CN111690024B - Loganin derivative iridoid compound and extraction method and application thereof - Google Patents

Loganin derivative iridoid compound and extraction method and application thereof Download PDF

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CN111690024B
CN111690024B CN201910189304.XA CN201910189304A CN111690024B CN 111690024 B CN111690024 B CN 111690024B CN 201910189304 A CN201910189304 A CN 201910189304A CN 111690024 B CN111690024 B CN 111690024B
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CN111690024A (en
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王福生
杨敏
殷明
李逢逢
米丹
付晨
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Dali University
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Abstract

The invention relates to the technical field of active ingredient extraction, in particular to a cycloiridoid compound of a derivatives of a cycloxanthone and an extraction method and application thereof. The main structural characteristics of the loganin derivative iridoid compound provided by the invention are that hydroxyl groups on loganin beta-D-glucopyranose are acetylated to different degrees, and the 4' -hydroxyl group of the beta-D-glucopyranose is further esterified with 2',3' -dihydroxybenzoic acid, and the 2',3' -dihydroxyl is further glycosided with the beta-D-glucopyranose or alpha-D-glucopyranose. The results of screening the activity of the anti-Alzheimer disease drug by taking the inhibition of the activity of acetylcholinesterase and the inhibition of the generation of beta-amyloid (Abeta) as targets show that the compound provided by the invention can effectively inhibit the activity of acetylcholinesterase and reduce the generation of Abeta 40 and Abeta 42, and has obvious effects of preventing and treating Alzheimer disease and good research and development prospects.

Description

Loganin derivative iridoid compound and extraction method and application thereof
Technical Field
The invention relates to the technical field of active ingredient extraction, in particular to a cycloiridoid compound of a derivatives of a cycloxanthone and an extraction method and application thereof.
Background
About 400 gentian plants worldwide, 247 varieties and 41 varieties in China. There are 125 varieties in Yunnan, including 6 varieties, most of which are distributed in the northwest region of Yunnan. The gentian mainly contains iridoid glycoside, secoiridoid glycoside, flavonoid, triterpene compound and other components, and has obvious various physiological activities of anti-inflammatory, antioxidant, antifungal, antitumor, liver protecting, blood sugar reducing, wound healing and the like. Micro-seed radix Gentianae (Gentiana delavayi Franch) is a plant of Gentiana genus of Gentianaceae family, and is mainly distributed in Yunnan Kunming, heqing, jianchuan, eryuan and Sichuan south. At present, little research is carried out on gentiana microphylla, the activity of the extract is researched, and no report is made on the extraction and separation of the acteoside derivative iridoid compound from gentiana microphylla and the further research on the pharmaceutical activity of the compound.
Disclosure of Invention
The invention aims to provide a cycloiridoid compound of a derivative of the acteoside, and an extraction method and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a cycloiridoid compound of a cycloxanthone derivative, which has a structure shown in a formula I:
r in formula I 1 ～R 6 The combinations of (a) are as follows:
the invention provides an extraction method of a loganin derivative iridoid compound, which comprises the following steps:
(1) Extracting flowers of gentiana microphylla with an aqueous ethanol solution to obtain a total extract;
(2) Concentrating the total extract under reduced pressure until no alcohol smell exists, sequentially extracting the obtained extract water suspension with petroleum ether, chloroform and n-butanol, concentrating under reduced pressure, and collecting chloroform extract and n-butanol extract;
(3) Subjecting chloroform-methanol as eluent to silica gel column chromatography under gradient elution to obtain 5 components, denoted as D 1-5 ；
Will D 2 Subjecting the components to column chromatography to obtain compound 3；
Using chloroform-methanol as eluent, and subjecting D to gradient elution 3 Separating the components by silica gel column chromatography to obtain 5 components, which are marked as D 3.1-3.5 The method comprises the steps of carrying out a first treatment on the surface of the Chloroform-methanol is used as eluent, D is eluted under the condition of equal gradient 3.2 Separating the components by Sephadex LH-20 column chromatography to obtain 3 components, denoted as D 3.2.1-3.2.3 The method comprises the steps of carrying out a first treatment on the surface of the Chloroform-methanol is used as eluent, D is eluted under the condition of equal gradient 3.2.2 Subjecting the components to column chromatography separation to obtain a compound 2 and a compound 7 in sequence;
using water-methanol as eluent, and under gradient elution condition, D 4 Subjecting the components to MCI column chromatography to obtain 5 components in turn, denoted as D 4.1-4.5 The method comprises the steps of carrying out a first treatment on the surface of the Using chloroform-methanol as eluent, and subjecting D to gradient elution 4.5 Separating the components by silica gel column chromatography to obtain 5 components, which are marked as D 4.5.1-4.5.5 The method comprises the steps of carrying out a first treatment on the surface of the Will D 4.5.1 Subjecting the components to column chromatography separation, and recrystallizing the obtained fraction to obtain a compound 5; will D 4.5.2 Subjecting the components to column chromatography separation, and recrystallizing the obtained fraction to obtain a compound 6;
using chloroform-methanol as eluent, and subjecting D to gradient elution 5 Separating the components by silica gel column chromatography to obtain 6 components, which are marked as D 5.1-5.6 The method comprises the steps of carrying out a first treatment on the surface of the Will D 5.3 Subjecting the components to column chromatography separation to obtain a compound 8; will D 5.5 Subjecting the components to column chromatography separation to obtain a compound 4;
(4) Subjecting the n-butanol fraction extract to AB-8 macroporous adsorbent resin column chromatography under gradient elution with water-methanol as eluent to obtain 4 components, denoted as E 1-4 ；
Using chloroform-methanol as eluent, and subjecting E to gradient elution 2 Subjecting the components to silica gel column chromatography to obtain 5 components, denoted as E 2.1-2.5 The method comprises the steps of carrying out a first treatment on the surface of the Will E 2.5 Subjecting the components to column chromatography separation to obtain a compound 1;
the step (3) and the step (4) are defined in no time sequence.
Preferably, the volume ratio of petroleum ether, chloroform and n-butanol for extraction to the material to be extracted is independently (2-6): 1.
preferably, said D 2 The method for separating the components by column chromatography comprises the following steps:
petroleum ether-acetone is used as eluent, D is carried out under the gradient elution condition 2 Separating the components by silica gel column chromatography to obtain 5 components, which are marked as D 2.1-2.5 ；
Chloroform-methanol is used as eluent, D is eluted under the condition of equal gradient 2.3 Separating the components by Sephadex LH-20 column chromatography to obtain 3 components, denoted as D 2.3.1-2.3.3 ；
Methanol is used as eluent to carry out D 2.3.3 The component is subjected to Sephadex LH-20 column chromatography separation to obtain the compound 3.
Preferably, in the extraction of said compound 5, in particular with methanol as eluent, D 4.5.1 Subjecting the components to Sephadex LH-20 column chromatography, and recrystallizing the obtained fraction in methanol.
Preferably, in the extraction of said compound 6, in particular with methanol as eluent, D 4.5.2 Subjecting the components to Sephadex LH-20 column chromatography, and recrystallizing the obtained fraction in methanol.
