Patent Document

[0001]     This application is a divisional of U.S. application Ser. No. 10/186,504, filed Jun. 28, 2002. 
     
    
     FIELD OF THE INVENTION  
       [0002]     The invention relates to compositions that can ameliorate or prevent hypertension and are useful as dietary supplements (e.g., health drinks). These compositions contain yeast cells obtainable by growth in electromagnetic fields with specific frequencies and field strengths.  
       BACKGROUND OF THE INVENTION  
       [0003]     Hypertension is an insidious disease that affects many people. Hypertension is generally defined as an abnormally increased blood pressure. Hypertension is also a major indicator of the prognosis for future development of cardiovascular, cerebrovascular, and renal diseases. Certain risk factors, e.g., hypercholesterolemia, diabetes, smoking, and a familial history of vascular disease, in conjunction with hypertension, may predispose individuals to arteriosclerosis and consequent cardiovascular morbidity and mortality.  
         [0004]     There are two major types of hypertension: essential (primary or idiopathic) and secondary hypertension. Hypertension of unknown etiology is termed essential hypertension, which is a relatively common disease state in people. Essential hypertension is generally asymptomatic and has been associated with the early onset of coronary disease, kidney failure and stroke. A combination of factors including age, obesity, smoking, heredity, stress, and an aggressive, hyperactive personality are thought to contribute to the occurrence of this form of hypertension.  
         [0005]     Secondary hypertension is described as a symptom of a variety of underlying or primary diseases, such as renal vascular disease (e.g., atherosclerosis or stenosis of the renal artery) and renal parenchymal disease. Other disorders such as dysfunction of the adrenal cortex and medulla, atherosclerosis of the systemic arteries, and coarctation of the aorta may also produce secondary hypertension. The most common primary disorders leading to secondary hypertension are those that interfere with the supply of blood to the kidney. The resulting ischemia of renal tissue stimulates the secretion of renin, an enzyme that catalyzes the conversion of angiotensinogen to angiotensin I. Other enzymes in the body then convert angiotensin I to angiotensin II, the most potent vasoconstrictor known. Angiotensin II acts directly on the blood vessels and also acts as a physiologic stimulant to the adrenal gland and the production of aldosterone. Hence there is a twofold effect: (1) an increase in peripheral resistance due to vasoconstriction and a resulting elevation of blood pressure, and (2) a retention of sodium and water by the renal tubules in response to increased levels of serum aldosterone. The retained sodium and water increase blood volume, since they remain in the vascular system rather than being excreted by the kidney. The result is an increased cardiac output and an elevation of blood pressure.  
         [0006]     Drug therapy is a common approach to treatment of hypertension. In general, antihypertensive drugs include diuretics, beta-blockers, angiotensin converting enzyme (ACE) inhibitors, anti-sympaticotonica, beta-blockers, calcium-antagonists and vasodilatators. However, each of these available drugs, though generally effective in reducing hypertension, has side effects, such as potassium depletion, hyperglycemia, hypokalemia, depression, carbohydrate intolerance, tachycardia, and/or allergic skin rashes, and in more severe cases, vomiting, fever, diarrhea, angina, and cardiac failure.  
       SUMMARY OF THE INVENTION  
       [0007]     This invention is based on the discovery that certain yeast cells can be activated by electromagnetic fields having specific frequencies and field strengths to produce substances useful in treating hypertension (e.g., both essential and secondary hypertension). Compositions comprising these activated yeast cells can therefore be used as dietary supplements, in the form of health drinks or dietary pills (tablets or powder). For instance, these compositions can be used to alleviate (e.g., lower) high blood pressure (systolic and/or diastolic pressure) in a hypertensive human, or to prevent or postpone the onset of hypertension in a high risk individual (e.g., someone predisposed to hypertension because of his genetic background or life style).  
         [0008]     This invention embraces a composition comprising a plurality of yeast cells that have been cultured in an alternating electric field having a frequency in the range of about 11000 to 12000 MHz (e.g., 11500-11600 MHz) and a field strength in the range of about 180 to 500 mV/cm (e.g., 190-210, 210-250, 280-320, 320-350, 350-380, 380-420 or 420-450 mV/cm). The yeast cells are cultured for a period of time sufficient to activate said plurality of yeast cells to produce substances useful in treating hypertension in a subject. In one embodiment, the frequency and/or the field strength of the alternating electric field can be altered within the aforementioned ranges during said period of time. In other words, the yeast cells are exposed to a series of electromagnetic fields. An exemplary period of time is about 30-200 hours (e.g., 35-100 hours).  
         [0009]     Also included in this invention is a composition comprising a plurality of yeast cells that have been cultured under acidic conditions in an alternating electric field having a frequency in the range of about 11000 to 12000 MHz (e.g., 11510-11550 MHz) and a field strength in the range of about 200 to 500 mV/cm (e.g., 380-420 mV/cm). In one embodiment, the yeast cells are exposed to a series of electromagnetic fields. An exemplary period of time is about 30-100 hours (e.g., 35-80 hours).  
         [0010]     Yeast cells that can be included in this composition are available from the China General Microbiological Culture Collection Center (“CGMCC”), a depository recognized under the Budapest Treaty (China Committee for Culture Collection of Microorganisms, Institute of Microbiology, Chinese Academy of Sciences, Haidian, P.O. Box 2714, Beijing, 100080, China). Useful yeast species include, but are not limited to, those commonly used in food and pharmaceutical industries, such as  Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces rouxii, Saccharomyces sake, Saccharomyces uvarum, Saccharomyces sp., Schizosaccharomyces pombe , and  Rhodotorula aurantiaca . For instance, the yeast cells can be of the strain  Saccharomyces carlsbergensis  Hansen AS2.420, AS2.440, or AS2.444; or  Saccharomyces cerevisiae  Hansen AS2.375, AS2.501, AS2.502, AS2.503, AS2.504, AS2.535, AS2.558, AS2.560, AS2.561, AS2.562, or IFFI1048. Other useful yeast strains are illustrated in Table 1.  
         [0011]     This invention further embraces a composition comprising a plurality of yeast cells, wherein said plurality of yeast cells have been activated to treat hypertension in a subject. Included in this invention are also methods of making these compositions.  
         [0012]     Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting. Throughout this specification and claims, the word “comprise,” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. A subject includes a human and veterinary subject.  
         [0013]     Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.  
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0014]      FIG. 1  is a schematic diagram showing an exemplary apparatus for activating yeast cells using electromagnetic fields.  1 : yeast culture;  2 : container;  3 : power supply.  
         [0015]      FIG. 2  is a schematic diagram showing an exemplary apparatus for making yeast compositions of the invention. The apparatus comprises a signal generator and interconnected containers  1 ,  2  and  3 . 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0016]     This invention is based on the discovery that certain yeast strains can be activated by electromagnetic fields (“EMF”) having specific frequencies and field strengths to become highly efficient in producing substances that lower blood pressure (systolic and/or diastolic pressure) in a subject (e.g., a human subject). Compositions containing these activated yeast cells are therefore useful in the treatment of hypertension. Yeast compositions containing activated yeast cells can be used as dietary supplements, in the form of health drinks or dietary pills (tablets or powder).  
         [0017]     Since the activated yeast cells contained in the yeast compositions have been cultured to endure acidic conditions (pH 2.5-4.2), these cells can survive the gastric environment and pass on to the intestines. Once in the intestines, the yeast cells are ruptured by various digestive enzymes, and the anti-hypertensive substances are released and readily absorbed.  
         [0018]     Without being bound by any theory or mechanism, the inventor believes that EMFs activate or enhance the expression of a gene or a set of genes in yeast cells such that the yeast cells become active or more efficient in performing certain metabolic activities which lead to the desired anti-hypertensive effect.  
         [0000]     I. Yeast Strains Useful in the Invention  
         [0019]     The types of yeasts useful in this invention include, but are not limited to, yeasts of the genera  Saccharomyces, Schizosaccharomyces pombe  and  Rhodotorula.    
         [0020]     Exemplary species within the above-listed genera include, but are not limited to, those illustrated in Table 1. Yeast strains useful for this invention can be obtained from laboratory cultures, or from publically accessible culture depositories, such as CGMCC and the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209. Non-limiting examples of useful strains (with accession numbers of CGMCC) are  Saccharomyces carlsbergensis  Hansen AS2.420, AS2.440, and AS2.444;  Saccharomyces cerevisiae  Hansen AS2.375, AS2.501, AS2.502, AS2.503, AS2.504, AS2.535, AS2.558, AS2.560, AS2.561, AS2.562, and IFFI 1048. Other useful yeast strains are illustrated in Table 1.  
         [0021]     Although it is preferred, the preparation of the yeast compositions of this invention is not limited to starting with a pure strain of yeast. A yeast composition of the invention may be produced by culturing a mixture of yeast cells of different species or strains. The ability of any activated species or strain of yeasts to treat hypertension can be readily tested by methods known in the art. See, for instance, Examples 1 and 2.  
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         TABLE 1                       Exemplary Yeast Strains                     Saccharomyces cerevisiae  Hansen            ACCC2034   ACCC2035   ACCC2036   ACCC2037   ACCC2038       ACCC2039   ACCC2040   ACCC2041   ACCC2042   AS2. 1       AS2. 4   AS2. 11   AS2. 14   AS2. 16   AS2. 56       AS2. 69   AS2. 70   AS2. 93   AS2. 98   AS2. 101       AS2. 109   AS2. 110   AS2. 112   AS2. 139   AS2. 173       AS2. 174   AS2. 182   AS2. 196   AS2. 242   AS2. 336       AS2. 346   AS2. 369   AS2. 374   AS2. 375   AS2. 379       AS2. 380   AS2. 382   AS2. 390   AS2. 393   AS2. 395       AS2. 396   AS2. 397   AS2. 398   AS2. 399   AS2. 400       AS2. 406   AS2. 408   AS2. 409   AS2. 413   AS2. 414       AS2. 415   AS2. 416   AS2. 422   AS2. 423   AS2. 430       AS2. 431   AS2. 432   AS2. 451   AS2. 452   AS2. 453       AS2. 458   AS2. 460   AS2. 463   AS2. 467   AS2. 486       AS2. 501   AS2. 502   AS2. 503   AS2. 504   AS2. 516       AS2. 535   AS2. 536   AS2. 558   AS2. 560   AS2. 561       AS2. 562   AS2. 576   AS2. 593   AS2. 594   AS2. 614       AS2. 620   AS2. 628   AS2. 631   AS2. 666   AS2. 982       AS2. 1190   AS2. 1364   AS2. 1396   IFFI1001   IFFI1002       IFFI1005   IFFI1006   IFFI1008   IFFI1009   IFFI1010       IFFI1012   IFFI1021   IFFI1027   IFFI1037   IFFI1042       IFFI1043   IFFI1045   IFFI1048   IFFI1049   IFFI1050       IFFI1052   IFFI1059   IFFI1060   IFFI1062   IFFI1063       IFFI1202   IFFI1203   IFFI1206   IFFI1209   IFFI1210       IFFI1211   IFFI1212   IFFI1213   IFFI1214   IFFI1215       IFFI1220   IFFI1221   IFFI1224   IFFI1247   IFFI1248       IFFI1251   IFFI1270   IFFI1277   IFFI1287   IFFI1289       IFFI1290   IFFI1291   IFFI1292   IFFI1293   IFFI1297       IFFI1300   IFFI1301   IFFI1302   IFFI1307   IFFI1308       IFFI1309   IFFI1310   IFFI1311   IFFI1331   IFFI1335       IFFI1336   IFFI1337   IFFI1338   IFFI1339   IFFI1340       IFFI1345   IFFI1348   IFFI1396   IFFI1397   IFFI1399       IFFI1411   IFFI1413   IFFI1441   IFFI1443              Saccharomyces cerevisiae  Hansen Var. ellipsoideus (Hansen) Dekker            ACCC2043   AS2.2   AS2.3   AS2.8   AS2.53       AS2.163   AS2.168   AS2.483   AS2.541   AS2.559       AS2.606   AS2.607   AS2.611   AS2.612              Saccharomyces chevalieri  Guilliermond            AS2.131   AS2.213              Saccharomyces delbrueckii              AS2.285              Saccharomyces delbrueckii  Lindner ver. mongolicus       (Saito) Lodder et van Rij            AS2.209   AS2.1157              Saccharomyces exiguous  Hansen            AS2.349   AS2.1158              Saccharomyces fermentati  (Saito) Lodder et van Rij            AS2.286   AS2.343              Saccharomyces logos  van laer et Denamur ex Jorgensen            AS2.156   AS2.327   AS2.335              Saccharomyces mellis  (Fabian et Quinet) Lodder et kreger van Rij            AS2.195              Saccharomyces mellis  Microellipsoides Osterwalder            AS2.699              Saccharomyces oviformis  Osteralder            AS2.100              Saccharomyces rosei  (Guilliermond) Lodder et Kreger van Rij            AS2.287              Saccharomyces rouxii  Boutroux            AS2.178   AS2.180   AS2.370   AS2.371              Saccharomyces sake  Yabe            ACCC2045              Candida arborea              AS2.566              Candida lambica  (Lindner et Genoud) van. Uden et Buckley            AS2.1182              Candida krusei  (Castellani) Berkhout            AS2.1045              Candida lipolytica  (Harrison) Diddens et Lodder            AS2.1207   AS2.1216   AS2.1220   AS2.1379   AS2.1398       AS2.1399   AS2.1400              Candida parapsilosis  (Ashford) Langeron et Talice Var.       intermedia Van Rij et Verona            AS2.491              Candida parapsilosis  (Ashford) Langeron et Talice            AS2.590              Candida pulcherrima  (Lindner) Windisch            AS2.492              Candida rugousa  (Anderson) Diddens et Lodder            AS2.511   AS2.1367   AS2.1369   AS2.1372   AS2.1373       AS2.1377   AS2.1378   AS2.1384              Candida tropicalis  (Castellani) Berkhout            ACCC2004   ACCC2005   ACCC2006   AS2.164   AS2.402       AS2.564   AS2.565   AS2.567   AS2.568   AS2.617       AS2.637   AS2.1387   AS2.1397              Candida utilis  Henneberg Lodder et Kreger Van Rij            AS2.120   AS2.281   AS2.1180              Crebrothecium ashbyii  (Guillermond)       Routein ( Eremothecium ashbyii  Guilliermond)            AS2.481   AS2.482   AS2.1197              Geotrichum candidum  Link            ACCC2016   AS2.361   AS2.498   AS2.616   AS2.1035       AS2.1062   AS2.1080   AS2.1132   AS2.1175   AS2.1183              Hansenula anomala  (Hansen)H et P sydow            ACCC2018   AS2.294   AS2.295   AS2.296   AS2.297       AS2.298   AS2.299   AS2.300   AS2.302   AS2.338       AS2.339   AS2.340   AS2.341   AS2.470   AS2.592       AS2.641   AS2.642   AS2.782   AS2.635   AS2.794              Hansenula arabitolgens  Fang            AS2.887              Hansenula jadinii  (A. et R Sartory Weill et Meyer) Wickerham            ACCC2019              Hansenula saturnus  (Klocker) H et P sydow            ACCC2020              Hansenula schneggii  (Weber) Dekker            AS2.304              Hansenula subpelliculosa  Bedford            AS2.740   AS2.760   AS2.761   AS2.770   AS2.783       AS2.790   AS2.798   AS2.866              Kloeckera apiculata  (Reess emend. Klocker) Janke            ACCC2022   ACCC2023   AS2.197   AS2.496   AS2.714       ACCC2021   AS2.711              Lipomycess starkeyi  Lodder et van Rij            AS2.1390   ACCC2024              Pichia farinosa  (Lindner) Hansen            ACCC2025   ACCC2026   AS2.86   AS2.87   AS2.705       AS2.803              Pichia membranaefaciens  Hansen            ACCC2027   AS2.89   AS2.661   AS2.1039              Rhodosporidium toruloides  Banno            ACCC2028              Rhodotorula glutinis  (Fresenius) Harrison            AS2.2029   AS2.280   ACCC2030   AS2.102   AS2.107       AS2.278   AS2.499   AS2.694   AS2.703   AS2.704       AS2.1146              Rhodotorula minuta  (Saito) Harrison            AS2.277              Rhodotorula rubar  (Demme) Lodder            AS2.21   AS2.22   AS2.103   AS2.105   AS2.108       AS2.140   AS2.166   AS2.167   AS2.272   AS2.279       AS2.282   ACCC2031              Rhodotorula aurantiaca  (Saito) Lodder            AS2.102   AS2.107   AS2.278   AS2.499   AS2.694       AS2.703   AS2.704   AS2.1146              Saccharomyces carlsbergensis  Hansen            AS2.113   ACCC2032   ACCC2033   AS2.312   AS2.116       AS2.118   AS2.121   AS2.132   AS2.162   AS2.189       AS2.200   AS2.216   AS2.265   AS2.377   AS2.417       AS2.420   AS2.440   AS2.441   AS2.443   AS2.444       AS2.459   AS2.595   AS2.605   AS2.638   AS2.742       AS2.745   AS2.748   AS2.1042              Saccharomyces uvarum  Beijer            IFFI1023   IFFI1032   IFFI1036   IFFI1044   IFFI1072       IFFI1205   IFFI1207              Saccharomyces willianus  Saccardo            AS2.5   AS2.7   AS2.119   AS2.152   AS2.293       AS2.381   AS2.392   AS2.434   AS2.614   AS2.1189              Saccharomyces  sp.            AS2.311              Saccharomycodes ludwigii  Hansen            ACCC2044   AS2.243   AS2.508              Saccharomycodes sinenses  Yue            AS2.1395              Schizosaccharomyces octosporus  Beijerinck            ACCC2046   AS2.1148              Schizosaccharomyces pombe  Lindner            ACCC2047   ACCC2048   AS2.214   AS2.248   AS2.249       AS2.255   AS2.257   AS2.259   AS2.260   AS2.274       AS2.994   AS2.1043   AS2.1149   AS2.1178   IFFI1056              Sporobolomyces roseus  Kluyver et van Niel            ACCC2049   ACCC2050   AS2.19   AS2.962   AS2.1036       ACCC2051   AS2.261   AS2.262              Torulopsis candida  (Saito) Lodder            AS2.270   ACCC2052              Torulopsis famta  (Harrison) Lodder et van Rij            ACCC2053   AS2.685              Torulopsis globosa  (Olson et Hammer) Lodder et van Rij            ACCC2054   AS2.202              Torulopsis inconspicua  Lodder et Kreger van Rij            AS2.75              Trichosporon behrendii  Lodder et Kreger van Rij            ACCC2056   AS2.1193              Trichosporon capitatum  Diddens et Lodder            ACCC2056   AS2.1385              Trichosporon cutaneum  (de Beurm et al.) Ota            ACCC2057   AS2.25   AS2.570   AS2.571   AS2.1374              Wickerhamia fluorescens  (Soneda) Soneda            ACCC2058   AS2.1388                  
 
 II. Application of Electromagnetic Fields 
 
         [0022]     An electromagnetic field useful in this invention can be generated and applied by various means well known in the art. For instance, the EMF can be generated by applying an alternating electric field or an oscillating magnetic field.  
         [0023]     Alternating electric fields can be applied to cell cultures through electrodes in direct contact with the culture medium, or through electromagnetic induction. See, e.g.,  FIG. 1 . Relatively high electric fields in the medium can be generated using a method in which the electrodes are in contact with the medium. Care must be taken to prevent electrolysis at the electrodes from introducing undesired ions into the culture and to prevent contact resistance, bubbles, or other features of electrolysis from dropping the field level below that intended. Electrodes should be matched to their environment, for example, using Ag—AgCl electrodes in solutions rich in chloride ions, and run at as low a voltage as possible. For general review, see Goodman et al.,  Effects of EMF on Molecules and Cells , International Review of Cytology, A Survey of Cell Biology, Vol. 158, Academic Press, 1995.  
         [0024]     The EMFs useful in this invention can also be generated by applying an oscillating magnetic field. An oscillating magnetic field can be generated by oscillating electric currents going through Helmholtz coils. Such a magnetic field in turn induces an electric field.  
         [0025]     The frequencies of EMFs useful in this invention range from about 11000 to 12000 MHz(e.g., 11500-11600 MHz). Exemplary frequencies are 11508, 11515, 11521, 11528, and 11535 MHz. The field strength of the electric field useful in this invention ranges from about 180 to 500 mV/cm (e.g., 190-210, 210-250, 280-320, 320-350, 350-380, 380-420 or 420-450 mV/cm). Exemplary field strengths are 200, 223, 310, 316, 332, 364, 406, 413, 433, and 435 mV/cm.  
         [0026]     When a series of EMFs are applied to a yeast culture, the yeast culture can remain in the same container while the same set of EMF generator and emitters is used to change the frequency and/or field strength. The EMFs in the series can each have a different frequency or a different field strength; or a different frequency and a different field strength. Such frequencies and field strengths are preferably within the above-described ranges. Although any practical number of EMFs can be used in a series, it may be preferred that the yeast culture be exposed to a total of 2, 3, 4, 5, 6, 7, 8, 9 or 10 EMFs in a series.  
         [0027]     Although the yeast cells can be activated after even a few hours of culturing in the presence of an EMF, it may be preferred that the activated yeast cells be allowed to multiply and grow in the presence of the EMF(s) for a total of 30-200 hours (e.g., 35-100 hours).  
         [0028]      FIG. 1  illustrates an exemplary apparatus for generating alternating electric fields. An electric field of a desired frequency and intensity is generated by an AC source ( 3 ) capable of generating an alternating electric field, preferably in a sinusoidal wave form, in the frequency range of 10 to 20,000 MHz. Signal generators capable of generating signals with a narrower frequency range can also be used. If desirable, a signal amplifier can also be used to increase the output. The activation container ( 2 ) can be made from non-conductive metal material, e.g., plastics, glass or ceramic. The wire connecting the activation container ( 2 ) and the signal generator ( 3 ) is preferably a high frequency coaxial cable with a transmission frequency of at least 30 GHz.  
         [0029]     The alternating electric field can be applied to the culture by a variety of means, including placing the yeast culture ( 1 ) in close proximity to the signal emitters such as a metal wire or tube capable of transmitting EMFs. The metal wire or tube can be made of red copper, and be placed inside the container ( 2 ), reaching as deep as 3-30 cm. For example, if the fluid in the container ( 2 ) has a depth of 15-20 cm, 20-30 cm, 30-50 cm, 50-70 cm, 70-100 cm, 100-150 cm or 150-200 cm, the metal wire can be 3-5 cm, 5-7 cm, 7-10 cm, 10-15 cm, 15-20 cm, 20-30 cm and 25-30 cm from the bottom of the container ( 2 ), respectively. The number of electrode wires used depends on the volume of the culture as well as the diameter of the wires. The number of metal wires/tubes used can be from 1 to 10 (e.g., 2 to 3). It is recommended, though not mandated, that for a culture having a volume up to 10 L, metal wires/tubes having a diameter of 0.5 to 2.0 mm be used. For a culture having a volume between 10 L and 100 L, metal wires/tubes having a diameter of 3.0 to 5.0 mm can be used. For a culture having a volume in the range of 100-1000 L, metal wires/tubes having a diameter of 6.0 to 15.0 mm can be used. For a culture having a volume greater than 1000 L, metal wires/tubes having a diameter of 20.0 to 25.0 mm can be used.  
         [0030]     In one embodiment, the electric field is applied by electrodes submerged in the culture ( 1 ). In this embodiment, one of the electrodes can be a metal plate placed on the bottom of the container ( 2 ), and the other electrode can comprise a plurality of electrode wires evenly distributed in the culture ( 1 ) so as to achieve even distribution of the electric field energy. The number of electrode wires used depends on the volume of the culture as well as the diameter of the wires.  
         [0000]     III. Culture Media  
         [0031]     Culture media useful in this invention contain sources of nutrients assimilable by yeast cells. Complex carbon-containing substances in a suitable form, such as carbohydrates (e.g., sucrose, glucose, fructose, dextrose, maltose, xylose, cellulose, starches, etc.) and coal, can be the carbon sources for yeast cells. The exact quantity of the carbon sources utilized in the medium can be adjusted in accordance with the other ingredients of the medium. In general, the amount of carbohydrates varies between about 0.1% and 10% by weight of the medium and preferably between about 0.1% and 5% (e.g., about 2%). These carbon sources can be used individually or in combination. Amino acid-containing substances in suitable form (e.g., beef extract and peptone) can also be added individually or in combination. In general, the amount of amino acid containing substances varies between about 0.1% and 0.5% by weight of the medium and preferably between about 0.1% and 0.3% (e.g., about 0.25%). Among the inorganic salts which can be added to the culture medium are the customary salts capable of yielding sodium, potassium, calcium, phosphate, sulfate, carbonate, and like ions. Non-limiting examples of nutrient inorganic salts are (NH 4 ) 2 HPO 4 , KH 2 PO 4 , K 2 HPO 4 , CaCO 3 , MgSO 4 , NaCl, and CaSO 4 .  
         [0000]     IV. Electromagnetic Activation of Yeast Cells  
         [0032]     To activate or enhance the ability of yeast cells to produce substances beneficial for the treatment of hypertension (e.g., lowering of blood pressure), these cells can be activated by being cultured in an appropriate medium under sterile conditions at 20° C.-38° C., preferably at 28-32° C. (e.g., 30° C.) for a sufficient amount of time, e.g., 30-200 hours (e.g., 35-100 hours), in an alternating electric field or a series of alternating electric fields as described above.  
         [0033]     An exemplary culture medium is made by mixing 1000 ml of distilled water with 20 g of sucrose, 30 μg of vitamin B 3 , 60 μg of vitamin H, 30 μg of vitamin B 12 , 0.20 g of KH 2 PO 4 , 0.2 g of MgSO 4 .7H 2 O, 0.25 g of NaCl, 0.1 g of CaSO 4 .2H 2 O, 3.0 g of CaCO 3 .5H 2 O, and 2.5 g of peptone.  
         [0034]     An exemplary set-up of the culturing process is depicted in  FIG. 1 . Untreated yeast cells are added to a culture medium at 1×10 8  cells per 1000 ml of the culture medium. The yeast cells may be  Saccharomyces cerevisiae  Hansen AS2.561, or may be selected from any of the strains listed in Table 1. An exemplary activation process of the yeast cells involves the following sequence: the yeast cells are grown in the culture medium for 38-42 hours (e.g., 40 hours) at 28-32° C. and then exposed to (1) an alternating electric field having a frequency of 11508 MHz and a field strength in the range of 280-320 mV/cm (e.g., 310 mV/cm) for 16-22 hours (e.g., 20 hours); (2) then to an alternating electric field having a frequency of 11515 MHz and a field strength in the range of 280-320 mV/cm (e.g., 316 mV/cm) for 16-22 hours (e.g., 20 hours); (3) then to an alternating electric field having a frequency of 11521 MHz and a field strength in the range of 350-380 mV/cm (e.g., 364 mV/cm) for 20-25 hours (e.g., 23 hours); (4) then to an alternating electric field having a frequency of 11528 MHz and a field strength in the range of 420-450 mV/cm (e.g., 435 mV/cm) for 16-22 hours (e.g., 21 hours); and (5) finally to an alternating electric field having a frequency of 11535 MHz and a field strength in the range of 420-450 mV/cm (e.g., 433 mV/cm) for 15-22 hours (e.g., 15 hours). The activated yeast cells are then recovered from the culture medium by various methods known in the art, dried (e.g., by lyophilization) and stored at about 4° C. in powder form. The resultant yeast powder preferably contains no less than 10 10  cells/g activated yeast.  
         [0035]     Subsequently, the activated yeast cells can be evaluated for their ability to treat hypertension using standard methods known in the art, such as those described in Section VII.  
         [0000]     V. Acclimatization of Yeast Cells To the Gastric Environment  
         [0036]     Because the activated yeast cells of this invention must pass through the stomach before reaching the small intestine, where the effective components are released from these yeast cells, it is preferred that these yeasts be cultured under acidic conditions so as to acclimatize the cells to the gastric juice. This acclimatization process results in better viability of the yeast cells in the acidic gastric environment.  
         [0037]     To achieve this, the yeast powder containing activated yeast cells can be mixed with a highly acidic acclimatizing culture medium at 10 g (containing more than 10 10  activated cells per gram) per 1000 ml. The yeast mixture can then be cultured first in the presence of an alternating electric field having a frequency of 11528 MHz and a field strength in the range of 380-420 mV/cm (e.g., 406 mV/cm) at about 28 to 32° C. for 36-42 hours (e.g., 38 hours). The resultant yeast cells can then be further incubated in the presence of an alternating electric field having a frequency of 11535 MHz and a field strength in the range of 380-420 mV/cm (e.g., 413 mV/cm) at about 28 to 32° C. for 20-42 hours (e.g., 20 hours). The resulting acclimatized yeast cells are then recovered from the culture medium by various methods known in the art and are either dried and stored in powder form (≧10 10  cells/g) at room temperature or in vacuum at 0-4° C.  
         [0038]     An exemplary acclimatizing culture medium is made by mixing 700 ml fresh pig gastric juice and 300 ml wild Chinese hawthorn extract. The pH of acclimatizing culture medium is adjusted to 2.5 with 0.1 M hydrochloric acid (HCl) and 0.2 M potassium biphthalate (C 6 H 4 (COOK)COOH). The fresh pig gastric juice is prepared as follows. At about 4 months of age, newborn Holland white pigs are sacrificed, and the entire contents of their stomachs are retrieved and mixed with 2000 ml of water under sterile conditions. The mixture is then allowed to stand for 6 hours at 4° C. under sterile conditions to precipitate food debris. The supernatant is collected for use in the acclimatizing culture medium. To prepare the wild Chinese hawthorn extract, 500 g of fresh wild Chinese hawthorn is dried under sterile conditions to reduce water content (≦8%). The dried fruit is then ground (≧20 mesh) and added to 1500 ml of sterile water. The hawthorn slurry is allowed to stand for 6 hours at 4° C. under sterile conditions. The hawthorn supernatant is collected to be used in the acclimatizing culture medium.  
         [0000]     VI. Manufacture of Yeast Compositions  
         [0039]     To prepare the yeast compositions of the invention, an apparatus depicted in  FIG. 2  or an equivalent thereof can be used. This apparatus includes three containers, a first container ( 1 ), a second container ( 2 ), and a third container ( 3 ), each equipped with a pair of electrodes ( 4 ). One of the electrodes is a metal plate placed on the bottom of the containers, and the other electrode comprises a plurality of electrode wires evenly distributed in the space within the container to achieve even distribution of the electric field energy. All three pairs of electrodes are connected to a common signal generator.  
         [0040]     The culture medium used for this purpose is a mixed fruit extract solution containing the following ingredients per 1000 L: 300 L of wild Chinese hawthorn extract, 300 L of jujube extract, 300 L of  Schisandra chinensis  ( Turez )  Baill  seeds extract, and 100 L of soy bean extract. To prepare hawthorn, jujube and  Schisandra chinensis  ( Turez )  Baill  seeds extracts, the fresh fruits are washed and dried under sterile conditions to reduce the water content to no higher than 8%. One hundred kilograms of the dried fruits are then ground (≧20 mesh) and added to 400 L of sterile water. The mixtures are stirred under sterile conditions at room temperature for twelve hours, and then centrifuged at 1000 rpm to remove insoluble residues. To make the soy bean extract, fresh soy beans are washed and dried under sterile conditions to reduce the water content to no higher than 8%. Thirty kilograms of dried soy beans are then ground into particles of no smaller than 20 mesh, and added to 130 L of sterile water. The mixture is stirred under sterile conditions at room temperature for twelve hours and centrifuged at 1000 rpm to remove insoluble residues. Once the mixed fruit extract solution is prepared, it is autoclaved at 121° C. for 30 minutes and cooled to below 40° C. before use.  
         [0041]     One thousand grams of the activated yeast powder prepared as described above (Section V, supra) is added to 1000 L of the mixed fruit extract solution, and the yeast solution is transferred to the first container ( 1 ) shown in  FIG. 2 . The yeast cells are then cultured in the presence of an alternating electric field having a frequency of 11528 MHz and a field strength of about 420-450 mV/cm (e.g., 435 mV/cm) at 28-32° C. under sterile conditions for 12 hours. The yeast cells are further incubated in an alternating electric field having a frequency of 11535 MHz and a field strength of 420-450 mV/cm (e.g., 433 mV/cm). The culturing continues for another 10 hours.  
         [0042]     The yeast culture is then transferred from the first container ( 1 ) to the second container ( 2 ) (if need be, a new batch of yeast culture can be started in the now available the first container ( 1 )), and subjected to an alternating electric field having a frequency of 11528 MHz and a field strength of 320-350 mV/cm (e.g., 332 mV/cm) for 14 hours. Subsequently the frequency and field strength of the electric field are changed to 11535 MHz and 310-350 mV/cm (e.g., 310 mV/cm), respectively. The culturing process continues for another 12 hours.  
         [0043]     The yeast culture is then transferred from the second container ( 2 ) to the third container ( 3 ), and subjected to an alternating electric field having a frequency of 11528 MHz and a field strength of 210-250 mV/cm (e.g., 223 mV/cm) for 18 hours. Subsequently the frequency and field strength of the electric field are changed to 11535 MHz and 190-210 mV/cm (e.g., 200 mV/cm), respectively. The culturing continues for another 12 hours.  
         [0044]     The yeast culture from the third container ( 3 ) can then be packaged into vacuum sealed bottles for use as dietary supplement. The compositions may conveniently be formulated as health drinks. If desired, the final yeast culture can also be dried within 24 hours and stored in powder form. The dietary supplement can be taken three to four times daily at 30-60 ml per dose for a three-month period, preferably 10-30 minutes before meals and at bedtime.  
         [0045]     In some embodiments, the compositions of the invention can also be administered intravenously or peritoneally in the form of a sterile injectable preparation. Such a sterile preparation can be prepared as follows. A sterilized health drink composition is first treated under ultrasound (1000 Hz) for 10 minutes and then centrifuged at 4355 g for another 10 minutes. The resulting supernatant is adjusted to pH 7.2-7.4 using 1 M NaOH and subsequently filtered through a membrane (0.22 μm for intravenous injection and 0.45 μm for peritoneal injection) under sterile conditions. The resulting sterile preparation is submerged in a 35-38° C. water bath for 30 minutes before use.  
         [0046]     The yeast compositions of the present invention are derived from yeasts used in food and pharmaceutical industries. The yeast compositions are thus devoid of side effects associated with many pharmaceutical compounds.  
         [0000]     VII. Examples  
         [0047]     In order that this invention be more fully understood, the following examples are set forth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way.  
         [0048]     The activated yeast compositions used in the following examples were prepared as described above, using  Saccharomyces cerevisiae  Hansen AS2.561, cultured in the presence of an alternating electric field having the electric field frequency and field strength exemplified in the parentheses following the recommended ranges listed in Section IV, supra. Control (i.e., untreated) yeast compositions were those prepared in the same manner as described in Section VI, supra, except that the yeast cells were cultured in the absence of EMFs. Unless otherwise specified, all compositions of interest were administered to the animals by intragastric feeding.  
       EXAMPLE 1  
     Control of Essential Hypsertension in Wistar Rat  
       [0049]     To test the ability of the activated yeast compositions to reduce blood pressure, thirty-two healthy Wistar rats of average weight of about 200±20 g (8-10 months old, half of them male and the other half female) were randomly divided into four equal groups, Groups A, B, C and D. Blood pressure of each rat was measured with a manometer for three consecutive days. After three days, each rat was subcutaneously injected with 4 mg of testosterone propionate in 0.4 ml of sesame oil per day for fourteen consecutive days. At this point, the elevation in their blood pressure level was observed to be more than 1.3 kPa (10 mmHg).  
         [0050]     A composition of interest (2 ml/kg/day) was then administered to each rat for sixteen consecutive days, while their blood pressure level was monitored every four days. Rats in Groups A, B, C and D received distilled water, reserpine, the activated yeast composition and the control yeast composition, respectively. The results were summarized in Table 2.  
                                               TABLE 2                           Blood Pressure Readings of Wistar Rats                Blood Pressure (kPa)                            4                       Change           1           in Blood           Before   2   3   Pressure           Injection   After Injection   After   (Value           with   with   Administration   in Col. 3 −           Testosteronyl   Testosteronyl   of a   Value in       Group   17-propionate   17-propionate   Composition   Col. 2)               A   14.5 ± 0.41   17.4 ± 0.82   17.8 ± 0.56   −0.4 ± 0.26       B   14.7 ± 0.32   16.7 ± 0.56   15.3 ± 0.89    1.4 ± 0.33       C   14.3 ± 0.23   16.5 ± 0.52   12.3 ± 0.68    4.2 ± 0.16       D   14.6 ± 0.27   16.4 ± 0.54   17.5 ± 0.51   −1.1 ± 0.03                  
 
         [0051]     The results in Table 2 show that the activated yeast composition was more effective in reducing blood pressure than both the known anti-hypertensive agent reserpine and the control yeast composition.  
       EXAMPLE 2  
     Control of Renal Hypertension in Wistar Rats  
       [0052]     To test the ability of the activated yeast compositions to treat renal hypertension, thirty healthy male Wistar rats of average weight of about 250 to 300 g (12-14 months old) were randomly divided into three equal groups, Groups A, B and C. Under ether anesthesia, each rat was laid prone on an operating table and its posterior abdominal cavity was opened. The left kidney was separated from muscle tissues and the renal pedicle was clamped for the duration of the procedure (about four to five hours). Anesthesia pentobarbital (50 mg/kg) was injected into the abdominal cavity. The anesthetized rat was then turned over and its trachea was located and intubated. Heparin solution (0.1% heparin in saline, 0.15 ml/100 g body weight) was injected into the carotid artery through a small catheter, which was subsequently sealed. A sphygmomanometer was connected to the carotid artery. The femoral vein was located and separated from the surrounding tissue and inserted with a small catheter equipped with a syringe containing a composition of interest (0.15 ml/100 g body weight). The composition was then injected into the femoral vein within 10 seconds. Rats in Group A, B and C were injected with saline, the activated yeast composition and the control yeast composition, respectively. After the blood pressure readings stabilized, the clamp at the renal pedicle was released. The blood pressure readings were observed to gradually increase and the stabilized readings were recorded. The effects of the tested compositions on renal hypertension were summarized in Table 3.  
                                                                 TABLE 3                           Blood Pressure Readings of Male Wistar Rats                Blood Pressure (kPa)                    Before Administering a               Composition                Before       After   Monitoring           Release   After Release of   Administering   Duration       Group   of the Clamp   the Clamp   a Composition   (minutes)               A   15.2 ± 0.8   19.2 ± 1.7   19.8 ± 1.3   &gt;50       B   15.6 ± 0.9   19.6 ± 1.3   11.5 ± 1.4   &gt;50       C   15.4 ± 0.7   19.8 ± 1.2   18.7 ± 1.7   &gt;50                  
 
         [0053]     The results in Table 3 show that unlike the control yeast composition, the activated yeast composition was effective in treating renal hypertension, namely, lowering blood pressure.  
         [0054]     While a number of embodiments of this invention have been set forth, it is apparent that the basic constructions may be altered to provide other embodiments which utilize the compositions and methods of this invention.

Technology Category: 1