CN104274484B - Application of the LBP-X in the medicine for preparing treatment scheroma - Google Patents
Application of the LBP-X in the medicine for preparing treatment scheroma Download PDFInfo
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Abstract
The present invention relates to the new application of LBP-X, and in particular to application of the LBP-X in the medicine for preparing treatment scheroma, wherein the molecular weight of the LBP-X is more than 2000 dalton.Application of the present invention, wherein the external preparation that the medically acceptable auxiliary material that described medicine is LBP-X and conventional amount used is made, e.g., eye drops or gel.Medicine of the present invention has the advantages that colorless and odorless, non-stimulated to ocular mucous membrane.
Description
Technical field
The present invention relates to medicinal preparation, and in particular to contains the medicine from Lycium polysaccharide.
Background technology
Matrimony vine is the ripening fruits of matrimony vine of solanaceae plant, the effect of with liver-kidney tonifying, in matrimony vine LBP-X content compared with
Height, is one of its main active.Pharmacological research shows that LBP-X has antitumor, reducing blood lipid, hypoglycemic, protect liver, prevented
The bioactivity such as radiation and anti anoxia.At present, LBP-X is just gradually into pharmacological research and the focus of developing new drug.
Scheroma is also known as angle xerosis of conjunctiva, refers to that tear matter caused by any reason or amount are abnormal, or dynamics is abnormal
Caused tear film stability declines, and with ophthalmic uncomfortable and (or) the general name of a variety of diseases of ocular lesion tissue feature.It is dry
The most common eye surface diseases of illness in eye, have a strong impact on the quality of life of patient, it is burning sensation, foreign body sensation, drying that patient, which starts symptom,
And photophobia, aggravated with disease progression, ulcer of the cornea or even blindness finally occur.Currently a popular disease is learned and clinical investigation is shown,
The U.S. has 14.6% to suffer from scheroma in the crowd of 65-84 Sui；All continuous non-selection patients in each 30 of optometry clinic of Canada
Survey show that 28.7% patient has dry eyes symptom；The Japanese continuous outpatient's investigation knot of 8 Eye Centers 2127
Really, suffering from dry eyes (tear film and ocular are all abnormal) has 359 (17%).China someone is to 5136 progress questionnaires of ophthalmology out-patient
Investigation, there is scheroma 745 (14.52%).It is now recognized that its cause of disease is with using long eye working time, environmental pollution, wearing angle
The factors such as film contact lense, female incretion influence, social senilization are relevant, and the incidence of disease is in rising trend.With the hair of dry eyes
Sick rate more and more higher, the Clinics and Practices of dry eyes are increasingly paid attention to by people, and the research to dry eyes has become domestic external eyes
The focus of section's research.
Existing many scholars take different treatment methods to be studied at present, but there is no specific short so far, manually
The treatment that tear preparation mitigates symptom is relying primarily on for scheroma.Although artificial tears can mitigate symptom to some extent,
All cure the symptoms, not the disease, effect can not be lasting.
Scheroma belongs to " dry astringent eye, xerosis conjunctivitis " category in theory of traditional Chinese medical science, and " liver " has one's ideas straightened out in mesh, and liver-yin moistens eye-candy eyeball, and tear belongs to
Yin-fluid scope, deficiecny of liver-YIN can then cause the dry mesh of eyes losing moistening and lustre, mesh puckery.The Chinese patent drug of existing treatment scheroma is mainly oral system
Agent, such as Liuwei Dihuang Wan.But herbal mixture for oral administration works slowly, and is difficult to Unified Generalization application for the crowd of different constitutions.
The content of the invention
The technical problems to be solved by the invention are to provide the new application of LBP-X, i.e., the application in pharmacy.
Application of the above-mentioned LBP-X in pharmacy be specifically, LBP-X in the medicine for preparing treatment scheroma should
With wherein the molecular weight of the LBP-X is more than 2000 dalton (Da).
Application of the present invention, wherein described medicine is the medically acceptable auxiliary of LBP-X and conventional amount used
Expect the external preparation being made, e.g., eye drops or gel.
Above-mentioned external preparation, wherein, concentration 0.1mg/ml -50mg/ml of LBP-X in the eye drops；It is described solidifying
The weight/mass percentage composition of LBP-X is 0.1mg/ml -50mg/ml in jelly.
