TWI586359B - Use of extract composition of fruiting body of antrodia cinnamomea - Google Patents

Description

Use of Antrodia camphorata fruit body extracting composition

The invention relates to the use of extracts of the fruit body extract of Antrodia camphorata; in particular, it is used for treating red blood cells and/or platelet depression.

In the treatment of cancer, the use of chemotherapeutic drugs plays a very important role. However, in addition to poisoning cancer cells, chemotherapeutic drugs are also toxic to many normal cells, causing many side effects. In general, cancer cells are cells with rapid growth and division. The mechanism of chemotherapeutic drugs is usually related to the inhibition of cell division. However, many normal tissue cells in the body grow and divide rapidly, so chemotherapeutic drugs also affect The physiological functions of these cells are usually affected by chemotherapeutic drugs such as bone marrow hematopoietic cells, epithelial mucosal cells of the digestive tract, cells of the reproductive system, and hair follicle cells.

Among the aforementioned cells susceptible to chemotherapeutic drugs, bone marrow hematopoietic cells are an important cell, which is the main hematopoietic tissue, which has the characteristics of rapid growth and division, and is based on the sensitivity of bone marrow hematopoietic cells to chemotherapeutic drugs. Almost all of the "myelosuppression" side effects of drug treatment are caused by a decrease in the number of white blood cells, red blood cells, and platelets. The decrease in the number of white blood cells makes the patient susceptible to infection; the decrease in the number of red blood cells may lead to anemia, which may occur. Symptoms such as weakness and fatigue; lowering the number of platelets reduces the patient's coagulation function.

At this stage, depending on the state of myelosuppression, different treatment mechanisms are used. For the reduction of white blood cells, granulocyte-macrophage colony-stimulating factor (G-CSF) is used; for red blood cells, iron can be taken, if necessary, blood transfusion or erythropoietin (EPO); For platelet reduction, platelets are needed for treatment. However, the aforementioned treatments are related to the safety and supply considerations of blood preparations, and the erythropoiesis is expensive. The use of granulocyte-macrophage stimulating factors also causes many side effects, including bone soreness, muscle soreness, headache, fatigue, nausea, Vomiting, fever, insomnia, etc.; on biochemical tests, elevated alkaline phosphatase, elevated liver function, and elevated uric acid can be found; in addition, musculoskeletal system, skin, respiratory system, cardiovascular system, kidney Functions and so on have an impact.

Therefore, it is still necessary to develop a pharmaceutical product that can properly treat the side effects of bone marrow suppression in the treatment of cancer.

Antrodia cinnamomea is a unique fungus in Taiwan. It belongs to the fungi taxonomy of Basidiomycotina, Hymenomycetes, Aphyllophorales, Polyporaceae, and Phaeoporus. Antrodia's perennial fungus grows only on the inner wall of hollow core material of Taiwan's endemic burdock tree ( Cinnamomum kanehirai ). Its fruit body is annual to perennial, with bright red, orange or light cinnamon, and has a eucalyptus aroma.

Anthraquinone was first regarded as a hangover medicine by Taiwanese aborigines. It has the effect of relieving hangovers and contains triterpenoids, poly-saccharides and β-glucan. After extensive research, Niuzhizhi has been confirmed to have anti-B hepatitis virus, anti-cancer, immune regulation, anti-inflammatory, anti-oxidation, anti-angiogenesis, blood pressure lowering, blood lipid lowering, neuroprotection, antibacterial, whitening and other functions.

Antrodia camphorata is a kind of fungus. Its growth can be divided into different stages of mycelium, mycelium, fruit body and sporophyte. Its cultivation cost, composition and composition are different. The mycelium and the mycelium can be cultivated by liquid fermentation or solid culture, wherein the liquid fermentation is low in cost, the culture time is short, and the solid fermentation can obtain the mycelium similar to the shape of the fruit body, but the bacteria The triterpenoids contained in the silk and mycelium are extremely low, and the therapeutic effect is far less than the fruiting body; the fruiting body can only be collected in the field in the past, the price is expensive and the ecological cost is enormous, and the cultivation of the eucalyptus has been developed. The fruit body is cultivated by using the burdock host burdock eucalyptus as the medium, which is the most similar and effective method for the current collection of burdock.

Due to the wide variety of physiologically active ingredients contained in the body of A. annua, it is still the goal of the industry to develop its efficacy to expand the application of Antrodia camphorata.

The present invention provides a use of an extract of A. angustifolia fruiting body for the manufacture of a medicament for treating red blood cells and/or platelet depression, wherein the A. angustifolia fruit body extract composition comprises a carbon dioxide supercritical fluid extract of an Antrodia camphorata fruiting body.

The foregoing uses are preferably used to manufacture a medicament for treating red blood cells and/or low platelet counts with low white blood cells or for the manufacture of a medicament for treating red blood cells and/or low blood platelet weight loss.

The invention further provides a pharmaceutical composition for treating red blood cells and/or platelet hypoxia, comprising a therapeutically effective amount of an extract of Antrodia camphorata fruit body extract, wherein the Antrodia camphorata fruit body extracting composition comprises a carbon dioxide supercritical fluid extract of Antrodia camphorata fruiting body .

The pharmaceutical composition described above is preferably used to treat red blood cells and/or low platelet counts with low white blood cells or to treat red blood cells and/or platelet hypoxia with weight loss.

On the other hand, the aforementioned Antrodia camphorata fruit extracting composition further comprises a sesame extract.

