Inonotus obliquus mushroom related formulations, chemical compounds, compositions, and methods of making and using same

Abstract

Inonotus obliquus mushroom-based pharmaceutical and nutritional formulations, chemical compounds and pharmaceutical compositions, and methods of preparation. The formulations, compounds, and pharmaceutical compositions are useful for the treatment of diseases such as cancer when administered to humans or animals. The nutritional formulation comprises an inonotus obliquus mushroom reduzate, wherein the reduzate is formed in a reducing solvent; and an Inonotus obliquus mushroom extract, wherein the extract is formed in an extraction solvent. The reduzate and the extract are mixed together or combined according to methods known to those skilled in the art to form the nutritional formulation. The pharmaceutical preparation is a reaction product prepared by: preparing an inonotus obliquus mushroom reduzate in a reducing solvent under pressure; preparing an inonotus obliquus mushroom extract in an extraction solvent; mixing or combining the inonotus obliquus reduzate with the inonotus obliquus extract; inonotus obliquus preparations are mixed or combined with an esterification mixture or a predetermined amount of Manuka honey, the esterification mixture containing proline, fructose and fatty acids for the development of esterification compounds and comprising a sugar ester source capable of emulsifying in an otherwise immiscible liquid compound, the predetermined amount of manuka honey is sufficient to induce esterification and promote emulsification at a temperature capable of inducing a reaction between the ingredients of inonotus obliquus and the esterification mixture or ingredients of manuka honey; and combining the heterogeneous mixture with a medium chain triglyceride oil.

Description

Technical Field

[0001] The present invention generally relates to nutritional and pharmaceutical preparations based on Chaga mushroom, and also to chemical compounds and pharmaceutical compositions and methods for their preparation and use, and more particularly to preparations, chemical compounds and compositions useful for treating diseases such as cancer. Background Art

[0002] The following is a preliminary discussion of the present invention, which is not necessarily prior art and should not necessarily be construed as such.

[0003] Cancer is a leading cause of death worldwide. Additionally, in an increasingly younger population, the concerning new cancer diagnosis rates continue to threaten public health and quality of life. Current treatments for the population may be effective, but conventional treatments still have a significant failure rate, many of the currently available anti-cancer drugs are not target-specific and produce several side effects and complications, and the threat to long-term health and quality of life remains.

[0004] Skin diseases and various equine cancers are growing at an unprecedented rate in various species. It is projected that by 2030, equine cancer diagnoses will increase by 75%-90% per year. There are no long-term treatment options for horses with advanced cancer.

[0005] Therefore, there is an urgent need for new, effective and less toxic treatment methods for the treatment of cancer in humans and animals (including equines).

[0006] For hundreds of years, medicinal mushrooms and other natural products have been used around the world to treat various diseases and infections. Today, medicinal mushrooms are also used around the world to treat many types of diseases (including lung diseases) and many types of cancer. For over 30 years, medicinal mushrooms have been approved as a supplement to standard cancer treatment in Japan and China. In these countries, mushrooms have been used safely alone or in combination with radiation or various chemotherapeutic agents for a long time. In Asia, over 100 different types of mushrooms are used to treat cancer, including Ganoderma lucidum (relish), Trametes versicolor or Curious versicolor (turkey tail), Lentinus edodes (shiitake), and Grifola frondosa (maitake), etc. Since many mushrooms are edible and non-toxic, there is the possibility that one or more of their constituent compounds could provide an effective and non-toxic treatment for cancer.

[0007] Of particular interest is how mushrooms affect the immune system and whether they halt or slow tumor growth or kill tumor cells. It is thought that certain chemical compounds, such as polysaccharides (β-glucans), triterpenes, alkaloids, and other forms of biomolecules, enhance the immune system to directly combat cancer as well as exert direct anti-cancer functions on cancer cells themselves. Their potential use alone and as adjuncts to cancer therapy has emerged. It is also known that mushrooms complement chemotherapy and radiotherapy by counteracting the side effects of cancer, such as nausea, myelosuppression, anemia, and reduced resistance. Recently, many bioactive molecules, including anti-tumor agents, have been identified from various mushrooms. These bioactive molecules include polysaccharides, proteins, fats, ash, glycosides, alkaloids, volatile oils, tocopherols, phenols, flavonoids, carotenoids, folic acid, ascorbic acid enzymes, and organic acids. Polysaccharides such as β-D-glucan are the most widely known mushroom-derived compounds with anti-cancer and immunomodulatory properties. For example, polysaccharide K (PSK), a protein-bound polysaccharide found in turkey tail mushrooms, is an approved mushroom product used in Japan for the treatment of cancer and has been studied as an adjuvant therapy for gastric, breast, colorectal, and lung cancers.

[0008] A dietary supplement prepared from Trametes versicolor (turkey tail mushroom) has been shown to reduce the growth of hormone-responsive prostate cancer LNCaP cells. The crude extract of Trametes versicolor has been shown to inhibit the growth of many human cancer cell lines, including gastric cancer (7907), lung cancer (SPC), leukemia (MCL), and lymphoma (SLY). The polysaccharides of this mushroom have been shown to inhibit the proliferation of human liver cancer (QCY) cells in vitro and in vivo, which occurs with a decrease in the expression of apoptosis- and cell cycle-related genes p53, Bcl-2, and Fas.

[0009] The genus Ganoderma (commonly known as Reishi or Lingzhi) has been administered as an anti-cancer agent for centuries throughout Asia. Extracts of Ganoderma have been shown to reduce the viability of human gastric cancer cells. Ganoderic acid T (GA-T) has been shown to inhibit tumor invasion and metastasis in human colon cancer cell lines, while other ganoderic acids, including (GA-Me, GA-Mf, GA-S), have been shown to be cytotoxic to human colon cancer cells and reduce the growth of cell populations in human cancer cell lines. The natural glycopeptide LZ-D-4 and its sulfated derivative LZ-D purified from the fruiting bodies of Ganoderma have shown anti-tumor activity against murine lymphocytic leukemia in vitro.

[0010] D-glucans purified from Grifola frondosa (also known as Maitake or Hen-of-the-woods) have been shown to enhance the efficacy of cisplatin, checking the decrease in the number of immunocompetent cells (i.e., macrophages, DCs, and NK cells) in cisplatin-treated mice. The chemically sulfated polysaccharide (S-GAP-P) of a water-insoluble polysaccharide derived from Grifola frondosa has been shown to have an anticancer effect, inhibiting cell growth and inducing apoptosis when used in combination with 5-fluorouracil (5-FU) in human cancer cells. A polysaccharide peptide GFPPS1b isolated from cultured Grifola frondosa mycelia has been shown to have antitumor activity, inhibiting the proliferation of human gastric adenocarcinoma cells. The cells died of apoptosis, which was associated with a decrease in mitochondrial transmembrane potential, upregulation of Bax, downregulation of Bcl-2, and activation of caspase-3.

[0011] Several different additional mushroom types have become the subject of anticancer research. One of these, the Chaga mushroom (Inonotus obliquus), water and ethanol extracts have been shown to induce apoptosis in human colon cancer (DLD-1) cells, among other functions, by preventing tissue damage induced by reactive oxygen species (ROS). The water extract of Chaga has also been shown to arrest the cell cycle in the G0 / G1 phase in B16-F10 murine melanoma cells, causing not only apoptosis but also inducing cell differentiation. These effects are associated with downregulation of the expression levels of pRb, p53, and p27, and further show that the Chaga extract results in G0 / G1 cell cycle arrest, accompanied by a decrease in the expression levels of cyclin E / D1 and Cdk 2 / 4. In addition, the antitumor effect of the Chaga extract was evaluated in vivo in Balb / c mice. Intraperitoneal administration of the Chaga extract significantly inhibited the growth of tumor masses in B16-F10 cells implanted in the mice, resulting in a 3-fold inhibition at a dose of 20 mg / kg / day for 10 days. Ethanol extracts of the Chaga sclerotium and fruit body caused significant antitumor activities of 74.6% and 44.2%, respectively.

[0012] Pentacyclic triterpenoid betulinic acid occurs naturally in Phellinus igniarius and has been the subject of numerous studies for its anticancer properties due to its antitumor activity and ability to overcome resistance by inducing apoptosis in various human cancers. Its selective cytotoxicity against cancer was first described in 1995 in human melanoma both in vitro and in vivo. Since then, betulinic acid has been reported to be effective against many human cancers, including lung cancer, colon cancer, prostate cancer, and ovarian cancer. One study showed that normal cells remained unaffected by betulinic acid treatment. Betulinic acid has also been applied in vitro to childhood cancers, namely medulloblastoma, glioblastoma, Ewing's sarcoma, neuroblastoma, and leukemia. Accumulated experimental evidence indicates that betulinic acid treatment results in morphological changes in sensitive cells, such as cell shrinkage, DNA fragmentation, nuclear condensation, and membrane blebbing. Although the exact molecular mechanism of betulinic acid-induced apoptosis is not clear, some studies suggest that proteolytic cleavage of caspases, activation of the MAP kinase cascade, regulation of NF-κB signaling, generation of reactive oxygen species, and inhibition of topoisomerase I may all be contributing processes. In addition to these functions, betulinic acid has been found to reactivate mitochondria and induce apoptosis by releasing cytochrome C, a signaling protein that naturally induces apoptosis in response to irreparable cell damage.

[0013] The potential benefits of Phellinus igniarius mushroom need to be further explored, especially the potential oncology benefits. One limitation that has been discovered in the ongoing research on betulinic acid and publicly discussed in many research groups is the poor bioavailability of betulinic acid. Scientists speculate that this low bioavailability is caused by the poor water solubility of betulinic acid. The low bioavailability of betulinic acid severely limits its practical application as a therapeutic agent. Various attempts have been made to overcome this low bioavailability and have included self-nanoemulsifying drug delivery systems and spray gun technology, but none have provided a practical solution. Therefore, what is needed is betulinic acid or its derivatives that have sufficient bioavailability to provide a therapeutic effect when administered to humans and animals. Summary of the Invention

[0014] Provided herein are nutritional and pharmaceutical formulations, chemical compounds, and pharmaceutical compositions. Also provided are methods of preparing the formulations and methods of using the formulations, chemical compounds, and pharmaceutical compositions to treat diseases such as cancer.

[0015] One embodiment of the invention relates to a nutritional formulation and a method of preparing the nutritional formulation, wherein the formulation is a Phellinus igniarius mushroom reduct formed in a reducing solvent; and a Phellinus igniarius mushroom extract formed in an extraction solvent, and the reduct and the extract are mixed together, or combined according to methods known to those skilled in the art, to form a nutritional formulation.

[0016] Another embodiment of the present invention is a pharmaceutical preparation and a method for preparing the pharmaceutical preparation: preparing a chaga mushroom reduct in a reducing solvent under pressure; preparing a chaga mushroom extract in an extraction solvent; combining the chaga reduct with the chaga extract; combining the chaga preparation with an esterification mixture or a predetermined amount of Manuka honey, the esterification mixture containing proline, fructose, and fatty acids such as medium-chain triglyceride (MCT) oil or sunflower lecithin or other fatty acids of various carbon chain lengths, with and without phospholipids for developing esterified compounds, further comprising a sugar ester source capable of emulsifying within otherwise immiscible liquid compounds, the predetermined amount of Manuka honey being sufficient to cause esterification and promote emulsification at a temperature capable of inducing a reaction between the components of the chaga mushroom and the components of the esterification mixture or Manuka honey; and combining the heterogeneous mixture with medium-chain triglyceride to produce a pharmaceutical preparation.

[0017] Another embodiment is a chemical compound. The chemical compound is isolated from or synthesized for the pharmaceutical preparation and is optionally combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition.

[0018] Another embodiment is a method for treating cancer by administering to a human or animal one or more of the pharmaceutical preparation, the chemical compound, or the pharmaceutical composition.

[0019] The following are various additional concepts numbered for reference in other concepts.

[0020] Concept 1 - A chaga preparation containing: a chaga mushroom reduct, wherein the reduct is formed in a reducing solvent; and a chaga mushroom extract, wherein the extract is formed in an extraction solvent; wherein the reduct and the extract are mixed together.

[0021] Concept 2 - The preparation according to Concept 1, further containing a distillation residue fraction formed from a mixture of the reduct and the extract in a volume range of 1:1 to 1:10 extract:reduct.

[0022] Concept 3 - The preparation according to any one of Concepts 1 or 2, wherein the volume ratio of the reduct to the extract (the reduct:extract volume ratio) is in the range of 100:1 to 1:100.

[0023] Concept 4 - The preparation according to any one of Concepts 1 - 3, wherein the reduct:extract volume ratio is in the range of 20:1 to 1:10.

[0024] Concept 5 - The preparation according to any one of Concepts 1 - 4, wherein the reduct:extract volume ratio is in the range of 20:1 to 1:1.

[0025] Concept 6 - The preparation according to Concept 2, wherein the volume ratio of the reducing substance to the remaining fraction (reducing substance: remaining fraction volume ratio) is in the range of 100:1 to 1:100.

[0026] Concept 7 - The preparation according to Concept 2 or 6, wherein the reducing substance: remaining fraction volume ratio is in the range of 20:1 to 1:10.

[0027] Concept 8 - The preparation according to any one of Concepts 2, 6 or 7, wherein the reducing substance: remaining fraction volume ratio is in the range of 20:1 to 1:1.

[0028] Concept 9 - The preparation according to Concept 2, wherein the volume ratio of the extract to the remaining fraction (extract: remaining fraction volume ratio) is in the range of 100:1 to 1:100.

[0029] Concept 10 - The preparation according to Concept 2 or 9, wherein the extract: remaining fraction volume ratio is in the range of 10:1 to 1:10.

[0030] Concept 11 - The preparation according to any one of Concepts 2, 9 or 10, wherein the extract: remaining fraction volume ratio is in the range of 5:1 and 1:5.

[0031] Concept 12 - The preparation according to Concept 2, wherein the reducing substance, the extract and the distillation residue are present in the preparation in a volume ratio of about 5:1:1.

[0032] Concept 12 - The preparation according to any one of Concepts 1 - 12, wherein the reducing solvent has a dielectric constant greater than 50.

[0033] Concept 14 - The preparation according to any one of Concepts 1 - 13, wherein the reducing solvent has a dielectric constant greater than 60.

