Activation of AMPK by Agarwood Components

Abstract

The present invention discloses a use of a compound of formula I or a compound of formula II or a pharmaceutically acceptable salt thereof in preparing a medicine, food, beverage or dietary supplement for preventing and / or treating diabetes, obesity and / or complications thereof associated with AMPK activation, wherein R1 is a hydroxyl group or a C 1‑12 Alkoxy, R2 is hydrogen, R3 is hydrogen or C 1‑12 Alkoxy, R4 is hydrogen. The compound of the present invention can significantly activate the in vitro activity of AMPK and promote glucose uptake, and can be used in the preparation of medicines, foods, beverages or dietary supplements for diabetes, obesity and / or its complications related to AMPK activation. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the activation effect of agarwood components on AMPK. Background Art

[0002] Agarwood is a resinous wood formed by plants of the genus Aquilaria Lam. or Gyrinops Gaertn. of the family Thymelaeaceae under injury-induced defense response. In my country, agarwood medicinal materials mainly come from the plant Aquilaria sinensis (Lour.) Gilg of the family Thymelaeaceae. It is a traditional precious medicinal material commonly used in clinical practice. It has the effects of promoting qi and relieving pain, warming the middle and stopping vomiting, and promoting qi and relieving asthma. It is widely used in Chinese patent medicine, Tibetan medicine and Japanese Kampo medicine.

[0003] AMPK (Adenosine 5'-monophosphate (AMP)-activated protein kinase) is a serine / threonine kinase that is widely present in eukaryotic cells. AMPK has been found to be an important drug target for the treatment of various diseases. It is the main effector of metformin, a first-line clinical drug for the treatment of diabetes, and is also considered an important therapeutic target for controlling human diseases such as metabolic syndrome and cancer. AMP-activated protein kinase (AMPK) agonists have long been an international hotspot for the study of metabolic diseases such as obesity and diabetes.

[0004] Specifically, the AMPK signaling pathway is an energy sensor and regulator that plays an important role in promoting ATP production and inhibiting ATP consumption in various tissues. The kinase is activated in response to stresses that deplete the cellular ATP supply, such as hypoglycemia, hypoxia, ischemia, and heat shock. As a cellular energy sensor that can respond efficiently to low ATP levels, AMPK activation can actively regulate and replenish signaling pathways that supply cellular ATP. For example, AMPK activation can effectively promote GLUT4 transcription and translocation, which in turn leads to increased glucose uptake stimulated by insulin.

[0005] As a central regulator of lipid and glucose metabolism, AMPK is considered a key therapeutic target for the treatment of obesity, type 2 diabetes, and cancer. AMPK negatively regulates several core proteins in the ATP consumption process, such as CREB transcriptional coactivator (TORC2) and glycogen synthase, which can ultimately lead to downregulation or inhibition of gluconeogenesis, glycogen, lipid, and protein synthesis. AMP-activated protein kinase (AMPK) plays an important role in regulating cellular energy homeostasis. This enzyme is activated under low ATP conditions, usually caused by various stresses, and regulates signaling pathways that increase the available ATP supply.

[0006] In recent years, people have gained a lot of new insights into AMPK activation and the AMPK signaling pathway. New findings suggest that when glucose levels drop, AMPK can be activated before ATP levels drop, replenishing the potential ATP deficiency in a timely manner.

[0007] There are two main ways of indirect activation of AMPK. The more important one is to increase the ratio of AMP / ATP in cells. For example, berberine hydrochloride and metformin increase the ratio of AMP / ATP by inhibiting the respiratory chain to achieve the effect of indirect activation of AMPK, thereby producing therapeutic effects.