Figure 1 shows the effect of THP-1 cells on the secretion of granulocyte-macrophage colony-stimulating factor after treatment with different concentrations of Antrodia camphorata fruit extract composition (SR4) for 24 hours.

Figure 2 shows the effect of THP-1 cells on the secretion of stimulating factors in granular macrophage cell lines after treatment with different concentrations of Antrodia camphorata fruit extracts (SR4s) for 24 hours.

The present invention provides a use of an extract of A. angustifolia fruiting body for the manufacture of a medicament for treating red blood cells and/or platelet depression, wherein the A. angustifolia fruit body extract composition comprises a carbon dioxide supercritical fluid extract of an Antrodia camphorata fruiting body. The pharmaceutical composition comprising the extract composition of Antrodia camphorata can treat red blood cells and/or platelet hypoxia in a single body, the pharmaceutical composition comprising an effective amount of the extract composition of Antrodia camphorata fruit body and a pharmaceutically acceptable carrier as needed Or an excipient.

The invention will be more readily understood by reference to the following detailed description of the embodiments of the invention and the accompanying drawings. Before the use and extracts of the present invention are disclosed and described, it is to be understood that the invention is not limited to the particular method of preparation, the carrier or the formulation, or the compound of the present invention, unless otherwise specifically indicated by the scope of the claims. The particular mode of product or composition for topical, oral or parenteral administration is well known to those of ordinary skill in the art and can be varied. It is also understood that the terminology used herein is for the purpose of describing the particular aspects of the invention.

Unless otherwise stated, the following terms as used in the present invention are to be interpreted as having the following meanings.

The scope is generally expressed herein as "about" a particular value and/or to "about" another particular value. When such a range is expressed, it is meant to include a particular value and/or range to another particular value. Similarly, when values are expressed as approximations by the use of the word "about," it should be understood that a particular value can form another aspect. In addition, it should be understood that each endpoint of each range is significant, and one endpoint is related to and independent of the other endpoint.

"As appropriate" or "as appropriate" means that the subsequently described event or condition may or may not occur, and that the description includes the occurrence of the event or condition and the circumstances in which it did not occur. For example, "contains a medicament as appropriate" means that the medicament may or may not be present.

It must be noted that the singular forms "a" and "the" Therefore, unless the context requires otherwise, the singular terms shall include the plural and the plural terms shall include the singular.

The term "individual" as used herein means any animal, preferably a mammal, and more preferably a human. Examples of individuals include humans, non-human primates, rodents, guinea pigs, rabbits, sheep, pigs, goats, cows, horses, dogs, and cats.

The term "effective amount" of a compound as provided herein means that the amount of the compound is sufficient to provide the desired modulation of the desired function, such as gene expression, protein function, or induction of a particular type of response. As indicated below, the actual amount of need will vary from individual to individual, depending on the individual's disease condition, physical condition, age, sex, species and weight, characteristics of the composition, and formulation. The dosage regimen can be adjusted to induce an optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced depending on the urgency of the treatment situation. Therefore, it is difficult to specify the exact "effective amount". However, those of ordinary skill in the art will be able to determine the appropriate effective amount using routine experimentation.

The term "treating" as used herein means reversing, alleviating or ameliorating the condition or condition to which the term applies, or one or more symptoms of the condition or condition, or inhibiting its progression.

The term "carrier" or "excipient" as used herein refers to a carrier and/or diluent and/or adjuvant or vehicle that is not itself a therapeutic agent but is used to deliver a therapeutic agent to an individual. An agent, or any substance added to the formulation to improve the handling or storage properties of the formulation or to allow or facilitate the dosage unit of the composition to form a dosage unit (such as a capsule or lozenge) suitable for oral administration. Suitable carriers or excipients are well known to those of ordinary skill in the art of making pharmaceutical formulations or foods. The carrier or excipient may include, by way of example and not limitation, buffers, diluents, disintegrating agents, binders, adhesives, wetting agents, polymers, lubricants, slip agents, to mask or counteract undesirable taste. A substance, a flavoring agent, a dye, a fragrance, and a substance added to improve the appearance of the composition. Acceptable load Agents or excipients include citrate buffers, phosphate buffers, acetate buffers, bicarbonate buffers, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of calcium and sulfuric acid, Magnesium carbonate, talc, gelatin, gum arabic, sodium alginate, pectin, dextrin, mannitol, sorbitol, lactose, sucrose, starch, gelatin, cellulosic material (such as cellulose ester of alkanoic acid and cellulose) Alkyl esters), low melting waxes, cocoa butter, amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (eg serum albumin), ethylenediaminetetraacetic acid (EDTA), dimethyl hydrazine (DMSO), Sodium chloride or other salts, liposomes, mannitol, sorbitol, glycerin or powders, polymers (such as polyvinylpyrrolidone, polyvinyl alcohol and polyethylene glycol), and other pharmaceutically acceptable substance. The carrier should not destroy the pharmacological activity of the therapeutic agent and should be non-toxic when administered at a dose sufficient to deliver a therapeutic amount of the agent.

Preferably, the Antrostrata lucidum fruiting body extracting composition is contained in a composition. The composition according to the invention is preferably a food composition or a pharmaceutical composition.

