CN107573393A - The preparation of hypo-glycosylated Pu Luning a kind of and its application in anti-inflammatory suppressing panting calming medicine - Google Patents
The preparation of hypo-glycosylated Pu Luning a kind of and its application in anti-inflammatory suppressing panting calming medicine Download PDFInfo
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Abstract
The present invention relates to hypo-glycosylated Pu Luning preparation and its application in for anti-inflammatory suppressing panting calming medicine.Hypo-glycosylated Pu Luning is to be made by raw materials such as naringins by biological enzyme twice.By biological enzyme twice, solve Pu Luning water-soluble and fat-soluble poor problem, improve more than 75 times by hypo-glycosylated Pu Luningbipuluning solubility, bioavilability significantly improves, and Pu Luning can be allowed to be used widely.The hypo-glycosylated Pu Luning of the present invention has more preferable anti-inflammatory and antiasthmatic effect, evident in efficacy for the various asthma as caused by acute and chronic bronchitis and flu etc.；Excellent performance is also shown in terms of anti-inflammatory, has no toxic side effect, it is more notable the effect of than traditional anti-inflammatory suppressing panting calming medicine anti-inflammatory Asthma capsule, it is anti-inflammatory and ideal medicament of relievining asthma, possesses wide market prospects.
Description
Technical field
The present invention relates to the preparation of hypo-glycosylated Pu Luning a kind of and its application in anti-inflammatory suppressing panting calming medicine
Technical background
Cough, phlegm, asthma are the common symptons of respiratory disease, are common in acute and chronic bronchitis, sense in daily life especially
Caused by emitting etc., clinic be by cough, cough up phlegm with pant and recurrent exerbation characterized by, be due to that infection or non-infective agent are drawn
The trachea-bronchial epithelial cell mucous membrane and the acute and chronic nonspecific inflammation of surrounding tissue risen.The cough of category traditional Chinese medicine, phlegm and retained fluid, asthma model
Farmland, it is a kind of common disease, frequently-occurring disease.Particularly the elderly easily produces cough due to environmental factor and the influence of habits and customs
The bronchitis symptom such as cough, cough up phlegm, not carrying out treatment control during morbidity well, and be developing progressively as disease.Branch
San bronchial asthma (asthma) is common clinical and frequently-occurring disease, and the incidence of disease is in rising trend in recent years.Its pathogenesis is not yet complete
Illustrate.In view of the limitation of human trial, at present to asthma because, pathogenesis and treatment etc. research largely
Need to carry out by animal model, it is significant to establish good bronchial astehma animal model.
The characteristics of such medicine for having listed still has dosage more and takes inconvenience at present, and effect and fail to understand
It is aobvious.It is therefore desirable to continue to develop to take safety, curative effect and reliably relieving asthma anti-inflammatory novel drugs.Naringenin is flavone compound
Aglycon, be a kind of Flavonoid substances, have, antibacterial, anti-inflammatory, anticancer, spasmolysis and cholagogic treat angiocardiopathy, and drop courage is consolidated
The effect of alcohol etc..Research shows that chromocor compound has the effect of fine in terms of anti-inflammatory is relievingd asthma in treatment, therefore, prepares dissolving
Degree is high, and effect is good, the chromocor compound of anti-inflammatory of relievining asthma convenient to take is spirit of the invention.The present invention using mouse and rabbit as
Animal experimental model demonstrates effects of the hypo-glycosylated Pu Luning in anti-inflammatory is relievingd asthma, by the hypo-glycosylated Pu Luning of verification experimental verification
It is more more notable than traditional effect that only anti-inflammatory is relievingd asthma.
Chromocor compound is widely present in rutaceae, metabolism of the different types of flavone compound in human body
Approach is different with bioavilability, and aglycone-type chromocor compound is easy to the absorption and utilization of human body, can directly inhaled by small bowel
Take in into blood, and the chromocor compound of glucoside type is needed in enteric cavity in the presence of microorganism, passes through degraded and metabolic pathway
Aglycone-type flavones is converted into, can be just absorbed and utilized by the body.Therefore, aglycone-type chromocor compound has higher biological utilisation
Value and nutritive value.But solubility of the aglycone-type flavones typically in water is low, it is unfavorable for the biological utilisation and hair of aglycon flavones
Wave the drug effect of flavone aglycone.Therefore, to increase the dissolubility of aglycon flavones, scientific research personnel adds alkali such as in a solvent：In solution
It is middle to add NaOH, KOH etc., or add organic solvent such as：Ethanol, dimethyl sulfoxide (DMSO) etc., greatly improve the dissolving of aglycon flavones
Property, but highly basic and organic solvent are harmful, can limit the utilization of aglycon flavones.
