Nanotechnological approaches using curcumin and Withania somnifera: neuroprotection and antimicrobial activities

Tese de Doutoramento em Biologia de PlantasNeurological disorders contribute to 6.3% of the global burden of the diseases and this number is increasing every year due to aging population. Despite enormous efforts in brain and central nervous system related research, neurodegenerative diseases such as Alzheimer or Parkinson disease remain the world's leading cause of disability. Moreover, neurodegenerative diseases are multifactorial pathogenesis with a complex combination of genetic components and environmental factors. Although, several treatment methods are available to treat these conditions, they are associated with the risk of infection, high neurosurgical cost and limited drug availability due to the presence of blood–brain barrier (BBB). Herbal medicine for neurological problems has gained much attention in the recent years, because of the disadvantages of conventional therapies and increased patient compliance. Generally, the drugs obtained from natural sources are considered to be safe alternatives. Since ancient times, plant extracts have been used in the treatment of pathologic conditions of the central nervous system. As the methods to isolate active constituents from plant extracts have greatly improved, the scientific understanding of psychoactive plants has also advanced significantly. Curcuma species and Withania somnifera have been used as traditional therapeutic agents in Asian medicine to treat various common ailments and recently reported to possess antioxidant, antidepressant and neuroprotective effects. Although a number of herbal-based medicines have been screened and identified, still the treatment for the neurological disorders is not up to the required level. This is mainly because most of the herbal medicines exhibit poor bioavailability due to low absorption, fast metabolism, and rapid systemic elimination in the body. Although several approaches have been employed to overcome these problems, modification in the delivery method is a promising approach. In particular, employing herbal medicine based nanocarrier system to treat neurological disorder has gained specific interest because of its several advantages namely, the ability of nanoparticles to deliver the drug in a pre-determined rate at a particular site of action, providing high bioavailability and low toxicity. Compared to the conventional delivery methods, nanoparticle-mediated administration of drugs has been proven to be the ideal way to deliver drugs for neurological disorders. Due to these advantages, pharmaceutical scientists have started designing nanoparticle mediated drug delivery systems for herbal medicines with neurotherapeutic potential. Nanoencapsulation of bioactive compounds and extracts into the polymer matrix gives several advantages, including protection against degradation, enhancing the solubility and bioavailability. In this PhD thesis we have selected two important medicinal plants with proven antioxidant, antidepressant and neuroprotective effects namely W. somnifera and Curcuma sps for nanoencapsulation and subsequent cellular uptake with U251 glioma cells and biodistribution analysis in Zebrafish model. First we developed HPLC methods to identify withanolides and quantify curcumin respectively from W. somnifera extracts and Curcuma species . Extracts derived from different parts (leaves, roots and fruits) of W. somnifera were tested. W. somnifera extract with maximum amount of withanolides and curcumin (active principle of Curcuma sps) were selected for nanoencapsulation. We found that Purospher RP-18 e 5 µm column is ideal for efficient separation and simultaneous quantification of withanolides whereas, Purospher C-18 column was optimum for curcumin quantification from Curcuma sps extracts. HPLC quantification of different extracts of W. somnifera revealed that the methanolic extract obtained from leaf tissues contain the highest amount of withanolides compared to roots and fruits. Compared to C. aromatica, C. longa possess higher amount of curcumin. W. somnifera extract/curcumin loaded polycaprolactone (PCL) and methoxy polyethylene glycol- polycaprolactone (MPEG-PCL) nanoparticles were prepared by solvent displacement method. Prepared nanoparticles were characterized for their physico-chemical properties such as size, shape, in vitro drug release and encapsulation efficiency. After the physico-chemical characterization, we analyzed the in vitro cytotoxicity and cellular uptake of nanoparticles by U251 glioma cells. In addition, we evaluated the protective effect of W. somnifera extract and curcumin loaded nanoparticles against tBHP-induced insult in U251 glioblastoma cells, as a measure of neuroprotection. Biodistribution of W. somnifera /curcumin loaded PCL and MPEG-PCL nanoparticles in animal system were evaluated using zebrafish larvae as model organism. Physical characterization of the prepared nanoparticles revealed that MPEG-PCL nanoparticles were smaller in size compared to PCL nanoparticles irrespective of W. somnifera extract or curcumin encapsulation. Transmission scanning electron microscopy (TEM) images of nanoparticles revealed a round shape in general, although the surface of PCL nanoparticles appeared