CN116768836A - Compound with insulin resistance improving effect or medicinal salt thereof, and preparation method and application thereof - Google Patents
Compound with insulin resistance improving effect or medicinal salt thereof, and preparation method and application thereof Download PDFInfo
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- CN116768836A CN116768836A CN202210220097.1A CN202210220097A CN116768836A CN 116768836 A CN116768836 A CN 116768836A CN 202210220097 A CN202210220097 A CN 202210220097A CN 116768836 A CN116768836 A CN 116768836A
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 160
- 208000001072 type 2 diabetes mellitus Diseases 0.000 title claims abstract description 41
- 206010022489 Insulin Resistance Diseases 0.000 title claims abstract description 40
- 230000000694 effects Effects 0.000 title claims abstract description 32
- 150000003839 salts Chemical class 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title abstract description 19
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- 238000006366 phosphorylation reaction Methods 0.000 claims abstract description 16
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- 240000008397 Ganoderma lucidum Species 0.000 claims abstract description 9
- 235000001637 Ganoderma lucidum Nutrition 0.000 claims abstract description 9
- 230000003213 activating effect Effects 0.000 claims abstract description 8
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 6
- 108010034219 Insulin Receptor Substrate Proteins Proteins 0.000 claims abstract description 5
- 230000001737 promoting effect Effects 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 61
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 36
- 238000004440 column chromatography Methods 0.000 claims description 30
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- 238000000605 extraction Methods 0.000 claims description 15
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 claims description 15
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- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 1
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- BJEPYKJPYRNKOW-UHFFFAOYSA-N malic acid Chemical compound OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
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- 230000010534 mechanism of action Effects 0.000 description 1
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- XZWYZXLIPXDOLR-UHFFFAOYSA-N metformin Chemical compound CN(C)C(=N)NC(N)=N XZWYZXLIPXDOLR-UHFFFAOYSA-N 0.000 description 1
- 229960003105 metformin Drugs 0.000 description 1
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- 239000002674 ointment Substances 0.000 description 1
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
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- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/94—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom spiro-condensed with carbocyclic rings or ring systems, e.g. griseofulvins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
Landscapes
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Diabetes (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Emergency Medicine (AREA)
- Endocrinology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a compound with an insulin resistance improving effect or a medicinal salt thereof, a preparation method and application thereof, and relates to the technical field of chemical medicaments; the invention discloses a plurality of compounds which are extracted and separated from ganoderma lucidum and have the effect of improving insulin resistance, and the compounds have the effects of activating Akt or phosphorylation of insulin receptor substrate 1, promoting glucose uptake, reducing blood sugar and reducing fat in a dose-dependent manner, thus indicating the value of the compounds in the aspect of preparing medicaments for preventing or treating insulin resistance related diseases.
Description
Technical Field
The invention relates to the technical field of chemical medicines, in particular to a compound with an insulin resistance improving effect or a medicinal salt thereof, and a preparation method and application thereof.
Background
Insulin resistance (Insulin resistance) is one of the metabolic syndromes and is closely related to various diseases, such as diabetes, polycystic ovary syndrome, alzheimer's disease, nonalcoholic fatty liver disease, hypertension, atherosclerosis, obesity, etc. Currently, there are 11 drugs on the market that are currently approved globally for insulin resistance, 7 drugs in clinical third phase, 7 drugs in clinical second phase and 7 drugs in clinical first phase, and 13 drugs that have been terminated. Moreover, these drugs are often directed to metabolic diseases caused by insulin resistance, such as diabetes, and the cause thereof is insulin resistance, and these drugs are directed mainly to controlling blood sugar and delaying the occurrence of diabetic complications, and thus cannot completely cure diabetes. In addition, many of these drugs are chemical drugs and organic heterocyclic drugs, and many side effects such as gastrointestinal discomfort such as nausea and vomiting, acute kidney injury, vitamin B deficiency, etc. have been found in clinical use, and based on the above knowledge, it is necessary and urgent to find a safe drug capable of directly improving insulin resistance.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a compound with an insulin resistance improving effect or a medicinal salt thereof, and a preparation method and application thereof. The invention discloses a plurality of compounds which are extracted and separated from ganoderma lucidum and have the effect of improving insulin resistance, and the compounds have the effects of activating Akt phosphorylation or insulin receptor substrate 1 phosphorylation, promoting glucose uptake, reducing blood sugar and reducing fat in a dose-dependent manner.
In order to solve the technical problems, the invention provides the following technical scheme:
in a first aspect, there is provided a compound, or a pharmaceutically acceptable salt thereof, having an insulin resistance-ameliorating effect, for activating Akt phosphorylation and promoting glucose uptake; the compound has a structure shown in a formula I:
in a second aspect, there is provided a compound having an insulin resistance improving effect, or a pharmaceutically acceptable salt thereof, for activating insulin receptor substrate 1 phosphorylation and reducing blood glucose and fat weight; the compound has a structure shown in a formula II:
in the present invention, pharmaceutically acceptable salts of the above compounds may be: sodium salt, potassium salt, ammonium salt, amino acid salt, lactate, hydrochloride, phosphate, acetate, malate, citrate, aspartate, etc., the present invention is not particularly limited to the pharmaceutical salt.