[0008] As an important kinase in the body's energy regulation process, AMPK maintains the cell's own energy supply and nutrient replenishment by sensing the level of ATP in the body. Under low energy supply conditions, AMPK phosphorylates specific enzymes and sites, which can increase ATP production and reduce ATP consumption. In mammals, when AMPK is activated, its regulation acts on almost the entire physiological metabolic process of the organism, and plays an indispensable and important role in the four major physiological metabolic activities of protein metabolism, lipid metabolism, carbohydrate metabolism, autophagy and mitochondrial homeostasis. Genetic and pharmacological studies have shown that AMPK is necessary for the body to maintain glucose balance. Activation of AMPK can actively improve the metabolic imbalance caused by type 2 diabetes. However, the molecular mechanism of activating AMPK is very complex. Activating AMPK through drug molecules and finding AMPK activators that can efficiently activate AMPK and have low side effects are huge challenges in the treatment of diabetes.

[0009] However, there are no reports on diabetes, obesity and / or its complications associated with AMPK activation in the presence of giant-panwood-type 2-2-phenylethyl chromone compounds (referred to as "FTPECs") and 5,6,7,8-tetrahydroxy-2-(2-phenylethyl) chromone compounds (referred to as "THPECs") in agarwood. Summary of the invention

[0010] Based on this, the present invention provides a use of a compound of formula I or a compound of formula II or a pharmaceutically acceptable salt thereof in the preparation of a medicine, food, beverage or dietary supplement for preventing and / or treating diabetes, obesity and / or complications thereof associated with AMPK activation.

[0011]

[0012] Wherein, R1 is hydroxyl or C 1-12 Alkoxy, R2 is hydrogen, R3 is hydrogen or C 1-12 Alkoxy, R4 is hydrogen.

[0013] Furthermore, R1 is hydroxyl or C 1-8 Alkoxy, R2 is hydrogen, R3 is hydrogen or C 1-8 Alkoxy, R4 is hydrogen;

[0014] Furthermore, R1 is hydroxyl or C 1-3 Alkoxy, R2 is hydrogen, R3 is hydrogen or C 1-3 Alkoxy, R4 is hydrogen.

[0015] Further, the compound of formula I has a structure selected from the group consisting of:

[0016]

[0017] According to another aspect of the present invention, there is provided a use of a composition comprising the above-mentioned compound or a pharmaceutically acceptable salt thereof in the preparation of a medicine, food, beverage or dietary supplement for preventing and / or treating diabetes, obesity and / or its complications associated with AMPK activation.

[0018] Furthermore, the composition further comprises one or more drugs and / or extracts.

[0019] Furthermore, the medicine comprises a hypoglycemic agent.

[0020] Furthermore, the hypoglycemic drug is selected from one or more of the following: glipizide, gliclazide, glibenclamide, glibornamide, glimepiride, gliquidone, acarbose, voglibose, pioglitazone, sitagliptin, saxagliptin, vildagliptin, metformin, rosiglitazone and insulin.

[0021] According to another aspect of the present invention, there is provided a use of a preparation comprising the above-mentioned compound or a pharmaceutically acceptable salt thereof or the above-mentioned composition in the preparation of a medicine, food, beverage or dietary supplement for preventing and / or treating diabetes, obesity and / or its complications associated with AMPK activation.

[0022] Furthermore, the preparation further comprises one or more pharmaceutically acceptable excipients.

[0023] Furthermore, the auxiliary material is selected from one or more of the following: dispersants, wetting agents, adhesives, diluents, glidants, lubricants, sustained-release agents, plasticizers, disintegrants, inclusion agents, flavoring agents, opacifiers and antioxidants.

[0024] Furthermore, the preparation is in the form of tablets, pills, capsules, powders, injections, films, lozenges, granules or oral solutions.

[0025] Furthermore, the diabetes includes type I diabetes and / or type II diabetes.

[0026] Further, the complication is an acute complication and / or a chronic complication.

[0027] Furthermore, the acute complication is diabetic ketoacidosis, hyperosmolar non-ketotic diabetic coma and / or lactic acidosis.

[0028] Furthermore, the chronic complications are macrovascular complications, microvascular complications, neuropathy and / or diabetic foot.

[0029] Furthermore, the macrovascular complications are cardiovascular and cerebrovascular diseases, coronary heart disease, stroke and / or lower limb gangrene.

