CN106361994B - Alpha-glucosidase activity constituents for suppressing in Habenaria Ciliolaris Kranzl and its preparation method and application - Google Patents
Alpha-glucosidase activity constituents for suppressing in Habenaria Ciliolaris Kranzl and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to the alpha-glucosidase activity constituents for suppressing and its preparation method and application in a kind of Habenaria Ciliolaris Kranzl, belong to chemical field.The preparation method is the following steps are included: crushing Habenaria Ciliolaris Kranzl to its granularity is 420-580 μm；By the first solid-liquid ratio 20-100g:500-6000mL mixing extractant and smashed Habenaria Ciliolaris Kranzl, extracts 20-50h and obtain Habenaria Ciliolaris Kranzl crude extract；Supernatant is collected by filtration, is concentrated to give crude extract medicinal extract；Crude extract medicinal extract is dispersed in water by the second solid-liquid ratio 1g:1-6mL, the gradient elution load solution in macroporous adsorption resin chromatography column is collected and is concentrated active methanol eluting peak, obtains activity suppression component, and this method is simple, easy to operate.The alpha-glucosidase activity constituents for suppressing prepared through the method has stronger alpha-glucosaccharase enzyme inhibition activity, especially has good inhibiting effect to maltase activity.
Description
Technical field
The present invention relates to chemical fields, and the alpha-glucosidase activity inhibition group in particular to a kind of Habenaria Ciliolaris Kranzl
Point and its preparation method and application.
Background technique
Diabetes (Diabetes mellitus, DM) are that insulin content is insufficient and cause interior in a kind of blood of human body
The disease for secreting metabolic disorder can cause multiple systems, internal organs impaired characterized by blood glucose increases with glucose in urine, be that the mankind suffer from
The main reason for cardiovascular and cerebrovascular disease, blinding, amputation and renal failure.It clinically has been developed that a variety of hypoglycemic drugs, such as improves Portugal
The insulin secretion stimulators of grape sugared tolerance and insulin replies level improve glucose metabolism agent, prevent and delay illness
The aldose reductase inhibitor and insulin sensitizer of generation, wherein alpha-glucosidase restrainer has become oral drop at present
Sugared drug and the hot spot of development of functional food research.
Alpha-glucosidase restrainer can block the maltose and invertase for being present in mucous membrane of small intestine microvillose membrane surface
The activity of equal disaccharide hydrolase, make polysaccharide, oligosaccharides and the disaccharide digestion of intake become the processes of the monosaccharide such as glucose, fructose by
Resistance, absorption of the delaying human body to glucose, to reduce the peak value of blood glucose.Currently used for clinical alpha-glucosidase restrainer
Hypoglycemic medicine has acarbose (Acarbose), voigelibo (Voglibose) and Miglitol (Miglitol), these three objects
The structure of matter can play effective hypoglycemic effect similar to glucose, but expensive, have flatulence, vomiting, diarrhea etc. are secondary to make
With, and source is relatively simple, type is very little.Efficient, Small side effects alpha-glucosidase suppressions are thus found from natural plants
Preparation is hot spot studied both at home and abroad at present.
Bamboo shoot are closed in Habenaria Ciliolaris Kranzl (Habenaria Ciliolaris Kranzl) also known as hair Roripa Yu Fenghua or ocean, are belonged to dicotyledonous
Medicine orchid, be mainly grown in China Yangtze river basin and on the south the areas such as Hunan, Hubei, Sichuan, be rich in a variety of nutrition
Ingredient.It records that Habenaria Ciliolaris Kranzl is sweet, flat, kidney channel in " Xinhua's book on Chinese herbal medicine outline ", there is the effect of enriching yin and nourishing kidney.Recent years, research are found
Habenaria Ciliolaris Kranzl can be used for treating the deficiency of liver-yin and kidney-yin, have a dizzy spell, the diseases such as hypopsia, soreness and weakness of waist and knees.Domestic and foreign scholars are to open country at present
Sun close chemical component and its application study achieve certain progress, but also need system further investigation polysaccharide, flavonoids and
The structure and activity of polyphenols.Habenaria Ciliolaris Kranzl is resourceful, but fails always sufficiently to develop and use for a long time.
Summary of the invention
The purpose of the present invention is to provide a kind of preparation method of the alpha-glucosidase activity constituents for suppressing in Habenaria Ciliolaris Kranzl,
This preparation method is easy to operate, it is easy to accomplish, alpha-glucosidase activity constituents for suppressing content obtained is larger and anti alpha-grape
Glucosides enzyme effect is strong.
Another object of the present invention is to provide a kind of alpha-glucosidase activities being prepared by above-mentioned preparation method
Constituents for suppressing, which has good alpha-glucosaccharase enzyme inhibition, such as to maltose
Enzyme has apparent inhibitory effect.Its raw material is natural products, and toxic side effect is small, can be widely applied to various alpha-glucosidases
In inhibitor.
The third object of the present invention is to provide a kind of application of above-mentioned alpha-glucosidase activity constituents for suppressing, i.e., will
Alpha-glucosidase activity constituents for suppressing is applied in alpha-glucosidase restrainer as active constituent.