Application of the present invention, wherein the LBP-X can be extracted by the method that polysaccharide is extracted generally from plant
Arrive, the method that inventor recommends comprises the following steps：
(1) take matrimony vine to be extracted by solvent of pure water, then extract solution is used into molecular cut off for 2000 dalton
Milipore filter ultrafiltration, collects the filtered fluid of liquid feeding side；
(2) enzymolysis deproteinized is carried out toward addition neutral proteinase in filtered fluid, then, go out enzyme, centrifugation, takes supernatant；
(3) by DEAE- cellulose chromatographies post on supernatant, and pillar is rinsed with 0.1M~1M phosphate buffer, so
Coomassie Brilliant Blue detection albumen, discards the eluent that absorbance is more than 0.1 afterwards；
(4) on-line checking is carried out to the eluent left to evaporate photodetector, collects each crest segment efflux, and with benzene
Phenol sulfuric acid method detects the polyoses content of each crest segment efflux, and retention absorbance is more than 0.1 efflux；
(5) by the efflux of retention, using the nanofiltration membrane that molecular cut off is 200 dalton, tapping side is discarded
Filtered fluid, removes phosphate-buffered salt, then, the filtered fluid of liquid feeding side is concentrated, vacuum freeze drying, obtaining molecular weight is
More than the LBP-X of 2000 dalton.
The present invention has the following advantages that compared with prior art：
The present invention extracts polysaccharide component from matrimony vine, and eye drops or gel is made for eyes local topical, makes tear
Glandular secretion showed increased, corneal fluorescein dyeing are significantly reduced, and lachrymal gland lesion substantially mitigates, and goblet cell number is substantially recovered.
Secondly, LBP-X glues compared to other compositions in Chinese medicine such as flavones, saponin(e, alkaloid etc. with colorless and odorless, to ocular
The non-stimulated advantage of film.
Brief description of the drawings
Fig. 1~5 are each group fluorescein sodium stained positive reaction experiment result figure, wherein, Fig. 1 is normal group, and Fig. 2 is model
Group, Fig. 3 is positive controls, and Fig. 4 is LBP-X low dose group, and Fig. 5 is LBP-X high dose group.
Fig. 6~10 be light microscope under observe lachrymal gland aspect graph, wherein, Fig. 6 is normal group, and Fig. 7 is model group,
Fig. 8 is positive controls, and Fig. 9 is LBP-X low dose group, and Figure 10 is LBP-X high dose group.
Figure 10~15 are conjunctiva scanning electron microscope (SEM) photograph, wherein, Figure 11 is normal group, and Figure 12 is model group, and Figure 13 is positive right
According to group, Figure 14 is LBP-X low dose group, and Figure 15 is LBP-X high dose group.
Embodiment
Embodiment 1
1st, decocting cooking method prepares LBP-X
(1) matrimony vine 10kg is taken, crushed after being dried to coarse powder adds 10 times and 8 times of water, decocted 2 times, each 1h respectively；Merge 2
It is secondary to take liquid, it is concentrated under reduced pressure, is concentrated into 100L, tripod pendulum type batch centrifugal initial filter takes supernatant fluid filtrate 95L, and add 0.3% potassium sorbate
Anti-corrosion.
(2) ultrafiltration instrument is used, the instrument is equipped with rolling ultrafiltration membrane, can makes liquid with layer flow mode by rolled film, on film
The ultrafiltration hole of certain molecular weight is distributed, liquid is after closed cycle certain time, more than ultrafiltration molecular cut off and less than this point
The molecule of son amount is two parts liquid by milipore filter.This experiment takes the milipore filter that molecular cut off is 2000, by fructus lycii extracted solution
Ultrafiltration is carried out, small molecule Chinese medicinal ingredients of the elimination less than 2000 are such as：Pigment, trace element, alkaloid, vitamin, free sugar, ammonia
Base acid, flavones, saponin(e etc., the decoction part for taking molecular weight to be more than 2000 mainly contains polysaccharide and albumen.
(3) by molecular weight be more than 2000 decoction part, in 60 DEG C, under stirring condition add 5% fining agent ZTC B into
Point, continue to stir 15min, add 2.5% fining agent ZTC A compositions, be stored at room temperature 12h, tubular type centrifugation takes supernatant.