In a third aspect, there is provided a method for producing a compound having an insulin resistance improving effect as described in the first aspect, the method comprising the steps of:
(1) Pulverizing Ganoderma, reflux-extracting with organic solvent, concentrating under reduced pressure to obtain extract, suspending the extract in warm water, and extracting with ethyl acetate to obtain ethyl acetate extract;
(2) Separating ethyl acetate extract by MCI gel CHP 20P column chromatography, sequentially eluting with methanol-water solution gradient with volume ratio of 40:60, 45:55, 50:50, 55:45, 65:35, 75:25, 85:15, 90:10, 95:5, 100:0, detecting eluent by TLC, and combining the same flow fractions to obtain Fr.1-Fr.17 total flow sections;
(3) Separating Fr.4 by Sephadex LH-20 column chromatography, eluting with methanol, detecting by TLC after eluting sample, and mixing the same fractions to obtain 2 total flow sections Fr.4.1 and Fr.4.2;
(4) Fr.4.2 is separated by RP-18 column chromatography, methanol-water solution gradient elution is sequentially carried out by using the volume ratio of 20:80, 23:77, 28:72, 33:67, 38:62, 43:57, 48:42 and 53:47, TLC detection is adopted after sample elution, and the same fractions are combined to obtain 5 flow sections of Fr.4.2.1-Fr.4.2.5;
(5) Separating Fr.4.2.4 by Sephadex LH-20 column chromatography, eluting with methanol-water solution with volume ratio of 70:30, purifying by silica gel column chromatography, sequentially eluting with dichloromethane-methanol solution with volume ratio of 15:1,8:1,3:1, detecting by TLC after sample elution, and mixing the same fractions to obtain Fr.4.2.4.1-Fr.4.2.4.8 total flow sections;
(6) Subjecting Fr.4.2.4.3 to solid phase extraction, sequentially performing gradient elution with dichloromethane-methanol solution with volume ratio of 10:1,5:1 and 3:1, detecting by TLC after sample elution, and combining the same fractions to obtain Fr.4.2.3.1-Fr.4.2.4.3.4 total 4 flow sections;
(7) Fr.4.2.4.3.2 is separated by Sephadex LH-20 column chromatography, methanol is used as an eluent, and then semi-preparative HPLC is used for purification to obtain a compound, and the compound is subjected to chiral resolution to obtain the target compound shown in the formula I.
Further, in the step (1), the organic solvent is an ethanol solution, and the volume ratio of the ethanol solution to the ganoderma lucidum is 6:1; the reflux extraction times are 1-3 times and 3-5 times.
Preferably, the ethanol solution is an 80% ethanol solution; the number of reflux extractions was 2 and the number of extractions was 4.
Preferably, in step (2), the volume ratio is 40:60, 45:55, 50:50, 55:45, 65:35, 75:25, 85:15, 90:10, 95:5, 100:0 of methanol-water is used in an amount of 181L,136L,59L,136L, 96L,136L, respectively.
Preferably, in step (4), the volume ratio of methanol-water is 20:80, 23:77, 28:72, 33:67, 38:62, 43:57, 48:42, 53:47, all in an amount of 15-20L.
Further, in step (7), the conditions of the semi-preparative HPLC are: acetonitrile-water solution with mobile phase of volume ratio of 20:80 is used in 0-25min, acetonitrile-water solution with mobile phase of volume ratio of 25:75 is used in 25.1-60min, and flow rate is 3mL/min; the mobile phase water contains 0.05% TFA;
the eluting solvent for chiral resolution is n-hexane-ethanol with a volume ratio of 88:12, and the ethanol of the eluting solvent contains 0.05% of TFA.
In a fourth aspect, there is provided a method for producing a compound having an insulin resistance improving effect as described in the second aspect, the method comprising the steps of:
(1) Pulverizing Ganoderma, percolating with organic solvent, concentrating under reduced pressure to obtain extract, suspending the extract in warm water, and extracting with ethyl acetate to obtain ethyl acetate extract;
(2) Separating ethyl acetate extract by Sephadex LH-20 column chromatography, eluting with methanol, detecting by TLC, and mixing the same fractions to obtain 3 flow sections;
(3) Subjecting Fr.B to MCI gel CHP 20P column chromatography, sequentially eluting with methanol-water solution gradient with volume ratio of 40:60, 45:55, 50:50, 55:45, 65:35, 75:25, 85:15, 90:10, 95:5, 100:0, detecting by TLC after eluting, and combining the same flow components to obtain Fr.BA-Fr.BL total 12 flow sections;
(4) Subjecting Fr.BB to Sephadex LH-20 column chromatography, eluting with methanol, detecting by TLC, and mixing the same fractions to obtain 3 flow segments;
(5) Subjecting the Fr.BB2 to MCI gel CHP 20P column chromatography, sequentially carrying out gradient elution by using methanol-water solutions with volume ratios of 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 65:35, 75:25, 85:15, 90:10, 95:5 and 100:0, detecting by adopting TLC after elution, and combining the same fractions to obtain 7 flow sections of Fr.BB21-Fr.BB27;
(6) Subjecting the Fr.BB22 to YMC-GE ODS-A-HG column chromatography, sequentially eluting with methanol-water solution gradient of which the volume ratio is 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 65:35, 75:25, 85:15, 90:10, 95:5, 100:0, detecting by TC after eluting, and combining the same fractions to obtain 8 total flow fragments of Fr.BB221-Fr.BB228;
(7) Purification of fr.bb226 by first half prep HPLC gave the target compound as described in formula II.
Further, in the step (1), the organic solvent is an ethanol solution, and the volume ratio of the ethanol solution to the ganoderma lucidum is 10:1; the extraction flow rate is 230mL/min, and the extraction times are 3-5 times.
Preferably, the ethanol solution is a 95% ethanol solution; the extraction times are 4 times, and the extraction is equal volume extraction.
Further, in step (7), the first half of the preparative HPLC conditions are: the mobile phase is acetonitrile-water solution with volume ratio of 31:69, and water contains 0.05% of TFA; the flow rate was 3mL/min.
The preparation method of the compound shown in the formula III is referred to as the preparation method of the compound shown in the formula II, and the difference is that: when the target compound shown in the formula II is prepared in the step (7), purifying 1 component obtained at the same time by a second semi-preparative HPLC to obtain the target compound shown in the formula III.
Preferably, in the preparation method of the compound shown in formula III, the second semi-preparative HPLC condition is: the mobile phase is acetonitrile-water solution with volume ratio of 23:77, and water contains 0.05% of TFA; the flow rate was 3mL/min.
Preferably, in the preparation method of the invention, the ganoderma lucidum is produced by using Yunnan Baoshan, and the temperature of the warm water is between 25 ℃ and 40 ℃; the volumes of the eluent and the gradient eluent can be adaptively adjusted according to the specific weight of the sample and the experimental requirements.
