Patent Publication Number: US-5631001-A

Title: Antimicrobial grapefruit extract

Description:
This application is a division, of application Ser. No. 07/967,158, filed Oct. 27, 1992, now U.S. Pat. No. 5,425,944. 
    
    
     BACKGROUND AND SUMMARY OF THE INVENTION 
     The present invention is directed to an improved, multi-purpose disinfectant and, more particularly, to an extract of ground grapefruit seeds and pulp which is particularly useful as a bactericide, fungicide, virucide, and is believed to be particularly effective in the treatment of HIV infections. 
     Ascorbic acid (Vitamin C) is known to be a disinfectant which can be effective to kill various pathogenic microorganisms. There is some evidence that ascorbic acid may even be used to inhibit the activity and growth of the AIDS virus (HIV). 
     Recent studies indicate that ascorbic acid (Vitamin C) may be effective to inactivate certain viruses including the polio virus, Herpes virus, vaccinia virus and hepatitis virus. Also, the intracellular replication of rhino virus, Rous Sarcoma virus and human T cell leukemia virus have been shown to be suppressed by ascorbic acid. Some patients with poliomyelitis, hepatitis, and infectious mononucleosis have improved with a Vitamin C treatment. There is also some evidence that large doses of synthesized ascorbic acid (10-20 grams/day) may inhibit the activity and growth of HIV. 
     Ascorbic acid is found in many plants, particularly citrus fruits. For example, U.S. Pat. No. 4,021,548 teaches that grapefruit pulp from which a product, probably having an ascorbic acid content, can be extracted having antifungal and antibacterial properties. In U.S. Pat. No. 4,468,372, there is disclosed the use of a grapefruit seed extract, again probably containing ascorbic acid, impregnated into silicate pellets used to destroy air-borne bacteria, fungi and viruses in an airstream passing thereover. 
     In the present invention, however, it has been found that a certain extract produced from the reaction of a prescribed amount of glycerin with a substantially equal amount by weight of a ground mixture of 80:20 by weight of dried grapefruit seeds and pulp respectively results in a superior disinfectant that may be used as a bactericide, fungicide, and virucide, and even appears to be effective in the treatment of HIV infections. There appear to be two advantages to the extract according to the present invention. First of all, as the extract is formed of both seeds and pulp, the reaction product contains a significant quantity (14-15% by weight) of bioflavonoids along with ascorbic acid (approximately 16.5% by weight). Secondly, as the ascorbic acid is produced naturally, rather than by synthesis, it is significantly more stable. A common problem with synthetically formed ascorbic acid is that it has a shelf-life of only approximately 6-8 weeks. The resulting natural extract may be used either as a liquid, or it may be dehydrated, stored, and used in dry form. 
     In its broadest aspect, the present invention is directed to a certain grapefruit extract obtained from reacting a ground mixture of 80:20 dried grapefruit seeds:grapefruit pulp by weight with approxiamtely an equal weight of a glycerin solution and the extraction technique therefor. The resulting extract is useful as an effective disinfectant for use against such microorganisms as bacteria, fungi, and viruses (even HIV). The active constituents of the extract are a combination of natural ascorbic acid and bioflavonoids. The extraction process is carried out using approximately equal amounts of glycerin and the seed/pulp mixture at a temperature of approximately 150° C. 
     More particularly, the process according to the present invention includes the step of separating the seed and pulp of certain types of tree-ripened grapefruit from the remainder of the fruit and drying the seed and pulp for 24-48 hours at a temperature in the range of 150°-200° C. The seed and pulp are then tested for pesticides and only non-contaminated seed and pulp are selected for processing. The dried, non-contaminated seed and pulp is mixed at a ratio of 80:20 seed/pulp by weight. The mixture is then ground in a hammermill to small particles, whereupon the ground mixture is placed inside some type of mesh bag or perforated container which is then placed in a reaction vessel. Previously, a glycerin solution in an amount by weight approximately equal to the weight of the ground mixture has been placed in the vessel and heated to a temperature of at least approximately 150° C. The vessel is sealed and the glycerin solution is then circulated through the extraction chamber and past an external ultraviolet system and magnetic system which helps to stabilize the ingredients and to remove ferrous metallic particles therefrom. The glycerin circulation is continued at the same temperature for approximately 3-4 hours, whereupon the temperature is reduced to approximately 60° C. while the pressure within the chamber is increased to a range of 2,500-3,000 lbs/sq. inch. There results a syrup and a residue in the reaction chamber. The syrup is then passed through a force filter system having a 300-350 mesh nylon filter to obtain a heavy viscous lemon yellow liquid having a pH in the range of 2.5-3.0. This is the reaction product that is either then diluted and used in various applications or else dehydrated and used in various applications. 
     It is therefore an object of the present invention to provide a new disinfectant composition as the extraction product of grapefruit seeds and pulp. 
     It is a further object of this invention to provide an improved grapefruit extract which is effective as a disinfectant for various microorganisms. 
     It is yet another object of the present invention to provide an improved technique for extracting a disinfecting composition as the reaction product from the seeds and pulp of grapefruit. 
     Another object of the present invention is to provide an improved bactericide, fungicide and virucide. 
     Another object of the present invention is to provide a new treatment for treating HIV infections. 
     Another object of the present invention is to provide an effective treatment for salmonella and escherichia in fish and poultry. 
     Additional objects and advantages of the invention will become apparent from reading the following detailed description in conjunction with the examples given. The invention consists of the novel steps, methods, processes, procedures, compositions, products described. 
     In accordance with the present invention, the starting material for the reactive product is grapefruit seeds and pulp. The grapefruit selected is tree-ripened in a climate with cool days and nights which better establishes the trace elements and oils. While the grapefruit may be grown in a variety of locations, the climate must be suitable to provide substantial amounts of the active ingredients, so that the reactive products will be effective. Locations known to have such a climate include Africa, Florida, and Peru. Also, the grapefruit must be grown in an environment in which substantially few, if any, chemical pesticides are used. As will be seen hereinafter, seeds and pulp contaminated with pesticides must be disposed of and cannot be used. The following types of grapefruit have been determined to provide useful raw material: Albedo, Ducan, March, Thompson, Ruby Red and Shaddon. 
     The pulp is located immediately under the hard outer rind of the skin of a fresh grapefruit and is obtained by mechanically shaving the rind portion from the skin, after the juice and section skins have been previously removed. The separation of the rind from the inner pulp layer of the skin should be accomplished in such a manner that the inner pulp is not damaged. 
     The seed and pulp are then dried immediately through a drying process to remove moisture and water percentage. Exposure of the seed and pulp to a temperature in the range of 150°-200° C. for 24-48 hours develops a deep brown color and a good starting product. 
     The dry seeds are mixed in a weight ratio of 80% seed:20% pulp. The dried seed and pulp are ground in a hammermill into small particles and either proceed to testing or storage. Immediately prior to extraction, the ground mixture of seed and pulp, whether processed directly from the hammermill or from storage, is subjected to a test for pesticides. This test is accomplished according to conventional testing techniques on a Perkin-Elmer Atomic Absorbsion Unit provided with hollow cathode lamps, During the testing, seed and/or pulp contaminated with pesticide is removed. 
     In accordance with the present invention, the seed/pulp mixture is then reacted with glycerin at an elevated temperature. The reaction product is subjected to the influence of ultraviolet radiation and a magnetic treatment, which produces a stable reaction product free of ferromagnetic particles. Preferably, the glycerin extraction solution is provided in the reaction vessel in a weight quantity approximately equal to the weight of the ground mixture to be reacted. The glycerin is preferably U.S.P. grade 99.7%. 
    
