Abstract:
A method of blocking free radical processes in an animal which result in mediated pathology without deleterious pro-oxidant side reactions which comprises administering an extract of the fruit of the Emblica officinalis plant to effect such advantageous result, preferably in a use formulation at an active use level of 0.005 to 5% by weight of the formulation.

Description:
CROSS REFERENCE TO RELATED CO-PENDING U.S. PATENT APPLICATIONS 
     This application is a continuation-in-part of U.S. patent applications Ser. No. 09/251,917, filed Feb. 17, 1999, now U.S. Pat. No. 6,124,268 and Ser. No. 09/503,899, filed Feb. 15, 2000, now U.S. Pat. No. 6,235,721 by the same inventor as herein. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to a method of blocking free radical processes in an animal which result in mediated pathology without accompanying deleterious pro-oxidant side reactions, and, more particularly, to administration of an extract of the fruit of the Emblica officinalis plant to effect such result. 
     2. Description of the Prior Art 
     The biomedical literature has recognized that “free radicals” and other reactive species are involved in different human diseases. These species have been implicated in over 100 disorders, ranging from rheumatoid arthritis and haemorragic shock to cardiomyopathy and cystic fibrosis to gastrointestinal ischaemia, AIDS and even male pattern baldness. Some of the clinical conditions in which the involvement of free radicals is given in Table 1 below. 
     
       
         
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Category 
                 Examples 
               
               
                   
               
             
             
               
                 Inflammatory/immune 
                 Glomerulonephritis, vasculitis, 
               
               
                 injury 
                 Autoimmune diseases, rheumatoid arthritis, 
               
               
                   
                 hepatitis 
               
               
                 Ischaemia-reflow states 
                 Stroke, myocardial 
               
               
                   
                 infarction/arrythmias/angina/stunning, organ 
               
               
                   
                 transplantation, inflamed rheumatoid joint, 
               
               
                   
                 frostbite, Dupuytren&#39;s contracture, cocaine- 
               
               
                   
                 induced fetal damage 
               
               
                 Iron overload (tissue 
                 Idiopathic haemochromatosis, dietary 
               
               
                 and plasma) 
                 iron overload (Bantu), thalassaemia and 
               
               
                   
                 other chronic anaemias treated with multiple 
               
               
                   
                 blood transfusions, nutritional deficiencies 
               
               
                   
                 (kwashiorkor), alcoholism, multi-organ 
               
               
                   
                 failure, cardiopulmonary bypass, fulminant 
               
               
                   
                 hepatic failure, prematurity, alcohol-related 
               
               
                   
                 iron overload, cancer 
               
               
                   
                 chemotherapy/radiotherapy 
               
               
                 Radiation injury 
                 Consequences of nuclear explosions, 
               
               
                   
                 accidental exposure, radiotherapy or 
               
               
                   
                 exposure to hypoxic cell sensitizers or 
               
               
                   
                 radon gas; cataract 
               
               
                 Ageing 
                 Disorders of premature ageing, ageing 
               
               
                   
                 itself, age-related diseases, e.g. cancer 
               
               
                 Red blood cells 
                 Phenylhydrazine, primaquine and related 
               
               
                   
                 drugs, lead poisoning, protoporphyrin 
               
               
                   
                 photoxidation, malaria, sickle cell anaemia, 
               
               
                   
                 favism, Fanconi&#39;s anaemia, haemolytic 
               
               
                   
                 anaemia of prematurity, chemotherapy 
               
               
                 Respiratory tract 
                 Effects of cigarette smoke, snuff inhalation, 
               
               
                   
                 other smoke inhalation, emphysema 
               
               
                   
                 (COPD), hyperoxida, bronchopulmonary 
               
               
                   
                 dysplasia, exposure to air pollutants (03, 
               
               
                   