[0030] Furthermore, the microvascular complication is diabetic retinopathy and / or diabetic nephropathy.

[0031] Furthermore, the cells associated with diabetes and / or its complications associated with AMPK activation are human renal epithelial cell line cells.

[0032] Furthermore, the human kidney epithelial cell line cells are HEK293T cells.

[0033] Further, the compound is obtained from agarwood or agarwood extract.

[0034] Furthermore, this AMPK activation is an indirect activation mechanism.

[0035] Furthermore, the AMPK comprises an α2 subunit, a β1 subunit and a γ1 subunit.

[0036] Furthermore, the activation of AMPK occurs in muscle, liver and / or kidney tissues.

[0037] Beneficial effects of the present invention:

[0038] The compound of the present invention can significantly activate the in vitro activity of AMPK and promote glucose uptake, and can be used in the preparation of medicines, foods, beverages or dietary supplements for diabetes, obesity and / or its complications related to AMPK activation. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0040] Figure 1The figure is a schematic diagram of the in vitro activity results of agarwood ethanol extract in activating AMPK in 293T cells. The results are presented as mean ± SD (n = 3), compared with the solvent control (DMSO), **p < 0.01, ***p < 0.001.

[0041] Figure 2 Schematic diagram of the results of treating HEK 293T cells with different concentrations of PECs monomers and detecting pAMPK and AMPK levels in cell lysates using SDS-PAGE and Western Blot. Compared with the solvent control (DMSO), **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0042] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0043] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0044] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0045] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0046] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0047] As described in the background technology section, the giant-panwood-type-2-2-phenylethyl chromone compounds and 5,6,7,8-tetrahydroxy-2-(2-phenylethyl) chromone compounds in agarwood have not been reported to be related to diabetes, obesity and / or its complications associated with AMPK activation. In order to solve the above problems, the present invention provides a compound of formula I or a compound of formula II or a pharmaceutically acceptable salt thereof for use in the preparation of a medicine, food, beverage or dietary supplement for preventing and / or treating diabetes, obesity and / or its complications associated with AMPK activation,

[0048]

[0049]

[0050] Wherein, R1 is hydroxyl or C 1-12 Alkoxy, R2 is hydrogen, R3 is hydrogen or C 1-12 Alkoxy, R4 is hydrogen.

[0051] In a preferred embodiment, R1 is hydroxyl or C 1-8 Alkoxy, R2 is hydrogen, R3 is hydrogen or C 1-8 Alkoxy, R4 is hydrogen.

[0052] In a preferred embodiment, R1 is hydroxyl or C 1-3 Alkoxy, R2 is hydrogen, R3 is hydrogen or C 1-3 Alkoxy, R4 is hydrogen.

[0053] In a preferred embodiment, the compound of formula I has a structure selected from the group consisting of:

[0054]

[0055] The above compounds of the present invention are commercially available, extracted and separated from plants such as agarwood, or derived from chemical organic synthesis.

[0056] According to another aspect of the present invention, there is provided a use of a composition comprising the above-mentioned compound or a pharmaceutically acceptable salt thereof in the preparation of a medicine, food, beverage or dietary supplement for preventing and / or treating diabetes, obesity and / or its complications associated with AMPK activation.

[0057] In the present invention, the term "treatment" also includes "prevention", unless there is a specific description to the contrary. The terms "treatment" and "therapeutically" should be understood accordingly.

[0058] In the present invention, the term "treatment" includes alleviating, inhibiting or improving the symptoms or conditions of a disease; inhibiting the occurrence of complications; improving or preventing potential metabolic syndrome; inhibiting the occurrence of a disease or symptom, such as controlling the development of a disease or condition; alleviating a disease or symptom; reducing a disease or symptom; alleviating complications caused by a disease or symptom, or preventing or treating signs caused by a disease or symptom. As used herein, a composition or a preparation, after administration, can improve a disease, symptom or condition, especially improve its severity, delay the onset, slow the progression of the disease, or reduce the duration of the disease. Whether fixed or temporary administration, continuous or intermittent administration, can be attributed to or related to the administration.

