Drug – bio-affecting and body treating compositions – Extract or material containing or obtained from a...
Reexamination Certificate
2001-03-19
2002-08-27
Prats, Francisco (Department: 1651)
Drug, bio-affecting and body treating compositions
Extract or material containing or obtained from a...
C435S254100
Reexamination Certificate
active
06440420
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for extracting oleaginous substances from
Ganoderma lucidum
spores, which are germination-activated and epispore-broken. The extraction method involves the use of supercritical fluid carbon dioxide (“SCF—CO
2
”).
BACKGROUND OF THE INVENTION
Ganoderma (
Ganoderma lucidum
Leyss ex Fr. Karst) is a polyporous fungus. It belongs to the class of Basidiomycetes, the family of Polypolaceae, and the genus of Ganoderma. In Chinese folklore, Ganoderma has been regarded as a panacea, which is probably due to certain efficacy of Ganoderma in treating many diseases. Some of the known medicinal or therapeutic effects of Ganoderma include treating patients with chronic bronchitis, chronic viral hepatitis, coronary heart disease, granulocytopenia, chronic Keshan disease, neurasthenia, progressive muscular dystrophy, atrophic myotonia and certain neurological diseases (See e.g., Liu et al.,
Chinese Medical Journal
, 92:496-500 (1979)). There are also reports on Ganoderma as anti-HIV agent (See e.g., El-Mekkawy et al.,
Phytochemistry
, 49: 1651-1657 (1998); Min et al.,
Chem. Pharm. Bull
, 46: 1607-1612 (1998)), or for having anti-tumor, cardiovascular, antiviral, antibacterial, antiparasitic, and immune modulating activities (See e.g., Wasser et al., Critical Review in Immunology, 19:65-96 (1999)).
There are two major types of compounds found in Ganoderma which have been shown to be associated with the medicinal or therapeutic effects of Ganoderma. They are the polysaccharide compounds and the terpenoids. The polysaccharide compounds are primarily water-soluble. The terpenoids are oleaginous substances and are generally insoluble in water.
The polysaccharide compounds isolated from Ganoderma include hetero-&bgr;-glucans and their protein complexes (such as xyloglucans and acidic &bgr;-glucan-containing uronic acid, dietary fibers, lectins). The polysaccharides found in Ganoderma have been reported to possess anti-tumor and immune modulating effects (See Wasser et al., supra).
The Ganoderma terpenoids contain a lanostane skeleton. They are classified into several groups based on their carbon numbers and state of oxidation (Komoda et al.,
Chem. Pharm. Bull
., 33:4829-4835 (1985)). These Ganoderma terpenoids include lanostanine-type triterpenoids (e.g., ganoderic acids A, B, C
1
, C
2
, D
1
, D
2
, E
1
, E
2
, F, G, H, I, J, K
1
, K
2
, L, Ma, Mb, Mc, Md, Me, Mf, Mg, Mi, Mj, Mk, Mn, N, O, P, Q, S, T, U, V, W, X, Y, and Z), 7-O-methyl-ganoderic acid O, trideacetyl ganoderic acid T, ganoderenic acids A, B, C, D, E, F, G, H, I, ganolucidic acids A, B, C, D, and E, lucidenic acids A, B, C, D
1
, D
2
, E
1
, E
2
, F, G, H, I, J, K, L, M, ganoderiol type 1 (A, B, F) and type 2 (C, D, E, F, G, H, and I), ganoderal A and B, epoxyganoderiol A, B, C, lucidone A, B, C, furanoganoderic acid, and other terpenoid components. Ganoderma terpenoids (e.g., ganoderic acids R, T, U-Z) have been reported to inhibit growth of hepatoma cells in vitro (See Toth et al.,
Tetrahedron Lett
., 24:1081-1084 (1983)).
Ganoderma spores are tiny mist-like brown oval-shaped spores of (6~7) &mgr;m×(10~12) &mgr;m in sizes which are released at the pelius of mature
Ganoderma lucidum
. These spores contain the entire genetic materials and biological substances of Ganoderma. However, the wild Ganoderma spores are difficult to collect, particularly due to their short release period and low germination rate under unfavorable environmental conditions. Therefore, although it is known that the Ganoderma spores are of greater pharmaceutical values than the fruiting bodies of Ganoderma, due to difficulties associated with the collection of the Ganoderma spores, most of the studies on Ganoderma are conducted using the fruiting bodies of Ganoderma.
