CN115806483A - Phenolic heteroterpene compound (+) -Dayaolingzhiol M and application thereof in pharmacy and food - Google Patents
Phenolic heteroterpene compound (+) -Dayaolingzhiol M and application thereof in pharmacy and food Download PDFInfo
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- CN115806483A CN115806483A CN202111068740.5A CN202111068740A CN115806483A CN 115806483 A CN115806483 A CN 115806483A CN 202111068740 A CN202111068740 A CN 202111068740A CN 115806483 A CN115806483 A CN 115806483A
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Abstract
The invention discloses a phenolic heteroterpene compound (+) -Dayaolingzhiol M and application thereof in pharmacy and food, relating to the technical field of chemical drugs. The compound (+) -Dayaolingzhiol M is a novel compound which is separated and identified from ganoderma lucidum for the first time. The phenolic heteroterpene compound (+) -Dayaolingzhiol M has the effects of activating protein kinase B and adenylate activated protein kinase phosphorylation and activating glucose uptake dose-dependently, shows a certain effect on improving insulin resistance related diseases, and can be applied to preparation of medicaments for activating protein kinase B and AMPK phosphorylation and/or medicaments for treating insulin resistance related diseases and corresponding foods.
Description
Technical Field
The invention relates to the technical field of chemical medicines, in particular to a phenolic heteroterpene compound (+) -Dayaolingzhiol M and application thereof in pharmacy and food.
Background
Ganoderma lucidum is a famous and precious traditional Chinese medicine with a long history, and has been widely concerned by scholars at home and abroad for a long time due to the remarkable curative effect. Ganoderma contains polysaccharide, nucleoside, furan, sterol, alkaloid, triterpene, oil, amino acids, proteins, enzymes, organic germanium, and trace elements. Wherein the ganoderan has the effects of regulating immunity, reducing blood sugar, reducing blood lipid, resisting oxidation, resisting aging and resisting tumor; the triterpenes can reduce cholesterol and protect liver function; the Ganoderma preparation has tranquilizing, anticonvulsive, heart tonifying, arrhythmia relieving, blood pressure lowering, cough relieving, and asthma relieving effects; in addition, ganoderma has anticoagulant, platelet aggregation inhibiting and antiallergic effects.
In recent years, the reports of more and more structures and activities of ganoderma lucidum heteroterpenes suggest that the compounds have wide pharmacological activities, such as renal protection, anti-tumor, neuroprotection and the like.
AMPK (AMP-dependent protein kinase) is a receptor for changes in energy metabolism, and activation of AMPK can enhance glucose uptake, fatty acid oxidation and insulin sensitivity in the muscle and liver, and can reduce production of glucose, cholesterol and triglyceride, directly improving insulin resistance.
However, the research on whether the heteroterpene component in the ganoderma has the functions of activating protein kinase B (Akt) and adenylate activated protein kinase (AMPK) phosphorylation and improving the insulin resistance activity is not reported in documents.
Disclosure of Invention
The invention aims to solve the technical problem of providing ganoderma lucidum hetero-terpenoid compounds with the activity of activating AMPK phosphorylation and improving insulin resistance and application thereof in pharmacy and food, so as to solve the defects in the prior art.
In order to solve the above problems, the present invention proposes the following technical solutions:
in a first aspect, the present invention provides a phenolic heteroterpene compound having activated protein kinase B and adenylate activated protein kinase phosphorylation, said compound being designated (+) -Dayaolingzhiol M, said compound having the formula:
further, the compound (+) -Dayaolingzhiol M is obtained by extraction from Ganoderma lucidum, or by artificial synthesis.
In a second aspect, the present invention provides a pharmaceutical composition containing a phenolic heteroterpene compound, said pharmaceutical composition comprising the phenolic heteroterpene compound (+) -Dayaolingzhiol M of the first aspect, or a pharmaceutically acceptable derivative thereof and a salt thereof.
Further, the pharmaceutical composition is a pharmaceutical preparation, and consists of (+) -Dayaolingzhiol M, or a pharmaceutically acceptable derivative or salt thereof as an active ingredient and a pharmaceutically acceptable excipient.
The pharmaceutical preparation includes solid preparations, semi-solid preparations, liquid preparations, ophthalmic preparations and the like. Wherein the solid preparation comprises: tablets, capsules, pills, granules and the like; semisolid preparations include ointments, suppositories, and the like; the liquid formulation comprises: solutions, injections, sprays, and the like; the ophthalmic preparation comprises: eye drops, ophthalmic gels, and the like.