Preferably, in the extraction of said compound 8, in particular with methanol as eluent, D 5.3 The components are subjected to Sephadex LH-20 column chromatography separation.
Preferably, in the extraction of said compound 4, in particular chloroform-methanol is used as eluent, D 5.5 The components are subjected to Sephadex LH-20 column chromatography separation.
The invention provides an application of a loganin derivative iridoid compound or a pharmaceutically acceptable salt thereof in preparation of an anti-Alzheimer disease drug, wherein the loganin derivative iridoid compound is the loganin derivative iridoid compound in the technical scheme or extracted by the extraction method in the technical scheme.
Preferably, the anti-alzheimer disease drug contains at least one of compound 1, compound 5, compound 6, compound 7, compound 1 pharmaceutically acceptable salt, compound 5 pharmaceutically acceptable salt, compound 6 pharmaceutically acceptable salt and compound 7 pharmaceutically acceptable salt.
The invention provides an iridoid compound of a derivative of loganin, which is mainly structurally characterized in that hydroxyl groups on beta-D-glucopyranose of loganin are acetylated to different degrees, and the 4' -hydroxyl group of the beta-D-glucopyranose is further esterified with 2',3' -dihydroxybenzoic acid, and the 2',3' -dihydroxyl is further glycosidated with the beta-D-glucopyranose or the alpha-D-glucopyranose. The results of screening the activity of the Alzheimer's disease drug by taking the inhibition of the activity of acetylcholinesterase and the inhibition of the generation of beta-amyloid (Abeta) as targets show that the loganin derivative iridoid compound provided by the invention can effectively inhibit the activity of acetylcholinesterase and reduce the generation of Abeta 40 and Abeta 42, and the compounds have obvious effects of preventing and treating Alzheimer's disease and good research and development prospects.
The invention provides an extraction method of a loganin derivative iridoid compound, which takes flowers of micro-seed gentian as raw materials, and a series of loganin derivative iridoid compounds are obtained through a plurality of separation methods, thus providing a foundation for medicinal research.
Drawings
FIG. 1 is a graph showing the results of inhibition of extracellular Aβ40 and Aβ42 production by Compound 5;
FIG. 2 is a graph showing the results of inhibition of extracellular Aβ40 and Aβ42 production by Compound 6 (LL-48 represents Compound 6);
FIG. 3 is a graph showing the results of inhibition of extracellular Aβ40 and Aβ42 production by Compound 7 (LL-69 represents Compound 7);
FIG. 4 is a graph showing the effect of Compound 6 on the content of BACE1 in extracellular fluid (LL-48 represents Compound 6);
FIG. 5 is a graph showing the effect of Compound 6 on the Aβ degrading enzyme MME (LL-48 represents Compound 6).
Detailed Description
The invention provides a cycloiridoid compound of a cycloxanthone derivative, which has a structure shown in a formula I:
r in formula I 1 ～R 6 The combinations of (a) are as follows:
in the invention, the structural formulas of the compounds 1 to 8 are specifically shown as follows:
the invention provides an extraction method of a loganin derivative iridoid compound, which comprises the following steps:
(1) Extracting flowers of gentiana microphylla with an aqueous ethanol solution to obtain a total extract;
(2) Concentrating the extractive solution under reduced pressure until no alcohol smell exists, sequentially extracting the obtained extract water suspension with petroleum ether, chloroform and n-butanol, concentrating under reduced pressure, and collecting chloroform extract and n-butanol extract;
(3) Subjecting chloroform-methanol as eluent to silica gel column chromatography under gradient elution to obtain 5 components, denoted as D 1-5 ；
Will D 2 Subjecting the components to column chromatography separation to obtain a compound 3;
using chloroform-methanol as eluent, and subjecting D to gradient elution 3 Separating the components by silica gel column chromatography to obtain 5 components, which are marked as D 3.1-3.5 The method comprises the steps of carrying out a first treatment on the surface of the Chloroform-methanol is used as eluent, D is eluted under the condition of equal gradient 3.2 Separating the components by Sephadex LH-20 column chromatography to obtain 3 components, denoted as D 3.2.1-3.2.3 The method comprises the steps of carrying out a first treatment on the surface of the Chloroform-methanol is used as eluent, D is eluted under the condition of equal gradient 3.2.2 Subjecting the components to column chromatography separation to obtain a compound 2 and a compound 7 in sequence;
using water-methanol as eluent, and under gradient elution condition, D 4 Subjecting the components to MCI column chromatography to obtain 5 components in turn, denoted as D 4.1-4.5 The method comprises the steps of carrying out a first treatment on the surface of the Chloroform-methanol is used as eluent, D is eluted under the condition of equal gradient 4.5 Separating the components by silica gel column chromatography to obtain 5 components, which are marked as D 4.5.1-4.5.5 The method comprises the steps of carrying out a first treatment on the surface of the Will D 4.5.1 Subjecting the components to column chromatography separation, and recrystallizing the obtained fraction to obtain a compound 5; will D 4.5.2 Subjecting the components to column chromatography separation, and recrystallizing the obtained fraction to obtain a compound 6;
using chloroform-methanol as eluent, and subjecting D to gradient elution 5 Separating the components by silica gel column chromatography to obtain 6 components, which are marked as D 5.1-5.6 The method comprises the steps of carrying out a first treatment on the surface of the Will D 5.3 Subjecting the components to column chromatography separation to obtain a compound 8; will D 5.5 Subjecting the components to column chromatography separation to obtain a compound 4;
(4) Subjecting the n-butanol fraction extract to AB-8 macroporous adsorbent resin column chromatography under gradient elution with water-methanol as eluent to obtain 4 components, denoted as E 1-4 ；
Using chloroform-methanol as eluent, and subjecting E to gradient elution 2 Subjecting the components to silica gel column chromatography to obtain 5 components, denoted as E 2.1-2.5 The method comprises the steps of carrying out a first treatment on the surface of the Will E 2.5 Subjecting the components to column chromatography separation to obtain a compound 1;
the step (3) and the step (4) are defined in no time sequence.
The invention uses ethanol water solution to extract the flowers of gentiana microphylla to obtain total extract. In the present invention, the flowers of gentiana microphylla are preferably dried and crushed before use; the specific operation method of drying and crushing is not particularly limited, and the subsequent extraction can be ensured to be carried out smoothly. In the present invention, the method of extracting flowers of gentiana microphylla preferably comprises the steps of:
extracting flowers of gentiana microphylla with a first ethanol aqueous solution to obtain an extract A and medicinal residues; performing second extraction on the residues by adopting a second ethanol aqueous solution to obtain an extract B; combining the extract A and the extract B as a total extract.
In the present invention, the mass concentration of the first aqueous ethanol solution is preferably 93 to 97%, more preferably 94 to 96%; the ratio of the first ethanol aqueous solution to the flowers of gentiana rigescens is preferably (1-3) L:1kg, more preferably (2 to 3) L:1kg. In the present invention, the mass concentration of the second aqueous ethanol solution is preferably 40 to 70%, more preferably 50 to 60%; the dosage ratio of the second ethanol aqueous solution to the dregs is preferably (1-3) L:1kg, more preferably (2 to 3) L:1kg. In the present invention, the number of times of the first extraction and the second extraction is preferably 2 to 3 independently, and the time of each extraction is preferably 12 to 24 independently, and the first extraction and the second extraction are preferably performed under room temperature conditions.
After the total extract is obtained, the total extract is concentrated under reduced pressure until no alcohol smell exists, the obtained extract aqueous suspension is sequentially extracted by petroleum ether, chloroform and n-butanol, and the chloroform part extract and the n-butanol part extract are collected through reduced pressure concentration. In the present invention, the volume ratio of the petroleum ether, chloroform and n-butanol for extraction to the material to be extracted is preferably independently (2 to 6): 1, more preferably (3 to 5): 1. in the present invention, the petroleum ether fraction extract and the water fraction extract are also collected during the extraction process, and these two fractions are not involved in the subsequent further separation process and are not described here too much.
In the invention, the chloroform fraction extract is further separated to obtain compounds 2 to 7, which specifically comprise the following steps:
subjecting chloroform-methanol as eluent to silica gel column chromatography under gradient elution to obtain 5 components, denoted as D 1-5 . In the invention, the granularity of the silica gel used for the silica gel column chromatography separation is preferably 80-100 meshes; the gradient elution comprises five gradients which are sequentially carried out, and the composition of the eluent in each gradient is preferably 1:0, 3:4, 2:1, 1:4 and 0:1 according to the volume ratio of chloroform to methanol; the invention preferably monitors by thin layer chromatography, R f The components of similar or identical values are combined to finally give 5 components (D 1-5 ). In the subsequent column chromatography separation process, thin layer chromatography monitoring is adopted, and the method is not particularly limited in the subsequent related steps.
The invention further provides the D obtained by the method 2 The components are subjected to column chromatography separation to obtain the compound 3. In the present invention, the D 2 The method for separating the components by column chromatography preferably comprises the following steps:
petroleum ether-acetone is used as eluent, D is carried out under the gradient elution condition 2 Separating the components by silica gel column chromatography to obtain 5 components, which are marked as D 2.1-2.5 The method comprises the steps of carrying out a first treatment on the surface of the Wherein, the granularity of the silica gel used for the silica gel column chromatographic separation is preferably 300-400 meshes; the gradient elution comprises five gradients which are sequentially carried out, and the composition of the eluent in each gradient is preferably 1:0, 3:4, 2:1, 1:4 and 0:1 according to the volume ratio of petroleum ether to acetone;
chloroform-methanol is used as eluent, D is eluted under the condition of equal gradient 2.3 Separating the components by Sephadex LH-20 column chromatography to obtain 3 components, denoted as D 2.3.1-2.3.3 The method comprises the steps of carrying out a first treatment on the surface of the Wherein the volume ratio of chloroform to methanol is preferably 1:1;
methanol is used as eluent to carry out D 2.3.3 The component is subjected to Sephadex LH-20 column chromatography separation to obtain the compound 3.
The invention further provides the D obtained by the method 3 Subjecting the components to column chromatography separation to obtain a compound 2 and a compound 7; the method specifically comprises the following steps:
using chloroform-methanol as eluent, and subjecting D to gradient elution 3 Silica gel is carried out on the componentsSeparating by column chromatography to obtain 5 components, which are marked as D 3.1-3.5 The method comprises the steps of carrying out a first treatment on the surface of the Wherein, the granularity of the silica gel used for the silica gel column chromatographic separation is preferably 300-400 meshes; the gradient elution comprises five gradients which are sequentially carried out, and the composition of the eluent in each gradient is preferably 100:1, 80:1, 60:1, 40:1 and 20:1 according to the volume ratio of chloroform to methanol;
chloroform-methanol is used as eluent, D is eluted under the condition of equal gradient 3.2 Separating the components by Sephadex LH-20 column chromatography to obtain 3 components, denoted as D 3.2.1-3.2.3 The method comprises the steps of carrying out a first treatment on the surface of the Wherein the volume ratio of chloroform to methanol is preferably 1:1;
chloroform-methanol is used as eluent, D is eluted under the condition of equal gradient 3.2.2 Subjecting the components to column chromatography separation to obtain a compound 2 and a compound 7 in sequence; wherein the column chromatographic separation is preferably silica gel column chromatographic separation; the granularity of the silica gel used for the silica gel column chromatographic separation is preferably 300-400 meshes; the volume ratio of chloroform to methanol is preferably 30:1.
The invention further provides the D obtained by the method 4 Subjecting the components to column chromatography separation to obtain a compound 5 and a compound 6; the method specifically comprises the following steps:
using water-methanol as eluent, and under gradient elution condition, D 4 Subjecting the components to MCI column chromatography to obtain 5 components in turn, denoted as D 4.1-4.5 The method comprises the steps of carrying out a first treatment on the surface of the The gradient elution comprises five gradients which are sequentially carried out, and the composition of the eluent in each gradient is preferably 1:0, 3:4, 2:1, 1:4 and 0:1 according to the volume ratio of water to methanol;
using chloroform-methanol as eluent, and subjecting D to gradient elution 4.5 Separating the components by silica gel column chromatography to obtain 5 components, which are marked as D 4.5.1-4.5.5 The method comprises the steps of carrying out a first treatment on the surface of the Wherein, the granularity of the silica gel used for the silica gel column chromatographic separation is preferably 300-400 meshes; the gradient elution comprises five gradients which are sequentially carried out, and the composition of the eluent in each gradient is preferably 50:1, 40:1, 30:1, 15:1 and 0:1 according to the volume ratio of chloroform to methanol;
will D 4.5.1 Subjecting the components to column chromatography separation, and recrystallizing the obtained fraction to obtain a compound 5; wherein the column layerThe separation is preferably Sephadex LH-20 column chromatography, and the eluent is preferably methanol; the reagent used for the recrystallization is preferably methanol;
will D 4.5.2 Subjecting the components to column chromatography separation, and recrystallizing the obtained fraction to obtain a compound 6; wherein, the column chromatographic separation is preferably Sephadex LH-20 column chromatographic separation, and the eluent is preferably methanol; the reagent used for the recrystallization is preferably methanol.
The invention further provides the D obtained by the method 5 Subjecting the components to column chromatography separation to obtain a compound 4 and a compound 8; the method specifically comprises the following steps:
using chloroform-methanol as eluent, and subjecting D to gradient elution 5 Separating the components by silica gel column chromatography to obtain 6 components, which are marked as D 5.1-5.6 The method comprises the steps of carrying out a first treatment on the surface of the Wherein, the granularity of the silica gel used for the silica gel column chromatographic separation is preferably 300-400 meshes; the gradient elution comprises six gradients which are sequentially carried out, and the composition of the eluent in each gradient is preferably 10:1, 8:1, 6:1, 4:1, 2:1 and 0:1 according to the volume ratio of chloroform to methanol;
will D 5.3 Subjecting the components to column chromatography separation to obtain a compound 8; wherein, the column chromatographic separation is preferably Sephadex LH-20 column chromatographic separation; the eluent used is preferably methanol;
will D 5.5 Subjecting the components to column chromatography separation to obtain a compound 4; wherein, the column chromatographic separation is preferably Sephadex LH-20 column chromatographic separation; the eluent used is preferably chloroform-methanol, the volume ratio of chloroform to methanol being preferably 1:1.
In the invention, the n-butanol fraction extract is further separated to obtain a compound 1, which specifically comprises the following steps:
subjecting the n-butanol fraction extract to AB-8 macroporous adsorbent resin column chromatography under gradient elution with water-methanol as eluent to obtain 4 components, denoted as E 1-4 The method comprises the steps of carrying out a first treatment on the surface of the Wherein the gradient elution comprises five gradients which are sequentially carried out, and the composition of the eluent in each gradient is preferably 1:0, 3:4, 2:1, 1:4 and 0:1 according to the volume ratio of water to methanol;
using chloroform-methanol as eluent, and subjecting E to gradient elution 2 Subjecting the components to silica gel column chromatography to obtain 5 components, denoted as E 2.1-2.5 The method comprises the steps of carrying out a first treatment on the surface of the Wherein, the granularity of the silica gel used for the silica gel column chromatographic separation is preferably 300-400 meshes; the gradient elution comprises five gradients which are sequentially carried out, and the composition of the eluent in each gradient is preferably 20:1, 15:1, 10:1, 5:1 and 0:1 according to the volume ratio of chloroform to methanol;
will E 2.5 Subjecting the components to column chromatography separation to obtain a compound 1, wherein the column chromatography separation is preferably Sephadex LH-20 column chromatography separation; the eluent used is preferably methanol.
The invention provides application of an loganin derivative iridoid compound or a pharmaceutically acceptable salt thereof in preparation of an anti-Alzheimer disease drug, wherein the loganin derivative iridoid compound is prepared by the technical scheme or the extraction method.
In the present invention, the anti-alzheimer disease drug preferably contains at least one of compound 1, compound 5, compound 6, compound 7, compound 1 pharmaceutically acceptable salt, compound 5 pharmaceutically acceptable salt, compound 6 pharmaceutically acceptable salt and compound 7 pharmaceutically acceptable salt.
In the invention, the anti-Alzheimer disease drug takes the loganin derivative iridoid compound or pharmaceutically acceptable salt thereof as an active ingredient and also comprises a medicinal auxiliary material. In the invention, pharmaceutically acceptable salts of the loganin derivative iridoid compounds are preferably salts formed by the loganin derivative iridoid compounds and inorganic acid, organic acid, alkali metal, alkaline earth metal or basic amino acid; wherein the inorganic acid preferably comprises hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid or hydrobromic acid, the organic acid preferably comprises maleic acid, fumaric acid, tartaric acid, lactic acid, citric acid, acetic acid, methanesulfonic acid, p-toluenesulfonic acid, adipic acid, palmitic acid or tannic acid, the alkali metal preferably comprises lithium, sodium or potassium, the alkaline earth metal preferably comprises calcium or magnesium, and the basic amino acid preferably comprises lysine. In the present invention, the pharmaceutical excipients preferably include at least one of the following substances according to actions:
diluents or excipients: water;
filler: starch or sucrose;
a b mixture: cellulose derivatives, alginates, gelatin or polyvinylpyrrolidone;
a wetting agent: glycerol;
disintegrating agent: agar, calcium carbonate or sodium bicarbonate;
sodium absorption promoter: a quaternary ammonium compound;
and (2) a surfactant: cetyl alcohol;
adsorption carrier: kaolin or bentonite;
and (3) a lubricant: talc, calcium stearate, magnesium stearate or polyethylene glycol;
other adjuvants such as flavoring agent, sweetener, etc. can also be used.
The invention is not particularly limited to the formulation of the anti-Alzheimer disease drug, and can be specifically tablets, granules, capsules, oral liquid, injection, freeze-dried preparation for injection or powder injection and the like, and the preparation of the pharmaceutical formulations such as the tablets, the granules, the capsules, the oral liquid, the injection, the freeze-dried preparation for injection or the powder injection and the like is common knowledge in the field, namely, various pharmaceutical formulations prepared by the loganin derivative iridoid compound or pharmaceutically acceptable salts thereof and corresponding pharmaceutical excipients can be realized by the person skilled in the art.
In the present invention, the content of the active ingredient in the anti-Alzheimer's disease drug is preferably 0.1 to 99.5%, more preferably 0.5 to 95%, still more preferably 5 to 85%, still more preferably 25 to 75%.
The administration amount of the anti-Alzheimer's disease drug of the present invention may vary depending on the administration route, age and weight of the patient, type and severity of the disease to be treated, etc., and the daily dose thereof may be 0.01 to 10mg/kg body weight, preferably 0.1 to 5mg/kg body weight; can be administered one or more times.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. 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 be within the scope of the invention.
Example 1
Extracting the compounds 1 to 8, comprising the following steps:
drying flowers of gentiana microphylla, crushing, and mixing the crushed flowers of gentiana microphylla with 95wt% ethanol water solution according to the dosage ratio of 2L:1kg, soaking and extracting with 95wt% ethanol water solution for 3 times at room temperature for 20 hr each time, and mixing to obtain extract (A); the dosage ratio of the dregs to 70wt% ethanol water solution is 2L:1kg, soaking and extracting the residues with 70wt% ethanol water solution at room temperature for 2 times, each for 20 hours, and mixing to obtain an extract (B); combining the extract (A) and the extract (B), concentrating under reduced pressure until no alcohol smell exists, obtaining an extract water suspension, sequentially extracting with petroleum ether, chloroform and n-butanol with the volume ratio of 4 times, concentrating under reduced pressure, and recovering each extraction part to obtain an anhydrous extract petroleum ether part extract (C), a chloroform part extract (D), an n-butanol part extract (E) and a water part extract (F) respectively;
separating chloroform fraction extract (D) by silica gel (80-100 mesh) column chromatography (eluent chloroform-methanol gradient elution according to volume ratio of 1:0, 3:4, 2:1, 1:4 and 0:1), monitoring by thin layer chromatography, mixing similar or identical components, and sequentially obtaining 5 components (D) 1-5 ) The method comprises the steps of carrying out a first treatment on the surface of the Will D 2 Separating the components by silica gel (300-400 meshes) column chromatography (eluent petroleum ether-acetone is eluted according to the volume ratio of 1:0, 3:4, 2:1, 1:4 and 0:1), and finally obtaining 5 components (D) in sequence 2.1-2.5 ) The method comprises the steps of carrying out a first treatment on the surface of the Will D 2.3 Separating the components by Sephadex LH-20 column chromatography (eluent chloroform-methanol is eluted with equal gradient according to volume ratio of 1:1), and finally obtaining 3 components (D) 2.3.1-2.3.3 ) The method comprises the steps of carrying out a first treatment on the surface of the Will D 2.3.3 Separating the components by Sephadex LH-20 column chromatography (eluting solvent is methanol) to obtain compound 3;
will D 3 Separating the components by silica gel (300-400 meshes) column chromatography (eluent chloroform-methanol is eluted according to the volume ratio of 100:1, 80:1, 60:1, 40:1 and 20:1), and finally obtaining 5 components (D) in sequence 3.1-3.5 ) The method comprises the steps of carrying out a first treatment on the surface of the Will D 3.2 Separating the components by Sephadex LH-20 column chromatography (eluent chloroform-methanol is eluted with equal gradient according to volume ratio of 1:1), and finally obtaining 3 components (D) 3.2.1-3.2.3 ) The method comprises the steps of carrying out a first treatment on the surface of the Will D 3.2.2 Separating the components by silica gel (300-400 meshes) column chromatography (eluent chloroform-methanol is eluted according to the volume ratio of 30:1 and the like) to sequentially obtain a compound 2 and a compound 7;
will D 4 The fractions were separated by MCI column chromatography (eluent water-methanol gradient elution at volume ratios 1:0, 3:4, 2:1, 1:4 and 0:1) to finally give 5 fractions (D 4.1-4.5 ) The method comprises the steps of carrying out a first treatment on the surface of the Will D 4.5 Separating the components by silica gel (300-400 meshes) column chromatography (eluent chloroform-methanol is eluted according to the volume ratio of 50:1, 40:1, 30:1, 15:1 and 0:1), and finally obtaining 5 components (D) in turn 4.5.1-4.5.5 ) The method comprises the steps of carrying out a first treatment on the surface of the Will D 4.5.1 Separating the components by Sephadex LH-20 column chromatography (eluting solvent is methanol), and recrystallizing the obtained fraction in methanol to obtain compound 5; will D 4.5.2 Separating the components by Sephadex LH-20 column chromatography (eluting solvent is methanol), and recrystallizing the obtained fraction in methanol to obtain compound 6;
will D 5 Separating the components by silica gel (300-400 meshes) column chromatography (eluent chloroform-methanol is eluted according to the volume ratio of 10:1, 8:1, 6:1, 4:1, 2:1 and 0:1), and finally obtaining 6 components (D) in turn 5.1-5.6 ) The method comprises the steps of carrying out a first treatment on the surface of the Will D 5.5 Separating the components by Sephadex LH-20 column chromatography (eluent chloroform-methanol is eluted according to the volume ratio of 1:1 and the like) to obtain a compound 4; will D 5.3 Separating the components by Sephadex LH-20 column chromatography (eluting solvent is methanol) to obtain compound 8;
separating n-butanol fraction extract (E) by AB-8 macroporous adsorbent resin column chromatography (eluent water-methanol gradient elution according to volume ratio of 1:0, 3:4, 2:1, 1:4 and 0:1), and sequentially obtaining 4 components (E) 1-4 ) The method comprises the steps of carrying out a first treatment on the surface of the Will E 2 The components are separated by column chromatography of silica gel (300-400 meshes) (eluent chloroform-methanol according to the volume ratio of 20:1, 15:1, 10:1, 5:1 and 01 gradient elution) to finally obtain 5 components (E) 2.1-2.5 ) The method comprises the steps of carrying out a first treatment on the surface of the Will E 2.5 The components are separated by Sephadex LH-20 column chromatography (eluting solvent is methanol) to obtain the compound 1.
The spectrum data of the compounds 1 to 8 are specifically as follows:
compound 1,4' -O- (2, 3-hydroxy-benzoyl) loganin, yellow oil, molecular formula: c (C) 24 H 30 O 13 。HREIMS m/z 549.1859[M+Na] + (calcd549.1854)。
The compound 2,4'-O- (2, 3-dihydroxybenzoyl) -2',3',6' -triacylglutannin, white powder, molecular formula: c (C) 30 H 36 O 16 。HREIMS m/z 675.1898[M+Na] + (calcd 675.1896)。
(c 0.27,CHCl 3 ),IR(KBr)ν max 3493,2955,1757,1640,1373,1233,1075,901,750cm -1 ；UV(CHCl 3 )λ max 252,324nm。
Compound 3,2',3',6'-triacetyl-4' -O- (3-hydroxy-2-O- β -D-glucopyranosyl-benzoyl) loganin, white powder, molecular formula: c (C) 36 H 46 O 21 。HREIMS m/z 837.2431[M+Na] + (calcd 837.2424)。
(c 0.17,CH 3 OH),IR(KBr)ν max 3410,2932,1755,1627,1283,1234,1075,896,748cm -1 ；UV(CH 3 OH)λ max 214,218nm。
The compound 4,2',3',6'-triacetyl-4' -O- (3-hydroxy-2-O- α -D-glucopyranosylbenzoyl) loganin, white powder, molecular formula: c (C) 36 H 46 O 21 。HREIMS m/z 837.2424[M+Na] + (calcd 837.2424)。
(c 0.38,DMSO),IR(KBr)ν max 3406,2929,1753,1640,1464,1234,1075,900,789cm -1 ；UV(CH 3 OH)λ max 257,292nm。
Compound 5,2',3',6'-triacetyl-4' -O- (2-hydroxy-3-O- β -D-glucopyranosyl-benzoyl) loganin, white powder, molecular formula: c (C) 36 H 46 O 21 。HREIMS m/z 837.2426[M+Na] + (calcd 837.2424)。
(c 0.26,CH 3 OH),IR(KBr)ν max 3410,2932,1753,1638,1375,1234,1075,900,755cm -1 ；UV(CH 3 OH)λ max 217,220nm。
Compound 6,7,2',3',6'-tetraacetyl-4' -O- (3-hydroxy-2-O- β -D-glucopyranosylbenzoyl) loganin, white powder, molecular formula: c (C) 38 H 48 O 22 。HREIMS m/z 837.2424[M+Na] + (calcd 837.2424)。
(c 0.55,CH 3 OH),IR(KBr)ν max 3431,2934,1756,1633,1235,1070,901,758cm -1 ；UV(CH 3 OH)λ max 214,215nm。
Compound 7,4'-O- (2, 3-dihydroxybenzoyl) -2',3',6' -triacetylloganinyl pentacosanoate, colorless oil, molecular formula: c (C) 55 H 84 O 17 。HREIMS m/z 837.2424[M+Na] + (calcd 837.2423)。
(c 1.2,CHCl 3 ),IR(KBr)ν max 3410,2932,1725,1635,1466,1243,1072,756cm -1 ；UV(CHCl 3 )λ max 245,316nm。
Compound 8,2',6' -diacetyl-4' -O- (3-hydroxy-2-O-beta-D-glucopyranosyl-benzoyl) -loganin, pale yellow powderThe molecular formula: c (C) 34 H 44 O 20 。HREIMS m/z 795.2322[M+Na] + (calcd 795.2318)。
(c 0.43,CH 3 OH),IR(KBr)ν max 3410,2956,1636,1468,1289,1236,1075,898,758cm -1 ；UV(CH 3 OH)λ max 216nm。/>
Compounds 1 to 8 13 C-NMR 1 The H-NMR data are shown in tables 1 to 2:
TABLE 1 Compounds 1 to 8 13 C-NMR data
/>
a Recorded at 400MHz in CD 3 OD. b Recorded at 400MHz in CDCl 3 . c Recorded at 400MHz in DMSO-d6。
TABLE 2 Compounds 1 to 8 1 H-NMR data
/>
a Recorded at 400MHz in CD 3 OD. b Recorded at 400MHz in CDCl 3 . c Recorded at 400MHz inDMSO-d6。
Example 2
Cell viability experiments were performed on APP/PS1 double-transgenic CHO cells with compounds 1, 5-7 of example 1, wherein the relevant experimental principles, methods and results are as follows:
1. experimental principle: succinate dehydrogenase in the mitochondria of living cells reduces exogenous MTT to water insoluble blue-violet crystalline Formazan (Formazan) and deposits in cells, whereas dead cells do not. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and the light absorption value can be measured at 570nm wavelength by an enzyme-linked immunosorbent assay, so that the number of living cells can be indirectly reflected.
The Ellman method is to hydrolyze thiocholine iodide substrate by cholinesterase to generate free sulfhydryl, and colorless 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB) is reduced to yellow 5-mercapto-2-nitrobenzoic acid in the presence of sulfhydryl. Since 5-mercapto-2-nitrobenzoic acid has a maximum absorbance at 415nm, compounds can be screened for acetylcholinesterase (AchE) activity by detecting absorbance at 415 nm.
2. The experimental method comprises the following steps:
1. sample to be tested: compounds 1, 5 to 7 of example 1
2. And (3) cells: APP/PS1 double transfer CHO cell line was purchased from Shanghai Saiqi BioCo., ltd
3. The experimental reagents and instruments are shown in tables 3 to 4
TABLE 3 Experimental reagents
Table 4 Experimental apparatus
4. Cell resuscitating and culturing:
taking out frozen APP/PS1 double-transfer CHO cell line in liquid nitrogen, shaking in water bath at 37deg.C for 1min to dissolve, sterilizing the outer wall of the frozen tube with 75% alcohol, placing in a super clean bench, transferring into 15mL centrifuge tube with 1000 μl pipette, centrifuging at 1000r/min for 5min, discarding supernatant, repeatedly blowing suspension cells with complete culture medium, transferring cell suspension into 9cm culture dish, placing in constant temperature incubator (5% CO) 2 Cultured at 37 ℃) until the cells confluent to 80% for passaging.
5. Sample processing and preparation of culture medium
(1) Sample treatment:
the test sample and the control drug were added to dimethyl sulfoxide to give a concentration of 1mg/mL (100. Mu.g/. Mu.L), and the mother solutions were diluted with DMEM medium to give final concentrations: 100nmol/L, 10nmol/L, 1nmol/L, 0.1nmol/L and 0.01nmol/L for standby.
(2) Preparing a culture medium:
the DMEM culture medium, the fetal calf serum and the double antibody mixed solution are respectively added into a culture bottle according to the proportion of 89%, 10% and 1%, and are sealed, stored at 4 ℃ and cultured to obtain PS1/APP double-transfer CHO cells.
6. The specific operation steps are as follows:
(1) Effect of compounds 1, 5-7 on extracellular fluids aβ40 and aβ42 content: when PS1/APP double-transfer CHO cells in a culture dish are observed to be 80% -90% in advance by a microscope, the culture solution is poured out, and the culture solution is washed 3 times by 1% PBS. Adding about 2mL pancreatin, stopping digestion when observed to increase cell gap under microscope, adding 10% DMEM medium, blowing with suction tube for 30 times, making into single cell suspension, and regulating cell number to 1×10 with cell culture solution 5 mL -1 Cell suspension 1mL was added to each well of a 12-well round-bottomed cell culture plate. After 12h of incubation, the culture broth was aspirated, rinsed 1 time with DMEM broth, and 1mL of DMEM broth and 111. Mu.L of each concentration of the liquid drug were added (the final concentrations of Compounds 1 and 5 were low-dose 1. Mu. Mol/L and high-dose 10. Mu. Mol/L; the final concentrations of Compounds 6 and 7 and positive drug were low-dose 0.1nmol/L, medium-dose 1nmol/L and high-dose 10 nmol/L). A blank control group and a positive control group are additionally arranged. Each group had 3 duplicate wells. ContinuingAfter 24h of culture, 500. Mu.L of culture solution was taken per well, centrifuged, and the supernatant was taken and the extracellular Abeta 40 and Abeta 42 contents were determined according to the procedure of ELISA kit instructions.
(2) In order to study whether the compound provided by the invention influences the production of Abeta by inhibiting the key enzyme BACE1 in the APP shearing process, ELISA kits are used for detecting the influence of the compound with different concentrations on the BACE1 expression level in cell supernatant after treating PS1/APP CHO cells, in particular to study the influence of the compound 6 on the content of extracellular fluid BACE 1: when PS1/APP double-transfer CHO cells in the culture dish are observed to be 80% -90% in a microscopic washing mode, the culture solution is poured out, and the culture solution is washed 3 times by 1% PBS. Adding about 2mL pancreatin, stopping digestion when observed to increase cell gap under microscope, adding 10% DMEM medium, blowing with suction tube for 30 times, making into single cell suspension, and regulating cell number to 1×10 with cell culture solution 5 mL -1 Cell suspension 1mL was added to each well of a 12-well round-bottomed cell culture plate. After 12 hours of incubation, the culture solution was aspirated, washed 1 time with DMEM medium, and 1mL of DMEM medium and 111. Mu.L of each concentration of the liquid medicine were added (the final concentrations of Compound 6 and the positive drug were 100nmol/L, 10nmol/L, 1nmol/L, 0.1nmol/L, and 0.01 nmol/L), and a blank control group and a positive control group were additionally provided. Each group had 3 duplicate wells. After further incubation for 24h, 500. Mu.L of culture broth was taken per well and the content of BACE1 in the extracellular fluid was measured according to the procedure of ELISA kit instructions.
(3) The primary enzyme that degrades aβ protein is MME, whose gene deficiency can lead to the occurrence of AD. Therefore, whether the compound provided by the invention affects the level of Abeta by affecting the MME or not is studied by Western-blot, in particular, the influence of the compound 6 on Abeta degrading enzyme MME is studied: the cell culture medium was discarded and washed 3 times with 4℃pre-chilled PBS for 1min each. Each well (6-well plate) was added with 0.2mL of RIPA lysate (containing PMSF, protease inhibitor, phosphatase inhibitor, etc.), and lysed on ice for 20min. The cells were scraped off with a cell scraper, transferred to a 1mL centrifuge tube pre-chilled on ice, and lysed on a shaker for 20min. After centrifugation at 15000r/min and 4℃for 30min, the supernatant was collected and glass plates were used to prepare different concentrations of separation gel (according to different target protein molecular weights) and 5% concentration gel according to the usual laboratory procedure. After adding the medicinal liquid of each concentration (the final concentration of the compound 6 and the positive medicine is 100nmol/L, 10nmol/L, 1nmol/L, 0.1nmol/L and 0.01 nmol/L), the solution is subjected to constant-pressure electrophoresis at 60V for 30min, and after the protein is electrophoresed to the separation gel, the voltage is increased to 90V until the electrophoresis is finished. The NC membrane was removed and incubated overnight at 220mA constant flow membrane 120min, 5% BSA in TBST at room temperature for 1h or 4 ℃. Inside the antibody cassette, the NC membrane that has been blocked is added to the primary antibody and incubated overnight at 4 ℃. After incubation, the incubation was performed 3 times with TBST on a shaker at room temperature for 5min each. Secondary antibody (diluted 1:5000) was added and incubated for 2h at room temperature, and washed 3 times with TBST 5min each on a shaker at room temperature. Putting the mixture into a Tanon gel imager, adding ECL luminous solution, adjusting the exposure time according to the black and white degree of the protein strips, and taking a picture. And calculating the relative content of each group of target proteins in the experiment by taking the reference protein as a reference.
7. Tacrine and HuperineA are selected as positive control medicines, and an Ellman method is used for detecting the direct inhibition of the compound on the activity of acetylcholinesterase in vitro.
Positive control: (1) Tacrine: acetylcholinesterase with IC50=42.3 nM (Electrophorus electricus) (Santa Cruz), IC50=193.6 nM (electric heel, acta Pharmaceutica Sinica 2012,47 (7): 916-921), IC50=333 nM (ratbrainAChE, J.Med.Chem.2002,45, 2277-2282), IC50=125 nM (ratbranAChE,
neuropharmacology 38 (1999) 181-193, IC50 = 220nM (electric el, neuropharmacology journal, vol.1No.3 june.2011).
(2) HuperineA (stronger than Tacrine): actylcholinesenterase (G4 form), IC50:7nM (Ki), i50=220 nM (electric heel, neuropharmacology journal, vol.1No.3 june.2011).
3. Experimental results:
1. compound 1 and compound 5 inhibited aβ40, aβ42 and AchE activity results (see table 5):
TABLE 5 results of inhibition of Abeta 40, abeta 42 and inhibition of AchE activity by Compounds 1 and 5
2. Compound 6 and compound 7 inhibited aβ40, aβ42 and AchE activity results (see table 6):
TABLE 6 results of inhibition of Abeta 40, abeta 42 and inhibition of AchE activity by Compounds 6 and 7
Remarks: ++ infinity represents that inhibition of aβ40 and aβ42 production is dose independent from the administration concentration, but showed some inhibitory activity in the range of the dosing concentration of the study.
FIG. 1 is a graph showing the results of inhibition of extracellular Abeta 40 and Abeta 42 by Compound 5, FIG. 2 is a graph showing the results of inhibition of extracellular Abeta 40 and Abeta 42 by Compound 6 (LL-48 represents Compound 6), and FIG. 3 is a graph showing the results of inhibition of extracellular Abeta 40 and Abeta 42 by Compound 7 (LL-69 represents Compound 7).
As can be seen from FIGS. 1 to 3 and tables 5 to 6, the IC50 value of compound 6 for inhibiting the production of Abeta 40 by APP/PS1 double-transferred CHO cells is 0.052+ -0.023 nM by performing cell experiments on APP/PS1 double-transferred CHO cells with compounds 1, 5 to 7; IC50 values for inhibiting the production of Abeta 42 by APP/PS1 double-transferred CHO cells were 1.52+ -0.95 nM. Compound 7 inhibited APP/PS1 double-transformed CHO cells from producing aβ42 with an IC50 value of 99.91±31.65. Among them, AZD3293 is a positive drug, and the IC50 values of the Aβ40 and Aβ42 which inhibit the generation of the Aβ40 and Aβ42 by APP/PS1 double-transfer CHO cells are 204+ -0.67 nM and 102.88 + -9.33 nM respectively. Therefore, the compound 6 has good effect of inhibiting the production of extracellular Abeta 40 and Abeta 42, the compound 7 has good effect of inhibiting the production of extracellular Abeta 42, and the effects of the two compounds are better than those of positive medicines, so that the compound can be used for preparing medicines for reducing Abeta 40 and Abeta 42, and the compound has potential effects of preventing and treating Alzheimer's disease.
Furthermore, at concentrations of 1 μm and 10 μm, the inhibition rate of compound 5 to inhibit production of aβ40 by APP/PS1 bi-transformed CHO cells was 15.49±4.55% and 69.95 ±5.27%, respectively; the inhibition rate to Abeta 42 is 49.42+/-2.80% and 67.57 +/-14.43% respectively, which shows that the compound has potential effects of preventing and treating Alzheimer disease.
The results of measuring the acetylcholinesterase activity of the compound 1 by an Ellman method show that the compound 1 has obvious effect of inhibiting the acetylcholinesterase activity, and the IC50 value is 15.0+/-2.7 mu M (wherein, the positive medicine is Huperzine A: 223.2+/-6.1 nM; tacrine: 388.2+/-2.3 nM), which indicates that the compound has potential anti-Alzheimer disease effect.
FIG. 4 is a graph showing the effect of Compound 6 on the content of BACE1 in extracellular fluid (LL-48 represents Compound 6), and it is clear from FIG. 4 that Compound 6 inhibits the secretion of Abeta in PS1/APP double transfer CHO cells by BACE 1.
FIG. 5 is a graph showing the effect of Compound 6 on Abeta degrading enzyme MME (LL-48 represents Compound 6), and it is clear from FIG. 5 that Compound 6 increases the expression level of MME protein in PS1/APP double-transferred CHO cells in a dose-dependent manner. It is shown that it accelerates the degradation of aβ by MME, thus reducing the amount of aβ secreted into extracellular fluid by PS1/APP bi-transferred CHO cells.
Example 3
Preparation of tablets: compound 1 was obtained by extraction as in example 1, and the excipient was added in a weight ratio of 1:7 to the excipient, and the mixture was formulated into tablets by conventional methods.
Example 4
Preparation of oral liquid: compound 5 was obtained by extraction as in example 1 and formulated into oral liquid according to conventional methods.
Example 5
Preparation of capsules: extracting to obtain compound 6 according to the method of example 1, adding excipient according to the weight ratio of 9:1, and making into capsule according to conventional method.
Example 6
Preparation of granules: extracting to obtain compound 7 according to the method of example 1, adding excipient according to the weight ratio of 3:1, and making into granule according to conventional method.
Example 7
Preparation of injection: the compound 1 and 5-7 are obtained by extraction according to the method of the example 1, and injection liquid is prepared by adding water for injection, fine filtering, filling and sterilizing according to the conventional method.
Example 8
Preparation of sterile powder injection: the compound 1, 5-7 is extracted according to the method of example 1, the corresponding salt is prepared by using organic acid, the compound is dissolved in sterile water for injection according to the conventional method, the solution is stirred to be dissolved, the solution is filtered by a sterile suction filter funnel, is subjected to sterile fine filtration, is packaged in an ampoule, is subjected to sterile sealing after low-temperature freeze drying, and is prepared into sterile powder injection.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. Loganin derivative iridoid compound has a structure shown in formula I:
r in formula I 1 ～R 6 The combinations of (a) are as follows:
compound 1R 6 ＝H R 1 ＝H R 2 ＝H R 3 ＝H R 4 ＝OH R 5 ＝OH
Compound 2R 6 ＝H R 1 ＝Ac R 2 ＝Ac R 3 ＝Ac R 4 ＝OH R 5 ＝OH
Compound 3R 6 ＝H R 1 ＝Ac R 2 ＝Ac R 3 ＝Ac R 4 ＝β-D-Glu R 5 ＝OH
Compound 4R 6 ＝H R 1 ＝Ac R 2 ＝Ac R 3 ＝Ac R 4 ＝α-D-Glu R 5 ＝OH
Compound 5R 6 ＝H R 1 ＝Ac R 2 ＝Ac R 3 ＝Ac R 4 ＝OH R 5 ＝β-D-Glu
Compound 6R 6 ＝Ac R 1 ＝Ac R 2 ＝Ac R 3 ＝Ac R 4 ＝β-D-Glu R 5 ＝OH
Compound 7R 6 ＝Ac R 1 ＝Ac R 2 ＝Ac R 3 ＝Ac R 4 ＝OH R 5 ＝β-D-Glu
Compound 8R 6 ＝H R 1 ＝Ac R 2 ＝H R 3 ＝Ac R 4 ＝β-D-Glu R 5 ＝OH。
2. The extraction method of the loganin derivative iridoid compound as claimed in claim 1, comprising the following steps:
(1) Extracting flowers of gentiana microphylla with an aqueous ethanol solution to obtain a total extract;
(2) Concentrating the total extract under reduced pressure until no alcohol smell exists, sequentially extracting the obtained extract water suspension with petroleum ether, chloroform and n-butanol, concentrating under reduced pressure, and collecting chloroform extract and n-butanol extract;
(3) Subjecting chloroform-methanol as eluent to silica gel column chromatography under gradient elution to obtain 5 components, denoted as D 1-5 The method comprises the steps of carrying out a first treatment on the surface of the The gradient elution comprises five gradients which are sequentially carried out, wherein the composition of the eluent in each gradient is 1: 0. 3: 4. 2: 1. 1:4 and 0:1, a step of;
will D 2 Subjecting the components to column chromatography separation to obtain a compound 3;
using chloroform-methanol as eluent, and subjecting D to gradient elution 3 Separating the components by silica gel column chromatography to obtain 5 components, which are marked as D 3.1-3.5 The method comprises the steps of carrying out a first treatment on the surface of the Chloroform-methanol is used as eluent, D is eluted under the condition of equal gradient 3.2 Subjecting the components to SephadexLH-20 column chromatography to obtain 3 components, denoted as D 3.2.1-3.2.3 The method comprises the steps of carrying out a first treatment on the surface of the Chloroform-methanol is used as eluent, D is eluted under the condition of equal gradient 3.2.2 Subjecting the components to column chromatography separation to obtain a compound 2 and a compound 7 in sequence;
using water-methanol as eluent, and under gradient elution condition, D 4 Subjecting the components to MCI column chromatography to obtain 5 components in turn, denoted as D 4.1-4.5 The method comprises the steps of carrying out a first treatment on the surface of the Using chloroform-methanol as eluent, and subjecting D to gradient elution 4.5 Subjecting the components to silica gel column chromatography to obtain 5 in turnThe individual components, denoted as D 4.5.1-4.5.5 The method comprises the steps of carrying out a first treatment on the surface of the Will D 4.5.1 Subjecting the components to column chromatography separation, and recrystallizing the obtained fraction to obtain a compound 5; will D 4.5.2 Subjecting the components to column chromatography separation, and recrystallizing the obtained fraction to obtain a compound 6;
using chloroform-methanol as eluent, and subjecting D to gradient elution 5 Separating the components by silica gel column chromatography to obtain 6 components, which are marked as D 5.1-5.6 The method comprises the steps of carrying out a first treatment on the surface of the Will D 5.3 Subjecting the components to column chromatography separation to obtain a compound 8; will D 5.5 Subjecting the components to column chromatography separation to obtain a compound 4;
(4) Subjecting the n-butanol fraction extract to AB-8 macroporous adsorbent resin column chromatography under gradient elution with water-methanol as eluent to obtain 4 components, denoted as E 1-4 ；
Using chloroform-methanol as eluent, and subjecting E to gradient elution 2 Subjecting the components to silica gel column chromatography to obtain 5 components, denoted as E 2.1-2.5 The method comprises the steps of carrying out a first treatment on the surface of the Will E 2.5 Subjecting the components to column chromatography separation to obtain a compound 1;
the step (3) and the step (4) are defined in no time sequence.
3. The extraction method according to claim 2, wherein the volume ratio of petroleum ether, chloroform and n-butanol for extraction to the material to be extracted is independently (2 to 6): 1.
4. the extraction method according to claim 2, wherein the D is 2 The method for separating the components by column chromatography comprises the following steps:
petroleum ether-acetone is used as eluent, D is carried out under the gradient elution condition 2 Separating the components by silica gel column chromatography to obtain 5 components, which are marked as D 2.1-2.5 ；
Chloroform-methanol is used as eluent, D is eluted under the condition of equal gradient 2.3 Separating the components by Sephadex LH-20 column chromatography to obtain 3 components, denoted as D 2.3.1-2.3.3 ；
Methanol is used as eluent to carry out D 2.3.3 The component is subjected to Sephadex LH-20 column chromatography separation to obtain the compound 3.
5. The extraction method according to claim 2, wherein in extracting the compound 5, in particular with methanol as eluent, D 4.5.1 Subjecting the components to Sephadex LH-20 column chromatography, and recrystallizing the obtained fraction in methanol.
6. The extraction method according to claim 2, wherein in extracting the compound 6, in particular with methanol as eluent, D 4.5.2 Subjecting the components to SephadexLH-20 column chromatography, and recrystallizing the obtained fraction in methanol.
7. The extraction method according to claim 2, wherein in extracting the compound 8, in particular with methanol as eluent, D 5.3 The components are subjected to Sephadex LH-20 column chromatography separation.
8. The extraction method according to claim 2, wherein in extracting the compound 4, in particular chloroform-methanol is used as eluent, D 5.5 The components are subjected to Sephadex LH-20 column chromatography separation.
9. The application of loganin derivative iridoid compounds or pharmaceutically acceptable salts thereof in preparing anti-Alzheimer disease drugs, wherein the loganin derivative iridoid compounds are the loganin derivative iridoid compounds of claim 1 or the loganin derivative iridoid compounds extracted by the extraction method of any one of claims 2-8.
10. The use according to claim 9, wherein the anti-alzheimer's disease agent comprises at least one of compound 1, compound 5, compound 6, compound 7, a pharmaceutically acceptable salt of compound 1, a pharmaceutically acceptable salt of compound 5, a pharmaceutically acceptable salt of compound 6, and a pharmaceutically acceptable salt of compound 7.
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