The present invention solves its technical problem and adopts the following technical solutions to realize:
The present invention proposes a kind of preparation method of the alpha-glucosidase activity constituents for suppressing in Habenaria Ciliolaris Kranzl comprising following
Step: extract: crushing Habenaria Ciliolaris Kranzl to its granularity is 420-580 μm, using hydrophilic organic solvent as extractant, by the first solid-liquid ratio
20-100g:500-6000mL mixing extractant and smashed Habenaria Ciliolaris Kranzl, extract 20-50h obtain Habenaria Ciliolaris Kranzl crude extract.
Then separate: filtering Habenaria Ciliolaris Kranzl slightly takes object, collects supernatant, is concentrated to give crude extract medicinal extract.
Then it purifies: crude extract medicinal extract being dispersed in water by the second solid-liquid ratio 1g:1-6mL, load solution is obtained, with first
Alcohol-water solution is eluent, molten with the elution flow rate gradient elution loading of 0.5-2mL/min in macroporous adsorption resin chromatography column
Liquid is collected and is concentrated active methanol eluting peak, obtains activity suppression component.
The present invention also proposes that one kind prepares resulting alpha-glucosidase activity constituents for suppressing by above-mentioned preparation method.
The invention also provides a kind of above-mentioned alpha-glucosidase activity constituents for suppressing to prepare alpha-glucosidase suppression
Application in preparation.
It alpha-glucosidase activity constituents for suppressing in a kind of Habenaria Ciliolaris Kranzl of the embodiment of the present invention and preparation method thereof and answers
Beneficial effect is: by crushing to Habenaria Ciliolaris Kranzl, the contact surface increased between raw material and extractant is connect, and is improved and is leached
Speed.Using hydrophilic organic solvent as extractant, it both can guarantee that fast leaching velocity, the purity is high of extract, impurity were few, but also
Cost can be reduced.Can be made using 20-100g:500-6000mL as the first solid-liquid ratio active constituent extraction effect in Habenaria Ciliolaris Kranzl compared with
It is good；Extraction time is selected as 20-50h, can avoid because extraction time it is too short caused by active principle dissolution rate it is low, extraction time is too long again
Meeting is so that the excessive dissolution of impurity such as grease, tannin in plant cell, the defects of increasing the difficulty isolated and purified.With 1g:1-
6mL can be such that crude extract medicinal extract is well-dispersed in water as the second solid-liquid ratio；Using macroporous adsorption resin chromatography column combination first
Alcohol-water solution is as eluent, using 0.5-2mL/min as elution flow rate, the separation of the active constituent in load solution can be made to imitate
Fruit and recovery rate reach best.This method is easy to operate, it is easy to accomplish, the alpha-glucosidase activity in Habenaria Ciliolaris Kranzl prepared
Constituents for suppressing has good alpha-glucosaccharase enzyme inhibition, especially has apparent inhibitory effect to maltose.Due to
The raw material of the invention is natural products, and toxic side effect is small, therefore can be wide by the alpha-glucosidase activity constituents for suppressing that it is prepared
It is general to be applied in various alpha-glucosidase restrainers.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 is the high-efficient liquid phase chromatogram of the alpha-glucosidase activity constituents for suppressing in Habenaria Ciliolaris Kranzl.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Below to alpha-glucosidase activity constituents for suppressing in the Habenaria Ciliolaris Kranzl of the embodiment of the present invention and preparation method thereof and
Using being specifically described.
The preparation method of alpha-glucosidase activity constituents for suppressing in Habenaria Ciliolaris Kranzl provided in an embodiment of the present invention, can be first right
Raw material carries out pre-treatment, i.e., crushes Habenaria Ciliolaris Kranzl, to increase the contact area between raw material and extractant, improves leaching velocity.
Grinding particle size for example can be 420-580 μm, and the effective component dissolution rate in the particle size range in raw material is larger.Raw material is preferably adopted
Active constituent content highest from the fresh Habenaria Ciliolaris Kranzl of full-bloom stage, the Habenaria Ciliolaris Kranzl in this period is conducive to improve subsequent extracted object
In effective component content.
Preferably, before pulverising step, it the works such as can also be cleaned, cleaned and be dried to Habenaria Ciliolaris Kranzl raw material
Sequence.Wherein clean and clean can avoid other impurity to preparation effect interference, drying can partial destruction raw material cell membrane and
Cell wall is conducive to extract.Wherein, it is 20-45 DEG C, preferably 35-37 DEG C that drying temperature, which for example can choose,.Preferred temperature herein
It spends in range, the active constituent in raw material can retain to the greatest extent not by high temperature.
Preferably, the embodiment of the present invention use extraction, using hydrophilic organic solvent as extractant to pre-treatment after
Raw material extracts.Using rule of similarity, which can be selected from methanol-water solution or dehydrated alcohol-water
Any one in solution both can guarantee that fast leaching velocity, the purity is high of extract, impurity were few and at low cost.When selection nothing
When water-ethanol-aqueous solution is as extractant, volume fraction of the dehydrated alcohol in dehydrated alcohol-aqueous solution for example can be
70%-95%, preferably 90%, the polarity of ethanol solution is moderate under this volume fraction, so that the exhausted big portion in Habenaria Ciliolaris Kranzl
Point effective component is soluble in the solution, and it is less to extract very fast and impurity.In addition, acetone etc. also can be selected.