to be rough and porous. The entrapment efficacy of W. somnifera extract and curcumin was higher in MPEG-PCL compared to PCL. We observed an initial burst release followed by a slow extended release profile for both W. somnifera extract and curcumin irrespective of the polymer used for encapsulation. Treatment of U251 glioma cells with PCL and MPEG-PCL nanoparticles loaded with W. somnifera extract/ curcumin evidenced the efficient cellular uptake of nanoparticles. However, MPEG-PCL nanoparticles showed better internalization compared to PCL nanoparticles, irrespective of their W. somnifera extract/ curcumin load. Neuroprotection assay showed that both W. somnifera/ curcumin loaded nanoparticles protect the cells from oxidative damage. While the neuroprotective effect of W. somnifera extract increased in a dose dependent manner, curcumin was much effective in lower concentrations. Together, our results show that W. somnifera/ curcumin loaded MPEG-PCL nanoparticles possess significantly higher neuroprotective effect in U251 human glioma cells compared to the free drugs and their PCL counterparts. The in vivo localization of nanoparticles in Zebrafish model suggested that the MPEG-PCL nanoencapsulation had efficient and quicker delivery into the larvae compared with the free drug or PCL nanoparticles. Fluorescence imaging of nanoparticles revealed that the nanoparticles were distributed throughout the animal. However, in terms of fluorescence, the head region of animals treated with MPEG-PCL nanoparticles was more intense than that of PCL nanoparticles, indicating that the former might be accumulating in the brain region. Together our results suggest that delivery of W. somnifera extract/ curcumin as MPEG-PCL nanoparticles could enhance the neuroprotective activity. We have also tested the possibility of green synthesis of silver nanoparticles using W. somnifera extract and evaluated the biosynthesized silver nanoparticles for their antibacterial activity in a cream formulation. We developed a simple, fast and cost effective green synthesis technique for silver nanoparticles with potent antimicrobial activity using W. somnifera extract. We also found that catechin, p-couparic acid, luteolin-7-glucoside and a non-identified withanolide are the various compounds present in W. somnifera aqueous leaf extract responsible for green synthesis. The cream incorporated with silver nanoparticles showed antimicrobial activity against a wide variety of clinical isolates.As doenças neurodegenerativas representam 6,3% do gasto geral na saúde e este número tem vindo a aumentar continuamente, anualmente, devido ao envelhecimento da população. Apesar do imenso esforço feito na investigação de doenças foro neurológico, as doenças neurodegenerativas, tais como Alzheimer ou Parkinson, permanecem uma das maiores causas de incapacidade em humanos. Além disso, as doenças neurodegenerativas paresentam patogénese multifactorial, com uma complexa combinação de componentes genéticos e factores ambientais. Apesar de existirem alguns métodos usados no tratamente destas patologias, elas estão associadas a risco de infecção, custos elevados e biodisponibilidade reduzida devido à presença da barreira hematoencefálica (BEE). O uso de plantas medicinais em patologias do foro neurológico tem sido bastante estudado nos anos recentes, em virtude das desvantagens da terapia convencional e indo ao encontro da vontade dos pacientes. De um modo geral, os produtos farmacológicos derivados de plantas são considerados alternativas seguras. Desde tempos remotos que o uso de extractos de plantas tem sido utilizado no tratamento de patologias do foro neurológico. Assim como os dos métodos de extracção dos compostos de plantas têm evoluído significativamente, também o conhecimento científico da acção destes compostos a nível neurológico tem aumentado de forma substancial. A curcuma e Withania somnifera são utilizadas na medicina tradicional asiática no tratamento de várias patologias e recentemente foram evidenciadas as suas actividades antioxidante, antidepressiva e neuroprotectora. Apesar de algumas formulações, compostos e fitofármacos baseados em plantas terem sido identificados como potenciais no tratamento de várias neuropatologias o seu desenvolvimento ainda não está no nível requerido para a sua utilização. Uma das razões principais deve-se ao facto deste exibirem baixa biodisponibilidade devido à sua baixa absorção, metabolismo e eliminação rápida pelo corpo humano. Comparado com os métodos convencionais, a administração de fármacos via nanoparticulas tem provado ser uma alternativa eficaz para o uso em neuropatologias. Devido a estas vantagens, os pesquisadores têm vindo a desenvolver sistemas baseados em nanopartículas como veículos de extractos e compostos de plantas com actividade neurológica reconhecida, para o uso em neuropatologias. A nanoencapsulação de compostos e extractos bioactivos numa matriz polimérica apresenta várias vantagens, nomeadamente protecção contra a degradação, aumento da solubilidade e biodisponibilidade. Neste trabalho foram seleccionadas duas plantas medicinais importantes, com actividade antioxidante, antidepressivo e efeitos neuroprotetores comprovados, designadamente W. somnifera e Curcuma sp., para nanoencapsulação e posteriores estudos em