In a third aspect, the present invention provides a method for preparing a phenolic diterpene compound having activated protein kinase B and adenylate activated protein kinase phosphorylation according to the first aspect, comprising the steps of:
pulverizing Ganoderma lucidum 30kg, percolating with 95% ethanol 8-10 times volume fraction at extraction flow rate of 140ml/min, recovering ethanol under reduced pressure to obtain extract 2.1kg, suspending the extract in warm water, and isovolumetrically extracting with ethyl acetate for 3 times to obtain ethyl acetate 1.1kg;
1.1kg of ethyl acetate fraction was column chromatographed on MCI gel CHP 20P, with a methanol/water gradient elution of 40; 123.0g Fr.K silica gel column chromatography, petroleum etherAcetone was eluted with a gradient of 10; 99.0g Fr.K4 is separated by Sephadex LH-20 column chromatography and eluted by methanol to obtain 3 components, K41-K43;1.7g fr. K43 is subjected to vacuum liquid chromatography column chromatography, petroleum ether/acetone gradient elution 15; k435, 1.3g, was subjected to preparative thin layer chromatography eluting with petroleum ether/acetone 3;374.0mg Fr.K4353, R f =0.2, purification by semi-preparative HPLC, where acetonitrile/water 62, containing 0.05% TFA in water, gives 11 components, K4353A-K4353K;23.0mg Fr.K4353A was further purified by semi-preparative HPLC, methanol/water 74, containing 0.05% TFA in water, to give (. + -.) -Dayaolingzhiol M,1.3mg, t R =25.8min; the compound is a pair of enantiomers and is subjected to chiral resolution in the presence of 95% n-hexane/ethanol, 0.05% TFA in ethanol, to yield (+) -Dayaolingzhiol M0.7 mg, t R ＝33.0min。
In a fourth aspect, the invention also provides application of the phenolic diterpenoid compound which can activate protein kinase B and adenylate activated protein kinase phosphorylation in preparation of drugs which can activate protein kinase B and AMPK phosphorylation and/or drugs for treating insulin resistance related diseases and corresponding food.
In a fifth aspect, the invention also provides application of the pharmaceutical composition containing the phenolic diterpenoid compound in preparing medicaments for activating protein kinase B and AMPK phosphorylation and/or medicaments for treating diseases related to insulin resistance and corresponding foods.
Compared with the prior art, the invention can achieve the technical effects that:
1. the invention discloses a method for extracting and separating a novel compound (+) -Dayaolingzhiol M from ganoderma lucidum for the first time.
2. The compound (+) -Dayaolingzhiol M is a novel compound which is separated and identified from ganoderma lucidum for the first time.
3. The novel compound (+) -Dayaolingzhiol M provided by the invention has the effects of activating phosphorylation of protein kinase B (Akt) and adenylate activated protein kinase (AMPK) and activating glucose uptake in a dose-dependent manner, shows a certain effect on improving insulin resistance-related diseases, and can be applied to preparation of medicaments for activating phosphorylation of protein kinase B and AMPK and/or treatment of insulin resistance-related diseases and corresponding foods.
Drawings
FIG. 1 is the experimental results of the insulin resistance activity study of example 6 of the present invention.
Detailed Description
The technical solutions in the embodiments will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, wherein like reference numerals represent like elements in the drawings. It is apparent that the embodiments to be described below are only a part of the embodiments of the present invention, and not all of them. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It will be understood that the terms "comprises" and/or "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
It is also to be understood that the terminology used in the description of the embodiments of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of embodiments of the invention. As used in the description of embodiments of the present invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
Example 1
Extraction separation and structure identification of compound (+) -Dayaolingzhiol M
Pulverizing Ganoderma 30kg, percolating with 95% ethanol 8 times volume fraction at 140ml/min, recovering ethanol under reduced pressure to obtain extract 2.1kg, suspending the extract in warm water, and isovolumetrically extracting with ethyl acetate for 3 times to obtain ethyl acetate 1.1kg.