Pu Luning is mono-glycosylated naringenin, and pharmacological research both domestic and external shows：Pu Luning, which has, to be sterilized, is anti-oxidant, anti-
A variety of pharmacological activity such as viral, anti-ischemic, antitumor, anti-inflammatory, antiallergy (Grzechulska J, ApplCatal B, 2002,
36(1):45-51；Daghrir R, Ind Eng Chem Res, 2013,52 (10):3581-3599).Pu Luning initially by
Extraction obtains in the folk medicine mountain peach of South Korea, and it is distributed mainly on the plant such as Caesalpiniaceae, the rose family, Euphorbiaceae in nature
In thing.Its content in nature is few and recovery rate is low, but because of water-soluble, fat-soluble difference, it is more difficult to obtain, it is expensive.At present
Pu Luning preparation method mainly has two kinds, chemical synthesis and enzymatic hydrolysis aurantiin method.Synthesized relative to chemical method
Pu Luning, severe reaction conditions, product separation it is cumbersome and to environmental hazard it is big the shortcomings that；Utilize enzyme process (α-L- rhamnosidases
Cut away obtain after naringin rhamnose glycosyl Pu Luning (Hu Qunfang Modern Food Science and Technology 2015,
Vol.31, No.1)) mild condition, high conversion rate, safety and environmental protection that naringin prepares Pu Luning are hydrolyzed, therefore it is prepared by studying enzyme method
Pu Luning has very high application value.But Pu Luning dissolubilities and fat-soluble all extremely low (＜ 10mg/L) in water, this is significantly
Pu Luning application is limited, makes Pu Luning bioavilabilities relatively low.
It is contemplated that the Pu Luning solubility in water is improved, to improve Pu Luning bioavilability.Therefore, originally
Invention obtains more than 75 times that hypo-glycosylated Pu Luning solubility is Pu Luning, the half-life period of pharmaceutical preparation by 2 enzyme process
There is obvious extension, hypo-glycosylated Pu Luning bioavilabilities improve more than 10 times.Specific implementation method is as follows：
A kind of hypo-glycosylated Pu Luning, it is characterised in that chemical structural formula is as shown in I：
Wherein
R1=H,
Wherein n=1
~3；
R2=H or Rha；
Hypo-glycosylated Pu Luning is prepared from high-glycosylation parent, and its structural formula is as shown in II：
R1=H,
N=4~10；
R2=H, Rha；
High-glycosylation Pu Luning is prepared from the citruses such as naringin, naringenin, Pu Luning or grapefruit abstract；To Pu Luning
During high-glycosylation, glycosylase be UDP- glycosyl transferases etc. obtain high-glycosylation Pu Luning (degree of glycosylation be 4-10 sugar
Base).High glycosyl Pu Luning alpha-L-Rhamnosidases, glucoamylase, the poly- enzyme of xylose prepare hypo-glycosylated Pu Luning (glycosyls
Change degree is less than 4 glycosyls).
The content of the invention
First purpose of the invention is, there is provided hypo-glycosylated Pu Luning preparation method.
Second purpose of the invention is, there is provided a hypo-glycosylated Pu Luning is to acute and chronic bronchitis and flu
The preparation method of the medicine of the acute diseases such as the inflammation Deng caused by, asthma.
The hypo-glycosylated Pu Luning of the present invention is realized by approach once.
1. prepare hypo-glycosylated Pu Luning dry powder
(1) naringin ultrafiltration solution is prepared
The molar concentration that sodium hydroxide or potassium hydroxide solution are dissolved in by 50~98% naringin of mass fraction is 0.1
In~1mol/L, 30min, i.e. naringin solution are handled under the conditions of 30~100 DEG C.The naringin solution prepared is pumped into retention point
Son amount carries out hyperfiltration treatment in 1000~2000Da poly (ether sulfone) film ultrafilter, it is 0.1~0.3MPa to control ultrafiltration pressure,
Stop when the 1/10~1/20 of the chromocor compound liquor capacity that ultra-filter retentate volume is reduced in Exocarpium Citri Grandis, receive respectively
Collect ultrafiltration filtered solution and ultra-filter retentate, as the ultrafiltration filtered solution of collection, naringin ultrafiltration solution.
(2) high-glycosylation Pu Luning solution is prepared
After the completion of (1) step, glucoside transferase is added in ultrafiltration filtered solution：The mass ratio of cyclodextrin is 1:20~80 throw
Entering into reaction vessel, 25~70 DEG C of constant temperature stir 1~20h, are then pumped into 4000~5000Da poly (ether sulfone) film ultrafilter,
Ultra-filtration and separation reclaims under conditions of temperature is not less than 15mL/min, 0.6~1.1MPa of operating pressure not higher than 50 DEG C, flow velocity,
It is mainly glucoside transferase in trapped fluid, returns in glucoside transferase container and reuse.It is height to the ultrafiltrate of collection
Glycosylate Pu Luning solution.