The Antrodia camphorata fruit extract composition can be added to a conventional food composition (also a ready-to-eat food or drink or its precursor) during the food manufacturing process. Almost all of the food compositions may be added to the Antrodia camphorata fruit extract composition according to the present invention. The food composition according to the present invention may comprise, but is not limited to, confectionery, baked goods, ice cream, dairy products, desserts and flavors, snacks, meat substitutes, snack foods, soups, Pasta, noodles, canned foods, frozen foods, dried foods, chilled foods, fats, baby foods, soft foods, or bread spreads or mixtures thereof.

The pharmaceutical compositions of this invention may be administered topically or systemically by any method known in the art including, but not limited to, intramuscular, intradermal, intravenous, subcutaneous, intraperitoneal, intranasal, oral, mucosal or External route of administration. Suitable routes of administration, methods of formulation, and schedule of administration can be determined by those of ordinary skill in the art. In the present invention, the pharmaceutical composition can be formulated in various ways according to the corresponding administration route, such as liquid solution, suspension, emulsion, syrup, lozenge, nine-dose, capsule, sustained release formulation, powder, powder Granules, ampoules, injectable solutions, infusion solutions, kits, ointments, lotions, liniments, creams or combinations thereof. Where necessary, it may be sterilized or mixed with any pharmaceutically acceptable carrier or excipient, many of which are known to those of ordinary skill in the art.

As used herein, the term "burdock body extract composition" refers to a composition comprising an extract of the body extract of Antrodia camphorata, specifically a carbon dioxide supercritical fluid extract comprising the body of A. niger.

The "B. chinensis" as described in the present invention is a unique fungus in Taiwan. It belongs to the fungi taxonomy of Basidiomycotina, Hymenomycetes, Aphyllophorales, Polyporaceae, and thin pores. An endophytic fungus of the genus Antrodia, whose scientific name is Antrodia cinnamomea , Ganoderma comphoratum , Antrodia camphorata or Taiwanofungus camphoratus .

The "B. chinensis fruiting body" as used in the present invention refers to a multicellular sexual sporulation structure produced by Antrodia camphorata, which is accompanied by a sporulation-producing ascus and a load, and the fruiting body is a sexual stage in the life history of Antrodia camphorata. From year to year, it is bright red, orange or light cinnamon with a eucalyptus aroma. The A. angustifolia fruiting body according to the present invention may be obtained from burdock tree or cultured from Burdock eucalyptus, preferably from burdock eucalyptus.

The carbon dioxide supercritical fluid extract of the Antrodia camphorata fruit body according to the present invention refers to an extract obtained by extracting an Anthracis sinensis fruit body with a carbon dioxide supercritical fluid. The supercritical flow system means that when the critical temperature and the critical pressure are exceeded, the properties of the gas and the liquid will be similar, and finally a homogeneous state of the fluid will be achieved. Supercritical fluids are similar in gas compressibility and have liquid-like fluidity. They can be used for extraction. Commercially available supercritical fluid extraction equipment is also available. For example, NATEX, SEPAREX, UHDE and Taichao are commercially available. The supercritical fluid extraction equipment, its model specifications are generally expressed in the capacity of the extraction tank, from 500g to 2000kg, can be selected It is necessary to select a suitable supercritical fluid extraction device.

In a specific embodiment of the present invention, the extraction pressure of the carbon dioxide supercritical fluid extract of the Antrodia camphorata fruit body is from about 150 bar to about 1000 bar; preferably from about 200 bar to about 800 bar; more preferably from about 350 bar to About 600bar.

In a specific embodiment of the present invention, the extraction temperature of the carbon dioxide supercritical fluid extract of the Antrodia camphorata fruit body is from about 25 ° C to about 80 ° C; preferably from about 35 ° C to about 70 ° C; more preferably From about 40 ° C to about 65 ° C.

In a specific embodiment of the present invention, the carbon dioxide flow rate of the carbon dioxide supercritical fluid extract of the Antrodia camphorata fruit body is from about 20 kg/h to about 70 kg/h; preferably from about 30 kg/h to about 60 kg/h. More preferably, it is from about 35 kg/h to about 55 kg/h.

In one embodiment of the invention, the extraction time of the carbon dioxide supercritical fluid extract of the Antrodia camphorata fruit body is from about 40 minutes to about 120 minutes; preferably from about 50 minutes to about 100 minutes.

In one embodiment of the invention, the carbon dioxide supercritical fluid extract of the Antrodia camphorata fruit body is extracted in the presence of a co-solvent such as from about 1% to about 10% 95% ethanol.

In a specific embodiment of the present invention, in the extraction of the carbon dioxide supercritical fluid extract of the Antrodia camphorata fruit body, the weight ratio of the Antrodia camphorata fruit body to the carbon dioxide supercritical fluid is from about 10:1 to about 1:10; preferably. It is from about 5:1 to about 1:5; more preferably from about 3:1 to 1:3.

In a preferred embodiment of the invention, the system of burdock chinensis is first comminuted into small pieces, and more preferably, the small pieces are pulverized into powder. The manner of dicing and/or shredding is well known to those of ordinary skill in the art to which the invention pertains.

Preferably, the supercritical fluid extraction method further comprises a concentration step. In a preferred embodiment, the carbon dioxide supercritical fluid extract of the A. angustifolia fruiting body is subjected to a supercritical fluid extraction of carbon dioxide as a simulated moving bed (Simulated Moving) Bed, referred to as SMB) and carbon 18 solid adsorbent are concentrated, and components with a Henry's constant of 2.8 or more are collected. According to the simulated moving bed of the present invention, the active ingredient of Antrodia camphorata is separated and concentrated according to the polar characteristic (Henry's constant H) of the active ingredient of Antrodia camphorata, and the embodiment thereof is as described in the "concentration step" of the Republic of China Patent No. I487531. The relevant content is hereby incorporated by reference.

