Germination activated Ganoderma lucidum spores and method...

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Reexamination Certificate

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Reexamination Certificate

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ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to bioactive substances which are extracted from germination activated spores of
Ganoderma lucidum Leyss ex Fr. Karst.
It further relates to methods for culturing and producing such substances. Finally, it relates to the use and methods of using the bioactive substances produced by germination activated ganoderma spores for treating patients or mammals with immunological disorders, cancer, AIDS, hepatitis, diabetes, and cardiovascular diseases. The bioactive substances can also be used to prevent or inhibit free radical oxidation and hepatotoxic effect.
BACKGROUND OF THE INVENTION
Ganoderma (
Ganoderma lucidum Leyss ex Fr. Karst
) is a polyporous fungus. It belongs to the class Basidiomycetes, the family Polypolaceae, and the genus Ganoderma. Since ancient times, ganoderma has been praised as a miracle fungus for its capability of prolonging human life. It is believed that the medicinal effects of ganoderma lie upon the natural or bioactive substances it produces which can stimulate or modulate the neuro-endocrino-immuno system of human body to fight off diseases. Ganoderma is also well known for its antitumor and immune enhancing properties, (Kim et al.,
Int. J. Mol. Med.
(1999), 4(3):273-277), cardiovascular effects (Lee et al.,
Chem. Pharm. Bull.
(1990), 38:1359-1364), as well as free radical scavenging and antihepatotoxic activities (Lin et al.,
J. Ethnopharmacol.,
(1995), 47(1):33-41). Two substances extracted from ganoderma have been reported to especially relate to the medicinal effects of ganoderma. They are triterpene and polysaccharide, although so far no clinical evidence has been provided which supports the claims of medicinal effects of these substances. (Mekkawy et al.,
Phytochemistry,
(1998), 49(6):1651-1657; Wasser et al.,
Crit. Rev. Immunol.,
(1999), 19(1):65-96).
Ganoderma is the most rare and valuable herb in Chinese medicine. It is known in China for over 5,000 years as “ling zhi”. There are a variety of ganoderma, for instance,
G.. lucidum
(red),
G.. applanatum
(brown),
G.. tsugae
(red),
G.. sinense
(black), and
G.. oregonense
(dark brown). However, due to the fact that wild types of ganoderma only grow naturally and very rarely on aged trees in steep mountains, research which requires a constant supply of high quantity and quality of ganoderma has rarely been conducted.
Not until recently, after the development of artificial cultivation techniques, methods for artificially cultivating
Ganoderma lucidum
(Fr.) Karst have been developed. (see e.g., U.S. Pat. No. 4,472,907). The newly developed cultivation methods allow researchers to produce sufficient amount of ganoderma for the studies.
Although it is believed that the spores of ganoderma represent the essence of ganoderma because they contain all the bioactive substances of ganoderma, most of the ganoderma studies are conducted using the fruit body or mycelium of ganoderma as experimental materials. Ganoderma spores are rarely studied.
Ganoderma spores are tiny and mist-like spores of 5~8 &mgr;m in sizes which have extremely hard and resilient, double-layer epispores, thus making them difficult to break open. The ganoderma spores normally scatter at the pelius of mature ganoderma. When mature, the ganoderma spores are ejected from the pileus. Such ejected ganoderma spores are collectively called “spore powders”. In the wild, the “spore powders” are difficult to collect because of the following reasons: (1) the germination rate (i.e., about 3-15%) of the spores is extremely low; (2) the ejection period is relatively short (i.e., approximately 10 days per lifecycle); and (3) some environmental factors, such as wind and rain, may also hinder the collection of the spores. In addition, the substances of the collected spores are difficult to extract due to the resiliency of the epispores.
In recent years, there have been reports which disclose methods for breaking the cell walls of the
Ganoderma lucidum
spores. For example, JP52041208 discloses the extraction of effective components of spores of “shiitake” by efficient mechanical breaking of the cell membranes. JP2240026A discloses the use of solvent to break open the ganoderma spores. CN1134306 discloses a method for producing sporoderm-broken
Ganoderma lucidum
sporopollen which combines water soaking, air-drying and microwave heating treatment. CN1165032 discloses a method for breaking the skin of spore powder of
Ganoderma lucidum
with a skin-dissolving enzyme such as lysozyme, snail enzyme, cellulase and hemicellulase, followed by freezing and melting in enzymolized liquid, and ultrasound.
With the improvement of the spore breaking techniques, more research which directed to the studies of the ganoderma spores has been undertaken. However, the improvement of the spore breaking techniques does not overcome the shortcoming of the low germination rate of the spores. In fact, due to the low germination rate, most of the studies on ganoderma spores are conducted using the extraction of bioactive substances from spores representing an array of dormant to various germination stages. Because the spores at different stages of the lifecycle produce different kinds and/or proportions of bioactive substances, each batch of the mixture of the spores thus contains different active ingredients. The results from such studies are apparently meaningless since no proper controls can be provided.
In the invention to be present below, a germination activation method will be introduced. This method can successfully activate the dormant ganoderma spores and increase the germination rate of the ganoderma spores to more than 95%. The present invention also provides a unique spore breaking method which not only allows for the high recovery of the bioactive substances in the spores, but also successfully preserves the functional activities of the bioactive substances. Finally, the present invention provides clinical studies which demonstrate the significance of the bioactive substances extracted from the germination activated ganoderma spores in treating patients or mammals with immunological disorders, cancer, AIDS, hepatitis, diabetes, and cardiovascular diseases. The bioactive substances can also be used to prevent or inhibit free radical oxidation and hepatotoxic effect.
SUMMARY OF THE INVENTION
The present invention provides a method which can significantly increase the germination rate of the spores of
Ganoderma lucidum.
When the dormant spores of
Ganoderma lucidum
are activated by germination, they produce bioactive substances which contain, but are not limited to, active genes and promoters, active enzymes, sterols, cytokines, interferons, lactone A, ganoderma acid A, triterpenes, polysaccharides, vitamins, superoxide dismutases (SOD), vitamin E, glycoproteins, growth factors, etc. These bioactive substances, in their entirety, demonstrate superb medicinal effects.
This method requires soaking the ganoderma spores in a solution to induce germination. The solution used for this purpose includes clear or distilled water, saline solution, and any nutritional solution. Examples of the nutritional solution include coconut juice, 1-5% malt extract solution, 0.5-25% extracts of the ganoderma sporocarps or capillitia, 0.1-5% of culture solution containing biotin, 0.1-3% of culture solution containing monobasic potassium phosphate and magnesium sulfate. One or more of the above listed nutritional solution can be used, with the amount added being 0.1-5 times of the weight of the ganoderma spores. The spores are soaked in the solution for 30 minutes to 8 hours at 20-43° C., preferably for 2 to 4 hours and between 25 and 35° C.
The germination induced ganoderma spores are placed in a well-ventilated culture box which is controlled with constant temperature and constant humidity to activate the synthesis of bioactive substances. The relative humility is at 65-98%, preferably 85-97%, and the temperature is at 18-48° C., preferably at 25-35° C.
The present invention also provides a method for producing sp

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