(3) hypo-glycosylated Pu Luning solution is prepared
It is pumped into when high-glycosylation Pu Luning solution is concentrated into original 1/3 in reactor, then adds activity and be not less than
100U/mg glucosides enzyme solutions, high-glycosylation Pu Luning solution：Glucosides enzyme solutions=1:0.005~1.In 25~70 DEG C of constant temperature
1~20h is stirred, is then pumped into 1000~1500Da poly (ether sulfone) film ultrafilter, is not less than in temperature not higher than 50 DEG C, flow velocity
Ultra-filtration and separation reclaims under conditions of 15mL/min, 0.6~1.1MPa of operating pressure, is mainly glycosidase in trapped fluid, returns to
Reused 6-8 times in glycosidase container.To the ultrafiltrate of collection, as hypo-glycosylated Pu Luning solution.
Glucoside transferase of the activity of glucoside transferase used not less than 100U/mg, i.e. cyclodextrin glucose glycosides transferase,
One or more kinds of mixtures in malt-base transferase, 4- phloroses transferase, alpha-amylase；Glycosyl used is,
Glucose, UDPG, UDP- xyloses, cyclodextrin, soluble starch, fructose etc..
Glycosidase activity is not less than 100U/mg, and the enzyme of selection is with alpha-L-Rhamnosidase, glucase, glucose
The poly- enzyme of amylase, xylose, alpha-L-Rhamnosidase or their immobilization product.
(4) hypo-glycosylated Pu Luning dry powder is prepared
After the completion of (3) step, trapped fluid is depressurized under conditions of 40~50 DEG C of temperature, 0.09~0.l of vacuum MPa
Paste is concentrated into, hypo-glycosylated Pu Luning dry powder is obtained by being freeze-dried or being spray-dried.
After hypo-glycosylated, the comprehensive utilization of naringin can reach 90%, obtain the purity of hypo-glycosylated Pu Luning dry powder
More than 98.3% can be reached.
The hypo-glycosylated Pu Luning of present invention drug effect is obtained by experimental program once.
1. prepare hypo-glycosylated Pu Luning acceptable preparations in medicine.
Hypo-glycosylated Pu Luning described in the present invention can contain 0.1- in the medicine for preparing anti-inflammatory and relievining asthma, its composition
The hypo-glycosylated Pu Luning of 10%wt.Described hypo-glycosylated Pu Luning medicines can by glycosylate Pu Luning monomer compositions, or
It is made up of hypo-glycosylated Pu Luning and other active ingredients or conventional manner auxiliary material.
Above-mentioned hypo-glycosylated Pu Luning medicines are customary adjuvants by selecting general formulation or are not added with auxiliary material, with normal
Rule method is prepared into the pharmaceutical preparation of required different dosage forms.The auxiliary material of addition can be solid, semisolid or liquid substance,
Carrier, excipient or medium as hypo-glycosylated Pu Luning.Therefore, hypo-glycosylated Pu Luning pharmaceutical preparations can be piece
It is agent, pulvis, sachets, elixir, supensoid agent, emulsion, solution, syrup, aerosol, inhalant, soft or hard shell capsules, sterile
The various formulations such as parenteral solution.
The preparation of hypo-glycosylated Pu Luning medicines includes：Capsule, its inclusion contain 0.5-99.5%wt low sugar base
Change Pu Luning or generally by the hypo-glycosylated Pu Luning no less than 0.5%wt and other active ingredients or various customary adjuvant groups
Into；Tablet, the hypo-glycosylated Pu Luning containing 0.5-99.5%wt in its composition；Can be by hypo-glycosylated no less than 0.1%wt
Pu Luning forms with other active ingredients or various customary adjuvants；Inhalant, its inclusion contain 0.1-100%wt low sugar base
The hypo-glycosylated Pu Luning and other active ingredients or various customary adjuvant groups for changing Pu Luning or generally can be no less than 0.1%wt
Into；Itself and pulvis, sachets, elixir, supensoid agent, emulsion, solution, syrup, aerosol, soft or hard shell capsules, sterile note
The various formulations of liquid, which are penetrated, in it 0.5-10%wt hypo-glycosylated Pu Luning and acceptable auxiliary material.
2. the present invention is to carry out evaluating drug effect by the following method.
Inventor carries out the experiment of relievining asthma of experimental animal small white mouse to hypo-glycosylated Pu Luning.As a result show：It is hypo-glycosylated
Pu Luning compared with blank control group, has significant extension, statistically to stimulating mouse to cause the tolerance time of asthma
There were significant differences (P ＜ 0.05)；Compared with positive control anti-inflammatory Asthma capsule, tolerance time extends, and curative effect is compared with positive control
Medicine anti-inflammatory Asthma capsule is notable.