Red blood cells and/or platelet hypoxia according to the present invention refers to red blood cells and/or platelet hypoxia caused by any cause, such as congenital or acquired, or caused by other diseases or drugs. In a preferred embodiment of the present invention, the red blood cells and/or platelets are low in red blood cells and/or platelets due to drugs; preferably, the drugs are drugs for treating tumors and having myelosuppressive side effects, for example, A chemotherapeutic drug for the treatment of cancer. The term "chemotherapeutic drug" as used in the present invention means a drug which inhibits the division of cancer cells. The term "myelosuppression" as used in the present invention refers to a side effect of the normal physiological condition of bone marrow hematopoietic cells affected by the use of a chemotherapeutic drug, and the symptoms include a decrease in the number of white blood cells, red blood cells, and/or platelets.

In a specific embodiment of the invention, the tumor system includes, but is not limited to, malignant lymphoma, Wilm's tumor, head and neck squamous cell carcinoma, bone cancer, hypercalcemia of the bone metastatic lesion, or squamous carcinoma.

In a specific embodiment of the invention, the drug comprises, but is not limited to, Chlorambucil, Cytarabine, Dactinomycin, Bleomycin, Puka Pilcamycin, Etoposide, nitrosourea or cyclophosphamid; preferably cyclophosphamide.

In a preferred embodiment of the present invention, the Antrodia camphorata fruit extracting composition further comprises a sesame extract. The "sesame" as used in the present invention, also known as flax, fat hemp, and linseed, is a flax plant of the genus Hemp, and its scientific name is Sesamum indicum , and preferably refers to the seed of the sesame plant. The "sesame extract" as used in the present invention refers to a mixture obtained by extracting active ingredients of sesame seeds, and the extraction method thereof includes, but not limited to, solvent extraction, supercritical fluid extraction, thermal extraction, solid phase extraction, preferably super Critical fluid extraction, more preferably carbon dioxide supercritical fluid extraction. In a preferred embodiment of the present invention, the sesame extract is sesamin, and the term "sesamin" as used in the present invention means a component of a fat-soluble antioxidant sesame lignan in sesame.

The sesame and Antrodia camphorata according to the present invention may be separately extracted and combined into the extract composition of the Antrodia camphorata fruit body, for example, a sesame extract and a carbon dioxide supercritical fluid extract of the Antrodia camphorata fruit body, respectively, and then added with a carrier/excipient as needed. The extract composition of the burdock fruit body is obtained. In a preferred embodiment of the present invention, the sesame extract and the carbon dioxide supercritical fluid extract of the Antrodia camphorata fruit body are mixed in a weight ratio of from about 1:3 to about 3:1; more preferably from about 1: 2 to about 2:1. On the other hand, the sesame and the burdock fruit body according to the present invention may be combined with a carbon dioxide supercritical fluid extraction, and the carrier/excipient may be added as needed to prepare the burdock fruit body extract composition. In a preferred embodiment of the present invention, the sesame and Antrodia camphorata fruit body body mixture ratio is from about 1:3 to about 3:1; more preferably from about 1:2 to about 2:1.

Preferably, the Antrodia camphorata fruit extracting composition according to the present invention can treat red blood cells and/or platelet hypopoxia and leukopenia, to treat the side effects of myelosuppression in all directions, and to improve the conventional use of granulocyte-macrophage stimulating factor. At the same time, although the number of neutrophils, mononuclear cells and lymphocytes in the peripheral blood is increased, the problems of hemoglobin and platelet count are lowered, and the side effects caused by the granulating macrophage cell stimulating factor are also avoided.

In a specific embodiment of the present invention, the treatment of red blood cells and/or platelet hypoxia complicated with leukopenia includes, but is not limited to, promoting an increase in the total number of white blood cells, promoting an increase in the number of granulocytes, promoting an increase in the number of neutrophils, and an increase in the number of lymphocytes. Promote the increase of mononuclear ball number and increase the secretion of granulocyte-macrophage colony-stimulating factor.

In another aspect, the Antrodia camphorata fruit body extracting composition according to the present invention is treatable Red blood cells and / or platelets are low with weight loss. Since cancer patients are almost always combined with weight loss, the Antrodia camphorata fruit body extract composition according to the present invention can treat bone marrow suppression together with weight loss treatment, and more broadly assists in the treatment of cancer chemicals.

In the BALB/c mouse experimental model of the present invention, the efficacy test of the mycelium-inhibiting side effect of cyclophosphamide (CTX) was tested against the extract of the body extract of A. sinensis. The mice were individually IP-based on the 0th day. The chemotherapeutic drug CTX 300 mg/kg was administered by intraperitoneal injection to induce a model of myelosuppression in the experimental animals; the extract of the Antrodia camphorata fruiting body was administered orally (PO) on the first day to the fourth day, respectively. The results showed that the toxicity of chemotherapeutic drugs of Shit CTX caused the number of red blood cells in each group to drop to a low point on the fourth day, while the oral extract of A. angustifolia fruit extract could increase the number of red blood cells by 1.19 times and continued on the 7th day. The number of red blood cells increased by 1.23 times; on the other hand, the application of CTX on day 7 caused a significant decrease in the number of platelets in mice. Compared with day 0, CTX caused a decrease in platelet count of about 50%; The Antrodia camphorata fruit extract composition has the ability to protect the number of platelets.

