Abstract:
A method of extracting proteins and peptides from nano pearl powder comprising the steps of preparing a solution containing uniformly dissolved nano pearl powder; rotating the solution to form a suspension; sifting the suspension; separating a first organic compound extract of pearl having a molecular weight more than a predetermined molecular weight and a second organic compound extract of pearl having a molecular weight less than the predetermined molecular weight from the sifted suspension respectively; and activating a first gel filter to obtain pearl proteins from the first organic compound extract of pearl, and activating a second gel filter to obtain pearl peptides from the second organic compound extract of pearl respectively.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    1. Field of Invention 
         [0002]    The invention relates to pearl extracts and more particularly to a method of extracting proteins and peptides from pearl powder (e.g., nano pearl powder). 
         [0003]    2. Description of Related Art 
         [0004]    Pearl powder used as cosmetics or food is well known. Further, pearl powder preparation methods are disclosed in prior patents. For example, U.S. Pat. No. 7,393,402 discloses a pure pearl powder preparation method. Thus, it is desirable to provide a novel method of extracting proteins and peptides from pearl powder. 
       SUMMARY OF THE INVENTION 
       [0005]    It is therefore one object of the invention to provide a method of extracting proteins and peptides from pearl powder (e.g., nano pearl powder). 
         [0006]    The above and other objects, features and advantages of the invention will become apparent from the following detailed description taken with the accompanying drawings. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0007]      FIG. 1  is a flowchart depicting a process of extracting proteins and peptides from pearl powder according to the invention; 
           [0008]      FIG. 2  is an exploded view of Centriprep Centrifugal Filter; 
           [0009]      FIG. 3  depicts ammonium sulfate deposit according to the invention; 
           [0010]      FIG. 4  is a side elevation of gel filter according to the invention; 
           [0011]      FIG. 5  plots protein distribution percentage versus fraction for nano pearl powder utilized in the invention; 
           [0012]      FIG. 6  plots protein distribution percentage versus fraction for micro pearl powder utilized in the invention; 
           [0013]      FIG. 7  is an SDS-PAGE picture showing deposited proteins being stored in fraction 1 to fraction 6 according to the invention; 
           [0014]      FIG. 8  is an SDS-PAGE picture showing deposited proteins being stored in fraction 7 to fraction 13 according to the invention; 
           [0015]      FIG. 9  is an SDS-PAGE picture showing proteins extracted from micro pearl powder having a molecular weight more than 5 kDa, proteins extracted from micro pearl powder having a molecular weight less than 5 kDa, proteins extracted from nano pearl powder having a molecular weight more than 5 kDa, and proteins extracted from nano pearl powder having a molecular weight less than 5 kDa according to the invention; 
           [0016]      FIG. 10  plots cell mobility (% of control) versus mg/mL for pearl 1 to 4 utilized in the invention; 
           [0017]      FIG. 11  plots inhibition (%) versus time (minute) for pearl 1 to 4 and arbutin utilized in the invention; 
           [0018]      FIG. 12  is a flowchart depicting a process of evaluating activation capabilities of the first organic compound extract of pearl and the second organic compound extract of pearl according to the invention; and 
