CN117018091A - Activation of AMPK by Phoebe cheilis extract - Google Patents

Abstract

The invention discloses an application of photinia extract in preparing medicines, foods, beverages or dietary supplements for preventing and/or treating diabetes, obesity and/or complications thereof related to AMPK activation. The photinia fraseri extract disclosed by the invention can remarkably activate the in-vitro activity of the AMPK and promote glucose uptake, and can be applied to preparation of medicines, foods, beverages or dietary supplements for diabetes mellitus, obesity and/or complications thereof related to the activation of the AMPK.

Description

Activation of AMPK by Phoebe cheilis extract

Technical Field

The invention relates to the technical field of medicines, in particular to an activation effect of a photinia extract on AMPK.

Background

The agalloch (agalloch) is an agalloch produced by a special species of aquilaria sinensis (Aquilaria sinensis (lour.) Gilg) and is recorded in a plurality of documents as high-quality medicinal agalloch, however, the agalloch (agalloch) is very rare for a long time due to the rare and historical reasons of the resources of the agalloch (agalloch). In recent years, as the artificial cultivation technology of the phoenix is mature, the phoenix (agilawood) is primarily developed into an industry and is still vigorously developed. However, the Qilan (agilawood) industry currently lacks quality standards and has insufficient development and utilization for the Qilan (agilawood) industry.

AMPK (adenosine monophosphate activated protein kinase, adenosin 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 treating various diseases, which is a major effector of metformin, a first-line clinical drug for treating diabetes, and is also considered as an important therapeutic target for controlling human diseases such as metabolic syndrome, cancer and the like. AMP-activated protein kinase (AMPK) agonists have long been used as international hot spots for the study of metabolic diseases such as obesity and diabetes.

Specifically, the AMPK signal pathway is an energy sensor and regulator, and plays an important role in promoting ATP production in various tissues and suppressing ATP consumption. The kinase is activated in response to stress that depletes cellular ATP supplies, such as hypoglycemia, hypoxia, ischemia, and heat shock. AMPK acts as a cellular energy sensor capable of efficiently responding to low ATP levels, and activation of AMPK can actively regulate and supplement the signaling pathway of cellular ATP supply. For example, AMPK activation is effective in promoting GLUT4 transcription and translocation, which in turn leads to increased glucose uptake by the body stimulated by insulin.

AMPK is considered as a key therapeutic target for the treatment of obesity, type two diabetes and cancer as a central regulator of lipid and sugar metabolism. AMPK down-regulates several core proteins during ATP consumption, such as CREB transcriptional coactivator (TORC 2) and glycogen synthase, can ultimately lead to gluconeogenesis, down-regulation or inhibition of 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, typically caused by various stresses, and modulates signal pathways that increase the available ATP supply.

In recent years, there have been many new findings on both AMPK activation and AMPK signal paths. The new findings suggest that AMPK can be activated before ATP levels drop when glucose levels drop, timely supplementing potential ATP deficiency.

There are two main indirect activation modes of AMPK known at present. More importantly, the ratio of AMP/ATP in cells, such as berberine hydrochloride and metformin, is increased by inhibiting respiratory chain to achieve the effect of indirectly activating AMPK, thereby generating curative effect.

AMPK is an important kinase in the process of regulating energy of a body, and maintains energy supply and nutrition supply of the cell by sensing the level of ATP in the body. Under the condition of low energy supply, the AMPK phosphorylates specific enzyme and site, can increase ATP generation and reduce ATP consumption. In mammals, when AMPK is activated, its regulation acts on almost the whole physiological metabolic process of living bodies, and has an indispensable important role in 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 essential for the body to maintain glucose balance. The activation of AMPK can actively improve the metabolic imbalance caused by type two diabetes mellitus, however, the molecular mechanism for activating AMPK is very complex, and the activation of AMPK through drug molecules, the finding of AMPK activators which can activate AMPK with high efficiency and low side effect, is a great challenge for treating diabetes mellitus.

Whereas extracts containing megalopsis-2-2 phenethyl chromones (abbreviated as "FTPECs") and/or 5,6,7, 8-tetrahydroxy-2- (2-phenethyl) chromones (abbreviated as "THPECs") in the phoenix have not been reported in relation to diabetes, obesity and/or complications thereof associated with AMPK activation.

Disclosure of Invention

Based on this, the present invention provides a use of a photinia extract for the preparation of a medicament, food, beverage or dietary supplement for the prevention and/or treatment of diabetes, obesity and/or complications thereof associated with AMPK activation.

Further, the extract of the photinia serrulata comprises a separating part of the megalopum type-2-2 phenethyl chromone compounds and/or a separating part of the 5,6,7, 8-tetrahydroxy type-2- (2-phenethyl) chromone compounds.

Further, the phoenix extract is prepared by a preparation method comprising the following steps:

(1) Weighing a proper amount of Phoebe cheilis sample, and adding alcohol solution with a certain mass/volume ratio;

(2) Extracting after soaking at room temperature; and

(3) Cooling and filtering, removing solvent and drying to obtain the extract.

Further, the mass/volume ratio is in g/ml or kg/L.

Further, the mass/volume ratio is from 1:50 to 300, such as from 1:75 to 200, such as from 1:90 to 150, such as about 1:100.

Further, the alcohol is ethanol.

Further, the alcohol solution has a volume percentage concentration of 80 to 100%, for example, about 95%.

Further, the room temperature soaking time is 0.2 to 1 hour, for example, about 0.5 hour.

Further, the extraction is reflux extraction or ultrasonic extraction, such as micro-boiling reflux extraction.

Further, the extraction time is 0.5h to 2h, for example about 1h.

Further, the drying is freeze drying or spray drying.

Further, the preparation method further comprises suspending the extract in water, and extracting with dichloromethane to obtain the isolation part of the grandma wood type-2-phenethyl chromone compound and/or the isolation part of the 5,6,7, 8-tetrahydroxy type-2- (2-phenethyl) chromone compound.

Further, before extraction with the dichloromethane, extraction was performed with petroleum ether.

Further, the preparation method further comprises the following steps:

(4) Adding the photinia extract into a solvent with a certain mass/volume ratio for dissolution, then adding dry silica gel powder with a certain mass/mass ratio, stirring and volatilizing the solvent, and loading the mixture into a silica gel chromatographic column by a dry method;

(5) Eluting with an eluent, wherein the eluent comprises dichloromethane and/or methane; and

(6) Collecting the fractions to obtain the separating part of the grandma wood type-2-2 phenethyl chromone compound and/or the separating part of the 5,6,7, 8-tetrahydroxy type-2- (2-phenethyl) chromone compound.

Further, the mass/volume ratio is in g/ml or kg/L.

Further, the mass/volume ratio is from 1:10 to 50, such as from 1:15 to 30, such as about 1:22.

Further, the unit of the mass/mass ratio is g/g or kg/kg.

Further, the mass/mass ratio is 1 to 5:10, for example about 1:7.