[0034] Concept 15 - The preparation according to any one of Concepts 1 - 14, wherein the reducing solvent has a dielectric constant greater than 70.

[0035] Concept 16 - The preparation according to any one of Concepts 1 - 15, wherein the extraction solvent has a dielectric constant less than 50.

[0036] Concept 17 - The preparation according to any one of Concepts 1 - 16, wherein the extraction solvent has a dielectric constant less than 40.

[0037] Concept 18 - The preparation according to any one of Concepts 1 - 17, wherein the extraction solvent has a dielectric constant less than 30.

[0038] Concept 19 - The preparation according to any one of Concepts 1 - 18, wherein the extraction solvent has a first dielectric constant and the reduction solvent has a second dielectric constant, and the second dielectric constant is at least 10 greater than the first dielectric constant.

[0039] Concept 20 - The preparation according to any one of Concepts 1 - 19, wherein the extraction solvent has a first dielectric constant and the reduction solvent has a second dielectric constant, and the second dielectric constant is at least 20 greater than the first dielectric constant.

[0040] Concept 21 - The preparation according to any one of Concepts 1 - 20, wherein the extraction solvent has a first dielectric constant and the reduction solvent has a second dielectric constant, and the second dielectric constant is at least 30 greater than the first dielectric constant.

[0041] Concept 22 - The preparation according to any one of Concepts 1 - 21, which further contains a honey component.

[0042] Concept 23 - The preparation according to Concept 22, wherein the honey component contains Manuka honey.

[0043] Concept 24 - The preparation according to any one of Concepts 22 and 23, wherein the honey component is present in the preparation at a weight ratio of reducing substance:honey in the range of 10:0.1 to 10:10.

[0044] Concept 25 - The preparation according to any one of Concepts 22 - 24, wherein the honey component is present in the preparation at a weight ratio of reducing substance:honey in the range of 10:0.1 to 10:2.

[0045] Concept 26 - The preparation according to any one of Concepts 22 - 25, wherein the honey component is present in the preparation at a weight ratio of reducing substance:honey of about 10:1.

[0046] Concept 27 - The preparation according to any one of Concepts 1 - 26, which further contains a carrier.

[0047] Concept 28 - The preparation according to Concept 26, wherein the carrier contains medium-chain triglycerides.

[0048] Concept 29 - The preparation according to Concept 26, wherein the carrier contains coconut oil.

[0049] Concept 30 - The preparation according to any one of Concepts 27 - 29, wherein the carrier is present in the preparation at a weight ratio of reducing substance:carrier in the range of 10:0.1 to 1:50.

[0050] Concept 31 - The preparation according to any one of Concepts 27 - 30, wherein the carrier is present in the preparation at a weight ratio of reductant:carrier in the range of 10:0.1 to 10:2.

[0051] Concept 32 - The preparation according to any one of Concepts 27 - 31, wherein the carrier is present in the preparation at a reductant:carrier weight ratio of about 10:1.

[0052] Concept 33 - A method for preparing a preparation by: preparing a chaga reductant in a reducing solvent; preparing a chaga extract in an extraction solvent; and mixing the chaga reductant with the chaga extract to produce the preparation.

[0053] Concept 34 - The method according to Concept 33, further comprising preparing a distillation residue fraction from the mixture of the chaga reductant and the chaga extract, the chaga reductant and chaga extract being present in the mixture in a volume range of 1:1 to 1:10 extract:reductant; and mixing the chaga reductant and the chaga extract by mixing the chaga reductant, the chaga extract, and the distillation residue fraction.

[0054] Concept 35 - The method according to any one of Concepts 33 - 34, wherein mixing the chaga reductant and the chaga extract comprises mixing between 100 parts by volume of reductant:1 part by volume of extract and 1 part by volume of reductant:100 parts by volume of extract.

[0055] Concept 36 - The method according to any one of Concepts 33 - 35, wherein mixing the chaga reductant and the chaga extract comprises mixing between 20 parts by volume of reductant:1 part by volume of extract and 1 part by volume of reductant:10 parts by volume of extract.

[0056] Concept 37 - The method according to any one of Concepts 33 - 36, wherein mixing the chaga reductant and the chaga extract comprises mixing between 20 parts by volume of reductant:1 part by volume of extract and 1 part by volume of reductant:1 part by volume of extract.

[0057] Concept 38 - The method according to Concept 34, wherein mixing the chaga reductant and the remaining distillation fraction comprises mixing between 100 parts by volume of reductant and 1 part by volume of the remaining distillation fraction and 1 part by volume of reductant and 100 parts by volume of the remaining distillation fraction.

[0058] Concept 39 - The method according to Concept 34 or Concept 38, wherein mixing the chaga reductant and the remaining distillation fraction includes mixing between 20 parts by volume of the reductant and 1 part by volume of the remaining distillation fraction and 1 part by volume of the reductant and 10 parts by volume of the remaining distillation fraction.

[0059] Concept 40 - The method according to Concept 34, 38 or 39, wherein mixing the chaga reductant and the remaining distillation fraction includes mixing between 20 parts by volume of the reductant and 1 part by volume of the remaining distillation fraction and 1 part by volume of the reductant and 1 part by volume of the remaining distillation fraction.

[0060] Concept 41 - The method according to Concept 34 or any one of Concepts 38 - 40, wherein mixing the chaga extract and the remaining distillation fraction includes mixing between 100 parts by volume of the extract and 1 part by volume of the remaining distillation fraction and 1 part by volume of the extract and 100 parts by volume of the remaining distillation fraction.

[0061] Concept 42 - The method according to Concept 34 or any one of Concepts 38 - 41, wherein mixing the chaga extract and the remaining distillation fraction includes mixing between 10 parts by volume of the extract and 1 part by volume of the remaining distillation fraction and 1 part by volume of the extract and 10 parts by volume of the remaining distillation fraction.

[0062] Concept 43 - The method according to Concept 34 or any one of Concepts 38 - 42, wherein mixing the chaga extract and the remaining distillation fraction includes mixing between 5 parts by volume of the extract and 1 part by volume of the remaining distillation fraction and 1 part by volume of the extract and 50 parts by volume of the remaining distillation fraction.

[0063] Concept 44 - The method according to Concept 34, wherein the reductant, the extract and the distillation remaining fraction are present in the preparation in a volume ratio of about 5:1:1.

[0064] Concept 45 - The method according to any one of Concepts 33 - 44, wherein the reducing solvent has a dielectric constant greater than 50.

[0065] Concept 46 - The method according to any one of Concepts 33 - 45, wherein the reducing solvent has a dielectric constant greater than 60.

[0066] Concept 47 - The method according to any one of Concepts 33 - 46, wherein the reducing solvent has a dielectric constant greater than 70.

[0067] Concept 48 - The method according to any one of Concepts 33 - 47, wherein the extraction solvent has a dielectric constant less than 50.

[0068] Concept 49 - The method according to any one of Concepts 33 - 48, wherein the extraction solvent has a dielectric constant of less than 40.

[0069] Concept 50 - The method according to any one of Concepts 33 - 49, wherein the extraction solvent has a dielectric constant of less than 30.

[0070] Concept 51 - The method according to any one of Concepts 33 - 50, wherein the extraction solvent has a first dielectric constant and the reduction solvent has a second dielectric constant, and the second dielectric constant is at least 10 greater than the first dielectric constant.

[0071] Concept 52 - The method according to any one of Concepts 33 - 51, wherein the extraction solvent has a first dielectric constant and the reduction solvent has a second dielectric constant, and the second dielectric constant is at least 20 greater than the first dielectric constant.

[0072] Concept 53 - The method according to any one of Concepts 33 - 52, wherein the extraction solvent has a first dielectric constant and the reduction solvent has a second dielectric constant, and the second dielectric constant is at least 30 greater than the first dielectric constant.

[0073] Concept 54 - The method according to any one of Concepts 33 - 53, further comprising providing a honey component, and wherein mixing the chaga reduction with the chaga extract further comprises mixing the honey component with the chaga reduction and the chaga extract.

[0074] Concept 55 - The method according to any one of Concepts 33 - 54, wherein the honey component contains Manuka honey.

[0075] Concept 56 - The method according to any one of Concepts 33 - 55, wherein the honey component is present in the formulation at a weight ratio of reduction product:honey in the range of 10:0.1 to 10:10.

[0076] Concept 57 - The method according to any one of Concepts 33 - 56, wherein the honey component is present in the formulation at a weight ratio of reduction product:honey in the range of 10:0.1 to 10:2.

[0077] Concept 58 - The method according to any one of Concepts 33 - 57, wherein the honey component is present in the formulation at a weight ratio of reduction product:honey of about 10:1.

[0078] Concept 59 - The method according to any one of Concepts 33 - 58, further comprising providing a carrier, and wherein mixing the chaga reduct with the chaga extract further comprises mixing the carrier with the chaga reduct and the chaga extract.

[0079] Concept 60 - The method according to any one of Concept 59, wherein the carrier comprises medium - chain triglycerides.

[0080] Concept 61 - The method according to Concept 59 or 60, wherein the carrier comprises coconut oil.

[0081] Concept 62 - The method according to any one of Concepts 59 - 61, wherein the carrier is present in the formulation at a reduct:carrier weight ratio in the range of 10:0.1 to 1:50.

[0082] Concept 63 - The method according to any one of Concepts 59 - 62, wherein the carrier is present in the formulation at a reduct:carrier weight ratio in the range of 10:0.1 to 10:2.

[0083] Concept 64 - The method according to any one of Concepts 59 - 63, wherein the carrier is present in the formulation at a reduct:carrier weight ratio of about 10:1.

[0084] Concept 65 - A method of treating a mammal, comprising:

[0085] Administering to a mammalian patient a formulation according to any one of Concepts 1 - 32 at a dose in the range of 0.1 ounce per 1000 pounds of patient body weight per day to 1 ounce per pound of patient body weight per day.

[0086] Concept 66 - The method according to Concept 65, wherein the formulation is administered to the patient once a day.

[0087] Concept 67 - The method according to Concept 65, wherein the formulation is administered to the patient at least twice a day.

[0088] Concept 68 - The method according to any one of Concepts 65 - 67, wherein the mammalian patient is a human patient.

[0089] Concept 69 - The method according to any one of Concepts 65 - 67, wherein the mammalian patient is a horse patient.

[0090] Concept 70 - The method according to any one of Concepts 65 - 69, wherein administering the formulation comprises orally administering the formulation.

[0091] Concept 71 - The method according to any one of Concepts 65 - 69, wherein administering the preparation comprises topically administering the preparation.

[0092] Concept 72 - The method according to any one of Concepts 65 - 69, wherein administering the preparation comprises orally administering the preparation or topically administering the preparation.

[0093] Concept 73 - A chemical compound that is useful for treating a disease such as cancer when administered to a mammal. The chemical compound can be isolated from the pharmaceutical preparation or synthesized.

[0094] Concept 74 - A pharmaceutical composition comprising a chemical compound in a pharmaceutically acceptable carrier. When administered to a mammal, the composition is useful for treating a disease such as cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] The present invention can be more fully understood by considering the following detailed description of various embodiments of the invention in conjunction with the accompanying drawings, in which:

[0096] Figures 1-7 The results of in vitro tests are shown to understand the cytotoxic effect of the preparation on three types of cancer cell lines. The in vitro tests are performed on cancer cell lines from the ATCC cell bank. The cell lines tested are polygonal melanoma, triple-negative breast cancer, and liver cancer cells. The cytotoxic effect, apoptosis induction, and inhibition of ATP utilization by cancer cells are all shown to be consistent with the results of several in vitro studies.

[0097] Figure 1 is a bar graph showing the inhibition of ATP production by the novel product on polygonal melanoma.

[0098] Figure 2 is a bar graph showing the relative ADP levels from ATP inhibition; this is an indicator of a type of cell death.

[0099] Figure 3 is a bar graph showing the cytotoxic properties of the novel product on polygonal melanoma.

[0100] Figure 4 is a bar graph showing the inhibition of ATP production by the novel product on triple-negative breast cancer.

[0101] Figure 5 is a bar graph showing the relative ADP levels from ATP inhibition; this is an indicator of a type of cell death.

[0102] Figure 6 is a bar graph showing the cytotoxic properties of the novel product on triple-negative breast cancer.

[0103] Figure 7It is a bar graph showing the inhibition of ATP production by the novel product against liver cancer.

[0104] Figure 8 It is a bar graph showing the relative ADP levels from ATP inhibition; this is an indicator of a type of cell death.

[0105] Figure 9 It is a bar graph showing the cytotoxic properties of the novel product against liver cancer.

[0106] Figure 10 It is a bar graph showing the quantitative ATP levels in HepG2 cells after treatment with the sample.

[0107] Figure 11 It provides data that has been logarithmically transformed to better visualize the Figure 9 data.

[0108] Figure 12 It is a bar graph showing the evaluation of HepG2 cell viability.

[0109] Figure 13 It provides data that has been logarithmically transformed to better visualize the Figure 12 data.

[0110] Figure 14 It is an IC 50 curve for examining cell viability in HepG2 cells treated with sample 5.

[0111] Figure 15 It is an IC 50 curve for examining cell viability in HepG2 cells treated with sample 6.

[0112] Figure 16 It is a bar graph showing the relative ATP levels in MDA-MB-231 cells after treatment with sample 5 or 6.

[0113] Figure 17 It provides data that has been logarithmically transformed to better visualize the Figure 16 data.

[0114] Figure 18 It is for showing the evaluation of cell viability of MDA-MB-231 cells using the CellQuanti-Blue TM cell viability assay.

[0115] Figure 19 It provides data that has been logarithmically transformed to better visualize the Figure 18 data.

[0116] Figure 20 It provides the IC of sample 5 using MDA-MB-231 cells50 Evaluated graph.

[0117] Figure 21 It provides the IC of sample 6 with MDA-MB-231 cells 50 Evaluated graph.

[0118] Figure 22 It is a bar graph showing the quantitative ATP levels of SK-Mel 28 cells after treatment with samples 5 and 6.

[0119] Figure 23 It provides the data that has been logarithmically transformed to better visualize the data Figure 22 graph of the data.

[0120] Figure 24 It is a bar graph showing the cell viability assessment of SK-Mel 28 cells using the CellQuanti-Blue cell viability assay.