células U251 (de glioma) e análise de biodistribuição no modelo do peixe zebra. Metodologias de HPLC foram desenvolvidas para identificar a curcumina (princípio activo de Curcuma sp.) e whitanólidos presentes em extractos de Curcuma sp e W. somnifera, respectivamente. Extractos obtidos de diferentes partes (folhas, raízes e frutos) de W. somnifera foram testados. Os extractos contendo a máxima quantidade de withanólidos e curcumina foram seleccionados para nanoencapsulação. Identificou-se que a coluna Purospher RP18 (5Xm) é a ideal para uma separação eficiente e a quantificação simultânea dos withanólidos e para uma eficiente quantificação de curcumina em extractos de Curcuma sp.. A quantificação por HPLC de diferentes extratos de W. somnifera revelou que o extrato metanólico obtido a partir de tecidos foliares contêm a maior quantidade de withanólidos, por comparação com raízes e frutos. Em comparação com C. aromatica, a C. longa possui maior quantidade de curcumina. Extractos de W. somnifera / curcumina foram incorporados em nanopartículas de policaprolactona (PCL) e polietilenoglicol metoxi-policaprolactona (MPEG-PCL) usando o método de deslocamento de solvente. As nanopartículas preparadas foram caracterizado pelas suas propriedades físico-químicas, tais como tamanho, forma, libertação do fármaco e a eficiência de encapsulação. Após a caracterização físico-química, analisou-se a citotoxicidade in vitro e a absorção celular das nanopartículas usando células de glioma U251. Além disso, avaliou-se o efeito protector das nanopartículas contendo extracto de W. somnifera ou curcumina contra o insulto oxidativo induzida por t-BHP, como uma medida de neuroproteção. A biodisponibilidade destas nanopartículas contendo o extracto de de W. somnifera ou curcumina foi avaliada ainda num modelo animal, usando alevins de peixe zebra. A caracterização física das nanopartículas revelou que as nanopartículas de MPEG - PCL foram menores em dimensão em comparação com as nanopartículas de PCL, independentemente do encapsulamento ter utilizado extracto de W. somnifera ou curcumina. A microscopia de varrimento electrónico de transmissão (TEM) das nanopartículas revelou uma forma redonda, em geral , embora a superfície das nanopartículas de PCL parece ser áspero e poroso. A eficácia de aprisionamento do extracto de W. somnifera e curcumina foi maior em MPEG-PCL, quando comparado com o PCL. Observou-se uma libertação rápida. inicial, seguida de um perfil de libertação lenta e prolongada para ambos extractos, W. somnifera e curcumina, independentemente do polímero utilizado para a encapsulação. O tratamento de células U251 (giloma) com nanopartículas de PCL e MPEG-PCL carregadas com extracto de W. somnifera ou curcumina evidenciou uma absorção celular eficiente de nanopartículas. No entanto, as nanopartículas de MPE-PCL mostraram uma melhor internalização em comparação com as nanopartículas de PCL, independentemente da sua carga com extracto de W. somnifera ou curcumina. Ensaios de neuroproteção mostraram que nanopartículas contendo extracto de W. somnifera ou curcumina protegem as células dos danos oxidativos. Enquanto o efeito neuroprotector do extracto W. somnifera aumentou de uma forma dependente da dose, a curcumina foi muito eficaz em concentrações mais baixas. Em conjunto, os nossos resultados mostraram que em células de glioma humano U251 as nanopartículas de MPEG-PCL carregadas com extracto de W. somnifera ou curcumina possuem, significativamente. um maior efeito neuroprotector em comparação com os fármacos livres ou seus homólogos encapsulados em PCL . A localização in vivo das nanopartículas no modelo de peixe zebra sugeriu que a nanoencapsulação em MPEGPCL teve uma biodisponibilidade mais eficiente e rápida quando comparado com o fármaco livre ou em nanopartículas de PCL. Imagiologia de fluorescência das nanopartículas revelou que as mesmas encontram-se distribuídas por todo o animal. No entanto, a região da cabeça dos animais tratados com nanopartículas de MPEG-PCL era mais intensa do que com nanoparticulas de PCL. Os resultados sugerem que a entrega de extracto de W. somnifera / curcumina por nanopartículas de MPEG-PCL poderá aumentar a atividade neuroprotetora. Também foi testada a possibilidade de biossíntese de nanopartículas de prata usando o extrato de W. somnifera e avaliou-se a sua atividade antibacteriana numa formulação de creme. Foi desenvolvido um método rápido, de baixo custo e eficaz para a síntese verde de nanopartículas de prata com potente atividade antimicrobiana utilizando o extrato de W. somnifera. Também foi evidenciado que entre os vários compostos presentes no extracto de W. somnifera, a catequina, ácido p-couparico, luteolina -7- glicosídeo e um withanolido (não identificada) são os responsáveis pela biossíntese das nanopartículas. O creme incorporando nanopartículas de prata mostrou actividade antimicrobiana contra uma ampla variedade de isolados clínicos (bactérias e leveduras).A tese de doutoramento aqui apresentada foi desenvolvida no âmbito de financiamento pela Fundação para a Ciência e Tecnologia (FCT) através de uma bolsa individual de doutoramento, com a referência SFRH/BD/72809/2010 (no âmbito do QREN - POPH - Tipologia 4.1 - Formação Avançada, comparticipado pelo Fundo Social Europeu e por fundos nacionais do MCTES)