Further, the solvent is a mixture of an alcohol solution and methylene chloride.

Further, the alcohol is ethanol.

Further, the alcohol solution has a volume percentage concentration of 80 to 100%, for example, about 95%.

Further, the volume ratio of the alcohol solution to the dichloromethane is about 3:2.

Further, the dissolving is performed under ultrasonic conditions.

Further, the time of the ultrasound is 0.5 to 2 minutes, for example, about 1 minute.

Further, the eluent comprises dichloromethane, dichloromethane: methanol = about 40: about 1 and/or dichloromethane: methanol = about 1: about 1.

Further, the elution sequence is 0.5 to 8 column volumes eluted with dichloromethane, with dichloromethane: methanol = about 40: about 1 elution 0.5 to 8 column volumes, and with dichloromethane: methanol = about 1: about 1 elute 0.5 to 8 column volumes.

Further, the order of elution was about 4 column volumes eluted with dichloromethane, with dichloromethane: methanol = about 40: about 1 elution about 3 column volumes, 3 fractions were collected, about 1 column volume per fraction, and dichloromethane: methanol = about 1: about 1 elution about 1 column volume.

Further, with methylene chloride: methanol = about 40: the intermediate stream eluted at about 1 was separated into the grand disk wood-2-2 phenethyl color ketone compound separation sites.

Further, with methylene chloride: methanol = about 1: about 1 column volume of about 1 elution corresponds to a flow split into 5,6,7, 8-tetrahydroxy-2- (2-phenethyl) chromone compound separation sites.

According to another aspect of the present invention, there is provided a use of a composition comprising the above-mentioned photinia extract for the preparation of a medicament, food, beverage or dietary supplement for preventing and/or treating diabetes, obesity and/or complications thereof associated with AMPK activation.

Further, the composition further comprises one or more drugs and/or extracts.

Further, the medicament comprises a hypoglycemic agent.

Further, the hypoglycemic agent is selected from one or more of the following: glipizide, gliclazide, glibenclamide, glimepiride, gliquidone, acarbose, voglibose, pioglitazone, sitagliptin, saxagliptin, vildagliptin, metformin, rosiglitazone and insulin.

According to another aspect of the present invention, there is provided the use of a formulation comprising the above-mentioned extract of photinia fraseri or the above-mentioned composition for the preparation of a medicament, food, beverage or dietary supplement for the prevention and/or treatment of diabetes, obesity and/or complications thereof associated with AMPK activation.

Further, the formulation further comprises one or more pharmaceutically acceptable excipients.

Further, the auxiliary material is selected from one or more of the following: dispersing agents, wetting agents, binders, diluents, glidants, lubricants, slow-release agents, plasticizers, disintegrants, inclusion agents, flavoring agents, opacifiers and antioxidants.

Further, the preparation is in the form of tablet, dripping pill, capsule, powder, injection, film, lozenge, granule or oral liquid.

Further, the diabetes includes type I diabetes and/or type II diabetes.

Further, the complications are acute complications and/or chronic complications.

Further, the acute complication is diabetic ketoacidosis, hypertonic non-ketotic diabetic coma and/or lactic acidosis.

Further, the chronic complications are macrovascular complications, microvascular complications, neuropathy and/or diabetic foot.

Further, the macrovascular complications are cardiovascular and cerebrovascular diseases, coronary heart disease, cerebral apoplexy and/or lower limb gangrene.

Further, the microvascular complication is diabetic retinopathy and/or diabetic nephropathy.

Further, the cells associated with diabetes and/or complications thereof associated with AMPK activation are human kidney epithelial cell line cells.

Further, the human kidney epithelial cell line cell is a HEK293T cell.

Further, the AMPK activation is an indirect activation mechanism.

Further, the AMPK contains an α2 subunit, a β1 subunit and a γ1 subunit.

Further, the activation of AMPK occurs in muscle, liver and/or kidney tissues.

According to another aspect of the present invention, there is provided a method for preparing a phoenix extract, the method comprising the steps of:

(1) Weighing a proper amount of Phoebe cheilis sample, and adding alcohol solution with a certain mass/volume ratio;

(2) Extracting after soaking at room temperature;

(3) Cooling, filtering, removing solvent, and drying to obtain the extract;

(4) Adding the photinia extract into a solvent with a certain mass/volume ratio for dissolution, then adding dry silica gel powder with a certain mass/mass ratio, stirring and volatilizing the solvent, and loading the mixture into a silica gel chromatographic column by a dry method;

(5) Different gradients of dichloromethane were used: eluting and flushing with methanol; and

(6) Collecting the fractions to obtain the separating part of the grandma wood type-2-2 phenethyl chromone compound and/or the separating part of the 5,6,7, 8-tetrahydroxy type-2- (2-phenethyl) chromone compound.

The invention has the beneficial effects that:

the photinia fraseri extract disclosed by the invention can remarkably activate the in-vitro activity of the AMPK and promote glucose uptake, and can be applied to preparation of medicines, foods, beverages or dietary supplements for diabetes mellitus, obesity and/or complications thereof related to the activation of the AMPK.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it will be apparent that the drawings in the following description are only some embodiments of the present invention, and that other drawings can be obtained according to these drawings by those skilled in the art without departing from the scope of the claimed invention.

FIG. 1 is a peak-out chromatogram of a mixed control solution.

FIG. 2 is a graph showing the results of in vitro activity of ethanol extract of Photinia serrulata in 293T cells to activate AMPK. Where the results are presented as mean ± SD (n=3), p <0.01, p <0.001 compared to solvent control (DMSO).

Detailed Description

The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Unless otherwise defined, all technical and scientific terms and abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains or to which this term applies. Although any methods, conditions, materials, or materials similar or equivalent to those disclosed herein can be used in the practice of the present invention, the preferred methods, conditions, materials, or materials are described herein.

The invention is intended to cover all alternatives, modifications and equivalents, which may be included within the art of the invention as defined by the appended 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 invention is in no way limited to the description of methods and materials.

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.

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

As described in the background section, the extract of Photinia serrulata containing megalopum-2-2 phenethyl chromone and/or 5,6,7, 8-tetrahydroxy-2- (2-phenethyl) chromone did not show problems reported in relation to diabetes, obesity and/or complications thereof associated with AMPK activation. In order to solve the above problems, the present invention provides a use of a photinia fraseri extract in the preparation of a medicament, food, beverage or dietary supplement for preventing and/or treating diabetes, obesity and/or complications thereof associated with AMPK activation.

In a preferred embodiment, the extract of Photinia comprises a grandis-type-2-phenethyl chromone compound separation site and/or a 5,6,7, 8-tetrahydroxy-2- (2-phenethyl) chromone compound separation site.

In a preferred embodiment, the extract of phoenix is prepared by a preparation method comprising the steps of:

(1) Weighing a proper amount of Phoebe cheilis sample, and adding alcohol solution with a certain mass/volume ratio;

(2) Extracting after soaking at room temperature; and

(3) Cooling and filtering, removing solvent and drying to obtain the extract.