Extractant is mixed with smashed Habenaria Ciliolaris Kranzl raw material with the first liquid ratio for example 20-100g:500-6000mL,
It obtains to extract.Preferably, the first liquid ratio such as can be 20g:800mL, the activity under this ratio, in Habenaria Ciliolaris Kranzl
Constituents extraction effect is best.
Further, it will be extracted to extract.Preferably, in the embodiment of the present invention in room temperature (20-30 DEG C), often
Pressure treats extract with magnetic stirring apparatus and is stirred extraction 20-50h, obtains Habenaria Ciliolaris Kranzl crude extract.It can be made because extraction time is too short
Low at active principle dissolution rate, extraction time is too long and can make the excessive dissolutions of impurity such as grease, tannin in plant cell,
Increase the difficulty isolated and purified, therefore extraction time is preferably for 24 hours.
In addition, extracting mode can also be using Microwave Extraction, ultrasonic wave extraction, supercritical fluid extraction etc..
Habenaria Ciliolaris Kranzl crude extract is filtered, such as can be centrifugal filtration, the supernatant after collecting separation.After filtering
Residue repeat to extract 2 times according to above-mentioned extraction step, merge 3 supernatants extracted after separation, be concentrated to get crude extract
Medicinal extract.By weight percentage, the weight of the crude extract medicinal extract accounts for the 10-40% of the weight of Habenaria Ciliolaris Kranzl raw material.Preferably,
In the embodiment of the present invention for example can by supernatant in 45-55 DEG C water-bath, revolving speed be 30-50 turn/min and vacuum degree be
Rotary evaporation under conditions of 0.07-0.09MPa, remove organic phase in ethyl alcohol, obtain soaking crude extract paste, under this condition into
Capable reduced pressure is conducive to that the structure of active constituent in Habenaria Ciliolaris Kranzl is avoided to be destroyed in recycling design.
Further, crude extract medicinal extract is purified.Preferably, the upper quadrat method in the embodiment of the present invention is preferably wet
Crude extract medicinal extract is dissolved in after solvent and carries out loading again by method loading, to improve purification effect.Specifically, can be by second
Crude extract medicinal extract is dispersed in water by liquid ratio for example 1g:1-6mL, and wherein distilled water for example can be selected in water, also can be selected super
Pure water obtains load solution, then carries out purifies and separates to load solution using the column chromatography method in chromatography.It is specific and
Speech, chromatographic principle is that different material is utilized to distribute in the selectivity of different phase, with mobile phase to the mixing in stationary phase
Object is eluted, and different substances can be moved along stationary phase at different rates in mixture, is finally reached the effect of separation.Make
To be preferred, the chromatographic column in the present embodiment selects macroporous adsorption resin chromatography column, a kind of substantially object height of absorption
By the adsorption phenomenon that active force is unequal and generates, this absorption property is due to Van der Waals force for dispersion or surface molecular
Or generate the result of hydrogen bond.Simultaneously because the substance that the porous structure of macroporous absorbent resin keeps it different to molecular size has sieve
It is elected to be use.By above-mentioned this absorption and screening principle, organic compound according to the difference of adsorption capacity and the size of molecular weight,
The different purposes such as elute and reach separation, purifying, removal of impurities, concentration through certain solvent on macroporous absorbent resin.
Preferably, the internal diameter of above-mentioned macroporous adsorption resin chromatography column is 4.5cm, and filler is added to example into chromatographic column
It is best to the purification effect of crude extract medicinal extract under this condition such as 30-35cm.Filler is preferably HP-20, the filling adsorption of the type
Amount is high, particle is uniform, mechanical strength is good, non-breakable and residue is few.
Further, eluent is added into macroporous adsorption resin chromatography column, and gradient elution is carried out to load solution.Its
In, for eluent for example selected from alcohol-water solution, considering cost and separating effect, alcohol-water solution is preferably methanol-water solution.
Preferably, during gradient elution, methanol is followed successively by 0% by content of the volume percent in methanol-water solution,
20%, 40%, 60%, 80% and 100%.Specifically, using the pure water of 600-800mL100% as the first eluent pair first
Load solution carries out first time elution, obtains the first elution fraction；Then, the first for being 20% with 400-600mL methanol volume content
Alcohol-water solution carries out second to load solution as the second eluent and elutes, and obtains the second elution fraction；With 400-600mL first
The methanol-water solution that alcohol volume content is 40% carries out third time elution to load solution as third eluent, obtains third and washes
De- component；The methanol-water solution that 400-600mL methanol volume content is 60% is used to carry out as the 4th eluent to load solution
4th elution, obtains the 4th elution fraction；Use the methanol-water solution that 400-600mL methanol volume content is 80% as the 5th
Eluent carries out the 5th elution to load solution, obtains the 5th elution fraction；Finally, use the pure methanol solution of 400-600mL as
6th eluent carries out the 6th elution to load solution, obtains the 6th elution fraction.Wherein, elution flow rate for example can be 0.5-
2mL/min, preferably 1mL/min, the separating effect of active constituent and recovery rate are best under this flow velocity.
