CN105859803B - A kind of preparation method of galloyl glucose - Google Patents
A kind of preparation method of galloyl glucose Download PDFInfo
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- CN105859803B CN105859803B CN201610297817.9A CN201610297817A CN105859803B CN 105859803 B CN105859803 B CN 105859803B CN 201610297817 A CN201610297817 A CN 201610297817A CN 105859803 B CN105859803 B CN 105859803B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
- C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
- C07H13/08—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals directly attached to carbocyclic rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
Abstract
The present invention provides a kind of preparation methods of galloyl glucose.The preparation method of galloyl glucose of the present invention include crush, slightly carry, extract, high speed adverse current chromatogram enrichment, purifying are refined, collection and drying and other steps.The preparation method of galloyl glucose of the present invention is more time saving than traditional column chromatography, economical, quick, efficient; according to actual needs; not only the chemical constituent of galloyl glucose can have been obtained, but also can be further purified to obtain the monomeric compound of 4-6 kind galloyl glucoses.This method provides new plant origin for a large amount of preparations of galloyl glucose compounds, while providing experimental basis for related drug development.
Description
Technical field
The present invention relates to Separation of Natural Products technical fields, in particular to a kind of system of galloyl glucose
Preparation Method.
Background technology
Galloyl glucose is a kind of condensed tannin monomeric compound, is widely present in plant, is had multi-party
The bioactivity in face.Wherein five galloyl glucoses have anti-oxidant, antitumor, antiviral, anti-inflammatory, hypoglycemic, drop blood
Fat Deng Huoxing [Li Huichen, Hu Wei, fourth well five galloyl glucose antineoplastic action progress of forever;J]Modern swollen
Tumor medicine, 2014, (9):1672-4992.], [Five-O- galloyl-B-D- glucose of 1,2,3,4,6- is as preparation anti-current
Feel the Yong Tu ] of drug;(Authorization Notice No. CN101618043B) reports five galloyl glucoses answering in terms of anti influenza
With value.Fa Mingzhuanli [Yong Tu ] of the active parts of gallnut in the drug for preparing anti-ulcerative colitis;(Authorization Notice No.
CN101862351B) report application value of the active parts of gallnut in terms of anti-ulcerative colitis, activity therein at
It includes tetra--O- galloyl-beta-D-glucoses of 1,2,3,6-, five-O- galloyl-β-D- grapes of 1,2,3,4,6- to divide
Sugar , [The application of galloyl glucose glycoside derivative and Yao Wuzuhewu ] for treating hyperuricemia;(application number
CN104940216A application value of such compound in terms for the treatment of hyperuricemia, Ling Wai, [) are reported;Polyhydroxy is not eaten
The Xin Yongtu ] of sub- acyl-beta-D-Glucose derivative;(Authorization Notice No. CN101209254B) also reported such compound pair
The preventive and therapeutic effect of helicobacter pylori, and point out 1,2,3,6- tetra--O- galloyl-beta-D-glucoses are most preferred compound.
These researchs prompt galloyl glucose compounds are likely to become a kind of natural products with huge potentiality to be exploited,
Thus actively finding and develop such new compound resource becomes the important research direction of scientists.
The chemical constitution of such compound is complicated, and dissolubility is poor, and contains multiple phenolic hydroxyl groups, and conventional pillar layer separation is pure
Very strong suction-operated can be generated on solid dielectric surface by changing, and not only cause that the rate of recovery is very low, purity is not high, but also separating technology
It is cumbersome, experimental period is very long.There are document report (Ren Yulin, et al.J Med Chem.2006,49 (9):2829), can lead to
Cross chemically synthesized method and prepare such compound, but due to complex process, it is expensive the shortcomings of make its application by very big
Limitation.
In view of this, special propose the present invention.
Invention content
The purpose of the present invention is to provide a kind of preparation method of galloyl glucose, the galloyl Portugal
The preparation method skill of grape sugar is simple, quick, and separation cycle is short, and target product yield is high, sample nondestructive loses, good separating effect, single
Body preparation amount is big.
In order to realize that the above-mentioned purpose of the present invention, spy use following technical scheme：
A kind of preparation method of galloyl glucose is isolated and purified using high speed adverse current chromatogram from walnut
Galloyl glucose chemical constituent and monomeric compound.
The preparation method of galloyl glucose of the present invention is detached using high speed adverse current chromatogram from walnut pure
Change galloyl glucose chemical constituent and monomeric compound；With silica gel, high speed unlike the chromatographic techniques such as efficient liquid phase
Adverse current chromatogram is not required to any solid-state carrier, therefore is avoided that surface of solid phase carriers reacts and cause the dirt of sample with sample
The harmful effects such as dye, inactivation, denaturation and Irreversible Adsorption.Also have simultaneously applied widely, easy to operate, separating rate it is fast,
The advantages such as favorable reproducibility, sample injection volume are big, expense is low, can efficiently solve the defect of traditional separation method.Side of the present invention
Method separation condition is mild, and separative efficiency is high, can the chemical constituent of fast enriching galloyl glucose and further same
When refined no less than 4 galloyl glucose monomeric compounds, for new drug development and clinical application provide technological guidance with
Material base.