[0059] In a preferred embodiment, the composition further comprises one or more drugs and / or extracts.

[0060] In a preferred embodiment, the medicament comprises a glucose-lowering drug.

[0061] In a preferred embodiment, the glucose-lowering drug is selected from one or more of the following: glipizide, gliclazide, glibenclamide, glibornamide, glimepiride, gliquidone, acarbose, voglibose, pioglitazone, sitagliptin, saxagliptin, vildagliptin, metformin, rosiglitazone and insulin.

[0062] According to another aspect of the present invention, there is provided a use of a preparation comprising the above-mentioned compound or a pharmaceutically acceptable salt thereof or the above-mentioned composition in the preparation of a medicine, food, beverage or dietary supplement for preventing and / or treating diabetes, obesity and / or its complications associated with AMPK activation.

[0063] In a preferred embodiment, the preparation further comprises one or more pharmaceutically acceptable excipients.

[0064] In a preferred embodiment, the preparation of the present invention contains at least one pharmaceutically acceptable excipient in an amount of 0.00001 to 50 wt.%, or 0.0001 to 10 wt.%, or 0.0001 to 5 wt.%, or 0.005 to 1 wt.%, or 0.1 to 20 wt.%, or 0.5 to 15 wt.%, or 1 to 5 wt.%, based on the weight of the preparation.

[0065] In the present invention, the term "pharmaceutically acceptable" refers to a substance, such as a carrier or diluent, that does not abrogate the biological activity or properties of the compound and is relatively non-toxic, i.e., a substance that does not cause undesirable biological effects or interact in a deleterious manner with any of its components when administered to an individual.

[0066] In the present invention, the term "pharmaceutically acceptable excipient" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with subjects and active ingredients (i.e., capable of inducing the desired therapeutic effect without causing any undesirable local or systemic effects), which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).

[0067] In a preferred embodiment, the auxiliary material is selected from one or more of the following: dispersants, wetting agents, binders, diluents, glidants, lubricants, sustained-release agents, plasticizers, disintegrants, inclusion agents, flavoring agents, opacifiers and antioxidants.

[0068] Those skilled in the art will know how to select specific chemical substances within the scope of the above-mentioned auxiliary material category.For example, the dispersant can be selected from one or more of the following: cross-linked sodium carboxymethyl cellulose, sodium starch glycolate and pre-gelatinized corn starch.The wetting agent can be selected from one or more of the following: polyoxymethylene stearate, poloxamer, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil, polyoxyethylene alkyl ether, polysorbate such as polysorbate 80, cetyl alcohol, glycerol fatty acid ester (such as triacetin, glyceryl monostearate and the like), polyoxyethylene fatty acid ester, polyethylene glycol fatty acid ester, sodium lauryl sulfate, sorbitol fatty acid ester, sucrose fatty acid ester, polyoxyethylene ether, benzalkonium chloride, polyoxyethylene castor oil and docusate sodium.The adhesive can be selected from one or more of the following: polyvinyl pyrrolidone, hydroxypropyl cellulose, polyethylene glycol and methylcellulose. The diluent may be selected from one or more of the following: powdered sugar, starch, compressible starch, lactose, dextrin, mannitol, sorbitol, microcrystalline cellulose, calcium sulfate and calcium carbonate. The glidant is micropowder silica gel. The lubricant may be selected from one or more of the following: calcium stearate, talc, magnesium stearate, stearic acid and colloidal silicon dioxide. The sustained-release agent may be selected from one or more of the following: sodium carboxymethylcellulose, low-substituted hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, hydroxyethyl cellulose, gum arabic, gelatin and shellac. The plasticizer may be dibutyl sebacate and / or various citrates. The disintegrant may be selected from one or more of the following: carboxymethyl cellulose, carboxymethyl cellulose calcium salt and sodium carboxymethyl cellulose. The inclusion agent is hydroxypropyl beta-cyclodextrin. The flavoring agent may be selected from one or more of the following: sorbitol, glucose, mannose, sucrose and lactose. The opacifier is titanium dioxide. The antioxidant may be selected from one or more of the following: sodium bisulfite, sodium metabisulfite, sodium sulfite and sodium thiosulfite.

