Patent Application
20050106705
The invention relates to compositions that can ameliorate or prevent hyperlipemia and are useful as dietary supplements (e.g., health drinks) or medication. These compositions contain yeast cells obtainable by growth in electromagnetic fields with specific frequencies and field strengths.
Hyperlipemia is a state of higher than normal blood concentration of lipid components, such as cholesterols, neutral fats, phospholipids or free fatty acids that are in the form of water-soluble lipoproteins. Hyperlipemia is caused by abnormal lipoprotein metabolism. A prolonged hyerlipmic status has been linked to diseases of the circulatory system such as arteriosclerosis, myocardial infarction, angina pectoris, cerebral infarction, apoplexy, coronary diseases, cerebrovascular disorders, high blood pressure, and obesity.
Conventional therapeutic agents for the treatment of hyperlipemia include clofibrate, clinofibrate, phenofibrate, bezafibrate and the like, probucol and nicotinic acid. However, the clofibrate-type drugs are accompanied by adverse side effects such as formation of gallstone, muscular disorders, hepatic dysfunctions and gastrointestinal disorders. Moreover, these drugs must be administered in a large quantity to obtain a certain clinical effect. As a result, severe side effects are frequently observed. There remains a need for an effective agent for treating hyperlipemia.
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 hyperlipidemia (for example, regulating triglyceride and/or cholesterol levels). Compositions comprising these activated yeast cells can therefore be used as medication or 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 lipid concentration in a hyperlipmic human, or to prevent or postpone the onset of hyperlipemia in a high risk individual (e.g., someone predisposed to hyperlipemia because of his genetic background or life style).
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 7000 to 13000 MHz (e.g., 7500-8000, 10000-10500, and/or 12400-12800 MHz) and a field strength in the range of about 200 to 450 mV/cm (e.g., 220-240, 270-290, 300-330, 310-340, 320-350, 340-370, 350-380, 360-390, 370-400, 390-430, and/or 420-450 mV/cm). The yeast cells are cultured for a period of time sufficient to activate said plurality of yeast cells to treat hyperlipemia 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 80-150 hours (e.g., 100-130 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 12000 to 13000 MHz (e.g., 12400-12800 MHz) and a field strength in the range of about 200 to 450 mV/cm (e.g., 310-340 and/or 350-380 mV/cm). In one embodiment, the yeast cells are exposed to a series of electromagnetic fields. An exemplary period of time is about 40-80 hours (e.g., 50-66 hours).
Yeast cells that can be included in this composition can be derived from parent strains publically available 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, those commonly used in food and pharmaceutical industries, such as Saccharomyces cerevisiae (e.g., Hansen and Hansen Var. ellipsoideus), Saccharomyces carlsbergensis, Saccharomyces rouxii, Saccharomyces sake, Saccharomyces uvarum, Saccharomyces sp., Schizosaccharomyces pombe, Rhodotorula glutinis and Rhodotorula aurantiaca. For instance, the yeast cells can be of the strain Saccharomyces cerevisiae Hansen AS2.560 or ACCC2038, Saccharomyces sp. AS2.3 11, Schizosaccharomyces pombe Lindner AS2.259, Saccharomyces sake Yabe ACCC2045, Saccharomyces uvarum Beijer IFFI1036, Saccharomyces rouxii Boutroux AS2.371, Saccharomyces cerevisiae Hansen Var. ellipsoideus AS2.559, Saccharomyces carlsbergensis Hansen AS2.440, or Rhodotorula glutinis (Fresenius) Harrison AS2.704. Other useful yeast strains are illustrated in Table 1.
This invention further embraces a composition comprising a plurality of yeast cells, wherein said plurality of yeast cells have been activated to treat hyperlipemia in a subject. Included in this invention are also methods of making the above compositions.
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.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
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 triglyceride and cholesterol levels in a subject. Compositions containing these activated yeast cells are useful in the treatment of hyperlipemia. Yeast compositions containing activated yeast cells can be used as medication or as dietary supplements, in the form of health drinks or dietary pills (tablets or powder).
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-hyperlipemic substances are released and readily absorbed.
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, 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 cerevisiae Hansen AS2.560 and ACCC2038, Saccharomyces sp. AS2.311, Schizosaccharomyces pombe Lindner AS2.259, Saccharomyces sake Yabe ACCC2045, Saccharomyces uvarum Beijer IFF11036, Saccharomyces rouxii Boutroux AS2.371, Saccharomyces cerevisiae Hansen Var. ellipsoideus AS2.559, Saccharomyces carlsbergensis Hansen AS2.440, and Rhodotorula glutinis (Fresenius) Harrison AS2.704. Other useful yeast strains are illustrated in Table 1.
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 hyperlipemia can be readily tested by methods known in the art. See, for instance, Examples 1 and 2.
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.,
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 7000-13000 MHz (e.g., 7500-8000, 10000-10500, and/or 12400-12800 MHz). Exemplary frequencies are 7858, 7873, 10072, 12623, and 12642 MHz. The field strength of the electric field useful in this invention ranges from about 200 to 450 mV/cm (e.g., 220-240, 270-290, 300-330, 310-340, 320-350, 340-370, 350-380, 360-390, 370-400, 390-430, and/or 420-450 mV/cm). Exemplary field strengths are 228, 276, 284, 305, 317, 320, 332, 346, 363, 374, 384, and 407 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 7500-8000, 10000-10500, and 12400-12800 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 activated yeast cells be allowed to multiply and grow in the presence of the EMF(s) for a total of 80-150 hours (e.g., 100-130 hours).