                 NO 2 , SO 2  diesel exhaust), ARDS, mineral 
               
               
                   
                 dust pneumoconiosis, asbestos 
               
               
                   
                 carcinogenicity, bleomycin toxicity, paraquat 
               
               
                   
                 toxicity, skatole toxicity, asthma, cystic 
               
               
                   
                 fibrosis 
               
               
                 Heart and cardiovascular 
                 Alcohol cardiomyopathy, Keshan 
               
               
                 System 
                 disease (selenium deficiency), 
               
               
                   
                 artherosclerosis, anthracycline 
               
               
                   
                 cardiotoxicity, cardiac iron overload 
               
               
                 Kidney 
                 Autoimmune nephrotic syndromes, 
               
               
                   
                 aminoglycoside nephrotoxicity, heavy metal 
               
               
                   
                 nephrotoxicity (Pb, Cd, Hg), 
               
               
                   
                 myoglobin/haemoglobin damage, 
               
               
                   
                 haemodialysis, transplant storage/rejection 
               
               
                 Gastrointestinal tract 
                 Betel nut-related oral cancer, liver injury 
               
               
                   
                 caused by endotoxins or halogenated 
               
               
                   
                 hydrocarbons (e.g. bromobenzene, CCI 4 ), 
               
               
                   
                 exposure to diabetogenic agents, 
               
               
                   
                 pancreatitis, NSAID-induced gastrointestinal 
               
               
                   
                 tract lesions, oral iron poisoning 
               
               
                 Brain/nervous system/ 
                 Hyperbaric oxygen, vitamin E 
               
               
                 Neuromuscular disorders 
                 deficiency, exposure to neurotoxins, 
               
               
                   
                 Alzheimer&#39;s disease, Parkinson&#39;s disease, 
               
               
                   
                 Huntington&#39;s chorea, stroke, neuronal 
               
               
                   
                 ceroid lipofuscinoses, allergic 
               
               
                   
                 encephalomyelitis, aluminium overload, 
               
               
                   
                 sequelae of traumatic injury, muscular 
               
               
                   
                 dystrophy, multiple sclerosis, amyotrophic 
               
               
                   
                 lateral sclerosis, Guam dementia; may also 
               
               
                   
                 occur during preservation of fetal dopamine- 
               
               
                   
                 producing cells for transplantation 
               
               
                 Eye 
                 Cataract, ocular haemorrhage, 
               
               
                   
                 degenerative retinal damage/macular 
               
               
                   
                 degeneration, retinopathy of prematurity 
               
               
                   
                 (retrolental fibroplasia), photic retinopathy, 
               
               
                   
                 penetration of metal objects 
               
               
                 Skin 
                 UV radiation, thermal injury, porphyria, 
               
               
                   
                 hypericin, exposure to other 
               
               
                   
                 photosensitizers, contact dermatitis, 
               
               
                   
                 baldness 
               
               
                   
               
               
                 Abbreviations: ARDS, adult respiratory syndrome; COPD, chromic obstructive pulmonary disease; NSAID, non-steroidal anti-inflammatory drug  
               
             
          
         
       
     
     These pathologies have been alleviated with antioxidants which function as blockers of such radical process. However, an antioxidant cannot distinguish between radicals that play a useful physiologic role and those that are harmful. Moreover, antioxidant compounds not only function as antioxidants, but they may have pro-oxidant action as well. Examples of antioxidants which also exhibit pro-oxidant activity are given below: 
     Vitamin C 
     Vitamin C is a hydrophilic vitamin with well-known antioxidant properties; however Vitamin C also can act as a pro-oxidant in the following manner. Specifically, the combination of Vitamin C with Fe 3−  or Fe 2+  ions causes intense oxidation of polyunsaturated fatty acids (PUFAs). The mechanism of such pro-oxidation is as follows: 
     The Vitamin C radical (dehydroascorbate radical anion, Vit C.), a relatively non-reactive species, can decay by disproportionation resulting in the production of Vitamin C and dehydroascorbate (DHA), thereby terminating the propagation of free radical reactions: 
      2Vit C.+2H + →Vit C+DHA 
     Alternately, the vitamin C radical may reduce another Fe &#39;+  ion: 
     