Further, six above-mentioned elution fractions are collected respectively, and test six elution fractions to alpha-glucosaccharase
The inhibitory effect of enzyme, concentration wherein have the methanol eluting peak of optimum activity to get active component.Preferably, it can also utilize
High performance liquid chromatography carries out preliminary species analysis to the active component.
The present invention also provides a kind of alpha-glucosidase activity inhibition groups being prepared in Habenaria Ciliolaris Kranzl using the above method
Point.By weight percentage, the weight of the alpha-glucosidase activity constituents for suppressing for example accounts for Habenaria Ciliolaris Kranzl in the embodiment of the present invention
The 0.8-3% of the weight of raw material.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
It is 420 μm that fresh Habenaria Ciliolaris Kranzl, which is crushed to granularity, and using methanol-water solution as extractant, wherein methanol is in methanol-water
Percentage by volume in solution is 90%.Extractant is mixed with smashed Habenaria Ciliolaris Kranzl by the first solid-liquid ratio 20g:500mL, is obtained
To extract.Under 20 DEG C, condition of normal pressure, extract is treated with magnetic stirring apparatus and is stirred extraction 50h, Habenaria Ciliolaris Kranzl is obtained and slightly mentions
Object.The crude extract is subjected to centrifugal filtration, collects and separate supernatant, and residue is repeated to extract according to above-mentioned extracting mode
2 times, merge 3 resulting supernatants of difference, and by supernatant in 45 DEG C of water-bath, the revolving speed and 0.07MPa of 50 turns/min
Vacuum degree condition under rotary evaporation, obtain crude extract medicinal extract, the crude extract medicinal extract weight be Habenaria Ciliolaris Kranzl raw material 10%.Using
Wet process loading is dispersed crude extract medicinal extract in distilled water for 1g:1mL by the second solid-liquid ratio, obtains load solution.The internal diameter is taken to be
The macroporous adsorption resin chromatography column of 4.5cm and be 30cm to HP-20 filler to packed height is added inside it.It is molten with methanol-water
Liquid is followed successively by 0%, 20%, 40%, 60%, 80% and 100% according to volume content of the methanol in eluent as eluent
Gradient elution is carried out to load solution.Wherein, the effluent volume of gradient elution be respectively 600mL, 400mL, 400mL,
400mL, 400mL and 400mL, elution flow rate 0.5mL.Elution fraction after collecting elution, tests six elution fractions pair
The inhibitory effect of alpha-glucosidase, test data are shown in Table 1.The wherein optimal methanol eluting peak of inhibitory effect is concentrated, obtains active
Component, the weight of the active component account for the 0.8% of Habenaria Ciliolaris Kranzl raw material weight.
Embodiment 2
Fresh Habenaria Ciliolaris Kranzl is cleaned, is cleaned, it is 580 μm that granularity is crushed to after drying at 20 DEG C, with dehydrated alcohol-
Aqueous solution is extractant, and wherein percentage by volume of the dehydrated alcohol in dehydrated alcohol-aqueous solution is 70%.By the first solid-liquid ratio
100g:6000mL mixes extractant with smashed Habenaria Ciliolaris Kranzl, obtains to extract.Under 30 DEG C, condition of normal pressure, stirred with magnetic force
Mix device treat extract be stirred extract 20h, obtain Habenaria Ciliolaris Kranzl crude extract.The crude extract is subjected to centrifugal filtration, collect and is divided
It repeats to extract 2 times according to above-mentioned extracting mode from supernatant, and by residue, merges 3 resulting supernatants of difference, by supernatant
Liquid rotary evaporation under 55 DEG C of water-bath, the revolving speed of 30 turns/min and the vacuum degree condition of 0.08MPa obtains crude extract leaching
Cream, the crude extract medicinal extract weight are the 15% of Habenaria Ciliolaris Kranzl raw material.It is that 1g:6mL will be mentioned slightly by the second solid-liquid ratio using wet process loading
Object medicinal extract is scattered in ultrapure water, obtains load solution.Take the macroporous adsorption resin chromatography column that internal diameter is 4.5cm and to inside it plus
Entering HP-20 filler to packed height is 35cm.Methanol-water solution is used to contain as eluent according to volume of the methanol in eluent
Amount is followed successively by 0%, 20%, 40%, 60%, 80% and 100% pair of load solution and carries out gradient elution.Wherein, gradient elution
Effluent volume is respectively 800mL, 600mL, 600mL, 600mL, 600mL and 600mL, elution flow rate 2mL.After collecting elution
Elution fraction, test six elution fractions to the inhibitory effect of alpha-glucosidase, test data is shown in Table 1.Concentration is wherein
The optimal methanol eluting peak of inhibitory effect, obtains active component, the weight of the active component accounts for the 1.2% of Habenaria Ciliolaris Kranzl raw material weight.
Embodiment 3
Fresh Habenaria Ciliolaris Kranzl is cleaned, is cleaned, it is 500 μm that granularity is crushed to after drying at 45 DEG C, with dehydrated alcohol-
Aqueous solution is extractant, and wherein percentage by volume of the dehydrated alcohol in dehydrated alcohol-aqueous solution is 95%.By the first solid-liquid ratio
60g:3200mL mixes extractant with smashed Habenaria Ciliolaris Kranzl, obtains to extract.Under 25 DEG C, condition of normal pressure, stirred with magnetic force
Mix device treat extract be stirred extract 35h, obtain Habenaria Ciliolaris Kranzl crude extract.The crude extract is subjected to centrifugal filtration, collect and is divided
It repeats to extract 2 times according to above-mentioned extracting mode from supernatant, and by residue, merges 3 resulting supernatants of difference, by supernatant
Liquid rotary evaporation under 50 DEG C of water-bath, the revolving speed of 40 turns/min and the vacuum degree condition of 0.09MPa obtains crude extract leaching
Cream, the crude extract medicinal extract weight are the 20% of Habenaria Ciliolaris Kranzl raw material.It is that 1g:4mL will be mentioned slightly by the second solid-liquid ratio using wet process loading
Object medicinal extract is scattered in distilled water, obtains load solution.Take the macroporous adsorption resin chromatography column that internal diameter is 4.5cm and to inside it plus
Entering HP-20 filler to packed height is 33cm.Methanol-water solution is used to contain as eluent according to volume of the methanol in eluent
Amount is followed successively by 0%, 20%, 40%, 60%, 80% and 100% pair of load solution and carries out gradient elution.Wherein, gradient elution
Effluent volume is respectively 700mL, 500mL, 500mL, 500mL, 500mL and 500mL, elution flow rate 2.2mL.Collect elution
Elution fraction afterwards tests six elution fractions to the inhibitory effect of alpha-glucosidase, and test data is shown in Table 1.It is concentrated
The middle optimal methanol eluting peak of inhibitory effect, obtains active component, the weight of the active component accounts for the 2% of Habenaria Ciliolaris Kranzl raw material weight.
Embodiment 4
Fresh Habenaria Ciliolaris Kranzl is cleaned, is cleaned, it is 560 μm that granularity is crushed to after drying at 37 DEG C, with dehydrated alcohol-
Aqueous solution is extractant, and wherein percentage by volume of the dehydrated alcohol in dehydrated alcohol-aqueous solution is 90%.By the first solid-liquid ratio
20g:800mL mixes extractant with smashed Habenaria Ciliolaris Kranzl, obtains to extract.Under 28 DEG C, condition of normal pressure, magnetic agitation is used
Device treats extract and is stirred extraction 48h, obtains Habenaria Ciliolaris Kranzl crude extract.The crude extract is subjected to centrifugal filtration, collects and separates
Supernatant, and residue is repeated to extract 2 times according to above-mentioned extracting mode, merge 3 resulting supernatants of difference, by supernatant
The rotary evaporation under the vacuum degree condition of 55 DEG C of water-baths, the revolving speed of 40 turns/min and 0.09MPa obtains crude extract medicinal extract,
The crude extract medicinal extract weight is the 40% of Habenaria Ciliolaris Kranzl raw material.It is 1g:3mL by crude extract by the second solid-liquid ratio using wet process loading
Medicinal extract is scattered in distilled water, obtains load solution.Take the macroporous adsorption resin chromatography column that internal diameter is 4.5cm and to being added inside it
HP-20 filler to packed height is 32cm.Use volume content of the methanol-water solution as eluent according to methanol in eluent
It is followed successively by 0%, 20%, 40%, 60%, 80% and 100% pair of load solution and carries out gradient elution.Wherein, gradient elution is washed
De- liquid product is respectively 700mL, 500mL, 500mL, 500mL, 500mL and 500mL, elution flow rate 1mL.After collecting elution
Elution fraction tests six elution fractions to the inhibitory effect of alpha-glucosidase, and test data is shown in Table 1.Concentration wherein presses down
The optimal methanol eluting peak of effect processed, obtains active component, the weight of the active component accounts for the 3% of Habenaria Ciliolaris Kranzl raw material weight.
Embodiment 5
Fresh Habenaria Ciliolaris Kranzl is cleaned, is cleaned, it is 520 μm that granularity is crushed to after drying at 35 DEG C, with dehydrated alcohol-
Aqueous solution is extractant, and wherein percentage by volume of the dehydrated alcohol in dehydrated alcohol-aqueous solution is 90%.By the first solid-liquid ratio
40g:1500mL mixes extractant with smashed Habenaria Ciliolaris Kranzl, obtains to extract.Under 28 DEG C, condition of normal pressure, stirred with magnetic force
Mix device treat extract be stirred extraction for 24 hours, obtain Habenaria Ciliolaris Kranzl crude extract.The crude extract is subjected to centrifugal filtration, collect and is divided
It repeats to extract 2 times according to above-mentioned extracting mode from supernatant, and by residue, merges 3 resulting supernatants of difference, by supernatant
Liquid rotary evaporation under 50 DEG C of water-bath, the revolving speed of 40 turns/min and the vacuum degree condition of 0.09MPa obtains crude extract leaching
Cream, the crude extract medicinal extract weight are the 25% of Habenaria Ciliolaris Kranzl raw material.It is that 1g:4mL will be mentioned slightly by the second solid-liquid ratio using wet process loading
Object medicinal extract is scattered in ultrapure water, obtains load solution.Take the macroporous adsorption resin chromatography column that internal diameter is 4.5cm and to inside it plus
Entering HP-20 filler to packed height is 33cm.Methanol-water solution is used to contain as eluent according to volume of the methanol in eluent
Amount is followed successively by 0%, 20%, 40%, 60%, 80% and 100% pair of load solution and carries out gradient elution.Wherein, gradient elution
Effluent volume is respectively 700mL, 500mL, 500mL, 500mL, 500mL and 500mL, elution flow rate 1mL.After collecting elution
Elution fraction, test six elution fractions to the inhibitory effect of alpha-glucosidase, test data is shown in Table 1.Concentration is wherein
The optimal methanol eluting peak of inhibitory effect, obtains active component, the weight of the active component accounts for the 1.5% of Habenaria Ciliolaris Kranzl raw material weight.
Embodiment 6
Fresh Habenaria Ciliolaris Kranzl is cleaned, is cleaned, it is 550 μm that granularity is crushed to after drying at 32 DEG C, with dehydrated alcohol-
Aqueous solution is extractant, and wherein percentage by volume of the dehydrated alcohol in dehydrated alcohol-aqueous solution is 90%.By the first solid-liquid ratio
20g:800mL mixes extractant with smashed Habenaria Ciliolaris Kranzl, obtains to extract.Under 28 DEG C, condition of normal pressure, magnetic agitation is used
Device treats extract and is stirred extraction for 24 hours, obtains Habenaria Ciliolaris Kranzl crude extract.The crude extract is subjected to centrifugal filtration, collects and separates
Supernatant, and residue is repeated to extract 2 times according to above-mentioned extracting mode, merge 3 resulting supernatants of difference, by supernatant
The rotary evaporation under the vacuum degree condition of 50 DEG C of water-baths, the revolving speed of 40 turns/min and 0.09MPa obtains crude extract medicinal extract,
The crude extract medicinal extract weight is the 26% of Habenaria Ciliolaris Kranzl raw material.It is 1g:3mL by crude extract by the second solid-liquid ratio using wet process loading
Medicinal extract is scattered in distilled water, obtains load solution.Take the macroporous adsorption resin chromatography column that internal diameter is 4.5cm and to being added inside it
HP-20 filler to packed height is 33cm.Use volume content of the methanol-water solution as eluent according to methanol in eluent
It is followed successively by 0%, 20%, 40%, 60%, 80% and 100% pair of load solution and carries out gradient elution.Wherein, gradient elution is washed
De- liquid product is respectively 700mL, 500mL, 500mL, 500mL, 500mL and 500mL, elution flow rate 1mL.After collecting elution
Elution fraction tests six elution fractions to the inhibitory effect of alpha-glucosidase, and test data is shown in Table 1.Concentration wherein presses down
The optimal methanol eluting peak of effect processed, obtains active component, the weight of the active component accounts for the 1.55% of Habenaria Ciliolaris Kranzl raw material weight.
Embodiment 7
Fresh Habenaria Ciliolaris Kranzl is cleaned, is cleaned, it is 560 μm that granularity is crushed to after drying at 36 DEG C, with dehydrated alcohol-
Aqueous solution is extractant, and wherein percentage by volume of the dehydrated alcohol in dehydrated alcohol-aqueous solution is 90%.By the first solid-liquid ratio
20g:800mL mixes extractant with smashed Habenaria Ciliolaris Kranzl, obtains to extract.Under 28 DEG C, condition of normal pressure, magnetic agitation is used
Device treats extract and is stirred extraction for 24 hours, obtains Habenaria Ciliolaris Kranzl crude extract.The crude extract is subjected to centrifugal filtration, collects and separates
Supernatant, and residue is repeated to extract 2 times according to above-mentioned extracting mode, merge 3 resulting supernatants of difference, by supernatant
The rotary evaporation under the vacuum degree condition of 50 DEG C of water-baths, the revolving speed of 40 turns/min and 0.09MPa obtains crude extract medicinal extract,
The crude extract medicinal extract weight is the 27% of Habenaria Ciliolaris Kranzl raw material.It is 1g:3mL by crude extract by the second solid-liquid ratio using wet process loading
Medicinal extract is scattered in distilled water, obtains load solution.Take the macroporous adsorption resin chromatography column that internal diameter is 4.5cm and to being added inside it
HP-20 filler to packed height is 33cm.Use volume content of the methanol-water solution as eluent according to methanol in eluent
It is followed successively by 0%, 20%, 40%, 60%, 80% and 100% pair of load solution and carries out gradient elution.Wherein, gradient elution is washed
De- liquid product is respectively 700mL, 500mL, 500mL, 500mL, 500mL and 500mL, elution flow rate 1mL.After collecting elution
Elution fraction tests six elution fractions to the inhibitory effect of alpha-glucosidase, and test data is shown in Table 1.Concentration wherein presses down
The optimal methanol eluting peak of effect processed, obtains active component, the weight of the active component accounts for the 1.6% of Habenaria Ciliolaris Kranzl raw material weight.
Test example 1
Test method is inhibited using following alpha-glucosidase activity, to resulting 6 after gradient elution in embodiment
A elution fraction and active component carry out inhibiting rate test.
Test method: from SIGMA company order rat small intestine acetone extraction powder, with phosphate buffer (PBS, 0.1M,
PH7.0) homogenized removes supernatant after centrifugation；The mixed liquor homogenized for using PBS+10%Triton again, obtains after centrifugation
Supernatant.Supernatant dialysis membrane is dialysed 24 hours, rat small intestine alpha-glucosaccharase enzyme solutions are obtained.Rat small intestine α-Portugal
The thick enzyme Maltose hydrolysis activity of polyglycoside enzyme is 0.3U/mL.Wherein enzyme activity unit is defined as: 37 DEG C, under the conditions of pH7.0,
1 minute 1 μm of ol maltose of decomposition under the action of enzyme is, it is specified that be an enzyme activity unit (U).
Measuring determinand includes: rat small intestine alpha-glucosaccharase to the reaction solution of rat small intestine maltose inhibiting effect
Enzyme solutions (0.1mL, 0.3U/mL), tested material (0.1mL is dissolved in 20% dimethyl sulfoxide), maltose solution (0.3mL, 4mM wheat
Bud sugar is dissolved in 0.1M, the phosphate buffer of pH7.0).After the above reaction solution is reacted 15min in 37 DEG C of incubators, use
The Tris-HCl of 0.75mL terminates reaction.Using glucose oxidizing process (GOD method), light absorption value of the reaction solution at 490nm is measured,
Alpha-glucosidase inhibitory activity is calculated with the content of glucose.Calculation formula is as follows:
Enzyme activity inhibiting rate (%)=[1- (ASample-ASample background)/(ABlank-ABlank background)] × 100%.
Wherein: ABlankFor the light absorption value not plus after tested material reaction, ABlank backgroundFor not plus tested material and enzyme reaction light absorption value,
ASampleFor the light absorption value that tested material reaction is added, ASample backgroundFor the absorbance that the not enzyme reaction of tested material is added.
Test result is as shown in Table 1 and Table 2.
Inhibiting rate of the different elution fractions to maltose that 1 concentration of table is 5mg/mL
By table 1, it can be concluded that, 6 elution fractions in 7 groups of embodiments are the 5th elution fraction to rat small intestine malt
The inhibitory effect of carbohydrase is best, therefore the elution fraction is active component, and each elution fraction of embodiment 7 is compared with remaining 6 groups realities
Elution fraction in example is applied to the inhibiting effect of maltose more preferably.
Inhibiting rate of the active component component to maltose under 2 various concentration of table
Table 2 is the test carried out with inhibiting rate of the active component of various concentration in embodiment 7 to maltose, by table 2
As a result the IC of the active component is obtained50Value is 0.74mg/mL.
Commercially available different alpha-glucosidase restrainers are chosen as control, IC50Respectively 0.89mg/mL, 1.2mg/
ML and 1.44mg/mL.By comparison it can be concluded that, alpha-glucosaccharase enzyme activity in Habenaria Ciliolaris Kranzl prepared by the embodiment of the present invention
Property constituents for suppressing have stronger maltose inhibitory effect.
Therefore, the present invention also provides a kind of alpha-glucosidase activity constituents for suppressing of above-mentioned preparation to prepare phlorose
Application in glycosides enzyme inhibitor.Alpha-glucosidase activity constituents for suppressing in the embodiment of the present invention is to rat small intestine phlorose
The active inhibiting rate IC of Maltose hydrolysis in glycosides enzyme50Value is characterized as 0.74mg/mL.Preferably, because it is to rat small intestine malt
The inhibitory effect of carbohydrase is obvious, therefore can be applied in rat small intestine maltose inhibitor.Again because of the alpha-glucosidase
Activity suppression component comes from natural plants, and toxic side effect is small, can also be used in various health care products.
Test example 2
Using following HPLC analytical method, component content analysis is carried out to the active component in embodiment.
Analysis method: ODS-3 chromatographic column (4.6 × 150mm, 5 μm)；Flow velocity, 1.0mL/min；Column temperature, 30 DEG C；Detect wave
It is long, 254nm；Gradient eluent: 0min, 0% methanol (0.1% formic acid)；10min, 30% methanol (0.1% formic acid)；25min,
45% methanol (0.1% formic acid)；40min, 60% methanol (0.1% formic acid)；50-60min, 100% methanol (0.1% formic acid),
Its result is as shown in Figure 1.
As seen from Figure 1, the active material in the active component be mainly distributed on peak retention time be 21min,
At 22min, 24min, 33.5min and 38min, that is, illustrate alpha-glucosidase activity inhibition group obtained in the embodiment of the present invention
The substance that active function is played in point is present in substance corresponding to the above peak retention time.
In conclusion the embodiment of the present invention is increased between raw material and extractant by crushing to Habenaria Ciliolaris Kranzl raw material
Contact surface connect, improve leaching velocity.Using hydrophilic organic solvent as extractant, it both can guarantee that leaching velocity was fast, extract
Purity is high, impurity are few, and can also reduce cost.It can make in Habenaria Ciliolaris Kranzl using 20-100g:500-6000mL as the first solid-liquid ratio
Active constituent extraction effect it is preferable；Extraction time is selected as 20-50h, can avoid because extraction time it is too short caused by active principle it is molten
Extracting rate is low, and extraction time is too long and the excessive dissolution of impurity, the increases such as grease, tannin in plant cell can be made to isolate and purify
Difficulty the defects of.Using 1g:1-6mL as the second solid-liquid ratio, crude extract medicinal extract can be made to be well-dispersed in distilled water；Using big
Macroporous adsorbent resin chromatographic column combination methanol-water solution is as eluent, using 0.5-2mL/min as elution flow rate, loading can be made molten
The separating effect and recovery rate of active constituent in liquid reach best.Filler is selected from HP-20, and adsorbance is high, particle is uniform, machine
Tool intensity is good, non-breakable and residue is few, can further improve purification effect.This method is easy to operate, it is easy to accomplish, preparation
Alpha-glucosidase activity constituents for suppressing in Habenaria Ciliolaris Kranzl out has good alpha-glucosaccharase enzyme inhibition, especially to wheat
Bud carbohydrase has apparent inhibitory effect.Since the raw material of the invention is natural products, toxic side effect is small, therefore prepared by it
Alpha-glucosidase activity constituents for suppressing can be widely applied in various alpha-glucosidase restrainers.
Embodiments described above is a part of the embodiment of the present invention, instead of all the embodiments.Reality of the invention
The detailed description for applying example is not intended to limit the range of claimed invention, but is merely representative of selected implementation of the invention
Example.Based on the embodiments of the present invention, obtained by those of ordinary skill in the art without making creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.
Claims (9)
1. a kind of preparation method of the alpha-glucosidase activity constituents for suppressing in Habenaria Ciliolaris Kranzl, which is characterized in that it includes following
Step:
Extract: crushing Habenaria Ciliolaris Kranzl to its granularity is 420-580 μm, using hydrophilic organic solvent as extractant, by the first solid-liquid ratio
20-100g:500-6000mL mixes the extractant and the smashed Habenaria Ciliolaris Kranzl, extracts 20-50h, obtains Habenaria Ciliolaris Kranzl and slightly mention
Object；
Separation: it filters the Habenaria Ciliolaris Kranzl and slightly takes object, collect supernatant, be concentrated to give crude extract medicinal extract；
Purifying: the crude extract medicinal extract is dispersed in water by the second solid-liquid ratio 1g:1-6mL, load solution is obtained, with methanol-water
Solution is eluent, and loading described in the elution flow rate gradient elution in macroporous adsorption resin chromatography column with 0.5-2mL/min is molten
Liquid is collected and is concentrated active methanol eluting peak, obtains activity suppression component；
The extractant is selected from methanol-water solution or dehydrated alcohol-aqueous solution.
2. preparation method according to claim 1, which is characterized in that first solid-liquid ratio is 20g:800mL.
3. preparation method according to claim 1, which is characterized in that the time of the extraction is for 24 hours.
4. preparation method according to claim 1, which is characterized in that the filler in the macroporous adsorption resin chromatography column
Selected from HP-20.
5. the preparation method according to claim 4, which is characterized in that the internal diameter of the macroporous adsorption resin chromatography column is
4.5cm, the packed height of the filler are 33cm.
6. preparation method according to claim 1, which is characterized in that during the gradient elution, methanol presses volume hundred
Content of the score meter in the methanol-water solution is followed successively by 0%, 20%, 40%, 60%, 80% and 100%.
7. preparation method according to claim 1, which is characterized in that the elution flow rate is 1mL/min.
8. a kind of alpha-glucosidase activity constituents for suppressing, which is characterized in that it is by the described in any item wild sun of claim 1-7
The preparation method of alpha-glucosidase activity constituents for suppressing in conjunction is prepared.
9. alpha-glucosidase activity constituents for suppressing according to claim 8 is in preparing alpha-glucosidase restrainer
Using.
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|---|---|---|---|---|
| CN1839939A (en) * | 2006-01-12 | 2006-10-04 | 上海大学 | Method for extracting alpha- glucosidase inhibitor from traditional Chinese cinnamomum cassia |
| CN101254238A (en) * | 2008-03-26 | 2008-09-03 | 清华大学 | Method for extracting alpha-glucosidase restraining agent effective ingredient from plants |
| CN101371869A (en) * | 2007-08-24 | 2009-02-25 | 北京北大维信生物科技有限公司 | Inhibitor originated from alpha-glucosidase of natto and preparation method thereof |
| CN104432361A (en) * | 2013-09-13 | 2015-03-25 | 吴渭钟 | Method using habenaria ciliolaris kranzl for making health care beverage |
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|---|---|---|---|---|
| CN1839939A (en) * | 2006-01-12 | 2006-10-04 | 上海大学 | Method for extracting alpha- glucosidase inhibitor from traditional Chinese cinnamomum cassia |
| CN101371869A (en) * | 2007-08-24 | 2009-02-25 | 北京北大维信生物科技有限公司 | Inhibitor originated from alpha-glucosidase of natto and preparation method thereof |
| CN101254238A (en) * | 2008-03-26 | 2008-09-03 | 清华大学 | Method for extracting alpha-glucosidase restraining agent effective ingredient from plants |
| CN104432361A (en) * | 2013-09-13 | 2015-03-25 | 吴渭钟 | Method using habenaria ciliolaris kranzl for making health care beverage |
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