Test anxiety levels of board exam going students in Tamil Nadu, India

The latest report by the National Crime Records Bureau has positioned Tamil Nadu as the Indian state with highest suicide rate. At least in part, this is happening due to exam pressure among adolescents, emphasizing the imperative need to understand the pattern of anxiety and various factors contributing to it among students. The present study was conducted to analyze the level of state anxiety among board exam attending school students in Tamil Nadu, India. A group of 100 students containing 50 boys and 50 girls from 10th and 12th grades participated in the study and their state anxiety before board exams was measured by Westside Test Anxiety Scale. We found that all board exam going students had increased level of anxiety, which was particularly higher among boys and 12th standard board exam going students. Analysis of various demographic variables showed that students from nuclear families presented higher anxiety levels compared to their desired competitive group. Overall, our results showing the prevalence of state anxiety among board exam going students in Tamil Nadu, India, support the recent attempt taken by Tamil Nadu government to improve student's academic performance in a healthier manner by appointing psychologists in all government schools

Anti-ulcer (ulcer-preventive) activity of ficus arnottiana miq. (moraceae) leaf methanolic extract

Problem statement: In spite of being one of the well-known medicinal plants used in Indian traditional medicine to treat several ailments, studies pertaining to the pharmacological properties of Ficus arnottiana are very scarce. We studied the anti-ulcer activity and acute toxicity of Ficus arnottiana leaf methanolic extract for the first time. Approach: Freshly collected F. arnottiana leaves were dried, powdered and extracted in methanol. To study the anti-ulcer activity, Wistar rats were orally administered with different doses of the extract (0, 250 and 500 mg kg-1 body weight day-1) or with the reference drug omeprazole (8 mg kg-1) for 10 days. After induction of ulcer using 5 mL kg-1 ethanol, stomachs of these animals were analyzed for gastric volume, ulcer area and gross pathological changes. Results: Our results showed that F. arnottiana methanolic extract could prevent ulcer in rats in a dose-dependent manner. Histological studies revealed that the extract had mucoprotective activity. The extract did not show any acute toxicity even at the dose of 5000 mg kg-1 indicating that the extract has no lethal effect. Preliminary phytochemical screening of this extract identified the presence of important secondary metabolites like flavonoids and tannins. Conclusion/Recommendations: From this study, it is clear that F. arnottiana leaf extract had significant anti-ulcer activity in animal models. It had muco-protective activity and gastric antisecretary activity. The extract is non-toxic even at relatively high concentrations.(undefined