       
         Vit C.+Fe 3+→DHA+Fe   2+   
       
     
     During oxidation of Vitamin C, H 2 O 2  is also formed: 
     
       
         Vit C+O 2 →DHA+H 2 O 2    
       
     
     The reduction of Fe 3+  thus appears to be the probable cause for the pro-oxidant action of Vitamin C. The degree of ferric ion reduction may therefore determine the prevalence of Vitamin C acting either as an antioxidant or as a pro-oxidant. 
     Vitamin E 
     The propagation reaction of the oxidative breakdown of PUFAs, indicated as LH, is shown below, where K 1  is the equilibrium constant:                           
     In which LOO .  indicates lipid peroxyl radicals. 
     The lipid-soluble Vitamin E thus owes its antioxidant activity to trapping of LOO . , which in turn is reduced to stable LOOH (lipid hydroperoxides), as follows:                           
     Vitamin E itself is a mixture of four lipid-soluble tocopherols (designated as α, β, γ and δ); α-tocopherol being the most active with respect to trapping of peroxyl radicals. The resonance stabilization of Vit E derived from α-tocopherol renders it less reactive than LOO . , therefore, Vitamin E is a good antioxidant. 
     In contrast, Vitamin E, if present at a relatively high concentration, can induce deleterious radical formation by a side reaction with LOOH, thus functioning also as a pro-oxidant. 
     
       
         Vit E+LOOH→Vit E . +LO . +H 2 O 
       
     
     Superoxide Dismutase (SOD) 
     Intra-articular administration of Cu/Zn-containing SOD has been used to prevent free radical damage. However, H 2 O 2 , a reaction product of O 2   −  dismutation, can inactivate SOD. Therefore, in the presence of H 2 O 2 , SOD will act as a pro-oxidant. 
     Glutathione (GSH) 
     Thiol (SH) groups are essential in the protection against the deleterious effects of reactive oxygen species. The tripeptide GSH (γ-Glu-Cys-GLY) is the pivot in various protective systems. In addition, the SH group is important for the function of many proteins. To protect the SH groups of proteins, high concentrations of the reducing GSH are required. However, it is difficult to estimate the level of GSH needed for this function since thiols may exhibit both antioxidant and pro-oxidant actions. The pro-oxidant activity of the thiol GSH can be simply explained as involving the reduction of Fe 2+ . Hence, a generally accepted antioxidant, such as GSH, may possess deleterious pro-oxidant activity under certain conditions. 
     Accordingly, it is an object of this invention to provide a method of blocking free radical processes which result in mediated pathology without deleterious pro-oxidant side reactions. 
     Another object of the present invention is to provide advantageous antioxidant activity to block free radical processes without accompanying pro-oxidant side reactions by administration of an extract of the fruit of the Emblica officinalis plant, optionally including one or more additional antioxidants. 
     A feature of the invention is the provision of a use formulation containing an active use level of said extract in the amount of about 0.005 to 5% by weight of said formulation. 
     These and other objects and features of the invention will be made apparent from the following description thereof. 
     SUMMARY OF THE INVENTION 
     What is described herein is a method of blocking free radical processes in an animal which result in mediated pathology without deleterious pro-oxidant side reactions. The method comprises administering an extract of the fruit of the Emblica officinalis plant to effect such advantageous result. In practice, a use formulation containing an active use level of said extract in an amount of about 0.005 to 5% by weight of the formulation, is utilized for such administration. Optionally one or more additional antioxidants may be included in the formulation. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The antagonist for blocking free radical processes in humans according to the invention is an extract blend, hereinafter referred to as “Extract Blend (EB)”, which is isolated in stable form from the fruit of the Emblica officinalis plant, as described in detail in U.S. Pat. No. 6,124,268. The extraction process includes treating the finely-pulped fruit with a dilute aqueous or alcoholic-water salt solution, e.g. a 0.1 to 5% (w/w) sodium chloride solution, or the like, preferably at about 70° C. ±5° C., or with a buffer solution, e.g. 0.1 to 5% (w/w) of sodium citrate/citric acid, or the like, filtering and drying, to provide the extract in powder form. 
     The extract includes the active constituents Emblicanin-A and -B, which are gallic/ellagic acid derivatives of 2-keto-glucono-δ-lactone, in an amount, by weight, of about 35-55%; as well as Punigluconic acid, or 2,3-di-O-galloyl-4,6-(S)-hexahydroxydiphenoyl gluconic acid (about 4-15%); Pedunculagin, or 2,3,4,6-bis-(S)-hexahydroxydiphenoyl-D-glucose (about 10-20%); Rutin, or flavanol-3-glycoside or 3′,4′,5,7-tetrahydroxy-flavone-3-)-rhamnoglucoside (about 5-15%); and low-to-medium molecular weight tannoids of gallic/ellagic acid (about 10-30%); gallic acid (about 0-5%) and ellagic acid (0-5%). 
     Experimental Results 
     1. Antioxidant Activity 
     Several test materials at concentrations of 2.5, 5, 10, 20, 40 and 80 μg/mL were separately added to a control, DPPH (diphenyl picryl hydrazyl) radical solution (100 μM in absolute ethyl alcohol) in a cuvette. After 20 minutes, the absorbance (optical density) of the mixture was measured at a wavelength of 570 nm and compared to that of the control sample. The degree of scavenging of the DPPH radical by each test sample was calculated by comparing the absorbance of the test sample and the diluent control reaction mixture. The results are shown in Table 2 below, a lower IC value indicating a better antioxidant effect on the DPPH radical. 
     