In a preferred embodiment, the mass/volume ratio is in g/ml or kg/L.

In a preferred embodiment, the mass/volume ratio is from 1:50 to 300, such as from 1:75 to 200, such as from 1:90 to 150, such as about 1:100.

In a preferred embodiment, the alcohol is ethanol.

In a preferred embodiment, the alcohol solution has a volume percent concentration of 80 to 100%, for example about 95%.

In a preferred embodiment, the room temperature soak time is from 0.2 hours to 1 hour, for example about 0.5 hours.

In a preferred embodiment, the extraction is a reflux extraction or an ultrasonic extraction, such as a micro-boiling reflux extraction.

In a preferred embodiment, the extraction time is from 0.5h to 2h, for example about 1h.

In a preferred embodiment, the drying is freeze-drying or spray-drying.

In a preferred embodiment, the preparation method further comprises suspending the extract of Photinia serrulata in water and extracting with dichloromethane to obtain the isolation site of the grandma type-2-2 phenethyl chromone compound and/or the isolation site of the 5,6,7, 8-tetrahydroxy type-2- (2-phenethyl) chromone compound.

In a preferred embodiment, petroleum ether is used for extraction prior to extraction with the dichloromethane.

In a preferred embodiment, the method of preparation further comprises the steps of:

(4) Adding the photinia extract into a solvent with a certain mass/volume ratio for dissolution, then adding dry silica gel powder with a certain mass/mass ratio, stirring and volatilizing the solvent, and loading the mixture into a silica gel chromatographic column by a dry method;

(5) Eluting with an eluent, wherein the eluent comprises dichloromethane and/or methane; and

(6) Collecting the fractions to obtain the separating part of the grandma wood type-2-2 phenethyl chromone compound and/or the separating part of the 5,6,7, 8-tetrahydroxy type-2- (2-phenethyl) chromone compound.

In a preferred embodiment, the mass/volume ratio is in g/ml or kg/L.

In a preferred embodiment, the mass/volume ratio is from 1:10 to 50, such as from 1:15 to 30, such as about 1:22.

In a preferred embodiment, the mass/mass ratio is in g/g or kg/kg.

In a preferred embodiment, the mass/mass ratio is from 1 to 5:10, for example about 1:7.

In a preferred embodiment, the solvent is a mixture of an alcohol solution and methylene chloride.

In a preferred embodiment, the alcohol is ethanol.

In a preferred embodiment, the alcohol solution has a volume percent concentration of 80 to 100%, for example about 95%.

In a preferred embodiment, the volume ratio of the alcoholic solution to the dichloromethane is about 3:2.

In a preferred embodiment, the dissolution is performed under ultrasonic conditions.

In a preferred embodiment, the time of the ultrasound is between 0.5 and 2 minutes, for example about 1 minute.

In a preferred embodiment, the eluent comprises dichloromethane, dichloromethane: methanol = about 40: about 1 and/or dichloromethane: methanol = about 1: about 1.

In a preferred embodiment, the sequence of elution is 0.5 to 8 column volumes eluted with dichloromethane, with dichloromethane: methanol = about 40: about 1 elution 0.5 to 10 column volumes, and with dichloromethane: methanol = about 1: about 1 elute 0.5 to 10 column volumes.

In a preferred embodiment, the order of elution is about 4 column volumes eluted with dichloromethane, with dichloromethane: methanol = about 40: about 1 elution about 3 column volumes, 3 fractions were collected, about 1 column volume per fraction, and dichloromethane: methanol = about 1: about 1 elution about 1 column volume.

In a preferred embodiment, methylene chloride is used: methanol = about 40: the intermediate stream eluted at about 1 was separated into the grand disk wood-2-2 phenethyl color ketone compound separation sites.

In a preferred embodiment, methylene chloride is used: methanol = about 1: about 1 column volume of about 1 elution corresponds to a flow split into 5,6,7, 8-tetrahydroxy-2- (2-phenethyl) chromone compound separation sites.

In the present invention, "about" means a value within + -5% of a specific value. For example, "about 1:100" includes + -5% of 1:100, or from 1:95 to 1:105, including 1:95 and 1:105;

"about 95" includes 95.+ -. 5%, or from 90.25 to 99.75, inclusive of 90.25 and 99.75;

"about 0.5" includes + -5% of 0.5, or from 0.475 to 0.525, including 0.475 and 0.525;

"about 1" includes + -5% of 1, or from 0.95 to 1.05, including 0.95 and 1.05; "about 1:22" includes + -5% of 1:22, or from 1:20.9 to 1:23.1, including 1:20.9 and 1:23.1;

"about 1:7" includes + -5% of 1:7, or from 1:6.65 to 1:7.35, including 1:6.65 and 1:7.35; "about 3" includes + -5% of 3, or from 2.85 to 3.15, including 2.85 and 3.15;

"about 2" includes + -5% of 2, or from 1.9 to 2.1, including 1.9 and 2.1; "about 4" includes + -5% of 4, or from 3.8 to 4.2, including 3.8 and 4.2; "about 40" includes + -5% of 40, or from 38 to 42, inclusive of 38 and 42.

According to another aspect of the present invention, there is provided a use of a composition comprising the above-mentioned photinia extract for the preparation of a medicament, food, beverage or dietary supplement for preventing and/or treating diabetes, obesity and/or complications thereof associated with AMPK activation.

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

In a preferred embodiment, the medicament comprises a hypoglycemic agent.

In a preferred embodiment, the hypoglycemic agent is selected from one or more of the following: glipizide, gliclazide, glibenclamide, glimepiride, gliquidone, acarbose, voglibose, pioglitazone, sitagliptin, saxagliptin, vildagliptin, metformin, rosiglitazone and insulin.

According to another aspect of the present invention, there is provided the use of a formulation comprising the above-mentioned extract of photinia fraseri or the above-mentioned composition for the preparation of a medicament, food, beverage or dietary supplement for the prevention and/or treatment of diabetes, obesity and/or complications thereof associated with AMPK activation.

In the present invention, the term "treatment" also includes "prophylaxis" unless there is a specific description of the contrary. The terms "therapeutic" and "therapeutic" should be understood accordingly.

In the present invention, the term "treatment" includes alleviation, inhibition or amelioration of symptoms or conditions of the disease; inhibit the occurrence of complications; improving or preventing underlying metabolic syndrome; inhibiting the occurrence of a disease or condition, such as controlling the progression of a disease or condition; alleviating a disease or symptom; causing the disease or symptom to subside; alleviating complications caused by diseases or symptoms, or preventing or treating signs caused by diseases or symptoms. As used herein, a composition or formulation, upon administration, may result in an improvement in a disease, symptom, or condition, particularly an improvement in severity, delay of onset, slow progression, or decrease in duration of the condition. Whether stationary or temporary, continuous or intermittent, may be due to or associated with administration.

In a preferred embodiment, the formulation further comprises one or more pharmaceutically acceptable excipients.

