Use of Antrodia cinnamomea Compounds for the Preparation of Pharmaceutical Compositions for Treating Diabetes Mellitus

Abstract

To provide a novel application of compounds derived from Antrodia camphorata.SOLUTION: A compound derived from Antrodia camphorata, represented by formula (1) below, or a pharmaceutically acceptable salt thereof, reduces blood glucose levels and blood lipids in diabetic patients.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the use of Antrodia cinnamomea compounds, in particular to the use of Antrodia cinnamomea compounds for preparing a pharmaceutical composition for treating diabetes. [Background technology]

[0002] Antrodia camphorata, also known as Antrodia camphorata, Red Antrodia camphorata, or Red Antrodia camphorata, is a perennial mushroom belonging to the Aphyllophorales and Polypoaceae families. It is a fungus unique to Taiwan, and grows only on the inner walls of the rotten heartwood of the Cinnamoum kanehirai Hay, a protected tree species in Taiwan. In addition to the extremely low distribution of Cinnamoum kanehirai, wild Antrodia camphorata, which cannot grow without parasitizing the Cinnamoum kanehirai Hay, has become even rarer due to the extremely low distribution of Cinnamoum kanehirai and the fact that the fruiting bodies grow very slowly and only grow between June and October, making it very expensive.

[0003] Among the many components of Antrodia cinnamon, research on triterpene compounds has been most advanced. Triterpene compounds are a general term for natural compounds that are hexagonal or pentagonal and consist of 30 carbon atoms. The bitter taste of Antrodia cinnamomea is mainly due to triterpene compounds. In 1995, Cherng et al. discovered that the extract of Antrodia cinnamomea fruiting body contained three new triterpene compounds with ergostane skeleton, antcin A, antcin B, and antcin C (Cherng, IH, and Chiang, HC 1995. Three new triterpenoids from Antrodia cinnamomea. J. Nat. Prod. 58:365-371).

[0004] Chen et al. discovered three triterpene compounds, zhankuic acid A, zhankuic acid B, and zhankuic acid C, by extracting the fruiting bodies of Antrodia cinnamomea with ethanol (Chen, CH, and Yang, SW 1995. New steroid acids from Antrodia cinnamomea, -a fungus parasitic on Cinnamomum micranthum. J. Nat. Prod. 58:1655-1661).

[0005] In 1995, Chiang et al. also discovered three new triterpene compounds, namely, antrocin, 4,7-dimethoxy-5-methyl-1,3-benzodioxole, and 2,2',5,5'-tetramethoxy-3,4,3',4'-bi- methylenedioxy-6,6'- dimethylbiphenyl, from the fruit body extract (Chiang, HC, Wu, DP, Cherng, IW, and Ueng, CH 1995. A sesquiterpene lactone, phenyl and biphenyl compounds from Antrodia cinnamomea. Phytochemistry. 39:613-616).

[0006] In 1996, Cherng et al. used a similar analytical method to discover four more novel triterpene compounds: antcin E, antcin F, methyl antcinate G, and methyl antcinate H (Cherng, IH, Wu, DP, and Chiang, HC 1996. Triteroenoids from Antrodia cinnamomea. Phytochemistry. 41:263-267).

[0007] Yang et al. discovered two new compounds based on the ergostane skeleton, namely, zhankuic acid D and zhankuic acid E, and three new compounds based on the lanostane skeleton, namely, 15 α-acetyl-dehydrosulphurenic acid, dehydroeburicoic acid, and dehydrosulphurenic acid (Yang, SW, Shen, YC, and Chen, CH 1996. Steroids and triterpenoids of Antrodia cinnamomea-a fungus parasitic on Cinnamomum micranthum. Phytochemistry. 41:1389-1392).

[0008] Diabetes mellitus is a chronic endocrine metabolic disease that is a metabolic disorder of sugar, fat, and protein caused by an absolute or relative deficiency in insulin secretion, and is clinically classified into two types, type I and type II. Type I is insulin-dependent diabetes mellitus (IDDM), which is mostly caused by autoimmunity, and patients are unable to secrete insulin due to damage to the beta cells of the pancreatic islets, resulting in an absolute insulin deficiency.