[0069] These excipients are preferably pharmaceutically inert, or may have a synergistic or additive effect to enhance the therapeutic activity of the pharmaceutical composition, and the above-mentioned excipients are only listed. The excipients actually used in the present invention are not limited to the above-mentioned excipients and can be adjusted according to actual conditions to achieve the effects of the present invention.

[0070] In a preferred embodiment, the preparation is in the form of tablets, pills, capsules, powders, injections, films, lozenges, granules or oral solutions.

[0071] In a preferred embodiment, the diabetes includes type I diabetes and / or type II diabetes.

[0072] In a preferred embodiment, the complication is an acute complication and / or a chronic complication.

[0073] In a preferred embodiment, the acute complication is diabetic ketoacidosis, hyperosmolar non-ketotic diabetic coma and / or lactic acidosis.

[0074] In a preferred embodiment, the chronic complications are macrovascular complications, microvascular complications, neuropathy and / or diabetic foot.

[0075] In a preferred embodiment, the macrovascular complications are cardiovascular and cerebrovascular diseases, coronary heart disease, stroke and / or lower limb gangrene.

[0076] In a preferred embodiment, the microvascular complication is diabetic retinopathy and / or diabetic nephropathy.

[0077] In a preferred embodiment, the cells associated with diabetes and / or its complications associated with AMPK activation are human kidney epithelial cell lineage cells.

[0078] In a preferred embodiment, the human kidney epithelial cell line cell is HEK293T cell.

[0079] In a preferred embodiment, the compound is obtained from agarwood or an agarwood extract.

[0080] In a preferred embodiment, the AMPK activation is an indirect activation mechanism.

[0081] In a preferred embodiment, the AMPK comprises an α2 subunit, a β1 subunit and a γ1 subunit.

[0082] In a preferred embodiment, the activation of AMPK occurs in muscle, liver and / or kidney tissue.

[0083] According to another aspect of the present invention, there is provided the above-mentioned compound, the above-mentioned composition or the above-mentioned preparation for preventing and / or treating diabetes, obesity and / or complications thereof associated with AMPK activation in a subject.

[0084] According to another aspect of the present invention, a method for preventing and / or treating diabetes, obesity and / or complications thereof associated with AMPK activation is provided, comprising administering an effective amount of the above compound, the above composition or the above preparation to the subject.

[0085] In the present invention, the term "subject" is a mammal. The mammal may be a human, non-human primate, mouse, rat, dog, cat, horse or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects representing models of diabetes, obesity and / or complications thereof associated with AMPK activation. Preferably, the subject is a human.

[0086] The "effective amount" of the composition or preparation used in the present invention can obtain the desired treatment and / or prevention effect. The amount effective for this purpose will depend on, for example, the pharmaceutical composition, the mode of administration, the stage and severity of the disease to be treated, the individual's body weight and overall health, and the judgment of the prescribing physician. The dosage can be given once a week, or once every two days or every day, or even several times a day. The dosage unit can be given in a short period of time (e.g., weeks to months) or longer periods of time (months to years).

[0087] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0088] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0089] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0090] Example