In a preferred embodiment, the formulation of the invention contains 0.00001 to 50wt.%, or 0.0001 to 10wt.%, or 0.0001 to 5wt.%, or 0.005 to 1wt.%, or 0.1 to 20wt.%, or 0.5 to 15wt.%, or 1 to 5wt.% of at least one pharmaceutically acceptable auxiliary material, calculated to the weight of the formulation.

In the present invention, the term "pharmaceutically acceptable" refers herein to a material, such as a carrier or diluent, that does not cause the biological activity or nature of the compounds in the extract to disappear, and that is relatively non-toxic, e.g., the administration of a material to an individual that does not cause unwanted biological effects or interact in a deleterious manner with any of the components of the extract in which it is contained.

In the present invention, the term "pharmaceutically acceptable excipients" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient (i.e., capable of eliciting a desired therapeutic effect without eliciting any undesired local or systemic effects), as is well known in the art (see, e.g., remington's Pharmaceutical sciences. Mediated by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995).

In a preferred embodiment, the adjuvant is selected from one or more of the following: dispersing agents, wetting agents, binders, diluents, glidants, lubricants, slow-release agents, plasticizers, disintegrants, inclusion agents, flavoring agents, opacifiers and antioxidants.

The person skilled in the art will know how to select a specific chemical within the above-mentioned auxiliary material category. For example, the dispersant may be selected from one or more of the following: croscarmellose sodium, sodium starch glycolate and pregelatinized corn starch. The wetting agent may be selected from one or more of the following: polyoxymethylene stearate, poloxamer, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hydrogenated castor oil, polyoxyethylene alkyl ethers, polysorbates such as polysorbate 80, cetyl alcohol, glycerin fatty acid esters (such as triacetin, glycerin monostearate and the like), polyoxyethylene fatty acid esters, polyethylene glycol fatty acid esters, sodium lauryl sulfate, sorbitol fatty acid esters, sucrose fatty acid esters, polyoxyethylene ethers, benzalkonium chloride, polyoxyethylene castor oil, and sodium docusate. The binder may be selected from one or more of the following: polyvinylpyrrolidone, hydroxypropyl cellulose, polyethylene glycol and methylcellulose. The diluent may be selected from one or more of the following: sugar powder, starch, compressible starch, lactose, dextrin, mannitol, sorbitol, microcrystalline cellulose, calcium sulfate and calcium carbonate. The glidant is micro powder 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 slow release agent can be selected from one or more of the following: sodium carboxymethyl cellulose, low substituted hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, hydroxyethyl cellulose, acacia, 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 betacyclodextrin. The flavouring 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 bisulphite, sodium metabisulfite, sodium sulfite and sodium thiosulfate.

The auxiliary materials are preferably inert to drugs or can have synergistic or additive effects to enhance the therapeutic activity of the pharmaceutical composition, and the auxiliary materials are only listed, so that the auxiliary materials actually used in the invention are not limited to the auxiliary materials and can be adjusted according to the actual situation, and the effects of the invention can be achieved.

In a preferred embodiment, the formulation is in the form of a tablet, drop pill, capsule, powder, injection, film, lozenge, granule or oral liquid.

In a preferred embodiment, the diabetes comprises type I diabetes and/or type II diabetes.

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

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

In a preferred embodiment, the chronic complication is macrovascular complications, microvascular complications, neuropathy and/or diabetic foot.

In a preferred embodiment, the macrovascular complication is cardiovascular and cerebrovascular disease, coronary heart disease, cerebral apoplexy and/or lower limb gangrene.

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

In a preferred embodiment, the cells associated with diabetes mellitus and/or complications thereof associated with AMPK activation are human kidney epithelial cell line cells.

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

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

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

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

According to another aspect of the present invention, there is provided the above-mentioned extract of photinia fraseri, 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.

According to another aspect of the present invention, there is provided a method for preventing and/or treating diabetes, obesity and/or complications thereof associated with AMPK activation, comprising administering to the subject an effective amount of the above-mentioned photinia extract, the above-mentioned composition or the above-mentioned formulation.

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 may be advantageously used as subjects representing models of diabetes, obesity and/or complications thereof associated with AMPK activation. Preferably, the subject is a human.

An "effective amount" of a composition or formulation used in the present invention may achieve the desired therapeutic and/or prophylactic effect. The amount effective for this use will depend, for example, on the pharmaceutical composition, the mode of administration, the stage and severity of the disease being treated, the weight and general health of the individual, and the discretion of the prescribing physician. The dose may be administered once a week, or for two days or once a day, or even several times a day. Dosage units may be administered for a short period (e.g., weeks to months) or longer period (months to years).

According to another aspect of the present invention, there is provided a method for preparing a phoenix extract, the method comprising the steps of:

(1) Weighing a proper amount of Phoebe cheilis sample, and adding alcohol solution with a certain mass/volume ratio;

(2) Extracting after soaking at room temperature;

(3) Cooling, filtering, removing solvent, and drying to obtain the extract;

(4) Adding the photinia extract into a solvent with a certain mass/volume ratio for dissolution, then adding dry silica gel powder with a certain mass/mass ratio, stirring and volatilizing the solvent, and loading the mixture into a silica gel chromatographic column by a dry method;

(5) Different gradients of dichloromethane were used: eluting and flushing with methanol; and

(6) Collecting the fractions to obtain the separating part of the grandma wood type-2-2 phenethyl chromone compound and/or the separating part of the 5,6,7, 8-tetrahydroxy type-2- (2-phenethyl) chromone compound.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not noted, are generally according to conventional conditions or conditions suggested by manufacturers.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

The above-mentioned features of the invention, or of the embodiments, may be combined in any desired manner. All of the features disclosed in this patent specification may be combined with any combination of the features disclosed in this specification, and the various features disclosed in this specification may be substituted for any alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the disclosed features are merely general examples of equivalent or similar features.

Examples

1. Materials and instruments

1.1 materials

Human kidney embryo epithelial cell line HEK293T cells (deposited at the university of Qinghai university, prop. From American type culture Collection, american Type Culture Collection, ATCC) and 3T3-L1 mouse fat precursor cells (from American type culture Collection, american Type Culture Collection, ATCC); anti-AMPK phosphorylation and anti-AMPK total antibodies (Cell signaling, usa); antibodies against beta-actin (Beijing BI Co.); PBS phosphate buffer (Hyclone); south america fetal bovine serum (Gibco); a mixture of green streptomycin and double antibody (Hyclone); trypsin (trypsin); PVDF membrane (Millipore, usa); glucose uptake kit (Promega); DMEM high sugar medium (Gibco); OPTI-MEM medium (Hyclone); mycoplasma inhibitors (scientific); dexamethasone (DEX), dimethyl sulfoxide (DMSO), insulin (Insulin), 1-methyl-3-isobutanol xanthine (IBMX), 5' -ATP disodium salt, 5' -AMP disodium salt, 5' -ADP disodium salt, metformin (Met), berberine hydrochloride (BBR), and Compund C were all derived from Sigma, USA. The Phoebe cheilis sample is identified by the professor Zhang Zhijie of Chinese medical institute of Chinese medical science and is stored in the crude drug center drug storage room of Chinese medical science. Methanol (lot number: 20230504, beijing city, general fine chemical company). Dichloromethane (lot number 20230515, tianjin Fuyu fine chemical Co., ltd.). Silica gel powder (200-300 mesh, qingdao ocean chemical plant). Absolute ethyl alcohol (lot number: 20230603, beijing city, general fine chemical company). Chromatography columns, glass rods, beakers and measuring cylinders are commercially available.

