02-01-2013, 10:01 AM
Treatment of Cancer on human Melanoma Cell lines by Ricinus Communis extract
[attachment=46084]
ABSTRACT
Ricin protein which is present in the extract of Ricinus communis is a heterodimeric glycoprotein composed of a catalytically active 32kDaA-chain (RTA) linked by a disulfide bond to a 34 kDaB-chain (RTB), a galactose and N-acetylgalactosamine-specific lectin. The molecule enters through endocytosis and undergo retrograde translocation to the Golgi apparatus/endoplasmic reticulum. At this point, the subunits dissociate and a portion of the RTA the cytosol where it inactivates ribosomes by depurinating a single adenine nucleotide. This depurination prevents binding of elongation factors, which leads to the inhibition of protein synthesis.
INTRODUCTION
Skin cancer, the most common of all human cancers accounts for a half of all cancers diagnosed and is reported to be increasingly annually. Melanoma is the most malignant entity of all skin cancers. It arises generally from moles (naevi). The peak incidence is in the fifth decade of life, although, the young and the elderly can also develop the disease. Its incidence has been rising in the last few decades in almost all geographic areas, although it varies in magnitude between different continents. The registered incidence in Australia was 80/100.00 in 2012 [1] compared to a registered incidence of 16.1/100.00 in the Netherlands in 2003. [2] every 1 man in 49 and every 1 woman in 75). In 2003, it estimate 3500 new cases and 6500 deaths, in 2009, it estimates 6,700 new cases and 8, 6500 deaths, implicated alarming increase in melanoma worldwide. In spite of advances in medicine, still there is no cure for melanoma except early surgical intervention. So this deadly disease demands attention from investigators worldwide. In recent past, investigators have proposed several novel targets and therapies for management of melanoma; however, more investigation is needed to make effective and safe treatment of melanoma. With keeping these strategies and understanding in mind we proposed present study, here we explore one of the prevalent herbs (Ricinus communis) against this deadly disease called melanoma.
By using Maceration Technique
The coarsely powdered dried leaves of selected plant were placed in a container with 1000ml petroleum ether solvent for defating and permissible to stand at room temperature for a period of at least 3 days with recurrent agitation until the soluble matter has dissolved. Similarly, the defatted leaf extract was further placed in the same container after utterly washing of container with distilled water. The leaf extract was soaked in the container with five volumes of distilled water and the pH was adjusted to 4.0 by adding dilute acetic acid. The suspension was homogenized at a maximum speed in a grinder mixture at 40C in ten sequences of 1 min. each with 30 min. interval and left over night. The homogenate was then centrifuged at 800g for 10 min. Percentage yield of aqueous extract was found [5], [6], which was stored in refrigerator having 100C for supplementary use. Supernatant was collected and divided into two volumes. One part was preserved at 40C and other was condensed at 450C in water bath.
Purification of Crude extract.
The second part of crude (supernatant preserved at 40C) was adjusted to 60% ammonium sulphate saturation, left over night at 4oC and centrifuged at 10,000g for 15 min. in refrigerated centrifuge. Pellet was collected and dissolved in 0.005M sodium phosphate and exhaustively dialyzed against PBS till complete removal of ammonium sulphate. The dissolved pellet was concentrated by lyophilization and referred as crude drug.[7] The weight of crude drug obtained in lyophilized form was determined and the percentage yield was calculated. A crude extract obtained from the leaves of plant through above technique, was subjected to purification by further tests and different chromatographic techniques like Thin layer chromatography, Column chromatography etc.
Principle of the assay
The MTT Cell Proliferation and Viability Assay is a safe, sensitive, in vitro assay for the measurement of cell proliferation or, when metabolic events lead to apoptosis or necrosis, a reduction in cell viability. Cells are cultured in flat-bottomed, 30- well tissue culture plates, the cells are treated as per experimental design and incubation times are optimized for each cell type and system. The tetrazolium compound MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) is added to the wells and the c ells are incubated. MTT is reduced by metabolically active cells to insoluble purple formazen dye crystals. Detergent is then added to the wells, solubilizing the crystals so the absorbance can be read using a spectrophotometer.