       
         
               
               
               
             
           
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                 Test Material 
                 IC 50  (μg/mL)* 
               
               
                   
                   
               
             
             
               
                   
                 Ascorbic acid (AA) 
                 18.41 ± 3.88 
               
               
                   
                 Pycnogenols (PG) (Marker) 
                 27.33 ± 4.44 
               
               
                   
                 α-Tocopheryl acetate (TA) 
                 50.55 ± 7.03 
               
               
                   
                 Extract Blend (EB) 
                 12.82 ± 2.01 
               
               
                   
                 EB + AA (1:1, w/w) 
                  8.88 ± 1.77 
               
               
                   
                 EB + TA (1:1, w/w) 
                 10.22 ± 2.03 
               
               
                   
                   
               
               
                   
                 *Values are mean ± SEM (n = 10 to 12)  
               
             
          
         
       
     
     The results show that Extract Blend alone had a more pronounced antioxidant effect in controlling oxidation than any of the other antioxidant test compounds. Mixtures of EB and AA or TA also were significantly better than any of the individual compounds. 
     Exact Blend thus is a very potent antioxidant agent (and not a pro-oxidant) at all concentrations as evident from the reduction of DPPH radical to DPPH 2  under the Udenfriend reaction conditions. The mechanistic sequence of this reaction is shown below: 
     When ascorbic acid (AA) is in low concentration: 
     
       
         EDTA Fe 3+ EDTA+AA→Fe 2+ +AA .   
       
     
     
       
         2 AA . →AA+DHAA (dihydro ascorbic acid) 
       
     
     
       
         Fe 2+ +H 2 O 2 →Fe 3 + . OH+ − OH 
       
     
     
       
         DPPH . +AA→DPPH 2  +AA .   
       
     
     
       
         DPPH . + . OH⇄DPPH .  . . .  . OH  (i) (ii) Spin-pair 
       
     
     The decrease in absorbance observed herein is due to the shift in equilibrium to (I), which is indicated by the appearance of a new shoulder at a lower wavelength (λ˜520 nm). DPPH 2 + . OH [after dissociation as in (I)] to DPPH . +H 2 O causes an increase in the optical density to 0.764. 
     When ascorbic acid is in high concentration: 
     
       
         EDTA Fe 3+ EDTA+AA→Fe 2+ +AA .   
       