Control: linalool (lot number: 111980-201904, purity not less than 98%) was purchased from chinese food and drug assay institute; 6-hydroxy-2- (2-phenethyl) chromone (lot: AF21042704, purity not less than 98%), isoagalloch eaglewood tetrol (lot: AF21042702, purity not less than 98%), 8-chloro-2- (2-phenethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochromone (lot: AF210328076, purity not less than 95%), 4' -methoxy agalloch eaglewood tetrol (lot: AF21042706, purity not less than 90%), and 6, 7-dimethoxy-2- (2-phenethyl) chromone (lot: AF22030801, purity not less than 98%) were all purchased from Chengdu Biotech Co., ltd; 6, 7-dimethoxy-2- [2- (4-methoxyphenyl) ethyl ] chromone (lot: STC8600105, purity. Gtoreq.98%), 6-methoxy-2- (2-phenethyl) chromone (lot: STC6680105, purity. Gtoreq.98%) and 6-methoxy-2- [2- (3-methoxyphenyl) ethyl ] chromone (lot: STC6690105, purity. Gtoreq.98%) were all purchased from Shanghai Shimadder biotechnology Co., ltd; 2- (2-phenethyl) chromone (lot number PS020524, purity > 98%) was purchased from Chenopodium biotechnology Co., ltd.

1.2 instruments

science-IID ultrasonic cell disruption apparatus (Ningbonew Zhi biological Co.), DYCZ-24DN electrophoresis apparatus (Beijing Liuyi instrument Co.), ELITE II carbon dioxide constant temperature incubator (Revco Co.), YP4002N electronic balance (Shanghai Qinghai instrument Co., ltd.), SK7210HP ultrasonic cleaner (Shanghai Kochia ultrasonic instruments Co., ltd.), LC-20T high performance liquid chromatograph (Shimadzu Co., ltd.), N-1100 rotary evaporator (Shanghai Ailang instrument mailbox Co.).

2. Experimental method

2.1 pharmaceutical preparation

2.1.1 preparation of Phoebe cheilis extract

Precisely weighing 5g of the photinia sample, adding 500ml of 95% ethanol, soaking for half an hour at room temperature, and extracting for 1 hour by micro-boiling reflux. Filtering with 8 layers of gauze and 0.45 μm microporous membrane after cooling, rotary evaporating the filtrate to remove ethanol, and lyophilizing to obtain QINAN extract. Adding DMSO to dissolve, and preparing into 250mg/ml Phoebe maculata extract, and refrigerating in refrigerator. When cells are administered, the extract of Phoebe cheilis is dissolved in DMSO, and the final concentration of DMSO is 0.1% without toxicity to cells.

After the photinia extract was suspended in water, it was extracted with Petroleum Ether (PE), dichloromethane and ethyl acetate (EtOAc), n-butanol in this order.

2.1.2 preparation of the separation site of the megalopum-2-phenethyl chromone and the separation site of the 5,6,7, 8-tetrahydroxy-2- (2-phenethyl) chromone

2.1.2.1 preparation method 1

(1) The chromatographic column is fixed on the iron stand, and the piston of the chromatographic column is closed;

(2) Placing a small piece of cotton at the bottom of the chromatographic column, and then placing quartz sand with the height of 1-2 cm;

(3) 20mL of methylene chloride (1/3-1/4 column volume of liquid) was poured into the column;

(4) Opening the piston, beating the column body by the ear washing ball, and discharging bubbles in cotton and quartz sand;

(5) Closing the piston, and reserving liquid (1/3-1/4 column volume of liquid) in the column;

(6) Weighing 5.06g of silica gel powder, diluting with 15-20mL of dichloromethane, and stirring to form paste;

(7) Opening a piston, and pouring the stirred silica gel powder homogenate into a chromatographic column at one time;

(8) The ear washing ball strikes the column body to enable the silica gel to sink and compact (finally, the volume of the silica gel column is 14 mL);

(9) After the silica gel is compacted, closing the piston when the liquid flows to a position 1-2cm away from the silica gel surface;

(10) 0.26g of the Phoebe cheilis extract powder (from the preparation of 2.1.1 Phoebe cheilis extract) is weighed, dissolved in 4mL of solvent (3 mL of 95% ethanol, 1mL of dichloromethane) and sonicated for 1min;

(11) Sucking the sample solution by using a 1mL rubber head dropper, and slowly dripping the sample solution into a silica gel chromatographic column along the column wall;

(12) After loading was completed, dichloromethane with different gradients was used: eluting and flushing with methanol;

(13) Elution order: (1) dichloromethane: methanol=1: 0 (three column volumes) 3 fractions (1 column volume for each fraction) were collected (2) dichloromethane: methanol=1: 1 (three column volumes) 3 fractions (each fraction about 1 column volume), (3) methylene chloride: methanol=0: 1 (three column volumes) 1 fraction was collected;

(14) Separating 5ml of each flow, placing the flow in a rotary evaporator for spin drying, and then re-dissolving the flow with 5ml of 95% ethanol;

(15) The re-dissolved solution is filtered by a microporous filter membrane with the thickness of 0.22 mu m and enters a high-efficiency liquid phase.

2.1.2.2 preparation method 2

(1) The chromatographic column is fixed on the iron stand, and the piston of the chromatographic column is closed;

(2) Placing a small piece of cotton at the bottom of the chromatographic column, and then placing quartz sand with the height of 1-2 cm;

(3) 20mL of methylene chloride (1/3-1/4 column volume of liquid) was poured into the column;

(4) Opening the piston, beating the column body by the ear washing ball, and discharging bubbles in cotton and quartz sand;

(5) Closing the piston, and reserving liquid (1/3-1/4 column volume of liquid) in the column;

(6) Weighing 5.13g of silica gel powder, diluting with 15-20mL of dichloromethane, and stirring to form paste;

(7) Opening a piston, and pouring the stirred silica gel powder homogenate into a chromatographic column at one time;

(8) The ear washing ball strikes the column body to enable the silica gel to sink and compact (finally, the volume of the silica gel column is 14 mL);

(9) After the silica gel is compacted, closing the piston when the liquid flows to a position 2-3cm away from the silica gel surface;

(10) 0.23g of the extract powder of Phoebe cheilis (from the preparation of the extract of Phoebe cheilis of "2.1.1") was weighed, dissolved in 5mL of solvent (3 mL of 95% ethanol, 2mL of dichloromethane) and sonicated for 1min;