The anti-inflammatory that inventor carries out experimental animal small white mouse to hypo-glycosylated Pu Luning is tested.As a result show：It is hypo-glycosylated
Pu Luning is excellent to the performance of mice ear antiphlogistic effects, compared with blank control group, there is significant increase, statistically has aobvious
Write difference (P ＜ 0.05).Compared with positive control medicine aspirin, mice ear antiphlogistic effects are dramatically increased, counted
There were significant differences on (P ＜ 0.05), and curative effect is notable compared with positive control medicine aspirin.Illustrate that hypo-glycosylated Pu Luning has
The effect of anti-inflammatory.
It is demonstrated experimentally that low base saccharification Pu Luning not only has antiinflammatory action of relievining asthma very well, and do not have in mouse experiment
Toxicity.Results of animal shows that, when animal is administered orally in the base saccharification Pu Luning of 2000mg/kg dosage, animal has no poison
Property reaction, the dosage equivalent to people's taking dose be 4-6g bases be saccharified Pu Luning/kg body weight.
Hypo-glycosylated Pu Luning medicines of the present invention are in the hypo-glycosylated Pu Luning/kg body weight/days of 0.1-700mg, tool
There are good antiphlogistic effects of relievining asthma, preferable daily dose is the hypo-glycosylated Pu Luning/kg body weight/days of 1-100mg.
In summary, illustrate that base saccharification Pu Luning has good antiphlogistic effects of relievining asthma, can treat well in clinic by
In asthma caused by acute and chronic bronchitis and flu etc..Therefore, available for preparing anti-inflammatory suppressing panting calming medicine.
Brief description of the drawings
Fig. 1：Hypo-glycosylated Pu Luning granules solubility
With reference to case study on implementation, the present invention is described further.
In each embodiment involved material materials with the solid in when component content and solid, liquid and liquid and
The ratio of liquid and solid is calculated with wt/wt (mass ratio), v/v (volume ratio), wt/v (weight/volume) respectively, unless otherwise
Explanation.
Embodiment 1：Prepare hypo-glycosylated Pu Luning
(1) naringin ultrafiltration solution is prepared
The molar concentration that sodium hydroxide or potassium hydroxide solution are dissolved in by 50~98% naringin of mass fraction is 0.1
In~1mol/L, 30min, i.e. naringin solution are handled under the conditions of 30~40 DEG C.The naringin solution prepared is pumped into retention point
Son amount carries out hyperfiltration treatment in 1000~2000Da poly (ether sulfone) film ultrafilter, it is 0.1~0.3MPa to control ultrafiltration pressure,
Stop when the 1/10~1/20 of the chromocor compound liquor capacity that ultra-filter retentate volume is reduced in Exocarpium Citri Grandis, receive respectively
Collect ultrafiltration filtered solution and ultra-filter retentate, as the ultrafiltration filtered solution of collection, naringin ultrafiltration solution.
(2) high-glycosylation Pu Luning solution is prepared
After the completion of (1) step, glucoside transferase is added in ultrafiltration filtered solution：The mass ratio of cyclodextrin is 1:20~80 throw
Entering into reaction vessel, 25~70 DEG C of constant temperature stir 1~20h, are then pumped into 4000~5000Da poly (ether sulfone) film ultrafilter,
Ultra-filtration and separation reclaims under conditions of temperature is not less than 15mL/min, 0.6~1.1MPa of operating pressure not higher than 50 DEG C, flow velocity,
It is mainly glucoside transferase in trapped fluid, returns in glucoside transferase container and reuse.It is height to the ultrafiltrate of collection
Glycosylate Pu Luning solution.
(3) hypo-glycosylated Pu Luning solution is prepared
It is pumped into when high-glycosylation Pu Luning solution is concentrated into original 1/3 in reactor, then adds activity and be not less than
100U/mg glucosides enzyme solutions, high-glycosylation Pu Luning solution：Glucosides enzyme solutions=1:0.005~1.In 25~70 DEG C of constant temperature
1~20h is stirred, is then pumped into 1000~1500Da poly (ether sulfone) film ultrafilter, is not less than in temperature not higher than 50 DEG C, flow velocity
Ultra-filtration and separation reclaims under conditions of 15mL/min, 0.6~1.1MPa of operating pressure, is mainly glycosidase in trapped fluid, returns to
Reused 6-8 times in glycosidase container.To the ultrafiltrate of collection, as hypo-glycosylated Pu Luning solution.
Glucoside transferase of the activity of glucoside transferase used not less than 100U/mg, i.e. cyclodextrin glucose glycosides transferase,
One or more kinds of mixtures in malt-base transferase, 4- phloroses transferase, alpha-amylase；Glycosyl used is,
Glucose, UDPG, UDP- xyloses, cyclodextrin, soluble starch.
