CN109942649A - A kind of indoles glycosides compound and its purification methods and uses - Google Patents
A kind of indoles glycosides compound and its purification methods and uses Download PDFInfo
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- CN109942649A CN109942649A CN201910359489.4A CN201910359489A CN109942649A CN 109942649 A CN109942649 A CN 109942649A CN 201910359489 A CN201910359489 A CN 201910359489A CN 109942649 A CN109942649 A CN 109942649A
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- -1 indoles glycosides compound Chemical class 0.000 title claims abstract description 59
- 229930182470 glycoside Natural products 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title abstract description 17
- 238000000746 purification Methods 0.000 title abstract description 4
- 239000000284 extract Substances 0.000 claims abstract description 48
- 241001517312 Calanthe Species 0.000 claims abstract description 25
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 63
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 48
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 41
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 36
- 238000000605 extraction Methods 0.000 claims description 34
- 238000010828 elution Methods 0.000 claims description 30
- 239000010410 layer Substances 0.000 claims description 29
- 239000003814 drug Substances 0.000 claims description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 26
- 235000013402 health food Nutrition 0.000 claims description 25
- 239000002904 solvent Substances 0.000 claims description 25
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 22
- 235000019441 ethanol Nutrition 0.000 claims description 18
- 238000000926 separation method Methods 0.000 claims description 18
- 238000010898 silica gel chromatography Methods 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 17
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 12
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- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 6
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- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
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- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a kind of indoles glycosides compound and its purification methods and uses, the present invention a kind of isolated indoles glycosides compound, molecular formula C from the rhizome of plant tasselled calanthe for the first time24H32N2O12；Its structure, which is one group, has chiral isomers.Special hydrophilic chromatographic column is used in the present invention, it can be isolated and purified to obtain well, has method simple and effective, the high feature of obtained compound purity.The present invention can extract from tasselled calanthe dry rhizome and isolate indoles glycosides compound, which can promote cell that can resist hepatitis, anti-inflammatory and anti-gastric-ulcer to the intake and conversion of glucose.
Description
Technical field
The invention belongs to medicine and field of health care food, be related to a kind of indoles glycosides compound and its extraction separation method and
Using.
Background technique
Very big change has occurred along with the industrialization and life modernization, people's lives mode in the whole world.Due to life
The flat raising of running water, the change of dietary structure, the rhythm of life being becoming tight day and much few more dynamic life styles sat etc. it is many because
Element, global diabetes morbidity rapid development, diabetes have become the third-largest after tumour, cardiovascular pathological changes seriously threaten
The chronic disease of human health.For whole world diabetic more than 1.2 hundred million people, China patients occupy the second in the world at present, because
The research and development of this diabetes medicament are very urgent.The disadvantages of drug of clinical treatment diabetes is big there is toxic side effect at present,
And the advantages such as natural botanical medicine is widely distributed with its, and material is easy to get and cheap, and toxic side effect is small are valued by people,
It has broad application prospects.
Orchid family (Orchidaceae) Calanthe (Calanthe) plant tasselled calanthe (Calanthe fimbriata
Franch even, the place of production includes Shaanxi, Gansu, Yunnan, Sichuan, Tibet and platform for pseudobulb and herb), alias meat lotus ring or nine sons
Also there are the distribution of the plant in the provinces and regions such as gulf in Sillim and Japan.It is frequently grown the mountainous region hayashishita and Cao Po in 1500-3500 meters of height above sea level
On.Its is lightly seasoned, micro-pungent, hardship, cool in nature.It is slightly poisonous.Return stomach, liver, lung channel.With clearing heat and detoxicating, swelling and pain relieving, stasis of blood and other effects is dissipated.
Currently, focusing primarily upon ornamental value, cultivation technique to the research of entire Calanthe plant both at home and abroad and educating
Kind of method etc., and to the research of its effective component and pharmacological activity then rare report.
Summary of the invention
In order to overcome the disadvantages of the above prior art, the purpose of the present invention is to provide a kind of indoles glycosides compound and its
Purification methods and uses can extract from tasselled calanthe and isolate indoles glycosides compound, which has anti-
Hyperglycemia, anti-hepatitis, anti-inflammatory and gastric mucosal protective effect.
In order to achieve the above object, the present invention is achieved by the following scheme:
A kind of indoles glycosides compound disclosed by the invention, the molecular formula of the indoles glycosides compound are C24H32N2O12；
Its structural formula is as shown in following formula 1, formula 2:
Formula 1 and formula 2 chiral isomer each other.
The invention also discloses the extraction separation methods of above-mentioned indoles glycosides compound, comprising the following steps:
1) tasselled calanthe dry rhizome is taken, several times, extracting solution is concentrated under reduced pressure combined extract refluxing extraction, obtains
To total medicinal extract or concentrate；
2) total medicinal extract is suspended in water, obtains medicinal extract liquid, by medicinal extract liquid or concentrate successively through petroleum ether, ethyl acetate
Organic layer is isolated afterwards several times with extracting n-butyl alcohol, and solvent is removed under reduced pressure and obtains each extract layer；
3) extracting n-butyl alcohol layer is splined on silicagel column, using chloroform-methanol system as eluent, by volume (100:
0) gradient elution~(0:100) is carried out, efflux is detected, by effluent volume than the fraction merging for 7:3, is removed molten
Agent obtains crossing post part for the first time；
4) first time is crossed into post part and is splined on reversed-phase silica gel chromatography column, using methanol-water system as eluent, by volume
Gradient elution is carried out than (20:80)~(100:0), by effluent volume than the fraction merging for 35:65, solvent is removed, obtains
Post part is crossed for the second time；
5) post part will be crossed for second and is splined on reversed-phase silica gel chromatography column again, using methanol-water system as eluent, by body
Product carries out gradient elution than (5:95)~(100:0), by effluent volume than the fraction merging for 20:80, removes solvent, obtains
Third time crosses post part；
6) third time is crossed into post part and is splined on high performance liquid chromatography separation column, carried out isocratic elution with mobile phase, obtain
Indoles glycosides compound.