[0121] Figure 25 It provides the data that has been logarithmically transformed to better visualize the data Figure 24 graph of the data.

[0122] Figure 26 It provides the IC of sample 5 with SK-Mel 28 cells 50 Evaluated graph.

[0123] Figure 27 It provides the IC of sample 6 with SK-Mel 28 cells 50 Evaluated graph.

[0124] Figure 28 It is a graph showing the quantitative ATP levels of HCT-116 cells after treatment with sample 7.

[0125] Figure 29 It is a graph showing the quantitative ATP levels of HCT-116 cells after treatment with sample 8.

[0126] Figure 30 It provides the IC of sample 7 with HCT-116 cells 50 Evaluated graph.

[0127] Figure 31 It provides the IC of sample 8 with HCT-116 cells 50 Evaluated graph.

[0128] Figure 32 It is a graph showing the quantitative ATP levels of HEPG2 cells after treatment with sample 7.

[0129] Figure 33 It is a graph showing the quantitative ATP levels of HEPG2 cells after treatment with sample 8.

[0130] Figure 34 It is a graph providing the IC of sample 7 with HEPG2 cells 50 for evaluation.

[0131] Figure 35 It is a graph providing the IC of sample 8 with HEPG2 cells 50 for evaluation.

[0132] Figure 36 It is a graph showing the quantitative ATP levels of MDA-MB-231 cells after treatment with sample 7.

[0133] Figure 37 It is a graph showing the quantitative ATP levels of MDA-MB-231 cells after treatment with sample 8.

[0134] Figure 38 It is a graph providing the IC of sample 7 with MDA-MB-231 cells 50 for evaluation.

[0135] Figure 39 It is a graph providing the IC of sample 8 with MDA-MB-231 cells 50 for evaluation.

[0136] Figure 40 It is a graph showing the quantitative ATP levels of SK-Mel 28 cells after treatment with sample 7.

[0137] Figure 41 It is a graph showing the quantitative ATP levels of SK-Mel 28 cells after treatment with sample 8.

[0138] Figure 42 It is a graph providing the IC of sample 7 with SK-Mel 28 cells 50 for evaluation.

[0139] Figure 43 It is a graph providing the IC of sample 8 with SK-Mel 28 cells 50 for evaluation.

[0140] Figure 44 Provides an enlarged view of the chemical reaction synthesis pathway shown in Figure 58 for four different possible reaction products resulting from the first proposed theoretical reaction between betulinic acid (BA) and methylglyoxal (MGO).

[0141] Figure 45 Provides four different possible products resulting from the first proposed theoretical reaction between betulinic acid and methylglyoxal Figure 58An enlarged view of the chemical reaction synthesis pathway shown in, which shows three additional intermediate compounds, Compounds 2, 3, and 4.

[0142] Figure 46 Provided are Figure 58 an enlarged view of the chemical reaction synthesis pathway shown in.

[0143] Figure 47 Provided Figure 46 is an enlarged view of one of the four different possible products (Compound 5).

[0144] Figure 48 Provided Figure 46 is an enlarged view of one of the four different possible products (Compound 6).

[0145] Figure 49 Provided Figure 46 is an enlarged view of one of the four different possible products (Compound 7).

[0146] Figure 50 Provided Figure 46 is an enlarged view of one of the four different possible products (Compound 8).

[0147] Figure 51 Provided Figure 58 is an enlarged view of additional components of the chemical reaction synthesis pathway shown in.

[0148] Figure 52 Provided Figure 58 is an enlarged view of possible products of the chemical reaction synthesis pathway shown in.

[0149] Figure 53 Provided Figure 58 is an enlarged view of yet another possible product (Compound 10) of the chemical reaction synthesis pathway shown in.

[0150] Figure 54 Provided are Figure 58 an enlarged view of the major product of the chemical reaction synthesis pathway shown in, Compound 11 (also referred to herein as the compound represented by Formula 1).

[0151] Figure 55 Provided are Figure 58An enlarged view of the main product of the chemical reaction synthesis pathway shown in the figure - compound 12 (also referred to herein as the compound represented by formula 2).

[0152] Figure 56 Provide NMR data measurements that show unique electromagnetic signals with frequencies that are unique characteristics of the magnetic field of a specific nucleus; show the presence of specific characteristics of the compound of formula 1.

[0153] Figure 57 Provide NMR data measurements that show unique electromagnetic signals with frequencies that are unique characteristics of the magnetic field of a specific nucleus; show the presence of specific characteristics of the compound of formula 2.

[0154] Figure 58 Show the chemical reaction synthesis pathways of four different products, all of which are the results of the proposed theoretical reactions. Compounds 1 - 4 show the possible reactions and product results of betulinic acid (BA) and methylglyoxal (MGO) in an isolated environment. Compounds 5 - 8 show the potential product results from the proposed reactions between the above - mentioned BA, MGO, and fatty acids present in MCT oil. Compounds 9 - 12 show the potential product results from the proposed reactions between betulinic acid, proline, lecithin, and fructose.

[0155] While the present invention is suitable for various modifications and alternative forms, details thereof have been shown by way of example in the drawings and will be described in detail. However, it should be understood that the present invention is not intended to limit the present invention to the specific embodiments described. On the contrary, the present invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention as defined by the appended claims. Detailed Description

[0156] Nutritional and pharmaceutical formulations, chemical compounds, and pharmaceutical compositions containing said chemical compounds are described herein. Also described are methods for preparing said formulations, chemical compounds, and pharmaceutical compositions containing said compounds, and methods for using said formulations to treat diseases such as cancer.

[0157] One embodiment of the present invention relates to a nutritional formulation, wherein the formulation is a chaga mushroom reduct, wherein the reduct is formed in a reducing solvent; and a chaga mushroom extract, wherein the extract is formed in an extraction solvent, and wherein the reduct and the extract are mixed together, or combined according to methods known to those skilled in the art, to form a nutritional formulation.

[0158] Another embodiment of the present invention is a pharmaceutical preparation and a method for preparing a pharmaceutical preparation by: preparing a chaga mushroom reduct in a reducing solvent under pressure; preparing a chaga mushroom extract in an extraction solvent; mixing or combining the chaga reduct with the chaga extract; mixing or combining the chaga preparation with an esterification mixture or a predetermined amount of manuka honey, the esterification mixture containing proline, fructose, and fatty acids such as medium-chain triglyceride (MCT) oil or sunflower lecithin or other fatty acids of various carbon chain lengths, with and without phospholipids for developing esterified compounds, further comprising a sugar ester source capable of emulsifying within otherwise immiscible liquid compounds, the predetermined amount of manuka honey being sufficient to cause esterification and promote emulsification at a temperature capable of inducing a reaction between the components of the chaga mushroom and the components of the esterification mixture or manuka honey; and combining the heterogeneous mixture with medium-chain triglycerides.

[0159] The most likely type of reaction to be induced first is called the Maillard reaction, also known as the non-enzymatic browning reaction, and is responsible for forming intermediates that would normally form known end products but are redirected into a novel reaction with betulinic acid and possibly other components found in the chaga mushroom reduct and extract to form novel compounds as described herein. The Maillard reaction begins with a reaction between a reducing sugar and various types of amino acids when certain reaction parameter conditions are met. Two of the several intermediates formed during the onset of this reaction are called Amadori and Heyns products. The complete elucidation of the Maillard reaction remains unknown, but various possible outcomes have been studied as described herein, and measurements to confirm the formation of these products have also been achieved as described herein.

[0160] Another embodiment is a chemical compound. The chemical compound is isolated from or synthesized in the pharmaceutical preparation and optionally combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition.

[0161] Another embodiment is a method for treating cancer by administering to a human or animal one or more of the pharmaceutical preparation, the chemical compound, or the pharmaceutical composition.

[0162] Pharmaceutical preparation

[0163] The pharmaceutical preparations provided herein are based on the chaga mushroom, in some preparations together with honey, particularly manuka honey, and in some preparations together with medium-chain triglyceride oil, and are administered to mammalian patients suffering from various cancers. Mammalian patients include human patients and animal patients.

[0164] Although the ability of many different types of mushrooms to affect the cancer process has been examined, none have proven particularly successful, neither as a primary therapy nor as an adjuvant therapy, and most mushroom-based preparations have stalled in the laboratory. In the in vitro studies discussed in this article, the typical preparation methods of these chaga extracts were measured for a novel chaga preparation. The chaga preparation was superior to other common preparation methods.

[0165] One way to expand the potential usefulness of mushrooms in counteracting the effects of cancer is to combine the mushroom with another ingredient. Of particular interest is the exploration of combinations of chaga mushrooms with honey, particularly Manuka honey, and medium-chain triglyceride oil. In some embodiments, the preparation uses a chaga extract and a chaga reductate mixed with Manuka honey. In some embodiments, the chaga extract and the chaga reductate can be distilled before adding the honey. The preparation can be administered orally. In other embodiments, particularly where the preparation is to be administered topically, the preparation can be mixed with coconut oil or an alternative medium-chain glyceride.

[0166] Chaga mushroom

[0167] The most interesting compound that can be extracted from chaga mushrooms is betulinic acid (also referred to herein as "BA"). Betulinic acid is a pentacyclic triterpenoid that has recently been shown to have anti-cancer properties but lacks any clinical results. Betulinic acid itself has limited bioavailability, with a molar mass of 456.7 g / mol and a boiling point of 550 °C. Past studies of betulinic acid have pointed out potential limiting factors in its use, including its molecular size, poor water solubility, and low bioavailability. Betulinic acid is bound to chitin in its native organic form in chaga mushrooms, which is the main component of the cell wall in fungi. Therefore, extracting betulinic acid from chaga mushrooms must include releasing betulinic acid from its chitin binding.

[0168] Betulinic acid has become the subject of many studies and has been shown to induce apoptosis and fragment DNA by inhibiting topoisomerase in the mitochondria of cancer cells. It has also been proposed that betulinic acid has the ability to reverse the Warburg effect, a modified form of metabolism found in cancer cells that favors specialized fermentation of the aerobic respiration pathway preferred by most other cells in the body. During this fermentation process, the end product of glycolysis, pyruvate, is converted to lactate or ethanol while producing a lower amount of ATP than in the citric acid cycle. However, it allows cancer cells to convert glucose and glutamine into biomass by avoiding catabolic oxidation to carbon dioxide, thus preserving carbon-carbon bonds and promoting anabolism. The mechanism by which betulinic acid can interrupt the Warburg effect is currently unknown, but the induction of cellular respiration and the reversal of the glucose fermentation process (the main way cancer cells utilize glucose) may be a meaningful adjunct process against cancer cell growth.

[0169] In addition to these potential functions, it is known that betulinic acid, along with other bioactive compounds found in chaga mushrooms, breaks down lactate at a high rate. The removal of lactate may help combat the acidic, immune system-unfavorable environment created by cancer cells: a relatively high amount of lactate creates a more acidic environment for cancer cells, which favors a lower pH level than healthy cells. Some literature suggests that lactate can also help cancer cells avoid detection by the immune system, allowing them to evade destruction by immune cells. There are specific immune cells that can detect and destroy cancer cells when activated. Some of these types of cells are T cells that are specifically programmed to destroy cancer cells. When these immune cells can recognize cancer cells, they are fully capable of destroying them.

[0170] It has also been proposed that betulinic acid can preferably generate an oxidative stress burden in cancer cells, and this stress burden may be the factor by which betulinic acid inhibits topoisomerase, thus inhibiting DNA production while seemingly being harmless to surrounding healthy cells.

[0171] Also of interest is the high level of superoxide dismutase (SOD) in both chaga extracts and chaga reducts. SOD can be used to disproportionate reactive oxygen species (ROS) generated by the preferred mode of action of cancer cells and a part of the cell damage cycle, a part of which induces signal transduction that may be related to the spread of metastatic disease. Normal healthy cells produce their own SOD to manage oxidative stress from free radical oxygen species to maintain cell health, but cancer cells become unable to manage cell damage and lose the ability to signal apoptosis due to cell damage. The high content of SOD provided in the formulation can help specific cancer cells repair damage and re-establish normal metabolic functions.

[0172] There are potentially additional compounds in Chaga mushroom that can benefit the host's immune cells in their ability to combat cancer progression.

[0173] Manuka honey

[0174] Manuka honey is a monofloral or multifloral honey produced from the nectar of the Manuka plant (Leptospermum scoparium), which is native to southeastern Australia and New Zealand.

[0175] A test for monofloral Manuka honey adopted by the New Zealand Ministry for Primary Industries (see https: / / www.mpi.govt.nz / food-business / honey- bee-products-processing- requirements / manuka - honey - testing / ) is that the honey has the following five characteristics:

[0176] · Contains 3 - phenyl lactic acid at a level greater than or equal to 400 mg / kg;

[0177] · Contains 2′ - methoxyacetophenone at a level greater than or equal to 5 mg / kg;

[0178] · Contains 2 - methoxybenzoic acid at a level greater than or equal to 1 mg / kg;

[0179] · Contains 4 - hydroxy phenyl lactic acid at a level greater than or equal to 1 mg / kg; and

[0180] · The DNA level from Manuka pollen is less than Cq36, which is approximately 3 fg / μL.

[0181] A test for multifloral Manuka honey also adopted by the New Zealand Ministry for Primary Industries is that the honey has the following five characteristics:

[0182] · Contains 3 - phenyl lactic acid at a level greater than or equal to 20 mg / kg but less than 400 mg / kg;

[0183] · Contains 2′ - methoxyacetophenone at a level greater than or equal to 1 mg / kg;

[0184] · Contains 2 - methoxybenzoic acid at a level greater than or equal to 1 mg / kg;

[0185] · Contains 4 - hydroxy phenyl lactic acid at a level greater than or equal to 1 mg / kg; and

[0186] · The DNA level from Manuka pollen is less than Cq36, which is approximately 3 fg / μL.

[0187] When the term "manuka honey" is used herein, it refers to honey that has passed at least the DNA test for multifloral manuka honey or monofloral manuka honey as described in the previous two paragraphs (where the DNA level from manuka pollen is less than Cq36, which is approximately 3 fg / μL).