For leukopenia, when mice were given a single dose of CTX (300 mg/kg), the number of white blood cells in mice decreased to the lowest point on the fourth day; on the seventh day, the number of white blood cells began to rise. According to the analysis of these two observation points (Day4, Day7), the number of white blood cells in each group of mice fed with Antrodia camphorata fruit extracts was also reduced to the lowest point on the 4th day (the ratio of white blood cells decreased to 86.7~91.5). %); the mice in each group began to rise on the 7th day after the CTX was applied on the 0th day. The white blood cells of the CTX group rose about 3 times on the 7th day, and the treatment of the composition of the extract of the Antrodia camphorata fruit body significantly promoted the increase of the number of white blood cells.

To further analyze the effects of the changes of granulocyte, neutrophil, mononuclear and lymphocyte changes in the subgroups of the extracts of A. nigra. The results showed that a single dose of CTX (300 mg/kg) on day 0 caused the mice to have the lowest number of granulocytes and neutrophils on day 4; granules were observed on day 7. Number of white blood cells The test article A. angustifolia fruit body extract composition has a 2x significance to promote the ability of granular white blood cell to lift; on the other hand, on the 7th day, the body extract composition of A. angustifolia can increase the number of neutrophils by 2.93 times. In addition, the chemotherapeutic drug CTX also caused a significant decrease in the number of lymphocytes and mononuclear cells on the fourth day, and also found that the oral extract of the extract of A. serrata on the 7th day can increase the number of lymphocytes by 2.49 times; In the performance of the number of single-nuclear spheres, the extract of the Antrodia camphorata fruiting body had the potential to promote the rise of the single-nuclear ball on the 7th day.

For weight loss, mice given a single dose of CTX (300 mg/kg) showed a decrease in body weight (about 10%) on the third day compared with the blank group. The mice were fed with the extract of the Antrodia camphorata fruit body extract composition, and the weight gain was observed. Therefore, the Astragalus lucidum fruit body extract composition of the present invention can treat low body weight.

In order to increase the secretion of stimulating factors of granular macrophage cell lines, mononuclear cells were induced by LPS to produce cytokine GM-CSF, which was used as a positive control group. Using this test platform, the effect of the extract composition of Antrodia camphorata on the production of cytokine GM-CSF was investigated. The measured OD450nm absorbance was converted into a percentage by ELISA, and the GM-CSF secreted by the THP-1 cell itself was used. The amount of production was regarded as 100%. In the positive control group, the amount of cytokine GM-CSF produced by THP-1 cells was increased by 1.6 times on average. When THP-1 cells were treated with different concentrations of test samples, the extract of A. angustifolia fruit body extract was combined. The content of GM-CSF was also increased with the increase of the concentration of the extract composition of Antrodia camphorata fruit body. From this result, it was found that the concentration of the test substance A. angustifolia fruit body extract composition showed a dose-dependent relationship with the amount of GM-CSF produced, and the high concentration of 100 μg/ml of the extract body composition of Antrodia camphorata could induce GM-CSF. The amount of production increased by about 1.3 times.

The following non-limiting examples are intended to assist those of ordinary skill in the art to practice the invention. The examples are not to be construed as limiting the invention in any way. Those having ordinary skill in the art to which the present invention pertains may without departing from the spirit or scope of the invention. Modifications and variations of the embodiments discussed herein are still within the scope of the invention.

Instance

Antrodia camphorata fruit body extract composition

Carbon dioxide supercritical extract of Astragalus lucidum fruiting body (SR4)

Take 105 grams of Antrodia camphorata fruit body, crush into particles with a particle size of about 5 mm square, and place it in the extraction tank of the supercritical fluid extraction equipment. The internal pressure of the extraction tank is set to 350 bar, the internal temperature is 50 ° C, and the auxiliary solvent. 95% ethanol, and fed at a rate of 3 ml per minute, wherein the weight ratio of the body of the Antrodia camphorata to the supercritical fluid of carbon dioxide is 20:80 to 40:60, and the amount of supercritical fluid of carbon dioxide is 35 to 55 kg/h). Since the extract of Antrodia camphorata is in the form of a paste, which still contains 95% ethanol, the ethanol in the extract of Antrodia camphorata is removed in an oven set at a temperature of 30-35 ° C for subsequent concentration steps.

The concentration step is to separate the components of different Henry's constants in the extract of Antrodia camphorata by using a simulated moving bed as a continuous feeding purification platform; this example simulates the stationary phase (Stationary phase, SP for short) of the moving bed. Carbon 18 column, the mobile phase (MP) of this example is a mixture of water and methanol, the water contains 0.05% acetic acid, and the mobile phase dissolves the extract of Antrodia camphorata, the weight ratio of the water and methanol Preferably, the ratio of water to methanol is preferably 22:78, and the selective phase of the mobile phase is greater than 1.3, which is capable of separating the groups of different polarity characteristics more effectively. Part A and component B.

The simulated moving bed of this embodiment comprises at least three separation regions, which are sequentially α, β and γ regions, wherein the rear end of the α region is provided with a first discharge port O _α (referred to as “Extract outlet”), after the γ region The end is provided with a second discharge port O _γ (referred to as Raffinate outlet), and the feed port I (referred to as a feed inlet) is disposed between the β and γ regions.