Glycosidase activity is not less than 100U/mg, and the enzyme of selection is with alpha-L-Rhamnosidase, glucase, glucose
The poly- enzyme of amylase, xylose, alpha-L-Rhamnosidase or their immobilization product.
(4) hypo-glycosylated Pu Luning dry powder is prepared
After the completion of (3) step, trapped fluid is depressurized under conditions of 40~50 DEG C of temperature, 0.09~0.l of vacuum MPa
Paste is concentrated into, hypo-glycosylated Pu Luning dry powder, after hypo-glycosylated, naringin are obtained by being freeze-dried or being spray-dried
Comprehensive utilization can reach 90%.
Embodiment 2：Hypo-glycosylated Pu Luning purity analysis
1st, liquid phase analysis condition：Chromatographic column (18 250 × 4.6mm of model C)；Flow velocity：1.0ml/min；Mobile phase：0.5%
Acetum：Acetonitrile=7:3；Detection wavelength：346nm；Sample size:10μl.
2nd, the preparation of mobile phase：0.5% ml of acetum 1000 is prepared with ultra-pure water and glacial acetic acid (reagent is pure), is used
Liquid phase is filtered to being visible by naked eyes impurity, ultrasonic half an hour bubble removing with nutsch filter；500ml acetonitriles (analysis is pure) are taken, use liquid phase
Filtered with nutsch filter to being visible by naked eyes impurity, ultrasonic half an hour bubble removing.
3rd, the preparation and analysis of concentration known sample solution：5.0mg samples are taken to be dissolved in the DMSO solutions of 10ml 50% respectively
In, concussion makes to be completely dissolved.Every kind of strength solution 1ml is filtered in liquid-phase inlet bottle with needle and syringe cartridge type membrane filter
In, do high-efficient liquid phase analysis.
4th, sample purity is analyzed：Take the sample solution of any one concentration to do high-efficient liquid phase analysis, pass through peak area percent
Calculated purity.
It is 98.3% that can obtain glycosylating Pu Luning purity from peak area.
Embodiment 3：Hypo-glycosylated Pu Luning medicines cause cavy to relieving asthma pharmacological experiment
SPF level cavys are put into sprayer unit case, ultrasonic ultrasonic delay line memory constant pressure spray into 2% acecoline and
0.1% histamine mixed liquor aerosol 15s.50 cavys for asthma reaction occur in selection 150s are tested.Drawn according to cavy
Asthma incubation period is randomly divided into the basic, normal, high dosage group of model control group group, positive drug group, given the test agent (equivalent to quantity
1.875,3.75,7.5 times), 10/group, male and female half and half.Each group animal puts cavy after 1h according to 10ml/kg gastric infusions
Enter in sprayer unit case, 1 every time.Mixed by the acecoline of similarity condition constant-pressure atomization 2% and 0.1% histamine during screening
Close liquid 15s.Record spraying starts the time (being defined to twitch, fall) occurred to symptom, as the latent time after administration 1h.
Second day, each group animal continued gastric infusion, and after continuous 6d, last dose 1h, record causes the latent time of asthma.
The invention formulation of table one influences (X ± S) to Experimental Asthma In Guinea-pigs caused by histamine
| Group | Number of animals (only) | Incubation period (S) |
| Physiological saline | 10 | 104.6±32.9 |
| Positive controls1 | 10 | 231.7±95.1* |
| Small dose group of the present invention | 10 | 249.5±107.4* |
| Middle dose group of the present invention | 10 | 271.3±113.0** |
| Heavy dose of group of the present invention | 10 | 291.8±127.3** |
Note：1Positive controls medicine is the kind loquat leaf extract of thought；* P ＜ 0.01；* P ＜ 0.001
Cavy can cause anthropoid " asthma " model of class, this hair after respiratory tract sucks a certain amount of histamine phosphate
The basic, normal, high dosage group of bright given the test agent (1.875,3.75,7.5 times equivalent to quantity) official is micro-, and give after cavy can be with
" asthma " symptom caused by anti-animal tissue's amine, the extension of tumble phase is shown as, there is highly significant meaning compared with physiological saline group
Justice, it is statistically analyzed respectively P ＜ 0.01, P ＜ 0.001 and P ＜ 0.001.