The ethyl acetate fraction (1.1 kg) was column-chromatographed with MCI gel CHP 20P, methanol/water (40, about 110l,45, about 110l,50, about 52l,55, about 74l,60, about 74l, 40, about 74l,70, about 89l, 75. K (123.0 g) was subjected to silica gel column chromatography, and petroleum ether/acetone (10. K4 (99.0 g) was separated by Sephadex LH-20 column chromatography and eluted with methanol to give 3 fractions, K41-K43.Fr. K43 (1.7 g) was subjected to Vacuum Liquid Chromatography (VLC) column chromatography, gradient elution with petroleum ether/acetone (15, 1, 10. Fr.k435 (1.3 g) was chromatographed by Preparative Thin Layer Chromatography (PTLC) eluting with petroleum ether/acetone (3). Fr.K4353 (374.0 mg) (R) f = 0.2) purification by semi-preparative HPLC (acetonitrile/water with 0.05% TFA in water, 62). Fr.k4353a (23.0 mg) was further purified by semi-preparative HPLC (methanol/water with 0.05% TFA in water, 74 R =25.8 min). This compound is a pair of enantiomers and was subjected to chiral resolution (n-hexane/ethanol with 0.05% TFA in ethanol, 95 R ＝33.0min)。
The results of the identification of the obtained compounds were as follows:
the compound, (+) -Dayaolingzhiol M, yellow gum, [ alpha ]] D 25 +7.4(c 0.18,MeOH)；CD(MeOH)Δε 204 –0.42,Δε 231 +0.50,Δε 257 +0.23,Δε 313 –0.10；UV(MeOH)λ max (logε)367(2.89),258(3.18),228(3.48)nm；HRESIMS m/z 399.1782[M+Na] + (calcd for C 21 H 28 O 6 Na,399.1784)； 1 H and 13 C NMR data are shown in Table 1.
Method for producing compound (+) -Dayaolingzhiol M of Table 1 1 H and 13 c NMR data
Example 2:
the compound (+) -Dayaolingzhiol M in example 1 is added with injection solvent according to the conventional method, fine filtered, encapsulated and sterilized to prepare injection.
Example 3:
the compound (+) -Dayaolingzhiol M in example 1 can be made into tablets by conventional method with various pharmaceutical excipients.
The preparation method comprises the following steps: using the compound (+) -Dayaolingzhiol M of example 1 as a pharmaceutically active ingredient and several excipients as auxiliary ingredients for preparing a combined pharmaceutical tablet, tablet samples each containing 1-100mg of the pharmaceutical ingredient were prepared in a certain ratio.
Example 4:
the compound (+) -Dayaolingzhiol M of example 1 can be made into capsules by conventional methods with various pharmaceutical excipients.
Preparation of pharmaceutical combination capsule formulation containing as an effective ingredient (+) -Dayaolingzhiol M compound of example 1: the compound (+) -Dayaolingzhiol M in example 1 is used as a pharmaceutical active ingredient, and several excipients are used as auxiliary ingredients for preparing combined pharmaceutical capsules, and capsule preparations containing 1-100mg of the compound ingredient in each capsule are prepared according to a certain proportion.
Example 5:
the compound (+) -Dayaolingzhiol M1 part obtained in example 1 and 10 parts of non-dairy creamer were mixed and made into a solid beverage according to a conventional method.
Example 6:
insulin resistance Activity study
Cell Count Kit-8, ECL Kit and Glucose Colorimetric/Fluorometric Assay Kit were used to test AKT and AMPK phosphorylation, and Glucose uptake activity in this experiment. The method comprises the following specific steps:
AKT and AMPK phosphorylation:
1. various compounds, including (+) -Dayaolingzhiol M, were dissolved separately in DMSO to 20mM stock solutions.
C2C12 cell line was grown in DMEM containing 10% FBS (total bone serum) until contact inhibition, and the cell culture medium was changed to fresh DMEM containing 2% horse serum (horse serum), 100U/mL penicilin and 100. Mu.g/mL streptomycin, and incubated at 37 ℃ for 4 days.
3. Insulin resistance was simulated by adding 100nM insulin to the induction medium for 24h while the cells were differentiating. After 24h of compound treatment, total protein was extracted from the cell lines and a quantitative protein sample was used.
4. Equal amounts of protein extracts were separated by 8% SDS-PAGE and transferred to PVDF membrane. Membranes were blocked with 5% bovine serum albumin, then incubated with the indicated antibodies overnight at 4 ℃ and then with horseradish peroxidase (HRP) conjugated secondary antibody at room temperature.
5. The bands were visualized and measured by ECL kit. Densitometric analysis of immunoblot results was performed using ImageJ software.
Glucose uptake:
c2c12 cells were incubated in DMEM (low-sugar) for 6h.
2. The compound was added to high-glucose DMEM, treated with the compound for 30min, and then incubated with insulin (100 nmol/L) for 4h.