[0188] Although not wishing to be bound by the following, several hypothetical reaction and mechanism of action are described below. Manuka honey can contribute symbiotically to the function of the formulation. A particularly interesting finding is that manuka contains a high level of methylglyoxal, which is known to be produced as a by-product of several metabolic pathways (primarily glycolysis) in living organisms. While endogenous methylglyoxal in animals has been attributed to the formation of advanced glycation end products (or AGEs), which are used as biomarkers in the neutralization of aging and the development of many degenerative diseases, studies have shown that methylglyoxal derived from honey (such as manuka honey) does not cause an increase in advanced glycation end products in healthy individuals. In addition, methylglyoxal in manuka honey has been shown to have antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), attributing it to a "self-defense" mechanism through living cells. This may endow methylglyoxal with the potential to make cancer cells more sensitive to the functions of betulinic acid and other compounds found in manuka honey and chaga mushrooms.

[0189] Manuka honey, along with other honeys, contains high concentrations of fructose and amino acids, as well as other molecular compounds formed during the production of honey. Fructose, which is found in high concentrations, is one of several types of reducing sugars. Many different amino acids have also been found in varying concentrations in Manuka honey and other types of honey. The ability of fructose and amino acids to react together in an induced reaction has been demonstrated. This type of reaction requires certain parameters and does not occur spontaneously outside of these reaction parameters. This type of recorded reaction is called the Maillard reaction and occurs when a condensation reaction takes place between a reducing sugar and an amino acid. Both fructose and glucose are capable of acting as reducing sugars during the Maillard reaction with amino acids, first undergoing a condensation reaction to form intermediate products called Amadori and Heyns products, which are two examples of several types of intermediate products formed during the Maillard reaction. During the formation of these intermediate products, methylglyoxal is one of the reactive intermediates that contribute to the formation of the end products of the typical Maillard reaction. The formation of novel products with fructose-amine-triterpenes or other components results in a function that potentially interacts with cancer cells through glucose receptors and potentially other cell-signaling receptors. This possible novel product may retain a function-structure similar to that observed in other glucoside-like therapeutic agents found in nature and prepared in pharmaceutical laboratories. The hypothesis presented herein is that the possible novel product may have a similar structure-function and thus be readily consumed by cancer cells, making betulinic acid and other compounds more bioavailable.

[0190] The antimicrobial properties of Manuka honey may also play a role in restricting the proliferation of microorganisms that can inhibit the immune system's destruction of cancer cells. There is limited understanding of how certain microorganisms act as catalysts for the synthesis of betulinic acid during the growth stages of wild chaga mushrooms, and this has also been found in attempts to convert betulinol to betulinic acid in various laboratory work. Thus, providing raw materials via chaga extracts, in addition to the "blueprint" of betulinic acid and chaga mushroom extracts, may lead to the microbial community of the immune system itself helping or even accelerating the process of synthesizing higher amounts of betulinic acid and utilizing this process against cancer cells. This process may be hindered by opportunistic microorganisms, and interestingly, some of these microbial species, such as members of the Enterbacter family, like Helicobacter pylori, have been listed as human carcinogens. It is hypothesized here that the antimicrobial function of Manuka honey also contributes to creating a friendly microbial environment that better promotes the synthesis and reaction of betulinic acid and other compounds. This function can also promote an increase in the bioactivity of betulinic acid and allow healthy immune cells to better utilize betulinic acid and other compounds.

[0191] Another property of Manuka honey is its effective anti-biofilm property, which may be important due to the property of biofilm production by hostile microorganisms. One of the many not fully understood effects against biofilms is the reversal of gene mutations in microorganisms that have become resistant to antimicrobial therapy, making them more susceptible to eradication by the antimicrobial function of Manuka honey itself. Bee defensin is ubiquitous in all honeys, but Manuka honey shows some unique peroxidase-independent protective functions. This may be important because Manuka honey compounds do not disrupt friendly microorganisms that can be used for synthetic action and protection of healthy cells.

[0192] Manuka honey has a relatively low pH, around 3.5 - 4.5, which contrasts with the higher pH of Chaga mushroom extract and reductants. Additionally, the lower pH of the honey may contribute to the action of the formulation against cancer cells as it may be less affected by one of the main defense mechanisms of cancer cells, which is to lower the pH level to escape immune cell attack. This can also allow Manuka honey to inhibit microbial growth, stimulate the bactericidal action of macrophages in the host immune system, and in chronic wounds, reduce protease activity and increase fibroblast activity and oxygenation.

[0193] In addition to the functions discussed previously, recent studies have demonstrated that Manuka honey can exert anti-proliferative effects against cancer cells. These anti-cancer properties can involve different processes, including induction of apoptosis in cancer cells through depolarization of the mitochondrial membrane, inhibition of cyclooxygenase-2 by various components such as flavonoids, release of cytotoxic H2O2, and scavenging of reactive oxygen species (or ROS). The main mechanism by which Manuka honey exerts its anti-proliferative effect is through the activation of the mitochondrial apoptosis pathway, involving the stimulation of initiator caspase-9, which determines the activation of the executioner caspase-3. This last function is also shared by Chaga mushroom. Additionally, it can induce apoptosis via the activation of PARP, induction of DNA fragmentation, and loss of Bcl-2 expression.

[0194] In vivo, Manuka honey has been shown to be effective in reducing tumor volume and supporting tumor cell apoptosis in a murine melanoma model, alleviating colonic inflammation in rats with inflammatory bowel disease, restoring lipid peroxidation, and improving antioxidant parameters. Studies have shown that Manuka honey has neither harmful effects associated with advanced glycation end products nor harmful effects associated with changes in gut microbiota homeostasis. This is important as it relates to the unknown nature of how methylglyoxal is associated with the progression of certain chronic diseases.

[0195] carrier

[0196] Coconut oil or alternative medium-chain triglycerides can be used to carry the formulation and biochemically assist its function, especially in the case of topically applied formulations. When the formulation is applied to open skin lesions as well as subcutaneous tumors, a protective layer is added with medium-chain triglycerides. The carrier is preferably a soft solid, which can be applied as a paste or a liquid, and can preferably dissolve chaga mushroom extract / reduct and honey. In some embodiments, the carrier / formulation mixture can be sprayed onto the area to be treated. Coconut oil is a white solid fat that melts at a temperature of about 25 °C to form a clear, thin liquid oil. Alternative medium-chain triglycerides, such as palm oil, can be liquid at room temperature.

[0197] Preparation of Chaga Mushroom Extract

[0198] The following method can be used to prepare chaga mushroom extract. The chaga mushroom is chopped to increase the surface area exposed to the liquid. In some embodiments, the chaga mushroom is chopped such that 90% of the particles have a maximum size of less than 7 mm, preferably less than about 5 mm, more preferably less than about 3 mm, and even more preferably less than about 2 mm in maximum size.

[0199] The chopped chaga mushroom is then mixed with an extraction solvent. The extraction solvent generally includes a non-aqueous solvent and can be a solvent commonly used for food extraction. Examples of non-aqueous solvents include short-chain alcohols such as ethanol, short-chain diols such as propylene glycol, short-chain acids such as formic acid, acetic acid or lactic acid, short-chain ketones such as acetone, or short-chain esters such as ethyl acetate or n-butyl acetate. If a solvent that is toxic to the patient is used, it can be removed using standard chemical treatment means. The extraction solvent can be present in an amount such that the dry volume of the chopped chaga mushroom is about half the volume of the extraction solvent, but more or less can be used.

[0200] Water can be included in the extraction solvent, where the ratio of non-aqueous solvent to water in the extraction solvent is between about 5:95 and 100:0. Preferably, the ratio of non-aqueous solvent to water is in the range of 95:5 to 60:40, and more preferably in the range of 90:10 to 70:30. In other words, the chaga mushroom is mixed into a liquid containing at least one non-aqueous solvent. The extraction solvent dissolves the components of chaga mushroom released from the mushroom into the solvent. Non-aqueous solvents tend to dissolve the more lipophilic components of chaga mushroom, while water tends to dissolve the less lipophilic components of chaga mushroom because water is generally more polar than non-aqueous solvents. It should be understood that other non-aqueous solvents can be used.

[0201] The dielectric constant ε of the extraction solvent is preferably less than 50, more preferably less than 40, and can even be less than 30. For example, in the case where the liquid solvent is 100% ethanol, the dielectric constant εethanol is about 24.5. In a mixture of miscible liquids, the dielectric constant can be calculated by taking the volume average of the dielectric constants. For example, in a liquid containing 90 vol% ethanol and 10 vol% water (εwater = 80.1), the dielectric constant of the mixture is given by (0.9xεethanol)+(0.1xεwater) = 30.2. The dielectric constant of a liquid is an indicator of the polarity of the solvent mixture.

[0202] Larger values indicate a higher solvent polarity, which may result in the extraction of less lipophilic components of Chaga mushroom. Lower dielectric constant values indicate a lower solvent polarity, in which case the solvent can more effectively extract more lipophilic components from Chaga mushroom. The solvents used can be protic or aprotic. A mixture of ethanol and water is a mixture of two protic solvents.

[0203] Then the Chaga / liquid mixture is allowed to stand for a predetermined time suitable for extracting the components of the mushroom. In some embodiments, the mixture is allowed to stand at room temperature for more than 48 hours, more than one week, preferably more than one month, more preferably more than two months, and even more preferably more than three months. The mixture can be covered or sealed to prevent evaporation of the liquid components. If the mixture is maintained at an elevated temperature, such as 90°F, 100°F or higher, for example up to about 160°F, and / or if the mixture is stirred, for example using a magnetic stirrer, the extraction time can be shorter. The temperature of the mixture is kept below the boiling point of the liquid.

[0204] Another method of forming the extract is to use an ultrasonic extraction method, in which ultrasonic waves are introduced into the Chaga / liquid mixture. The ultrasonic waves generated by an ultrasonic probe or other suitable ultrasonic generator travel through the liquid, creating alternating high-pressure / low-pressure regions, which can cause acoustic cavitation. This in turn can lead to local extreme temperatures and pressures, heating / cooling rates, pressure differences, and high shear forces. When the cavitation bubbles implode on the surface of the Chaga part, the mass transfer from the Chaga part into the liquid is enhanced. Under ultrasonic extraction, the Chaga / liquid mixture is exposed to ultrasonic waves for a predetermined period of time, typically from a few minutes to several hours, to transfer the components from the Chaga to the liquid. Ultrasonic extraction can be carried out at room temperature or at an elevated temperature.

[0205] The mixture is then filtered to remove solids. Any suitable filtration method can be used depending on how the liquid extract is to be used. For example, if the liquid extract is to be administered orally or topically, the presence of some small chaga mushroom particles (usually <1 mm) may be acceptable, and a filter as coarse as a tea strainer may be acceptable. On the other hand, if the extract is to be sprayed onto a recipient, particles up to 1 mm may clog the spraying device, and a finer filtration method, such as using filter paper, can be used. The extract is preferably stored in an acid-resistant container, such as a glass container.

[0206] Chaga mushroom extract generally has a pH level ranging from 4 - 9. Typically, in the case of longer extraction times and less polar liquids, the pH of the chaga mushroom extract is lower.

[0207] Method for preparing chaga mushroom reductant

[0208] The chaga mushroom is reduced by immersing chopped chaga mushroom in water for a period of time at an elevated temperature, or by cycling through elevated temperatures multiple times. In one method, the chaga mushroom portion is exposed to boiling or significantly elevated temperatures. The chaga mushroom is chopped such that 90% of the particles have a maximum size of less than about 75 mm, preferably less than about 50 mm, more preferably less than about 30 mm, and even more preferably less than about 20 mm in their maximum dimension. The chaga mushroom can also be chopped as finely as discussed above for the extract, e.g., such that 90% of the particles have a maximum size of less than 7 mm, less than about 5 mm, less than about 3 mm, and even less than about 2 mm in their maximum dimension. A reductant of chaga mushroom is prepared using a solvent containing at least water and optionally another liquid present, but the solvent used in the reductant is more polar than the solvent used in the extract.

[0209] In one method, the chopped chaga mushroom is preferably placed in a reducing solvent such as water at a volume ratio of about 1:40 to about 1:2. Preferably, the chopped chaga mushroom is covered by the reducing solvent. The reducing solvent can be brought to a boil and then cooled again in a cycle. During the boil / cool cycle, the reducing solvent is brought to a strong boil for a period of time and held at an elevated temperature for a period of time, and then cooled. In some embodiments, the reducing solvent is brought to a strong boil for 1 - 10 minutes, more preferably 4 - 5 minutes, then to a slow boil for about 10 - 60 minutes, preferably 15 - 45 minutes, more preferably 20 - 30 minutes, where the rest of the cycle allows the water to cool. Exemplary cycles include heating to boiling, achieving a rolling boil for about 4 - 5 minutes, reducing to a slow boil for about 20 - 30 minutes, and cooling for the rest of the cycle. The cycle can take, for example, one hour or longer.

[0210] In another method of forming the reduct, the chopped chaga is heated to an elevated but non-boiling temperature, such as 160°F or higher, for a period of time, such as 24 - 96 hours, 3 - 7 days or even longer.

[0211] The reducing solvent may include a mixture of different liquid solvents. The dielectric constant ε of the reducing solvent is preferably greater than 50, more preferably greater than 60, and may even be greater than 70. For example, in the case where the liquid solvent is 100% water, the dielectric constant εwater is approximately 80.1. Here, the dielectric constant value is provided as the DC voltage dielectric constant. In a mixture of miscible liquids, the dielectric constant can be calculated by taking the volume average of the dielectric constants. For example, in a liquid containing 90 vol% water and 10 vol% ethanol, the dielectric constant of the mixture is given by (0.9xεwater)+(0.1xεethanol) 74.5. The dielectric constant of the reducing solvent is greater than the dielectric constant of the extraction solvent, such as greater by more than 10, greater by more than 20, or even greater by more than 30. In an example where the extraction solvent is 9:1 parts ethanol:water by volume, the dielectric constant is approximately 30.2, while in a reducing solvent containing 9:1 parts water:ethanol by volume, the dielectric constant is approximately 74.5, with a difference of approximately 34.

[0212] The mixture is then filtered to remove solids. Filtration can be carried out after cooling the water. Any suitable filtration method can be used depending on how the liquid reduct is to be used. For example, if the liquid reduct is to be administered orally or topically, the presence of some small particles (usually <1mm) may be acceptable. On the other hand, if the reduct is to be sprayed onto a recipient, particles up to 1mm may clog the spraying device, and a finer filtration method, such as using filter paper, can be used.