Embodiment 4：Influence of the hypo-glycosylated Pu Luning medicines to Asthmatic Rats
SPF level SD rats, 180-220g, single sex.Quarantine it is qualified after, 60 rats are randomly divided into Normal group
10 and model group 60.Model group rats are in the 0th day intraperitoneal injection 0.6%OVA- Alum sensitization liquid 1mL/ of experiment；Yu Shi
Test the 7th, 14 day intraperitoneal injection 0.2%OVA-Alum sensitization liquid 1mL/ only；Rats in normal control group gives the physiology salt of equivalent
Water.In experiment the 15th day, model group rats sucked 2.0ppm ozone 1h daily, continuous 7 days.In experiment the 21st day, model group was big
Mouse is randomly divided into model control group, 1 group of positive drug (aluminium hydroxide is adjuvant), the basic, normal, high dosage group of given the test agent, 10/
Group.In addition to Normal group, each group rat according to 10mL/kg body weight gastric infusions, is administered after 30min rat being placed in mist respectively
Change in inlet box and 30min is excited with 1%OVA solution, continuous 7 days.Last dose 1h, sacrificed by exsanguination after rat anesthesia, ligation are left
Lung, with the cold 5%NaHCO of 5mL3The lobe of the lung on the right side of lavation, is repeated 3 times, recovery BAL fluid (BALF).In 4 DEG C,
1500rpm, 10min is centrifuged, collect supernatant.ICAM-1, VCAM-1, L-selectin assay in ELISA detections BALF,
The expression of the left lung tissue of histopathologic examination and SABC detection lung tissue NF- κ B p65.
Influence of the hypo-glycosylated Pu Luning medicines of table two to Asthmatic Rats
It is one of key character of asthma that NF- κ B p65 cytokine levels, which increase,.As knowable to table result, with control group phase
Than significantly increasing by the horizontal nothing of asthma group NF- κ B p65 cell factors ICAM-1, VCAM- 1 of adjuvant of aluminium hydroxide.And with
Hypo-glycosylated Pu Luning is asthma group NF- κ B p65 cell factors ICAM-1, VCAM-1, the L-selectin contents level of adjuvant
It is significantly higher than itself corresponding control group, also significantly greater than using aluminium hydroxide as adjuvant and without adjuvant asthma group
The pharmacological experiment of the hypo-glycosylated Pu Luning medicines paraxylene induced mice ear swelling of embodiment 5
SPF level NIH mouse, male, 60.It is at random model control group, positive controls (aspirin), test medicine
Basic, normal, high dosage group, 10/group.Each group mouse is according to 20ml/kg body weight gastric infusions, 1 times/day, continuous 2 days.Last is given
After medicine 0.5h, auris dextra gives distilled water in mouse right ear coating dimethylbenzene 0.05ml/ only.Mouse is put to death after 2h, cuts left and right ear
Piece, round auricle is laid at the same position of left and right ear respectively with diameter 9mm card punch, two auricle weight differences of measurement are swelling.
Three hypo-glycosylated Pu Luning of table influences on mouse ear swelling test
Positive drug (aspirin), hypo-glycosylated Pu Luning basic, normal, high dosage group, ear swelling caused by paraxylene
There is obvious inhibitory action (P<0.05~0.01), wherein, it is most obvious with hypo-glycosylated Pu Luning middle and high dosage group effect.
The experiment shows that hypo-glycosylated Pu Luning has obvious anti-inflammatory activity.
The measure of the hypo-glycosylated Pu Luning granules dissolution rate of embodiment 6
The foundation of 1 analysis method
The determination of Detection wavelength：Claim hypo-glycosylated general Shandong peace right amount of auxiliary materials respectively, suitable concentration is configured to methanol
Solution, and using methanol as blank control, be scanned in the range of 200~700nm.As a result show there is low sugar base at 346nm
Change Pu Luning maximum absorption band；And auxiliary material is noiseless to hypo-glycosylated Pu Luning measure here.Therefore, select
346nm is as measure wavelength.Standard curve：Precision claims hypo-glycosylated Pu Luning appropriate, and the molten of series concentration is configured to methanol
Liquid, trap is determined at 346nm, linear regression is carried out to trap (A) with concentration (C).
The assay method of 2 hypo-glycosylated Pu Luning dissolution rates
Weighing contains the solid particle of general Shandong 83nmol (50mg), and contains the hypo-glycosylated general peaceful 83nmol's in Shandong
Solid particle samples carry out Dissolution Rate Testing.Every group of 3 parts of sample parallel determination, carried out by 2015 editions the second methods of Chinese Pharmacopoeia.It is molten
Go out the distilled water that medium is 900mL, temperature is 37 ± 0.5 DEG C, and rotating speed is 100 ± 3rpm.Taken respectively in 3,6,9,12,15min
Sample 5mL and the dissolution medium for filling into same volume, sample is through 0.8 μm of filtering with microporous membrane.Subsequent filtrate dilution is taken at 346nm
Determine trap, the hypo-glycosylated Pu Luning of calculatingization dissolution rate.