[0091] 1. Materials and Instruments

[0092] 1.1 Materials

[0093] Human renal embryonic epithelial cell line HEK293T cells (collected by the School of Life Sciences, Tsinghua University, originally purchased from the American Type Culture Collection, ATCC) and 3T3-L1 mouse preadipocytes (purchased from the American Type Culture Collection, ATCC); anti-AMPK phosphorylation and anti-AMPK total antibodies (Cell Signalling Company); anti-β-actin antibody (Beijing BI Company); PBS phosphate buffer (Hyclone); South American fetal bovine serum (Gibco); penicillin-streptomycin double antibody mixture (Hyclone); trypsin; PVDF membrane (Millipore Company, USA); glucose uptake kit (Promega); DMEM high glucose medium (Gibco); OPTI-MEM medium (Hyclone); mycoplasma inhibitor (ScienCell); dexamethasone (DEX), dimethyl sulfoxide (DMSO), insulin (Insulin), 1-methyl-3-isobutyrolxanthine (IBMX), 5'-ATP disodium salt, 5'-AMP disodium salt, 5'-ADP disodium salt, metformin (Met), berberine hydrochloride (BBR) and Compound C were all from Sigma Company, USA. The agarwood samples were identified by Zhang Zhijie, a researcher at the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, and stored in the medicinal material storage room of the Center for Crude Drugs of the Institute of Chinese Materia Medica.

[0094] Agarwood tetraol (batch number: 111980-201904, purity ≥98%, MW: 318.32 g / mol, code A130) was purchased from China Food and Drug Inspection Institutes.

[0095]

[0096] 8-Chloro-2-(2-phenylethyl)-5,6,7-trihydroxy-5,6,7,8-tetrahydrochromone (batch number: AF210328076, purity ≥98%, MW: 336.8 g / mol, code number A124) was purchased from Chengdu Aifa Biotechnology Co., Ltd.

[0097]

[0098] 6-Hydroxy-2-(2-phenylethyl)chromone (batch number: AF21042704, purity ≥98%, MW: 266.29 g / mol, code number A83) was purchased from Shanghai Stand Biotechnology Co., Ltd.

[0099]

[0100] 6-Methoxy-2-(2-phenylethyl)chromone (batch number: STC6680105, purity ≥98%, MW: 280.3 g / mol, code number A84) was purchased from Shanghai Stand Biotechnology Co., Ltd.

[0101]

[0102] 6-Methoxy-2-[2-(3'-methoxyphenyl)ethyl]chromone (batch number: STC6690105, purity ≥98%, MW: 310.3 g / mol, code number A86) was purchased from Shanghai Stand Biotechnology Co., Ltd.

[0103]

[0104] 1.2 Instruments

[0105] Scientz-IID ultrasonic cell disruptor (Ningbo Xinzhi Biological Company), DYCZ-24DN electrophoresis apparatus (Beijing Liuyi Instrument Factory), ELITE II carbon dioxide constant temperature incubator (Revco Company).

[0106] 2. Experimental Methods

[0107] 2.1 Drug preparation

[0108] Weigh 5g of agarwood sample accurately, add 500ml of 95% ethanol, soak at room temperature for half an hour, and then extract under low boiling reflux for 1 hour. After cooling, filter with 8 layers of gauze and 0.45μm microporous filter membrane, remove the solvent by rotary evaporation, add DMSO to dissolve, prepare 250mg / ml agarwood extract, and store in refrigerator for later use. When administering to cells, the agarwood extract is dissolved in DMSO, and the final concentration of DMSO is 0.1%, which is non-toxic to cells.

[0109] Accurately weigh 10 mg of each monomer compound (A130, A84, A124, A83, A86, BBR), add DMSO solution to dissolve, and make up to 10 ml volumetric flask to prepare 1 mg / ml test sample.

[0110] 2.2 Culture and drug treatment of human renal epithelial cell line HEK293T cells

[0111] HEK293T cells were cultured in a pre-prepared DMEM complete medium containing 10% fetal bovine serum and 1% double antibody. The temperature in the CO2 cell culture incubator was 37°C and the carbon dioxide content was 5%. The culture medium was replaced every 24 hours during the cell culture process. Drug treatment was performed when the cell density reached about 80%. When treating cells, different concentrations of each monomer compound (A130, A84, A124, A83, A86, BBR) were added to the pre-prepared complete medium (DMEM + 0.5% fatty acid-free bovine serum albumin (BSA) + 1% double antibody) and the cells were collected after culturing for 1 hour.