     
     
       
         Fe 2+ +H 2 O 2 →Fe 3+ + . OH+ − OH 
       
     
     
       
           . OH+AA→AA . + − OH 
       
     
     
       
         Fe 3+ +AA→Fe 2+ +AA .   
       
     
     
       
         2AA . →AA+DHAA 
       
     
     
       
         DPPH . +Fe 2+ ⇄DPPH .  . . . Fe 2+   (iii) (iv) comparatively stable spin-pair 
       
     
     The appreciable decrease in the optical density value at λ570 nm is due to a shift in the equilibrium to (iv). 
     In the case of EB, there is a significant reduction in the optical density due to the formation of type (iv) spin-pair with Emblicanin-A resulting in further reduction of DPPH .  to DPPH 2 . 
     2. Polymerization of Methylmethacrylate (MMA) to Polymethytlmethacrylate (PMMA) 
     Scope of the Method 
     Soft spin radicals do not initiate/augment polymerization of MMA into PMMA. The fact that the pro-oxidant activity of AA at low concentrations is quite prominent is indicated by the significant augmentation of polymerization at low concentration of AA (1.0 mg)+H 2 O 2  via the OH radical (Table 3). Thus, the pro-oxidant effect of a low concentration of AA could be systemically quite dangerous due to distinct involvement of OH radicals. This determination supports the superior antioxidant effect of EB under all circumstances. 
     Method 
     MMA (1 ml, ˜980 mg) was added to a solution of ferrous sulfate (15 mg) in double distilled water. N 2  was passed through the mixture for 30 min. followed by H 2 O 2 . The onset of polymerization (the induction period in minutes) was noted. Polymerization was completed in about 24 hours at 35±2° C. The resulting precipitate was collected, washed and dried at 50° C. until a constant weight was obtained (See Table 3). 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Polymerization of Methylmethacrylate to 
               
               
                 Polymethylmethacrylate in Presence or Absence of 
               
               
                 Extract Blend (EB) or Ascorbic Acid by Fenton&#39;s Reagent 
               
             
          
           
               
                   
                 Induction Time 
                 Yield of PMMA 
                 % Conversion of 
               
               
                 Sample 
                 (in Min.) 
                 (in MG.) 
                 MMA → PMMA 
               
               
                   
               
             
          
           
               
                 Control 
                 0 
                 668 
                 68.1 
               
               
                 +AA (1 mg) 
                 0 
                 772 
                 78.7 ↑ 
               
               
                 +AA (2 mg) 
                 0 
                 692 
                 69.8 ↑ 
               
               
                 +AA (3 g) 
                 0 
                 670 
                 68.2 ↑ 
               
               
                 +AA (4 mg) 
                 0 
                 432 
                 44.0 
               
               
                 +AA (5 mg) 
                 0 
                 227 
                 23.1 
               
               
                 +AA (10 mg) 
                 0 
                 83 
                 8.4 
               
               
                 +EB (1 mg) 
                 20 
                 124 
                 12.6 ↓ 
               
               
                 +EB (2 mg) 
                 65 
                 62 
                 6.3 ↓ 
               
               
                 +EB (3 mg) 
                 120 
                 Traces 
                 &lt;1 ↓ 
               
               
                 +EB (4 mg) 
                 &gt;5 hr 
                 0 
                 0 
               
               
                 +EB (5 mg) 
                 &gt;5 hr 
                 0 
                 0 
               
               
                 +AA +EB 
                 130 
                 44 
                 4.4 
               
               
                 (1 + 1 mg) 
               
               
                 +AA +EB 
                 138 
                 22 
                 2.2 
               
               
                 (1 + 2 mg) 
               
               
                 +AA +EB 
                 &gt;5 hr 
                 0 
                 0 
               
               
                 (1 + 3 mg) 
               
               
                   
               
             
          
         
       
     