(11) Adding 1.59g of dry silica gel powder into the dissolved sample solution, uniformly stirring by using a glass rod, placing the mixture in a fume hood for overnight, and completely volatilizing the solvent;

(12) Pouring silica gel powder of the adsorbed sample into a silica gel chromatographic column;

(13) Beating the column body by using an ear washing ball to enable the silica gel powder of the sample to sink;

(14) After loading was completed, dichloromethane with different gradients was used: eluting and flushing with methanol;

(15) Elution order: (1) dichloromethane: methanol=1: 0 (4 column volumes, 2 fractions were collected, 2 column volumes each), (2) dichloromethane: methanol=40: 1 (3 column volumes, 3 fractions were collected, 1 column volume per fraction), (3) dichloromethane: methanol=1: 1 (3 column volumes, 3 fractions were collected, 1 column volume per fraction), (4) dichloromethane: methanol=0: 1 (3 column volumes, 1 fraction was collected).

(16) 5ml of each stream was taken and dried by spin-drying on a rotary evaporator and then reconstituted with 5ml of 95% ethanol.

(17) The re-dissolved solution is filtered by a microporous filter membrane with the thickness of 0.22 mu m and enters a high-efficiency liquid phase.

2.1.2.3 preparation method 3

(1) Weighing 0.23g of Phoebe cheilis extract powder (from preparation of 2.1.1 Phoebe cheilis extract), dissolving with 5mL of solvent (i.e. absolute ethanol), and performing ultrasonic treatment for 1min;

(2) The dissolved sample solution was applied to an MCI column to separate fractions of the photinia extract at different ethanol-water (water, 30%, 50%, 70%, 95% ethanol) ratios.

2.2 preparation and detection of control solution

Taking a proper amount of reference substances of agalloch eaglewood tetrol, isoagalloch eaglewood tetrol, 8-chloro-2- (2-phenethyl) -5,6, 7-trihydroxy-5, 6,7, 8-tetrahydrochysene chromone, 6, 7-dimethoxy-2- (phenethyl) chromone, 6-hydroxy-2- (2-phenethyl) chromone, 6-methoxy-2- [2- (3-methoxy) phenethyl ] chromone, 6, 7-dimethoxy-2- [2- (4-methoxy) phenethyl ] chromone, 2- (2-phenethyl) chromone and 4' -methoxyagalloch eaglewood tetrol, precisely weighing, fully dissolving and preparing into a reference substance mother solution of 1mg/ml by using absolute ethyl alcohol, and filtering with a 0.22 mu m filter membrane.

Chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler; acetonitrile is taken as a mobile phase A,0.1% formic acid solution is taken as a mobile phase B, and the elution gradient condition is measured to be 0-20min,20-25% A;20-60min,25-65% A;60-65min,65-75% A;65-70min,75-95% A. The column temperature is 30 ℃, the flow rate is 1ml/min, and the sample injection quantity is 10 μl.

2.3 culture and pharmaceutical treatment of human Kidney epithelial cell line HEK293T cells

HEK293T cells were cultured in pre-formulated DMEM complete medium containing 10% fetal bovine serum and 1% diabody, CO ₂ The temperature in the cell incubator is 37 ℃, the carbon dioxide content is 5 percent, the culture medium is replaced every 24 hours in the cell culture process, and the medicine treatment can be carried out when the cell density reaches about 80 percent. When cells are treated, drugs with different concentrations and different extraction sites are added into a complete culture medium (DMEM+0.5% without fatty acid Bovine Serum Albumin (BSA) +1% dual antibody) prepared in advance, and the cells are collected after culturing for 1 h.

2.4 SDS-polyacrylamide gel electrophoresis and Western blot detection

After the administration, cells were collected and lysed by adding a cell lysate containing a protease inhibitor and a phosphatase inhibitor at 4℃for 2 hours, and then the cell lysate was centrifuged to obtain a supernatant, and the supernatant was subjected to a treatment with beta-mercaptoethanol for 30 minutes and boiled for 5 minutes to denature proteins. The Cell lysate was subjected to SDS-PAGE gel electrophoresis separation, the separated proteins were transferred onto PVDF membrane by wet transfer method, then blocked with 5% skimmed milk powder TBST solution for 2h at room temperature, then incubated overnight at 4℃with anti-AMPK antibody (Cell Signaling) and anti-p-AMPK antibody (Cell Signaling), then incubated with goat anti-rabbit horseradish peroxidase-labeled secondary antibody (Santa Cruz) for 1 h at room temperature, after full washing of the membrane, the luminophore was added, the photographic film was exposed in darkroom, and the image data was photographed and processed.

The total amount of AMPK is used as a reference, the NIH Image J software is used for analyzing and obtaining the gray values of pAMPK and AMPK western blotting bands at different concentration points in the graph, the ratio pAMPK/AMPK is used for representing the phosphorylation level of AMPK, the GraphPad Prism 6.0 is used for carrying out statistical analysis and mapping on pAMPK/AMPK, p is less than 0.01, and p is less than 0.001, so that the difference has obvious statistical significance.

2.5 glucose uptake-in vitro analysis

The glucose uptake assay was performed using a Promega glucose uptake assay kit, and the glucose uptake capacity of the mature 3T3-L1 adipocytes after drug treatment was measured using the kit. The kit was removed from the-20 ℃ refrigerator and thawed. According to the use instructions of the kit, the neutralizing buffer solution and the stopping buffer solution in the kit are stored at 4 ℃, and the rest components are stored at-20 ℃. The experimental method is as follows:

(1) Cell treatment

3T3-L1 cells were induced to mature adipocytes by day 7. Changing liquid, removing maintenance liquid, adding whole culture medium containing medicine, treating for 24 hr, and treating at 37deg.C with 5% CO ₂ Is cultured in a cell culture box.

(2) Desugaring treatment

After 24h of drug treatment, the culture medium is removed, and the sugar-free, serum-free and antibiotic-free culture medium containing the drug is added for starvation treatment for 2 to 3h, and the temperature is 37 ℃ and the concentration of CO is 5 percent ₂ And (5) culturing the cells in a cell culture box.

(3) Insulin activation

Removing the medium in (2), adding an insulin containing 10. Mu.g/mLPBS solution, 5% CO at 37deg.C ₂ Is activated for 10min, insulin is removed and washed 2-3 times with PBS.

(4) Preparing 2DG6P detection reaction liquid

The reaction solution was prepared 1h in advance. The preparation was carried out in accordance with the respective set of ratios provided in the kit instructions, in accordance with a volume of 100. Mu.L/sample.