    
     The following specific examples are presented to afford a better understanding of the present invention to those skilled in the art. It is to be understood that these examples are intended to be illustrative only and are not intended to limit the invention in any way. 
     EXAMPLE 1 
     Two lbs. (908 grams) of glycerin U.S.P. 99.7% is placed in a stainless steel cylinder which includes an inlet and an outlet tube therein. In the center there is a stainless steel shaft which moves up and down and includes a stirring rod. When the glycerin reaches its elevated temperature of 150° C., two lbs. (908 grams) of the ground seed/pulp mixture is placed in a stainless wire mesh sack and suspended in the stainless steel cylinder, which is then close and sealed. The reaction vessel includes a  1/2&#34; bottom outlet and a  1/2&#34; top inlet. The outlet system is connected to a stainless steel pump which transfers the liquid through an ultraviolet treatment stage and a magnetic treatment stage. The ultraviolet system consists of two circular 5&#34; tubes. The conduit carrying the recirculated liquid passes through the center of the two tubes. The ultraviolet tubes short spectrum is 254 manometers, 2537Å, and the long spectrum is 365 manometers, 3650Å. The magnetic gauss strength is 350. 
     The reaction process begins by circulating the glycerin through the system at a temperature of 150° C. for 3-4 hours. The temperature is then reduced to 60° C. and the pressure within the tank is increased from atmospheric to 2,500-3,000 lbs. of hydraulic force. This pressure forces the trace elements from the seed and pulp into the extraction liquid. After the extraction is completed, the liquid is cooled and a residue remains. The residue may be used and mixed into fertilizer for gardening application. The extracted liquid appears to be a deep brown syrup. the liquid is then filtered through a force filter system of 300-350 mesh nylon to achieve the final extract. The extract has the following properties: 
     