In a fifth aspect, there is provided a pharmaceutical composition comprising a compound having an insulin resistance improving effect according to the first and/or second aspects or a pharmaceutically acceptable salt thereof and/or a compound having an insulin resistance improving effect of the structure shown in formula III or a pharmaceutically acceptable carrier and/or adjuvant;
the pharmaceutical composition of the present invention, which uses at least one of the compounds of the first and second aspects or pharmaceutically acceptable salts thereof as the main active ingredient, does not exclude changes in the formulation system and the mode of administration, derivatives obtained by simple chemical modification of the above-mentioned compounds, and combinations of multiple compounds (i.e., using one or more of the above-mentioned three compounds), and the like.
In the present invention, the compounds provided herein can be formulated as active ingredients in non-toxic, inert and pharmaceutically acceptable carrier media; the formulated drug may be administered by conventional routes including, but not limited to, oral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.
When the pharmaceutical composition of the present invention is in the form of a medicament for oral administration, it contains a safe and effective amount of the above-mentioned compound of the present invention or a pharmaceutically acceptable carrier and/or adjuvant thereof, and the orally administered medicament can be formulated into conventional dosage forms such as tablets, pills, powders, granules, capsules, emulsions, syrups, ointments, suppositories and the like; in the present invention, the carrier and/or the auxiliary agent are not particularly limited.
The pharmaceutical composition of the present invention may also be prepared into injection, which may be prepared into injection with water for injection, physiological saline and glucose water in sterile environment through conventional process.
In a sixth aspect, there is provided use of a compound having an insulin resistance improving effect or a pharmaceutically acceptable salt thereof according to the first or second aspect, a compound having an insulin resistance improving effect of a structure shown in formula III or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to the fifth aspect in the preparation of a medicament for activating Akt phosphorylation and/or treating diseases associated with insulin resistance.
In particular, the above-mentioned related diseases such as metabolic syndrome and type II diabetes which are liable to be caused by insulin resistance.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses novel compounds (formula I and formula II) with an insulin resistance improving effect, wherein the compounds shown in the formula I can activate Akt phosphorylation and promote glucose uptake in a dose-dependent manner, the compounds shown in the formula II and formula III can activate insulin receptor substrate 1 phosphorylation and reduce blood sugar and fat weight, and the values of various compounds provided by the invention in the aspect of preparing medicaments for preventing or treating insulin resistance related diseases are suggested.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a schematic diagram showing the results of the cell viability assay of compound (+) -Spiroganodermaine G of the invention;
FIG. 2 is a schematic representation of the test results of the AKT phosphorylation assay of compound (+) -Spiroganodermaine G of the invention;
FIG. 3 is a schematic diagram showing the results of glucose uptake assay of compound (+) -Spiroganodermaine G of the present invention;
FIG. 4 is a graph showing the results of the glucose uptake assay and dosage of compound (+) -Spiroganodermaine G of the present invention;
FIG. 5 is a schematic diagram showing the results of cell viability assays of the compounds (+) -Spiroapplanatumine K and (+) -Spirolingzhine D of the present invention;
FIG. 6 is a schematic representation of the results of a test for improved p-IRS1 expression by compounds (+) -Spiroapplanatumine K and (+) -Spirolingzhine D of the invention;
FIG. 7 is a graph showing the change in body weight of mice in different experimental groups in an insulin resistant mouse model of the present invention;
FIG. 8 is a graph showing the trend of change in body weight of mice in different experimental groups in an insulin resistant mouse model according to the present invention;
FIG. 9 is a graph showing the trend of variation in feeding of mice in different experimental groups in an insulin resistant mouse model according to the present invention;
FIG. 10 is a graphical representation of the change in blood glucose after glucose injection in mice of different experimental groups in an insulin resistant mouse model of the invention;
FIG. 11 is a graph showing the weight change of mouse liver, epididymis, subcutaneous and brown fat in different experimental groups of the insulin resistant mouse model of the present invention;
FIG. 12 is a graph showing the trend of changes in liver, epididymis, subcutaneous and brown fat weights of mice from different experimental groups in an insulin resistant mouse model according to the present invention.
Detailed Description
For a more complete understanding of the present invention, reference is now made to the following descriptions taken in conjunction with the accompanying drawings and specific examples; it will be apparent that the described embodiments are only 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 fall within the scope of the invention.
The features, advantages and advantages of the present invention will become apparent to those skilled in the art from a reading of the present disclosure.
All percentages, fractions and ratios are by weight of the total composition of the present invention, unless otherwise specified. The term "weight content" is used herein to denote the symbol "%".
The term "comprising" is an open-ended expression, i.e., including what is indicated by the invention, but not excluding other aspects.
The term "pharmaceutical composition" means a mixture of one or more of the compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof, or in combination with other chemical components that improve insulin resistance.
The term "compound (+) -Spiroganodermaine G" is a compound of formula I, "compound (+) -Spiroapplanatumine K" is a compound of formula II, and "compound (+) -Spirolingzhine D" is a compound of formula III.
Example 1
The embodiment provides an extraction and separation method and structure identification of the compound.
1. The extraction and separation method of the compound (+) -Spiroganodermaine G comprises the following steps:
1.1, taking 500kg of ganoderma lucidum produced by Yunnan Baoshan, crushing, reflux-extracting with 6 times of 80% ethanol for 2 times, concentrating under reduced pressure to obtain 24.6kg of extract, suspending in warm water, and extracting with ethyl acetate for 4 times in equal volume to obtain 11.5kg of ethyl acetate extract;
ethyl acetate extract (11.5 kg) was separated by MCI gel CHP 20P column chromatography, followed by gradient elution with methanol/water (40:60, 181L,45:55, 136L,50:50, 59L,55:45, 136L,65:35, 136L,75:25, 136L,85:15, 136L,90:10, 136L,95:5, 96L,100:0, 136L), collecting the eluate, detecting the eluate by TLC, and combining the same fractions to obtain a total of 17 flow fractions fr.1-fr.17;
separating 1.3 and Fr.4 (78.7 g) by Sephadex LH-20 column chromatography, eluting with methanol, detecting by TLC after sample elution, and mixing the same fractions to obtain 2 total flow sections Fr.4.1 and Fr.4.2;
separating 1.4 and Fr.4.2 (72.4 g) by RP-18 column chromatography, sequentially eluting with methanol-water solution gradient with volume ratio of 20:80, 23:77, 28:72, 33:67, 38:62, 43:57, 48:42, 53:47, detecting by TLC after sample elution, and combining the same flow to obtain Fr.4.2.1-Fr.4.2.5 total flow sections;
separating 1.5 and Fr.4.2.4 (10.5 g) by Sephadex LH-20 column chromatography, eluting and purifying by using methanol-water solution with the volume ratio of 70:30, then subjecting to silica gel column chromatography, sequentially eluting by using dichloromethane-methanol solution with the volume ratio of 15:1,8:1 and 3:1, detecting by TLC after sample elution, and combining the same fractions to obtain Fr.4.2.4.1-Fr.4.2.4.8 total flow sections;
1.6 and Fr.4.2.4.3 (1.3 g) are subjected to Solid Phase Extraction (SPE), dichloromethane-methanol solution with volume ratio of 10:1,5:1 and 3:1 is used for gradient elution, TLC detection is adopted after sample elution, and Fr.4.2.4.3.1-Fr.4.2.4.3.4 is combined to obtain 4 flow sections;
1.7, fr.4.2.4.3.2 (550.4 mg) were separated by Sephadex LH-20 column chromatography with methanol as eluent, followed by semi-preparative HPLC [ acetonitrile/water containing 0.05% TFA,20:80 (0-25 min), 25:75 (25.1-60 min), flow rate 3mL/min]Purification to give the compound, which was subjected to chiral resolution (eluting solvent was 88:12 by volume of n-hexane-ethanol; ethanol contains 0.05% TFA) to give the target compound (+) -Spiroganodermaine G (1.7 mg, t) R ＝8.8min)。
Notably, the compound purified by semi-preparative HPLC is a pair of enantiomers and therefore requires chiral resolution to afford the target compound (+) -Spiroganodermaine G.
2. Extraction and isolation of compound (+) -Spiroapplanatumine K and compound (+) -Spirolingzhine D
2.1, 50kg of ganoderma lucidum produced in Yunnan province, percolating and extracting with 10 times of 95% ethanol by volume fraction, wherein the extraction flow rate is 230ml/min, recovering ethanol under reduced pressure to obtain 2.7kg of extract, suspending the extract in warm water, and extracting with ethyl acetate for 4 times by equal volume to obtain 2.4kg of ethyl acetate extract;
separating ethyl acetate extract by Sephadex LH-20 column chromatography, eluting with methanol as eluent, detecting by TLC, and mixing the same fractions to obtain 3 flow sections;
(3) Fr.B (813.7 g) is subjected to MCI gel CHP 20P column chromatography, methanol-water solution gradient elution with the volume ratio of 40:60, 45:55, 50:50, 55:45, 65:35, 75:25, 85:15, 90:10, 95:5 and 100:0 is sequentially used, TLC detection is adopted after elution, and the same fractions are combined to obtain 12 flow sections of Fr.BA-Fr.BL;
(4) Subjecting Fr.BB (20.4 g) to Sephadex LH-20 column chromatography, eluting with methanol as eluent, detecting by TLC, and mixing the same fractions to obtain Fr.BB1-Fr.BB3 total 3 flow sections;
(5) Fr.BB2 (14.8 g) is subjected to MCI gel CHP 20P column chromatography, and then methanol-water solution gradient elution is sequentially carried out with the volume ratio of 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 65:35, 75:25, 85:15, 90:10, 95:5 and 100:0, after elution, TLC detection is adopted, and the same fractions are combined to obtain 7 flow sections of Fr.BB21-Fr.BB27;
(6) Fr.BB22 (5.3 g) is subjected to YMC-GE ODS-A-HG column chromatography, and then subjected to gradient elution by methanol-water solution with the volume ratio of 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 65:35, 75:25, 85:15, 90:10, 95:5 and 100:0, and after elution, the same fractions are combined to obtain 8 flow fragments of Fr.BB221-Fr.BB228;
(7) Fr.BB226 (1.5 g) was purified by semi-preparative HPLC (mobile phase: acetonitrile-water solution with volume ratio of 31:69, water containing 0.05% TFA at a flow rate of 3 mL/min) to give the title compound (+) -Spiroapplanatumine K (11.8 mg, t) R ＝11.0min)；
(8) The 1 fraction obtained at the same time was purified by semi-preparative HPLC (mobile phase: acetonitrile-water solution with volume ratio of 23:77, water containing 0.05% TFA at flow rate of 3 mL/min) to give the target compound (+) -spirringzhine D (39.3 mg, t) R ＝15.2min)。
The compounds (+) -Spiroganodermaine G, (+) -Spiroapplanatumine K and (+) -Spirolingzhine D prepared above were identified as follows.
The structural formulae of the compounds (+) -Spiroganodermaine G, (+) -Spiroapplanatumine K and (+) -Spirolingzhine D are as follows in order:
compound (+) -Spiroganodermaine G (1), yellow gum, [ alpha ]] D 25 +34.3(c 0.02,MeOH)；CD(MeOH)Δε 232 +6.50,Δε 256 –0.34,Δε 319 +0.51,Δε 360 –1.01；UV(MeOH)λ max (logε)370(3.58),254(3.86),220(4.15)nm；HRESIMS m/z 319.1178[M-H] - (calcd for C 17 H 19 O 6 ,319.1187)； 1 H and 13 C NMR data is shown in Table 1.
Compound (+) -Spiroapplanatumine K (2), yellow gum, [ alpha ]] D 25 +160.0(c 0.01,MeOH)；CD(MeOH)Δε 237 +3.91,Δε 320 –2.33,Δε 365 +3.92；UV(MeOH)λ max (logε)373(3.57),255(3.82),225(3.91)nm；HRESIMS m/z 319.1171[M+H] + (calcd for C 17 H 19 O 6 ,319.1176)； 1 H and 13 C NMR data is shown in Table 1.
Compound (+) -Spirolingzhine D (3), yellow gum, [ alpha ]] D 25 +148.0 (c 0.03, meOH), (+) -Spirolingzhine D is a known compound, which was confirmed by applicant in 2015 to be the same compound by careful comparison of HPLC retention time, NMR, optical rotation and circular dichroism spectrum, published on Phytochemistry.
Table 1: compounds (+) -Spiroganodermaine G (1) and (+) -Spiroapplanatumine K (2) 1 H and 13 c NMR data
Example 2:
the embodiment provides an injection, and the preparation method of the injection comprises the following steps:
the compound (+) -Spiroganodermaine G prepared in the example 1 is added with solvent for injection by a conventional method, subjected to fine filtration, encapsulated and sterilized to prepare injection.
Alternatively, the compound (+) -Spiroapplanatumine K prepared in example 1 is added with solvent for injection by a conventional method, subjected to fine filtration, encapsulated and sterilized to prepare injection.
Alternatively, the compound (+) -Spirolingzhine D prepared in example 1 is added with solvent for injection by a conventional method, subjected to fine filtration, encapsulated and sterilized, and then prepared into injection.
Alternatively, 2 or 3 compounds of (+) -Spiroganodermaine G, (+) -Spiroapplanatumine K and (+) -Spirolingzhine D prepared in example 1 are mixed according to any proportion, and the mixture is added with solvent for injection by a conventional method, finely filtered, encapsulated and sterilized to prepare injection.
Example 3:
the embodiment provides a pharmaceutical tablet, and the preparation method of the pharmaceutical tablet comprises the following steps:
the compound (+) -Spiroganodermaine G prepared in the example 1 can be prepared into tablets by a conventional method and various pharmaceutical excipients.
Specifically, the compound (+) -Spiroganodermaine G is used as a pharmaceutical active ingredient, one or more common excipients are used as auxiliary ingredients for preparing pharmaceutical tablets, and tablet samples containing 1-100mg of the pharmaceutical ingredient are prepared according to a certain proportion.
Alternatively, the compound (+) -Spiroapplanatumine K prepared in example 1 can be formulated into tablets by conventional methods with various pharmaceutical excipients.
Specifically, the compound (+) -Spiroapplanatumine K is used as a pharmaceutical active ingredient, one or more common excipients are used as auxiliary ingredients for preparing pharmaceutical tablets, and tablet samples containing 1-100mg of the pharmaceutical ingredient are prepared according to a certain proportion.
Alternatively, the compound (+) -Spirolingzhine D prepared in example 1 can be formulated into tablets by a conventional method with various pharmaceutical excipients.
Specifically, the compound (+) -Spirolingzhine D is used as a pharmaceutical active ingredient, one or more common excipients are used as auxiliary ingredients for preparing pharmaceutical tablets, and tablet samples containing 1-100mg of pharmaceutical ingredients are prepared according to a certain proportion.
Alternatively, 2 or 3 compounds of (+) -Spiroganodermaine G, (+) -Spiroapplanatumine K, (+) -Spirolingzhine D prepared in example 1 may be mixed in any ratio, and formulated with various pharmaceutical excipients by conventional methods to make into tablets.
Specifically, 2 or 3 compounds are taken from the compounds (+) -Spiroganodermaine G, (+) -Spiroapplanatumine K, (+) -Spirolingzhine D according to any proportion, mixed and used as pharmaceutical active ingredients, one or more common excipients are used as auxiliary ingredients for preparing a combined pharmaceutical tablet, and tablet samples containing 1-100mg of pharmaceutical ingredients are prepared according to a certain proportion.
Example 4:
the embodiment provides a pharmaceutical capsule, and the preparation method of the pharmaceutical capsule comprises the following steps:
the compound (+) -Spiroganodermaine G prepared in the example 1 is prepared into capsules by a conventional method and various pharmaceutical excipients.
Specifically, the compound (+) -Spiroganodermaine G is used as a pharmaceutical active ingredient, one or more common excipients are used as auxiliary ingredients for preparing pharmaceutical capsules, and capsule preparations containing 1-100mg of the compound ingredient in each capsule are prepared according to a certain proportion.
Alternatively, the compound (+) -Spiroapplanatumine K prepared in example 1 can be prepared into capsules by a conventional method with various pharmaceutical excipients.
Specifically, the compound (+) -Spiroapplanatumine K is used as a pharmaceutical active ingredient, one or more common excipients are used as auxiliary ingredients for preparing pharmaceutical capsules, and capsule preparations containing 1-100mg of the compound ingredient in each capsule are prepared according to a certain proportion.
Alternatively, the compound (+) -Spirolingzhine D prepared in example 1 can be prepared into capsules by a conventional method with various pharmaceutical excipients.
Specifically, the compound (+) -Spirolingzhine D is used as a pharmaceutical active ingredient, one or more common excipients are used as auxiliary ingredients for preparing pharmaceutical capsules, and capsule preparations containing 1-100mg of the compound ingredient in each capsule are prepared according to a certain proportion.
Alternatively, 2 or 3 compounds of the compound (+) -Spiroganodermaine G, (+) -Spiroapplanatumine K, (+) -Spirolingzhine D prepared in example 1 are mixed according to any proportion, and various pharmaceutical excipients are matched according to a conventional method to prepare capsules.
Specifically, 2 or 3 compounds are taken from the compounds (+) -Spiroganodermaine G, (+) -Spiroapplanatumine K, (+) -Spirolingzhine D according to any proportion, mixed and used as the active pharmaceutical ingredients, one or more common excipients are used as auxiliary ingredients for preparing the combined pharmaceutical capsules, and the capsule preparation containing 1-100mg of the compound ingredients in each capsule is prepared according to a certain proportion.
Example 5:
the embodiment provides a solid beverage, and the preparation method of the solid beverage comprises the following steps:
taking and uniformly mixing 1 part of (+) -Spiroganodermaine G parts of the compound (+) -Spiroganodermaine G parts of the vegetable fat powder prepared by the method of the example 1 and preparing a solid beverage according to a conventional method;
alternatively, the compound (+) -Spiroapplanatumine K parts by weight and 10 parts by weight of vegetable fat powder prepared in the method of example 1 are uniformly mixed and prepared into a solid beverage according to a conventional method;
alternatively, 1 part of (+) -Spirolingzhine D of the compound prepared in the method of the example 1 and 10 parts of vegetable fat powder are taken and uniformly mixed, and a solid beverage is prepared according to a conventional method;
alternatively, 1 part or 3 of the compound (+) -Spiroganodermaine G, (+) -Spiroapplanatumine K, (+) -Spirolingzhine D prepared by the method of example 1 were mixed with 10 parts of the vegetable fat powder in an arbitrary ratio, and then a solid beverage was prepared by a conventional method.
Example 6:
this example uses Cell Count Kit-8, ECL Kit and Glucose Colorimetric/Fluorometric Assay Kit to examine the activity of AKT phosphorylation, IRS1 phosphorylation and glucose uptake of the above compounds.
Related experimental test of compound (+) -Spiroganodermaine G:
cell viability assay the Cell viability assay uses a conventional Cell Count Kit-8 assay, comprising the following steps:
1. C2C12 cells (5X 10) 5 cells/mL) was inoculated into 96-well plates with fresh DMEM, and after overnight cell culture, treated with methanol or DMSO for 24 hours;
2. cell Count Kit-8 was then added to each well and incubated for 1h at 37 ℃;
3. the absorbance of each well was measured at 450nm using a microplate reader.
The test results of the cell viability assay of compound (+) -Spiroganodermaine G are shown in FIG. 1.
AKT phosphorylation assay:
1. dissolving the following compounds into 20mM stock solution by using DMSO respectively; the compound (+) -7 is the compound (+) -Spiroganodermaine G provided by the invention;
2. the C2C12 cell line was grown to contact inhibition in DMEM containing 10% FBS (fetal bovine serum), the cell culture medium was replaced with fresh DMEM containing 2% horse serum (horseum), 100U/mL penicillin and 100. Mu.g/mL streptomyin, and incubated at 37℃for 4 days;
3. insulin resistance was simulated by adding 100nM insulin to the induction medium for 24h while cell differentiation was occurring. After 24h of compound treatment, total protein was extracted from the cell line and quantitative protein samples were used;
4. an equal amount of the protein extract was separated by 8% SDS-PAGE and transferred to PVDF membrane. Blocking the membrane with 5% bovine serum albumin, then overnight with the indicated antibodies at 4 ℃, then incubating with horseradish peroxidase (HRP) -conjugated secondary antibodies at room temperature;
5. the bands were visualized and measured by ECL kit. Densitometric analysis was performed on immunoblotted results using ImageJ software.
The test results of the AKT phosphorylation assay of compound (+) -Spiroganodermaine G are shown in figure 2.
Glucose uptake assay:
1. incubating the differentiated C2C12 cells in DMEM (low sugar) for 6h;
2. adding the compound into high-sugar DMEM, and adding insulin (100 nmol/L) for continuous culture for 4 hours after the compound is treated for 30 min;
3. glucose content in the culture supernatant was determined using a glucose colorimetric/fluorometric kit.
The test results of the glucose uptake assay for compound (+) -Spiroganodermaine G are shown in FIG. 3.
Related experimental tests of compounds (+) -Spiroapplanatumine K and (+) -Spirolingzhine D:
cell viability assay
Cell viability assay see cell viability assay for compound (+) -Spiroganodermaine G; the test results of the cell viability assay for compounds (+) -Spiroapplanatumine K and (+) -Spirolingzhine D are shown in fig. 5.
In FIG. 5, 30A is the compound (+) -Spirolingzhine D and 30B is (+) -Spiroapplanatumine K.
Wherein, after C2C12 cells were treated with 20. Mu.M compound for 24 hours, the expression of p-IRS-1 was detected by Westernblot method, and the test results are shown in FIG. 6.
Insulin resistance mouse model:
1. c57BL6/J male mice (30), 6 weeks old, after adaptively feeding for 3 days, according to the weight, randomly dividing into 10 normal control groups (ND) and 20 high fat model groups (HFD), feeding 14-16 weeks with ND (10 kcal% fat) and HFD (60 kcal% fat), respectively, during which the growth state of the mice is closely concerned, the ingestion amount is recorded regularly with the weight of the mice, and the survival conditions of the two groups of mice are consistent.
2. The animals were fed with the high-fat diet for 3 months, and after 17D administration of the compound (+) -Spirolingzhine D (32.4 mg/kg) +(+) -Spiroapplanatumine K (7.6 mg/kg), the mice were closely focused and their weight changes and feeding changes were recorded. The test results are shown in fig. 7, 8 and 9.
3. After the end of the administration, glucose tolerance (glucose tolerance tests, GTT) mice were determined and glucose (20% solution, 2 g/kg) was administered by intraperitoneal injection 12 hours after fasting, and the blood glucose of the mice was measured with a glucometer at 0, 15, 30, 60 and 120min after glucose injection. The test results are shown in fig. 10.
After the end of administration, the liver, epididymis, subcutaneous and brown fat weights of the mice were measured by sampling materials, and the test results are shown in fig. 11 and 12.
Analysis of test results:
as shown in figures 1 and 5, compared with a blank control group, the compound (+) -Spiroganodermaine G, (+) -Spiroapplanatumine K, (+) -Spirolingzhine D group has no obvious change in cell viability, which shows that the compound provided by the invention has higher safety.
As shown in figure 2, compound (+) -Spiroganodermaine G was able to significantly activate AKT phosphorylation at a concentration of 40 μm.
As shown in FIGS. 3 and 4, C2C12 cells were treated with 40. Mu.M compound (+) -Spiroganodermaine G for 24 hours, and glucose uptake was measured with metformin (Met) as a positive control. The compound (+) -Spiroganodermaine G was found to improve glucose uptake by insulin stimulated cells and attenuate disruption of insulin signaling pathways in myotubes. #p <0.05 and #p <0.001 (compared to the control group not stimulated by insulin); * P <0.05 and P <0.01 (compared to the model group stimulated with insulin only).
As shown in fig. 6: 30A in FIG. 6 is (+) -Spirolingzhine D, and 30B in FIG. 6 is (+) -Spiroapplanatumine K. After C2C12 cells were treated with 20. Mu.M compound for 24 hours, the expression of p-IRS1 was detected by the Westernblot method. From fig. 6, the compound (+) -Spirolingzhine D improved insulin stimulation and attenuated disruption of insulin signaling pathways in myotubes (fig. 6, panel B). Compound (+) -Spiroapplanatumine K ameliorates disruption of the insulin signaling pathway in insulin-stimulated myotubes (panel C in fig. 6).
As shown in fig. 7-8: in fig. 7-8: 1 (32.4 mg/kg) +2 (7.6 mg/kg) is (+) -Spirolingzhine D (32.4 mg/kg) +(+) -Spiroapplanatumine K (7.6 mg/kg). As can be seen from FIG. 7, there was no significant change in ND body weight after 16 weeks of administration of the high fat diet, and both HFD group and HFD+ compound (+) -Spirolingzhine D (32.4 mg/kg) + (+) -Spiroapplanatumine K (7.6 mg/kg) group had elevated body weight (P < 0.05). As can be seen from fig. 8, the mice in the group with the compound (+) -Spirolingzhine D (32.4 mg/kg) +(+) -Spiroapplanatumine K (7.6 mg/kg) were given a significantly lower rate of body weight gain than the model group (P < 0.05) after 17 days of treatment.
As shown in fig. 9: in fig. 9: 1 (32.4 mg/kg) +2 (7.6 mg/kg) is (+) -Spirolingzhine D (32.4 mg/kg) +(+) -Spiroapplanatumine K (7.6 mg/kg). Effect of compound (+) -Spirolingzhine D (32.4 mg/kg) +(+) -Spiroapplanatumine K (7.6 mg/kg) on diet induced in mice by high fat diet. Close attention was paid to the ingestion amount of the 17D intraperitoneal injection compound (+) -Spirolingzhine D (32.4 mg/kg) +(+) -Spiroapplanatumine K (7.6 mg/kg), and as a result, it was confirmed that the ingestion amount of HFD was reduced compared with the ND group, and the ingestion amount of mice with hfd+ compound (+) -Spirolingzhine D (32.4 mg/kg) +(+) -Spiroapplanatumine K (7.6 mg/kg) was significantly lower than that of the HFD group with the increase of the administration time, and the difference was significant (P < 0.05). This result suggests that the compound may affect appetite and energy metabolism, etc. by affecting the hypothalamus.
As shown in fig. 10: in fig. 10:1 (32.4 mg/kg) +2 (7.6 mg/kg) is (+) -Spirolingzhine D (32.4 mg/kg) +(+) -Spiroapplanatumine K (7.6 mg/kg). After intraperitoneal injection of glucose, the islet sensitivity of ND group mice did not change significantly (P > 0.05). Compared with the ND group, the HFD group and the HFD+ compound (+) -Spirolingzhine D (32.4 mg/kg) + (plus-Spiroapplanatumine K) (7.6 mg/kg) have the advantages that the GTT is obviously increased from 15min later, the GTT reaches the highest value at 30min, but the blood glucose trend of the HFD+ compound (+) -Spirolingzhine D (32.4 mg/kg) + (plus-Spiroapplanatumine K (7.6 mg/kg) mice at different moments is obviously reduced, and the compound (+) -Spirolingzhine D (32.4 mg/kg) + (plus-Spiroapplanatumine K) (7.6 mg/kg) has better tolerance to glucose.
As shown in fig. 11 and 12: in fig. 11 and 12: 1 (32.4 mg/kg) +2 (7.6 mg/kg) is (+) -Spirolingzhine D (32.4 mg/kg) +(+) -Spiroapplanatumine K (7.6 mg/kg). The liver, epididymis, subcutaneous and brown adipose tissue weights of the HFD group were significantly increased (P < 0.05) compared to the ND group, and the HND+ compound (+) -Spirolingzhine D (32.4 mg/kg) +(+) -Spiroapplanatumine K (7.6 mg/kg) group mice had significantly decreased epididymis, subcutaneous and brown adipose weights (P < 0.05) after administration, confirming that the HND+ compound (+) -Spirolingzhine D (32.4 mg/kg) +(+) -Spiroapplanatumine K (7.6 mg/kg) was able to decrease adipose weight. This result suggests that compounds act through the insulin resistance signaling pathway, in combination with the above-described effects on the hypothalamus, suggesting a non-unique mechanism of action for these compounds.
In conclusion, the compound (+) -Spiroganodermaine G provided by the invention can obviously activate AKT phosphorylation at the concentration of 40 mu M, and has dose dependency on regulation of glucose uptake; the compounds (+) -Spiroapplanatumine K and (+) -Spirolingzhine D were both able to activate significantly activated IRS1 phosphorylation at 20 μm; mice fed compound (+) -Spirolingzhine D (32.4 mg/kg) +(+) -Spiroapplanatumine K (7.6 mg/kg) had significantly lower blood glucose and body weight than the control group, and epididymal, subcutaneous and brown fat weights were significantly reduced, demonstrating that compounds (+) -Spiroganodermaine G, (+) -Spiroapplanatumine K and (+) -Spirolingzhine D have a certain effect of improving insulin resistance activity.
When the compound provided by the invention is specifically applied to a therapeutic drug, the therapeutic drug takes one or more of the compounds or pharmaceutically acceptable salts thereof as a main active ingredient, and the conditions of changing a preparation system and an administration mode, using derivatives obtained by simply chemically modifying the compounds and multiple active substances and the like are not excluded.
The foregoing has described in detail the technical solutions provided by the embodiments of the present invention, and specific examples have been applied to illustrate the principles and implementations of the embodiments of the present invention, where the above description of the embodiments is only suitable for helping to understand the principles of the embodiments of the present invention; meanwhile, as for those skilled in the art, according to the embodiments of the present invention, there are variations in the specific embodiments and the application scope, and the present description should not be construed as limiting the present invention.