[0213] In some methods, a mixture of water and some other non-aqueous solvents (such as food-grade solvents) is used to form the reduct. Examples of non-aqueous solvents can be short-chain alcohols such as ethanol, short-chain diols such as propylene glycol, short-chain acids such as formic acid, acetic acid or lactic acid, short-chain ketones such as acetone, or short-chain esters such as ethyl acetate or n-butyl acetate. If a solvent that is toxic to the patient is used, it can be removed using standard chemical treatment means.

[0214] Method for preparing an extract / reduct mixture

[0215] The extract can be mixed with a reducing agent to produce an extract / reducing agent mixture (ER mixture). The volume ratio of the extract to the reducing agent used to form the ER mixture can cover a wide range, for example: 50:1 (i.e., 10 parts extract: 1 part reducing agent) to 1:50 (i.e., 1 part extract: 50 parts reducing agent), preferably 5:1 to 1:20, more preferably 1:1 to 1:10, and even more preferably 1:3 to 1:7. The volume ratio of the extract to the reducing agent in the ER mixture can be about 1:5. In some methods, the extract is added to the reducing agent in an acid-resistant container (such as a glass container).

[0216] The proposed reaction mechanism

[0217] Without wishing to be bound by the following proposed reaction mechanism theory, the Maillard reaction occurring in the method for preparing the above extract / reducing agent mixture is most likely to induce a condensation reaction. This may start with fructose and proline (amino acids present in high concentrations in honey), and the product of this reaction reacts with methylglyoxal / betulinic acid, as methylglyoxal is a precursor of protein glycation. When some intermediates are very reactive, the condensation reaction with betulinic acid / methylglyoxal, and then with one or more of the fatty acids, can also occur at certain stages of the Maillard reaction. If this occurs, it may result in the production of glycoside-like molecules from fructose / proline / methylglyoxal / betulinic acid and the fatty acid component. If possible, this can help explain the non-polar / polar indications found in the formulation, as some pH tests show various pH levels in the final summary. This can also explain the fluorescent properties of the formulation found in the first in vitro study, as due to absorbance, an alternative to fluorescence measurement must be used for the final measurement. If the resulting active ingredient has a glycoside quality and it survives intact in the digestive system, it may be attractive to the glucose receptors on cancer cells and promote binding.

[0218] Another theory of this reaction mechanism is that the Maillard reaction is initiated within the temperature range at which the above reaction mixture is incubated; the Maillard reaction has the ability to induce a condensation reaction, especially if fructose is involved as it reacts with the available carbonyl groups; the reaction between fructose and proline is highly likely, and the product of this reaction is commercially available from Toronto Research Chemicals; the reaction can also involve glycine or other amino acids; the Maillard reaction can protect the molecule from decomposition in the stomach and small intestine, allowing it to reach the cancer cells intact; the combination of polar and non-polar starting materials that react can promote a spherical structure, a structure that is thought to be observed in some laboratory tests of the substances provided herein; if the structure of the resulting molecule is a micellar structure, this can explain the observed hydrophobic / hydrophilic properties; the micellar structure can facilitate transport through the bloodstream in or around the small intestine, and this seems feasible given the observed particle size.

[0219] Method for preparing a pharmaceutical preparation

[0220] The preparation can be formed using any combination of the products listed above, including chaga extract, chaga reduct, distillate, and residual distillate. Additionally, the preparation can include honey (such as Manuka honey) and a carrier (such as coconut oil or other medium-chain triglycerides). For example, an oral formulation can include a suitable ratio of chaga extract, chaga reduct, and chaga distillate. Generally, the reduct is present in the oral formulation in an amount greater than the chaga extract or distillate, but this is not a requirement. For example, in the preparation, the ratio of reduct to extract can range from 100:1 to 1:100, preferably from 20:1 to 1:10, more preferably from 20:1 to 1:1. The ratio of reduct:residual distillate can also range from 100:1 to 1:100, preferably from 20:1 to 1:10, more preferably from 20:1 to 1:1. The ratio of extract:residual distillate can range from 100:1 to 1:100, preferably from 10:1 to 1:10, more preferably from 5:1 to 1:5. In a specific example, the weight ratio of chaga reduct, chaga extract, and distillate is 5:1:1. Other ratios can be used within the scope limitations discussed.

[0221] Additionally, if administered orally, the preparation can also include Manuka honey, usually but not necessarily in an amount less than the distillate or extract. For example, the weight ratio of chaga reduct, chaga extract, distillate, and Manuka honey in the preparation can be 10:2:2:1. Other ratios can be used. In particular, the weight ratio of reduct:honey can range from 10:0 to 10:10, or from 10:0.1 to 10:10, although ratios greater than about 10:2 can change the consistency of the preparation and also become expensive.

[0222] The preparation can also include a carrier, such as coconut oil or other medium-chain triglycerides, usually but not necessarily in an amount less than the distillate. The weight ratio of reduct:carrier can range from 10:0 to 10:50 or from 10:0.1 to 1:50, depending on the method of administering the preparation. For example, in a liquid preparation to be administered to horse feed, the weight ratio of reduct:carrier can be from 10:0 to about 10:2. In an example of a liquid preparation, the weight ratio of chaga reduct, chaga extract, distillate, and coconut oil in the preparation can be 10:2:2:1. Higher amounts of the carrier can be used for more solid preparations, such as ointments. In an example, the reduct:carrier weight ratio can be 10:10. Other ratios can be used.

[0223] The preparation may contain both manuka honey and a carrier (such as coconut oil or other medium-chain triglycerides). The amounts of manuka honey and the carrier may, but need not, be less than the amounts of the extract and the distillate. For example, the weight ratio of chaga reductant, chaga extract, distillate, manuka honey, and coconut oil in the preparation may be 10:2:2:1:1. Other ratios may be used.

[0224] Alternative preparations are provided for the esterification process of betulinic acid with fructose and proline to produce fructose-amine-triterpenes, and the alternative preparations are the reaction between the above-mentioned chaga extract and proline, fructose, and a fatty acid or other surfactant such as medium-chain triglyceride oil or sunflower lecithin. These are the isolated starting materials originally provided by manuka honey. The fully synthetic product is suspended in the original preparation, and all materials are food-grade and FDA-approved for sale in the original form.

[0225] Administration of the pharmaceutical preparation

[0226] The pharmaceutical preparation can be administered by any suitable means known to those skilled in the art. The pharmaceutical preparation is a liquid and can be administered orally or to solid food. For example, the preparation can be taken orally by the patient, or the preparation can contain additives such as sugar, salt, etc., which can be used to change the flavor to a more preferred taste of the patient. In other methods, the preparation can be added to another liquid that the patient drinks. For example, in the case of a human patient, the preparation can be added to beverages such as coffee, tea, carbonated soda, etc. In the case of an animal patient (such as a horse patient), the pharmaceutical preparation can be added to the animal's water. In other methods, the preparation can be mixed with food. For example, in the case of a human patient, the preparation can be added to soup, included in gravy served on meat, vegetables, or potatoes, or included in a sauce served with, for example, pasta or meat. In the case of an animal patient such as a horse patient, the preparation can be fed directly or sprayed on the horse's feed. When fed directly, additives can be included to mask the taste of the preparation. An example of a taste masking agent is sugar. The preparation is well-absorbed in granular feed. On the other hand, whole grain feed and hay do not absorb the preparation either, and in this case, the preparation can include a coating agent such as medium-chain triglyceride oil so that the preparation adheres to the feed.

[0227] In other methods, the formulation can be administered topically. In some cases, when administered topically, the formulation can be made more viscous, for example by adding a more viscous carrier such as coconut oil or other medium-chain triglyceride oils, or a thicker oil. A towel, cloth piece, or pad soaked in the formulation can be used to administer the formulation topically. Another method is to apply a pad, poultice, etc. that has been soaked in the formulation to the area to be treated and hold the pad, poultice, etc. in place against the skin. The pad, poultice, etc. can be held in place using, for example, an adhesive strip, bandage, or any other suitable method. When the formulation contains a relatively large proportion of carrier, the formulation can have the consistency of an ointment that can be spread over the area of interest.

[0228] In other methods, the formulation can be included in a gel, paste, or lotion that can be applied to the area to be treated.

[0229] In other methods, the formulation can be sprayed onto the area to be treated. In such cases, it may be preferred to include a formulation having a viscous carrier, and the formulation is filtered more finely than is required for oral administration to prevent clogging of the spraying device.

[0230] Regarding the administration of pharmaceutical compositions containing isolated or synthetic chemical compounds represented by Formula 1 and Formula 2, additional methods of administering the pharmaceutical formulation are provided below.

[0231] The pharmaceutical formulation can be administered to a patient at a suitable dosage level. For example, an oral formulation can be administered at a dosage of from 0.1 ounce / 1000 pounds (about 0.065 mL / kg) of patient weight to 1 ounce / 1 pound (about 6.5 mL / kg) of patient weight per day. In some embodiments, the pharmaceutical formulation is administered at a daily dosage of 1 ounce / 100 pounds of patient weight (about 0.65 mL / kg). The pharmaceutical formulation can be provided as a single daily dose or in two or more smaller doses during the day.

[0232] The pharmaceutical formulation can be administered in combination with other therapies. For example, a patient taking the pharmaceutical formulation as described herein can also receive a standard antibiotic regimen.

[0233] Chemical Compounds and Preparation Methods

[0234] Novel chemical compounds are provided herein. The novel compounds are isolated from or synthesized into the pharmaceutical formulation. The following chemical compounds have successfully demonstrated in vitro anti-cancer activity.

[0235] In one embodiment, the chemical compound is represented by the following Formula 1:

[0236]

[0237] wherein R1 - R3 are independently alkyl / alkane, alkene / alkenyl or alkyne / alkynyl having 1 to 20 carbon atoms; benzene / aromatic / phenyl, ether, amide, alkyl halide, amine (-amino), alcohol / hydroxyl / hydroxy (-OH), thiol, aldehyde, ketone, ester / ester quaternary ammonium salt, carboxylic acid (COOH), acid anhydride / acetic anhydride, acyl halide or methyl.

[0238] In another embodiment, the chemical compound is represented by Formula 1:

[0239]

[0240] wherein R1 - R3 are independently alkyl / alkane, alkene / alkenyl or alkyne / alkynyl having 1 to 20 carbon atoms; benzene / aromatic / phenyl, ether, amide, amine (-amino), alcohol / hydroxyl / hydroxy (-OH), thiol, aldehyde, ketone, ester / ester quaternary ammonium salt, carboxylic acid (COOH), acid anhydride / acetic anhydride or methyl.

[0241] In another embodiment, the chemical compound is represented by Formula 1:

[0242]

[0243] wherein R1 - R3 are hydroxyl groups.

[0244] In another embodiment, the chemical compound is represented by Formula 2:

[0245]

[0246] wherein R1 - R7 are independently alkyl / alkane, alkene / alkenyl or alkyne / alkynyl having 1 to 20 carbon atoms; benzene / aromatic / phenyl, ether, amide, alkyl halide, amine (-amino), alcohol / hydroxyl / hydroxy (-OH), thiol, aldehyde, ketone, ester / ester quaternary ammonium salt, carboxylic acid (COOH), acid anhydride / acetic anhydride, acyl halide or methyl.

[0247] In another embodiment, the chemical compound is represented by Formula 2:

[0248]

[0249] wherein R1 - R7 are independently alkyl / alkane, alkene / alkenyl or alkyne / alkynyl having 1 to 20 carbon atoms; benzene / aromatic / phenyl, ether, amide, amine (-amino), alcohol / hydroxyl / hydroxy (-OH), thiol, aldehyde, ketone, ester / ester quaternary ammonium salt, carboxylic acid (COOH), acid anhydride / acetic anhydride or methyl.

[0250] In another embodiment, the chemical compound is represented by Formula 2.

[0251]

[0252] wherein R1 - R7 are independently hydroxyl groups.

[0253] As described in more detail in the examples, the chemical compounds provided above were isolated from pharmaceutical formulations.

[0254] Analytical techniques such as HPLC, GC, mass spectrometry or NMR were used to confirm the isolate or synthetic compound as a betulinic acid derivative.

[0255] Using Figure 58 The chemical compounds were synthesized using the reaction pathways shown in. The compounds represented by Formula 1 were synthesized using a reaction temperature of 240 °C to 260 °C. The compounds represented by Formula 2 were synthesized using a reaction temperature of 240 °C to 260 °C.

[0256] Pharmaceutical compositions and preparation methods

[0257] The pharmaceutical compositions provided herein contain one or more of the above chemical compounds in combination with a suitable carrier. The pharmaceutical compositions are prepared by combining or mixing one or more of the chemical compounds provided above with a pharmaceutically acceptable carrier according to methods known to those skilled in the art.

[0258] Suitable carriers include artificial and biological delivery systems such as, but not limited to, liquids, gels, suspensions, emulsions, dendrimers, quantum dots, hydrogels, aerogels, foams and creams. Suitable carriers also include nano - drug delivery carriers such as, but not limited to, nanospheres, hydrogels with and without nanoparticles, nanocapsules, nanotubes and nanoparticles. Microsystems are also suitable carriers such as, but not limited to, patches and micropumps. Vesicles of biological or inert origin are also included in the list of suitable carriers such as, but not limited to, liposomes, aquasomes, niosomes, ethosomes, polymeric vesicles and cubosomes. In addition, carriers having a biological origin such that they provide a class of macromolecules include, but are not limited to, lipids, carbohydrate structures, proteins, peptides and nucleic acids.

[0259] Administration of the pharmaceutical composition

[0260] The pharmaceutical composition can be administered by any suitable means known to those skilled in the art. Suitable delivery systems include passive delivery systems, such as drug delivery via diffusion, or active delivery systems, such as drug delivery via digestion. The pharmaceutical composition can be administered orally in liquid form or in solid form (such as pills, capsules or powders); intravenously, topically, subcutaneously, vaginally or rectally, for example, with a medicated suppository; via ocular means, such as eye drops, ointments or medicated contact lenses; via transdermal means such as patches, via pulmonary means such as inhalers, via microelectrochemical systems, or via micropumps. Due to the potential of nanoparticle carriers, there are many administration options and are known to those skilled in the art.