         ______________________________________                                    
Appearance       Liquid/Heavy Viscous                                     
______________________________________                                    
Color (Gardner)  2, lemon yellow                                          
Specific gravity (25° C.)                                          
                 1.110                                                    
Density (lbs./gal.)                                                       
                 9.37                                                     
pH (25° F.)                                                        
                 2.5-3.0                                                  
Flash point (°F.)                                                  
                 292                                                      
Viscosity index  -23.2                                                    
Surface tension, 40.0                                                     
Avg. of 5 readings                                                        
(dynes/cm)                                                                
Apparent interfacial                                                      
                 5.0                                                      
tension (dynes/cm)                                                        
True corrected inter-                                                     
                 4.5                                                      
facial tension                                                            
(dynes/cm)                                                                
______________________________________                                    
 
    
     EXAMPLE 2 
     An analysis of the extract produced in accordance with Example 1 shows the following ingredients in the indicated amount: 
     
         ______________________________________                                    
Ingredient         Percentage                                             
______________________________________                                    
Active                                                                    
Vitamin C (Ascorbic acid)                                                 
                   16.5                                                   
Crude protein      2.0                                                    
Crude fat          1.0                                                    
Nitrogen-free extract                                                     
                   39.6                                                   
(includes Bioflavonoids                                                   
14-15%)                                                                   
Inert                                                                     
Glycerin           30.0                                                   
Mineral ash (max)  0.5                                                    
Crude fiber (max)  0.4                                                    
Moisture (max)     10.0                                                   
______________________________________                                    
 
    
     EXAMPLE 3 
     The disinfectant of Example 1 was used in an efficacy test to determine the effect of the extract on various bacteria, yeast and fungi, including staphylococcus aureus, streptococcus faecilis, bacillus SPP, escherichia coli (E coli), salmonella SPP, shigella SPP, pseudomonas aeruginosa, candida albicans, and aspergillus nigers. The bacteria and yeast were all those isolated from clinical specimens, while aspergillus niger strains were isolated from natural sources. 
     The bacteria were each cultured in tryptic soy broth (TSB) at 37° C. for 18 hours. The yeast and other fungi were each cultured on Sabouraud Dextrose Agar (SDA) at room temperature for 48 hours and 7 days, respectively. On the day of the experiment, the cultures of yeast and fungi were washed with 5 ml of Sabouraud dextrose broth (SDB). 
     In a first test, three kinds of utensils were applied. These included stainless cylinder cups, glass rods, and cotton swabs. The utensils were immersed in microbial cultures and then separately put into the extract solution at dilutions of 1:100, 1:1000, 1:2000, 1:5000, and 1:10,000. At the end of 5 and 15 minutes. and at the end of 1, 2 and 24 hours, and one time later, the utensils were observed for growth or no growth. The results are tabulated in Table 1 below. 
     