3 measurement results
Calibration curve equation C=16.3553A+0.4162, R2=0.9893, the range of linearity：1.765~67.803 μ g/
mL.The present invention use dissolution in vitro experiment to determine hypo-glycosylated Pu Luning in vitro molten using Pu Luning bulk drugs to compare
Artificial situation, the results showed that, dissolution is all right in vitro by hypo-glycosylated Pu Luning.It the results are shown in Table four
The dissolution rate of the hypo-glycosylated Pu Luning of table four-PVP K30 particle
As can be seen from Table IV, bulk drug Pu Luning is 13.13% in the percentage of 15min vitro cumulative dissolutions, and
The hypo-glycosylated Pu Luning of the present invention reaches the 96.33%- 99.41% of scalar in 3-15min, after manufactured glycosylation
Dissolution rate is above Pu Luning.Therefore glycosylation adds the extracorporeal releasing speed of medicine.
The measure of the hypo-glycosylated Pu Luning granules solubility of embodiment 7
Hypo-glycosylated Pu Luning granules are dissolved in water, and the size of solvability directly influences medicine in solution system and thin
The application of cell space system.Because Pu Luning stablizes in aqueous, we are using ultraviolet spectrophotometry to saturation state
The Pu Luning aqueous solution carries out the measure of solubility values.In this experiment, we prepare the 0.3g/L Pu Luning aqueous solution, according to
Absorbance is surveyed after certain proportion dilution and makes standard curve, and integration is carried out to the characteristic peak in 346 nm sections and is used as ordinate.Again
The light absorption value for the saturated solution dilution prepared is determined, the concentration of dilution is obtained by internal standard method, it is molten finally to calculate saturation
Liquid concentration.Canonical plotting such as Fig. 1.
Calibration curve equation is y=1624.2495A-18.96, R2=0.9935.Saturated solution is inhaled after 50 times of dilutions
Luminosity integrated value is 645.27, concentration 0.409g/L, then hypo-glycosylated Pu Luning solubility is 20.03g/L.
The toxicological experiment of the hypo-glycosylated Pu Luning medicines of embodiment 8
At 28 ± 1 DEG C of temperature, 70 ± 5% damp condition, 7-8 week old, healthy cleaning grade NIH mouse are chosen
20 male and female half and half, body weight is in 20-22g.Test in preceding and experiment observation period, feed and water sterilization by chow diet
Condition is raised.
Hypo-glycosylated Pu Luning is dissolved in 0.5%Tween80, concentration 300mg/ml, by the liquid oral administration
Mouse, dosage are 0.4ml/20g mouse weights.Observation post administration Isosorbide-5-Nitrae is given, 8,12 hours, is observed once per 12h later.See
Death condition is examined, records mouse weight change and other symptoms daily.10th day, the neck that breaks put to death mouse, takes each organ to enter
Row pathologic finding.
At the 10th day, whole mouse survivals, the hypo-glycosylated Pu Luning of 2.0g/kg dosage had no toxic reaction.Mouse is each
Organ pathologic finding is normal, does not find lesion, mouse weight has no mitigation in 10 days.Therefore, the low sugar base of the present invention is illustrated
Change Pu Luning medicines and have no toxicity when animal is administered orally.
The hypo-glycosylated general Shandong Yiganning capsule preparation of case study on implementation 9
Gelatine capsule is prepared into by following component proportioning：
| Composition | Component (%) |
| Dry starch | 35 |
| Hypo-glycosylated Pu Luning | 60 |
| Superfine silica gel powder | 5 |
| Amount to | 100 |
Auxiliary material is well mixed with hypo-glycosylated Pu Luning, is fitted into gelatine capsule, produces.Loading amount：100mg/ capsules.
The hypo-glycosylated Pu Luning tablets of embodiment 10
Matched by following component and prepare piece agent：
| Composition | Component |
| Hypo-glycosylated Pu Luning | 500g |
| Starch | 472.5g |
| Amylan (14%) | 25.0g |
| Magnesium stearate | 2.5g |
| Amount to | 1000g |
Hypo-glycosylated Pu Luning is well mixed with starch, adding starch slurry to continue stirring makes into softwood, handsome with 10 mesh nylon
Plasmid, 80-90 DEG C of aeration-drying, dry granular add magnesium stearate, by 12 mesh Sai whole grains, mix, are pressed into tablet.It there are 10000
Piece, every piece are weighed about as 0.1g.
The hypo-glycosylated Pu Luning inhalants of embodiment 11
Matched by following component and prepare piece agent：
| Composition | Component (g) |
| Hypo-glycosylated Pu Luning | 100 |
| Lactose | 500 |
| Poloxamer | 1 |
| Superfine silica gel powder | 10 |
| L-Leu | 0.5 |
| PEG400 (50%) aqueous solution | 300 |
| Amount to | 911.5 |
By hypo-glycosylated Pu Luning, lactose, poloxamer, L-Leu PEG400 aqueous dissolutions, then sprayed
Dry, gained spray-dried powders add superfine silica gel powder, are well mixed, are sub-packed in capsule dry-powder inhaling device.