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.
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.
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 (NH4)2HPO4, KH2PO4, K2HPO4, CaCO3, MgSO4, NaCl, and CaSO4.
To activate or enhance the ability of yeast cells to produce substances beneficial for the treatment of hyperlipemia (e.g., lowering of triglyceride and/or cholesterol levels), 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., 100-200 hours (e.g., 147-171 hours), in an alternating electric field or a series of alternating electric fields as described above.
An exemplary culture medium is made by mixing 950 ml of distilled water with 20 g of sucrose, 20 μg of vitamin B12, 40 μg of vitamin B6, 100 μg of vitamin E, 50 ml of fetal 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, and 2.5 g of peptone.
An exemplary set-up of the culturing process is depicted in
Subsequently, the activated yeast cells can be evaluated for their ability to treat hyperlipemia using standard methods known in the art, such as those described in Section VII.
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.
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 can then be cultured first in the presence of an alternating electric field having a frequency of 12623 MHz and a field strength in the range of 350-380 mV/cm (e.g., 363 mV/cm) at about 28 to 32° C. for 34-42 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 12642 MHz and a field strength in the range of 310-340 mV/cm (e.g., 320 mV/cm) at about 28 to 32° C. for 16-24 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 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 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 hydrogen phthalate (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. 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.
To prepare the yeast compositions of the invention, an apparatus depicted in
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.
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
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 the first container (A)), and subjected to an alternating electric field having a frequency of 12623 MHz and a field strength of 390-430 mV/cm (e.g., 407 mV/cm) for 24 hours. Subsequently the frequency and field strength of the electric field are changed to 12642 MHz and 360-390 mV/cm (e.g., 374 mV/cm), respectively. The culturing process 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 12623 MHz and a field strength of 270-290 mV/cm (e.g., 276 mV/cm) for 24 hours. Subsequently the frequency and field strength of the electric field are changed to 12642 MHz and 220-240 mV/cm (e.g., 228 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 for use as medicament or dietary supplement, e.g., in the form of health drinks, pills, or powder, etc. 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.
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 (≧18,000 Hz) for 10 minutes and then centrifuged 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. 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.
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 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.
The activated yeast compositions used in the following examples were prepared as described above, using Saccharomyces cerevisiae Hansen AS2.560 cells, 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. All compositions of interest were administered to the animals by intragastric feeding, unless otherwise specified.
To test the ability of the activated yeast compositions to control hyperlipemia, thirty healthy domesticated rabbits (Oryctolagus curiculus, 3.8-4.2 kg, half of them male and half of them female, 18-24 months old) were given regular rabbit feed for four weeks and then were randomly divided into three groups, Group A, B and C. A mixture of 0.2 mg/kg cholesterol+2 ml lard was administered to each rabbit once daily and a composition of interest at a dosage of 1.5 ml/kg was administered twice a day for eight consecutive weeks. Rabbits in Groups A, B and C were given the activated yeast composition, the control yeast composition and saline, respectively, in addition to cholesterol and lard. All rabbits were otherwise maintained the same way.
Prior to the administration of the mixture and the composition of interest, as well as at 2, 4, 6 and 8 weeks after the administration, blood samples were taken from the marginal vein of an ear of each rabbit and centrifuged at 3000 rpm to recover sera. The amount of serum cholesterol was measured by the method described in Table 2 below.
A standard curve of cholesterol was established and the concentration of cholesterol in the samples was determined according to the standard curve. The results were shown in Table 3.
The above results show that the activated yeast composition was more effective in lowering the cholesterol levels than the control yeast composition.
To test the ability of the activated yeast compositions to control hyperlipemia, forty healthy Wistar rats (120-140 g, half of them male and half of them female, 2-4 months old) were randomly divided into four groups, Groups A, B, C and D. Groups A, B and C rats were given high lipid content rat feed and Groups D rats were given regular rat feed for three weeks. A composition of interest at a dosage of 1.5 ml/100 g was then administered to each rat in Groups A, B and C once daily in conjunction with the high lipid content rat feed for another three consecutive weeks. Rats in Group A, B and C were given the activated yeast composition, the control yeast composition and saline, respectively, in addition to the high lipid content rat feed. Rats in Group D were given 1.5 ml/100 g saline and regular rat feed instead. The high lipid content rat feed was prepared by mixing 1% cholesterol, 0.5% bile salt, 0.2% methylthiouracil, 2% lard, 5% soybean, 1% egg, 2% ground dry yolk, 1% fish meal, and 87.3% regular rat feed with water to form a paste.
Twenty-four hours after the last administration of the composition, the rats were given no feed but water for another twelve hours. Blood samples were then taken from the carotid and jugular vein of the rat and centrifuged at 3000 rpm to recover sera. The amount of serum cholesterol was measured by the same method as described in Example 1. The amount of serum triglycerides was measured by the method described in Table 4 below.
A standard curve of triglyceride was established and the concentration of triglyceride in the samples was determined according to the standard curve. The results were shown in Table 5.
The above results show that the activated yeast composition was more effective in lowering the triglyceride and cholesterol levels than the control yeast composition.
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.
