CN110184317A - A kind of preparation method and application of trifloroside and derivative - Google Patents
A kind of preparation method and application of trifloroside and derivative Download PDFInfo
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- CN110184317A CN110184317A CN201910433385.3A CN201910433385A CN110184317A CN 110184317 A CN110184317 A CN 110184317A CN 201910433385 A CN201910433385 A CN 201910433385A CN 110184317 A CN110184317 A CN 110184317A
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- Prior art keywords
- trifloroside
- glycosylation
- derivative
- preparation
- reaction
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- RMBMLYUFYBZPCX-DZPSSIEHSA-N Trifloroside Natural products O=C(OC[C@H]1[C@@H](OC(=O)c2c(O)c(O[C@H]3[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O3)ccc2)[C@H](OC(=O)C)[C@@H](OC(=O)C)[C@H](O[C@H]2[C@@H](C=C)[C@H]3C(C(=O)OCC3)=CO2)O1)C RMBMLYUFYBZPCX-DZPSSIEHSA-N 0.000 title claims abstract description 113
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 21
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- VGEREEWJJVICBM-UHFFFAOYSA-N phloretin Chemical compound C1=CC(O)=CC=C1CCC(=O)C1=C(O)C=C(O)C=C1O VGEREEWJJVICBM-UHFFFAOYSA-N 0.000 claims abstract description 12
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- 238000001802 infusion Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
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- 238000012417 linear regression Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
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- 231100000956 nontoxicity Toxicity 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
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- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- IOUVKUPGCMBWBT-UHFFFAOYSA-N phloridzosid Natural products OC1C(O)C(O)C(CO)OC1OC1=CC(O)=CC(O)=C1C(=O)CCC1=CC=C(O)C=C1 IOUVKUPGCMBWBT-UHFFFAOYSA-N 0.000 description 1
- 235000019139 phlorizin Nutrition 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 201000009104 prediabetes syndrome Diseases 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000004224 protection Effects 0.000 description 1
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- 230000000384 rearing effect Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
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- 230000001954 sterilising effect Effects 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
Present disclose provides the preparation method and application of a kind of glycosylation trifloroside and derivative, and the method specific steps include: that raw material and glycosyl donor are put into reaction buffer；Feed liquid is after mixing；Then glycosidase and glycosyl transferase is added in reaction system；It is eventually adding dehydrogenase, the raw material is trifloroside, the extract of phloretin or conversion product and its derivative.The solubility of prepared glycosylation trifloroside in water is 300 times of conventional trifloroside water solubility in the method, the solubility of effective trifloroside also improves 50 times or more, it solves the problems, such as that trifloroside solubility in food, drug, cosmetics and health care product is difficult, it is difficult to absorb, considerably increases bioavailability.
Description
Technical field
This disclosure relates to bioengineering field, and in particular to the preparation method and application of a kind of trifloroside and derivative.
Background technique
Flavone compound has mammal and other types of cell due to its unique chemical structure many heavy
Physiology, the medicine healthy sofa wanted act on.Further investigation with domestic and international chemist to its structure-activity relationship, it was found that flavonoid
The mechanism of action of object Pharmacological provides theoretical foundation for its application in medicine, field of food, accelerates flavonoids
The development and utilization of compound.
Trifloroside (phloretin -4'- beta-glucosidase, p- phloridzin) sugariness is 300 times of sucrose, as sweetener by generation
Boundary's health organization and FAO (Food and Agriculture Organization of the United Nation) include, and have natural and safety.Trifloroside has good pharmacological action and life
Reason activity；Opening the firm equal trifloroside that describes in the literature has inhibition diabetes key enzyme (alpha-glucosidase and alpha-amylase)
Activity and inoxidizability；Amol Gupte etc. in the literature experiments have shown that trifloroside has inhibiting effect to nucleoside transporter, and
And it is better than hENT1 (mankind's balanced type nucleoside transporter) to the selectivity of Hcnt3 (mankind's concentration type nucleoside transporter), it shows
Show its potential application foreground in anticancer, antiviral, cardiovascular and neuroprotection field；Trifloroside also shows anti-angiogenic receipts
Contracting effect.(Gu Shuhua, Sun Weixin, Zhao Weimin wait a kind of a kind of three leaves of activity extract of trifloroside of and application thereof to Gu Shuhua
Activity extract of glycosides and application thereof [P] .CN 101874824 A, 2010.) in, carry out trifloroside extract for treating glycosuria
The swollen drug efficacy study of sick nephrosis, edema due to dysfunction of the liver, the results showed that trifloroside has protection kidney, liver cell and hypoglycemic, blood lipid and albuminuria
Effect.