[0261] The oral formulation of the pharmaceutical composition can be administered to a patient at a suitable dose level calculated to achieve the desired chemotherapeutic effect. In one embodiment, an oral formulation of a compound of Formula 1 or 2 having a concentration of 10 - 1000 μg / mL in an aqueous solution can be administered at a dose of 0.1 ounce / 1000 pounds body weight (about 0.065 mL / kg patient weight) to 1 ounce / 1 pound (about 6.5 mL / kg patient weight) per day. In some embodiments, the oral formulation is administered at a daily dose of 1 ounce / 100 pounds patient weight (about 0.65 mL / kg). The oral formulation can be provided as a single daily dose or in two or more smaller doses throughout the day. If the pharmaceutical composition exhibits increased potency, the drug dose is reduced accordingly. In another embodiment, the dose range of the pharmaceutical composition is 0.001 mL / 100 pounds body weight to 3 mL / 100 pounds body weight. In another embodiment, the dose of the pharmaceutical composition is 0.03 mL / 100 pounds body weight.

[0262] The pharmaceutical composition can be administered in combination with other therapies. For example, the pharmaceutical composition can be administered to a patient simultaneously or sequentially with a second therapeutic agent, which is, for example but not limited to, a chemotherapeutic drug or steroid, an antibiotic, a vitamin, antibody therapy, gene therapy, etc.

[0263] The present invention will now be further described with reference to the following non-limiting examples.

[0264] Examples

[0265] Example 1: Inonotus obliquus Ethanol Extract

[0266] Use a coffee grinder to chop the chaga mushroom into pieces smaller than about 3 mm. Mix one portion of the chopped chaga with two portions of ethanol. Add water to the ethanol / chaga mixture at a volume ratio of 70:30 ethanol / water. Then let the water / ethanol / chaga mixture stand at room temperature for a period of three months, at which time the mixture is filtered through a sieve with a pore size of about 1 mm to substantially remove all solid matter. After filtration, store the mixture in a glass jar as an ethanol extract.

[0267] Example 2: Chaga reductate

[0268] Use a coffee grinder to chop the chaga mushroom into pieces smaller than about 3 mm. Place about 1.5 cups of the chopped chaga in a jar containing about 4.8 liters of room temperature water. The water has been reverse osmosis filtered. Heat the water and bring it to a strong rolling boil for 4 - 5 minutes, then reduce the temperature to maintain a low boil for about 20 - 30 minutes. Remove the heat and let the water cool for about 30 minutes, such that the heating and cooling cycle takes about 1 hour. This cycle is repeated 5 - 6 times over a period of 5 - 6 hours. Filter the chaga / water mixture through a sieve with a pore size of about 1 mm to substantially remove all solid matter from the water. After cooling and filtration, store the mixture as chaga reductate.

[0269] Example 3: Extract / reductate mixture

[0270] Add 1 volume portion of the ethanol extract to 5 volume portions of the chaga reductate in a glass container to form an extract / reductate mixture. The extract / reductate mixture is also referred to hereinafter as the "oral formulation solution".

[0271] Example 4: Distillate

[0272] Heat the mixture to the temperature at which steam is first detected escaping from the mixture, thereby distilling the extract / reductate mixture. Cool and condense the material escaping from the mixture to form a distillate. The amount of distillate obtained from 384 ounces of the ER mixture is in the range of 30 - 45 ounces. Thus, the remaining fraction is present in an amount of 339 - 354 ounces.

[0273] Example 5: Oral formulation I

[0274] Mix the reductate, extract, and remaining distillate in a weight ratio of 5:1:1 to prepare Oral Formulation I.

[0275] Example 6: Oral formulation II

[0276] Mix the reductate, extract, remaining distillate, Manuka honey, and coconut oil in a weight ratio of 10:2:2:1:1 to prepare Oral Formulation II.

[0277] Example 7: Topical Preparation I

[0278] Equal volume portions of the remaining distillate and the ethanol extract were mixed together to form an intermediate mixture, and manuka honey was added to the intermediate mixture at a volume ratio of 8 parts of the intermediate mixture to 1 part of manuka honey. The mixture with honey was stirred to dissolve the honey. Then coconut oil was added to the honey-containing mixture (about 1 part of coconut oil by volume : 1 part of the honey-containing mixture) to prepare Topical Preparation I.

[0279] Example 8: Topical Preparation II

[0280] Preparation II was prepared using the same procedure as Preparation I, except that the intermediate mixture contained two parts of chaga reductant : one part of distillate and one part of ethanol extract. The active ingredient in all the preparations described in these examples is the chemical compound represented by Formula 1.

[0281] Example 9: Case Study

[0282] The above chaga-based preparations and mixtures were administered to a number of equine and human subjects suffering from different types of cancer. Each of the following case studies describes the manner in which the chaga-based preparations and mixtures were administered and the progression of the cancer after administration.

[0283] Case Study 1: Equine Subject 1 - Metastatic Melanoma

[0284] A nine-year-old grey Thoroughbred gelding was found to have a tumor growing in its esophageal region and was subsequently diagnosed with multiple metastatic melanoma tumors that invaded its esophagus and were growing rapidly. The size of the tumor at diagnosis was approximately 5 cm x 6 cm. Before the tumor progressed to compromise all functions of the esophagus, a consultation with experts resulted in a plan for euthanasia. There were no surgical options. The consulting veterinarian recommended against performing a biopsy that carried the risk of worsening the cancer cells and causing the cells to metastasize at an accelerated rate so as to know the most likely cause of the tumor accurately.

[0285] The patient began a chaga / manuka honey regimen in the hope that the regimen would slow the progression of the tumor and give the patient more time before euthanasia. The regimen consisted of an oral component and a topical component. The oral component included administering an oral formulation solution to the patient once a day for 90 days. The mixture was administered by adding it to the patient's feed. The topical component included administering Preparation I by spraying it on the tumor site every 12 - 24 hours using a manual pump sprayer.

[0286] After 90 days of treatment, the patient's owner confirmed to the consulting veterinarian via visual inspection that the tumor had regressed and there was no visual or physical evidence of the tumor. The patient continued to receive the oral formulation and the topical formulation for an additional 60 days. Within the two-year period following the first administration of the chaga / manuka honey regimen, the melanoma tumor did not recur.

[0287] Case Study 2: Equine Subject 2 - Squamous Cell Carcinoma

[0288] A 14-year-old spotted draft horse was identified as having a foul-smelling, non-healing, deep first lesion approximately 10 cm x 7.5 cm on the left side of the base of the tail, along with a visible second lesion approximately 20 mm wide distal to the first area. A third lesion approximately 18 mm wide formed by thickened skin was felt under the skin and was medial and distal to the second lesion. Biopsies of the two areas of the lesion were taken for histopathology. Initial examination of the preliminary sections revealed eosinophilic / granulomatous inflammation. Subsequently, it was confirmed that the lesion contained squamous cell carcinoma (SCC).

[0289] On day 0, those portions of the SCC tumor that were considered safely removable were surgically excised, with suspected unclean margins. The surgery left an open lesion approximately 7.5 cm x 10 cm x 28 cm. The surgeon was concerned about permanent nerve damage to the patient's tail due to the rapid growth of the SCC. On day 3, the patient began the chaga / manuka honey regimen. The topical formulation I was applied to the lesion site at least once every 48 hours for 77 days. The oral formulation I was administered daily during the last two weeks of the 77-day period by spraying 10 ounces of the solution on the patient's feed.

[0290] During treatment with the chaga / manuka honey regimen, the lesion improved rapidly. The lesion had significantly disappeared by day 59. The patient's primary treating physician confirmed via visual inspection that all SCC (confirmed and suspected) had been resolved.

[0291] Due to the area of concern, continued close monitoring and debridement were carried out. The involvement of nematodes at the lesion site required additional use of a skin ointment to prevent recurrence. Over a six-week period, the patient continued to receive a single 10-ounce dose of the oral formulation I every two days. Starting on day 134, the patient received a first course of antibiotics for three weeks to treat burns caused by nematode infection. The patient began a half-strength daily dose (5 ounces / day) on day 165 until approximately day 203, at which time the dose was restored to 10 ounces / day. The patient received a second course of antibiotics between days 186 and 207. As of day 223, the site of the original cancer lesion remained clear on visual inspection, and hair had begun to regrow at the lesion site. Case Study 3: Equine Subject 3 - Melanoma

[0292] A 10-year-old grey Irish gelding sports horse was diagnosed with multiple sarcoids and melanoma growths. On day 0, the melanoma was removed from the left neck, near the nuchal ligament. The sarcoid growth was removed from the right medial hock. The patient was given ceftiofur (antibiotic) and flunixin (non-steroidal anti-inflammatory drug). After treatment, some of the masses regressed while others regrew. When tumour regrowth was observed, the sutures were removed on day 21. Sarcoid implants were made in the left neck. The patient continued to have subcutaneous tumours around the neck area.

[0293] Around day 130, the patient began treatment with the Chaga / Mānuka honey protocol. Topical preparation I was applied to the tumours at the site of the neck lesions in the neck area at least every 48 hours. Oral preparation I was administered daily by spraying 10 ounces of the solution on the patient's feed. Around day 190, the patient's owner reported that the patient's response to the protocol was positive and that the tumours located on both sides of its neck and head had significantly regressed in size and shape. According to palpation, the remaining tumour masses were reported to have changed in feel. The remaining masses were reported to feel like "regular" melanoma masses, without palpable internal ridges and the mobile soft tissue around them that is typically associated with growing melanoma. The patient continued the Chaga / Mānuka honey protocol until around day 220.

[0294] Around day 365 and after, the patient returned to competition in the summer and autumn. Around day 585, the patient was still alive at the time of writing.

[0295] Case study 4: Human subject 1 - Prostate cancer

[0296] The patient's PSA was measured at 66.75 on day 0, with concern for bone lesions. On day 42, the patient was diagnosed with prostate cancer, Gleason score 7, and the cancer was classified as moderately graded. The patient initially started hormone therapy in the hope that this would slow the progression of the prostate cancer. The patient was also informed about the bone lesions on his scans. On day 42, the patient started taking degarelix. On day 72, the patient started taking bicalutamide. Around day 92, the patient's PSA was measured at 64. On day 112, the patient was diagnosed with basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). The BCC and SCC sites were surgically removed around day 122.

[0297] The patient began voluntary daily use of the chaga / manuka honey formula at around day 92 by adding approximately 2 ounces of oral preparation I to a cup of coffee. The patient also voluntarily began taking other herbal supplements. The patient continued to experience recurrent kidney infections (which may be due to the location and size of the prostate tumor) until around day 145. The patient also had a catheter placed on day 0 and continued to use the catheter until day 145. On day 154, the patient's PSA was measured at 13.25.

[0298] At around day 154, the patient was able to remove the catheter and has not required catheter placement since then. At around day 214, the patient's PSA was measured at 6.4. The patient's PSA remained below 8 until a measurement around day 440 confirmed a PSA level of 11.9. As a result, the patient stopped taking the oral formula solution around day 381. The patient resumed the chaga / manuka honey regimen around day 440.

[0299] After day 440, the patient's cancer diagnosis was updated to benign, and he continued the chaga / manuka honey regimen. On day 560, the patient's PSA was measured at 6.25 and his health status was maintained.

[0300] Case Study 5: Human Subject 2 - Colorectal Cancer

[0301] A male patient, around 74 years old, was diagnosed with stage IV colorectal cancer that had metastasized to the liver, lungs, and kidneys. The cancer was considered inoperable. After two years of chemotherapy treatment (including experimental treatment), the patient entered hospice care, received palliative care, and was given a life expectancy of 2 - 6 months. One month after entering hospice care, the patient began a regimen of oral preparation II. From days 1 - 7, 8 ounces of preparation II were administered to the patient daily. On day 8 and subsequently, 4 ounces of oral preparation II were administered to the patient daily. The patient's "cancerous cough" subsided during the first two weeks of treatment and disappeared by day 15. It was reported that the patient had improved appetite, reduced pain, improved sleep cycle, and improved vision by around day 45. Around day 55, the patient began walking two miles daily. Around day 105, laboratory test results confirmed that the patient's CEA marker had decreased from 875 measured on day 0 to 9.1.

[0302] Case Study 6: Human Patient with Breast Cancer Metastatic to Leptomeningeal Carcinomatosis

[0303] LMC is a 100% lethal diagnosis, with a survival rate of less than 8 weeks without chemotherapy treatment, and in the case of treatment for 2 - 3 months, up to 12 months in some cases. Six weeks after the LMC diagnosis, a patient who opted out of chemotherapy care due to lack of long-term survival rate began taking an oral preparation II at 5 - 32 ounces per day. From day 0 - 120, the patient maintained normal life functions. Test results around day 125 showed normal standard CBC markers and normal CEA markers. The patient remained stable at the 300th day in the past, with daily life functions.

[0304] Example 10: In Vitro Cell Viability and ATP / ADP Ratio Study

[0305] Materials: The cells SK Mel 28, MDA-MB-231, and HepG2 used in this study were originally obtained from ATCC (American Type Culture Collection, Manassas, VA) and maintained according to the following protocol. Five samples tested in this study were prepared. Samples 1 - 4 are various components of the final preparation represented by Formula 1. Thus, this example is for comparing the anticancer activities of these various components for preparing the compound of Formula 1 with the chemical compound of Formula 1 itself to show the increased bioavailability and function of the chemical compound of Formula 1.

[0306] Sample 1 is the reducing component for preparing the pharmaceutical composition; Sample 2 is the extract component for preparing the pharmaceutical composition, Sample 3 is the distillate produced only during the preparation of the pharmaceutical composition; Sample 4 is a mixture of the reducing component, extract, distillate, and the chemical compound represented by Formula 1. Samples 5 and 6 are chemical compounds represented by Formula 1 at different concentrations, where Sample 6 is at a higher concentration of Formula 1 than Sample 5. Materials ELDT-100 (Lot number: CC01A25) and CQBL-05K (Lot number: CB08A31) were provided by BioAssay Systems (Hayward, CA).