(4) PH to 6.0~7.0 is adjusted, the Bacillus subtilis neutral protease progress enzymolysis for adding 0.5%~1% removes egg
In vain, then, go out enzyme, centrifugation, takes supernatant；Neutral proteinase is the Submerged fermentation of bacillus subtilis 1398 through seed selection,
A kind of endo protease refined by advanced extraction process, can be by macro-molecular protein under certain temperature, pH value
It is decomposed into the products such as polypeptide and amino acid.Digested 1~2 hour at 60 DEG C, remove a part of albumen, 90 DEG C of 10min go out enzyme, tubular type
Centrifuge high speed centrifugation, takes supernatant 10L.
(5) 10L supernatants are subjected to anion-exchange chromatography analysis by DEAE- celluloses, efflux contains neutral polysaccharide,
And anion-containing polysaccharide, and protein absorption is on post.Pillar is rinsed with 0.1M~1M phosphate buffer again, will be flowed out
Liquid collects 50 bottles altogether by mono- bottle of collection of 200ml.Liquid is gone out to every bottle of liquid albumen is detected with Coomassie Brilliant Blue respectively, with phenol sulphur
Acid system detect polysaccharide, collect and merge absorbance be more than 0.1 saccharide portion, remove absorbance be more than 0.1 protein content compared with
High part.30 bottles are collected in 50 bottles.
(6) by molecular sieve resin on the amalgamation liquid containing polysaccharide, to evaporate photodetector on-line checking, each crest segment is collected
Efflux, detects polyoses content with Phenol sulfuric acid procedure to every bottle of collection liquid, protein content is detected with Coomassie Brilliant Blue, collect and inhale
The high part of polyoses content of the luminosity more than 0.1, removes the protein content upper section that absorbance is more than 0.1.
(7) nanofiltration instrument is used, nanofiltration desalination is carried out by 200 NF membrane of molecular cut off, removes and add in column chromatography
The phosphate composition entered, while also filtering off most of water, reaches the purpose of concentration.
(8) method for taking vacuum freeze drying, when powdered LBP-X 100g.
2nd, polyoses content and molecular weight identification
Phenol sulfuric acid procedure detection LBP-X content is 95%, and LBP-X molecule is determined with molecular sieve resin chromatography scheme
Amount distribution is the Da of 2000Da -2,000,000, using dextran standard as control.
Embodiment 2
1st, microwave extraction method prepares LBP-X
(1) matrimony vine 10kg is taken, crushed after being dried to coarse powder, using pure water as solvent, amount of water is respectively 10 times of crude drug amount,
8 times of volumes, with 1KW~20KW Microwave Extraction Apparatus Microwave Extraction 2 times, extract 5~30 minutes, merge 2 times and take liquid, subtract respectively
Pressure concentration, is concentrated into 100L, Aqueous extracts and the ratio fed intake are 10：1, tripod pendulum type batch centrifugal initial filter takes supernatant fluid filtrate 95L, and add
0.3% potassium sorbate anti-corrosion.
(2) step (2) is same as Example 1 to (8).
2nd, polyoses content and molecular weight identification
The identical method of embodiment 1 is used to measure LBP-X content obtained by this example for 97%, point of LBP-X
Son amount is the Da of 2000Da -2,000,000.
Embodiment 3
1st, ultrasonic extraction method prepares LBP-X
(1) matrimony vine 10kg is taken, crushed after being dried to coarse powder, using pure water as solvent, amount of water is respectively 10 times of crude drug amount,
8 times of volumes, are extracted 2 times with ultrasonic extraction instrument, extracted 5~30 minutes respectively, merged 2 times and take liquid, be concentrated under reduced pressure, be concentrated into
100L, Aqueous extracts and the ratio fed intake are 10：1, tripod pendulum type batch centrifugal initial filter takes supernatant fluid filtrate 95L, and add 0.3% potassium sorbate
Anti-corrosion.
(2) step (2) is same as Example 1 to (8).
2nd, polyoses content and molecular weight identification
Use the identical method of embodiment 1 measure this example obtained by freeze-dried powder LBP-X content for 96%, molecular weight is
The Da of 2000Da -2,000,000.