                                           TABLE 1                                 
__________________________________________________________________________
Table 1 Efficacy of DF-100 (liquid) as a disinfectant on stainless        
cylinder cups, glass rods and cotton swabs                                
contaminated microorganisms (40 isolates tested)                          
Stainless cups          Glass rods         Cotton swabs                   
Cumulative No. (%) of microorganisms being killed at*                     
Dilution                                                                  
     5 m                                                                  
        15 m                                                              
           1 h                                                            
              2 h                                                         
                 24 h&gt;                                                    
                     24 h                                                 
                        5 m                                               
                           15 m                                           
                              1 h                                         
                                 2 h                                      
                                    24 h&gt;                                 
                                        24 h                              
                                           5 m                            
                                              15 m                        
                                                 1 h                      
                                                    2 h                   
                                                       24                 
                                                           24             
__________________________________________________________________________
                                                           h              
1:100                                                                     
     85.0                                                                 
        95.0                                                              
           100                                                            
              100                                                         
                 100 100                                                  
                        85.0                                              
                           97.5                                           
                              100                                         
                                 100                                      
                                    100 100                               
                                           82.5                           
                                              92.5                        
                                                 95.0                     
                                                    100                   
                                                       100 100            
1:1,000                                                                   
     60.0                                                                 
        77.5                                                              
           87.5                                                           
              90.0                                                        
                 100 100                                                  
                        65.0                                              
                           82.5                                           
                              92.5                                        
                                 92.5                                     
                                    100 100                               
                                           55.0                           
                                              70.0                        
                                                 77.5                     
                                                    85.0                  
                                                       100 100            
1:2,000                                                                   
     57.5                                                                 
        67.5                                                              
           77.5                                                           
              85.0                                                        
                 95.0                                                     
                     100                                                  
                        57.5                                              
                           70.0                                           
                              77.5                                        
                                 85.0                                     
                                    95.0                                  
                                        100                               
                                           37.5                           
                                              52.5                        
                                                 70.0                     
                                                    77.5                  
                                                       90.0               
                                                           100            
1:5,000                                                                   
     47.5                                                                 
        65.0                                                              
           70.0                                                           
              80.0                                                        
                 95.0                                                     
                     100                                                  
                        50.0                                              
                           65.0                                           
                              72.5                                        
                                 80.0                                     
                                    95.0                                  
                                        100                               
                                           22.5                           
                                              40.0                        
                                                 62.5                     
                                                    77.5                  
                                                       82.5               
                                                           100            
1:10,000                                                                  
     40.0                                                                 
        47.5                                                              
           62.5                                                           
              90.0                                                        
                 90.0                                                     
                     100                                                  
                        40.0                                              
                           47.5                                           
                              62.5                                        
                                 70.0                                     
                                    90.0                                  
                                        100                               
                                           7.5                            
                                              15.0                        
                                                 32.5                     
                                                    57.5                  
                                                       80.0               
                                                           100            
__________________________________________________________________________
 *The results were observed as growth and no growth in broth and on agar  
 media                                                                    
 
    
     The effect of organic matter on the efficacy of the extract was also done by using blood as the representative. The experiment was carried out on the contaminated stainless cylinder cups. For each growth culture, it was divided into two parts. One part was added with 5% human blood, while another part remained untreated. Afterwards, the test was conducted as described above. The results of this test are set forth in Table 2. 
     
                                           TABLE 2                                 
__________________________________________________________________________
Table 2 Efficacy of dF-100 (liquid) as a disinfectant on stainless        
cylinder cups                                                             
contaminated with microornisms in the absence and presence of blood (20   
isolates tested)                                                          
Absence of blood        Presence of blood                                 
Cumulative nO. (%) of microorganisms being killed at*                     
Dilution                                                                  
     5 m                                                                  
        15 m                                                              
           1 h                                                            
              2 h                                                         
                 24 h&gt;                                                    
                     24 h                                                 
                        5 m                                               
                           15 m                                           
                              1 h                                         
                                 2 h                                      
                                    24 h&gt;                                 
                                        24 h                              
__________________________________________________________________________
1:100                                                                     
     80.0                                                                 
        90.0                                                              
           100                                                            
              100                                                         
                 100 100                                                  
                        80.0                                              
                           90.0                                           
                              100                                         
                                 100                                      
                                    100 100                               
1:1,000                                                                   
     65.0                                                                 
        80.0                                                              
           95.0                                                           
              95.0                                                        
                 100 100                                                  
                        65.0                                              
                           75.0                                           
                              90.0                                        
                                 90.0                                     
                                    100 100                               
1:2,000                                                                   
     60.0                                                                 
        65.0                                                              
           85.0                                                           
              90.0                                                        
                 100 100                                                  
                        65.0                                              
                           65.0                                           
                              85.0                                        
                                 90.0                                     
                                    100 100                               
1:5,000                                                                   
     50.0                                                                 
        60.0                                                              
           80.0                                                           
              90.0                                                        
                 95.0                                                     
                     100                                                  
                        50.0                                              
                           65.0                                           
                              75.0                                        
                                 90.0                                     
                                    100 100                               
1:10,000                                                                  
     45.0                                                                 
        45.0                                                              
           60.0                                                           
              85.0                                                        
                 95.0                                                     
                     100                                                  
                        45.0                                              
                           45.0                                           
                              65.0                                        
                                 80.0                                     
                                    95.0                                  
                                        100                               
__________________________________________________________________________
 *The results were observed as growth and no growth in broth and on agar  
 media                                                                    
 