12 hypo-glycosylated Pu Luning of embodiment bioavilability experiment
1st, sample preparation：Take respectively Pu Luning numberings be 1. 2. two samples with glycosylation Pu Luning numberings；
2nd, test method：Experimental animal is using body weight about 2kg healthy rabbits 40 (by Zhongshan University's Experimental Animal Center
There is provided), animal is randomly divided into four groups, and after advance fasting 12h, gavage gives above-mentioned sample respectively, dosage be 300mg/kg (quite
In 70kg Coming-of-Age Days taking dose 4g), the intensive content for determining the general hypo-glycosylated Pu Luning of Shandong peace in blood plasma after sample is given,
To peak time half an hour after, detection blood peak concentration of drug (Cmax)；
After test specimen gavage terminates, be discontinued a period of time, be metabolized completely to animal vivo sample, be then injected intravenously to
The dose sample solution such as give, and determine sample size in blood plasma (μ g/mL), in this, as reference data；
It the results are shown in Table seven：
Table seven：Hypo-glycosylated Pu Luning bioavilabilities
| Test specimen | ① | ② |
| Dosage (g) | 0.6 | 0.6 |
| Sample size (%) | 2.66 | 2.60 |
| Sample dosage (mg) | 16.00 | 15.60 |
| Vein gives sample plasma sample concentration (μ g/mL) | 72.91 | 70.16 |
| Orally give sample peak concentration C max (μ g/mL) in sample blood plasma | 10.98 | 65.33 |
| Absorptivity (%) | 15.06 | 93.12 |
Result of the test shows：Absorptivity is high in hypo-glycosylated Pu Luning (2. number sample) of the present invention, for Pu Luning (1.
Number sample) 6.18 times, its bioavilability greatly improves.
Claims (10)
1. a kind of hypo-glycosylated Pu Luning, it is characterised in that chemical structural formula is as shown in I：
Wherein：
R1=H, Wherein n=1~3；
R2=H or Rha；
2. according to described in claim 1, hypo-glycosylated Pu Luning is prepared from high-glycosylation parent, and its structural formula is as shown in II：
R1=H, Wherein n=4~10；
R2=H or Rha；
3. according to described in claim 2, high-glycosylation Pu Luning preparing raw materials from citruses such as naringin, naringenin, Pu Luning or
Grapefruit abstract.
4. according to described in claim 2, high-glycosylation Pu Luning (degree of glycosylation is 4-10 glycosyl) is shifted by glycosyl
Enzyme is come what is be prepared, and wherein glycosyl transferase is cyclodextrin glycosyltransferase and UDP- glycosyl transferases.
5. according to described in claim 4, wherein UDP- glycosyl transferases include UDPG based transferase, UDP- xylosyls turn
Move enzyme, UDP- rhamnosyltransferases, UDP- galactosyltransferases, it is a kind of in UDP- transfructosylases or/and both and
Combination of the above.
6. according to described in claim 2, high-glycosylation Pu Luning is with glycosylase, alpha-L-Rhamnosidase, glucase, Portugal
The poly- enzyme of grape saccharogenic amylase, xylose, the poly- enzyme of fructose etc. prepare hypo-glycosylated Pu Luning (degree of glycosylation is less than 4 glycosyls).
7. as claimed in claim 1, applications of the hypo-glycosylated Pu Luning in anti-inflammatory, suppressing panting calming medicine.
8. as claimed in claim 7, applications of the hypo-glycosylated Pu Luning in anti-inflammatory, suppressing panting calming medicine, it is characterised in that low sugar base
Change Pu Luning and pharmaceutically acceptable carrier, or hypo-glycosylated Pu Luning as active ingredient and other effectively into
Divide or conventional pharmaceutical aids forms.
9. a kind of Pharmaceutical composition, it includes compound described at least one claim 1 or its is pharmaceutically acceptable
Salt and crystal, and its pharmaceutically acceptable carrier, excipient, diluent, assistant agent, medium or combinations thereof.
10. as claimed in claim 6, it is characterized in that being the hypo-glycosylated Pu Luning that medicine contains 0.001-100%wt, preferably
0.01-50%wt hypo-glycosylated Pu Luning, more preferably 0.1-10%wt hypo-glycosylated Pu Luning.
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| CN110156855A (en) * | 2019-05-23 | 2019-08-23 | 广东金骏康生物技术有限公司 | Glycosylated flavanoid and its preparation method and application |
| CN112358515A (en) * | 2020-05-30 | 2021-02-12 | 中国农业科学院作物科学研究所 | Application of naringenin (4-O-methyl) glucoside compound in preparation of anti-inflammatory or lipid-lowering drugs |
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