The biological substances within the Ganoderma spores which give rise to the therapeutic effects of Ganoderma are stored within the double-layered epispores of
Ganoderma lucidum
. However, these epispores have compact structure, which are extremely rigid and resilient. Therefore, it is very difficult to break-open the epispore layers of the Ganoderma spores and release the biological substances therein using conventional extraction methods.
There have been reports on methods for breaking the epispores of Ganoderma spores. For example, Japanese Patent No. JP52041208 discloses an extraction method for breaking Ganoderma spores using mechanical force. Chinese Patent No. CN1134306 teaches a method for breaking the sporoderm of the Ganoderma spores by soaking the spores in water, followed by microwave-heating. Chinese Patent No. CN1165032 teaches a method for breaking the cell wall of
Ganoderma lucidum
spores by digesting the spores with skin-dissolving enzymes such as lysozyme, snail enzyme, cellulase, or hemicellulase, followed by ultrasonic breakage of the cell walls at 20-50° C. However, these methods use a mixed batch of spores collected from different stages of the Ganoderma lifecycle. It is known that the spores at different stages of the lifecycle produce different kinds and/or proportions of the biological substances, which may or may not possess the high level of therapeutic effects as expected. Therefore, the sporoderm-broken spores produced by these methods display inconsistent results and their respective medicinal effects vary.
There have also been reports on isolation or separation of the oleaginous substances (e.g., the terpenoids) from Ganoderma, most involving the use of organic solvents. For example, Min et al.,
Chem. Pharm. Bull
., supra, disclose the isolation of lanostane-type triterpenes using column chromatography of a CHCl
3
-soluble fraction of the methanol extract of the Ganoderma spores. Lin et al.,
J. Chromatography
, 410: 195-200 (1987) disclose the separation of oxygenated triterpenoids from
Ganoderma lucidum
by high-performance liquid chromatography of a methanolic extract of
Ganoderma lucidum
. These methods are unsatisfactory due to complex extraction procedures and low yield of the oleaginous substances.
In the present invention, a method for extracting the oleaginous substances from Ganoderma spores is provided. The Ganoderma spores to be used in the present invention are germination-activated to ensure that the biological substances are maximally produced. The epispores of the germination-activated Ganoderma spores are broken by a mechanical means to release the biological substances. Finally, the oleaginous substances of the biological substances are separated by a supercritical fluid carbon dioxide (SCF—CO
2
) extraction method. The present invention has the advantage of producing high yield of oleaginous substances from Ganoderma (i.e., the yield of the oleaginous substances is about 37% by weight of the entire biological substances released from Ganoderma). The oleaginous substances isolated based on the present method retain the special fragrance of Ganoderma and are without solvent residue and strange odor.
SUMMARY OF THE INVENTION
The present invention provides a method for extracting the oleaginous substances from the sporoderm-broken Ganoderma spores using a supercritical fluid carbon dioxide (SCF—CO
2
) extraction method. The Ganoderma spores are germination-activated and sporoderm-broken.
To obtain the germination-activated Ganoderma spores, the Ganoderma spores are first soaked in a nutritional solution which is suitable for inducing germination. The nutritional solution for germination purpose includes, but is not limited to, an immersed solution of Ganoderma fruiting body, a biotin solution, water, and an immersed solution of Ganoderma mycelium. The immersed solution of Ganoderma fruiting body or Ganoderma mycelium is preferably 0.5 to 25% by weight; the preferred biotin solution is 0.1 to 0.5% by weight. The ratio between the volume of the nutritional solution and the weight of Ganoderma spores is about 0.01 to 5 times. The preferred soaking time is between 10 minutes to 10 hours. The preferred soaking temperature is between 16° C. and 43° C.
The germination-induced Ga
Chung Peter Chee-Keung
Huang Xiao-Ni
Liu Xin
Chao Fei-Fei
Coe Susan D.
Prats Francisco
Venable Baetjer Howard & Civiletti LLP
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