Preferably, in step 1), refluxing extraction number is 1~6 time, every time 1~3h；
For ethyl alcohol in refluxing extraction using methanol, water or volume fraction 10~95% as extractant, tasselled calanthe is dry
The mass volume ratio of dry rhizome and extractant is 1kg:(1~5) L；When the second that extractant is methanol or volume fraction 10~95%
When alcohol, solvent recovery in obtained extracting solution is obtained into total medicinal extract；When extractant is water, by the volume concentration of extracting solution to 1/
10~1/6, obtain concentrate.
Preferably, in step 2), total medicinal extract is suspended in water, the volume ratio of total medicinal extract and water is 1:1~1:4；
When extraction, successively after petroleum ether and ethyl acetate extraction, using extracting n-butyl alcohol；Extraction is to wait bodies every time
Product extraction；Every kind of solvent extracts 1~6 time respectively.
Preferably, every 300~800mL collects a fraction after gradient elution in step 3)；In step 4) after gradient elution
Every 100~300mL collects a fraction；Every 100~300mL collects a fraction after gradient elution in step 5).
Preferably, in step 6), the flow velocity of isocratic elution is 3~5mL/min；
Mobile phase selects methanol-water system or acetonitrile-water system, and the volume ratio of first alcohol and water is 25 in methanol-water system:
75；The volume ratio of acetonitrile and water is 35:65 in acetonitrile-water system.
The invention also discloses above-mentioned indoles glycosides compounds in preparing antidiabetic medicine and/or health food
Using.
The invention also discloses above-mentioned indoles glycosides compounds to prepare answering in anti-inflammatory drug and/or health food
With.
The invention also discloses above-mentioned indoles glycosides compounds to prepare answering in Antihepatitis medicament and/or health food
With.
The invention also discloses above-mentioned indoles glycosides compounds in preparing gastric mucosal protection drug and/or health food
Application.
Compared with prior art, the invention has the following advantages:
The present invention a kind of isolated indoles glycosides compound, molecule from the rhizome of plant tasselled calanthe for the first time
Formula is C24H32N2O12；Its structure, which is one group, has chiral isomers.
Indoles glycoside compound activity obtained in the present invention is preferable, but the content in plant is not high, and polarity is larger,
It is difficult to be enriched with conventional chromatographic process and isolated.Special hydrophilic chromatographic column is used in the present invention, it can be by it well
It isolates and purifies to obtain, has method simple and effective, the high feature of obtained compound purity.
The accumulation of the triglycerides of indoles glycoside compound on fatty cell in the present invention has facilitation, and having will be thin
Extracellular glucose intake cellular transformation is the facilitation of triglycerides, tests prove that, the compounds of this invention at 3 μM just
There is conspicuousness facilitation, there is concentration-dependent relation, and the compound has the characteristics that efficient, low toxicity, be expected to exploitation at new
Antidiabetic medicine, or be used to prepare with prevent and treat diabetes effect health food.
Indoles glycosides compound in the present invention has anti-inflammatory effect, tests prove that, to mouse RAW264.7 small cell
With good anti-inflammatory effect, which has the characteristics that efficient, low toxicity, is expected to develop into new anti-inflammatory drug, Huo Zheyong
There is the health food for preventing and treating inflammation in preparation.
Indoles glycosides compound in the present invention has antihepatitic activity, tests prove that, HepG2.2.15 cell is produced
Raw HBsAg and HBeAg has good inhibiting effect, which has the characteristics that efficient, low toxicity, be expected to develop into new
Antihepatitis medicament, or be used to prepare with the health food for preventing and treating hepatitis.
Indoles glycosides compound in the present invention has gastric mucosal protective effect, tests prove that, to alcohol induced small
Mouse gastric ulcer has certain resistant function, which has the characteristics that efficient, low toxicity, be expected to develop into new anti-gastric-ulcer
Drug, or be used to prepare with the health food for preventing and treating gastric ulcer.
Detailed description of the invention
Fig. 1 is the compounds of this invention 11H-NMR map；
Fig. 2 is the compounds of this invention 113C-NMR map；
Fig. 3 is the DEPT map of the compounds of this invention 1；
Fig. 4 is the compounds of this invention 11H-1H COSY map；
Fig. 5 is the HSQC map of the compounds of this invention 1；
Fig. 6 is the HMBC map of the compounds of this invention 1；
Fig. 7 is the HR-ESI-MS map of the compounds of this invention 1.
