Potent Immunostimulatory from Microalgae

Abstract

Immunostimulatory polysaccharides can be preferentially extracted from food-grade microalgae using an aqueous ethanol extraction procedure. The resulting preparations exhibit extremely potent immunostimulatory activity. The preferential extraction of these immunostimulatory polysaccharides is dependent on the concentration of ethanol used and the extraction temperature. The most efficient conditions are 50% ethanol concentration at temperatures between 60° and 70° C. The isolated polysaccharide preparations are potentially useful as a botanical or pharmaceutical preparation to improve immune function.

1 . An immunostimulatory  Aphanizomenon flos - aquae  preparation isolated from  Aphanizomenon flos - aquae , wherein the preparation is isolated by extraction of the  Aphanizomenon flos - aquae  with a solvent comprising a mixture of water and alcohol, and wherein said preparation comprises one or more immunostimulatory  Aphanizomenon flos - aquae  polysaccharides having an apparent molecular weight above approximately 2 million Daltons.  
   
   
       2 . An immunostimulatory  Chlorella  preparation isolated from  Chlorella , wherein the preparation is isolated by extraction of the  Chlorella  with a solvent comprising a mixture of water and alcohol, and wherein said preparation comprises one or more immunostimulatory  Chlorella  polysaccharides having an apparent molecular weight above approximately 2 million Daltons.  
   
   
       3 . An immunostimulatory  Spirulina  preparation isolated from  Spirulina , wherein the preparation is isolated by extraction of the  Spirulina  with a solvent comprising a mixture of water and alcohol, and wherein said preparation comprises one or more immunostimulatory  Spirulina  polysaccharides having an apparent molecular weight above approximately 2 million Daltons.  
   
   
       4 . The immunostimulatory preparation of  claim 1  wherein the immunostimulatory activity is manifested by monocyte/macrophage activation.  
   
   
       5 . The immunostimulatory preparation of  claim 2  wherein the immunostimulatory activity is manifested by monocyte/macrophage activation.  
   
   
       6 . The immunostimulatory preparation of  claim 3  wherein the immunostimulatory activity is manifested by monocyte/macrophage activation.  
   
   
       7 . The immunostimulatory preparation of  claim 2  wherein the microalgae are those of  Chlorella pyrenoidosa.    
   
   
       8 . The immunostimulatory preparation of  claim 3  wherein the microalgae are those of  Spirulina platensis.    
   
   
       9 . A pharmaceutical composition comprising the immunostimulatory preparations of  claim 1  and a pharmaceutically acceptable carrier or excipient.  
   
   
       10 . A pharmaceutical composition comprising the immunostimulatory preparations of  claim 2  and a pharmaceutically acceptable carrier or excipient.  
   
   
       11 . A pharmaceutical composition comprising the immunostimulatory preparations of  claim 3  and a pharmaceutically acceptable carrier or excipient.  
   
   
       12 . A pharmaceutical composition comprising the immunostimulatory preparations of  claim 4  and a pharmaceutically acceptable carrier or excipient.  
   
   
       13 . A pharmaceutical composition comprising the immunostimulatory preparations of  claim 5  and a pharmaceutically acceptable carrier or excipient.  
   
   
       14 . A pharmaceutical composition comprising the immunostimulatory preparations of  claim 6  and a pharmaceutically acceptable carrier or excipient.  
   
   
       15 . A pharmaceutical composition comprising the immunostimulatory preparations of  claim 7  and a pharmaceutically acceptable carrier or excipient.  
   
   
       16 . A pharmaceutical composition comprising the immunostimulatory preparations of  claim 8  and a pharmaceutically acceptable carrier or excipient.  
   
   
       17 . A dietary supplement comprising the immunostimulatory preparations of  claim 1  and an acceptable carrier or excipient for dietary supplements.  
   
   
       18 . A dietary supplement comprising the immunostimulatory preparations of  claim 2  and an acceptable carrier or excipient for dietary supplements.  
   
   
       19 . A dietary supplement comprising the immunostimulatory preparations of  claim 3  and an acceptable carrier or excipient for dietary supplements.  
   
   
       20 . A dietary supplement comprising the immunostimulatory preparations of  claim 4  and an acceptable carrier or excipient for dietary supplements.  
   
   
       21 . A dietary supplement comprising the immunostimulatory preparations of  claim 5  and an acceptable carrier or excipient for dietary supplements.  
   
   
       22 . A dietary supplement comprising the immunostimulatory preparations of  claim 6  and an acceptable carrier or excipient for dietary supplements.  
   
   
       23 . A method of enhancing immune function in an individual in need of such treatment, comprising administering to said individual an effective amount of the pharmaceutical composition of any one of claims  9 ,  10  or  11 .  
   
   
       24 . The method of  claim 23 , wherein the individual is suffering from a viral, bacterial or fungal infection.  
   
   
       25 . The method of  claim 23 , wherein the individual is suffering from cancer.  
   
   
       26 . The method of  claim 23 , wherein the individual is suffering from an immune deficiency.  
   
   
       27 . The method of  claim 23 , wherein the individual is a human being.  
   
   
       28 . The method of  claim 23 , wherein the individual is an animal.  
   
   
       29 . A method of enhancing immune function in an individual in need of such treatment, comprising administering to said individual an effective amount of the dietary supplement of any one of claims  17 ,  18  or  19 .  
   
   
       30 . The method of  claim 29 , wherein the individual is suffering from a viral, bacterial or fungal infection.  
   
   
       31 . The method of  claim 29 , wherein the individual is suffering from cancer.  
   
   
       32 . The method of  claim 29 , wherein the individual is suffering from an immune deficiency.  
   
   
       33 . The method of  claim 29 , wherein the individual is a human being.  
   
   
       34 . The method of  claim 29 , wherein the individual is an animal.  
   
   
       35 . A process to obtain a preparation from food-grade microalgae enriched for immunostimulatory polysaccharides, comprising the steps of: 
 (a) producing an extract by extracting the microalgae with a solvent comprising water or a mixture of water and at least one lower alkyl alcohol where the alkyl portion is from 1 to 4 carbon atoms at an extraction temperature of between about 4 degrees C. to 100 degrees C.;    (b) optionally concentrating the extract to a small volume where a large volume makes a concentration step desirable;    (c) precipitating the polysaccharide preparation out of the extract by precipitation means;    (d) separating the precipitated polysaccharide preparation by separation means; and    (e) washing the precipitate of (d) with 95% alcohol.    
   
   
       36 . The process of  claim 38  wherein the alcohol used in the extraction is ethanol.  
   
   
       37 . The process of  claim 35  wherein the alcohol used in the extraction is methanol.  
   
   
       38 . The process of  claim 35  wherein the alcohol used in the extraction is isopropanol or propanol.  
   
   
       39 . The process of any one of claims  35 - 38  wherein the preferred alcohol concentration in (a) is from 20-80%.  
   
   
       40 . The process of any one of claims  35 - 38  wherein the preferred temperature of extraction is between 40 and 80 degrees C.  
   
   
       41 . The process of any one of claims  35 - 38  wherein the microalgae are those of  Aphanizomenon flos - aquae.    
   
   
       42 . The process of any one of claims  35 - 38  wherein the microalgae are those of  Chlorella pyrenidosa.    
   
   
       43 . The process of any one of claims  35 - 38  wherein the microalgae are those of  Spirulina platensis.    
   
   
       44 . The process of any one of claims  35 - 38  wherein the concentration step (b) is carried out (when needed) by evaporation of the solvent, preferably under reduced pressure.  
   
   
       45 . The process of any one of claims  35 - 38  wherein the concentration step (b) is carried out (when needed) by freeze drying.  
   
   
       46 . The process of any one of claims  35 - 38  wherein the concentration step (b) is carried out (when needed) by dialysis.  
   
   
       47 . The process of any one of claims  35 - 38  wherein the polysaccharide preparation is precipitated in step (c) by the addition of ethanol to a final concentration of about 80% ethanol.  
   
   
       48 . The process of any one of claims  35 - 38  wherein the polysaccharide preparation is precipitated in step (c) by cooling the extract.  
   
   
       49 . The process of any one of claims  35 - 38  wherein the polysaccharide preparation is precipitated in step (c) by the addition of a salt.  
   
   
       50 . The process of  claim 49  wherein the salt is ammonium sulfate.  
   
   
       51 . The process of any one of claims  35 - 38  wherein the precipitated polysaccharide preparation is separated in step (d) by filtration.  
   
   
       52 . The process of any one of claims  35 - 38  wherein the precipitated polysaccharide preparation is separated in step (d) by centrifugation.  
   
   
       53 . The process of any one of claims  35 - 38  wherein the precipitated polysaccharide preparation is washed in step (e) by 95% ethanol.  
   
   
       54 . The process of any one of claims  35 - 38  further comprising purifying the precipitate by dissolving the precipitate in water and removing substantially all components of less than approximately 100,000 daltons molecular mass by ultrafiltration.  
   
   
       55 . The process of any one of claims  35 - 38  further comprising purifying the precipitate by dissolving the precipitate in water and removing substantially all components of less than approximately 2 million daltons molecular mass by size exclusion column chromatography.  
   
   
       56 . A method of treating an individual with an immunostimulatory polysaccharide preparation in order to provide to the individual a stimulation of monocyte/macrophage activity comprising administering to the individual an effective amount of a polysaccharide preparation extracted from food-grade microalgae in combination with an acceptable carrier.  
   
   
       57 . The method of  claim 56  wherein the immunostimulatory polysaccharide preparation is administered to enhance wound healing.  
   
   
       58 . The method of  claim 56  wherein the immunostimulatory polysaccharide preparation is administered to treat cancer.  
   
   
       59 . The method of  claim 56  wherein the immunostimulatory polysaccharide preparation is administered to treat immune deficiency.  
   
   
       60 . The method of  claim 56  wherein the immunostimulatory polysaccharide preparation is administered to treat a viral, bacterial or fungal infection.  
   
   
       61 . The method of  claim 56  wherein the individual is a human being.  
   
   
       62 . The method of  claim 56  wherein the individual is an animal.  
   
   
       63 . The method of  claim 56  wherein the microalgae are those of  Aphanizomenon flos - aquae.    
   
   
       64 . The method of  claim 56  wherein the microalgae are those of  Chlorella pyrenoidosa.    
   
   
       65 . The method of  claim 56  wherein the microalgae are those of  Spirulina platensis.    
   
   
       66 . The process of  claim 39  wherein the microalgae are those of  Aphanizomenon flos - aquae.    
   
   
       67 . The process of  claim 39  wherein the microalgae are those of  Chlorella pyrenidosa.    
   
   
       68 . The process of  claim 39  wherein the microalgae are those of  Spirulina platensis.    
   
   
       69 . The process of  claim 39  wherein the preferred temperature of extraction is between 40 and 80 degrees C.  
   
   
       70 . The process of  claim 39  wherein the concentration step (b) is carried out (when needed) by evaporation of the solvent, preferably under reduced pressure.  
   
   
       71 . The process of  claim 39  wherein the concentration step (b) is carried out (when needed) by freeze drying.  
   
   
       72 . The process of  claim 39  wherein the concentration step (b) is carded out (when needed) by dialysis.  
   
   
       73 . The process of  claim 39  wherein the polysaccharide preparation is precipitated in step (c) by the addition of ethanol to a final concentration of about 80% ethanol.  
   
   
       74 . The process of  claim 39  wherein the polysaccharide preparation is precipitated in step (c) by the addition of a salt.  
   
   
       75 . The process of  claim 74  wherein the salt is ammonium sulfate.  
   
   
       76 . The process of  claim 39  wherein the polysaccharide preparation is precipitated in step (c) by cooling the extract.  
   
   
       77 . The process of  claim 39  wherein the precipitated polysaccharide preparation is separated in step (d) by filtration.  
   
   
       78 . The process of  claim 39  wherein the precipitated polysaccharide preparation is separated in step (d) by centrifugation.  
   
   
       79 . The process of  claim 39  wherein the precipitated polysaccharide preparation is washed in step (e) by 95% ethanol.  
   
   
       80 . The process of  claim 39  further comprising purifying the precipitate by dissolving the precipitate in water and removing substantially all components of less than approximately 100,000 daltons molecular mass by ultrafiltration.  
   
   
       81 . The process of  claim 39  further comprising purifying the precipitate by dissolving the precipitate in water and removing substantially all components of less than approximately 2 million daltons molecular mass by size exclusion column chromatography.  
   
   
       82 . The process of  claim 40  wherein the microalgae are those of  Aphanizomenon flos - aquae.    
   
   
       83 . The process of  claim 40  wherein the microalgae are those of  Chlorella pyrenidosa.    
   
   
       84 . The process of  claim 40  wherein the microalgae are those of  Spirulina platensis.    
   
   
       85 . The process of  claim 40  wherein the concentration step (b) is carried out (when needed) by evaporation of the solvent, preferably under reduced pressure.  
   
   
       86 . The process of  claim 40  wherein the concentration step (b) is carried out (when needed) by freeze drying.  
   
   
       87 . The process of  claim 40  wherein the concentration step (b) is carried out (when needed) by dialysis.  
   
   
       88 . The process of  claim 40  wherein the polysaccharide preparation is precipitated in step (c) by the addition of ethanol to a final concentration of about 80% ethanol.  
   
   
       89 . The process of  claim 40  wherein the polysaccharide preparation is precipitated in step (c) by the addition of a salt.  
   
   
       90 . The process of  claim 89  wherein the salt is ammonium sulfate.  
   
   
       91 . The process of  claim 40  wherein the polysaccharide preparation is precipitated in step (c) by cooling the extract.  
   
   
       92 . The process of  claim 40  wherein the precipitated polysaccharide preparation is separated in step (d) by filtration.  
   
   
       93 . The process of  claim 40  wherein the precipitated polysaccharide preparation is separated in step (d) by centrifugation.  
   
   
       94 . The process of  claim 40  wherein the precipitated polysaccharide preparation is washed in step (e) by 95% ethanol.  
   
   
       95 . The process of  claim 40  further comprising purifying the precipitate by dissolving the precipitate in water and removing substantially all components of less than approximately 100,000 daltons molecular mass by ultrafiltration.  
   
   
       96 . The process of  claim 40  further comprising purifying the precipitate by dissolving the precipitate in water and removing substantially all components of less than approximately 2 million daltons molecular mass by size exclusion column chromatography.