Trifloroside has the function of removing free radical, anti-inflammatory and promoting blood circulation, but the solubility of trifloroside is low, cannot be fine
Ground is absorbed and utilized by small intestine, has seriously affected the bioavilability of trifloroside.The solubility and bioavilability of trifloroside are improved,
Trifloroside really can be realized into industrialization, there is great meaning to the application and development of trifloroside.
Summary of the invention
Purpose of this disclosure is to provide the preparation methods of a kind of glycosylation trifloroside and derivative, to reach raising trifloroside
Solubility, to increase bioavailability.
To achieve the above object, technical solution is as follows:
A kind of preparation method glycosylating trifloroside and derivative, specific steps include:
Raw material and glycosyl donor are put into reaction buffer, feed liquid after mixing, is then added in reaction system
Glycosidase and glycosyl transferase.
The raw material is trifloroside, the extract of phloretin or conversion product and its derivative.
The glycosyl donor is glucose, UDP-glucose, xylose, UDP- xylose, galactolipin, UDP- galactolipin, red moss
Sugar, alpha-cyclodextrin, beta-cyclodextrin, starch, xylan, sucrose, maltodextrin or rhamnose, the concentration of the glycosyl donor are 1-
10g/L。
The NaCl buffering that the reaction buffer is disodium hydrogen phosphate-sodium citrate buffer solution or mass fraction is 0.9%
Liquid, wherein the mass fraction of the disodium hydrogen phosphate-sodium citrate buffer solution is 0.05-1%.
The ratio of the raw material and reaction buffer is (1:10)-(1:50), and the pH value of the reaction buffer is 3.1-
6.6, reaction temperature is 35-75 DEG C, and the time of the reaction is 1-24h.
The glycosidase is rhamnosidase, glucuroide, and glycosyl transferase is glucosyltransferase, xylosyl
The combination of one or both of transferase and galactosyltransferase.
The glycosidase of the addition and the ratio of glycosyl transferase are (10:90)-(90:10), described two enzyme total amounts
Mass fraction is the 10%-40% of adding raw materials, and the mass fraction advanced optimizes as 10%-20%.
According to the structure of glycosylation trifloroside and derivative obtained by the above method are as follows:
Wherein: R=Glu, Rha, Xyl or Gal；N=1-9, structure advanced optimize as R=Glu or Rha；N=1-5.
A kind of pharmaceutical composition, comprising above-mentioned glycosylation trifloroside and derivative, pharmaceutically acceptable salt, carrier,
Excipient, diluent, medium or their combination.
The glycosylation trifloroside and derivative are preventing and treating pernio, are removing free radical, activating microcirculation and removing stasis medicinal, anti-inflammatory and quiet
Application in terms of arteries and veins varicose.
Application of the glycosylation trifloroside on skin care item, food, drug and health care product.
The beneficial effect of the disclosure is mainly reflected in: provide it is a kind of glycosylation trifloroside and derivative preparation method and
Using, the solubility of prepared glycosylation trifloroside in water is 300 times of conventional trifloroside water solubility in the method,
The solubility of effective trifloroside also improves 50 times or more, and it is molten in food, drug, cosmetics and health care product to solve trifloroside
Solution degree is difficult, absorbs difficult problem, considerably increases bioavailability.
Detailed description of the invention
The HPLC liquid phase figure of Fig. 1 glycosylation trifloroside.
Fig. 2 phloretin solubility matched curve.
Fig. 3 glycosylates trifloroside and trifloroside solubility comparison diagram.
Fig. 4 blood glucose curve.
Fig. 5 glycosylates trifloroside and Vc antiphlogistic effects comparison diagram.
Specific embodiment
Following steps are only to illustrate the technical solution of the disclosure, rather than its limitations；Although referring to These steps
The disclosure is described in detail, but those skilled in the art should understand that: it still can be to aforementioned each step
Technical solution documented by rapid is modified, or equivalent substitution of some or all of the technical features；And these
It modifies or replaces, the range of each step technique scheme of the disclosure that it does not separate the essence of the corresponding technical solution.
Embodiment 1
A kind of preparation method glycosylating trifloroside and its derivative, specific steps include:
Using trifloroside as raw material, reaction buffer is disodium hydrogen phosphate-sodium citrate buffer solution that mass fraction is 1%,
The pH5.4 of reaction buffer, 60 DEG C of reaction temperature, the reaction time controls 6h, and the ratio of raw material and reaction buffer is 1:20；So
After add glucuroide: in glycosyltransferase=20:80 reaction system, the mass fraction of enzyme be investment trifloroside
15%, there are also glucose 30g/L in the reaction system.In the reaction system, polysaccharide trifloroside, yield 98.5% are obtained.Knot
Fruit such as Fig. 1.From fig. 1, it can be seen that the reaction can effectively prepare polysaccharide trifloroside, due to the number of the glycosyl of polysaccharide trifloroside link
Amount is different, and appearance time will appear the peak once reduced in the figure of HPLC.
Embodiment 2
A kind of preparation method glycosylating trifloroside and its derivative, specific steps include:
Using trifloroside as raw material, reaction buffer is disodium hydrogen phosphate-sodium citrate buffer solution that mass fraction is 1%,
The pH6 of buffer, 56 DEG C of reaction temperature, the reaction time controls 8h, and the ratio of raw material and reaction buffer is 1:50；Then anti-
Answer and rhamnosidase is added portionwise in system: glucosyltransferase=15:80, the mass fraction of enzyme are investment trifloroside
16%, rhamnose 5g/L.In the reaction system, more rhamnose triflorosides (n=1-5), yield 96.1% are obtained.
Embodiment 3
A kind of preparation method glycosylating trifloroside and its derivative, specific steps include:
Using trifloroside as raw material, reaction buffer is disodium hydrogen phosphate-sodium citrate buffer solution that mass fraction is 1%,
The pH5.5 of buffer, 60 DEG C of reaction temperature, the reaction time controls 10h, and the ratio of raw material and reaction buffer is 1:10；Then
Glucuroide is added in the reaction system: glycosyltransferase=15:85, the mass fraction of enzyme are investment trifloroside
20%, xylose 30g/L.In the reaction system, xylan trifloroside (n=1-5) is obtained, yield is 97.9%.
Embodiment 4
A kind of preparation method glycosylating trifloroside and its derivative, specific steps include:
Using trifloroside as raw material, reaction buffer is disodium hydrogen phosphate-sodium citrate buffer solution that mass fraction is 1%,
The pH5 of buffer, 60 DEG C of reaction temperature, the reaction time controls 12h, and the ratio of raw material and reaction buffer is 1:25.Then exist
Glucuroide is added in reaction system: galactolipin glycosyl transferase=25:70, the mass fraction of enzyme are investment trifloroside
15%, galactolipin 20g/L.In the reaction system, galactosan trifloroside (n=1-5) is obtained, yield is 98.5%.
Embodiment 5 glycosylates trifloroside solubility test
The size of compound solvability directly influences drug in the application of solution system and cell system.This experiment benefit
The measurement of solubility values is carried out with aqueous solution of the HPLC method to the glycosylation trifloroside of saturation state.It is accurate respectively in this experiment
The phloretin for measuring 10mg, 50mg, 100mg, 200mg, 1000mg, is placed in 100mL volumetric flask, DMSO is added to be diluted to scale,
It shakes up, obtaining a series of concentration is respectively 0.1mg/mL, 0.5mg/mL, 1mg/mL, the trifloroside mark of 2mg/mL, 10mg/mL
Quasi- solution, is analyzed with HPLC, is integrated to the characteristic peak in the section 283nm, and peak area is recorded.It is vertical sit with the concentration of trifloroside
Mark, peak area is abscissa mapping, and carries out linear regression.
Calibration curve equation 1:y=0.00151A+0.00966, R2=0.9999 is trifloroside matched curve, such as Fig. 2.
Then glycosylation trifloroside and 0.2g trifloroside prepared by 0.2g embodiment 1 are added separately in 2mL water phase, are set
It in continuous oscillation 72h on 25 ± 1 DEG C of constant temperature oscillators, is transferred in centrifuge tube after taking-up, 8000r/min is centrifuged 15min, takes
0.45 μm of filtering with microporous membrane of clear liquid, and within methanol dilution to the range of linearity, three leaves of glycosylation are measured using HPLC method
The solubility of glycosides and trifloroside in water, as a result such as Fig. 3.
According to the linear of Fig. 3 the result shows that: the solubility for glycosylating trifloroside is 600 times or so of trifloroside.
The measurement of the glycosylation trifloroside urgency toxicity of embodiment 6
At 28 ± 1 DEG C of temperature, 70 ± 5% damp condition, 7~8 week old, healthy cleaning grade NIH mouse are chosen
20 half male and half females divide 2 groups of male and female each 5, and weight is in 20-22g.By feed and water sterilization, the test preceding observation period with test
It is interior, raised by chow diet condition.
Glycosylation trifloroside prepared by embodiment 1 is dissolved in 0.5%Tween80, concentration 500mg/mL, by the liquid
Body oral administration mouse is dosage by mouse weight 0.02ml/g.Observe Isosorbide-5-Nitrae after administration, 8,12h, every 12h observation later
Once.Death condition is observed, records mouse weight variation and other symptoms daily.It 10th day, is put to death in a manner of disconnected neck
Mouse takes each organ to carry out pathologic finding.
At the 10th day, the glycosylation trifloroside of whole mouse survivals, 0.02ml/g dosage had no toxic reaction.Each device of mouse
Official's pathologic finding is normal, does not find lesion, mouse weight has no mitigation in 10 days.Therefore, illustrate glycosylation three of the invention
Leaf glycosides has no toxicity when animal is administered orally.
Embodiment 7 glycosylates the anti-inflammatory experiment of trifloroside
1. experimental material
5-6 weeks female Balb/c mouse of SPF grade of the experimental animal from Guangdong Province's Experimental Animal Center；1 institute of embodiment
Preparation glycosylation trifloroside；TPA-99.99%, pharmaceutical college, Zhongshan University；Acetone, Guangzhou chemical reagent work；Methylene chloride, Guangzhou reagent
Factory；DMSO, Guangzhou chemical reagent work, brufen, Aladdin industrial group, the U.S..
2. experimental method
1) 40 Balb/c mouse are randomly divided into five groups, are acetone blank group, TPA control group, curcumin group, thickness respectively
Plain phenol and compound group, every group of 1 mouse, are repeated 3 times.
2) acetone blank group and TPA control group use the solution (methylene chloride: acetone=80:20) of 15 μ L autogamys uniform respectively
Two ears of mouse are applied to, and glycosylates trifloroside and is uniformly applied to two ears of mouse.
3) after 6min, acetone blank group is uniformly applied to two ears of mouse with acetone: and TPA control group, three leaves of glycosylation
Glycosides group is then uniformly applied to two ear two sides of mouse with 15 μ L TPA respectively and causes inflammation.
4) after 6h, mouse is put to death, then cuts mouse ears in time, and with the punch of diameter 6mm respectively same
Round auricle is laid at position.
5) weigh, be feminine gender with acetone blank group, using the difference of experimental group auricle weight and blank group auricle weight as
Swelling.
6) inhibiting rate=(1- administration group swelling/control group swelling) × 100%.
3. analysis of experimental results
In order to study inhibiting effect of the glycosylation trifloroside to the TPA mice auricle swelling induced, selected glycosylation three
The working concentration of leaf glycosides is 0.75 μM.By to glycosylation trifloroside to the Inhibition test of mice auricle swelling the study found that list
Pure smearing TPA inducing mouse auricle edema causes the average weight of Mice Auricle to be increased weight by 5.3mg to 11.5mg, and glycosylates
To the inhibiting effect of mice auricle swelling all showed differents, the inhibiting rate range for glycosylating trifloroside is trifloroside
91.7%-92.6%.
It is apparent that glycosylation trifloroside plays inhibiting effect to the mice auricle swelling that TPA is induced.By results of animal table
Bright, the inhibiting rate for glycosylating trifloroside is respectively 92.6% and 91.7%, and the inhibiting rate of brufen and NSAIDs are 95.3% He
86.1%.
Embodiment 8 glycosylates influence of the trifloroside to alimentary obesity C57BL/6J mouse glycolipid metabolism
1. experimental material
1.1 experimental animals and condition
C57BL/6J mouse, male, SPF grades, 4 week old are provided by Zhongshan University's Experimental Animal Center.Credit number:
SCXK (Guangdong) 2009~0004.Rearing conditions: 20-25 DEG C of temperature, humidity 55 ± 1%, 12h/12h light dark cycle, for 24 hours certainly
It is drunk water by diet.
1.2 key agents and reagent
Glycosylating trifloroside is prepared by embodiment 1；Simvastatin, Hangzhou Mo Shadong pharmaceutical Co. Ltd；Standard feed
And high lipid food, it is provided by Guangdong Province's animal center；Triglycerides (TG) kit, the middle raw north control limited public affairs of biotechnology share
Department provides；Total cholesterol (TC) kit, Zhongsheng Beikong Biological Science & Technology Co., Ltd. provide；Direct high-density lipoprotein gallbladder
Sterol (HDL-C) assay kit, Zhongsheng Beikong Biological Science & Technology Co., Ltd.；Direct low density lipoprotein cholesterol
(LDL-C) assay kit, Zhongsheng Beikong Biological Science & Technology Co., Ltd.；Free fatty acid microdetermination kit, Puli
Lema gene Technology Co., Ltd.；Superior type blood sugar test paper, company, Roche Group, Switzerland provide Luo Kang entirely.
2. experimental method and interpretation of result
2.1 the selected of animal, grouping and processing
C57BL/6J male mice randomly selects 10 as Normal group, feed with common standard feed (feed formula:
Crude protein 18.5%, crude fat 4.5%, carbohydrate 61%.Energy ratio: crude protein 21%, crude fat 11%, carbon hydrate
Object 68%, 3.5 Kcal/g of gross energy), remaining mouse gives high glucose and high fat feed (feed formula: crude protein 27%, crude fat
24%, carbohydrate 37%；Energy ratio: protein 23 %, fat 46%, carbohydrate 31%, 4.7 Kcal/ of gross energy
g).After feeding 12 weeks, reference weight is greater than the average weight ± 1.78 times standard deviation normally organized and greater than the 20% of normal type
Standard, choose qualified obesity mice, be divided into model control group, positive drug Simvastatin according to the uniform principle of weight
Group glycosylates trifloroside dosage group, and every group 10, animal is marked respectively between group and in group.Normal group gives common feeding
Material, remaining each group continue to give high glucose and high fat forage feed.The prepared glycosylation trifloroside of embodiment 1 is configured to distilled water
1000mg/L, Simvastatin are configured to 100mg/L (ready-to-use) with distilled water.
Glycosylate the dosage of trifloroside:, it by mouse weight 100ml/kg, is administered once daily, successive administration 14 weeks.
Simvastatin drug dose is administered once daily by mouse weight 0.3ml/kg, and successive administration 14 weeks.
Model control group and Normal group gavage isometric physiological saline daily, are administered once daily, successive administration 14
Week.
To the weight of each group mouse detects before administration and after administration, to observe the changes of weight of separate groups of mice.
2.2 Mouse oral glucose tolerance tests (OGTT)
After mouse fasting 12h (can't help water), by each medicine group corresponding dosage gastric infusion, trifloroside group, model are glycosylated
Control group, Normal group and Simvastatin group give solvent, and every group 6.After 1h, each group is with the filling of 2.5g/kg glucose solution
Stomach takes tail vein blood glucose meter to measure blood glucose value after 0,30,60,90,120,150,180min, draws blood glucose curve
And area under calculated curve, such as Fig. 4.
3. interpretation of result
Weight detection is carried out to the mouse of each group, as a result such as table 2, finds the model group Mice Body compared with Normal group
(P < 0.01) is dramatically increased again.Since administration the 9th week, Simvastatin, glycosylation trifloroside mouse weight were begun to decline, and were given
Simvastatin group after medicine, glycosylation trifloroside group mouse weight have with model control group compared with conspicuousness reduction (P <
0.01), there is antiobesity action.
The variation of 1 mouse weight of table
Model control group blood glucose is noticeably greater than normal group (P < 0.01), and each group mouse is to blood glucose caused by glucose after administration
Increasing has different degrees of reduction effect, and can be substantially reduced Area under the curve of blood glucose (P < 0.05), illustrate Simvastatin and
Glycosylation trifloroside can improve the impaired glucose tolerance of obesity mice, have and be used as hypoglycemic drug application prospect.
Influence (X ± S) of 2 drug of table to oral glucose tolerance
Embodiment 9 glycosylates trifloroside oxidation resistance
The glycosylation trifloroside (for prepared by embodiment 1) for taking 0.1ml various concentration respectively, using methanol as the sample of solvent
Solution is added 2 × 10 in test tube-4DPPH solution (methanol preparation) 3.9mL of mol/L is uniformly mixed, dark place reaction
After 30min, its absorbance (A is measured at 517nmi), replace sample solution to measure blank absorbency (A with solvent0), with methanol
Absorbance (A is measured instead of DPPH solutionj).Clearance rate is as the following formula:
It calculates: clearance rate (%)=[1- (Ai-Aj)/A0]×100
As a result such as Fig. 4, as shown in Figure 4, in the glycosylation trifloroside concentration gradient of 10-90 μ g/ml, DPPH solution
Clearance rate shows good linear relationship, and when reaching 80 μ g/ml, and clearance rate reaches stable state, and up to 97% is left
The right side, it is essentially identical with 100 μ g/ml Vc effects.
The bioavilability experiment of 10 trifloroside of embodiment and glycosylation trifloroside
Experimental animal uses the healthy rabbits 40 (being provided by Zhongshan University's Experimental Animal Center) of weight about 2kg, animal
It is randomly divided into 2 groups, after preparatory fasting 12h, glycosylation trifloroside (for prepared by embodiment 1) and trifloroside are given in stomach-filling respectively,
Dosage is 300mg/kg (being equivalent to 70kg Coming-of-Age Day taking dose 4g), gives intensively to measure in blood plasma after sample and glycosylates three leaves
The content of glycosides and trifloroside, until peak time half an hour after, is detected blood peak concentration of drug (Cmax).
It after test specimen stomach-filling, is discontinued a period of time, until animal vivo sample is metabolized completely, then intravenous injection is given
Equal dose samples solution is given, and measures sample size in blood plasma (μ g/mL), in this, as reference data.It as a result, can be with such as table 3
Find out that absorptivity is high in glycosylation trifloroside of the present invention, is 10.54 times of trifloroside, bioavilability greatly improves.
Table 3: the bioavilability of glycosylation trifloroside and trifloroside
| Laboratory sample | Glycosylate trifloroside group | Trifloroside group |
| Dosage (g) | 1.2 | 0.6 |
| Sample size (%) | 50 | 50 |
| Sample dosage (mg) | 600 | 300 |
| Vein gives sample plasma sample concentration (μ g/mL) | 120.61 | 112.9 |
| It takes orally to sample peak concentration C max (μ g/mL) in sample blood plasma | 96.54 | 12.85 |
| Absorptivity (%) | 73.15 | 6.94 |
Embodiment 11 glycosylates the sugariness and mouthfeel experiment of trifloroside
Professional sense organ evaluation and test person's (every test group 9 or 12 assessment persons) the Lai Jinhang sweetener composition sweet taste characteristic of group is surveyed
It comments.Following characteristic is to assess on 1 to 5 in specification:
1) similar to sucrose, (1=is similar to sucrose；5=is not similar to sucrose completely)；
2) (1=is bitter not at all for bitter taste；5=is extremely bitter)；
3) (1=does not have peculiar smell to peculiar smell；5=peculiar smell is extremely heavy)；
4 |) (1=is extremely slow for sugariness generation；5=is exceedingly fast)；
5) sweet taste demurrage, (1=was extremely short；5=is extremely long).
As a result such as table 4, it can be seen that either stagnant in bitter taste, sugariness generation, peculiar smell, sweet taste by glycosylated trifloroside
It stays and is all better than ReA, Abbas's sweet tea, trifloroside, closer to sucrose.
The sweet taste characteristic evaluating result of the different sweeteners of table 4
Claims (10)
1. the preparation method of a kind of glycosylation trifloroside and derivative, which is characterized in that the method specific steps include: will be former
Material and glycosyl donor are put into reaction buffer；Feed liquid is after mixing；Then glycosidase and glycosyl is added in reaction system
Transferase；It is eventually adding dehydrogenase, the raw material is trifloroside, the extract of phloretin or conversion product and its derivative.
2. the preparation method of glycosylation trifloroside according to claim 1 and derivative, which is characterized in that the glycosyl
Donor is glucose, UDP-glucose, xylose, UDP- xylose, galactolipin, UDP- galactolipin, erythrose, alpha-cyclodextrin, β-ring
Dextrin, starch, xylan, sucrose, maltodextrin or rhamnose, the concentration of the glycosyl donor are 1-10g/L.
3. the preparation method of glycosylation trifloroside according to claim 1 and derivative, the reaction buffer is phosphorus
The NaCl buffer that sour disodium hydrogen-sodium citrate buffer solution or mass fraction are 0.9% the, wherein disodium hydrogen phosphate-lemon
The mass fraction of sour sodium buffer is 0.05-1%.
4. the preparation method of glycosylation trifloroside according to claim 1 and derivative, which is characterized in that the raw material
Ratio with reaction buffer is (1:10)-(1:50), and the pH value of the reaction buffer is 3.1-6.6, reaction temperature 35-
75 DEG C, the time of the reaction is 1-24h.
5. the preparation method of glycosylation trifloroside according to claim 1 and derivative, which is characterized in that the sugar
Glycosides enzyme is rhamnosidase, glucuroide, and glycosyl transferase is glucosyltransferase, xylosyltransferase and galactosyl
The combination of one or both of transferase.
6. the preparation method of glycosylation trifloroside according to claim 1 and derivative, which is characterized in that the addition
Glycosidase and glycosyl transferase ratio be (10:90) to (90:10) described two enzyme total amounts mass fraction be addition original
The 10%-40% of material, the mass fraction advanced optimize as 10%-20%.
7. sugar prepared by the preparation method of glycosylation trifloroside and derivative described in a kind of claims 1-6 any one
Base trifloroside and its derivative, which is characterized in that the structure of the glycosylation trifloroside and derivative are as follows:
Wherein: R=Glu, Rha, Xyl or Gal；N=1-9, structure advanced optimize as R=Glu or Rha；N=1-5.
8. a kind of pharmaceutical composition, which is characterized in that include glycosylation trifloroside described in claims 7 and derivative, medicine
Acceptable salt, carrier, excipient, diluent, medium or their combination on.
9. the glycosylation trifloroside and derivative of claims 7 are preventing and treating pernio, are removing free radical, promoting blood circulationization
Application in terms of the stasis of blood, anti-inflammatory and varication.
10. application of the glycosylation trifloroside of claims 7 on skin care item, food, drug and health care product.
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