Publicação
Nanotechnological approaches using curcumin and Withania somnifera: neuroprotection and antimicrobial activities
| Resumo: | Neurological 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. |
|---|---|
| Autores principais: | Marslin, Gregory |
| Ano: | 2015 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Neurological 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. |
|---|