Procedure
Centrifuge an aliquot of cell suspension being tested for viability 5 min at 100×g and discard supernatant. The size of the aliquot depends on the approximate number of cells present. The aliquot should contain a convenient number of cells to count in a haemocytometer when suspended in 1 ml PBS and then diluted again by mixing with 0.4% trypan blue (e.g., 5×105cells/ml). re-suspend the cell pellet in 1ml PBS or serum-free complete medium. Serum proteins stain with trypan blue and can produce misleading results. Determinations must be made in serum-free solution. Mix 1 part of 0.4% trypan blue and 1 part cell suspension (dilution of cells). Allow mixture to incubate 3 min. at room temperature. Cells should be counted within 3 to 5 min of mixing with trypan blue, as longer incubation periods will lead to cell death and reduced viability counts. Mixing can be performed in a well of a microtiter plate or a small plastic tube using 10 t0 20μl each of cell suspension and trypan blue. Apply a drop of the trypan blue/cell mixture to a haemocytometer. Place the haemocytometer on the stage of a binocular microscope and focus on the cells. Count the unstained (viable) and stained (nonviable) cells separately in the haemocytometer.
RESULTS
Plant material was collected and shade dried. The weight of fresh sample and dried powder was determined and percentage loss of water was calculated. The percentage of loss in weight of plant materials on drying was 34.75 %( Table-3). Organoleptic property is a part of an evaluation parameter of any pharmaceutical drug for phtyochemical screening. According to Table-4, organoleptic observation of dried powder of Ricinus Communis leaves revealed that powder of drugs was Greenish in color and possessed characteristic aromatic odour and bitter in taste. Extraction of defatted powder (Pet. Ether) was carried out with acidified water of pH 4.0 by using maceration technique. The extracted crude was further purified by centrifugation and organoleptic evaluation in respect to semisolid extract of Ricinus communis leaves, extract was white in color and pungent in odour (Table-5).
CONCLUSION
In conclusion, our data confirm that extracts of Ricinus communis posses a ricin protein which shows anti-tumor activity against human melanoma cell lines. Our results confirming that survival rate of human melanoma cells increases when treated with ricin protein. So it is finally
concluded that, the Ricinus communis display activity that encourages their development as cancer therapeutics.
[attachment=46084]
ABSTRACT
Ricin protein which is present in the extract of Ricinus communis is a heterodimeric glycoprotein composed of a catalytically active 32kDaA-chain (RTA) linked by a disulfide bond to a 34 kDaB-chain (RTB), a galactose and N-acetylgalactosamine-specific lectin. The molecule enters through endocytosis and undergo retrograde translocation to the Golgi apparatus/endoplasmic reticulum. At this point, the subunits dissociate and a portion of the RTA the cytosol where it inactivates ribosomes by depurinating a single adenine nucleotide. This depurination prevents binding of elongation factors, which leads to the inhibition of protein synthesis.
INTRODUCTION
Skin cancer, the most common of all human cancers accounts for a half of all cancers diagnosed and is reported to be increasingly annually. Melanoma is the most malignant entity of all skin cancers. It arises generally from moles (naevi). The peak incidence is in the fifth decade of life, although, the young and the elderly can also develop the disease. Its incidence has been rising in the last few decades in almost all geographic areas, although it varies in magnitude between different continents. The registered incidence in Australia was 80/100.00 in 2012 [1] compared to a registered incidence of 16.1/100.00 in the Netherlands in 2003. [2] every 1 man in 49 and every 1 woman in 75). In 2003, it estimate 3500 new cases and 6500 deaths, in 2009, it estimates 6,700 new cases and 8, 6500 deaths, implicated alarming increase in melanoma worldwide. In spite of advances in medicine, still there is no cure for melanoma except early surgical intervention. So this deadly disease demands attention from investigators worldwide. In recent past, investigators have proposed several novel targets and therapies for management of melanoma; however, more investigation is needed to make effective and safe treatment of melanoma. With keeping these strategies and understanding in mind we proposed present study, here we explore one of the prevalent herbs (Ricinus communis) against this deadly disease called melanoma.
By using Maceration Technique
The coarsely powdered dried leaves of selected plant were placed in a container with 1000ml petroleum ether solvent for defating and permissible to stand at room temperature for a period of at least 3 days with recurrent agitation until the soluble matter has dissolved. Similarly, the defatted leaf extract was further placed in the same container after utterly washing of container with distilled water. The leaf extract was soaked in the container with five volumes of distilled water and the pH was adjusted to 4.0 by adding dilute acetic acid. The suspension was homogenized at a maximum speed in a grinder mixture at 40C in ten sequences of 1 min. each with 30 min. interval and left over night. The homogenate was then centrifuged at 800g for 10 min. Percentage yield of aqueous extract was found [5], [6], which was stored in refrigerator having 100C for supplementary use. Supernatant was collected and divided into two volumes. One part was preserved at 40C and other was condensed at 450C in water bath.
Purification of Crude extract.
The second part of crude (supernatant preserved at 40C) was adjusted to 60% ammonium sulphate saturation, left over night at 4oC and centrifuged at 10,000g for 15 min. in refrigerated centrifuge. Pellet was collected and dissolved in 0.005M sodium phosphate and exhaustively dialyzed against PBS till complete removal of ammonium sulphate. The dissolved pellet was concentrated by lyophilization and referred as crude drug.[7] The weight of crude drug obtained in lyophilized form was determined and the percentage yield was calculated. A crude extract obtained from the leaves of plant through above technique, was subjected to purification by further tests and different chromatographic techniques like Thin layer chromatography, Column chromatography etc.
Principle of the assay
The MTT Cell Proliferation and Viability Assay is a safe, sensitive, in vitro assay for the measurement of cell proliferation or, when metabolic events lead to apoptosis or necrosis, a reduction in cell viability. Cells are cultured in flat-bottomed, 30- well tissue culture plates, the cells are treated as per experimental design and incubation times are optimized for each cell type and system. The tetrazolium compound MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) is added to the wells and the c ells are incubated. MTT is reduced by metabolically active cells to insoluble purple formazen dye crystals. Detergent is then added to the wells, solubilizing the crystals so the absorbance can be read using a spectrophotometer.
Procedure
Centrifuge an aliquot of cell suspension being tested for viability 5 min at 100×g and discard supernatant. The size of the aliquot depends on the approximate number of cells present. The aliquot should contain a convenient number of cells to count in a haemocytometer when suspended in 1 ml PBS and then diluted again by mixing with 0.4% trypan blue (e.g., 5×105cells/ml). re-suspend the cell pellet in 1ml PBS or serum-free complete medium. Serum proteins stain with trypan blue and can produce misleading results. Determinations must be made in serum-free solution. Mix 1 part of 0.4% trypan blue and 1 part cell suspension (dilution of cells). Allow mixture to incubate 3 min. at room temperature. Cells should be counted within 3 to 5 min of mixing with trypan blue, as longer incubation periods will lead to cell death and reduced viability counts. Mixing can be performed in a well of a microtiter plate or a small plastic tube using 10 t0 20μl each of cell suspension and trypan blue. Apply a drop of the trypan blue/cell mixture to a haemocytometer. Place the haemocytometer on the stage of a binocular microscope and focus on the cells. Count the unstained (viable) and stained (nonviable) cells separately in the haemocytometer.
RESULTS
Plant material was collected and shade dried. The weight of fresh sample and dried powder was determined and percentage loss of water was calculated. The percentage of loss in weight of plant materials on drying was 34.75 %( Table-3). Organoleptic property is a part of an evaluation parameter of any pharmaceutical drug for phtyochemical screening. According to Table-4, organoleptic observation of dried powder of Ricinus Communis leaves revealed that powder of drugs was Greenish in color and possessed characteristic aromatic odour and bitter in taste. Extraction of defatted powder (Pet. Ether) was carried out with acidified water of pH 4.0 by using maceration technique. The extracted crude was further purified by centrifugation and organoleptic evaluation in respect to semisolid extract of Ricinus communis leaves, extract was white in color and pungent in odour (Table-5).
CONCLUSION
In conclusion, our data confirm that extracts of Ricinus communis posses a ricin protein which shows anti-tumor activity against human melanoma cell lines. Our results confirming that survival rate of human melanoma cells increases when treated with ricin protein. So it is finally
concluded that, the Ricinus communis display activity that encourages their development as cancer therapeutics.