Signaling pathways influencing tumor microenvironment and their exploitation for targeted drug delivery

In the recent years, the "tumor microenvironment" has been receiving growing attention due to its involvement in neoplastic transformation, tumor growth, invasion, and protection of tumor cells from host immune response. All these events are facilitated by chemical signals produced by the tumor as well as the surrounding stromal cells. This review is divided into two main parts in which the first part discusses the receptor tyrosine kinase (RTK)-mediated growth factor signaling, steroid hormone (SH) signaling, ancient signaling pathways, and other molecules that are involved in tumorigenesis and how they interact with each other to create a complex tumor microenvironment. In the second part, we bring together the recent nanocarrier-mediated drug delivery approaches to target the signaling pathways/molecules present in the tumor microenvironment.Foundation for Science and Technology (FCT) [(SFRH/BPD/89493/2012]; FCT [SFRH/BD/72809/2010]; Portuguese Government; FCT national funds (PIDDAC) [PTDC/AGR-GPL/119211/2010, PEst-C/AGR/UI4033/2011]; European Fund for Regional Development (FEDER) through COMPETE Operational Programme Competitive Factors (POFC)info:eu-repo/semantics/publishedVersio

Delivery as nanoparticles reduces imatinib mesylate-induced cardiotoxicity and improves anticancer activity

Clinical effectiveness of imatinib mesylate in cancer treatment is compromised by its off-target cardiotoxicity. In the present study, we have developed physically stable imatinib mesylate-loaded poly(lactide-co-glycolide) nanoparticles (INPs) that could sustainably release the drug, and studied its efficacy by in vitro anticancer and in vivo cardiotoxicity assays. MTT (methylthiazolyldiphenyl-tetrazolium bromide) assay revealed that INPs are more cytotoxic to MCF-7 breast cancer cells compared to the equivalent concentration of free imatinib mesylate. Wistar rats orally administered with 50 mg/kg INPs for 28 days showed no significant cardiotoxicity or associated changes. Whereas, increased alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase levels, and reduced white blood cell, red blood cell, and hemoglobin content were observed in the animals administered with free drug. While the histological sections from hearts of animals that received INPs did not show any significant cardiotoxic symptoms, loss of normal architecture and increased cytoplasmic vacuolization were observed in the heart sections of animals administered with free imatinib mesylate. Based on these results, we conclude that nano-encapsulation of imatinib mesylate increases its efficacy against cancer cells, with almost no cardiotoxicity

Antimicrobial activity of cream incorporated with silver nanoparticles biosynthesized from Withania somnifera

We report on the antimicrobial activity of a cream formulation of silver nanoparticles (AgNPs), biosynthesized using Withania somnifera extract. Aqueous extracts of leaves promoted efficient green synthesis of AgNPs compared to fruits and root extracts of W. somnifera. Biosynthesized AgNPs were characterized for their size and shape by physical-chemical techniques such as UV-visible spectroscopy, laser Doppler anemometry, transmission electron microscopy, scanning electron microscopy, atomic force microscopy, X-ray diffraction, and X-ray energy dispersive spectroscopy. After confirming the antimicrobial potential of AgNPs, they were incorporated into a cream. Cream formulations of AgNPs and AgNO3 were prepared and compared for their antimicrobial activity against human pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, Proteus vulgaris, Escherichia coli, and Candida albicans) and a plant pathogen (Agrobacterium tumefaciens). Our results show that AgNP creams possess significantly higher antimicrobial activity against the tested organisms.This work was supported by Fundacao para a Ciencia e Tecnologia (FCT), projects (PTDC/AGR-ALI/105169/2008 and PTDC/AGR-GPL/119211/2010). Gregory Marslin is supported by a FCT PhD fellowship (SFRH/BD/72809/2010).info:eu-repo/semantics/publishedVersio

Poly(D,L-lactic-co-glycolic acid) nanoencapsulation reduces erlotinib-Induced subacute toxicity in rat