The three separation regions α, β and γ are respectively composed of two columns c, and the columns c of the three separation regions are in communication with each other, and the column c is filled with the stationary phase, in particular, the particles of the stationary phase have pores therebetween. Passing the mobile phase, and causing the mobile phase to sequentially flow through the column c of the α, β, and γ regions in the same direction, and the stationary phase is switched by a feed port switching device at a feed port After T _sw , the relative position of the feed port I in the three separation regions is changed, so that the stationary phase is simulated to move in the other direction with respect to the moving phase.

According to the triangulation theory of the simulated moving bed, the components to be separated include: the component A having a Henry's constant of 2.8 or more and the component B having a Henry's constant of 2.8 or less, and the three separate regions α, β, and γ, the group The net mass flux F _{A of Part A} and the net mass flux F _{B of the component B} meet the conditions of Table 1, moving the component A to the area α, and the component B moves to the area γ, and each The flow rate ratio n of the three separation regions α, β, and γ (the flow rate ratios of the respective separation regions α, β, and γ are n _α , n _{β ,} and n _{γ , respectively} ) and the polarity of the component to be separated (ie, component A) The Henry's constant is H _A and the Henry's constant of component B is H _B ). The flow rate ratios n _α , n _β and n _γ should meet the conditions as shown in Table 1. The Henry's constant H of each component is based on the formula. I calculated that T0 is the residence time of the material not adsorbed by the stationary phase flowing through the column (T0=1.71), Tr is the residence time of the component to be separated, and ε is the porosity of the column used in the example (0.412) .

The simulated moving bed of this example is pressurized by a liquid pump (HITACHI L-2130), and the moving phase flows in the same direction in the column of each zone, and the feed port switching time T _{sw of} this example is set to simulate The stationary phase is directed to the opposite direction to the moving phase, and the Antrodia camphorata extract is separated according to the conditions shown in Table 2, and the component A having the Henry's constant of 2.8 or more is obtained at the first discharge port O _α . The second discharge port O _γ obtains the component B having a Henry's constant of 2.8 or less, that is, the carbon dioxide supercritical extract (SR4) of the body of the Antrodia camphorata.

Antrodia camphorata fruit extract composition (SR4s) contains carbon dioxide supercritical extract of burdock fruit body and sesame extract

Take 250 grams of sesame seeds and place them in the extraction tank of the supercritical fluid extraction equipment. The internal pressure of the extraction tank is set to 350 bar, the internal temperature is 50 ° C, the auxiliary solvent is 95% ethanol, and the rate is 3 ml per minute. The ratio of the weight of the sesame to the carbon dioxide supercritical fluid is 50:91, since the sesame extract is in the form of a paste, which still contains 95% ethanol, and the paste-like sesame is extracted by an oven set at a temperature of 30-35 ° C. The ethanol in the liquid is removed to obtain a sesame extract.

The sesame extract obtained as described above and the carbon dioxide supercritical extract (SR4) of the Antrodia camphorata fruit body were formulated at a weight ratio of 7:9 to obtain Antrodia camphorata fruit body extract composition (SR4s).

Therapeutic effect of the extract composition of Antrodia camphorata

Test materials and methods

[Animal mode]

BALB/c mice were administered with the chemotherapeutic drug cyclophosphamide (CTX) to induce bone marrow suppression induced by chemotherapeutic drugs, and whether the test substances improved the myelosuppressive effect produced by the chemotherapeutic drugs.

[experiment method]

The experimental animals were administered orally (PO) to the test substance, and the changes in the number of white blood cells and subgroups of granular white blood cells, lymphocytes, red blood cells and platelets were mainly analyzed.

[test substance]

The carbon dioxide supercritical extract (SR4) and the cattle obtained from the aforementioned Astragalus lucidum fruiting body Antrodia camphorata fruit extract composition (SR4s).

[experimental design]

Test drug and configuration

Chemotherapeutic drug CTX (Sigma Chemical Co. CAS Number: 6500-19-2 (St Louis, MO, USA)), granulocyte-macrophage stimulating factor (G-CSF, Filgrastim, Cat. 08502A, Kirin Brewery Co.) Ltd., Japan). CTX was added to physiological saline to a final concentration of 45 mg/mL as an intraperitoneal injection (IP) dosage form, and each mouse was given 300 mg per kilogram of body weight. The test substances SR4s and SR4 were respectively dissolved in DMSO (Dimethyl sulfoxide, purchased from sigma) at a concentration of 125 mg/mL; the concentration of the test substance in each group was 12.5 mg/mL, diluted ten times with secondary water, and the final concentration was 1.25 mg/mL. Oral reagents are 10 mg/kg per kilogram of body weight.

Experimental animal feeding and grouping

The experimental animals in this experiment were BALB/c mice: 6-8 weeks old, male, all animals were purchased from Lesco Biotech Co., Ltd., reared at 25±2°C, humidity range 40-70%, 12 hours light / Dark alternate, its drinking water is not limited. After the purchase, the mice were given an adaptation period of one week. After weighing, the rats were randomly divided into groups, so that there was no difference in the average body weight between the groups. The animals were divided into 12 groups and five groups each.