     Low concentrations of ascorbic acid thus augmented polymerization of MMA→PMMA (via  . OH radical; pro-oxidant activity), whereas EB, either significantly inhibited (1-3 mg), or totally prevented (4-5 mg) the polymerization reaction. The induction time of polymerization in the presence of EB thus is significantly higher than that of ascorbic acid which is instantaneous. Mixtures of EB and AA actually reversed the pro-oxidant activity of AA at low concentrations Accordingly, EB is a very potent antioxidant agent at all concentrations and devoid of pro-oxidant activity. 
     3. Prevention of DNA Strand Scission 
     The DNA strand scission was investigated by a method published by T. Ozawa, et al [ Biochem. Mol. Biol. Int.  31, 455-46, 1993]. The method consists of addition of hydrogen peroxide (30%, final concentration 25 mM) to a mixture of Cu(en) 2  (final concentration 0.25 mM) and 0.5 mcg of PBR 322 plasmid DNA solution (Takara Co., Japan). The hydroxyl radical, generated from Cu(en) 2 -hydrogen peroxide reaction, caused DNA strand scission. 
     At physiological pH, EB significantly suppressed the DNA strand-scission by hydroxyl radicals produced from the reaction of Cu(en) 2  and hydrogen peroxide. Both Vitamin C and a blend of Vitamins C/E (1:30), w/w) accelerated DNA strand-scission compared to the control value (Table 4). The protective effect of EB is due to: (1) Captodative action on the generation of hydroxyl radical by chelation of Cu ions from a Cu(en) 2 -complex. In contrast, both Vitamin C and a blend of Vitamins C/E produced loose and partially chelated Cu ions, which, by an Udenfriend-type reaction, further accelerated the DNA strand-scission (Table 4). The importance of DNA strand-scission in cellular damage is well known. EB thus helps to maintain the integrity of DNA against oxidative stress. It is well documented that Vitamin E, also under certain conditions, also acts as a pro-oxidant. Hence, the augmentation of DNA strand-scission (instead of protection) by Vitamin C/E blend is self-explanatory. 
     
       
         
               
             
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Comparative Suppressive Effects 
               
               
                 of DNA Strand Scission 
               
             
          
           
               
                   
                 Test Run 
               
             
          
           
               
                   
                 TYPES OF DNA 
                 1 
                 2 
                 3 
                 4 
                 5 
               
               
                   
                   
               
               
                   
                 Native form of 
                 90 
                 — 
                 80 
                  5 
                 — 
               
               
                   
                 Supercoiled DNA (SC) 
               
               
                   
                 Open circular form 
                 10 
                 60 
                 20 
                 65 
                 40 
               
               
                   
                 Of DNA (OC) 
               
               
                   
                 Linear form of DNA (LIN, 
                 — 
                 40 
                 — 
                 30 
                 60 
               
               
                   
                 Produced after breakage) 
               
               
                   
                   
               
               
                   
                 Test Run  
               
               
                   
                 1. DNA alone (without OH radical). Note: LIN absent and OC is intermediate to LIN  
               
               
                   
                 2. DNA + OH radical [from Cu(en) 2 -hydrogen peroxide)  
               
               
                   
                 3. DNA + OH radical + EB. Note: LIN absent and OC is only 20%  
               
               
                   
                 4. DNA + OH radical + Vitamin C/E blend. Note: Depletion of SC concentration  
               
               
                   
                 5. DNA + OH radical + Vitamin C. Note: Significant increase in LIN value indicative of DNA strand scission.  
               
             
          
         
       
     
     The extract blend may be administered to an animal such as a human in a use formulation containing about 0.005 to 5% by weight of the formulation. Optionally other known antioxidants, such as ascorbic acid, tocopherols, pycnogenols, glutathione, and the like, may be included in the formulation. 
     Suitable use formulations may be in the form of tablets, syrups, elixirs, lotions, cremes or gels, and administered for pharmaceutical, nutritional or personal care applications. Pharmaceutical formulations preferably are in the form of tablets, syrup, elixir or capsules while personal care compositions for skin care, for example are solutions, lotions, crème or gel. 
     While the invention has been described with particular reference to certain embodiments thereof, it will be understood that changes and modifications may be made which are within the skill of the art. Accordingly, it is intended to be bound only by the following claims, in which: