CN107875196A - Complex enzyme, extract solution system, medicament, extract and preparation method thereof, application - Google Patents
Complex enzyme, extract solution system, medicament, extract and preparation method thereof, application Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/60—Moraceae (Mulberry family), e.g. breadfruit or fig
- A61K36/605—Morus (mulberry)
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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Abstract
A kind of complex enzyme, extract solution system, medicament, extract and preparation method thereof, application, belong to field of medicaments.Complex enzyme is used for as the proenzyme material that enzyme extraction is carried out to combination Chinese herbal medicine.Foregoing combination Chinese herbal medicine includes counting in parts by weight：15~100 parts of Radix et Rhizoma Gynurae divaricatae, 25~120 parts of mulberry leaf.Complex enzyme mainly includes mixture.Mixture contains the following components counted in parts by weight：0.1~2 part of pectase；Proteinase-10 .1~2.5 part；Cellulase and hemicellulase amount to 0.1~5 part, and the enzymatic activity of any of mixture enzyme is more than or equal to 50,000 U/g.A kind of complex enzyme proposed by the present invention can be obtained to Radix et Rhizoma Gynurae divaricatae and the extraction effect of mulberry leaf with preferable Quality Control under mild conditions.
Description
Technical field
The present invention relates to field of medicaments, in particular to a kind of complex enzyme, extract solution system, medicament, extract and its
Preparation method, application.
Background technology
Diabetes (diabetes mellitus, DM) are one group comprehensive as caused by E＆H factor collective effect
Sign, its basic pathology feature be insulin secretion definitely or relative deficiency, or peripheral tissues cause with sugar to insulin insensitivity
Based on metabolic disorder, including fat, a kind of systemic disease of protein metabolism disorder.Many researchs confirm that adiposis patient is early
Before exception occurs in glycometabolism, disorders of lipid metabolism is there is, more with different degrees of high fat of blood.It is in the prior art to use more
Diabetes treatment by western medicine, still, for the patient of diabetes and hyperlipidemia complications, conventional Western medicine is difficult to obtain preferable effect.
Pseudo-ginseng be considered as it is a kind of containing the Chinese medicine that can treat diabetes composition, still, existing research based on pseudo-ginseng and corresponding
Medicament it is also not ideal to the therapeutic effect of diabetes.
The content of the invention
Based on the deficiencies in the prior art, the present invention proposes a kind of complex enzyme, extract solution system, extract and its preparation side
Method, application.Obtained by method proposed by the present invention and merged based on the extract of Chinese medicine for diabetes, especially diabetes
High fat of blood has preferable therapeutic effect.
What the present invention was realized in：
A kind of the first aspect of the present invention, there is provided complex enzyme.Complex enzyme is used as carrying out enzyme to combination Chinese herbal medicine
The proenzyme material of extraction.Wherein combining Chinese herbal medicine includes counting in parts by weight：15~100 parts of Radix et Rhizoma Gynurae divaricatae, 25~120 parts of mulberry leaf.
Complex enzyme mainly includes mixture, and mixture contains the following components counted in parts by weight：0.1~2 part of pectase；Protease
0.1~2.5 part；Cellulase and hemicellulase amount to 0.1~5 part, and the enzymatic activity of any of mixture enzyme is more than
Or equal to 50,000 U/g.
A kind of the second aspect of the present invention, there is provided extract solution system.Extract solution system includes above-mentioned complex enzyme, and multiple
Synthase is dispersed in the liquid that pH value is 3.5~7.5, temperature is 35~85 DEG C, and compound enzyme dosage is combination Chinese herbal medicine dry weight
0.1%-2.5%.
The third aspect of the present invention, there is provided a kind of method for preparing extract, extract therein are by foregoing group
Chinese herbal medicine is closed to extract and obtain in extract solution system.Preparing the method for extract includes：Extraction substrate is added into extract solution system
In；After enzyme digestion reaction, carry out separation of solid and liquid and collect separating liquid, extraction substrate is made by combination Chinese herbal medicine, the use of complex enzyme
Measure to extract the 0.1~2.5% of substrate dry weight.
The fourth aspect of the present invention, there is provided a kind of extract, it is prepared by above-mentioned extracting method.
A kind of medicament being prepared by foregoing extract.
A kind of extract as the aforementioned is in the application for preparing in treating diabetes and hyperlipidemia complications medicine.
Beneficial effect：
The biological enzymolysis extractive technique that the present invention uses, reaction condition is gentle, and step is simple, has safe, biological
It is active high, energy consumption is low, it is disposable, facilitate the features such as quality control.By to Radix et Rhizoma Gynurae divaricatae and mulberry leaf extracted
Thing can be used for producing preferable therapeutic effect to the illness of diabetes, especially diabetes and hyperlipidemia complications.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment
Condition person, the condition suggested according to normal condition or manufacturer are carried out.Agents useful for same or the unreceipted production firm person of instrument, it is
The conventional products that can be obtained by commercially available purchase.
Complex enzyme, extract solution system, extract below for the embodiment of the present invention and preparation method thereof, using being had
Body explanation：
The conventional means such as water body or alcohol extracting are usually taken for the medicinal ingredient in Chinese herbal medicine in the prior art, still,
In the view of at present, so some means are all or more or pure different the defects of less.For example, it may be possible to existing medicinal ingredient extraction
Yield it is not high, insufficient, impurity excessively needs to carry out relatively more complicated aftertreatment technology.
It is in the ascendant as a kind of new more excellent selection, enzyme extractive technique.
Enzyme is caused by organism living cells, with a kind of special biocatalyst existing for protein form.Some enzymes
Plant cell wall can be decomposed under normal temperature, normal pressure and gentle acid-base condition, improved by a relatively large margin in natural plants effectively
The recovery rate of composition, improve filtyration velocity and purification effect in production process, improve product purity and the quality of preparation.Biology
Zymolysis technique includes Enzymatic Extraction and the aspect of enzyme process separation and purification two.The technology is to extract basis in traditional natural plant composition
Upper progress, it can be completed using traditional extraction equipment, easy to operate, safe and efficient, cost is cheap.
Enzymatic Extraction is the composition according to plant cell wall, has the characteristics of high specificity using enzyme reaction, selection
Corresponding enzyme, the constituent (cellulose, hemicellulose and pectic substance) of cell membrane is hydrolyzed or degraded, destroys cell membrane knot
Structure, intracellular composition dissolved, be suspended or peptization is in solvent, so as to reach extraction purpose, and be advantageous to improve composition
Recovery rate.Contain protein in many natural plants, solidified using protein heat during decocting method, influence decocting for extract component
Go out, such as add protease, it is possible to the breaks down proteins in natural plants are separated out, can so improve the recovery rate of composition.
Enzyme process removal of impurities be separation and purification new method, the method be according to the species of impurity, property in Nature inorganic bone liquid,
Corresponding enzyme is targetedly used, these impurity are decomposed or removed, to improve the clarity of fluid product, improve product
Stability.Natural plants Aqueous extracts are in addition to containing extract component, also containing starch, protein, pectin, natural gum, resin, mucus
Matter etc., the presence of these compositions often makes extract solution be in suspension, and influences the filtyration velocity of extract solution, to implement to remove for this
Miscellaneous, conventional method has centrifugal process, fining agent method, alcohol deposition method, macroreticular resin absorbing method, ion-exchange, miillpore filter filtration
Method and ultrafiltration.Enzyme reaction has the selectivity of height, therefore determines the high efficiency of enzyme solution removal of impurities.
And normally, the medicinal ingredient in Chinese medicine is relatively complicated, and content compares height, extraction of the prior art
Means are difficult to obtain preferable extraction effect.Also just because of the complexity of the composition in Chinese medicine, and enzyme itself particularity
The limitation of matter and its use condition so that when carrying out the acquisition of medicinal ingredient using enzyme extracting mode, no small barrier be present
Hinder, although improved effect can be obtained in certain some aspects, still remain to be further improved its extraction effect.
Based on described above, a kind of new method that medicinal ingredient is extracted from Chinese medicine and involved original are inventors herein proposed
Material, medicinal material etc..
On the one hand, just it has been observed that enzyme has suitable specificity (selectivity), have preferably reaction to specific material and live
Property.When using specific Chinese medicine, the selection extraction effect that more targetedly enzyme can obtain.On the other hand, respectively
Being used in combination for kind medicinal ingredient, is all played in various degree in occupation mode, dosage and existing way etc. to final drug effect
Influence.It is different preferably to be considered with the use of needs with composition due to the complexity of various composition in Chinese medicine, with
Avoid the occurrence of to the unexpected injury of human body.And the complexity of the medicinal ingredient of Chinese medicine so that single medicinal ingredient extraction and
Difficulty is obtained, or even at most of conditions, it is also futile to recognize specific medicinal ingredient material exactly.Therefore, recognize
Know using suitable different medicinal material compatibilities, and combine the selection of extraction process, obtain the extraction of substance with more satisfactory effect
Need to be difficult to expected work and make great efforts to overcome the obstacle of prior art.
In the present invention, it is proposed that a kind of Chinese medicine composition used that is combined, and enzyme is used based on the Chinese medicine composition
The mode of extraction is to obtain the extraction of substance with preferable medicinal effects.It shall demonstrate the instant invention in more detail below.
Foregoing Chinese medicine composition will hereinafter be substituted with " combination Chinese herbal medicine " and will be quoted.Combination Chinese herbal medicine includes pressing
Parts by weight meter：15~100 parts of Radix et Rhizoma Gynurae divaricatae, 25~120 parts of mulberry leaf.In some instances, the weight of Radix et Rhizoma Gynurae divaricatae and mulberry leaf
Than for 1:1.It is highly preferred that the weight of Radix et Rhizoma Gynurae divaricatae and mulberry leaf ratio is 2:2 or 4:1.
Wherein, Radix et Rhizoma Gynurae divaricatae [Gynura divaricata (L.) DC] is composite family gynura segetum platymiscium, is otherwise known as white
(hundred) sub- dish.Its root, stem, leaf can be used as medicine.Its root nature and flavor is sweet, cool, has the effect of clearing heat and cooling blood, eliminating stasis to subdue swelling, cures mainly branch
Tracheitis, pulmonary tuberculosis, uterine bleeding carbuncle swells, scald, traumatic injury, knife wound bleeding etc.；Its stem, the salty micro-pungent of leaf nature and flavor, cold, poisonous, tool
There are heat-clearing, relaxing muscles and tendons, hemostasis, eliminating the phlegm, cure mainly pertussis, rheumatalgia, fracture, traumatic hemorrhage, too fat to move boil etc..Mulberry leaf
It is conventional medicinal and edible Chinese medicine kind.Mulberry leaf [Mori Folium] are Moraceae [Moraceae] Morus [MorusL] plant mulberries
The dried leaf of [MorusalbaL].Its cold in nature, sweet-bitter flavor, there is the effect of dispelling wind and heat from the body, clearing away the lungheat and moisturizing, clear liver and improve vision
Based on combination of the above Chinese herbal medicine, a kind of complex enzyme for being used to extract medicinal ingredient is suggested.Complex enzyme be used to make
To carry out the proenzyme material of enzyme extraction to combination Chinese herbal medicine.Complex enzyme mainly includes a kind of mixture of enzyme by polymorphic type.Mixing
Thing contains the following components counted in parts by weight：0.1~2 part of pectase；Proteinase-10 .1~2.5 part；Cellulase and hemicellulose
Plain enzyme amounts to 0.1~5 part, and the enzymatic activity of any enzyme in the mixture is more than or equal to 50,000 U/g.Complex enzyme can be with
By the way that four kinds of foregoing enzymes are obtained by mixing by weight proportion.In preferred at some and alternative example, mixture contains weight
Amount ratio is 2:2:1:1 or 2:1:0.5:0.5 cellulase, hemicellulase, pectase and protease.
It has been observed that complex enzyme is used as carrying out combination Chinese herbal medicine the proenzyme material of enzyme extraction, it is being used for carrying out
During extraction, complex enzyme is often used in a kind of specific extraction system.Therefore, the invention also provides one kind is based on
The extract solution system of complex enzyme.Extract solution system can be a kind of liquid dispersion system, and including solid and liquid, solid can be with
It is dissolved in liquid, or is present in a kind of appropriate dispersity in liquid.
Extract solution system includes complex enzyme, and complex enzyme be dispersed in pH value be 3.5~7.5, temperature be 35~85 DEG C
In liquid, the solid-liquid ratio of complex enzyme and water is 1:6-10.In other words, extract solution system comprises at least liquid and complex enzyme, and multiple
Synthase is dispersed in described liquid.In addition, based on enzymatic activity and keep even improving medicinal ingredient in Chinese medicine
Activity, recovery rate, purity etc. consider that it is necessary to select a kind of appropriate enzyme environmental condition or parameter.
For example, in embodiments of the present invention, complex enzyme disperses to make in a liquid the pH value for the liquid dispersion system to be formed to be 3.5
~7.5, temperature is 35~85 DEG C.Preferably, the pH value of liquid dispersion system be 3.9, temperature be 40 DEG C.It is highly preferred that liquid point
The pH value for dissipating system is 4.6, temperature is 50 DEG C.Or in some instances, the pH value of liquid dispersion system is 5.8, temperature 57
℃.Or in other examples, the pH value of liquid dispersion system is 6.6, temperature is 76 DEG C.The pH value of liquid dispersion system and
Temperature can be obtained by various appropriate means, in some cases, the pH value and temperature of liquid dispersion system, can be passed through
Additional substance and/or heat energy is provided realized.
As a kind of optional and preferable alternative solution, complex enzyme is dispersed in the water that pH value is 5.5, temperature is 55 DEG C
In.That is, in a kind of such scheme, in extract solution system is water for receiving by the liquid of scattered complex enzyme.In addition,
In extract solution system, compound enzyme dosage be it is described combination Chinese herbal medicine dry weight 0.1-2.5%, preferably 0.8~1.2%.Or
Person, compound enzyme dosage are the a% of the combination Chinese herbal medicine dry weight, and, a is by 0.3,0.8,0.9,1.8,1.6,2.0,2.3
The interval range value that limits of any one point value or both.
Using combination of the above Chinese herbal medicine and complex enzyme, the invention also provides extract.Described extract can be by
For treating diabetes, diabetes and hyperlipidemia complications illness is especially treated.Medicament can be made based on such a extract
For treating diabetes.Medicament can be tablet, decoction, capsule etc..Normally extract is supported on carrier, such as figuration
Agent or other pharmaceutic adjuvants.
Further, said extracted thing can obtain in the following manner.For example, by combination Chinese herbal medicine in above-mentioned extract solution
Extracted in system.
Extracting method includes：
Step S101, extraction substrate is added in extract solution system.
Wherein, extraction substrate is made by combination Chinese herbal medicine.Normally, extraction substrate is broken section, powder for combining Chinese herbal medicine
Or stamping etc..More preferably, extraction substrate is made by the following method：Dried combination Chinese herbal medicine is crushed and obtained
Powder, then powder lot dispersing in water and soak, the solid-liquid ratio of powder and water reaches 1:6-10.Wherein, in dried combination
Herbal medicine can be Radix et Rhizoma Gynurae divaricatae by buying commercially available drying (as dried in the shade naturally) and mulberry leaf coordinate and formed；Or it will carry on the back in vain
After pseudo-ginseng and mulberry leaf heat drying, proportioning is carried out by weight proportion and is formed.
Furthermore it is possible to the particle diameter of pulverized combination Chinese herbal medicine is limited, such as using the screen cloth with default aperture
Sieving, remove oversize.In a kind of alternative obtained specific example, 60 mesh sieves are crossed.In the combination after crushing that is soaked in water
During herbal medicine, water consumption is preferably that the solid-liquid ratio of powder and water is 1:6-10.As more preferable selection, first with the 3 of powder weight
The water immersion (preferably such as 40~60 DEG C of warm water) of quilt, soak time are 1 hour.Then it is 1 to add water regulation to solid-liquid ratio:6-10
(preferably 1:8), wherein material refers to dry combined traditional Chinese medicine material.It is added after appropriate degree is dipped into, then by complex enzyme
In.
Step S102, after enzyme digestion reaction, carry out separation of solid and liquid and collect separating liquid.The dosage of complex enzyme is is extracted bottom
The 0.1~2.5% of thing dry weight.
Enzyme digestion reaction can be a kind of reaction of reflexive progress, or is adjusted by other means, for example, in enzyme
Continue adjustment ring border pH value and/or temperature (55 DEG C of preferably pH5.5, temperature) in solution preocess.Further, liquid can also be entered
Row stirring, such as 1000~5000r/min.The enzyme digestion reaction time is preferably 2 hours, complex enzyme (wherein, the enzymatic activity of any enzyme
>=5 ten thousand U/g) be added into liquid to carry out solid-liquid split before time interval be preferably 2 hours.
Further, the medicinal ingredient that is extracted into liquid such as it is foregoing be collected in separating liquid, the also medicine of part
Depended in the dregs of a decoction with composition, therefore, considered based on making full use of for Chinese herbal medicine of combination, then by caused by solid-liquid separation step points
Water extraction is carried out with boiling boiling from solid, filters and collects filtrate.Also the medicinal ingredient of part can be obtained in obtained filtrate.
Separating liquid and filtrate can be merged, and powdered processing is carried out after then being inactivated to enzyme, powdered to obtain
Extract.Wherein, the mode of inactivation is usually adopted to heat such as to boil, or can use other known for inventor
Mode is inactivated, or pH regulations, particular ray illumination etc..Preferably, the method for powdered processing is spray drying, more preferably
Ground, the EAT of spray drying is less than 180 DEG C, such as 150 DEG C, 130 DEG C, 100 DEG C；Outlet temperature below 120 DEG C,
Such as 102 DEG C, 88 DEG C, 75 DEG C.
The extraction operation more than, the extract of acquisition are light brown dried powder, mildly bitter flavor.
It should be noted that, although having been described above the part by weight of four kinds of enzymes in complex enzyme, still, medicine extraction is being carried out
When, because the activity of enzyme leaves difference, therefore, the specifically used of each enzyme is preferably adjusted according to its activity, and reaches
The weight of active enzyme (enzymatic activity is more than or equal to 50,000 U/g) reaches foregoing part by weight.
The pharmaceutical research content of the extract obtained below for the present invention.
The experimental study that Radix et Rhizoma Gynurae divaricatae prescription influences on diabetes and hyperlipidemia complications rat model includes：(1) Radix et Rhizoma Gynurae divaricatae
Influence experiment of the prescription to mouse starch-bearing sugar tolerance.(2) Radix et Rhizoma Gynurae divaricatae prescription is to diabetes through animal experimentation blood
The influence of sugar.(3) Radix et Rhizoma Gynurae divaricatae prescription merges the influence of high blood lipid model rat blood sugar to diabetes model.(4) Radix et Rhizoma Gynurae divaricatae
Prescription merges the influence of high blood lipid model rat fat to diabetes model.
1. influence experiment of the Radix et Rhizoma Gynurae divaricatae prescription to mouse starch-bearing sugar tolerance
For evaluation Radix et Rhizoma Gynurae divaricatae and effect of the mulberry leaf formulation in terms of blood glucose-control, design has carried out different prescriptions and extraction
The testing program that method is adjusted to starch-bearing mouse blood sugar.
1.1 test material
1.1.1 medicine
Radix et Rhizoma Gynurae divaricatae water extraction folk prescription A：There is provided by Chengdu bio tech ltd of Sai Nuo Linkages, character：Yellowish-brown powder,
Gas micro-perfume, mildly bitter flavor.
Radix et Rhizoma Gynurae divaricatae enzymolysis folk prescription B：There is provided by Chengdu bio tech ltd of Sai Nuo Linkages, character：Light yellowish brown powder
End, gas micro-perfume, mildly bitter flavor.
Mulberry leaf water extraction folk prescription C：There is provided by Chengdu bio tech ltd of Sai Nuo Linkages, character：Yellowish-brown powder, gas are micro-
It is fragrant.
Mulberry leaf enzymolysis folk prescription D：There is provided by Chengdu bio tech ltd of Sai Nuo Linkages, character：Yellowish-brown powder, gas are micro-
It is fragrant.
Two above water extraction folk prescription technique (A, C)：Medicinal material adds water to cover medicinal material surface, soaks 1 hour, extraction two
Secondary, each 8 times of water of dosing material amount boil extraction 2 hours as solvent.
In two above enzymolysis folk prescription technique (B, D) and the present invention, using mixing of the complex enzyme to Radix et Rhizoma Gynurae divaricatae and mulberry leaf
Medicinal herbs are handled identical to make the technique of extract.
Gliquidone, specification：30mg,
0.9% sodium chloride injection：Kelun Pharm Ind Co., Ltd., Sichuan produces, 250ml：2.25g
1.1.2 reagent
Soluble starch, specification：500g, the production of Chengdu Ke Long chemical reagents factory.
Glucose kit Zhongsheng Beikong Biological Science ＆ Technology Co., Ltd. produces.
1.1.3 animal
Kunming mouse, all-male, 18~22g of body weight, 70, provided by Sichuan Academy of Medical Sciences's Experimental Animal Center,
Meet healthy primary animal, animal productiong licensing：SCXK (river) 2013-15.
1.1.4 instrument
BASIC semiautomatic biochemistry analyzers, Sichuan Mei Sheng Medical Devices Co., Ltd produce.
Three use electric heat constant temperature water tank：Model HH.S21-NI6, the production of Beijing Chang'an scientific instrument factory.
Electronic analytical balance：FA1004 types, upper current chart level instruments and meters Co., Ltd production.
TDL-80-2B medical centrifuges, Anting Scientific Instrument Factory, Shanghai's production.
Adjustable pipette (FinnPipette Colour 4027), specification：100~1000ul, sequence number：ZX29155,
Thermoelectricity (Shanghai) Instrument Ltd..
1.2 methods and result
Take healthy Kunming mouse 70,18~22g of body weight, before experiment fasting can't help water 12 hours, each group is pressed during experiment
Medicine listed by table 1 and dosage gavage single administration, 1 hour after administration, except blank control group gives starch-bearing, starch-bearing agent
Measure as 5g/kg.
Eye socket angular vein blood sampling in 1 hour, determines blood glucose, compares each group blood glucose value, the results are shown in Table 2 after starch-bearing.
The dosage regimen of table 1
The starch-bearing result of the test of table 2
Note：Administration group * P compared with model group<0.05, * * P<0.01, * * * P<0.001
1.3 conclusion (of pressure testing)
Result of the test is shown：Each administration group can reduce the rise of mouse blood sugar after starch-bearing compared with model group, its
In, Radix et Rhizoma Gynurae divaricatae enzymolysis folk prescription B blood glucose values are minimum, but tetra- prescriptions of ABCD do not have significant difference.For the white back of the body of further evaluation
The quality of notoginseng composition blood sugar decreasing effect, continue the experimental study that alloxan causes rat diabetes model.
2. influence of the Radix et Rhizoma Gynurae divaricatae prescription to diabetic model rats blood glucose
2.1 test material
2.1.1 medicine
Same 1.1.1
2.1.2 animal
SD kind rats, all-male, 180~220g of body weight, 50, provided by Sichuan Academy of Medical Sciences's Experimental Animal Center,
Meet healthy primary animal, animal productiong licensing：SCXK (river) 2013-15.
2.2 methods and result
The problem of experiment is due to experimental animal quantity, is split as four progress, investigates Radix et Rhizoma Gynurae divaricatae, mulberry leaf, peppery respectively
Extracting method, dosage, the compound compatibility ratio of wooden leaf.
2.2.1 extracting method is investigated
Body weight 180~220g, SD kind rat is taken, all-male, after water 12h is can't help in fasting, every rat is injected intraperitoneally by body weight
Alloxan 200mg/kg, 72h tail vein bloods determine blood glucose after injection.After blood glucose rise, with blood sugar level layering packet 10
Only/group, medicine and dosage as shown in table 3, difference gastric infusion, 1 times/day, continuous 4 weeks.Take SD similar in 10 body weight simultaneously
Rat is as Normal group, the gastric infusion only as shown in table 3,1 times/day, continuous 4 weeks.Every 1 week tail vein takes blood in experiment,
2500rpm/min centrifuges serum, and measure blood sugar effects are shown in Table 4.
The dosage regimen of table 3
The Radix et Rhizoma Gynurae divaricatae of table 4, mulberry leaf Different Extraction Method successive administration surrounding blood glucose value
Note：Compared with model group, * * P ＜ 0.01, * P ＜ 0.05
2.2.1.1 experimental result
Result of the test is shown：Each administration group is compared with model group, the rise of rat blood sugar caused by reducing alloxan,
Wherein, Radix et Rhizoma Gynurae divaricatae enzymolysis folk prescription B effects are best, and enzymatic extraction method is better than water extraction, but tetra- prescriptions of ABCD do not have conspicuousness
Difference.Continue investigating for dosage.
2.2.2 the investigation of dosage
Body weight 180~220g, SD kind rat is taken, all-male, after water 12h is can't help in fasting, every rat is injected intraperitoneally by body weight
Alloxan 200mg/kg, 72h tail vein bloods determine blood glucose after injection.After blood glucose rise, with blood sugar level layering packet 10
Only/group, medicine and dosage as shown in table 5, difference gastric infusion, 1 times/day, continuous 4 weeks.Take SD similar in 10 body weight simultaneously
Rat is as Normal group, the gastric infusion only as shown in table 5,1 times/day, continuous 4 weeks.Every 1 week tail vein takes blood in experiment,
2500rpm/min centrifuges serum, determines blood glucose.It the results are shown in Table 6.
The dosage regimen of table 5
The Radix et Rhizoma Gynurae divaricatae of table 6, mulberry leaf various dose method successive administration surrounding blood glucose value
Note：Compared with model group, * * P ＜ 0.01, * P ＜ 0.05
2.2.2.1 experimental result
Result of the test is shown：Each administration group is compared with model group, the rise of rat blood sugar caused by reducing alloxan,
Wherein, the zymolyte of two medicinal materials has a certain amount to imitate relation.Continue the investigation of compound proportion.
2.2.3 the investigation of proportion compatibility
Body weight 180~220g, SD kind rat is taken, all-male, after water 12h is can't help in fasting, every rat is injected intraperitoneally by body weight
Alloxan 200mg/kg, 72h tail vein bloods determine blood glucose after injection.After blood glucose rise, with blood sugar level layering packet 10
Only/group, medicine and dosage as shown in table 7, difference gastric infusion, 1 times/day, continuous 4 weeks.Take SD similar in 10 body weight simultaneously
Rat is as Normal group, the gastric infusion only as shown in table 5,1 times/day, continuous 4 weeks.Every 1 week tail vein takes blood in experiment,
2500rpm/min centrifuges serum, determines blood glucose, the results are shown in Table 8.
The dosage regimen of table 7
The Radix et Rhizoma Gynurae divaricatae of table 8, mulberry leaf different ratio successive administration surrounding blood glucose value
Note：Compared with model group, * * P ＜ 0.01, * P ＜ 0.05
3. influence of the Radix et Rhizoma Gynurae divaricatae prescription to diabetes and hyperlipidemia complications rat model blood glucose
3.1 experiment material
3.1.1 medicine and reference substance
See 1.1.1
The morbidity machine of high fat of blood animal model caused by the rise of Alloxan diabetic animal models blood fat is with high lipid food
Reason completely it is different, and gliquidone clinic have be directed to hyperglycaemia merge high fat of blood case, therefore this experiment selection this medicine for
Positive control drug.
3.1.2 reagent
Blood sugar kit, T-CHOL kit (enzyme process), triglyceride reagent box (enzyme process), HDL courage are consolidated
Alcohol kit, LDL-C kit
3.1.3 instrument
MS-500E semi-automatic biochemical analyzers
TDL-80-2B medical centrifuges
3.1.4 animal
Same 2.1.2
3.1.5 high-calorie feed
Basal feed 92%, 2% sucrose, 3% lard, 2% cholesterol and 1% hyocholic acid sodium mix.
3.2 methods and result
Alloxan 150mg/kg is injected intraperitoneally by body weight in rat, causes Alloxan diabetes models, successful to modeling
Rat is fed with high-calorie feed (compound method is shown in 3.1.5), the synchronous medicine as listed by table 9 and dosage difference gastric infusion, 1 time/
Day, continuous 4 weeks.SD rats similar in 10 body weight are taken as Normal group simultaneously, the gastric infusion only as shown in table 5,1 time/
Day, continuous 4 weeks.Serum, measure serum blood glucose, T-CHOL, triglycerides, height are taken in the 2nd week of administration and the 4th weekend rat-tail
Density lipoprotein-cholesterol, LDL-C content.It the results are shown in Table 10.
The dosage regimen of table 9
The Radix et Rhizoma Gynurae divaricatae prescription of table 10 merges the influence of hyperlipemia rat TG value, T-chol values, HDL values, LDL values to hyperglycaemia
Note：Compared with model group, * * P ＜ 0.01, * P ＜ 0.05
To sum up, the biological enzymolysis extractive technique that the present invention uses, water can be used as Extraction solvent.Extracting method step
Simply, mild condition, the extract of acquisition can obtain suitable with the diabetes indication class medicament that uses in the prior art
Effect.And based on described extracting method, a kind of alternative medicament of proposition has that use cost is low, bioactivity is high
The advantages of, and due to manufacture craft is safe, energy consumption is low, it is disposable, facilitate quality control, beneficial to volume production is carried out, reduction makes
The use cost of user.
With reference to embodiments to the complex enzyme of the present invention, extract solution system, extract and preparation method thereof, using make into
The detailed description of one step.
Embodiment 1
A kind of medicinal extract using multiplex-enzyme extraction from Chinese medicine.
Described Chinese medicine includes 25 parts of 15 parts of Radix et Rhizoma Gynurae divaricatae, the mulberry leaf of parts by weight meter.Complex enzyme by by cellulase,
Hemicellulase, pectase and protease are according to part by weight 2:2:1:1 carries out compatibility.
Extracting method is as follows.
The first step, medicinal material pre-treatment：Radix et Rhizoma Gynurae divaricatae, mulberry leaf, crushing and the sieve of 60 mesh medicines excessively is standby, then weighed by formula
Medicinal material, the water of 3 times of amount medicinal materials is added, is soaked 1 hour.
Second step, enzymolysis and extraction：Water is added, reaches 1 to solid-liquid ratio:6-10, then adjust pH to 3.5-7.5, heating
To 35-85 DEG C, then add complex enzyme and carry out enzymolysis and extraction.Wherein solid-liquid ratio most preferably 1:8, optimal pH 5.5, optimum extraction
Temperature is 55 DEG C, and mixing speed is 1000-5000r/min in extraction process.Complex enzyme is by cellulase, hemicellulase, fruit
Glue enzyme, protease composition, its ratio are 2:2:1:1 or 2:1:0.5:0.5, compound enzyme dosage is extraction substrate dry weight
0.1%-2.5%, optimum amount 0.8-1.2%, the U/g of the enzymatic activity of each single enzyme in complex enzyme >=50,000.Extraction time is 2
Hour.
3rd step, filtering：Enzyme digestion reaction mixture is crossed into 200 mesh medicines sieve, takes filtrate standby.
4th step, dregs of a decoction processing：The dregs of a decoction add 4 times of amount water, boil 1 hour and extract, and then 200 mesh medicines excessively, which sieve, is filtered
Liquid.
5th step, inactivation：Merge filtrate twice, 10min inactivations are boiled in heating.
6th step, spray drying：At 180 DEG C, outlet temperature is controlled at 120 DEG C, is finally extracted for EAT control
Thing.
The medicinal extract raw material of preparation in embodiment 2-8, technological process such as embodiment 1, the main distinction is recited in
In table 11.
The extract extraction conditions of table 11
Note：Dosage refers to that compound enzyme dosage accounts for the ratio of combination Chinese herbal medicine dry weight in table.
Comparative example 1
Radix et Rhizoma Gynurae divaricatae water extraction：Radix et Rhizoma Gynurae divaricatae adds water to cover medicinal material surface, soaks 1 hour, extracts twice, each dosing
8 times of water of material amount boil extraction 2 hours, filtering obtains filtrate, and drying is solid as solvent.
Radix et Rhizoma Gynurae divaricatae digests：Technique is referring to embodiment 1, the group 4 in specific extract solution system, spray drying such as table 11.
Mulberry leaf water extraction：Mulberry leaf add water to cover medicinal material surface, soak 1 hour, extract twice, each 8 times of dosing material amount
Water boils extraction 2 hours, filtering obtains filtrate, and drying is solid as solvent.
Mulberry leaf digest：Technique is referring to embodiment 1, the group 3 in specific extract solution system, spray drying such as table 11.
Radix et Rhizoma Gynurae divaricatae mulberry leaf water extraction：Radix et Rhizoma Gynurae divaricatae (2 parts by weight) and mulberry leaf (1 parts by weight) plus water cover medicinal material surface i.e.
Can, soak 1 hour, twice, each 8 times of water of dosing material amount boil extraction 2 hours, filtering obtains filtrate, does as solvent for extraction
Dry is solid.
Radix et Rhizoma Gynurae divaricatae mulberry leaf digest：Radix et Rhizoma Gynurae divaricatae (2 parts by weight) and mulberry leaf (1 parts by weight).Technique is referring to embodiment 1, tool
Group 3 in body extract solution system, spray drying such as table 11.
The Different Extraction Method product yield of table 12 and index components contrast
Although illustrate and describing the present invention with specific embodiment, but will be appreciated that without departing substantially from the present invention's
Many other change and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
- A kind of 1. complex enzyme, it is characterised in that for as to combination Chinese herbal medicine carry out enzyme extraction proenzyme material, in the combination Herbal medicine includes counting in parts by weight：15~100 parts of Radix et Rhizoma Gynurae divaricatae, 25~120 parts of mulberry leaf, the complex enzyme mainly include mixing Thing, the mixture contain the following components counted in parts by weight：0.1~2 part of pectase；Proteinase-10 .1~2.5 part；Fiber Plain enzyme and hemicellulase amount to 0.1~5 part, and the enzymatic activity of any of described mixture enzyme is more than or equal to 50,000 U/g.
- A kind of 2. extract solution system, it is characterised in that the extract solution system includes complex enzyme as claimed in claim 1, and The complex enzyme is dispersed in the liquid that pH value is 3.5~7.5, temperature is 35~85 DEG C, and the compound enzyme dosage is described Combine the 0.1%-2.5% of Chinese herbal medicine dry weight.
- 3. extract solution system according to claim 2, it is characterised in that the complex enzyme is dispersed in pH value as 5.5, temperature Spend in the water for 55 DEG C.
- 4. the extract solution system according to Claims 2 or 3, it is characterised in that in the extract solution system, the combination The solid-liquid ratio of Chinese herbal medicine and the liquid is 1:8.
- A kind of 5. method for preparing extract, it is characterised in that the extract is will in such as right by the combination Chinese herbal medicine Ask and extract and obtain in the extract solution system any one of 2~4, methods described includes：Extraction substrate is added into the extraction In liquid system；After enzyme digestion reaction, carry out separation of solid and liquid and collect separating liquid, the extraction substrate is by the combination Chinese herbal medicine system , the dosage of the complex enzyme is the 0.1~2.5% of the extraction substrate dry weight.
- 6. according to the method for claim 5, it is characterised in that solid water will be separated caused by the solid-liquid separation step Carry out water extraction is boiled, filters and collects filtrate；Methods described also includes：After filtering and collecting filtrate, the separating liquid and the filtrate are alternatively merged, is gone out Powdered processing is carried out after work；Preferably, the method for powdered processing is spray drying, it is highly preferred that the EAT of spray drying is less than 180 DEG C, Outlet temperature is below 120 DEG C.
- 7. according to the method for claim 5, it is characterised in that the extraction substrate is made by the following method：Will The dried combination Chinese herbal medicine, which crushes, obtains powder, and then the powder lot dispersing in water and soaks, the material of powder and water Liquor ratio is 1:6-10.
- A kind of 8. extract, it is characterised in that method of the extract according to any one of claim 5-7 prepare and Into.
- 9. a kind of medicament, it is characterised in that the medicament is prepared by extract such as according to claim 8.
- 10. a kind of extract as claimed in claim 8 is for preparing the application in treating diabetes and hyperlipidemia complications medicine.
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