[0307] Methods: SK-MEL-28 and HepG2 cell lines from ATCC were grown in T75 culture flasks with ATCC-recommended medium supplemented with antibiotics (10 v% FBS, 1 w% streptomycin / penicillin, Eagle's Minimum Essential Medium) and grown in an incubator at 5% CO2 and 37 °C. The MDA-MB-231 cell line was obtained from ATCC and grown in T75 culture flasks with complete medium (10 v% FBS, 1 w% streptomycin / penicillin, Roswell Park Memorial Institute 1640 medium) as described by Huang Z et al. (Onco Targets Ther. 2020;13:5395-5405) and supplemented with antibiotics and grown in an incubator at 5% CO2 and 37 °C. The cell lines were grown to 70-80% confluence before being seeded in 96-well plates for ELDT-100 and CQBL-05K.

[0308] For EnzyLight TM For the ATP assay (EATP-100, BioAssay, Hayward, CA), the SK Mel 28 and MDA-MB-231 cell lines were seeded at a density of 5,000 cells / well, while the HEPG2 cells were seeded at a density of 2,500 cells / well in 96-well white opaque tissue culture plates and then treated with various samples at 1-fold, 0.1-fold, 0.01-fold, and 0.001-fold dilutions for 48 hours. Compound dilutions were prepared in diH2O. After 48 hours, the medium was removed, then ATP reagent was added, and readings were taken on a photometer after one (1) minute. Blanks were obtained by processing and measuring cell-free complete medium, and cells not treated with one of the samples were used as controls.

[0309] For CellQuanti-Blue TM For the cell viability assay (CQBL-05K, BioAssay, Hayward, CA), the cell lines were seeded at a density of 16,000 to 20,000 cells / well in 96-well clear-bottom black tissue culture plates. (Each experiment was plated at a uniform cell density.) The cells were then treated with one of the various dilutions of the sample and incubated for 48 hours. Compound dilutions were prepared in diH2O. Then CellQuanti-Blue reagent was added to the wells and incubated at 37 °C for 1 hour. Fluorescence was then measured at 530 nm excitation and 590 nm emission. Blanks were obtained via cell-free complete medium and by treating cells with reconstituted 1 w% saponin.

[0310] Calculate the standard error using error propagation methods (see www.chem.libretexts.org / Bookshelves / Analytical_Chemistry / Supplemental_Modules_(Analytical_Chemistry) / Quantifying_Nature / Significant_Digits / Propagation_of_Error.). Read the samples on a MolecularDevices SpectraMax M2 instrument (BioAssay, Hayward, CA).

[0311] Results: The results of the ATP / ADP ratio studies and cell viability studies for each cell line and each of the five samples, including the compound represented by Formula 1, are shown in the figures below.

[0312] SK-Mel 28: The SK-Mel 28 cell line showed significant inhibition of ATP levels with 1-fold concentrations of Samples 1, 2, 4, and 5, where the ATP levels recovered as the sample levels decreased ( Figure 1 ). Figure 1 The relative ATP levels of SK Mel 28 cells after treatment with the compound are provided.

[0313] Plate SK Mel 28 cells as described in the method and then grow for 24 hours before measuring the relative ATP and ADP levels. The relative luminescence (RLU A measurement) of SK Mel 28 cells represents the relative ATP levels present in the cells.

[0314] Sample 3 showed no effect in the SK-Mel 28 cell line. Unfortunately, the ADP portion of the ATP / ADP ratio assay was less effective, so the clear picture of apoptosis relative to necrosis (necroptosis) was less clear ( Figure 2 ). Figure 2 The relative ADP levels of SK Mel 28 cells after treatment with the compound are provided.

[0315] Plate SK Mel 28 cells as described in the method and then grow for 24 hours before measuring the relative ATP and ADP levels. The relative luminescence (RLU C–RLU B measurement) of SK Mel 28 cells represents the relative ADP levels present in the cells after treatment with the compound.

[0316] The cell viability assessment showed a trend similar to the ATP levels but with a clearer gradation ( Figure 3)。For example, 0.1-fold and 0.01-fold of Compound 5 have relative ATP values within the same range, but the cell viability assays showed distinct differences at all three concentrations. Figure 3 Provided is the use of CellQuanti-Blue TM Cell viability assay for the assessment of the cell viability of SK-Mel 28 cells, which shows the percentage of viability relative to untreated cells.

[0317] MDA-MB-231: The MDA-MB-231 cell line showed a very significant inhibition of ATP levels with 1-fold concentrations of Samples 1, 2, 4, and 5, where the ATP levels recovered as the sample levels decreased ( Figure 4 )。 Figure 4 Provided is the relative ATP level of MDA-MB-231 cells after treatment with the samples. Plate MDA-MB-231 cells as described in the method and then grow for 24 hours before measuring the relative ATP and ADP levels. The relative luminescence (RLU A measurement) of MDA-MB-231 cells represents the relative ATP level present in the cells. Sample 3 showed no effect in the MDA-MB-231 cell line. The ADP portion of the ATP / ADP ratio assay was more effective than SK-Mel28, but a clear picture of relative apoptosis to necrosis (necroptosis) could not be obtained at 24 hours ( Figure 5 )。 Figure 5 Provided is the relative ADP level of MDA-MB-231 cells after treatment with the samples. Plate MDA-MB-231 cells as described in the method and then grow for 24 hours before measuring the relative ATP and ADP levels. The relative luminescence (RLU C–RLU B measurement) of MDA-MB-231 cells represents the relative ADP level present in the cells after compound treatment.

[0318] Suspected at 1-fold samples, we see necrosis (necroptosis), but at lower concentrations, we start to see apoptosis. The cell viability assessment showed an unusual trend for Samples 2, 3, and 4, with opposing evidence relationships ( Figure 6 )。 Figure 6 Provided is the cell viability assessment of MDA-MB-231 cells using the CellQuanti-Blue cell viability assay. The percentage of viability relative to untreated cells is shown. For Sample 5, the normal relationship is clearly that cell viability increases as the sample concentration decreases. The behavior of Sample 1 was inconsistent with the other four samples and did not show an obvious trend in cell viability.

[0319] HepG2: The HepG2 cell line showed a significant inhibition of ATP levels with 1-fold concentrations of Compounds 1, 2, 4, and 5, where the ATP levels recovered as the sample levels decreased (Figure 7 )。 Figure 7 The relative ATP levels of HepG2 cells after treatment with the samples were provided. HepG2 cells were plated as described in the method and then grown for 24 hours before measuring the relative ATP and ADP levels. The relative luminescence (RLU A measurement) of HepG2 cells represents the relative ATP levels present in the cells. Sample 3 showed no effect in the HepG2 cell line. The ADP portion of the ATP / ADP ratio determination was more effective for the HepG2 cell line ( Figure 8 )。 Figure 8 The relative ADP levels of HepG2 cells after treatment with the samples were provided. HepG2 cells were plated as described in the method and then grown for 24 hours before measuring the relative ATP and ADP levels. The relative luminescence (RLU C–RLU B measurement) of HepG2 cells represents the relative ADP levels present in the cells after sample treatment. Signs of apoptosis were present at 0.1-fold and 0.01-fold concentrations, and possible necrosis (necroptosis) was present at 1-fold concentration. The cell viability assessment showed a trend similar to the ATP levels of Samples 1, 4, and 5, but Sample 2 again showed an inverse relationship with cell viability and the samples, where a decrease in the samples led to a decrease in cell viability ( Figure 9 )。 Figure 9 The cell viability assessment of HepG2 cells using the CellQuanti-Blue cell viability assay was provided. The percentage of viability relative to untreated cells was shown.

[0320] Conclusion: In the ATP assay, all three cell lines (SK-Mel 28, MDA-MB-231, and HepG2) showed similar behavior towards all five samples. Samples 1, 2, 4, and 5 at 1-fold dilution inhibited ATP production, and ATP production recovered as the sample dilution increased. Among the three cell lines, the ADP component was less clear in terms of apoptosis relative to necrosis (necroptosis), where only HepG2 gave reasonable performance. The interpretation of cell viability showed opposite behavior for Samples 2, 3, and 4 compared to that observed in the MDA-MB-231 cell line.

[0321] Example 11: In Vitro Cell Viability and ATP Studies

[0322] Materials: The cells SK Mel 28, MDA-MB-231, and HepG2 used in this study were originally obtained from ATCC (American Type Culture Collection, Manassas, VA) and maintained according to the following protocol. The samples tested in this study were prepared and stored at 4 °C until use. Samples 5 and 6 are compounds represented by Formula 1 at different concentrations, where Sample 6 has a 10% higher concentration of the chemical compound represented by Formula 1 than Sample 5. The materials EATP-100 (Lot No.: CC11A01) and CQBL-05K (Lot No.: CC10A03) were provided by BioAssay Systems (Hayward, CA).

[0323] Methods: The SK-MEL-28 and HepG2 cell lines obtained from ATCC were grown in T75 culture flasks with ATCC-recommended medium (10 v% FBS, 1 w% streptomycin / penicillin, Eagle's Minimum Essential Medium) supplemented with antibiotics and grown in an incubator at 5% CO2 and 37 °C. The MDA-MB-231 cell line from ATCC was grown in T75 culture flasks using the complete medium (10 v% FBS, 1 w% streptomycin / penicillin, Roswell Park Memorial Institute 1640 medium) described by Huang Z et al. (Onco Targets Ther. 2020;13:5395-5405) and supplemented with antibiotics and grown in an incubator at 5% CO2 and 37 °C. The cell lines were grown to 70-80% confluence before being seeded in 96-well plates for ELDT-100 and CQBL-05K.

[0324] For EnzyLight TM ATP assay (EATP-100, BioAssay, Hayward, CA), the SK Mel 28 and MDA-MB-231 cell lines were seeded at a density of 5,000 cells / well, while the HepG2 cells were seeded at a density of 2,500 cells / well in 96-well white opaque tissue culture plates and then treated with 1-fold, 0.1-fold, 0.01-fold, and 0.001-fold dilutions of the samples for 48 hours. The compound dilutions were prepared in diH2O. After 48 hours, the medium was removed, then the ATP reagent was added, and the readings were taken on a photometer after one (1) minute. The blank was obtained by processing and measuring the cell-free complete medium, and the cells not treated with the sample were used as the control. For CellQuanti-Blue TMCell viability assay (CQBL-05K, BioAssay, Hayward, CA), the cell lines were seeded in 96-well clear-bottom black tissue culture plates at a density of 16,000 to 20,000 cells / well. (Each experiment was plated at a uniform cell density.) The cells were then treated with various dilutions of the sample and incubated for 48 hours. Compound dilutions were prepared in diH2O. Then CellQuanti-Blue reagent was added to the wells and incubated at 37 °C for 1 hour. Fluorescence was then measured at 530 nm excitation and 590 nm emission. Blanks were obtained via cell-free complete medium and by treating cells with reconstituted 1% saponin. Standard error was calculated using the error propagation method (see www.chem.libretexts.org / Bookshelves / Analytical_Chemistry / Supplemental_Modules_(Analytical_Chemistry) / Quantifying_Nature / Significant_Digits / Propagation_of_Error.). Samples were read on a Molecular Devices SpectraMax M2 instrument (BioAssay, Hayward, CA).

[0325] Results: The results of the ATP studies and cell viability studies for each cell line and each of the five samples are shown in the figures below.

[0326] HepG2: The HepG2 cell line demonstrated inhibition of ATP levels with 1x concentrations of Samples 5 and 6, with ATP levels recovering as the sample levels decreased ( Figure 10 ). The data were log-transformed to better visualize the data ( Figure 11 ). The cell viability assessment showed a trend similar to the ATP levels for the two samples ( Figure 12 and 13 ). By cell viability measurement, Sample 6 demonstrated greater cytotoxicity compared to Sample 5, especially at lower doses. Relative IC50 values were obtained for Sample 5 and Sample 6, with Sample 6 being relatively more potent ( Figure 14 and 15 ).

[0327] MDA-MB-231: The MDA-MB-231 cell line demonstrated significant inhibition of ATP levels with 1x concentrations of Samples 5 and 6, with ATP levels recovering as the sample levels decreased ( Figure 16 and 17)。Due to reasons that could not be determined, ATP quantification was proven challenging (the ATP standard curve was a problem), so only relative ATP levels are shown. For Samples 5 and 6, the expected relationship was clearly that cell viability increased as the sample concentration decreased ( Figure 18 and 19 ). IC50 assays showed that Sample 6 had greater cytotoxicity in MDA-MB-231 cells compared to Sample 5 ( Figure 20 and 21 ).

[0328] SK-Mel 28: The SK-Mel 28 cell line demonstrated inhibition of ATP levels with 1-fold concentrations of Compounds 5 and 6, with ATP levels recovering as the sample levels decreased ( Figure 22 and 23 ). As in previous studies, the ATP pictures were less clear than those for HepG2 and MDA-MB-231 ( Figure 24 and 25 ). Cell viability assessment was less clear, which was contrary to previous work that showed a clear effect of Sample 5 on cell viability ( Figure 26 and 27 ). IC50 assessment indicated that the samples were less toxic to SK-Mel 28 cells compared to MDA-MB-231.

[0329] Conclusion: Samples 5 and 6 demonstrated similar behavior in the MDA-MB-231 and HepG2 cell lines, which was consistent with the results obtained in the study described in Example 10 above. The behavior of the SK-MEL 28 cell line was more erratic and did not replicate the previous results, except for the ATP assay, which did show some similarity to previous work. This was most likely due to the decomposition timeline of the samples. The SK-Mel study was conducted last. In all cases, as shown by the IC50 curves, Sample 6 was more effective than Sample 5. (It should be noted that the IC50 values obtained were relative values compared to the samples at 1-fold concentration.) One of the challenges complicating the work was synchronizing the cell lines with sample availability, as the samples were somewhat unstable in nature. There were also difficulties with the HepG2 cell line, which made it necessary to obtain fresh cells again from the ATCC.

[0330] Example 12: In Vitro Cell Viability and ATP Studies - Drug Research

[0331] Materials: The cell lines HCT-116, SK Mel 28, MDA-MB-231, and HepG2 used in this study were obtained from ATCC (American Type Culture Collection, Manassas, VA) and maintained according to the following protocol. Samples for this study were prepared and stored at 4 °C until use. Samples 7 and 8 were compounds represented by Formula 2 at different concentrations, where Sample 7 had a 10% higher concentration of the chemical compound represented by Formula 1 than Sample 8. The materials EATP-100 (lot number: CD07A13) and CQBL-10K (lot number: CC10A03) were provided by BioAssay Systems (Hayward, CA).