Preferably, the preparation method of above-mentioned a kind of galloyl glucose, includes the following steps：
(1) the walnut powder after crushing is added to the aqueous solution of alcohol, refluxing extraction, the filtering of gained extracting solution subtracts
Pressure is concentrated into no alcohol taste, obtains walnut extract；
(2) walnut extract obtained by the step (1) is dissolved in water, with solvent extraction with remove it is fat-soluble at
Point, it is concentrated under reduced pressure and organic solvent volatilizees completely, remaining water layer solution is freeze-dried again, obtains walnut crude separation object；
(3) walnut crude separation object high speed adverse current chromatogram obtained by the step (2) is enriched with galloyl Portugal
Grape carbohydrate chemistry component；Solvent system is made of alkane-aliphatic ester-fatty alcohol-water, and the upper phase of solvent system is stationary phase, under
It is mutually mobile phase；
(4) galloyl glucose chemical constituent obtained by the step (3) is further purified with high speed adverse current chromatogram
It is refined, obtain galloyl glucose monomeric compound；Solvent system is made of alkane-aliphatic ester-fatty alcohol-water, molten
The upper phase of agent system is stationary phase, and lower phase is mobile phase.
The preparation method of galloyl glucose of the present invention is detached using high speed adverse current chromatogram from walnut pure
Change galloyl glucose chemical constituent and monomeric compound, simple for process, quick, separation cycle is short, substantially reduces point
From the time.The method of the present invention target product yield is high, sample nondestructive loses, good separating effect, both can be from recklessly according to actual needs
Separation prepares the chemical composition of galloyl glucose in peach family plant seed, and further can refine no less than 4 by Simultaneous purification
A galloyl glucose monomeric compound.The method of the present invention monomer preparation amount is big；According to required galloyl glucose
Quality, can adjust by the sample size of separation sample, countercurrent chromatography instrument can disposably isolate hundreds of milli
Gram even gram-grade object.
Preferably, the former plant be Juglandaceae walnut, preferably include walnut (Juglans regia L.),
Juglans mandshurica (Juglans mandshurica Maxim.), Chinese walnut (Juglans cathayensis Dode), numb walnut
It is one or more in (Juglans hopeiensis Hu) and complex peach (Juglans sigillata Dode).
The method of the present invention increases a large amount of plant origins for preparing galloyl glucose；According to the literature, nutgall
Acyl glucose class compound is typically found in moutan bark, Chinese herbaceous peony, Chinese gall, radix scutellariae, aloe, cassia seed, chrysanthemum, Cortex Eucommiae, big
In the plants such as Huang, this method is for the first time using walnut as raw material, with such compound of high-speed countercurrent chromatography mass production.
Preferably, the volume fraction of alcohol is 10%-90%, preferably 50%- in the aqueous solution of alcohol described in step (1)
70%；The mass ratio of the aqueous solution of the walnut powder and alcohol is 1:8-50, preferably 1:8-10；Return time is 2-
4h, preferably 2-3 hour.
Using specific determining alcohol, usage ratio and return time, help fully to extract the nutgall in walnut
Acyl glucose class compound.
Preferably, alcohol described in step (1) includes methanol, ethyl alcohol or propyl alcohol, further preferably includes ethyl alcohol.
Preferably, filter residue obtained by step (1) is repeated into step (1) refluxing extraction, filtration step, after merging gained filtrate,
It is concentrated under reduced pressure into no alcohol taste, obtains walnut extract.
Preferably, filter residue obtained by step (1) is repeated into 1-3 step (1) refluxing extraction, filtration step, further preferably
To be repeated 2 times, to improve raw material availability, the galloyl glucose compounds in walnut are fully extracted.
Preferably, solvent described in step (2) includes petroleum ether or n-hexane.
Preferably, the volume ratio of each raw material is alkane in solvent system described in step (3)：Aliphatic ester：Fatty alcohol：Water
=(1-2)：(3-5)：(1-2)：(3-5), preferably alkane：Aliphatic ester：Fatty alcohol：Water=1：2：1：2；Alkane include just oneself
Alkane or hexamethylene preferably include n-hexane, and aliphatic ester includes ethyl acetate or methyl acetate, preferably includes ethyl acetate, fat
Fat alcohol includes methanol, ethyl alcohol or propyl alcohol, preferably includes methanol or normal propyl alcohol.
Using specific solvent system ingredient and usage ratio, help to promote galloyl glucose chemical constituent
Concentration effect improves bioaccumulation efficiency.
Preferably, engine speed is 750~850rpm/min, preferably 800rpm/min in step (3)；Flow rate of mobile phase
For 1.5~3.0mL/min, preferably 2.0mL/min.
Using specific engine speed and flow rate of mobile phase, it can ensure the enrichment of galloyl glucose chemical constituent
Effect, and improve bioaccumulation efficiency.
Preferably, the volume ratio of each raw material is alkane in solvent system described in step (4)：Aliphatic ester：Fatty alcohol：Water
=(0.5-1.5)：(8-12)：(0.5-1.5)：(8-12), preferably alkane：Aliphatic ester：Fatty alcohol：Water=1：9：1：9；Alkane
Hydrocarbon includes n-hexane or hexamethylene, preferably includes n-hexane, and aliphatic ester includes ethyl acetate or methyl acetate, preferably includes second
Acetoacetic ester, fatty alcohol include normal propyl alcohol, isopropanol or n-butanol, preferably include normal propyl alcohol.
Using specific solvent system ingredient and usage ratio, help to promote the pure of galloyl glucose substance
Change refining effect, improves purifying purification efficiency, obtain galloyl glucose monomeric compound.