Fig. 8 is the compounds of this invention 21H-NMR map；
Fig. 9 is the compounds of this invention 213C-NMR map；
Figure 10 is the DEPT map of the compounds of this invention 2；
Figure 11 is the compounds of this invention 21H-1H COSY map；
Figure 12 is the HSQC map of the compounds of this invention 2；
Figure 13 is the HMBC map of the compounds of this invention 2；
Figure 14 is the HR-ESI-MS map of the compounds of this invention 2.
Specific embodiment
In order to enable those skilled in the art to better understand the solution of the present invention, below in conjunction in the embodiment of the present invention
Attached drawing, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is only
The embodiment of a part of the invention, instead of all the embodiments.Based on the embodiments of the present invention, ordinary skill people
The model that the present invention protects all should belong in member's every other embodiment obtained without making creative work
It encloses.
It should be noted that description and claims of this specification and term " first " in above-mentioned attached drawing, "
Two " etc. be to be used to distinguish similar objects, without being used to describe a particular order or precedence order.It should be understood that using in this way
Data be interchangeable under appropriate circumstances, so as to the embodiment of the present invention described herein can in addition to illustrating herein or
Sequence other than those of description is implemented.In addition, term " includes " and " having " and their any deformation, it is intended that cover
Cover it is non-exclusive include, for example, the process, method, system, product or equipment for containing a series of steps or units are not necessarily limited to
Step or unit those of is clearly listed, but may include be not clearly listed or for these process, methods, product
Or other step or units that equipment is intrinsic.
The invention will be described in further detail with reference to the accompanying drawing:
The method disclosed by the invention for extracting indoles glycosides compound from tasselled calanthe, comprising the following steps:
1) the tasselled calanthe dry rhizome for taking certain mass (kg) is tasselled calanthe dry root quality 1~5 with volume
The methanol of amount, ethyl alcohol or water (L) heating and refluxing extraction 1~6 time near respective boiling point that volume fraction is 10~95% again, often
Secondary 1~3 hour, when extractant is the ethyl alcohol of methanol or volume fraction 10~95%, it is molten that removing is recovered under reduced pressure in combined extract
Agent obtains total medicinal extract, and when extractant is water, combined extract and by its volume concentration to 1/10 to ten/6ths is obtained
To concentrate；
2) total medicinal extract is suspended in water, the volume ratio of total medicinal extract and water is 1:1~1:4, obtain medicinal extract liquid, medicinal extract liquid or
Concentrate is successively extracted with isometric organic solvent respectively, wherein with petroleum ether to medicinal extract liquid or concentration when extracting for the first time
Liquid carries out equal-volume extraction, and extraction is to isolate last organic layer extracted every time later, by remaining water layer with having
Solvent is extracted next time in equal volume；Every kind of solvent extracts 1~6 time, combining extraction liquid, and normal pressure or vacuum distillation remove
Organic solvent respectively obtains each extract layer and water layer.Organic solvent includes petroleum ether, ethyl acetate and n-butanol etc., and is extracted
Order is solvent first small with polarity, then the organic solvent big with polarity, petroleum ether and ethyl acetate can save.
3) extracting n-butyl alcohol layer is taken, by using separation methods such as column chromatographic purifyings, obtains indoles glycoside of the invention
Close object.
Column chromatography includes following three phases:
First stage: extracting n-butyl alcohol layer is splined on silicagel column, using chloroform-methanol system as eluent, by volume
Gradient elution is carried out than (100:0)~(0:100), every 300~800mL collects a fraction, TLC detection is carried out to efflux,
Merge identical fraction, 15 fractions are obtained, is respectively designated as FrB1-FrB15, normal pressure or evaporated under reduced pressure solvent, takes therein
FrB12 fraction, i.e. effluent volume cross post part than the fraction for 7:3, as first time；
Second stage: crossing post part for first time, be splined on reversed-phase silica gel chromatography column, using methanol-water system as elution
Liquid, (20:80)~(100:0) carries out gradient elution by volume, and every 100~300mL collects a fraction, carries out to efflux
TLC detection, merges identical fraction, solvent is removed under reduced pressure, obtain 12 fractions, be respectively designated as FrB12.1-FrB12.12, will
FrB12.2 fraction, i.e. effluent volume cross post part than the fraction for 35:65, as second；
Phase III: post part will be crossed second, and will be splined on reversed-phase silica gel chromatography column, using methanol-water system as elution
Liquid, (5:95)~(100:0) carries out gradient elution by volume, and every 100~300mL collects a fraction, carries out to efflux
TLC detection, merges identical fraction, solvent is removed under reduced pressure, obtain 16 fractions, be respectively designated as FrB12.2.1-
FrB12.2.16, by FrB12.2.13 fraction, i.e. effluent volume crosses post part as third time than the fraction for 20:80；
Fourth stage: third time is crossed into post part, is splined on high performance liquid chromatography separation column, isolated indoles glycoside
Close object.
Wherein, high performance liquid chromatography separation column is the Megres C18 column of Jiangsu Chinese nation, and high performance liquid chromatography separation is to use
Differential refraction detector carries out isocratic elution by 3~5mL/min using methanol-water system or acetonitrile-water system as mobile phase.
The volume ratio of first alcohol and water is 25:75 in methanol-water system；The volume ratio of acetonitrile and water is 35:65 in acetonitrile-water system.