           [0019]      FIG. 13  is a flowchart depicting a process of further evaluating activation capabilities of the first organic compound extract of pearl and the second organic compound extract of pearl according to the invention. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0020]    Referring to  FIG. 1 , a flow chart of the invention is illustrated. Nano pearl powder  1  (e.g., 20 g) is poured into water (e.g., 200 ml) to be uniformly mixed by activating a mixer  20  in room temperature for 12 hours. Next, pour the uniformly mixed solution into a rotational container  30  which is adapted to rotate in about 3,000 rpm. Hence, a suspension  11  with nano pearl powder is obtained. The suspension  11  is next sifted to separate the coarse from the fine particles. A Centriprep Centrifugal Filter  40  is employed to filter out first organic compound extract of pearl  12 A having a molecular weight more than 5 kDa and second organic compound extract of pearl  12 B from the sifted suspension  11  having a molecular weight less than 5 kDa respectively. 
         [0021]    For the first organic compound extract of pearl  12 A, a gel filter  50  is employed to obtain sifted pearl proteins  13 A. For the second organic compound extract of pearl  12 B, a gel filter  50  is employed to obtain sifted pearl peptides  13 B. 
         [0022]    Referring to  FIG. 2  in conjunction with  FIG. 1 , the Centriprep Centrifugal Filter  40  comprises a cylindrical shell  41  having a full marker  410  and an open top, the shell  41  being filled with the suspension  11  with the coarse being sifted out, a stepped-diameter liquid reservoir  42  adapted to fit in the shell  41  and comprising a bottom sieve  420 , an upper shoulder  421 , and a longitudinal discharge opening  422 , a retaining ring  43  secured to the top of the shell  41  with the discharge opening  422  passing through, and a cap  44  sealingly secured to the retaining ring  43 . 
         [0023]    The Centriprep Centrifugal Filter  40  is capable of rotating in about 3,000 rpm. Hence, the suspension  11  may be sifted through the sieve  420 . As a result, the first organic compound extract of pearl  12 A having a molecular weight more than 3 kDa and the second organic compound extract of pearl  12 B having a molecular weight less than 3 kDa can be obtained. 
         [0024]    The gel filter involves the following steps. The prepared pearl solution (e.g., 10 mg/1 mL) is dropped into ammonium sulfate solution after shaking. Hence, protein is deposited. The ammonium sulfate solution is contained in a 50 mL tube which is then rotated in about 14,000 rpm at 4° C. for about 40 minutes. The deposited material is collected prior to pouring into a two-liter container full of water. Water in the container is replenished every 12 hours. Protein is filtered out of the gel after 48 hours. This process may be best described by referring to  FIG. 3 . Ammonium sulfate is neutral and is capable of absorbing much water. That is, the absorbed water will bind with ions of ammonium sulfate to expose non-polar zones of protein molecules. Hence, protein molecules may be combined together to deposit. 
         [0025]    Referring to  FIG. 4  in conjunction with  FIG. 1 , the gel filter may be done by means of a gel filter  50 . The gel filter  50  comprises a cylindrical shell  51  filled with porous gel particles  52  such as polydextran gel. Coarse particles are separated from the shell  51  by quickly flowing out of the shell  51 . Fine particles are contained in the shell  51  prior to slowly flowing out of the shell  51 . Hence, coarse and fine particles are separated. Gel may be implemented as polydextran gel (e.g., Sephadex G25). Sephadex G25 is adapted to sift out molecules having a molecular weight less than 5 kDa. Hence, protein molecules in pearl powder can be sifted into one group having a molecular weight more than 5 kDa and the other grouped having a molecular weight less than 5 kDa. 
         [0026]    The filtered protein is mixed with 10 mM KH 2 PO 4  in a container. Next, pour 1 mL mixture into a tube called fraction. There are 15 tubes, i.e., fraction 1 to fraction 15 being obtained. 
         [0000]    
       