Preferably, engine speed is 750~850rpm/min, preferably 800rpm/min in step (4)；Flow rate of mobile phase
For 1.5~3.0mL/min, preferably 2.5mL/min.
Using specific engine speed and flow rate of mobile phase, it can ensure the purifying essence of galloyl glucose substance
Effect processed, and improve purifying purification efficiency.
Compared with prior art, beneficial effects of the present invention are：
The preparation method of galloyl glucose of the present invention is detached using high speed adverse current chromatogram from walnut pure
Change galloyl glucose chemical constituent and monomeric compound, simple for process, quick, separation cycle is short, substantially reduces point
From the time.The method of the present invention target product yield is high, sample nondestructive loses, good separating effect, both can be from recklessly according to actual needs
Separation prepares the chemical composition of galloyl glucose in peach family plant seed, and further can refine no less than 4 by Simultaneous purification
A galloyl glucose monomeric compound.The method of the present invention monomer preparation amount is big；According to required galloyl glucose
Quality, can adjust by the sample size of separation sample, countercurrent chromatography instrument can disposably isolate hundreds of milli
Gram even gram-grade object.The method of the present invention increases a large amount of plant origins for preparing galloyl glucose；According to document
Report, galloyl glucose compounds are typically found in moutan bark, Chinese herbaceous peony, Chinese gall, radix scutellariae, aloe, cassia seed, chrysanthemum
In the plants such as flower, Cortex Eucommiae, rheum officinale, this method is for the first time using walnut as raw material, with high-speed countercurrent chromatography mass production
Such compound.
Description of the drawings
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art are briefly described, it should be apparent that, in being described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, other drawings may also be obtained based on these drawings.
Fig. 1 is the chromatogram that 1 high speed adverse current chromatogram of the embodiment of the present invention detaches galloyl glucose chemical constituent；
Fig. 2 is the high-efficient liquid phase chromatogram of 1 galloyl glucose chemical constituent of the embodiment of the present invention；
Fig. 3 is the chromatogram that 1 high speed adverse current chromatogram of the embodiment of the present invention detaches galloyl glucose monomeric compound；
Fig. 4 a- Fig. 4 d are the high performance liquid chromatography of 14 kinds of galloyl glucose compounds of gained of the embodiment of the present invention
Figure；
Fig. 5 is the chromatogram that 2 high speed adverse current chromatogram of the embodiment of the present invention detaches galloyl glucose chemical constituent；
Fig. 6 is the high-efficient liquid phase chromatogram of 2 galloyl glucose chemical constituent of the embodiment of the present invention；
Fig. 7 is the chromatogram that 2 high speed adverse current chromatogram of the embodiment of the present invention detaches galloyl glucose monomeric compound；
Fig. 8 a- Fig. 8 d are the high performance liquid chromatography of 24 kinds of galloyl glucose compounds of gained of the embodiment of the present invention
Figure.
Specific implementation mode
Technical scheme of the present invention is clearly and completely described below in conjunction with the drawings and specific embodiments, but
Be it will be understood to those of skill in the art that it is following described embodiments are some of the embodiments of the present invention, rather than it is whole
Embodiment is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.Based on the embodiments of the present invention, ability
The every other embodiment that domain those of ordinary skill is obtained without making creative work, belongs to guarantor of the present invention
The range of shield.The person that is not specified actual conditions in embodiment, carries out according to conventional conditions or manufacturer's recommended conditions.Agents useful for same
Or production firm person is not specified in instrument, is the conventional products that can be obtained by commercially available purchase.
The present invention provides it is a kind of quickly, the method that efficiently separates galloyl glucose compounds, that is, apply height
The method that fast counter current chromatography isolates and purifies galloyl glucose from walnut.This method separation condition is mild,
Separative efficiency is high, can fast enriching galloyl glucose chemical constituent and further refined simultaneously no less than 4
Galloyl glucose monomeric compound provides technological guidance and material base for new drug development and clinical application.
The method that the present invention provides a kind of largely to prepare galloyl glucose from walnut, including it is as follows
Step：
(1) walnut extract is prepared：
Dry walnut (any position) is taken, it is appropriate to crush.Material powders are weighed, 8~50 times of weight of addition
In the ethanol solution that volume fraction is 10~90%, 2~4h of refluxing extraction, filtration；Filter residue is repeated the above steps, then extracts 2
Secondary, merging filtrate is concentrated under reduced pressure into medicinal extract shape, obtains walnut extract.
(2) degreasing and drying of extract：
Not influence subsequent freeze-drying, after adding suitable quantity of water to be suspended extract made from above-mentioned steps, it is put into extraction
In container, while isometric petroleum ether is added, extracts 3-5 times to remove liposoluble constituent, collect water layer solution, be concentrated under reduced pressure
Organic solvent volatilization is complete, the walnut extract after degreasing is obtained, then freeze-dried thick to get walnut
Isolate.
(3) high-speed countercurrent chromatography is enriched with galloyl glucose chemical constituent：
A. the preparation of solvent system and sample solution：
The solvent system of high speed adverse current chromatogram (HSCCC) is made of alkane-aliphatic ester-fatty alcohol-water, and wherein alkane can
Select n-hexane or hexamethylene, aliphatic ester that ethyl acetate or methyl acetate can be selected, methanol or ethyl alcohol, alkane can be selected in fatty alcohol
The proportioning of hydrocarbon-aliphatic ester-fatty alcohol-water is (1-2)：(3-5)：(1-2)：(3-5) was stood at room temperature after shake well
Night.Using the preceding stationary phase for taking upper phase respectively as HSCCC, mobile phase of the lower phase as HSCCC, ultrasound degassing 30min.Take step
Suddenly the walnut crude separation object in (2), it is spare with the upper and lower phase sample dissolution of same volume.