The indoles glycosides compound that the above method is prepared, structural formula are as follows:
Indoles glycosides compound in the present invention is preparing the application in antidiabetic medicine and/or health food.
The present invention has found in the research for carrying out chemical component and pharmacological activity to tasselled calanthe, therefrom isolated
Indoles glycosides compound has the function of improving fat cell insulin sensitivity well, can remarkably promote fat cell and take the photograph
Enter glucose and be converted into triglycerides, therefore, is expected to be developed into the antidiabetic medicine with insulin resistance is improved
And/or health food.
Indoles glycosides compound in the present invention is preparing the application in anti-inflammatory drug and/or health food: demonstrate,proving through test
It is bright, there is good anti-inflammatory effect to mouse RAW264.7 small cell, which has the characteristics that efficient, low toxicity, therefore has
It hopes exploitation at new anti-inflammatory drug, or is used to prepare with the health food for preventing and treating inflammation.
Indoles glycosides compound in the present invention is preparing the application in Antihepatitis medicament and/or health food: through testing
It proves, the HBsAg and HBeAg generate to HepG2.2.15 cell has good inhibiting effect, which has efficient, low
The feature of poison, is expected to develop into new Antihepatitis medicament, or is used to prepare with the health food for preventing and treating hepatitis.
Indoles glycosides compound in the present invention is preparing the application in gastric mucosal protection drug and/or health food: warp
Test proves there is certain resistant function to alcohol induced mouse gastric ulcer, which has the spy of efficient low toxicity
Point, therefore be expected to develop into new medicament for anti-gastric ulcer, or be used to prepare with the health food for preventing and treating gastric ulcer.
Embodiment 1
1, the extraction and separation of indoles glycosides compound
1) tasselled calanthe dry rhizome 10kg is taken, the methanol for being 5 times of quality of tasselled calanthe dry rhizome with volume
Heating and refluxing extraction 4 times, 3 hours every time, solvent was recovered under reduced pressure in combined extract, obtained total medicinal extract；
2) total medicinal extract is suspended in 4 times of amount water, with petroleum ether equal-volume extraction 4 times, then through ethyl acetate and positive fourth
Alcohol equal-volume extraction 4 times, extract layer respectively obtains petroleum ether layer, chloroform layer, ethyl acetate layer, n-butanol after solvent is removed under reduced pressure
Layer.
3) extracting n-butyl alcohol layer 70g is taken, first by using silica gel column chromatography, chloroform/methanol is by volume (v/v)
100:0~0:100 carries out gradient elution, and every 500mL collects a fraction, is obtained 15 after TLC is examined and known the identical fraction of merging
A fraction (FrB1-FrB15).
4) the wherein inverted silica gel column chromatography separation of the 12nd fraction FrB12, uses MeOH/H2O is by volume (v/v)
100:0~0:100 carries out gradient elution, and every 200mL collects a fraction, obtains 12 after TLC is examined and known the identical fraction of merging
Fraction (FrB12.1-FrB12.12).
5) wherein the 2nd fraction FrB12.2 is splined on reversed-phase silica gel chromatography column, uses MeOH/H2O is by volume (v/v)
(5:95)~(100:0) carries out gradient elution, and every 200mL collects a fraction, after TLC detection inspection is known and merges identical fraction
To 16 fractions (FrB12.2.1-FrB12.2.16).
6) then FrB12.2.13 is purified with half preparative high-performance liquid chromatographic, and using Megres C18 column, mobile phase is first
Alcohol-water solution (25:75, v/v), flow velocity 3.0mL/min obtain 1 (t of compoundR=30min) and 2 (t of compoundR=
34min)。
The present invention passes through physicochemical constant and Modern spectroscopy technological means (HR-ESI-MS, 1D-NMR, 2D-NMR) identificationization
Close the structure of object, compound 1 are as follows:
(R)-5-(3-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-((((2R,3R,4S,5S,6R)-3,
4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)tetrah-
ydro-2H-pyran-2-yl)oxy)-1H-indol-2-yl)pyrrolidin-2-one；
Compound 2 are as follows:
(S)-5-(3-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-((((2R,3R,4S,5S,6R)-3,
4,5-trihydroxy-6-(hydroxymethyl)tetrahy-dro-2H-pyran-2-yl)oxy)methyl)
tetrahydro-2H-pyran-2-yl)oxy)-1H-indol-2-yl)pyrrolidin-2-one。
2, the Structural Identification of indoles glycosides compound
1) Structural Identification of noval chemical compound 2
The Structural Identification correlation map of compound 2 is referring to Fig. 8~Figure 14；
Compound 2 is colorless oil, [α]D=-11 (c 0.39, MeOH), HR-ESIMS show quasi-molecular ion peak
For [M+H]+at m/z 541.2035 (calcd541.2028), it is thus determined that molecular formula is C24H32N2O12.Fig. 8's and Fig. 91H-NMR and13In C-NMR, chemical shift δH7.69 (d, J=7.9Hz, 1H), 7.27 (d, J=8.1Hz, 1H), 7.08 (t, J
=7.6Hz, 1H), 6.99 (t, J=7.5Hz, 1H) and chemical shift δC135.10,134.07,128.91,123.06,
122.25,120.08,118.77,112.35 signal, which is shown, contains a 2,3- disubstituted indole structure in the compound.Two
A anomeric proton δH4.68 (d, J=7.6Hz, 1H), 4.30 (d, J=7.7Hz, 1H) and their corresponding carbon signal δC106.27,
104.83 show that there are two sugared units in molecule.With GC analysis shows this two molecular saccharides is glucose after sour water solution, and configuration
For beta comfiguration, this can also determine that from the coupling constant of anomeric proton, respectively 7.6 and 7.7Hz.In HMBC spectrum, outside glucose
Anomeric proton δH6 mesomethylene carbon δ of 4.30 (d, J=7.7Hz, 1H) and inside glucoseC69.45 is related, therefore outside Portugal
1 of grape sugar is connected in 6 of inside glucose.Meanwhile the anomeric proton δ of inside glucoseH4.68 (d, J=7.6Hz, 1H) with
Carbon δ on indole ringC134.07 (C-3) are related, show that sugar chain is connected in 3 of indole ring.?1H-1In H COSY, H-10 and H-
11 is related, and H-11 is related to H-12, and H-10, H-11 and H-12 be equal and δ in HMBC spectrumC181.03 (C-13) are related, then tie
It closes to contain in molecular formula and shows to contain a gamma-lactam in the molecule there are two nitrogen-atoms.Again because HMBC spectrum in H-10 with
C-2 is related, then shows that C-2 is connected with C-10.