Experimental design of chemotherapeutic drug CTX and test substance

On the 0th day, the experimental mice were given the chemotherapy drug CTX 300mg/kg by IP intraperitoneal injection on the 0th day, which induced the model of myelosuppression in the experimental animals; the SR4s and SR4 of each test group started from the 1st to the 4th day respectively. The test substance is administered orally (PO). During the test, the body weight of the mice was weighed and recorded every three days; blood was collected on days 0, 4 and 7 and blood changes were observed.

Hematology analysis

The experimental animal was hand-held in the form of Lancet (facial vein in Mice), using a blood collection needle to poke into the back of the mouse's face, and collect it by a centrifuge tube. Blood, after collecting enough blood, use a sterile gauze to hold the bleeding port for 10-30 sec to stop bleeding. The collected blood was gently patted around the collected mice, 20 μL of blood was taken out, and diluted by a cell dilution, and then subjected to quantitative blood cell detection by XT-1800i blood analyzer (SYSMEX).

result

SR4s, SR4 treatment of CTX-induced red blood cells and low platelet count

As shown in Table 1 and Table 2, the toxicity of chemotherapeutic drugs of CTX will cause the number of red blood cells in each group to drop to a low point on the fourth day, while oral SR4s can increase the number of red blood cells by 1.19 times, and SR4 can increase the number of red blood cells. Up to 1.18 times, the number of red blood cells continued to increase on the 7th day. Oral SR4s increased the number of red blood cells by 1.17 times, and SR4 increased the number of red blood cells by 1.23 times. The effect of a single dose of CTX (300 mg/kg) on the number of platelets in mice. As shown in Table 2, the application of CTX on day 7 caused a significant decrease in the number of platelets in mice. Compared with day 0, CTX caused a decrease in the number of platelets by about 50%; the tube was fed with different concentrations of test substance SR4s, Each of the SR4 groups has the potential to protect the number of platelets, especially with the ability of test substance SR4 to protect the number of platelets.

SR4s, SR4 treatment of CTX-induced white blood cells and their subgroups of granulocytes, neutrophils, mononuclear cells and lymphocytes decreased

From Tables 1 and 2, it was found that when a single dose of CTX (300 mg/kg) was administered to mice, the number of white blood cells in the mice decreased significantly to the lowest point on the fourth day; on the seventh day, the number of white blood cells began to rise. . According to the analysis of these two observation points (Day4, Day7), the number of white blood cells in the mice fed the test objects SR4s and SR4 also fell to the lowest point on the fourth day (the ratio of white blood cell decline was about 86.7~91.5%). The mice in each group began to rise on the 7th day after the CTX was applied on the 0th day. From the results, it was found that the white blood cells of the CTX group rose about 3 times on the 7th day, and the test substances SR4s and SR4 had a significant tendency to promote the increase of the number of white blood cells (the blood cell bombs were 7 and 5 times respectively).

The effects of the test substances SR4s and SR4 on the changes of subpopulations of granular white blood cells, neutrophils, mononuclear cells and lymphocytes were further analyzed. From Tables 1 and 2, it was shown that a single dose of CTX (300 mg/kg) was applied on day 0, which caused the mice to have the lowest number of granular white blood cells and neutrophils on the fourth day; It can be observed that the number of granular white blood cells begins to rise; while the test substance SR4s has a 2x significance to promote the ability of granular white blood cells to rise; on the other hand, oral administration of SR4 on the 7th day can increase the number of neutrophils to 2.93. Times, SR4s can increase the number of neutrophils by 1.99 times.

In addition, the chemotherapeutic drug CTX also caused a significant decrease in the number of lymphocytes and mononuclear cells on the fourth day. On the 7th day, oral administration of SR4 increased the number of lymphocytes by 2.49 times; in the performance of single-nuclear balls, the test On the 7th day, SR4s had the potential to promote single-nuclear ball bombing.

SR4s, SR4 treatment of CTX-induced weight loss

The effects of the chemotherapeutic drugs on the body weight of the test animals showed that mice in each group given a single dose of CTX (300 mg/kg) were compared with the blank group, and the mice administered CTX were on the third day. The body weight begins to decrease (approximately 10% reduction), so the toxicity of a single dose of CTX (300 mg/kg) of its chemotherapeutic drug will cause a decrease in the body weight of the test animals, while the mice fed the test substance SR4 observe By the weight gain, the extract of the Antrodia camphorata fruit body of the present invention can treat low body weight.

Effect of burdock fruit body extract composition on granulating macrophage cell stimulating factor

Experimental materials and methods

[Experimental Materials]

Cell line: THP-1 cell line [ATCC-TIB-202].

Reagents required for cell culture and experiments: RPMI-1640 medium, fetal calf serum (FCS) and 2-mercaptoethanol. Lipopolysaccharide (LPS, purchased from Sigma), Human GM-CSF ELISA kit (Biolegend), and the like.

[experiment method]

THP-1 cell line culture:

THP-1 cells are a suspension-human monocytic leukemia cell line, cultured in RPMI medium containing 10% FCS and 2-mercaptoethanol, and cultured at 37 ° C, 5% CO ₂ Inside the incubator. The medium solution was changed every about 2-3 days, and when the cells in the flask were over 80%, subculture was performed. At the time of the experiment, the number of cells was counted by a Vi-cell cell counter, and the appropriate number of cells was used for the experiment.

Experimental material preparation

Both SR4 and SR4s were dissolved in DMSO at a concentration of 125 mg/ml, stored at 4 °C until use, and diluted to the desired concentration.

The preparation of LPS (concentration: 10 mg/ml), the LPS was dissolved in 1 x PBS, and the mixture was stored at -20 ° C for use. During the experiment, dilute to the desired concentration.