    
     The effects of pH on the efficacy of the extract was then tested by treating the extract with 0.1N HCl and 0.1N Na0H to provide a pH of 3, 5, 7, 9 and 11, respectively. These treated preparations were diluted with water of the following ratios: 1:100, 1:1000 and 1:10,000. The experiment was carried out on the contaminated cups. The further procedure was done as described previously. For control, water was also treated with 0.1N NCl and 0.1N Na0H to give the same pH as above, after which treated water was diluted with water in a ratio of 1:100. The results are tabulated in Table 3. 
     
                                           TABLE 3                                 
__________________________________________________________________________
Table 3 Efficacy of DF-100 (liquid) treated at different pH as a          
disinfectant on stainless cylinder cups                                   
contaminated with microorganisms (16 isolates tested)                     
1:100                   1:1,000              1:10,000                     
Cumulative No. (%) of microorganisms being killed at*                     
pH 5 m                                                                    
      15 m                                                                
         1 h                                                              
            2 h                                                           
               24 h&gt;                                                      
                   24 h                                                   
                       5 m 15 m                                           
                               1 h                                        
                                  2 h                                     
                                     24 h&gt;                                
                                         24 h                             
                                            5 m                           
                                               15 m                       
                                                  1 h                     
                                                     2 h                  
                                                        24                
                                                            24            
__________________________________________________________________________
                                                            h             
3  93.8                                                                   
      93.8                                                                
         100                                                              
            100                                                           
               100 100 56.3                                               
                           81.3                                           
                               87.5                                       
                                  100                                     
                                     100 100                              
                                            31.3                          
                                               50.0                       
                                                  62.5                    
                                                     75.0                 
                                                        81.3              
                                                            100           
5  87.5                                                                   
      100                                                                 
         100                                                              
            100                                                           
               100 100 56.3                                               
                           75.0                                           
                               87.5                                       
                                  100                                     
                                     100 100                              
                                            43.8                          
                                               62.5                       
                                                  75.0                    
                                                     75.0                 
                                                        81.3              
                                                            100           
7  93.8                                                                   
      100                                                                 
         100                                                              
            100                                                           
               100 100 56.3                                               
                           75.0                                           
                               93.8                                       
                                  100                                     
                                     100 100                              
                                            18.8                          
                                               31.3                       
                                                  31.3                    
                                                     31.3                 
                                                        56.3              
                                                            100           
9  87.5                                                                   
      93.8                                                                
         100                                                              
            100                                                           
               100 100 56.3                                               
                           75.0                                           
                               87.5                                       
                                  93.8                                    
                                     100 100                              
                                            18.8                          
                                               31.3                       
                                                  31.3                    
                                                     37.5                 
                                                        43.8              
                                                            100           
11 87.5                                                                   
      93.8                                                                
         100                                                              
            100                                                           
               100 100 56.3                                               
                           75.0                                           
                               87.5                                       
                                  100                                     
                                     100 100                              
                                            43.8                          
                                               62.5                       
                                                  75.0                    
                                                     81.3                 
                                                        93.8              
                                                            100           
__________________________________________________________________________
 *The results were observed as growth and no growth in broth and on agar  
 media                                                                    
 