B. high speed adverse current chromatogram is enriched with galloyl glucose chemical constituent：
The upper phase of solvent system is injected in high-speed counter-current chromatograph separating pipe first, is adjusted after upper phase is full of entire pipeline
Whole 750~850rpm/min of engine speed, then lower phase is pumped into 1.5~3.0mL/min flow velocitys, wait for mobile phase from column outlet stream
Go out, after two-phase reaches dynamic equilibrium in separating pipe, the sample solution in (3) a is injected by sampling valve.It examines at a wavelength of 280 nm
It surveys, records chromatogram, collect that maximum fraction of UV absorption, which is concentrated under reduced pressure after removing organic solvent, then pass through
Freeze-drying is to get galloyl glucose chemical constituent.
C. the analysis and identification of galloyl glucose chemical constituent
The galloyl glucose chemical composition being enriched in (3) b is analyzed and identified using efficient liquid phase：
Using Agilent SB-C18 chromatographic columns (Analytical HPLC Column 4.6 × 150mm, 5 μM), -0.1% trifluoro of acetonitrile
Acetic acid water (v/v) is that eluent gradient elutes [1-3min:5% acetonitrile；3-20min:5%-30% acetonitriles；20-24min:
30%-100% Yi Jings ], Detection wavelength 280nm records chromatogram, wherein there is 4-6 larger chromatographic peaks, they in 280nm and
There is prodigious UV absorption at 250nm, it is then very weak in the absorption of other wave bands, it is known that (3) that collected fraction is rich in not in b
Infanticide acyl glucose.
(4) high-speed countercurrent chromatography refines galloyl glucose monomeric compound：
A. the preparation of solvent system and sample solution
The solvent system of high speed adverse current chromatogram is made of alkane-aliphatic ester-fatty alcohol-water, and wherein alkane can be selected just
Hexane or hexamethylene, ethyl acetate can be selected in aliphatic ester or normal propyl alcohol, isopropanol or positive fourth can be selected in methyl acetate, fatty alcohol
The proportioning of alcohol, alkane-aliphatic ester-fatty alcohol-water is (0.5-1.5)：(8-12)：(0.5-1.5)：(8-12), shake well
It stands overnight at room temperature afterwards.Using the preceding stationary phase for taking upper phase respectively as HSCCC, mobile phase of the lower phase as HSCCC surpasses
Sound degassing 30min.Galloyl glucose chemical constituent made from step (3) b is taken, it is molten with the upper and lower phase of same volume
Sample is solved, it is spare.
B. high speed adverse current chromatogram detaches preparation process：
The upper phase of solvent system is injected in high-speed counter-current chromatograph separating pipe first, is adjusted after upper phase is full of entire pipeline
Whole 750~850rpm/min of engine speed, then lower phase is pumped into 1.5~3.0mL/min flow velocitys, wait for mobile phase from column outlet stream
Go out, after two-phase reaches dynamic equilibrium in separating pipe, the sample solution in (4) a is injected by sampling valve.It detects, remembers at 280nm
Chromatogram is recorded, the flow point of each monomeric compound of galloyl glucose is collected according to chromatogram.
C. the analysis and identification of galloyl glucose monomeric compound：
Purity detecting is carried out to gained galloyl glucose monomeric compound in (4) b using efficient liquid phase：Using peace
Prompt human relations SB-C18 chromatographic columns (Analytical HPLC Column 4.6 × 150mm, 5 μm), -0.1% trifluoroacetic acid water of acetonitrile
(v/v) it is that eluent gradient elutes [1-3min:5% acetonitrile；3-20min:5%-30% acetonitriles；20-24min:30%-100%
Yi Jing ], Detection wavelength 280nm, each flow point for measuring monomeric compound is simple spike, and purity is higher than 98%；It uses1H NMR
With13C NMR carry out Structural Identification to galloyl glucose monomeric compound.
Embodiment 1
A kind of preparation method of galloyl glucose, includes the following steps：
(1) Juglans mandshurica bark extract is prepared：
Dried Juglans mandshurica bark is taken, is crushed through pulverizer.Juglans mandshurica bark powder 1kg is weighed, 10 times of weight are added
Volume fraction be 70% ethanol water in, refluxing extraction 3h, filtering；Filter residue is repeated the above steps, then is extracted 2 times,
Merging filtrate is concentrated under reduced pressure into medicinal extract shape, obtains Juglans mandshurica bark extract 124g.
(2) degreasing and drying of Juglans mandshurica bark extract：
Juglans mandshurica bark extract 100g made from step (1) is taken, adds suitable quantity of water that 800ml suspensions are made, is put into liquid separation
In funnel, while petroleum ether 800mL is added and is extracted, be repeated 3 times, to take out liposoluble constituent, collects water layer solution, rotation
Evaporation is concentrated under reduced pressure into no alcohol taste, obtains the Juglans mandshurica bark extract after degreasing, then freeze-dried to get Juglans mandshurica bark
Crude separation object 70g.