(5) Preparing cell reaction liquid

Insulin treated cells and unactivated cells were carefully washed with PBS. Using 1mM2DG solution at 37℃and 5% CO ₂ The cells were treated in the cell incubator for 20 minutes, and half the volume of 2DG stop buffer was added to stop the glucose uptake reaction of the cells and lyse the cells. After shaking several times and complete lysis of the cells, the same volume of neutralization buffer as the stop buffer is added. All the above-mentioned liquids were collected in a centrifuge tube, centrifuged at 5000g for 5min at room temperature, and the supernatant was collected as a cell reaction solution required for the experiment.

(6) Detection of glucose uptake

100 mu L of cell reaction liquid and 100 mu L of 2DG6P detection reaction liquid are taken and placed into a 96-well plate to be fully mixed, incubated for 1h (room temperature) in a dark place, and the fluorescence intensity of each experimental group is detected on an Enspire multi-mode micro-well plate detector.

2.6 data processing and statistical analysis

Western blot banding analysis statistical analyses including one-way analysis of variance (ANOVA) and mean comparison were performed using NIH Image J Image analysis software using GraphPad Prism 6.0. * Indicating that p <0.05 indicates that the results are statistically significant, p <0.01 indicates that p <0.001 indicates that the results are statistically significant.

3. Results

3.1 Compound type determination

According to the peak-emitting pattern data of the mixed reference solution, as shown in fig. 1, it is known that the first 3 compounds emitting peaks are THPECs compounds with large polarity, and the later compounds emitting peaks are FTPECs compounds.

3.2 optimization of preparation method of separation site

The results show that the required relevant active sites (i.e. THPECs class compound separation site and FTPECs class compound separation site) are mainly distributed in dichloromethane: methanol (1:0) first fraction and dichloromethane: methanol (1:1) in the second fraction, since the loading was a wet loading of the dissolved sample with 95% ethanol, methylene chloride resulted: some of the more polar material in the first fraction eluted with methanol (1:0) also eluted partially.

Based on the above results, the loading method was further optimized, as shown in "2.1.2.2 preparation method 2".

The results show that the relevant active sites required are mainly distributed in methylene chloride: methanol (40:1) second fraction (this fraction was distilled to dryness to give FTPECs-like compound separation site) and dichloromethane: methanol (1:1) the first fraction (this fraction was distilled to dryness to give THPECs-like compounds as separation sites) was eluted with dichloromethane: methanol (1:0) elution, little eluting of sample in silica gel column, so dichloromethane was used: methanol (40:1) was further eluted.

The preparation method realizes very effective separation of THPECs compounds and FTPECs compounds in the Phoebe extract, and unexpected technical effects are obtained.

The technical scheme related to the 2.1.2.3 preparation method 3 cannot separate THPECs compounds from FTPECs compounds in the Phoebe fraseri extract.

3.3 Activity verification of Phoebe cheilis

To examine whether the Phoebe extract was able to activate AMPK, we treated HEK293T cells with different concentrations of Phoebe extract for 1 hour, and then examined pAMPK (Thr-172) and AMPK levels in cell lysates using SDS-PAGE and Western Blot (Western immunoblotting). As shown in FIG. 2, pAMPK levels in cells were dose-dependently elevated after treatment with Photinia extract. The Western Blot analysis results show that both the phoebe extract are capable of activating AMPK in HEK293T cells in vitro, both have AMPK activating activity and activate in an activity-dose dependent manner. The AMPK activating ability of the extract of photinia fraseri is even stronger than that of the positive drug metformin at a concentration of 120 μg/ml.

AMPK activating ability of the photinia extract is concentrated at the dichloromethane extraction site.

3.4 glucose uptake-in vitro analysis

Insulin sensitivity is closely related to the glucose uptake capacity of cells. In order to study the effect of different isolated sites of the Phoebe cheilis extract on glucose uptake by mature 3T3-L1 adipocytes, we first induced differentiated fat precursor cells to mature adipocytes over 7-8 days, observed that the cell state was good, and that a large number of lipid droplets appeared.

Then, the extracts of the Phoebe chebylonicae at different separation sites (namely, the separation site of the THPECs compounds and the separation site of the FTPECs compounds) are added with DMSO to prepare mother liquor for cell experiments, and cells are treated at the concentration of 20 mug/ml, so that the non-insulin-dependent glucose uptake is detected. Mature 3T3-L1 cells were treated with different isolated fractions of the Photinia extract at equal concentrations, respectively, using 1. Mu.M BBR as positive control. To verify that the samples promote glucose uptake by cells by activating AMPK, we treated cells with 20uM AMPK inhibitor complete C simultaneously with the drug. After 24h treatment, the cells were starved with sugar-free, serum-free, double antibody-free medium containing the same drug for 2h.

And finally removing starvation culture medium, adding 2DG (glucose substitute) in a glucose uptake detection kit into cells, taking 2DG by the cells after 20 minutes, detecting the uptake of glucose by using a Promega glucose detection kit, collecting and analyzing data, carrying out statistical analysis on the data by using GraphPad Prism 9.0 with the uptake of 2DG at a DMSO point as 1, and processing all the data to obtain data results of each group.

The results show that both isolated sites of the extract of photinia fraseri are able to promote glucose uptake in mature adipocytes. The THPECs separation part can promote the glucose uptake of cells by 1.2-1.6 times, the FTPECs separation part can promote the glucose uptake of cells by 1.4-1.8 times, the BBR serving as a positive control can promote by 1.44 times, and the FTPECs separation part shows a significant difference (p is less than 0.01) relative to the THPECs separation part. There was no significant change in cellular glucose uptake following simultaneous treatment with AMPK inhibitor Compound C and the sample.

The experimental results show that: the two separate sites of the extract of Phoebe cheilis are able to promote glucose uptake in a non-insulin dependent manner, and in the experimental group with Compound C, the two separate sites of the extract of Phoebe cheilis are not able to promote glucose uptake. Two isolated sites of the Phoebe cheilis extract were shown to promote glucose uptake non-insulin-dependent by activating AMPK, and the glucose uptake promoting effect was lost when AMPK activity was inhibited.

The foregoing has outlined rather broadly the more detailed description of embodiments of the invention in order that the detailed description of the principles and embodiments of the invention may be implemented in conjunction with the detailed description of embodiments of the invention that follows. Meanwhile, based on the idea of the present invention, those skilled in the art can make changes or modifications on the specific embodiments and application scope of the present invention, which belong to the protection scope of the present invention. In view of the foregoing, this description should not be construed as limiting the invention.

Claims (10)

1. Use of a photinia extract for the preparation of a medicament, food, beverage or dietary supplement for the prevention and/or treatment of diabetes, obesity and/or complications thereof associated with AMPK activation.

2. The use according to claim 1, characterized in that the extract of the photinia fraseri comprises a separation site of megalopum-2-phenethyl chromone compounds and/or a separation site of 5,6,7, 8-tetrahydroxy-2- (2-phenethyl) chromone compounds.