    
     The overall results of this example revealed that the extract possesses an antimicrobial activity even though diluted up to 1:10,000, as it shows a disinfective property on either contaminated stainless cups, glass rods or cotton swabs tested. The killing effect of this preparation is better on smooth surface utensils such as the stainless steel cups and glass rods rather than the rough ones, such as cotton swabs. According to the results obtained, the extract shows a very good antimicrobial activity when diluted to 1:100 (100% of the tested microorganisms were killed within 1-2 hours) and a good activity when it is diluted to 1:1000 (100% of tested microorganisms are killed within 24 hours). 
     When blood cultures were applied to the utensils, the extract showed no different effect from those utensils applied with normal cultures. In the pH testing, it was found that all pH&#39;s (pH 3, 5, 7, 9 and 11) tested to demonstrate some antimicrobial activity even up to dilutions of 1:10,000. when the pretreated liquid is diluted to either 1:100 or 1:1000, no significant difference is present as a result of pH differences. However, when the extract is diluted to 1:10,000, a lower disinfective property of the extract with the pH 7 and 9 was noticed. 
     EXAMPLE 4 
     The following test is based upon observation of undocumented studies and expected results in the treatment of HIV infections. The extract of Example 1 is diluted to a mouth rinse in the range of 50-100 ppm. Human subjects who have tested positive for HIV are utilized. The HIV infection is noted in the blood and the tonsils are also swollen. Approximately 100 mg. of the diluted extract (50 ppm) is used as a mouth rinse and swallowed twice a day for the first week. The concentration is then increased to 100 ppm and the same amount is used as a mouth rinse and swallowed twice a day up to three months or until the blood is clear and the tonsils stop swelling. Subsequently, the patients repeat a single dosage of the treatment periodically. 
     Patients so treated are clear of the HIV infection and have remained relatively clear thereof for a period of nine months. It is believed that the same treatment as described hereinabove is effective in the treatment of other viral infections as well. 
     EXAMPLE 5 
     As a result of earlier testing of the disinfectant described in applicant&#39;s U.S. Pat. No. 4,021,548, the following is believed to be a proper treatment against pathogenic microorganisms. The extract, when used at a dilution of about 1:1000 is added to the chilling bath through which poultry are passed for a period of approximately 5 minutes following evisceration. Results indicate that the extract lengthens the shelf-life by more than 24 hours at 22°/24° C. 
     When the extract, in dilutions of 240, 360, 420 and 720 ppm, is used to treat fish meal contaminated with salmonella (20,000 cfu/g) tests of the cultures after 3 and 6 days show no growth of salmonella. 
     Three batches of feed (artificially contaminated fish meal, naturally contaminated fish meal, and naturally contaminated feather meal) contaminated with salmonella were treated with the extract of Example 1 at dilutions of 180, 300, 600, and 900 ppm. The salmonella cultures are tested after 3 and 5 days. The results will show that salmonella may survive in batches treated with 300 ppm or less, however, no growth will be detected at 600 and 900 ppm. 
     Rinsing of fish and seafood prior to packaging and/or freezing for approximately 3 minutes at dilutions of 200 ppm reduces bacteria contamination to less than 300 cfu/gram and is highly effective in killing faecal coliform E. coli and staphylococcus aureus. 
     EXAMPLE 6 
     The extract of Example 1 was used in a test to determine the inhibitory effects of a 200 ppm solution for salmonella typhimurium and Escherichia coli under defining conditions. Under this procedure, the extract was diluted to a concentration of 200 ppm with sterile deionized water and 10 ml aliquots dispensed aseptically into sterile screw-cap test tubes. 0.1 ml of an 18 hour TSB culture of the test organisms was added to the test solution and to a sterile water control. Each tube was briefly vortexed to insure mixing and held at 35° C. 1.0 ml aliquots were removed at 1, 3, 5 10, 15 and 30 minute intervals and viable organisms determined by standard plate count methods (incubation for 48 hours at 35° C. using standard methods agar). 
     While both tests began with an organism count of 9.8×106 after 1 minute and at all the other intervals, the organism count had been reduced to less than 10 3 . 
     While various examples have been described in detail hereinabove, it is apparent the invention is not limited to the specific details described, but departures may be made from such details without departing from the scope of the invention which is set forth in the accompanying claims: