# Patent

## Title
Methods and compositions for treating vascular dementia

## Abstract

Compositions comprising a plurality of yeast cells, wherein said plurality of yeast cells have been cultured in the presence of an alternating electric field having a specific frequency and a specific field strength for a period of time sufficient to increase the capability of said plurality of yeast cells to treat vascular dementia in a mammal. Also included are methods of making such compositions and methods of treating vascular dementia.

## Background
FIELD OF THE INVENTION
The invention relates to compositions that treat vascular dementia and can be taken as dietary supplements or medication. The compositions comprise yeast cells obtainable by growth in electromagnetic fields with specific frequencies and field strengths.
BACKGROUND OF THE INVENTION
Vascular Dementia (VaD) is defined as the loss of cognitive function resulting from ischemic, ischemic-hypoxic, or hemorrhagic brain lesions as a result of cardiovascular diseases and cardiovascular pathologic changes. See, e.g., G. C. Roman, Med. Clin. North. Am., 86, pp. 477-99 (2002). VaD is a chronic disorder and the symptoms of VaD include cognitive loss, headaches, insomnia and memory loss. VaD may be caused by multiple strokes (MID or poststroke dementia) but also by single strategic strokes, multiple lacunes, and hypoperfusive lesions such as border zone infarcts and ischemic periventricular leukoencephalopathy (Binswanger's disease). See, G. C. Roman, supra. In Asian countries such as China, Japan and Korea, VaD is observed in over 60% of patients with dementia. Primary and secondary prevention of stroke and cardiovascular disease decreases the burden of VaD.
Treatment of VaD involves control of risk factors (i.e., hypertension, diabetes, smoking, hyperfibrinogenemia, hyperhomocystinemia, orthostatic hypotension, cardiac arrhythmias). See, G. C. Roman, supra. Researchers have also investigated whether hormone replacement therapy and estrogen replacement therapy could delay the onset of dementia in women. See, E. Hogervorst et al., Cochrane Database Syst. Rev., 3, CD003799 (2002). Although there has been evidence that aspirin is widely prescribed for VaD, there is very limited evidence that aspirin is effective in treating patients with VaD. See, P. S. Williams et al., Cochrane Database Syst. Rev., 2, CD001296 (2000). Nimodipine has been implicated as a drug demonstrating short-term benefits in VaD patients, but has not been justified as a long-term anti-dementia drug. See, J. M. Lopez-Arrieta and J. Birks, Cochrane Database Syst. Rev., 3, CD000147 (2002). Further, clinical efficacy data of piracetam does not support the use of this drug in the treatment of dementia or cognitive impairment. L. Flicker and G. Grimley Evans, Cochrane Database Syst. Rev., 2, CD001011 (2001). Thus, an agent that is effective in treating VaD is highly desired in the market.
SUMMMARY OF THE INVENTION
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 that assist in treating vascular dementia. The composition of this invention can be taken as dietary supplements in the form of health drinks or pills.
This invention embraces a composition comprising a plurality of yeast cells that have been cultured in the presence of an alternating electric field having a frequency in the range of about 10280 to 13000 MHz and a field strength in the range of about 200 to 500 mV/cm, as compared to yeast cells not having been so cultured. In one embodiment, the frequency of the culturing is in the range of about 10280 to 10400 MHz, 12320 to 12380 MHz or 12950 to 13000 MHz. In one embodiment, the field strength is in the range of about 200 to 400 mV/cm. The yeast cells are cultured in the alternating electric field for a period of time sufficient to increase the capability of said plurality of yeast cells to improve the memory of a mammal with vascular dementia, as compared to unactivated yeast cells. Preferably, the mammal is human. In one embodiment, the vascular dementia was induced by cerebral ischemia. In another embodiment, the vascular dementia was induced by blockage of the middle cerebral artery. 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 can be exposed to a series of electromagnetic fields. An exemplary period of time is about 80-140 hours.
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 12950-13000 MHz and a field strength in the range of about 240 to 460 mV/cm (e.g., 240-260, 320-350, 360-390 or 420-460 mV/cm). In one embodiment, the yeast cells are exposed to a series of electromagnetic fields. An exemplary period of time is about 80-190 hours.
Included in this invention are also methods of making the above compositions and methods of treating vascular dementia.
Yeast cells that can be included in this composition can all be derived from the China General Microbiological Culture Collection Center (“CGMCC”) (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, Schizosaccharomyces pombe, Saccharomyces sake, Saccharomyces uvarum, Saccharomyces rouxii, Saccharomyces carlsbergensis, Rhodotorula aurantiaca and Saccharomyces cerevisiae. For instance, the yeast cells can be of the strain Saccharomyces cerevisiae Hansen IFF11340. In another embodiment, the yeast cells are from the strains selected from the group consisting of Saccharomyces cerevisiae Hansen AS 2.501, AS2.502, AS2.503, AS2.504, AS2.535, AS2.558, AS2.560, AS2.561 and AS2.562. Other useful yeast species are illustrated in Table 1.
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.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.



BRIEF DESCRIPTION OF THE DRAWINGS
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.
FIG. 2 is a schematic diagram showing an exemplary apparatus for making yeast compositions of the invention. The apparatus comprises a signal generator (such as models 83721B and 83741A manufactured by HP) and interconnected containers A, B and C.



DETAILED DESCRIPTION OF THE INVENTION
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 produce agents useful in improving the memory of a mammal with vascular dementia. Yeast compositions containing activated yeast cells can be used as medication, or as dietary supplements in the form of, e.g., health drinks or dietary pills.
Since the activated yeast cells contained in these yeast compositions have been cultured to endure acidic conditions of pH 2.5-4.2, the compositions are stable in the stomach and can pass on to the intestines. Once in the intestines, the yeast cells are ruptured by various digestive enzymes, and the agents useful in improving memory are released and readily absorbed.
I. Yeast Strains Useful in the Invention
The types of yeasts useful in this invention include, but are not limited to, yeasts of the genera Saccharomyces, Schizosaccharomyces and Rhodotorula. 
Exemplary species within the above-listed genera include, but are not limited to, the species illustrated in Table 1. Yeast strains useful in 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 the accession numbers of CGMCC) are illustrated in Table 1. In general, yeast strains preferred in this invention are those used for fermentation in the food and wine industries. As a result, compositions containing these yeast cells are safe for human consumption. 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.







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
AS2.1146







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

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.
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.
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.
The frequencies of EMFs useful in this invention range from about 10280 to 13000 MHz (e.g., 10280 to 10400, 12320 to 12380 and 12950 to 13000 MHz). Exemplary frequencies include 10300, 10312, 12348, 12963 and 12987 MHz. The field strength of the electric field useful in this invention ranges from about 200 to 500 mV/cm (e.g., 240-260, 270-290 and 330-480 mV/cm). Exemplary field strengths include 256, 282, 332, 337, 343, 356, 367, 372, 382, 416, 435 and 461 mV/cm.
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, 10, 11, 12, 13 or more EMFs in a series. In one embodiment, the yeast culture is exposed to a series of EMFs, wherein the frequency of the electric field is alternated in the range of about 10280 to 10400, 12320 to 12380 and 12950 to 13000 MHz.
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 compositions comprising activated yeast cells be allowed to multiply and grow in the presence of the EMF(s) for a total of 80-140, 90-136 and 80-190 hours.
FIG. 1 illustrates an exemplary apparatus for generating alternating electric fields. An electric field of a desired frequency and intensity can be generated by an AC source (3) capable of generating an alternating electric field, preferably in a sinusoidal wave form, in the frequency range of 5 to 20,000 MHz. Signal generators capable of generating signals with a narrower frequency range can also be used. If desired, a signal amplifier can also be used to increase the output. The culture container (2) can be made from a non-conductive material, e.g., glass, plastic or ceramic. The cable connecting the culture container (2) and the signal generator (3) is preferably a high frequency coaxial cable with a transmission frequency of at least 20 GHz. In one embodiment, the transmission frequency is 30 GHz.
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 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 mm be used. For a culture having a volume of 10-100 L, metal wires/tubes having a diameter of 3 to 5 mm can be used. For a culture having a volume of 100-1000 L, metal wires/tubes having a diameter of 6 to 15 mm can be used. For a culture having a volume greater than 1000 L, metal wires/tubes having a diameter of 20-25 mm can be used.
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.
III. Culture Media
Culture media useful in this invention contain sources of nutrients that can be assimilated by yeast cells. Complex carbon-containing substances in a suitable form (e.g., carbohydrates such as sucrose, glucose, dextrose, maltose, starch, xylose; mannitol) can be the carbon sources for yeast cells. The exact quantity of the carbon sources can be adjusted in accordance with the other ingredients of the medium. In general, the amount of carbon-containing substances varies between about 0.5% and 10% by weight of the medium, and preferably between about 1% and 5%, and most preferably between about 1.0-2.5%. These carbon sources can be used individually or in combination. Vitamins can also be added to the medium, for example, Vitamin D, Vitamin B22, Vitamin E or Vitamin B6. Among the inorganic salts which can be added to a 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 (NH4)2HPO4, CaCO3, KH2PO4, K2HPO4, MgSO4, NaCl, and CaSO4.
IV. Electromagnetic Activation of Yeast Cells
To activate or enhance the innate ability of yeast cells to improve memory, these cells can be cultured in an appropriate medium under sterile conditions at 20° C.-35° C. (e.g., 28-32° C.) for a sufficient amount of time, e.g., 80-140, 90-136, 80-190 hours, in an alternating electric field or a series of alternating electric fields as described above.
An exemplary set-up of the culture process is depicted in FIG. 1 (see above). An exemplary culture medium contains the following in per 1000 ml of sterile water: 6 g of sucrose, 12 g of mannitol, 70 μg of Vitamin D, 50 μg of Vitamin B12, 40 μg of Vitamin E, 90 μg of Vitamin B6, 50 ml of bovine serum, 0.20 g of KH2PO4, 0.25 g of MgSO4.7H2O, 0.3 g of NaCl, 0.2 g of CaSO4.2H2O, 4.0 g of CaCO3.5H2O, 2.5 g of peptone. Yeast cells of the desired strains are then added to the culture medium to form a mixture containing 1×108 yeast cells per 1000 ml of culture medium. The yeast cells can be of any of the strains listed in Table 1. In one embodiment, the strain is Saccharomyces cerevisiae Hansen IFFI1340. The mixture is then added to the apparatus of FIG. 1.
The activation process of the yeast cells involves the following steps: 1) maintaining the temperature of the activation apparatus at 20-35° C., (e.g., 28-32° C.), culturing the yeast cells for 28 hours; 2) applying an electric field having a frequency of about 10300 MHz and a field strength of 240-260 mV/cm (e.g., about 256 mV/cm) for 16 hours; 3) then applying an electric field having a frequency of about 10312 MHz and a field strength of 330-360 mV/cm (e.g., about 343 mV/cm) for 36 hours; 4) then applying an electric field having a frequency of about 12348 MHz and a field strength of 350-380 mV/cm (e.g., about 367 mV/cm) for 32 hours; 5) then applying an electric field having a frequency of about 12963 MHz and a field strength of 370-400 mV/cm (e.g., about 382 mV/cm) for 36 hours; 6) then applying an electric field having a frequency of about 12987 MHz and a field strength of 330-360 mV/cm (e.g., about 337 mV/cm) for 16 hours; and 7) finally lyophilizing the compositions comprising activated yeast cells to form a powder and storing the powder at 4° C. Preferably, the concentration of the lyophilized yeast cells is more than 1010 cells/g.
V. Acclimatization of Yeast Cells to the Gastric Environment
Because the yeast compositions 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 yeast cells be cultured under acidic conditions to acclimatize the cells to the gastric juice. This acclimatization process results in better viability of the yeasts in the acidic gastric environment.
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 1010 activated cells per gram) per 1000 ml. The yeast mixture is then cultured first in the presence of an alternating electric field having a frequency of about 12963 MHz and a field strength of 390-430 mV/cm (e.g., about 416 mV/cm) at about 28 to 32° C. for 28-36 hours (e.g., about 32 hours). The resultant yeast cells are further incubated in the presence of an alternating electric field having a frequency of about 12987 MHz and a field strength of 340-370 mV/cm (e.g., about 356 mV/cm) at about 28 to 32° C. for 16-28 hours (e.g., about 20 hours). The resulting acclimatized yeast cells are then dried and stored either in powder form (≧1010 cells/g) at room temperature or in vacuum at 0-4° C.
An exemplary acclimatizing culture medium is made by mixing 700 ml of fresh pig gastric juice and 300 ml of wild Chinese hawthorn extract. The pH of the acclimatizing culture medium is adjusted to 2.5 with 0.1 M hydrochloric acid and 0.2 M potassium biphthalate (C6H4(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. To prepare the wild Chinese hawthorn extract, 500 g of fresh wild Chinese hawthorn is dried under sterile conditions to reduce the water content (≦8%). The dried fruit is then ground (≧20 mesh) and added to 1500 ml of sterile water. The mixture is allowed to stand for 6 hours at 4° C. under sterile conditions. The supernatant is collected to be used in the acclimatizing culture medium.
VI. Manufacture of Yeast Compositions
To prepare the yeast compositions of the invention, an apparatus depicted in FIG. 2 or an equivalent thereof can be used. This apparatus includes a first container (A), a second container (B), and a third container (C), 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.
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 fruit extracts from Schisandra chinensis Baill (wu wei zi), and 100 L of soy bean extracts. To prepare hawthorn, jujube and wu wei zi 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, the solution is sterilized at 121° C. for 30 minutes, and cooled to 40° C. before use.
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 (A) shown in FIG. 2. The yeast cells are then cultured in the presence of an alternating electric field having a frequency of about 12963 MHz and a field strength of about 420-460 mV/cm (e.g., about 435 mV/cm) at 28-32° C. under sterile conditions for 32 hours. The yeast cells are further incubated in an alternating electric field having a frequency of about 12987 MHz and a field strength of 270-290 mV/cm (e.g., about 282 mV/cm). The culturing continues for another 12 hours.
The yeast culture is then transferred from the first container (A) to the second container (B) (if need be, a new batch of yeast culture can be started in the now available first container (A)), and subjected to an alternating electric field having a frequency of about 12963 MHz and a field strength of 400-420 mV/cm (e.g., about 416 mV/cm) for 24 hours. Subsequently the frequency and field strength of the electric field are changed to about 12987 MHz and 320-350 mV/cm (e.g., about 332 mV/cm), respectively. The culturing continues for another 12 hours.
The yeast culture is then transferred from the second container (B) to the third container (C), and subjected to an alternating electric field having a frequency of about 12963 MHz and a field strength of 360-390 mV/cm (e.g., about 372 mV/cm) for 24 hours. Subsequently the frequency and field strength of the electric field are changed to about 12987 MHz and 240-260 mV/cm (e.g., about 256 mV/cm), respectively. The culturing continues for another 12 hours.
The yeast culture from the third container (C) can then be packaged into vacuum sealed bottles of 30-50 ml or 100 ml for use as a dietary supplement, e.g., health drinks, or medication in the form of pills, powder, etc. The dietary supplement can be taken 3-4 times daily at 30-50 ml each time for a period of three months (10-30 minutes before meals and at bedtime). If desired, the final yeast culture can also be dried within 24 hours and stored in powder form.
In one embodiment, the compositions of the invention can also be administered intravenously or peritoneally in the form of a sterile injectable preparation. Such a sterile preparation is prepared as follows. A sterilized health drink composition is first treated under ultrasound (>=18,000 Hz) for 10 minutes and then centrifuged at 4355 rpm 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.
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.
In other embodiments, the compositions of the invention may also be formulated with pharmaceutically acceptable carriers to be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, suspensions or solutions.
EXAMPLES
The following examples are meant to illustrate the methods and materials of the present invention. Suitable modifications and adaptations of the described conditions and parameters which are obvious to those skilled in the art are within the spirit and scope of the present invention.
The activated yeast compositions used in the following experiments were prepared as described above, using Saccharomyces cerevisiae Hansen IFF11340 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. Control yeast compositions were those prepared in the same manner except that the yeast cells were cultured in the absence of EMFs. Unless otherwise indicated, all yeast compositions and the corresponding controls were administered to the animals by intragastric feeding.
Example 1
Effects on Rats with VaD Induced by Cerebral Ischemia
A large number of clinical studies have shown that blockage in the artery or vein can reduce the blood flow in the brain, thereby inducing cerebral ischemia. When under ischemic conditions for only a few minutes, brain cells can be severely damaged, leading to stroke or dementia and even death. Blockage in the arteria carotis is a major cause in cerebral ischemia. A goal of the treatment is to help the damaged brain cells gradually recover.
In this experiment, VaD is induced by ligation of the common artery on both sides of the neck for 4-12 minutes (the duration depends on the blood flow), which results in memory loss in rats. The change in memory of the rats after administering the activated yeast composition is monitored. The rat VaD model closely resembles human VaD.
Male Wistar rats that were 4-6 months old, weighing 180-200 g were provided by the Chinese Academy of Medical Sciences. Anesthesia of 100 healthy rats was performed by administering abdominally 35 mg/kg (body weight) of chloral hydrate. Then, the necks of the rats were cleaved in the center. Twelve rats were selected for the positive control group (CK1 group), of which the cleaved skin was sealed, and 2×104 unit/kg (body weight) of penicillin was injected into the buttocks of the rats to prevent infection. For the rest of the rats, the common artery on each side was separated, and clamped to control 50% of the blood flow. After 10 minutes, the clamp was removed and the blood flow in the artery recovered to normal conditions. Then, the cleaved skin of the rats was sealed, and 2×104 unit/kg (body weight) of penicillin was injected into the buttocks of the rats to prevent infection.
The rats were fed for two days, and the memory of the rats were monitored by using the water maze method. Rats that exceeded 85 seconds in completing the maze were selected. The selected rats were divided into three groups of 12 each, the test group (AY), the control yeast group (NY) and the saline control group (CK2).
Each rat in groups AY, NY, CK2 and CK1 was administered twice daily 1 ml of the activated yeast composition, the control yeast composition and saline (for both CK2 and CK1), respectively for 21 days. The rats were monitored on Day 7, 14 and 21 for the time required to complete the maze. The results are shown in Table 2.











TABLE 2






Time for






completion of
Time for
Time for
Time for



maze before
completion of
completion of
completion of



treatment
maze on Day
maze on Day
maze on Day



(seconds)
7 (seconds)
14 (seconds)
21 (seconds)


Group
(x ± SD)
(x ± SD)
(x ± SD)
(x ± SD)







AY
109.3 ± 11.7
82.5 ± 9.2
61.7 ± 7.4
52.4 ± 6.6


NY
107.4 ± 11.2
104.8 ± 10.3
103.2 ± 10.8
103.7 ± 11.4


CK2
108.7 ± 11.5
106.6 ± 9.7 
104.3 ± 9.2 
105.5 ± 11.2


CK1
58.8 ± 7.2
51.6 ± 6.8
50.6 ± 6.2
50.7 ± 6.4









The above experiment shows that compared to the groups treated with unactivated yeast composition (NY) or saline (CK2), the group treated with activated yeast composition (AY) demonstrates significant recovery of memory after 7 days, 14 days and 21 days of treatment. On Day 21, the memory of the rats is comparable to those of the rats in the positive control group (CK1).
Example 2
Effects on VaD Induced by Blockage of the Middle Cerebral Artery
In this experiment, paraffin oil is injected into the middle cerebral artery of rats. The paraffin oil mimicks the microparticles of thrombus and induces blockage of the blood vessel, leading to VaD in the rats. The VaD observed in the rat model is similar to that observed in humans. Through treatment, damaged brain cells recover, and the memory is improved. In this experiment, the Morris maze method was used to record the change in memory after treatment.
Male Sprague-Dawley rats that were 5-7 months old, weighing 220-250 g were provided by the Chinese Medical Science Academy. Anesthesia of 80 healthy rats was performed by administering abdominally 35 mg/kg (body weight) of soluble phenobarbital. Then, the necks of the rats were cleaved in the center. Fifteen rats were selected for the positive control group (CK1). The middle cerebral artery of these rats were slowly injected with 20 μl/kg (body weight) of saline for 10-15 minutes. For the rest of the rats, paraffin oil was slowly injected into the middle cerebral artery for 10-15 minutes at 20 μl/kg (body weight). The paraffin oil was sterilized at 121° C. and cooled to 35 to 38° C. before use. Then, the cleaved skin of the rats was sealed, and 2×104 unit/kg (body weight) of penicillin was injected into the buttocks of the rats to prevent infection.
The rats were fed for ten days, and the memory of the rats were monitored by the Morris maze method on Day 11. Rats that exceeded 100 seconds in locating the safety zone were selected. The selected rats were divided into three groups of 15 each, the test group (AY), the control yeast group (NY) and the saline control group (CK2).
Each rat in groups AY, NY, CK2 and CK1 was administered twice daily 1 ml of the activated yeast composition, the control yeast composition and saline (for both CK2 and CK1), respectively for 21 days. The rats were monitored on Day 7, 14 and 21 for the time required to locate the safety zone. The results are shown in Table 3.











TABLE 3






Time for
Time for
Time for
Time for



locating the
locating the
locating the
locating the



safety zone
safety zone on
safety zone on
safety zone on



before treatment
Day 7
Day 14
Day 21



(seconds)
(seconds)
(seconds)
(seconds)


Group
(x ± SD)
(x ± SD)
(x ± SD)
(x ± SD)







AY
146.4 ± 17.8
63.6 ± 9.7
32.6 ± 6.2
18.4 ± 3.6


NY
144.7 ± 18.2
138.2 ± 17.9
127.4 ± 16.4
129.3 ± 17.3


CK2
141.8 ± 16.8
133.4 ± 18.2
121.3 ± 19.7
130.2 ± 16.5


CK1
58.8 ± 7.2
51.6 ± 6.8
50.6 ± 6.2
50.7 ± 6.4









The above experiment shows that compared to the groups treated with unactivated yeast composition (NY) or saline (CK2), the group treated with activated yeast composition (AY) demonstrates significant recovery of memory after 7 days, 14 days and 21 days of treatment. The group treated with unactivated yeast composition does not demonstrate any effect on the rats compared to the saline control group. On Day 21, the memory of the rats is even better than that of the rats in the positive control group (CK1). Thus, the activated yeast composition helps rats with vascular dementia recover their memory.
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.

## Claims

1. A composition comprising a plurality of yeast cells, wherein said plurality of yeast cells are characterized by an increase in their capability to improve the memory of a mammal with vascular dementia as a result of having been cultured in the presence of an alternating electric field having a frequency in the range of about 10280 to 13000 MHz and a field strength in the range of about 200 to 500 mV/cm as compared to yeast cells not having been so cultured.

2. The composition of claim 1, wherein the range of the frequency is about 10280 to 10400, 12320 to 12380 or 12950 to 13000 MHz.

3. The composition of claim 1, wherein the range of the field strength is about 200 to 400 mV/cm.

4. The composition of claim 1, wherein said yeast cells are of the species selected from the group consisting of Saccharomyces sp, Schizosaccharomyces pombe, Saccharomyces sake, Saccharomyces uvarum, Saccharomyces rouxii, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Rhodotorula aurantiaca.

5. The composition of claim 1, wherein said yeast cells are derived from the strain deposited at the China General Microbiological Culture Collection Center with the accession number selected from the group consisting of Saccharomyces cerevisiae Hansen AS 2.501, AS2.502, AS2.503, AS2.504, AS2.535, AS2.558, AS2.560, AS2.561, AS2.562 and IFFI1340.

6. The composition of claim 5, wherein said strain is Saccharomyces cerevisiae Hansen IFFI1340.

7. The composition of claim 1, wherein the composition is in the form of a tablet, powder or health drink.

8. The composition of claim 1, wherein the composition is in the form of a health drink.

9. A method of preparing a yeast composition, comprising culturing a plurality of yeast cells in the presence of an alternating electric field having a frequency in the range of about 10280 to 13000 MHz and a field strength in the range of about 200 to 500 mV/cm to increase the capability of said plurality of yeast cells to improve the memory of a mammal with vascular dementia as compared to yeast cells not having been so cultured.

10. The method of claim 9, wherein the range of the frequency is about 10280 to 10400, 12320 to 12380 or 12950 to 13000 MHz.

11. The method of claim 9, wherein the range of the field strength is about 200 to 400 mV/cm.

12. A method of improving the memory of a subject with vascular dementia, comprising the step of administering to said subject the composition of any one of claims 1 to 6.

13. The method of claim 12, wherein the administration is through oral administration.