Tulio Marra Machado, Eduarda Pavan, Raira Luiza Paes, Adrielli Tenfen, Paulo Othavio de Araujo Almeida, Diogo Alexandre Siebert, Luciano Vitali, Antonio Macho, Domingos Tabajara de Oliveira Martins AbstractEthnopharmacological relevanceSorocea guilleminina Gaudich. is a tree or shrub endemic to Brazil. Its leaves are used in Brazilian folk medicine for the healing of wounds, stomach problems, inflammation and as diuretic. The present study evaluates the activity and action mechanisms of the healing properties of the aqueous extract of S. guilleminiana leaves (AESg), in experimental models in vivo and in vitro, as well as performs a phytochemical analysis of the extract.Materials and methodsThe AESg was prepared by infusion: Ten g of dry leaves powder in 1 L hot water, soaked for 15 min, filtered, lyophilized, and stored at −30 °C. Phytochemical analyses were realized by colorimetry and HPLC/ESI/MS. Its’ in vitro cytotoxicity was evaluated on fibroblastic N3T3 cells. The potential of the wound healing activity in vivo was evaluated using excision and incision wound rat models, by histopathology of the injured skin along with the determination of nitric oxide, cytokines (IL-1β, IL-10, and TNF-α), and antioxidant parameters (GSH, MPO and CAT). In vitro wound healing activity was also demonstrated in scratched N3T3 cells, by measuring the proliferation/migration rate.ResultsThe phytochemical analysis of the AESg revealed a strong presence of polar compounds, especially flavonoids (4 majoritarian), as well as terpenes and/or sterols (2 majoritarian). The AESg showed no toxicity in the N3T3 cell line (IC50 > 800 μg/mL). Topical treatment with the AESg showed an increase (p < 0.05) in wound contraction with 2 mg/g cream on days 5 and 9 (43.56% and 6.70% increase, respectively), and with 50 mg/g on days 7 and 9 (10.88% and 7.91%, respectively), compared to the vehicle (non-ionic neutral cream). Topical application of AESg (2 or 50 mg/g non-ionic cream) in incised wounds caused an increase in the force necessary for the rupture of the wound when compared to the vehicle group. No changes in cytokines (IL-1β, IL-10, or TNF-α) or NO accumulation was found with up to 50 mg/g AESg treatment. For antioxidant activity on the incision wound, an increase in GSH levels was denoted with the AESg use, at the lowest and highest dose (2 and 50 mg/g) by 75.86% and 61.20% respectively, when compared to the vehicle. Also, the CAT activity was accentuated by AESg at the highest dose (50 mg/g) by 85.87%. Finally, the AESg at all doses attenuated MPO activity significantly in the incision wound by 71.35%, 73.21%, 78.08%, respectively. In the scratch test on N3T3 cells, the treatment with AESg resulted also in an increase in fibroblast proliferation/migration rate, compared to the vehicle.ConclusionAESg is not cytotoxic. The results confirm the popular use of the leaf infusion of S. guilleminiana for the treatment of cutaneous wounds, possibly by stimulating the proliferation of fibroblasts with a consequent deposition of collagen, fastening rearrangement of collagen fibers, and greater transformation into myofibroblasts, essential in the healing process. Preliminary chemical analyzes of AESg revealed the presence mainly of phenolic compounds, being salicylic acid, gallic acid, pinocembrin and isoquercitrin the majoritarian ones.Graphical abstract |
Flavonoids extracted from mulberry (Morus alba L.) leaf improve skeletal muscle mitochondrial function by activating AMPK in type 2 diabetes Publication date: 10 February 2020 Source: Journal of Ethnopharmacology, Volume 248 Author(s): Qinghai Meng, Xu Qi, Yu Fu, Qi Chen, Peng Cheng, Xichao Yu, Xin Sun, Jingzhen Wu, Wenwen Li, Qichun Zhang, Yu Li, Aiyun Wang, Huimin Bian AbstractEthnopharmacological relevanceMulberry (Morus alba L.) leaves have been widely applied to controlling blood glucose as a efficacious traditional Chinese medicine or salutary medical supplement. The extracts of mulberry leaf suppress inflammatory mediators and oxidative stress, protect the pancreatic β-cells and modulate glucose metabolism in diabetic rats. Our previous studies and others have shown that mulberry leaf extract has excellent therapeutic effects on type 2 diabetes mellitus (T2DM), however, the underlying mechanism remains to be studied.Aim of the studySkeletal muscle insulin resistance (IR) plays an important role in the pathogenesis of T2DM. The aim of this study was to investigate the effects and mechanisms of Mulberry leaf flavonoids (MLF) in L6 skeletal muscle cells and db/db mice.Materials and methodsL6 skeletal muscle cells were cultured and treated with/without MLF for in vitro studies. For in vivo studies, the db/db mice with/without MLF therapy were used. Coomassie brilliant blue staining and α-SMA immunofluorescence staining were used to identify the differentiated L6 cells. Glucose level and ATP level of L6 myotubes were performed by optical density detection and cell viability was performed by MTT method. Mitochondrial membrane potential of L6 myotubes was detected by JC-1 fluorescent staining. ROS level of L6 myotubes was detected by DCFH-DA fluorescent staining. The body weight, food intake, and blood glucose of the mice were measured in different treatment days. Oral glucose tolerance test (OGTT), starch glucose tolerance test (STT) and insulin tolerance test (ITT) were performed in mice. Glycated hemoglobin, glycated serum protein, insulin, liver and muscle glycogen, total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-c) and low-density lipoprotein cholesterol (LDL-c) of the mice were detected by corresponding kit. The pathologic change of pancreas and skeletal muscle of mice were performed by H & E staining. Immunohistochemistry staining was used to detect the GLUT4 and p-AMPK expressions in skeletal muscle in mice. GLUT4, CPT-1, NRF1, COXIV, PGC-1α, and p-AMPK expression levels in L6 cells and mice were detected by western bolt assay.ResultsMLF and metformin significantly ameliorated muscle glucose uptake and mitochondrial function in L6 muscle cells. MLF also increased the phosphorylation of AMPK and the expression of PGC-1α, and up-regulated the protein levels of m-GLUT4 and T-GLUT4. These effects were reversed by the AMPK inhibitor compound C. In db/db mice, MLF improve diabetes symptoms and insulin resistance. Moreover, MLF elevated the levels of p-AMPK and PGC-1α, raised m-GLUT4 and T-GLUT4 protein expression, and ameliorated mitochondrial function in skeletal muscle of db/db mice.ConclusionsMLF significantly improved skeletal muscle insulin resistance and mitochondrial function in db/db mice and L6 myocytes through AMPK-PGC-1α signaling pathway, and our findings support the therapeutic effects of MLF on type 2 diabetes.Graphical abstract |
Healing effects of Cornus mas L. in experimentally induced ulcerative colitis in rats: From ethnobotany to pharmacology Publication date: 10 February 2020 Source: Journal of Ethnopharmacology, Volume 248 Author(s): Ipek Süntar, Can Kerem Cevik, Ali Osman Çeribaşı, Alper Gökbulut AbstractEthnopharmacological relevanceThe ethnobotanical studies conducted in Turkey and other countries have revealed that Cornus mas L., from the family Cornaceae have been used against stomachache, diarrhea and colitis.Aim of the study: The objective the present study is to determine the possible activity of C. mas in experimentally induced ulcerative colitis in rats and to identify its phytochemical feature. Materials and methods2,4,6-Trinitrobenzene sulfonic acid-induced colitis model was induced in rats. The rats were orally treated with different doses (50, 100, 200 and 400 mg/kg) of C. mas 80% methanol extract for 14 days. Increase in body weight, consumed amount of feed, form of the stool, presence of rectal prolapse were followed every day. At the end of the experiment, colon tissues were removed and wet weights for each animal were measured and colon damages were scored. Total antioxidant and total oxidant status, cytokine (TNF-α and IL-1β) and protein levels of colon tissues were evaluated and histopathological analyses were carried out. After the detection of the effective dose as 400 mg/kg, the aqueous methanol extract was fractionated by using liquid-liquid fractionation technique and the sub-extracts were also tested for in vivo biological activities. High Performance Liquid Chromatography analyses were conducted to determine the phytochemical profile of the active crude extract and n-butanol sub-extract.ResultsAmount of feed consumed per day and increase in body weight were the lowest in the control group, while those values were determined to be the highest in 80% methanol extract (at 400 mg/kg dose), n-butanol sub-extract and reference groups. Following colitis induction, it was determined that the fecal form was yellow-slippery in all groups and returned to normal after the treatment with C. mas extracts. Rectal prolapse score was less in the extract (400 mg/kg) and n-butanol sub-extract treated groups. Total antioxidant, total oxidant status, cytokine and protein levels were found to be in parallel with macroscopic findings. 80% methanol extract (400 mg/kg) and n-butanol sub-extract provided the best healing according to the wet weight measurements and colon damage scoring performed on the removed colon tissues. These findings supported the results of histopathological analysis. According to the chromatographic analysis, ellagic acid was determined in both extracts and its amount was quantified.ConclusionsThe present study has verified the ethnomedical use of C. mas for the treatment of ulcerative colitis.Graphical abstract |
ΩτοΡινοΛαρυγγολόγος Medicine by Alexandros G. Sfakianakis,Anapafseos 5 Agios Nikolaos 72100 Crete Greece,00302841026182,00306932607174,
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