Therefore compound 2 is accredited as:
(S)-5-(3-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-((((2R,3R,4S,5S,6R)-3,
4,5-tri-hydroxy-6-(hydroxymethyl)tetrahy-dro-2H-pyran-2-yl)oxy)methyl)
tetrahydro-2H-pyran-2-yl)oxy)-1H-indol-2-yl)pyrrolidin-2-one。
It is one and has no noval chemical compound reported in the literature.Its structural formula is as follows:
Nuclear magnetic data is shown in Table 1.
(2) Structural Identification of noval chemical compound 1
The Structural Identification correlation map of compound 1 is referring to FIG. 1 to FIG. 7；
Compound 1 is colorless oil, [α]D=+9 (c 0.14, MeOH), HR-ESIMS show quasi-molecular ion peak
For [M+H]+at m/z 541.2026 (calcd541.2028), it is thus determined that molecular formula is C24H32N2O12.Compound 11H-
NMR and13C-NMR and compound 2 it is closely similar, then by DEPT,1H–1H-COSY, HMQC and HMBC spectrum and optically-active discovery
Compound 1 and compound 2 are corresponding isomers, and specific analytic method is identical, determines compound 1 are as follows:
(R)-5-(3-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-((((2R,3R,4S,5S,6R)-3,
4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)
tetrahydro-2H-pyran-2-yl)oxy)-1H-indol-2-yl)pyrrolidin-2-one。
It is one and has no noval chemical compound reported in the literature.1 structural formula of compound is as follows:
Nuclear magnetic data is shown in Table 1.
Compound 1 and 2 in 1 present invention of table1H NMR and13C NMR data
3, fat cell fat of triglyceride accumulation experiment
Experimental method:
3T3-L1 cell (5.0 × 104Cells/well it) is inoculated in 48 orifice plates, after 24 hours, differential medium is added
(DMEM containing 10%FBS be (high glucose:4500mg/L, 1 μM of dexamethasone, the IBMX of 0.5mM and 5 μ g/mL's
Insulin) culture 3 days, then culture medium change into maintain culture medium (containing 10%FBS DMEM (high glucose:
The insulin of 4500mg/L and 5 μ g/mL) be further cultured for 2 days after, replace fresh maintenances culture medium and continue culture 2 days, at the 8th day, suction
Culture medium is removed, 200 μ L distilled water, ultrasonication, in triglycerides liquid reagent box measurement clasmatosis liquid is added in every hole
TG content.Sample is dissolved in DMSO, is added in replacement culture medium every time, wherein final concentration of the 0.1% of DMSO,
Troglitazone is used as positive reference compound.Numerical value is expressed as TG content value added MEAN ± SEM compared with the control group
(n=4) p < 0.01 .*p < 0.05, * * (compared with the control group).
Experimental result is shown in Table 2.
TG cumulative effect of 2. compound 1 of table to 3T3-L1 fat cell
Experimental result: it is in table 2 statistics indicate that, the present invention in compound 1 and 2 all have well promote fat cell
The effect for accumulating triglycerides, just has at 3 μM conspicuousness facilitation (p < 0.01), and with the increase of concentration, promotes
Effect also enhances therewith, implies that with concentration-dependent relation.
In conjunction with the above experiment and its experimental result, show that the indoles glycosides compound in the present invention has extremely strong improvement rouge
Fat cell promotes the intake and conversion to glucose to the sensibility of insulin, therefore being expected to exploitation is new antidiabetic
Object；Or it is used to prepare the health food for preventing and treating diabetes.
4, extracorporeal anti-inflammatory is tested
Experimental method:
By RAW264.7 cell inoculation in 96 orifice plates, LPS (lipopolysaccharides) is added afterwards for 24 hours in culture.Then different component is other
The sample (the compounds of this invention 1 and 2, positive drug Indomethacin) of various concentration is added, blank group is added isometric
vehicle.50 μ L cell culture fluids mix isometric Griess reagent I and are added in above-mentioned 96 orifice plate, are subsequently added into Griess examination
Agent II.Microplate reader measures OD value of each group sample under 546nm wavelength, calculates NO using following equation and generates inhibiting rate, is used in combination
SPSS software calculates the half-inhibitory concentration (IC of tested sample50Value, μM).