Testing of cytokine GM-CSF using THP-1 cells as a platform

THP-1 cells were 4×10 ⁵ cells/well and seeded into 12-well plates. After 6 hours of culture, they were divided into control group (0 μg/ml), LPS group (1 μg/ml) and experimental group (different concentrations were added respectively). Drug treatment, 0.01, 0.1, 1, 10, 100 μg / ml), each triple-coated, cultured in an incubator at 37 ° C, 5% CO ₂ for 24 hours. The next day, the cells were taken out from the incubator, centrifuged (1000 rpm, 5 minutes, 4 ° C), and the cell supernatant was collected, and the content of the cytokine GM-CSF released in the cells was measured by a GM-CSF ELISA kit.

Human GM-CSF ELISA

Cytokine GM-CSF analysis was performed using a Human GM-CSF ELISA kit (Biolegend). The capture antibody was coated with 100 μl/well on a 96-well plate and allowed to stand at 4 ° C for 12-16 hours. The next day, the reagent and the sample to be tested were placed at room temperature, and warmed up; the previously coated 96-well The plate was washed with a washing buffer, and blocked with 200 μl/well, allowed to stand at room temperature for 1 hour, and then washed with a washing buffer; the standard solutions were 500, 250, 125, 62.5, 31.2, 15.6, 7.8 pg, respectively. /ml; The sample to be tested and the serially diluted standard solution were separately added to a 96-well plate and allowed to stand at room temperature for 2 hours; after washing, the detection antibody was added and allowed to stand at room temperature for 1 hour; after washing, 100 μl/well was added. The substrate (coloring reagent A+B) was reacted in the dark at room temperature for 30 minutes, and the reaction was terminated by adding a stop solution. The absorbance of O.D. 450 nm was measured by an ELISA reader, and the measured O.D. value was calculated by interpolation using the R-squared of its standard curve to calculate the concentration of the cytokine GM-CSF by interpolation.

result

Mononuclear cells were induced by LPS to produce cytokine GM-CSF as a positive control group. Using this test platform, the effects of test samples SR4s and SR4 on the production of cytokine GM-CSF were investigated. The results are shown in Fig. 1 and Fig. 2.

The measured OD450nm absorbance was converted to a percentage by ELISA, and the amount of GM-CSF secreted by the THP-1 cell line itself was taken as 100%, while the positive control group was stimulated by THP-1 cells via the LPS. The average amount of CSF production was increased by about 1.6 times. When THP-1 cells treated different concentrations of test sample SR4s, it was found that the amount of GM-CSF produced increased with the increase of SR4s concentration. From this result, it was found that the concentration of the test substance SR4s and the amount of GM-CSF produced showed a dose-dependent relationship, and the high concentration of SR4s of 100 μg/ml could induce an increase of GM-CSF production by about 1.3 times.

The above-described embodiments are merely illustrative of the principles and effects of the invention, and are not intended to limit the invention. Modifications and variations of the embodiments described above will be apparent to those skilled in the art without departing from the spirit of the invention. The scope of the invention should be as set forth in the appended claims.

Claims (10)

The invention relates to a medicament for extracting a composition of an extract of Antrodia camphorata, which is used for the manufacture of a medicament for treating red blood cells and/or platelet depression, wherein the extract composition of Antrodia camphorata comprises a carbon dioxide supercritical fluid extract of Antrodia camphorata fruit body, wherein the Antrodia camphorata fruit body The carbon dioxide supercritical fluid extract is concentrated in a supercritical fluid extraction of carbon dioxide, concentrated by a simulated moving bed and a carbon 18 solid adsorbent, and a component having a Henry's constant of 2.8 or more is collected. The use of claim 1, wherein the carbon dioxide supercritical fluid extract of the Antrodia camphorata body is extracted at a pressure of from about 150 bar to about 1000 bar and at a temperature of from about 25 ° C to about 80 ° C. The use of claim 1, wherein the red blood cells and/or platelets are low in red blood cells and/or platelets due to drugs. The use of claim 3, wherein the drug is a drug for treating a tumor and having a myelosuppressive side effect. The use of claim 4, wherein the tumor is selected from the group consisting of malignant lymphoma, Wilm's tumor, head and neck squamous cell carcinoma, bone cancer, hypercalcemia of bone metastasis lesions, and testicular cancer. The use of claim 4, wherein the drug is selected from the group consisting of Chlorambucil, Cytarabine, Dactinomycin, Bleomycin, and pucamycin ( Pilcamycin), a group of Etoposide, nitrosourea, and cyclophosphamid. The use of claim 1, wherein the burdock fruit body extracting composition further comprises a sesame extract, wherein the sesame extract is obtained by extracting sesame with a carbon dioxide supercritical fluid. The use of any of claims 1 to 7 for the manufacture of a medicament for treating red blood cells and/or low platelet counts with low white blood cells. The use of claim 8, wherein the treatment of red blood cells and/or platelet hypoxia complicated by leukopenia is selected from the group consisting of promoting an increase in the total number of white blood cells, promoting an increase in the number of granulocytes, promoting an increase in the number of neutrophils, increasing the number of lymphocytes, and promoting the single. The number of nuclear globules increases and increases the secretion of granulocyte-macrophage colony-stimulating factor. The use of any of claims 1 to 7 for the manufacture of a medicament for treating red blood cells and/or low platelet weight loss.