[0332] Methods: The SK Mel 28 and HepG2 cell lines were obtained from ATCC and grown in T75 culture flasks with ATCC-recommended medium supplemented with antibiotics (10 v% FBS, 1 w% streptomycin / penicillin, Eagle's Minimum Essential Medium) and grown in an incubator at 5% CO2 and 37 °C. The MDA-MB-231 cell line was obtained from ATCC and grown in T75 culture flasks using the complete medium described by Huang Z et al. (Onco Targets Ther. 2020;13:5395-5405) and supplemented with antibiotics (10 v% FBS, 1 w% streptomycin / penicillin, Roswell Park Memorial Institute 1640 medium) and grown in an incubator at 5% CO2 and 37 °C. The HCT-116 cell line was obtained from ATCC and grown in T75 culture flasks with ATCC-recommended medium supplemented with antibiotics (10 v% FBS, 1 w% streptomycin / penicillin, McCoy's 5a Modified Medium) and grown in an incubator at 5% CO2 and 37 °C. The cell lines were grown to 70-80% confluence before being seeded in 96-well plates for EATP-100 and CQBL-10K assays.

[0333] The cells were treated with Samples 7 and 8 at the following concentrations for 48 hours (compound dilutions were prepared in diH2O):

[0334] Sample 7: 1372.2, 457.4, 152.5, 50.8, 16.9, 5.65, and 1.88 μM

[0335] Sample 8: 1571.8, 523.9, 174.6, 58.2, 19.4, 6.47, and 2.16 μM

[0336] For EnzyLight TMATP assay (EATP-100, BioAssay, Hayward, CA), SK Mel 28 and MDA-MB-231 cell lines were seeded at a density of 5,000 cells / well, while HepG2 and HCT-116 cell lines were seeded at a density of 2,500 cells / well in 96-well white opaque tissue culture plates.

[0337] After 48 hours, the medium was removed, then ATP reagent was added, and readings were taken on a photometer after one (1) minute. A blank was obtained by measuring cell-free complete medium, and cells untreated with any sample were used as controls.

[0338] For CellQuanti-Blue TM Cell viability assay (CQBL-10K, BioAssay, Hayward, CA), MDA-MB-231, HCT-116 and HepG2 cell lines were seeded at a density of 10,000 cells / well, while SK MEL 28 cell line was seeded at a density of 20,000 cells / well in 96-well clear bottom black tissue culture plates. Cells were then treated with samples at the various dilutions indicated above and incubated for 48 hours. CellQuanti-Blue reagent was added directly to the wells and incubated at 37 °C for 1 hour. Fluorescence was measured at 530 nm excitation and 590 nm emission. Blanks were obtained via cell-free complete medium and by treating cells with reconstituted 1 w% saponin.

[0339] Data were analyzed using Microsoft Excel. The data were log-transformed for plotting in GraphPad Prism 4.0 (BioAssay, Hayward, CA). Standard error was calculated using the error propagation method according to www.chem.libretexts.org / Bookshelves / Analytical Chemistry / Supplemental Modules(Analytical Chemistry) / Quantifying_Nature / Significant_Digits / Propagation_of_Error).

[0340] Samples were read on a Molecular Devices SpectraMax M2 instrument (BioAssay, Hayward, CA).

[0341] Results: A summary of the IC50 results for the cell viability and ATP studies is shown in the table below.

[0342]

[0343] HCT-116: The HCT-116 cell line showed inhibition of ATP levels by Samples 7 and 8 ( Figure 28 and 29 ). Cell viability assessment showed a trend opposite to the ATP levels ( Figure 30 and 31 ). For ATP measurement, compared with Sample 8, Sample 7 showed evidence of a lower IC50 value. Conversely, according to cell viability measurement, compared with Sample 7 with a lower IC50 value, Sample 8 showed greater cytotoxicity.

[0344] HepG2: The HepG2 cell line showed inhibition of ATP levels by Compounds 7 and 8 ( Figure 32 and 33 ). Cell viability assessment showed a trend similar to the ATP levels of both samples ( Figure 34 and 35 ). By cell viability measurement, compared with Sample 7, Sample 8 showed greater cytotoxicity. The IC50 values of Samples 7 and 8 were obtained, with Sample 8 being more effective.

[0345] MDA-MB-231: The MDA-MB-231 cell line showed significant inhibition of ATP levels by Samples 7 and 8 ( Figure 36 and 37 ). IC50 determination showed that compared with Sample 7, Sample 8 had greater cytotoxicity in MDA-MB-231 cells ( Figure 38 and 39 ).

[0346] SK-Mel 28: The SK Mel 28 cell line showed inhibition of ATP levels by Samples 7 and 8 ( Figure 40 and 41 ). IC50 assessment of cell viability showed that compared with the other three cell lines, the two samples were more toxic to SK-MEL 28 cells ( Figure 42 and 43 ).

[0347] The two samples 7 and 8 studied in this work showed similar behavior in all four cell lines. When treated with Sample 7, the ATP levels were lower, but based on cytotoxicity, Sample 8 was more effective than Sample 7, as shown by the IC50 curves measured using CQBL-05K. Notably, even at the lowest level of sample treatment, the ATP levels in HepG2 were significantly lower than those in the other three cell lines. It is also worth noting that significantly lower IC50 values were observed for the cell viability determination of SK Mel 28 compared with the other three cell lines.

[0348] Example 13: NMR Observation

[0349] The chemical structure of the compound represented by Formula 1 was confirmed by NMR analysis as shown in Figure 56 and 57 . The percentage yield was consistent with the estimates in the published literature for the extract after the reaction of the starting materials, and the samples analyzed showed consistent measurements of the percentage yield, which improved upon previous research attempts.

[0350] The amount of the compound produced by the reaction was sufficient for the proposed activity of the compound, and when properly separated and concentrated, led to a higher percentage yield of the compound sufficient for clinical use.

[0351] The major H peak was selected for quantification because it was the peak visible in the sample. The azeotropic nature of the water / ethanol extract explained why air drying did not allow the solid to progress to complete dryness, but did suffice well enough to concentrate the sample to more clearly identify the peaks. Deuterated methanol was used as the solvent. The betulinic acid minor peak found in the reference sample was hidden by a group of peaks of ethanol. Most importantly, the major betulinic acid peak was easily seen and quantified.

[0352] After reviewing this specification, various modifications, improvements, and equivalents will be apparent to those skilled in the art to which this invention pertains. The claims are intended to cover these modifications, improvements, and equivalents. Those skilled in the art will recognize or be able to ascertain using only routine experimentation many equivalents to the specific embodiments of the invention described herein. These equivalents are intended to be covered by the following claims.

[0353] Example 14: Preparation of the Chemical Compound Represented by Formula 1

[0354] The chemical compound represented by Formula 1 was prepared and isolated as follows.

[0355] 1. Prepare a chaga reductant using a ratio of 100 L of reverse osmosis filtered water and 1.5 - 2 pounds of chopped and ground chaga.

[0356] 2. Heat the water / chaga mixture to approximately 160°F - 190°F and hold under a slight vacuum in a glass reactor vessel for 3 - 7 days.

[0357] * Up to 5 volume % of the reductant is lost by evaporation / vaporization.

[0358] 3. Once the chaga reductant has condensed for 3 - 7 days, transfer the chaga reductant from the glass reactor to stainless steel still equipped with a copper helmet.

[0359] 4. Then distill the chaga reductant for 3 - 7 hours and produce 1 - 5 gallons of distillate. The distillation is carried out at approximately 210°F + / - 10°F.

[0360] 5. After distillation, the remaining fraction that has not been converted to the distillate is used for the reaction. The volume of this remaining fraction is approximately 80% of the starting volume. The temperature of the fraction is maintained at about 190°F + / - 10°F during the reaction.

[0361] 6. Once the reductant is removed from the still, it is mixed in the following amounts for the final reaction:

[0362] 4 gallons of fraction

[0363] 250 - 400 g of Manuka honey

[0364] 115 - 120 mL of MCT oil

[0365] 5 mL of 70 / 30 H2O / Ethanol Chaga mushroom extract

[0366] All the ingredients are mixed together in a stainless - steel container at the start of the reaction. The initial indication that the reaction has occurred is the emission of a "glow", the formation of micelles, and the evolution of heat and gas. The mixture is then bottled to contain and induce a continuous reaction for 4 - 6 hours while the preparation is slowly cooled to room temperature. Preparation 1 is stored at ambient temperature for 3 - 4 weeks without degradation.

[0367] Example 15: Preparation of the chemical compound represented by Formula 2

[0368] The chemical compound represented by Formula 2 is prepared and isolated as follows.

[0369] ** The Chaga mushroom reductant - to - fraction prepared as in Example 14 is used as the starting material.

[0370] 1. In a 300 - mL Parr reaction vessel at room temperature, mix the following:

[0371] 200 mL of Chaga mushroom fraction

[0372] 15 g of proline

[0373] 15 g of fructose

[0374] 5 g of sunflower lecithin (or other FA surfactant)

[0375] 2. Gently stir the mixture. The starting materials begin to crystallize at room temperature.

[0376] 3. Seal the reaction vessel according to the manufacturer's guidelines.

[0377] 4. Heat the mixture in a completely sealed reaction chamber to 405°F + / - 15°F at 35 PSI + / - 10 PSI.

[0378] * Steps 2 - 4 take approximately 30 - 50 minutes

[0379] 5. Once the temperature / pressure is reached, remove the heat source and cool the vessel to a maximum of 90°F before degassing. **Wear PPE for all steps in the reaction**

[0380] 6. Once at the correct temperature, open the pressure relief valve and slowly degas the chamber.

[0381] 7. Open the vessel and separate the liquid from the solid crystalline phase. The liquid phase contains a certain concentration of the fully synthesized compound represented by Formula 2.

[0382] As described above, the present invention is applicable to pharmaceutical and nutritional preparations, chemical compounds and pharmaceutical compositions containing chemical compounds, and methods for their preparation and use. Accordingly, the present invention should not be considered limited to the above examples, but should be understood to cover all aspects of the present invention as clearly set forth in the appended claims.

Claims

1. A compound represented by Formula 1: Formula 1 wherein R1, R2 and R3 are independently selected from alkyl / alkane, alkene / alkenyl or alkyne / alkynyl having 1 to 20 carbon atoms; benzene / aromatic / phenyl, ether, amide, alkyl halide, amine (-amino), alcohol / hydroxyl / hydroxyl (-OH), thiol, aldehyde, ketone, ester, carboxylic acid (COOH), anhydride, acyl halide or methyl.

2. The compound according to claim 1, wherein at least one of R1, R2 and R3 is a hydroxyl group.

3. The compound according to claim 1, wherein R1, R2 and R3 are hydroxyl groups.

4. The compound represented by formula 2: wherein R1, R2, R3, R4, R5, R6 and R7 are independently selected from alkyl / alkane, alkene / alkenyl or alkyne / alkynyl having 1 to 20 carbon atoms; benzene / aromatic / phenyl, ether, amide, alkyl halide, amine (-amino), alcohol / hydroxyl / hydroxyl (-OH), thiol, aldehyde, ketone, ester, carboxylic acid (COOH), anhydride, acyl halide or methyl. The compound according to claim 4 , wherein at least one of R1-R7 is a hydroxyl group. The compound according to claim 4 , wherein R1-R7 are hydroxyl groups.

7. A pharmaceutical composition comprising a compound according to any one of claims 1 to 6 in a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, wherein the pharmaceutically acceptable carrier is selected from artificial and biological delivery systems, nano drug delivery vehicles, microsystems, vesicles of biological or inert origin, and biomacromolecules.

9. A method of treating a neoplastic disease in a mammalian subject, comprising administering to the subject an effective amount of a compound according to any one of claims 1 to 6 or a pharmaceutical composition according to any one of claims 7 to 8.

10. The method of claim 9, wherein the neoplastic disease is cancer.

11. The method of claim 10, wherein the cancer is melanoma, carcinoma, sarcoid, prostate cancer, colorectal cancer, breast cancer, liver cancer, and brain cancer.

12. The method of claim 11, wherein the cancer is squamous cell carcinoma, basal cell carcinoma, or Merkel cell carcinoma.

13. A nutritional preparation comprising a mixture of a Chaga mushroom reduce and a Chaga mushroom extract.

14. A method for preparing a nutritional formulation, comprising: forming a Chaga mushroom reduction in a reducing solvent; forming a Chaga mushroom extract in an extraction solvent; as well as The Chaga mushroom reduced material is mixed with the Chaga mushroom extract to prepare the nutritional preparation.

15. A pharmaceutical preparation comprising a reaction product, wherein the reaction product is a product formed by: reacting a mixture of a Chaga mushroom reduce and a Chaga mushroom extract with an esterification mixture comprising proline, fructose, and fatty acids and an emulsified sugar ester capable of emulsification in an otherwise immiscible liquid to facilitate emulsification at a temperature that causes a reaction between the mixture of the Chaga mushroom reduce and the Chaga mushroom extract and the esterification mixture to produce a reaction mixture; and combining the reaction mixture with a medium chain triglyceride oil to prepare the pharmaceutical preparation.

16. The method of claim 15, wherein the esterified mixture is Manuka honey.

17. A method for preparing a pharmaceutical preparation, comprising: preparing a Chaga mushroom reduction under pressure in a reducing solvent; preparing a Chaga mushroom extract in an extraction solvent; mixing the Chaga reduced product with the Chaga extract; reacting the Chaga reductase and Chaga extract mixture with an esterification mixture comprising proline, fructose, and fatty acids and an emulsified sugar ester capable of emulsification in an otherwise immiscible liquid to facilitate emulsification at a temperature that results in a reaction between the Chaga mushroom reductase and Chaga mushroom extract mixture and the esterification mixture to produce a reaction mixture; and combining the reaction mixture with a medium chain triglyceride oil to prepare the pharmaceutical preparation.

18. The method of claim 17, wherein the esterified mixture is manuka honey.

19. A method of treating a neoplastic disease in a mammalian subject, comprising administering to the subject an effective amount of the pharmaceutical formulation of claim 15.

20. The method of claim 19, wherein the neoplastic disease is cancer.

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