NO inhibiting rate (%)=[1-(sample sets/blank group)] × 100%.
Experimental result:
The cell RAW264.7 of 3 the compounds of this invention 1 and 2 pair LPS of table stimulation generates the inhibiting effect of NO
Interpretation of result: table 3 statistics indicate that, the present invention in compound 1 and 2 have good extracorporeal anti-inflammatory effect, it is right
Lipopolysaccharides LPS, which stimulates mouse RAW264.7 cell to generate NO, has good inhibiting effect, half-inhibitory concentration IC50Respectively
25.6 ± 1.3 μM, 30.8 ± 3.0 μM are superior to 39.8 ± 3.1 μM of positive control medicine Indomethacin.Illustrate in the present invention
Indoles glycosides compound 1 and 2 have preferable anti-inflammatory effect.
5, gastric mucosal protection is tested
The healthy Kunming male mice that weight is 20~25g or so is randomly divided into 9 groups, every group 8, i.e. blank control
Group, model control group, positive controls, 2 low, middle and high dose groups of 1 low, middle and high dose groups of compound and compound.Compound 1
Omeprazole 20mg/ is given with 2 each gastric infusion 20,40 of low, middle and high dose groups and 80mg/kg, positive controls stomach-filling
Kg, blank group and model group give isometric physiological saline.70% ethyl alcohol 0.1ml is given in stomach-filling after administration 1h, and blank group is given
The physiological saline of equivalent.It cuts open the belly after cervical dislocation puts to death mouse after 1h and quickly removes stomach, then fixed with 10% formaldehyde
10~15min is opened along big curved scissors and is completely flattened afterwards with normal saline flushing and blot moisture.Using vernier caliper to mouse
Ulcer area carries out accurate calculating: the calculating of ulcer index and inhibiting rate being unfolded by Guth standard.
Ulcer inhibition rate=(averaging model group UI- average experiment group UI)/averaging model group UI × 100%
Experimental result:
The inhibiting effect of the alcohol induced mouse gastric ulcer of 4 the compounds of this invention 1 and 2 pair of table
Interpretation of result: table 4 statistics indicate that, the present invention in compound 1 and 2 have certain anti-ulcer effect, it is right
Alcohol induced mouse gastric ulcer has certain resistant function, and wherein the anti-ulcer effect of compound 1 is better than compound 2
, illustrate that the indoles glycosides compound 1 and 2 in the present invention has certain anti-ulcer effect.
6. external anti-hepatitis experiment
HepG2.2.15 cell inoculation in 96 orifice plates, culture for 24 hours after different component not be added various concentration sample (
Invention compound 1 and 2, positive drug Lamivudine), isometric vehicle is added in blank group.Cell culture is collected in culture 6 days
Supernatant, uniformly detects HBsAg and HBeAg in supernatant with ELISA method, and after chromogenic reaction, microplate reader is read under 490nm
Absorbance value calculates 50% Drug inhibition concentration (IC50).
The inhibiting effect of 5 the compounds of this invention 1 and 2 pair HepG2.2.15 cell of table generation HBsAg and HBeAg
Interpretation of result: table 5 statistics indicate that, the compound 1 and 2 in the present invention has preferable external antihepatitic activity,
The HBsAg and HBeAg generate to HepG2.2.15 cell has good inhibiting effect, illustrates the indoles glycoside in the present invention
Closing object 1 and 2 has preferable antihepatitic activity.
Embodiment 2
1) tasselled calanthe dry rhizome 10kg is taken, with 95% that volume is 3 times of quality of tasselled calanthe dry rhizome
Ethyl alcohol heating and refluxing extraction 5 times, 2 hours every time, solvent was recovered under reduced pressure in combined extract, obtained total medicinal extract；
2) total medicinal extract is suspended in 3 times of amount water, with petroleum ether equal-volume extraction 4 times, then through chloroform, ethyl acetate and
N-butanol equal-volume extraction 4 times, extract layer, which is removed under reduced pressure after solvent, respectively obtains petroleum ether layer, chloroform layer, ethyl acetate layer, just
Butanol layer.
3) extracting n-butyl alcohol layer 70g is taken, first by using silica gel column chromatography, chloroform/methanol is by volume (v/v)
100:0~0:100 carries out gradient elution, and every 400mL collects a fraction, is obtained 15 after TLC is examined and known the identical fraction of merging
A fraction (FrB1-FrB15).
4) the wherein inverted silica gel column chromatography separation of the 12nd fraction FrB12, uses MeOH/H2O is by volume (v/v)
100:0~0:100 carries out gradient elution, and every 300mL collects a fraction, obtains 12 after TLC is examined and known the identical fraction of merging
Fraction (FrB12.1-FrB12.12).
5) wherein the 2nd fraction FrB12.2 is splined on reversed-phase silica gel chromatography column, uses MeOH/H2O is by volume (v/v)
(5:95)~(100:0) carries out gradient elution, and every 200mL collects a fraction, after TLC detection inspection is known and merges identical fraction
To 16 fractions (FrB12.2.1-FrB12.2.16).