3. And determining the glucose content in the culture supernatant by adopting a glucose colorimetric/fluorescent determination kit.
The results are shown in FIG. 1, where Compound (+) -5 is the Compound (+) -Dayaolingzhiol M.
In the drawings ## P<0.01 and # P<0.05 (compare to normal cell group stimulated with added insulin); #### P<0.0001 (compare to normal cell group without added insulin stimulation); * P<0.05 and P<0.01 (compared to insulin resistant cell group stimulated by addition of insulin) and<0.0001 (compare to normal cell group stimulated with added insulin); $$$$ P<0.0001 (compare to the group of insulin resistant cells stimulated with the addition of insulin).
The results show that the compound (+) -Dayaolingzhiol M can obviously activate AKT and AMPK phosphorylation and glucose uptake under the concentration of 20 mu M, and the regulation and control of the glucose uptake are in dose dependence, which indicates that the compound (+) -Dayaolingzhiol M has certain activity of improving insulin resistance.
In the above embodiments, the descriptions of the respective embodiments have respective emphasis, and for parts that are not described in detail in a certain embodiment, reference may be made to related descriptions of other embodiments.
While the invention has been described with reference to specific embodiments, the scope of the invention is not limited thereto, and various equivalent modifications and substitutions may be easily made by those skilled in the art within the technical scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (7)
1. A phenolic heteroterpene compound having activated protein kinase B and adenylate activated protein kinase phosphorylation, wherein said compound is designated (+) -Dayaolingzhiol M, and said compound has the structural formula:
2. a pharmaceutical composition comprising a phenolic heteroterpene compound, wherein the pharmaceutical composition comprises the phenolic heteroterpene compound (+) -Dayaolingzhiol M of claim 1, or a pharmaceutically acceptable derivative thereof and a salt thereof.
3. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition is a pharmaceutical formulation consisting of (+) -Dayaolingzhiol M, or a pharmaceutically acceptable derivative or salt thereof, as an active ingredient and a pharmaceutically acceptable excipient.
4. A method for producing the phenolic diterpene compound having the phosphorylation of activated protein kinase B and adenylate activated protein kinase according to claim 1, which comprises the steps of:
pulverizing Ganoderma lucidum 30kg, percolating with 95% ethanol 8-10 times volume fraction at extraction flow rate of 140ml/min, recovering ethanol under reduced pressure to obtain extract 2.1kg, suspending the extract in warm water, and isovolumetrically extracting with ethyl acetate for 3 times to obtain ethyl acetate 1.1kg;
1.1kg of ethyl acetate Fractions were column chromatographed with mcigel chp 20P, methanol/water eluting with a gradient of 40; k, silica gel column chromatography, petroleum ether/acetone gradient elution at 10, 1, 9; k4 (99.0 g) are separated by Sephadex LH-20 column chromatography and eluted by methanol to obtain 3 components, K41-K43; column chromatography in vacuum liquid chromatography at 1.7g fr.k43, eluting with a gradient of petroleum ether/acetone at 15; k435, preparative thin layer chromatography on petroleum ether/acetone 3;374.0mg Fr.K4353 f =0.2, purification by semi-preparative HPLC, where acetonitrile/water 62, containing 0.05% TFA in water, gives 11 components, K4353A-K4353K;23.0mg Fr.K4353A was further purified by semi-preparative HPLC, methanol/water 74, containing 0.05% TFA in water, to give (. + -.) -Dayaolingzhiol M,1.3mg, t R =25.8min; the compound is a pair of enantiomers and is subjected to chiral resolution in the presence of 95% n-hexane/ethanol, 0.05% TFA in ethanol, to yield (+) -Dayaolingzhiol M0.7 mg, t R ＝33.0min。
5. The phenolic diterpene compound having kinase B and adenylate activated protein kinase phosphorylation according to claim 1, wherein (+) -Dayaolingzhiol M is obtained by extraction from ganoderma lucidum or by artificial synthesis.
6. Use of the phenolic diterpene compounds activating phosphorylation of protein kinase B and adenylate activated protein kinase according to claim 1 for the preparation of medicaments activating phosphorylation of protein kinase B and AMPK and/or for the treatment of diseases associated with insulin resistance, and corresponding foods.
7. Use of the pharmaceutical composition containing phenolic diterpene compounds according to claim 2 or 3 for the preparation of medicaments for activating protein kinase B and AMPK phosphorylation and/or for the treatment of diseases associated with insulin resistance, and corresponding food products.
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