[0112] 2.3SDS-polyacrylamide gel electrophoresis and protein blotting

[0113] After the administration, the cells were collected and then added with a cell lysis solution containing protease inhibitors and phosphatase inhibitors for lysis at 4°C for 2 hours. The cell lysate was then centrifuged and the supernatant was taken. The sample was treated with β-mercaptoethanol for 30 minutes and boiled for 5 minutes to denature the protein. The cell lysate was separated by SDS-PAGE gel electrophoresis, and the separated proteins were transferred to a PVDF membrane by wet transfer, followed by blocking with a 5% skim milk powder TBST solution at room temperature for 2 hours, and then incubated with anti-AMPK antibody (Cell Signaling) and anti-p-AMPK antibody (Cell Signaling) at 4°C overnight, and then incubated with a goat anti-rabbit horseradish peroxidase-labeled secondary antibody (Santa Cruz) at room temperature for 1 hour. After the membrane was fully washed, a luminescent substance was added, and the membrane was exposed to a photosensitive film in a dark room, and then photographed and the atlas data was processed.

[0114] The total amount of AMPK was used as a reference. NIH Image J software was used to analyze and obtain the grayscale values ​​of pAMPK and AMPK protein immunoblot bands at different concentrations. The pAMPK / AMPK ratio represented the AMPK phosphorylation level. GraphPadPrism 6.0 was used to perform statistical analysis on pAMPK / AMPK. **p<0.01 and ***p<0.001 indicated that the differences were statistically significant.

[0115] 2.4 Glucose uptake - in vitro analysis

[0116] The glucose uptake test was completed using the Promega glucose uptake test kit, which was used to test the glucose uptake capacity of mature 3T3-L1 adipocytes treated with each monomer compound (A130, A84, A124, A83, A86). The kit was taken out of the -20°C refrigerator and thawed. According to the instructions for use of the kit, the neutralization buffer and stop buffer in the kit were stored at 4°C, and the remaining components were stored at -20°C. The experimental method is as follows:

[0117] (1) Cell treatment

[0118] 3T3-L1 cells were induced to become mature adipocytes on day 7. The medium was changed, the maintenance medium was removed, and the full medium containing drugs was added for 24 hours, and the cells were cultured in a cell culture incubator at 37°C and 5% CO2.

[0119] (2) Desugaring treatment

[0120] After the cells were treated with each monomer compound (A130, A84, A124, A83, A86) for 24 h, the culture medium was removed and starvation treatment was performed for 2-3 h by adding sugar-free, serum-free and antibody-free culture medium containing each monomer compound (A130, A84, A124, A83, A86) and culturing in a 37°C, 5% CO2 cell culture incubator.

[0121] (3) Insulin activation

[0122] The culture medium in (2) was removed, and a PBS solution containing 10 μg / mL insulin was added. The cells were activated in a cell culture incubator at 37° C. and 5% CO 2 for 10 min. The insulin was removed, and the cells were washed with PBS for 2-3 times.

[0123] (4) Preparation of 2DG6P detection reaction solution

[0124] The reaction solution was prepared 1 hour in advance, with a volume of 100 μL / sample and the proportions of each group provided in the kit instructions.

[0125] (5) Preparation of cell reaction solution

[0126] Carefully wash insulin-treated cells and unactivated cells with PBS. Treat with 1mM 2DG solution in a 37°C, 5% CO2 cell culture incubator for 20 minutes, add half the volume of 2DG stop buffer to terminate the glucose uptake reaction of the cells and lyse the cells. Shake several times, and after the cells are completely lysed, add the same volume of neutralization buffer as the stop buffer. Collect all the above liquids into a centrifuge tube, centrifuge at 5000g for 5 minutes at room temperature, collect the supernatant, and use it as the cell reaction solution required for the experiment.

[0127] (6) Detection of glucose uptake

[0128] 100 μL of cell reaction solution and 100 μL of 2DG6P detection reaction solution were placed in a 96-well plate and mixed thoroughly. The mixture was incubated in the dark for 1 h (room temperature). The fluorescence intensity of each experimental group was detected on the EnSpire multi-mode microplate reader.