(3) high-speed countercurrent chromatography is enriched with galloyl glucose chemical constituent：
A. the preparation of solvent system and sample solution
N-hexane is prepared in separatory funnel：Ethyl acetate：Methanol：Water two phase solvent system, volume ratio 1：2：1：
2, it is stood overnight at room temperature after shaking 30s.Upper and lower phase is released respectively using preceding, is deaerated using ultrasonic extractor
30min.The Juglans mandshurica bark crude separation object 1g in step (2) is taken, it is standby with the upper phase of 10mL and the lower phased soln sample of 10mL
With.
B. the process of high speed adverse current chromatogram enrichment galloyl glucose chemical constituent
First by the upper phase of solvent system quickly (20mL/min) injection high-speed counter-current chromatograph separating pipe, wait for that phase is filled
Engine speed 800rpm/min is adjusted after full entire pipeline, then lower phase is pumped into 2.0mL/min flow velocitys, waits for mobile phase from column outlet
Outflow after two-phase reaches dynamic equilibrium in separating pipe, injects the sample solution in a by sampling valve.It examines at a wavelength of 280 nm
It surveys, records chromatogram, the flow point rich in galloyl glucose is collected according to chromatogram, galloyl Portugal is made in this altogether
Grape carbohydrate chemistry component 80mg.High speed adverse current chromatogram figure is shown in Fig. 1.
C. the analysis and identification of galloyl glucose chemical constituent
Using high performance liquid chromatography to isolated galloyl glucose chemical constituent in b carry out constituent analysis and
Detection：Using Agilent SB-C18 chromatographic columns (Analytical HPLC Column4.6 × 150mm, 5 μM), acetonitrile -0.1%
Trifluoroacetic acid water (v/v) is that eluent gradient elutes [1-3min:5% acetonitrile；3-20min:5%-30% acetonitriles；20-24min:
30%-100% Yi Jings ], Detection wavelength 280nm records chromatogram, wherein there is 4 larger chromatographic peaks, they in 280nm and
There is prodigious UV absorption at 250nm, it is then very weak in the absorption of other wave bands, it is known that (3) that collected fraction is rich in not in b
Infanticide acyl glucose, high performance liquid chromatography detection figure are shown in Fig. 2.
(4) high-speed countercurrent chromatography refines galloyl glucose monomeric compound：
A. the preparation of solvent system and sample solution
N-hexane is prepared in separatory funnel：Ethyl acetate：Normal propyl alcohol：Water two phase solvent system, volume ratio 1：9：
1：9, it is stood overnight at room temperature after shaking 30s.Upper and lower phase is released respectively using preceding, is deaerated using ultrasonic extractor
30min.Galloyl glucose chemical constituent 40mg made from step (3) b is taken, with the upper phase of 4.5mL and the lower phase of 4.5mL
Sample dissolution, it is spare.
B. high speed adverse current chromatogram detaches preparation process
First by the upper phase of solvent system quickly (20mL/min) injection high-speed counter-current chromatograph separating pipe, wait for that phase is filled
Engine speed 800rpm/min is adjusted after full entire pipeline, then lower phase is pumped into 2.5mL/min flow velocitys, waits for mobile phase from column outlet
Outflow after two-phase reaches dynamic equilibrium in separating pipe, injects the sample solution in (4) a by sampling valve.At a wavelength of 280 nm
Detection records chromatogram, sees Fig. 3, and wherein 4 larger flow points are collected according to chromatogram.
C. the analysis and identification of galloyl glucose monomeric compound
Purity detecting is carried out to gained galloyl glucose monomeric compound in (4) b using efficient liquid phase：Using peace
Prompt human relations SB-C18 chromatographic columns (Analytical HPLC Column 4.6 × 150mm, 5 μm), -0.1% trifluoroacetic acid water of acetonitrile
(v/v) it is that eluent gradient elutes [1-3min:5% acetonitrile；3-20min:5%-30% acetonitriles；20-24min:30%-100%
Yi Jing ], Detection wavelength 280nm, each flow point for measuring monomeric compound is simple spike, and purity is above 98%, see Fig. 4 a,
4b, 4c and 4d；Through1H NMR and13The map analysis of C H NMR spectroscopies and document control, identification 4 fractions of gained are respectively that 1,6-O- bis- does not have
Infanticide acyl-beta-D- glucopyranoses (10.4mg), tri- galloyl-O- β-D- glucopyranoses (15.2mg) of 1,2,6-,
Five galloyl-O- β-D- of tetra- galloyl-O- β-D- glucopyranoses (20.3mg) of 1,2,3,6- and 1,2,3,4,6-
Glucopyranose (17.1mg), grape bglii fragment13C NMR datas are shown in Table 1.
1 compound of table13C NMR datas
| NO. | Compound 1a | Compound 2a | Compound 3b | Compound 4a |
| C-1 | 94.7 | 92.1 | 93.6 | 92.3 |
| C-2 | 73.7 | 72.7 | 71.8 | 70.7 |
| C-3 | 75.1 | 73.6 | 76.2 | 72.9 |
| C-4 | 70.2 | 69.6 | 69.6 | 68.3 |
| C-5 | 74.1 | 74.8 | 76.3 | 72.6 |
| C-6 | 63.5 | 62.9 | 63.8 | 61.6 |
aIn DMSO-d6Middle measurement；bIn Actone-d6Middle measurement.