Claims (10)
1. A compound having an insulin resistance improving effect or a pharmaceutically acceptable salt thereof, which is useful for activating Akt phosphorylation and promoting glucose uptake; the compound has a structure shown in a formula I:
2. a compound having an insulin resistance improving effect or a pharmaceutically acceptable salt thereof, which is used for activating phosphorylation of insulin receptor substrate 1 and reducing blood sugar and fat weight; the compound has a structure shown in a formula II:
3. a method for producing a compound having an insulin resistance improving effect as claimed in claim 1, comprising the steps of:
(1) Pulverizing Ganoderma, reflux-extracting with organic solvent, concentrating under reduced pressure to obtain extract, suspending the extract in warm water, and extracting with ethyl acetate to obtain ethyl acetate extract;
(2) Separating ethyl acetate extract by MCIgel CHP 20P column chromatography, sequentially eluting with methanol-water solution gradient with volume ratio of 40:60, 45:55, 50:50, 55:45, 65:35, 75:25, 85:15, 90:10, 95:5, 100:0, detecting eluent by TLC, and combining identical flow fractions to obtain Fr.1-Fr.17 total 17 flow sections;
(3) Separating Fr.4 by Sephadex LH-20 column chromatography, eluting with methanol, detecting by TLC after eluting sample, and mixing the same fractions to obtain 2 total flow sections Fr.4.1 and Fr.4.2;
(4) Fr.4.2 is separated by RP-18 column chromatography, methanol-water solution gradient elution is sequentially carried out by using the volume ratio of 20:80, 23:77, 28:72, 33:67, 38:62, 43:57, 48:42 and 53:47, TLC detection is adopted after sample elution, and the same fractions are combined to obtain 5 flow sections of Fr.4.2.1-Fr.4.2.5;
(5) Separating Fr.4.2.4 by Sephadex LH-20 column chromatography, eluting with methanol-water solution with volume ratio of 70:30, purifying by silica gel column chromatography, sequentially eluting with dichloromethane-methanol solution with volume ratio of 15:1,8:1,3:1, detecting by TLC after sample elution, and mixing the same fractions to obtain Fr.4.2.4.1-Fr.4.2.4.8 total flow sections;
(6) Subjecting Fr.4.2.4.3 to solid phase extraction, sequentially performing gradient elution with dichloromethane-methanol solution with volume ratio of 10:1,5:1 and 3:1, detecting by TLC after sample elution, and combining the same fractions to obtain Fr.4.2.3.1-Fr.4.2.4.3.4 total 4 flow sections;
(7) Fr.4.2.4.3.2 is separated by Sephadex LH-20 column chromatography, methanol is used as an eluent, and then semi-preparative HPLC is used for purification to obtain a compound, and the compound is subjected to chiral resolution to obtain the target compound shown in the formula I.
4. The method according to claim 3, wherein in the step (1), the organic solvent is an ethanol solution, and the volume ratio of the ethanol solution to the ganoderma lucidum is 6:1; the reflux extraction times are 1-3 times and 3-5 times.
5. A method according to claim 3, wherein in step (7), the conditions for semi-preparative HPLC are: acetonitrile-water solution with mobile phase of volume ratio of 20:80 is used in 0-25min, acetonitrile-water solution with mobile phase of volume ratio of 25:75 is used in 25.1-60min, and flow rate is 3mL/min; the mobile phase water contains 0.05% TFA;
the eluting solvent for chiral resolution is n-hexane-ethanol with a volume ratio of 88:12, and the ethanol of the eluting solvent contains 0.05% of TFA.
6. A method for producing a compound having an insulin resistance improving effect as claimed in claim 2, comprising the steps of:
(1) Pulverizing Ganoderma, percolating with organic solvent, concentrating under reduced pressure to obtain extract, suspending the extract in warm water, and extracting with ethyl acetate to obtain ethyl acetate extract;
(2) Separating ethyl acetate extract by Sephadex LH-20 column chromatography, eluting with methanol, detecting by TLC, and mixing the same fractions to obtain 3 flow sections;
(3) Subjecting Fr.B to MCI gel CHP 20P column chromatography, sequentially eluting with methanol-water solution gradient with volume ratio of 40:60, 45:55, 50:50, 55:45, 65:35, 75:25, 85:15, 90:10, 95:5, 100:0, detecting by TLC after eluting, and combining the same flow components to obtain Fr.BA-Fr.BL total 12 flow sections;
(4) Subjecting Fr.BB to Sephadex LH-20 column chromatography, eluting with methanol, detecting by TLC, and mixing the same fractions to obtain 3 flow segments;
(5) Subjecting the Fr.BB2 to MCI gel CHP 20P column chromatography, sequentially carrying out gradient elution by using methanol-water solutions with volume ratios of 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 65:35, 75:25, 85:15, 90:10, 95:5 and 100:0, detecting by adopting TLC after elution, and combining the same fractions to obtain 7 flow sections of Fr.BB21-Fr.BB27;
(6) Subjecting the Fr.BB22 to YMC-GE ODS-A-HG column chromatography, sequentially eluting with methanol-water solution gradient of which the volume ratio is 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 65:35, 75:25, 85:15, 90:10, 95:5, 100:0, detecting by TC after eluting, and combining the same fractions to obtain 8 total flow fragments of Fr.BB221-Fr.BB228;
(7) Purification of fr.bb226 by first half prep HPLC gives the intermediate and the target compound of formula II;
(8) The intermediate component is then purified by a second semi-preparative HPLC to afford the target compound of formula III.
7. The method according to claim 6, wherein in the step (1), the organic solvent is an ethanol solution, and the volume ratio of the ethanol solution to the ganoderma lucidum is 10:1; the extraction flow rate is 230mL/min, and the extraction times are 3-5 times.
8. The method of claim 6, wherein in step (7), the first half of the preparative HPLC conditions are: the mobile phase is acetonitrile-water solution with volume ratio of 31:69, and water contains 0.05% of TFA; the flow rate is 3mL/min;
the second semi-preparative HPLC conditions were: the mobile phase is acetonitrile-water solution with volume ratio of 23:77, and water contains 0.05% of TFA; the flow rate was 3mL/min.
9. A pharmaceutical composition comprising a compound having an insulin resistance-improving effect or a pharmaceutically acceptable salt thereof according to claim 1 and/or claim 2 and/or a compound having an insulin resistance-improving effect of the structure shown in formula III or a pharmaceutically acceptable carrier and/or adjuvant;
10. use of a compound having an insulin resistance improving effect or a pharmaceutically acceptable salt thereof according to claim 1 or 2, a compound having an insulin resistance improving effect of the structure shown in formula III or a pharmaceutically acceptable salt thereof, a pharmaceutical composition according to claim 9 for the manufacture of a medicament for activating Akt phosphorylation and/or treating diseases associated with insulin resistance.
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