[0129] 2.5 Data processing and statistical analysis

[0130] Western blot analysis was performed using NIH Image J software, and statistical analysis including one-way analysis of variance (ANOVA) and mean comparison was performed using GraphPad Prism 6.0. ** indicates P < 0.01 and *** indicates P < 0.001, indicating that the results are statistically significant.

[0131] 3. Results

[0132] 3.1 Verification of agarwood activity

[0133] In order to detect whether agarwood extract can activate AMPK, we treated HEK293T cells with different concentrations of agarwood ethanol extract for 1 hour, and then used SDS-PAGE and Western Blot (protein immunoblotting) to detect the levels of pAMPK (Thr-172) and AMPK in the cell lysate. Figure 1 As shown in the figure, after treatment with agarwood ethanol extract, the pAMPK level in the cells increased in a dose-dependent manner. Western Blot analysis results showed that agarwood ethanol extracts were able to activate AMPK in HEK293T cells in vitro, all had AMPK activation activity and activated in an activity-dose-dependent manner. At a concentration of 120 μg / ml, the AMPK activation ability of agarwood ethanol extract was similar to that of the positive drug metformin.

[0134] 3.2 Verification and comparison of the activity of monomer components of FTPECs and THPECs

[0135] In this experiment, we further compared the activity differences of the agarwood FTPECs and THPECs obtained by component analysis and screening. We selected four FTPECs monomer components and two THPECs for in vitro activity verification of AMPK agonist activity. According to the methods under "2.2 and 2.3", the selected FTPECs monomer components were used to treat cells respectively, and the pAMPK (Thr-172) and AMPK levels in the cell lysate were detected by SDS-PAGE and Western Blot (protein immunoblotting). Figure 2 As shown, compounds A84, A124, A83 and A86 all showed a dose-dependent relationship. The higher the administration concentration, the higher the phosphorylation level of AMPK172-threonine, which indicates that these compounds all have significant AMPK activation ability, while compound A130 did not show AMPK activation ability.

[0136] 3.3 Glucose uptake - in vitro analysis

[0137] The compounds A84, A124, A83 and A86 of the present invention can promote glucose uptake in 3T3-L1 adipocytes at a concentration of 1-600 mM. Compared with the DMSO control group, the glucose uptake rate (%) is greater than 100% and less than 350%.

[0138] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. Use of a compound of formula II or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating diabetes associated with AMPK activation, 2. Use of a composition comprising the compound or a pharmaceutically acceptable salt thereof as claimed in claim 1 in the preparation of a medicament for preventing and / or treating diabetes associated with AMPK activation.

3. The use according to claim 2, characterized in that: The composition further comprises one or more drugs and / or extracts.

4. The use according to claim 3, characterized in that The medicine comprises a glucose-lowering drug.

5. The use according to claim 4, characterized in that The hypoglycemic drug is selected from one or more of the following: glipizide, gliclazide, glibenclamide, glibornamide, glimepiride, gliquidone, acarbose, voglibose, pioglitazone, sitagliptin, saxagliptin, vildagliptin, metformin, rosiglitazone and insulin.

6. Use of a preparation comprising the compound or a pharmaceutically acceptable salt thereof according to claim 1 or the composition according to any one of claims 2 to 5 in the preparation of a medicament for preventing and / or treating diabetes associated with AMPK activation.

7. The use according to claim 6, characterized in that The formulation further comprises one or more pharmaceutically acceptable excipients.

8. The use according to claim 7, characterized in that The auxiliary material is selected from one or more of the following: dispersants, wetting agents, binders, diluents, glidants, lubricants, sustained-release agents, plasticizers, disintegrants, inclusion agents, flavoring agents, opacifiers and antioxidants.

9. The use according to claim 6, characterized in that: The dosage form of the preparation is tablet, pill, capsule, powder, injection, film, lozenge, granule or oral solution.

10. The use according to any one of claims 1 to 9, characterized in that The diabetes mellitus includes type I diabetes mellitus and / or type II diabetes mellitus.

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