Embodiment 2
A kind of preparation method of galloyl glucose, includes the following steps：
(1) walnut husk extract is prepared：
The walnut husk powder 1kg after dry smash is taken, the ethanol solution that the volume fraction of 8 times of weight is 80% is added
In, refluxing extraction 2h, filtering；Filter residue is repeated the above steps, then is extracted 2 times, merging filtrate is concentrated under reduced pressure into medicinal extract shape, obtains
Walnut husk extract 110g.
(2) degreasing and drying of walnut husk extract：
Walnut husk extract 100g made from step (1) is taken, adds suitable quantity of water that 800ml suspensions are made, is put into liquid separation
In funnel, while petroleum ether 800mL is added and is extracted, be repeated 3 times, to take out liposoluble constituent, collects water layer solution, rotation
Evaporation is concentrated under reduced pressure into no alcohol taste, obtains the walnut husk extract after degreasing, then freeze-dried to get walnut husk
Crude separation object 65g.
(3) high-speed countercurrent chromatography is enriched with galloyl glucose chemical constituent：
High speed adverse current chromatogram is enriched with galloyl glucose chemical constituent, and concrete operation method refers to example 1.Use body
Product is than being 1：2：1：2 n-hexane：Ethyl acetate：Methanol：Aqueous solvent system is 800rpm/min in engine speed, and flow velocity is
4.0mL/min obtains 92mg target components, through liquid under the conditions of Detection wavelength is 280nm from 1g walnut husk crude separation objects
It mutually detects, which is rich in galloyl glucose.High speed adverse current chromatogram chromatogram is shown in Fig. 5, high performance liquid chromatography inspection
Mapping is shown in Fig. 6.
(4) high-speed countercurrent chromatography refines galloyl glucose monomeric compound：
High-speed countercurrent chromatography refines galloyl glucose monomeric compound, and concrete operation method refers to example 1.Make
It is 1 with volume ratio：9：1：9 n-hexane：Ethyl acetate：Isopropanol：Aqueous solvent system is 800rpm/min in engine speed,
Flow velocity is that 4.0mL/min is purified under the conditions of Detection wavelength is 280nm from 92mg galloyl glucose chemical constituent kinds
To bis- tri- galloyl-O- β-D- glucopyranoses of galloyl-β-D- glucopyranoses 10.1mg, 1,2,6- of 1,6-O-
Tetra- five galloyl-O- of galloyl-O- β-D- glucopyranoses 17.2mg and 1,2,3,4,6- of 13.8mg, 1,2,3,6-
β-D- glucopyranose 10.5mg, are detected, the purity of this 4 compounds is all higher than 98% through efficient liquid phase.High speed adverse current chromatogram
Chromatogram is shown in that Fig. 7, high performance liquid chromatography detection figure are shown in Fig. 8 a- Fig. 8 d.
Comparative example 1
To make parallel comparison with embodiment 1, we take the drying coarse powder 1kg of Juglans mandshurica bark, with 10 times of 70% ethyl alcohol of amount
Refluxing extraction 3 times 2 hours every time, merges extracting solution, ethanol extract 130g is concentrated under reduced pressure to obtain.130g ethanol extracts are suspended in
Merge each extract layer respectively with isometric petroleum ether, chloroform, ethyl acetate, extracting n-butyl alcohol successively in deionized water, depressurizes
Concentration, obtains petroleum ether extract (13g), chloroform extract (11g), acetic acid ethyl ester extract (28g), n-butyl alcohol extract
(36g)。
Fully to extract galloyl glucose compounds, the contrast experiment is by acetic acid ethyl ester extract and n-butanol
Extract detaches again after merging.64 grams of extract after merging is crossed into macroreticular resin first and removes water-soluble soluble components, then is used
95% ethanol elution obtains 34 grams of eluates.Later by above-mentioned 34 grams of eluates by decompression silica gel column chromatography, polyamide column layer
Analysis, Sephadex LH-20 column chromatographies, opening ODS column chromatographies, repeatedly recrystallization and the equal chromatography means of preparation solution, finally obtain
The bis- tri- galloyl-O- β-D- pyrans Portugals galloyl-β-D- glucopyranoses 0.208g, 1,2,6- compound 1,6-O-
Tetra- five nutgall acyl of galloyl-O- β-D- glucopyranoses 0.361g and 1,2,3,4,6- of grape sugar 0.472g, 1,2,3,6-
Base-O- β-D- glucopyranoses 0.206g.
It, theoretically can be pure from 1 kilogram of Juglans mandshurica bark by conversion it can be found that using 1 the method for embodiment
Change obtains the bis- tri- galloyl-O- β-D- pyrans Portugals galloyl-β-D- glucopyranoses 0.903g, 1,2,6- 1,6-O-
Tetra- five nutgall acyl of galloyl-O- β-D- glucopyranoses 1.762g and 1,2,3,4,6- of grape sugar 1.319g, 1,2,3,6-
Base-O- β-D- glucopyranoses 1.484g.It can be seen that using the medicinal material of equivalent, the target product of embodiment 1, which must be measured, to be much larger than
Comparative example 1.
In addition, from the time, the galloyl glucose singulation of high-purity is prepared with 1 kilogram of Juglans mandshurica bark
Object is closed, 6-8 months time is needed using 1 the method for comparative example, and according to 1 the method for embodiment, it is only necessary to 40 days.