[0009] Type II is non-insulin-dependent diabetes mellitus (NIDDM), in which the patient's islet β cells have the ability to secrete insulin, but the insulin receptors of the target cells (corresponding cells) are resistant to or insensitive to insulin, so insulin is not released when needed, or the binding strength between the receptor and insulin is reduced. As a result, although the amount of insulin is normal, it cannot meet the need to maintain normal metabolism, causing an increase in blood glucose levels and an increase in urinary glucose; this situation is called relative insulin deficiency.

[0010] Taking the applicant's previous patent applications as examples, for example, Taiwan Patent I527583 "Pharmaceutical composition for promoting wound healing and use thereof", Taiwan Patent I626053 "Use of Antrodia cinnamon compounds for preparing a composition for promoting hair growth", China Patent CN104887661B "Pharmaceutical composition for promoting wound healing and use thereof", and China Patent CN201610313277.9 "Use of Antrodia cinnamon extract and compounds for preparing a composition for promoting hair growth", it is disclosed that the compound 4,7-dimethoxy-5-methyl-l,3-benzodioxole (abbreviated as SY1) purified from Antrodia cinnamon has the effects of promoting wound healing and hair growth, and has the potential for the development of related pharmaceutical compositions. However, other medical uses of the compound have not yet been investigated, and to date, no literature has disclosed uses of the compound related to improving type II diabetes, such as reducing blood glucose or blood lipids. Summary of the Invention [Problem to be solved by the invention]

[0011] Therefore, based on considerable ingenuity in the Antrodia cinnamomea compound, the inventors have, after intensive research and repeated testing, gradually found certain related applications, such as those related to the improvement of type II diabetes, such as reducing blood sugar and blood lipids, as disclosed in the present application. [Means for solving the problem]

[0012] The object of the present invention is to provide a use of an Antrodia cinnamon compound for preparing a pharmaceutical composition for treating diabetes, wherein the Antrodia cinnamon compound is a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof:

[0013] [ka]

[0014] Preferably, the pharmaceutical composition further comprises a pharmacologically acceptable carrier.

[0015] Preferably, the diabetes is type II diabetes.

[0016] Preferably, the present invention further includes the use for reducing blood glucose in a patient suffering from diabetes.

[0017] Preferably, the present invention further includes use for reducing blood lipids in a patient suffering from diabetes. Effect of the Invention

[0018] According to the present invention, there is provided a use of an Antrodia cinnamomea compound for preparing a pharmaceutical composition for treating diabetes. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows the results of an oral glucose tolerance test (OGTT). [Diagram 2] FIG. 1 shows the results of an oral glucose tolerance test (OGTT). [Diagram 3] FIG. 1 shows the results of a biochemical test for aspartate aminotransferase (AST). [Figure 4] FIG. 1 shows the results of alanine aminotransferase (ALT) biochemical testing. [Diagram 5] FIG. 1 shows the results of a biochemical test for low-density lipoprotein (LDL). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Preparation of Antrodia camphorata extract SY-C and compound SY-1 The mycelium, fruit body or mixture of Antrodia camphorata is extracted with water or an organic solvent to obtain an aqueous extract or an organic solvent extract of Antrodia camphorata. First, 1.0 kg of Antrodia camphorata mycelium, fruit body or mixture of both is extracted twice with 10 times the amount of alcohol, and the resulting extracts are combined and concentrated to obtain about 230 g of crude extract.

[0021] The crude extract was further partitioned and extracted three times with a dichloromethane / water (1:1) solution to separate it into about 102.6 g of a dichloromethane layer and about 127.4 g of an aqueous layer. Then, 6.0 g of the dichloromethane layer was taken and separated by silica gel column chromatography using solvents of n-hexane / dichloromethane (1:4), dichloromethane, and methanol / dichloromethane (5:95), and four fractions, LT-ED-1, LT-ED-2, LT-ED-3, and LT-ED-4, were obtained.

[0022] The above four fractions were obtained by combining the fractions obtained after eluting with n-hexane / dichloromethane (1:4) and dichloromethane, respectively, to obtain LT-ED-1, and further eluting with methanol / dichloromethane (5:95), the first fraction obtained was LT-ED-2, the second fraction obtained was LT-ED-3, and finally eluting with methanol to obtain LT-ED-4. The LT-ED-1 fraction was collected and became Antrodia cinnamon extract SY-C.

[0023] In order to further purify the active ingredient in Antrodia cinnamomea extract SY-C, the LT-ED-1 layer is taken out and separated and purified by silica gel column chromatography or preparative high performance liquid chromatography to purify the compound 4,7-dimethoxy-5-methyl-1,3-benzodioxole represented by the following formula (1) (i.e., SY-1).

[0024] [ka]

[0025] Here, the organic solvent may include, but is not limited to, alcohols (e.g., methanol, ethanol, or propanol), esters (e.g., ethyl acetate), alkanes (e.g., hexane), or haloalkanes (e.g., chloromethane, chloroethane). Among them, alcohols are preferred, and ethanol is more preferred. The weight ratio of the organic solvent to the mycelium, fruit body, or a mixture of both may be 15:1 to 5:1, and is preferably 12:1 to 8:1, but is not limited thereto. The ratio of dichloromethane / aqueous solution may be between 2:1 and 1:2, but is not limited thereto.

[0026] For the extraction of 4,7-dimethoxy-5-methyl-l,3-benzodioxole, please refer to Tu SH., J Agric Food Chem. 2012 Apr 11;60(14):3612-8.

[0027] In the past, related research, whether it is Antrodia cinnamon extract or purified SY-1 compound, has mainly focused on its antitumor effect, and there has been no research on the treatment of diabetes. In contrast, the present invention uses Antrodia cinnamon extract and purified compound SY-1 and its salts to carry out experiments, and has found and demonstrated that both have the effect of promoting insulin secretion, which is an unprecedented new use.

[0028] Mouse animal model of type II diabetes In this experiment, four-week-old male C57BL / 6JNarl mice were used from the National Animal Experiment Center (Taipei, Taiwan), and were housed in an environment of 23°C ± 2°C with a 12-hour light / 12-hour dark cycle.

[0029] The mice were given free access to water and food during the one-week adaptation period, and one week later, they were divided into groups and fed commercial diets 1320 (11% fat content) purchased from EJOY2 CORP. (New Taipei City, Taiwan) and D12451 (45% high fat diet) purchased from MEDGENE CO., LTD. (Taichung City, Taiwan) for 12 weeks of induction.

[0030] The amount of water and food was calculated weekly, the amount of urine in the mat was observed, and the condition of the mice was monitored to see if they went into a diabetic mode (data not shown).

[0031] During the experiment, the body weight and fasting blood glucose level of the mice are measured every week and the changes are recorded. The mean ± 3SD of the fasting blood glucose level of the mice is calculated. If the fasting blood glucose level exceeds the mean + 3SD of the normal diet group, 4 weeks of feeding tube administration is started, and an oral glucose tolerance test is performed by tail vein blood sampling in the last week of the experiment.

[0032] On the last day of the experiment, all animals (excluding unscheduled deaths) were fasted for 20 hours at the National Animal Experiment Center, euthanized using pure carbon dioxide, and the carcasses were necropsied, organs were weighed (liver), and gross examinations were performed including evaluation of the liver and pancreatic gland. At the time of final death, the majority of animal pathologies, including planned euthanasia and unscheduled deaths, were recorded.

[0033] Experimental Design and Methods Table 1: Treatment conditions for each experimental group and drug addition TIFF0007674546000003.tif49170

[0034] Oral glucose tolerance test (final week) Referring to Figures 1 and 2, Figures 1 and 2 respectively show the results of an oral glucose tolerance test (OGTT). The groups shown in Figures 1 and 2 correspond to the conditions shown in Table 1: Group A (NFD), Group B (NFD+5), Group C (NFD+20), Group D (HFD), E (HFD+5), F (HFD+10), G (HFD+20), and H (Glibenclamide). Here, NFD is a normal fat diet, HFD is a high fat diet, and glibenclamide is an oral hypoglycemic drug for type II (non-insulin dependent) diabetes patients.