3. Use according to claim 1 or 2, characterized in that the extract of phoenix is prepared by a preparation method comprising the steps of:

(1) Weighing a proper amount of Phoebe cheilis sample, and adding alcohol solution with a certain mass/volume ratio;

(2) Extracting after soaking at room temperature; and

(3) Cooling, filtering, removing solvent, and drying to obtain the extract;

preferably, the mass/volume ratio is in g/ml or kg/L;

preferably, the mass/volume ratio is from 1:50 to 300, such as from 1:75 to 200, such as from 1:90 to 150, such as about 1:100;

preferably, the alcohol is ethanol;

preferably, the alcohol solution has a volume percent concentration of 80 to 100%, for example about 95%;

preferably, the room temperature soak time is from 0.2h to 1h, for example about 0.5h;

preferably, the extraction is a reflux extraction or an ultrasonic extraction, such as a micro-boiling reflux extraction;

preferably, the extraction time is from 0.5h to 2h, for example about 1h;

Preferably, the drying is freeze-drying or spray-drying;

preferably, the preparation method further comprises suspending the photinia extract in water, and extracting with dichloromethane to obtain a grandma wood type-2-2 phenethyl chromone compound separation site and/or a 5,6,7, 8-tetrahydroxy type-2- (2-phenethyl) chromone compound separation site;

preferably, the extraction is performed using petroleum ether prior to the extraction using the dichloromethane.

4. Use according to claim 3, characterized in that the preparation method further comprises the steps of:

(4) Adding the photinia extract into a solvent with a certain mass/volume ratio for dissolution, then adding dry silica gel powder with a certain mass/mass ratio, stirring and volatilizing the solvent, and loading the mixture into a silica gel chromatographic column by a dry method;

(5) Eluting with an eluent, wherein the eluent comprises dichloromethane and/or methane; and

(6) Collecting the fractions to obtain a separating part of the grandma wood-2-2 phenethyl chromone compound and/or a separating part of the 5,6,7, 8-tetrahydroxy-2- (2-phenethyl) chromone compound;

preferably, the mass/volume ratio is in g/ml or kg/L;

preferably, the mass/volume ratio is from 1:10 to 50, such as from 1:15 to 30, such as about 1:22;

Preferably, the mass/mass ratio is in g/g or kg/kg;

preferably, the mass/mass ratio is from 1 to 5:10, for example about 1:7;

preferably, the solvent is a mixture of an alcohol solution and methylene chloride;

preferably, the alcohol is ethanol;

preferably, the alcohol solution has a volume percent concentration of 80 to 100%, for example about 95%;

preferably, the volume ratio of the alcoholic solution to the dichloromethane is about 3:2;

preferably, the dissolving is carried out under ultrasonic conditions;

preferably, the time of the ultrasound is 0.5 to 2min, for example about 1min;

preferably, the eluent comprises dichloromethane, dichloromethane: methanol = about 40: about 1 and/or dichloromethane: methanol = about 1: about 1;

preferably, the elution sequence is 0.5 to 8 column volumes eluted with dichloromethane, with dichloromethane: methanol = about 40: about 1 elution 0.5 to 10 column volumes, and with dichloromethane: methanol = about 1: eluting about 1 from 0.5 to 10 column volumes;

preferably, the order of elution is about 4 column volumes eluted with dichloromethane, with dichloromethane: methanol = about 40: about 1 elution about 3 column volumes, 3 fractions were collected, about 1 column volume per fraction, and dichloromethane: methanol = about 1: about 1 elution about 1 column volume;

Preferably, in dichloromethane: methanol = about 40: about 1, the eluted intermediate stream is divided into a grand disc wood type-2-2 phenethyl chromone compound separation part;

preferably, in dichloromethane: methanol = about 1: about 1 column volume of about 1 elution corresponds to a flow split into 5,6,7, 8-tetrahydroxy-2- (2-phenethyl) chromone compound separation sites.

5. Use of a composition comprising the extract of photinia fraseri of any one of claims 1 to 4 for the preparation of a medicament, food, beverage or dietary supplement for the prevention and/or treatment of diabetes, obesity and/or complications thereof associated with AMPK activation;

preferably, the composition further comprises one or more drugs and/or extracts;

more preferably, the medicament comprises a hypoglycemic agent;

still preferably, the hypoglycemic agent is selected from one or more of the following: glipizide, gliclazide, glibenclamide, glimepiride, gliquidone, acarbose, voglibose, pioglitazone, sitagliptin, saxagliptin, vildagliptin, metformin, rosiglitazone and insulin.

6. Use of a formulation comprising the photinia extract of any one of claims 1 to 4 or the composition of claim 5 for the preparation of a medicament, food, beverage or dietary supplement for the prevention and/or treatment of diabetes, obesity and/or complications thereof associated with AMPK activation;

Preferably, the formulation further comprises one or more pharmaceutically acceptable excipients;

more preferably, the auxiliary material is selected from one or more of the following: dispersing agents, wetting agents, binders, diluents, glidants, lubricants, slow-release agents, plasticizers, disintegrants, inclusion agents, flavoring agents, opacifiers and antioxidants;

still preferably, the formulation is in the form of tablet, dripping pill, capsule, powder, injection, film, lozenge, granule or oral liquid.

7. The use according to any one of claims 1 to 6, wherein the diabetes comprises type I diabetes and/or type II diabetes;

preferably, the complications are acute complications and/or chronic complications;

more preferably, the acute complication is diabetic ketoacidosis, hypertonic non-ketotic diabetic coma and/or lactic acidosis;

still more preferably, the chronic complications are macrovascular complications, microvascular complications, neuropathy and/or diabetic foot;

particularly preferably, the macrovascular complication is cardiovascular and cerebrovascular diseases, coronary heart disease, cerebral apoplexy and/or lower limb gangrene;

particularly preferably, the microvascular complication is diabetic retinopathy and/or diabetic nephropathy.

8. The use according to any one of claims 1 to 6, wherein the cells associated with diabetes mellitus and/or complications thereof associated with AMPK activation are human kidney epithelial cell line cells, such as HEK293T cells;

preferably, the AMPK activation is an indirect activation mechanism;

more preferably, the AMPK comprises an α2 subunit, a β1 subunit and a γ1 subunit.

9. The use according to any one of claims 1 to 6, wherein the activation of AMPK occurs in muscle, liver and/or kidney tissue.

10. A process for preparing a phoenix extract, the process comprising the steps of:

(1) Weighing a proper amount of Phoebe cheilis sample, and adding alcohol solution with a certain mass/volume ratio;

(2) Extracting after soaking at room temperature;

(3) Cooling, filtering, removing solvent, and drying to obtain the extract;

(4) Adding the photinia extract into a solvent with a certain mass/volume ratio for dissolution, then adding dry silica gel powder with a certain mass/mass ratio, stirring and volatilizing the solvent, and loading the mixture into a silica gel chromatographic column by a dry method;

(5) Different gradients of dichloromethane were used: eluting and flushing with methanol; and

(6) Collecting the fractions to obtain the separating part of the grandma wood type-2-2 phenethyl chromone compound and/or the separating part of the 5,6,7, 8-tetrahydroxy type-2- (2-phenethyl) chromone compound.