Comparative example 2
To make parallel comparison with embodiment 2, we take the drying coarse powder 1kg of walnut husk, with 10 times of 80% ethyl alcohol of amount
Refluxing extraction 3 times 2 hours every time, merges extracting solution, ethanol extract 103g is concentrated under reduced pressure to obtain.103g ethanol extracts are suspended in
Merge each extract layer respectively with isometric petroleum ether, chloroform, ethyl acetate, extracting n-butyl alcohol successively in deionized water, depressurizes
Concentration, obtains petroleum ether extract (14g), chloroform extract (8g), acetic acid ethyl ester extract (18g), n-butyl alcohol extract
(22g)。
Fully to extract galloyl glucose compounds, the contrast experiment is by acetic acid ethyl ester extract and n-butanol
Extract detaches again after merging.40 grams of extract after merging is crossed into macroreticular resin first and removes water-soluble soluble components, then is used
95% ethanol elution obtains 18 grams of eluates.Later by above-mentioned 18 grams of eluates by decompression silica gel column chromatography, polyamide column layer
Analysis, Sephadex LH-20 column chromatographies, opening ODS column chromatographies, repeatedly recrystallization and the equal chromatography means of preparation solution, finally obtain
The bis- tri- galloyl-O- β-D- pyrans Portugals galloyl-β-D- glucopyranoses 0.067g, 1,2,6- compound 1,6-O-
Tetra- five nutgall acyl of galloyl-O- β-D- glucopyranoses 0.144g and 1,2,3,4,6- of grape sugar 0.103g, 1,2,3,6-
Base-O- β-D- glucopyranoses 0.128g.
By converting it can be found that with 2 the method for embodiment, can theoretically be purified from 1 kilogram of walnut husk
Obtain bis- 0.722 gram of galloyl-β-D- glucopyranoses of 1,6-O-, the tri- galloyl-O- β-D- pyrans Portugals 1,2,6-
Tetra- five nutgall acyl of galloyl-O- β-D- glucopyranoses 1.23g and 1,2,3,4,6- of grape sugar 0.987g, 1,2,3,6-
Base-O- β-D- glucopyranoses 0.751g.It follows that using the medicinal material of equivalent, the target product of embodiment 2, which must be measured, to be much larger than
Comparative example 2.
In addition, from the time, the galloyl glucose singulation of high-purity is prepared with 1 kilogram of Juglans mandshurica bark
Object is closed, 6-8 months time is needed with 2 the method for comparative example, and according to 2 the method for embodiment, it is only necessary to 1 month.
The preparation method of galloyl glucose of the present invention is detached using high speed adverse current chromatogram from walnut pure
Change galloyl glucose chemical constituent and monomeric compound, simple for process, quick, separation cycle is short, substantially reduces point
From the time.The method of the present invention target product yield is high, sample nondestructive loses, good separating effect, both can be from recklessly according to actual needs
Separation prepares the chemical composition of galloyl glucose in peach family plant seed, and further can refine no less than 4 by Simultaneous purification
A galloyl glucose monomeric compound.The method of the present invention monomer preparation amount is big；According to required galloyl glucose
Quality, can adjust by the sample size of separation sample, countercurrent chromatography instrument can disposably isolate hundreds of milli
Gram even gram-grade object.The method of the present invention increases a large amount of plant origins for preparing galloyl glucose；According to document
Report, galloyl glucose compounds are typically found in moutan bark, Chinese herbaceous peony, Chinese gall, radix scutellariae, aloe, cassia seed, chrysanthemum
In the plants such as flower, Cortex Eucommiae, rheum officinale, this method is for the first time using walnut as raw material, with high-speed countercurrent chromatography mass production
Such compound.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that the above various embodiments is only used
To illustrate technical scheme of the present invention, rather than its limitations；It will be understood by those of ordinary skill in the art that：Without departing substantially from this hair
It, can be with technical scheme described in the above embodiments is modified, or to wherein in the case of bright spirit and scope
Some or all of technical characteristic carries out equivalent replacement；And these modifications or replacements, do not make the essence of corresponding technical solution
It departs from the scope of the technical solutions of the embodiments of the present invention；It is, therefore, intended that including belonging to the present invention in the following claims
All these substitutions and modifications in range.
Claims (25)
1. a kind of preparation method of galloyl glucose, which is characterized in that utilize high speed adverse current chromatogram from walnut
In isolate and purify galloyl glucose chemical constituent and monomeric compound；
Include the following steps：
(1) the walnut powder after crushing is added to the aqueous solution of alcohol, refluxing extraction, the filtering of gained extracting solution is depressurized dense
It is reduced to no alcohol taste, obtains walnut extract；
(2) walnut extract obtained by the step (1) is dissolved in water, with solvent extraction to remove liposoluble constituent,
It is concentrated under reduced pressure and organic solvent volatilizees completely, remaining water layer solution is freeze-dried again, obtains walnut crude separation object；
(3) walnut crude separation object high speed adverse current chromatogram obtained by the step (2) is enriched with galloyl glucose
Chemical constituent；Solvent system is made of alkane-aliphatic ester-fatty alcohol-water, and the upper phase of solvent system is stationary phase, lower phase
For mobile phase；
(4) galloyl glucose chemical constituent obtained by the step (3) is further purified with high speed adverse current chromatogram it is refined,
Obtain galloyl glucose monomeric compound；Solvent system is made of alkane-aliphatic ester-fatty alcohol-water, solvent
The upper phase of system is stationary phase, and lower phase is mobile phase；
The galloyl glucose monomeric compound is bis- galloyl-β-D- glucopyranoses of 1,6-O-, 1,2,6-
Three galloyl-O- β-D- glucopyranoses, tetra- galloyl-O- β-D- glucopyranoses of 1,2,3,6- or 1,2,3,4,
Five galloyl-O- β-D- glucopyranoses of 6-.
2. a kind of preparation method of galloyl glucose according to claim 1, which is characterized in that the juglans
Plant includes one or more in walnut, Juglans mandshurica, Chinese walnut, numb walnut and complex peach.
3. a kind of preparation method of galloyl glucose according to claim 1, which is characterized in that in step (1)
The volume fraction of alcohol is 10%-90% in the aqueous solution of the alcohol；The matter of the aqueous solution of the walnut powder and alcohol
Amount is than being 1：8-50；Return time is 2-4h.
4. a kind of preparation method of galloyl glucose according to claim 3, which is characterized in that in step (1)
The volume fraction of alcohol is 50%-70% in the aqueous solution of the alcohol.
5. a kind of preparation method of galloyl glucose according to claim 3, which is characterized in that in step (1)
The mass ratio of the aqueous solution of the walnut powder and alcohol is 1：8-10.
6. a kind of preparation method of galloyl glucose according to claim 3, which is characterized in that in step (1)
The return time is 2-3 hours.
7. a kind of preparation method of galloyl glucose according to claim 1, which is characterized in that in step (1)
The alcohol includes methanol, ethyl alcohol or propyl alcohol.
8. a kind of preparation method of galloyl glucose according to claim 1, which is characterized in that in step (1)
The alcohol includes ethyl alcohol.
9. a kind of preparation method of galloyl glucose according to claim 1, which is characterized in that in step (2)
The solvent includes petroleum ether or n-hexane.
10. a kind of preparation method of galloyl glucose according to claim 1, which is characterized in that in step (3)
The volume ratio of each raw material is alkane in the solvent system：Aliphatic ester：Fatty alcohol：Water=(1-2)：(3-5)：(1-2)：
(3-5)；Alkane includes n-hexane or hexamethylene, and aliphatic ester includes ethyl acetate or methyl acetate, fatty alcohol include methanol,
Ethyl alcohol or propyl alcohol.
11. a kind of preparation method of galloyl glucose according to claim 10, which is characterized in that step (3)
Described in solvent system the volume ratio of each raw material be alkane：Aliphatic ester：Fatty alcohol：Water=1：2：1：2.
12. a kind of preparation method of galloyl glucose according to claim 10, which is characterized in that step (3)
Described in alkane include n-hexane.
13. a kind of preparation method of galloyl glucose according to claim 10, which is characterized in that step (3)
Described in aliphatic ester include ethyl acetate.
14. a kind of preparation method of galloyl glucose according to claim 10, which is characterized in that step (3)
Described in fatty alcohol include methanol or normal propyl alcohol.
15. a kind of preparation method of galloyl glucose according to claim 1, which is characterized in that in step (3)
Engine speed is 750~850rpm/min；Flow rate of mobile phase is 1.5~3.0mL/min.
16. a kind of preparation method of galloyl glucose according to claim 15, which is characterized in that step (3)
Middle engine speed is 800rpm/min.
17. a kind of preparation method of galloyl glucose according to claim 15, which is characterized in that step (3)
Middle flow rate of mobile phase is 2.0mL/min.
18. a kind of preparation method of galloyl glucose according to claim 1, which is characterized in that in step (4)
The volume ratio of each raw material is alkane in the solvent system：Aliphatic ester：Fatty alcohol：Water=(0.5-1.5)：(8-12)：
(0.5-1.5)：(8-12)；Alkane includes n-hexane or hexamethylene, and aliphatic ester includes ethyl acetate or methyl acetate, fat
Alcohol includes normal propyl alcohol, isopropanol or n-butanol.
19. a kind of preparation method of galloyl glucose according to claim 18, which is characterized in that step (4)
Described in solvent system the volume ratio of each raw material be alkane：Aliphatic ester：Fatty alcohol：Water=1：9：1：9.
20. a kind of preparation method of galloyl glucose according to claim 18, which is characterized in that step (4)
Described in alkane include n-hexane.
21. a kind of preparation method of galloyl glucose according to claim 18, which is characterized in that step (4)
Described in aliphatic ester include ethyl acetate.
22. a kind of preparation method of galloyl glucose according to claim 18, which is characterized in that step (4)
Described in fatty alcohol include normal propyl alcohol.
23. a kind of preparation method of galloyl glucose according to claim 1, which is characterized in that in step (4)
Engine speed is 750~850rpm/min；Flow rate of mobile phase is 1.5~3.0mL/min.
24. a kind of preparation method of galloyl glucose according to claim 23, which is characterized in that step (4)
Middle engine speed is 800rpm/min.
25. a kind of preparation method of galloyl glucose according to claim 23, which is characterized in that step (4)
Middle flow rate of mobile phase is 2.5mL/min.
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Application publication date: 20160817 Assignee: Foshan Aiweidun Technology Co.,Ltd. Assignor: INSTITUTE OF SPECIAL ANIMAL AND PLANT SCIENCES OF CAAS Contract record no.: X2023980032401 Denomination of invention: A Preparation Method of Galloyl Glucose Granted publication date: 20181026 License type: Common License Record date: 20230223 |