[0035] As shown in the results, based on the results of the oral glucose tolerance test (OGTT) at week 16, the group to which the hypoglycemic drug glibenclamide was added showed a higher rise in fasting blood glucose when sugar water was not fed compared to all other groups. This shows that after 4 weeks of drug treatment, the blood glucose lowering effect of this drug was inferior to SY1 in the later stages of treatment.

[0036] Regarding the feeding of SY1, 30 minutes after feeding sugar water, SY1 has a significant effect on reducing blood glucose in the low-dose HFD+5 group and the medium-dose HFD+10 group. The blood glucose lowering effect of the HFD+5 group is equivalent to that of the HFD+10 group (n=6, the average blood glucose values ​​over 120 minutes for HFD+5 and HFD+10 are 155.83 and 155.33, respectively), and is more significant than the blood glucose lowering effect of the high-dose HFD+20 group and the hypoglycemic glibenclamide group.

[0037] ALT and AST biochemical analysis in blood There are two types of aminotransferases in the blood: alanine aminotransferase (ALT) and aspartate aminotransferase (AST). ALT is found mainly in hepatocytes, while AST is found mainly in the liver, myocardium, and muscles, as well as in red blood cells. When these cells are destroyed by various causes and become necrotic, ALT and AST in the cells are released into the blood.

[0038] Therefore, in cases of hepatitis, myocardial infarction, muscle inflammation, or hemolysis, AST levels may increase, and by detecting the degree of increase in these enzymes in a blood sample, the degree of cell destruction can be estimated. Similarly, an increase in ALT levels can be said to be due to liver inflammation, and liver function can be evaluated by testing the above two enzymes.

[0039] In this experiment, the National Laboratory Animal Center was commissioned to carry out the detection using a Hitachi 7080 blood biochemistry analyzer and related reagents. The results, shown in Figures 3 and 4, show the biochemistry analysis results of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the blood. Regarding liver function in the later stages of treatment, there was not much difference between the groups, but the liver function value of the Western medicine group increased, resulting in a significant difference. Long-term use of this Western medicine may cause minor damage to the liver, but this phenomenon was not observed in the SY1 group during the same treatment period.

[0040] Low-density lipoprotein (LDL) detection Low density lipoprotein (LDL) is one of the five main groups of lipoproteins. LDL is a large, spherical macromolecule that can transport hydrophobic molecules (lipids and cholesterol) throughout the body via the aqueous environment of the blood. LDL transfers fat molecules to cells and, when oxidized in the arterial wall, can cause atherosclerosis. Oxidized LDL is more likely to be retained by proteoglycans, and when they penetrate the endothelium, they are at risk for cardiovascular disease. Thus, high levels of LDL are associated with arterial inflammation and cardiovascular diseases (CVD), such as atherosclerosis, ischemic stroke, and myocardial infarction.

[0041] Regarding the LDL detection results, the LDL-lowering effect of the HFD+5 group was not significantly different from that of the Western medicine group, and was more effective than that of the HFD+10 group, proving that the HFD+5 group is the optimal dose for lowering blood sugar and LDL.

[0042] The use of Antrodia cinnamomea compound for preparing pharmaceutical composition for treating diabetes provided in the present invention is indeed industrially useful. However, the above-mentioned is only the preferred embodiment of the present invention, and those skilled in the art can make various other improvements based on the above description, but these modifications still fall within the spirit of the present invention and the scope of protection defined by the claims.

Claims

1. Use of an Antrodia cinnamomea compound for preparing a pharmaceutical composition for treating diabetes, wherein the Antrodia cinnamomea compound is a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof: Use of Antrodia camphorata compounds. 【Chemistry 1】

2. The pharmaceutical composition further comprises a pharmacologically acceptable carrier.

2. The use according to claim 1.

3. The diabetes is type II diabetes.

2. The use according to claim 1.

4. and further comprising using the composition to reduce blood glucose in a patient suffering from diabetes.

4. The use according to claim 3.

5. It further comprises being used to reduce blood lipids in a patient suffering from diabetes.

4. The use according to claim 3.

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