         
               
               
             
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
             
             
               
                   
                 Fraction 
               
             
          
           
               
                   
                 1 
                 2 
                 3 
                 4 
                 5 
               
               
                   
               
               
                 Protein 
                   2 ± 0.1 
                 2.5 ± 0.3 
                 2.6 ± 0.2 
                 12.4 ± 0.5  
                 18.8 ± 0.7  
               
               
                 (%) 
               
               
                   
               
             
          
           
               
                   
                 Fraction 
               
             
          
           
               
                   
                 6 
                 7 
                 8 
                 9 
                 10 
               
               
                   
               
               
                 Protein 
                 20.4 ± 0.3 
                 8.7 ± 0.4 
                 3.9 ± 0.1 
                 3.9 ± 0.2 
                 5.1 ± 0.3 
               
               
                 (%) 
               
               
                   
               
             
          
           
               
                   
                 Fraction 
               
             
          
           
               
                   
                 11 
                 12 
                 13 
                 14 
                 15 
               
               
                   
               
               
                 Protein 
                  5.2 ± 0.4 
                 4.3 ± 0.2 
                 4.5 ± 0.1 
                 3.1 ± 0.3 
                 2.7 ± 0.2 
               
               
                 (%) 
               
               
                   
               
             
          
         
       
     
         [0027]    Referring to  FIG. 5  and Table 1, protein separated from nano pearl powder is significant in fraction 4 to fraction 6 with a total protein distribution of about 51.6% and in fraction 10 to fraction 13 with a total protein distribution of about 10.3%. 
         [0000]    
       
         
               
               
             
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
             
           
               
                   
                 TABLE 2 
               
               
                   
                   
               
             
             
               
                   
                 Fraction 
               
             
          
           
               
                   
                 1 
                 2 
                 3 
                 4 
                 5 
               
               
                   
               
               
                 Protein 
                  2.6 ± 0.1 
                 3.1 ± 0.2 
                 3.5 ± 0.2 
                 14.1 ± 0.3  
                 22.9 ± 0.7  
               
               
                 (%) 
               
               
                   
               
             
          
           
               
                   
                 Fraction 
               
             
          
           
               
                   
                 6 
                 7 
                 8 
                 9 
                 10 
               
               
                   
               
               
                 Protein 
                 18.5 ± 0.6 
                 5.5 ± 0.7 
                 3.7 ± 0.5 
                 4.2 ± 0.3 
                 3.7 ± 0.4 
               
               
                 (%) 
               
               
                   
               
             
          
           
               
                   
                 Fraction 
               
             
          
           
               
                   
                 11 
                 12 
                 13 
                 14 
                 15 
               
               
                   
               
               
                 Protein 
                  3.1 ± 0.2 
                 3.8 ± 0.2 
                 3.2 ± 0.3 
                 3.7 ± 0.4 
                 4.6 ± 0.5 
               
               
                 (%) 
               
               
                   
               
             
          
         
       
     
         [0028]    Referring to  FIG. 6  and Table 2, protein separated from nano pearl powder is significant in fraction 4 to fraction 6 with a total protein distribution of about 55.5% and in fraction 15 with a protein distribution of about 4.6% but with no significant distribution from fraction 10 to fraction 14. It is found that nano pearl powder have higher protein distribution percentage than micro pearl powder in fraction 10 and fraction 11. This means that there are more protein molecules in nano pearl powder than that in micro pearl powder. 
         [0029]    Referring to  FIGS. 7 and 8 , SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) pictures after Sephadex G25 gel processing are shown. Peptides in nano pearl powder are processed by Sephadex G25. Next, it is stored in 15 tubes each having a volume of 1 mL. The tube is called fraction. Hence, fractions 1 to 15 are obtained. Fraction 4 to fraction 6 each has a molecular weight in the range of 6 kDa to 10 kDa (collectively called fraction A). Fraction 7 to fraction 9 each has a molecular weight in the range of 4 kDa to 6 kDa (collectively called fraction B). Fraction 10 to fraction 13 each has a molecular weight in the range of 2 kDa to 3.8 kDa (collectively called fraction C). 
         [0030]    Referring to  FIG. 1  again, the pearl powder deposit  1 A obtained from the rotating container  30  can be poured into a solution with 70% methyl alcohol. Next, it is mixed in a mixer  20 A for 12 hours at 4° C. Next, it is poured into the rotational container  30  which is adapted to rotate in about 3,000 rpm. 
         [0031]    The organic compounds obtained from the nano pearl powder and that obtained from the micro pearl powder according to the invention have the following characteristics. 
         [0032]    SDS-PAGE is served for verifying the filter function. The organic compounds in micro pearl powder is slightly yellow and that in nano pearl powder is white. The organic compounds having a molecular weight less than 5 kDa is thicker than that having a molecular weight more than 5 kDa. It is thus proved that it is possible of extracting organic compounds from pearl powder according to the invention. 
         [0033]    1 mg organic compound extract of pearl is poured into water to form a soluble organic compound extract of pearl. Next, Bradford protein binding assay is conducted to test protein. It is understood that Coomassie Brilliant Blue G-250 may bind with protein molecules of pearl extracts. Hence, Coomassie Brilliant Blue G-250 may change its color from red to blue after binding with protein molecules of pearl extracts. Thus, proteins can be easily observed if such occurs. 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                 Protein (μg)/organic compounds (mg) 
                 Protein percentage (%) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 Micro 
                 7.48 
                 0.75 
               
               
                 Nano 
                 14.43 
                 1.4 
               
               
                   
               
             
          
         
       
     
         [0034]    From Table 3, it is found that there is 14.43 μg of protein per 1 mg soluble organic compound extract of nano pearl. Further, the protein weight in soluble organic compound extract of micro pearl is less than that in soluble organic compound extract of nano pearl. There is 7.48 μg of protein per 1 mg soluble organic compound extract of micro pearl. 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
                 TABLE 4 
               
               
                   
                   
               
               
                   
                 Protein (μg)/organic compounds (mg) 
                 Protein percentage (%) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 Micro 
                 14.06 
                 1.4 
               
               
                 Nano 
                 16.84 
                 1.68 
               
               
                   
               
             
          
         
       
     
         [0035]    From Table 4, it is found that there is 16.84 μg of protein per 1 mg insoluble organic compound extract of nano pearl. Further, there is 14.06 μg of protein per 1 mg insoluble organic compound extract of micro pearl. 
         [0036]    By comparing Table 3 with Table 4, it is found that there are more proteins in insoluble organic compound extract of either micro or nano pearl than that in soluble organic compound extract of either micro or nano pearl. 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
                 TABLE 5 
               
               
                   
                   
               
             
             
               
                   
                 Pearl 1 
                 soluble organic compound extract 
                 micro 
                 &gt;5 kDa 
               
               
                   
                 Pearl 2 
                 soluble organic compound extract 
                 micro 
                 &lt;5 kDa 
               
               
                   
                 Pearl 3 
                 soluble organic compound extract 
                 nano 
                 &gt;5 kDa 
               
               
                   
                 Pearl 4 
                 soluble organic compound extract 
                 nano 
                 &lt;5 kDa 
               
               
                   
                   
               
             
          
         
       
     
         [0037]    Pearl powder solution is subjected to filter by employing a device called Centriprep YM3. As shown in Table 5, soluble organic compound extract of pearl is divided into two groups in which one has a molecular weight more than 5 kDa and the other has a molecular weight less than 5 kDa. In this experiment, growth rate of fiber cells in each of soluble organic compound extract of nano pearl and soluble organic compound extract of micro pearl is measured. Also, for pearl powder of the same grade growth rates of fiber cells in organic compounds having a molecular weight more than 5 kDa and in organic compounds having a molecular weight less than 5 kDa are measured. 
         [0038]    Referring to  FIG. 9 , it is an SDS-PAGE picture showing proteins extracted from micro pearl powder having a molecular weight more than 5 kDa, proteins extracted from micro pearl powder having a molecular weight less than 5 kDa, proteins extracted from nano pearl powder having a molecular weight more than 5 kDa, and proteins extracted from nano pearl powder having a molecular weight less than 5 kDa according to the invention. 
         [0000]    
       
         
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
               
                   
               
               
                 Hs 68 
                 0 mg/mL 
                 0.5 mg/mL 
                 1 mg/mL 
                 2 mg/mL 
                 4 mg/mL 
               
               
                   
               
             
             
               
                 Pearl 1 
                 100 ± 0% 
                   109 ± 1.6% 
                   114 ± 1.6% 
                   111 ± 1.6% 
                 117.75 ± 1.7%  
               
               
                 Pearl 2 
                 100 ± 0% 
                 110.7 ± 2.1% 
                 110.2 ± 1.7% 
                   101 ± 0.8% 
                   102 ± 0.8% 
               
               
                 Pearl 3 
                 100 ± 0% 
                 113.2 ± 1.7% 
                 130.2 ± 1.7% 
                 120.2 ± 2.2% 
                 119.7 ± 1.7% 
               
               
                 Pearl 4 
                 100 ± 0% 
                 126.7 ± 1.7% 
                   133 ± 0.8% 
                 103.5 ± 1.3% 
                 106.7 ± 1.7% 
               
               
                   
               
             
          
         
       
     
         [0039]    Referring to  FIG. 10  and Table 6, for nano pearl 4 of 1 mg/mL (i.e., concentration) which is soluble organic compounds having a molecular weight of less than 5 kDa extracted from nano pearl powder, the growth rate of fiber cells is about 133% after 24 hours. For pearl 3 of 1 mg/mL which is soluble organic compounds having a molecular weight of more than 5 kDa extracted from nano pearl powder, the growth rate of fiber cells is about 130% after 24 hours. 
         [0040]    As a comparison, for micro pearl 1 of 1 mg/mL which is soluble organic compounds having a molecular weight of more than 5 kDa extracted from micro pearl powder, the growth rate of fiber cells is about 144% after 24 hours. For micro pearl 2 of 1 mg/mL which is soluble organic compounds having a molecular weight of more than 5 kDa extracted from micro pearl powder, the growth rate of fiber cells is about 110% after 24 hours. Both growth rates are not significant. 
         [0041]    Concentration is increased to 2 mg/mL. It is found that the growth rate is decreased to about 111% for pearl 1, decreased to about 101% for pearl 2, decreased to about 120% for pearl 3, and decreased to about 103% for pearl 4 respectively. Next, concentration is further increased to 4 mg/mL. It is found that the growth rate is increased to about 117% for pearl 1, decreased to about 102% for pearl 2, decreased to about 119% for pearl 3, and increased to about 106% for pearl 4 respectively. 
         [0042]    It is thus concluded that growth rate of fiber cells for organic compounds is not proportional to concentration. 
         [0043]    Pearl powder solution is subjected to filter by employing a device called Centriprep YM3. Soluble organic compound extract of pearl is divided into two groups in which one has a molecular weight more than 5 kDa and the other has a molecular weight less than 5 kDa. In this experiment, effects of tyrosinase activation (i.e., dopachrome growth) caused by each of soluble organic compound extract of nono pearl powder and soluble organic compound extract of micro pearl powder are evaluated. Also, for pearl powder of the same effects of tyrosinase activation caused by organic compounds having a molecular weight more than 5 kDa and by organic compounds having a molecular weight less than 5 kDa are evaluated. 
         [0000]    
       
         
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
               
                   
               
               
                 Inhibition 
                   
                   
                   
                   
                   
               
               
                 (%) 
                 Pearl 1 
                 Pearl 2 
                 Pearl 3 
                 Pearl 4 
                 arbutin 
               
               
                   
               
             
             
               
                  0 minute 
                 0 ± 0 
                  0 ± 0 
                  0 ± 0 
                  0 ± 0 
                  0 ± 0 
               
               
                  5 minute 
                 9.7 ± 0.1 
                  6.8 ± 0.1 
                 17.6 ± 0.7 
                 10.5 ± 0.8 
                 36.5 ± 1.9 
               
               
                 10 minute 
                 14.3 ± 1.1  
                 12.3 ± 0.4 
                 20.8 ± 1.3 
                 13.8 ± 0.7 
                 48.6 ± 0.7 
               
               
                 15 minute 
                 15.2 ± 0.1  
                 14.2 ± 0.4 
                 23.5 ± 1.5 
                 15.7 ± 0.8 
                 54.3 ± 1.0 
               
               
                 30 minute 
                 13.6 ± 0.5  
                 14.0 ± 0   
                 24.7 ± 1.0 
                 14.3 ± 0.4 
                 54.7 ± 0.9 
               
               
                 45 minute 
                 5.7 ± 0   
                 11.5 ± 0.3 
                 20.9 ± 0.9 
                  9.2 ± 0.6 
                 37.1 ± 1.3 
               
               
                 60 minute 
                 2.8 ± 0.2 
                 7.72 ± 0.2 
                 19.3 ± 0.5 
                  4.7 ± 0.6 
                 21.6 ± 1.9 
               
               
                   
               
             
          
         
       
     
         [0044]    Referring to Table 7, it shows inhibition percentage of pearl extracts with respect to tyramine acid enzyme and that of arbutin with respect to tyramine acid enzyme. 
         [0045]    Referring to  FIG. 11 , it is a chart depicting inhibition percentage of pearl extracts (of 2 mg/mL) with respect to tyramine acid enzyme and that of arbutin (of 2 mg/mL) with respect to tyramine acid enzyme. 
         [0046]    Referring to  FIG. 11  and Table 7 again, experiment conditions are concentration of 1 mg/mL, temperature of 37° C., and pH value of 6.8. Next, tyramine acid of 20 mM is reacted with tyramine acid enzyme of 383 units/mL for 30 minutes. As a result, 24% inhibition of dopachrome growth is achieved. Further, 19% inhibition of dopachrome growth is achieved after one hour reaction. For the same amount of arbutin, 53% inhibition of dopachrome growth is achieved after 30 minutes of reaction. Further, 21% inhibition of dopachrome growth is achieved after one hour of reaction. Inhibition percentage of pearl 4 is about 45% of that of arbutin after 30 minutes of reaction. Further, inhibition percentage of pearl 4 is about the same as that of arbutin after one hour of reaction. 
         [0047]    For all of pearl 1, pearl 2, pearl 3, and pearl 4, their inhibition percentage of tyramine acid enzyme is in the range of 14% to 15% after 30 minutes of reaction. Further, their inhibition percentage of tyramine acid enzyme is in the range of 2% to 7% after one hour of reaction. 
         [0048]    Referring to  FIG. 12  in conjunction with  FIG. 1 , each of the first organic compound extract of pearl  12 A and the second organic compound extract of pearl  12 B is subjected to the following four steps. The first one is protein concentration assay in which Bradford assay is conducted. There is 14.06 μg of protein per 1 mg extract. The second one is inhibition percentage of tyramine acid enzyme. Further, it is compared with the inhibition percentage of tyramine acid enzyme of one commercial type of skin-whitening agent. Hence, the growth path of melanin can be stopped, thereby significantly increasing the inhibition percentage of tyramine acid enzyme. The third one is evaluation of cells. It is found that human skin cells growth is increased significantly. The fourth one is evaluation of UV (ultraviolet) absorption in which UVA (i.e., UV absorption of the first organic compound extract of pearl  12 A) and UVB (i.e., UV absorption of the second organic compound extract of pearl  12 B) are determined. 
         [0049]    Moreover, a fifth one is conducted after performing the third and fourth steps. The fifth one involves animal experiments, skin hurt experiments, etc. so as to determine its capability of skin protection. 
         [0050]    Referring to  FIG. 13 , for the first organic compound extract of pearl  12 A, a gel filter is employed to obtain sifted pearl proteins  13 A; and for the second organic compound extract of pearl  12 B, a gel filter  50  is employed to obtain sifted pearl peptides  13 B. Both the sifted pearl proteins  13 A and the sifted pearl peptides  13 B are combined to be subjected to an activation assay so as to obtain a single component with significant effect. Thereafter, SDS-PAGE, HPLC (high-performance liquid chromatography) analysis, and MALDI-TOP (matrix-assisted laser desorption/ionization-time of flight mass spectrometer) are performed respectively. 
         [0051]    While the invention herein disclosed has been described by means of specific embodiments, numerous modifications and variations could be made thereto by those skilled in the art without departing from the scope and spirit of the invention set forth in the claims.