Erlotinib-HCl is a quinazoline derivative used as a drug in the therapy of non-small-cell lung cancer. The present study was conducted to compare the subacute toxicity induced by Erlotinib-HCl delivered to rats as nanoparticles and as free drug. Wistar rats were orally administered with a daily dosage of 200 mg kg−1 Erlotinib-HCl either as free drug or as Poly(D,L-lactic-co-glycolic acid) (PLGA) encapsulated nanoparticles. After four weeks of treatment, the animals were analyzed for toxicological changes. Although nanoparticulate form of the drug did not induce any toxicity, free drug significantly reduced the levels of white blood cells (WBC), red blood cells (RBC) and haemoglobin, while increasing the levels of neutrophils and corpuscular haemoglobin. Moreover, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were significantly increased in the animals administered with free drug. Histopathological studies confirmed significant damage to the internal organs of animals treated with free drug. Whereas, the internal organs of animals treated with the drug encapsulated in PLGA nanoparticles were more or less similar to the healthy organs. Our results show that Erlotinib-HCl delivered in the form of nanoparticles has less toxic effect than the free drug in experimental rats

Construction and evaluation of a novel triple cell epitopebased polypeptide vaccine against cow mastitis induced by Staphylococcus aureus, Escherichia coli and Streptococcus

Purpose: To construct a novel triple cell epitope-based polypeptide vaccine against cow mastitis induced by Staphylococcus aureus, Escherichia coli and  Streptococcus and to reduce the use of antibiotics.Methods: Based on bioinformatics approach, a novel triple epitope-based polypeptide (CM-TEP) was designed and subjected to Ni-NTA flow resin purification. Purified CM-TEP was immunized into mice to prepare a polyclonal antibody. Pull-down assays and enzyme-linked immunosorbent assay (ELISA) were used to detect the interaction between CM-TEP antibodies and S. aureus, E. coli and Streptococcus. Active immunity mice and challenge of bacterial pathogens were used to detect immune protection of CM-TEP. Additionally, the optimal expressing conditions of CM-TEP strain were analyzed using orthogonal test design.Results: A novel cow mastitis triple cell epitope-based polypeptide (CM-TEP) with a MW of 36 kDa was designed, purified and used to immunize mice to prepare a  polyclonal antibody. Pull-down assays and ELISA data showed that CM-TEP  antibodies directly interacted with S. aureus, E. coli and Streptococcus. CM-TEP displayed a significant immune protective effect against infection by S. aureus (50 %, p < 0.05) and E. coli (54.54 %, p < 0.05) and provided some immune protective effect (30.78 %, p > 0.05) against Streptococcus. The optimum expressing conditions of CM-TEP were as follows: IPTG concentration of 0.3 mmol/L, strain OD600 value of 1, inducing temperature of 37 oC, and inducing time of 8 h.Conclusion: The findings suggest that epitope-based vaccine of CM-TEP may be a useful strategy for treating cow mastitis induced by S. aureus, E. coli and Streptococcus.Keywords: Cow mastitis, Epitope vaccine, Immunogenicity, Immune protectiv

Protective effect of wild Corni fructus methanolic extract against acute alcoholic liver injury in mice

Background: In Chinese folk medicine, Corni fructus (C. fructus) has traditionally been used to improve liver function, although the mechanism underlying its activity remains unclear. The aim of the present study was to evaluate the protective effects of wild C. fructus methanolic extract against acute alcoholic liver injury.Methods: Alcohol was administered to mice for three consecutive days, either alone or in combination with C. fructus methanolic extract (50, 100, or 200mg/kg body weight/d). Serum and liver tissue were collected from the animals and subjected to biochemical and histopathological analyses.Results:C. fructus significantly alleviated alcohol-induced liver injury by reducing serum alanine aminotransferase, aspartate aminotransferase, and thiobarbituric acid reactive species, inhibiting hydroxyl radicals (center dot OH), and increasing total superoxide dismutase, glutathione peroxidase, and glutathione in the liver (P<0.05). In addition, the C. fructus treatment inhibited the expression and activity of cytochrome P450 2E1 (P<0.05)Conclusions:C. fructus could be a promising natural substance for ameliorating acute alcohol-induced oxidative stress and hepatic injury.- This work was supported by the Construction Project of Shaanxi Collaborative Innovation Center (2015, Shaanxi Sci-tech University); High-End Foreign Experts Recruitment Program [Grant GDW20146100228]; and Key Construction Program of International Cooperation Base in S&T Shaanxi Province, China [Grant 2015SD0018].info:eu-repo/semantics/publishedVersio