6) then FrB12.2.13 is purified with half preparative high-performance liquid chromatographic, and using Megres C18 column, mobile phase is first
Alcohol-water solution (25:75, v/v), flow velocity 3.0mL/min obtain 1 (t of compoundR=30min) and 2 (t of compoundR=
34min)。
Embodiment 3
1) tasselled calanthe dry rhizome 10kg is taken, is added with the water that volume is 8 times of quality of tasselled calanthe dry rhizome
Circumfluence distillation 3 times, 2 hours every time, solvent was recovered under reduced pressure in combined extract, obtained total medicinal extract；
2) total medicinal extract is suspended in 1 times of amount water, with ethyl acetate equal-volume extraction 4 times, then again with bodies such as n-butanols
Product extraction 4 times, extract layer respectively obtains ethyl acetate layer, n-butanol layer after solvent is removed under reduced pressure.
3) extracting n-butyl alcohol layer 70g is taken, first by using silica gel column chromatography, chloroform/methanol is by volume (v/v)
100:0~0:100 carries out gradient elution, and every 600mL collects a fraction, is obtained 15 after TLC is examined and known the identical fraction of merging
A fraction (FrB1-FrB15).
4) the wherein inverted silica gel column chromatography separation of the 12nd fraction FrB12, uses MeOH/H2O is by volume (v/v)
100:0~0:100 carries out gradient elution, and every 200mL collects a fraction, obtains 12 after TLC is examined and known the identical fraction of merging
Fraction (FrB12.1-FrB12.12).
5) wherein the 2nd fraction FrB12.2 is splined on reversed-phase silica gel chromatography column, uses MeOH/H2O is by volume (v/v)
(5:95)~(100:0) carries out gradient elution, and every 200mL collects a fraction, after TLC detection inspection is known and merges identical fraction
To 16 fractions (FrB12.2.1-FrB12.2.16).
6) then FrB12.2.13 is purified with half preparative high-performance liquid chromatographic, and using Megres C18 column, mobile phase is second
Nitrile-aqueous solution (35:65, v/v), flow velocity 3.0mL/min obtain 1 (t of compoundR=28min) and 2 (t of compoundR=
31min)。
Embodiment 4
1) tasselled calanthe dry rhizome 10kg is taken, with 10% that volume is 4 times of quality of tasselled calanthe dry rhizome
Ethyl alcohol heating and refluxing extraction 4 times, 2 hours every time, solvent was recovered under reduced pressure in combined extract, obtained total medicinal extract；
2) total medicinal extract is suspended in 4 times of amount water, with ethyl acetate equal-volume extraction 3 times, then again with bodies such as n-butanols
Product extraction 3 times, extract layer respectively obtains ethyl acetate layer, n-butanol layer after solvent is removed under reduced pressure.
3) extracting n-butyl alcohol layer 70g is taken, first by using silica gel column chromatography, chloroform/methanol is by volume (v/v)
100:0~0:100 carries out gradient elution, and every 500mL collects a fraction, is obtained 15 after TLC is examined and known the identical fraction of merging
A fraction (FrB1-FrB15).
4) the wherein inverted silica gel column chromatography separation of the 12nd fraction FrB12, uses MeOH/H2O is by volume (v/v)
100:0~0:100 carries out gradient elution, and every 300mL collects a fraction, obtains 12 after TLC is examined and known the identical fraction of merging
Fraction (FrB12.1-FrB12.12).
5) wherein the 2nd fraction FrB12.2 is splined on reversed-phase silica gel chromatography column, uses MeOH/H2O is by volume (v/v)
(5:95)~(100:0) carries out gradient elution, and every 200mL collects a fraction, after TLC detection inspection is known and merges identical fraction
To 16 fractions (FrB12.2.1-FrB12.2.16).
6) then FrB12.2.13 is purified with half preparative high-performance liquid chromatographic, and using Megres C18 column, mobile phase is first
Alcohol-water solution (25:75, v/v), flow velocity 3.0mL/min obtain 1 (t of compoundR=30min) and 2 (t of compoundR=
34min)。
The invention discloses a kind of indoles glycosides compound and its applications.The indoles glycosides compound is from tasselled shrimp
Extracted in ridge orchid dry rhizome it is isolated, by experimental studies have found that such compound to the glycerol three of 3T3-L1 fat cell
Ester accumulation has facilitation, and implying that has facilitation to cellular uptake glucose, and such compound is extracted from wild plant
It is isolated, have the characteristics that high-efficiency low-toxicity, is expected to develop into drug and/or health food with antidiabetic effect.Its
Secondary, such indoles glycosides compound, which generates NO to lipopolysaccharides LPS stimulation mouse RAW264.7 cell, has good inhibiting effect,
I.e. they have good extracorporeal anti-inflammatory effect, are expected to develop into drug and/or health food with anti-inflammatory effect.Furthermore
Such indoles glycosides compound, which generates HBsAg and HBeAg to HepG2.2.15 cell, has good inhibiting effect, i.e. their tools
There is preferable external antihepatitic activity, is expected to develop into drug and/or health food with antihepatitic activity.In addition to this,
Indoles glycosides compound in the present invention has certain resistant function to alcohol induced mouse gastric ulcer, which has
Efficiently, less toxic feature, therefore be expected to develop into new medicament for anti-gastric ulcer, or being used to prepare, there is prevention and treatment stomach to burst
The health food of ulcer.
The above content is merely illustrative of the invention's technical idea, and this does not limit the scope of protection of the present invention, all to press
According to technical idea proposed by the present invention, any changes made on the basis of the technical scheme each falls within claims of the present invention
Protection scope within.
Claims (10)
1. a kind of indoles glycosides compound, which is characterized in that the molecular formula of the indoles glycosides compound is C24H32N2O12；It is tied
Structure formula is as shown in following formula 1, formula 2:
Formula 1 and formula 2 chiral isomer each other.
2. the extraction separation method of indoles glycosides compound described in claim 1, which comprises the following steps:
1) tasselled calanthe dry rhizome is taken, several times, extracting solution is concentrated under reduced pressure combined extract refluxing extraction, obtains total
Medicinal extract or concentrate；
2) total medicinal extract is suspended in water, obtains medicinal extract liquid, by medicinal extract liquid or concentrate successively through petroleum ether, ethyl acetate and just
Butanol, before immunoassay isolates organic layer afterwards several times, and solvent is removed under reduced pressure and obtains each extract layer；
3) extracting n-butyl alcohol layer is splined on silicagel column, using chloroform-methanol system as eluent, by volume (100:0)~
(0:100) carries out gradient elution, detects to efflux, by effluent volume than the fraction merging for 7:3, removes solvent,
It obtains crossing post part for the first time；
4) first time is crossed into post part and is splined on reversed-phase silica gel chromatography column, using methanol-water system as eluent, by volume
(20:80)~(100:0) carries out gradient elution, and by effluent volume than merging for the fraction of 35:65, removing solvent obtains the
It is secondary to cross post part；
5) post part will be crossed for second and is splined on reversed-phase silica gel chromatography column again, using methanol-water system as eluent, by volume
(5:95)~(100:0) carries out gradient elution, by effluent volume than the fraction merging for 20:80, removes solvent, obtains third
It is secondary to cross post part；
6) third time is crossed into post part and is splined on high performance liquid chromatography separation column, carried out isocratic elution with mobile phase, obtain indoles
Glycosides compound.
3. the extraction separation method of indoles glycosides compound according to claim 2, which is characterized in that in step 1), return
Flowing extraction time is 1~6 time, every time 1~3h；
Ethyl alcohol in refluxing extraction using methanol, water or volume fraction 10~95% is as extractant, tasselled calanthe dry root
The mass volume ratio of stem and extractant is 1kg:(1~5) L；When extractant is the ethyl alcohol of methanol or volume fraction 10~95%,
Solvent recovery in obtained extracting solution is obtained into total medicinal extract；When extractant be water when, by the volume concentration of extracting solution to 1/10~
1/6, obtain concentrate.
4. the extraction separation method of indoles glycosides compound according to claim 2, which is characterized in that, will in step 2)
Total medicinal extract is suspended in water, and the volume ratio of total medicinal extract and water is 1:1~1:4；
When extraction, successively after petroleum ether and ethyl acetate extraction, using extracting n-butyl alcohol；Extraction is to extract in equal volume every time
It takes；Every kind of solvent extracts 1~6 time respectively.
5. the extraction separation method of indoles glycosides compound according to claim 2, which is characterized in that gradient in step 3)
Every 300~800mL collects a fraction after elution；Every 100~300mL collects a fraction after gradient elution in step 4)；Step
It is rapid 5) in after gradient elution every 100~300mL collect a fraction.
6. the extraction separation method of indoles glycosides compound according to claim 2, which is characterized in that in step 6), etc.
The flow velocity of degree elution is 3~5mL/min；
Mobile phase selects methanol-water system or acetonitrile-water system, and the volume ratio of first alcohol and water is 25:75 in methanol-water system；
The volume ratio of acetonitrile and water is 35:65 in acetonitrile-water system.
7. indoles glycosides compound described in claim 1 is preparing the application in antidiabetic medicine and/or health food.
8. indoles glycosides compound described in claim 1 is preparing the application in Antihepatitis medicament and/or health food.
9. indoles glycosides compound described in claim 1 is preparing the application in gastric mucosal protection drug and/or health food.
10. indoles glycosides compound as described in claim 1 is preparing the application in anti-inflammatory drug and/or health food.
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| CN111647031A (en) * | 2020-05-22 | 2020-09-11 | 西安交通大学 | Novel alkaloid and extraction and separation method and application thereof |
| CN111690022A (en) * | 2020-05-22 | 2020-09-22 | 西安交通大学 | Alkaloid separated from radix Caulophylli, and separation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111518149A (en) * | 2020-05-22 | 2020-08-11 | 西安交通大学 | Alkaloid compound, preparation method and application |
| CN111647031A (en) * | 2020-05-22 | 2020-09-11 | 西安交通大学 | Novel alkaloid and extraction and separation method and application thereof |
| CN111690022A (en) * | 2020-05-22 | 2020-09-22 | 西安交通大学 | Alkaloid separated from radix Caulophylli, and separation method and application thereof |
| CN111690022B (en) * | 2020-05-22 | 2021-09-07 | 西安交通大学 | Alkaloid separated from radix Caulophylli, and separation method and application thereof |
| CN111647031B (en) * | 2020-05-22 | 2021-12-28 | 西安交通大学 | Alkaloid and extraction and separation method and application thereof |
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