Embodiment 4 (eye drops)
1st, the LBP-X 0.5g that Example 1 is obtained, is dissolved in 100ml pure water, and LBP-X concentration is 0.5%, is added
Sodium chloride 0.7g, osmotic pressure is determined to be isotonic using freezing point osmotic pressure gauge.
2nd, it is degerming with 0.22 μm of filtering with microporous membrane, it is filling into eye drops, mono- bottle of 10ml.
Embodiment 5 (gel)
Take 2 grams of Acritamer 940s, add in 100ml pure water, be swelled for 2% clear gel it is standby；What Example 1 was obtained
LBP-X 0.5g, is dissolved in 80ml pure water, and LBP-X concentration is 0.5%, adds 2% carbomer gel 10g, is added
0.1% ethylparaben, is mended with appropriate pure water to 100ml, is added triethanolamine and is adjusted PH to 7.0, forms gelinite, sterile point
Dress, is produced.
Embodiment 6 (pharmacodynamic experiment)
1 instrument and reagent, animal
1.1 medicines and reagent
Medicine：Matrimony vine sugar eye drops is the eye drops of embodiment 4.Sulfated compound chondroitin eye drops (Mentholatum medicine company
Co., Ltd), hydrochloric acid racemic Tangut Anisodus Radix parenteral solution (Hangzhou Minsheng Pharmaceutical Group Co).
1.2 experimental animal
SPF grades of mouse, provide, 18~22g of body weight, 50, male and female are each by Traditional Chinese Medicine University Of Guangzhou's Experimental Animal Center
Half.
1.3 laboratory apparatus
LYL-1 slit-lamp microscopes (Phenix Optical Instrument Co., Ltd.), light microscope, histotomy and dyeing are set
Standby, ESEM.
1.4 experimental situation
Guangzhou TCM Universities Experimental Animal Center, mouse cage tool, mouse feed and drinking water are through autoclaving.
2. method
The foundation of 2.1 treatment of experimental dry eye disease mouse model and GP TH
50 experiment mices (male and female half and half) are taken, 5 groups are divided into, each group is respectively：Normal group, model group, positive control
Group, LBP-X low dose group (0.1mg/ml), LBP-X high dose group (50mg/ml).Each group mouse nape part is subcutaneously noted
Radiosulfur acid atropine solution, each consumption is 0.6mg/0.3ml, three times a day (9am, 14pm, 19pm).The 7th of experiment the
My god, on the premise of atropine sulfate solution is injected, while giving positive controls mouse right eye gives sulfated compound chondroitin
Eye drops eye drip, three times a day (10am, 15pm, 20pm), the mouse right eye of blank group and normal group give physiological saline, real
Test a group mouse right eye and give herbal polysaccharide eye drops.In experiment the 7th, 21 days carry out lacrimal secretion measure and cornea fluorescent staining
Determine, and record data.
2.2 observation index and method of testing
2.2.1Schirmer I is tested
Check tear basal secretion situation.Method：It is smaller in view of mouse eye, take 5mm × 35mm tears detecting filters
Doubling halves, and cuts flat reflexed one end, is gently put under tested eye at conjunctival sac China and foreign countries 1/3, filter paper is taken out after 3min, filter paper is observed
Bar wetted length.
2.2.2 the inspection of Ocular surface damage
Fluorescein sodium stained positive reacts corneal epithelial defects.Experimental method：2% Fluress is dripped with dropper
Eye is gently cleaned in drawing physiological saline with syringe in mouse conjunctival sac, after 5min.Under slit-lamp microscope cobalt blue light
Observe coloring case.Methods of marking：Cornea is divided into 4 quadrants, be temporo top, temporo lower section, nose top, nasal downside etc. 4 as
Limit, each quadrant is according to 0~3 point of scoring, and each cornea scoring is 4 quadrant scoring sums.Check that each group measures small every time
Mouse right eye, record data.It is 0 to provide dye-free, have dyeing then divide it is light, in, weigh 3 grades, therefore totally 0~12 point.
2.2.3 pathological study
It is fixed that HE dyeing takes the conjunctiva of animal, cornea to be placed in neutral formalin.Routine paraffin wax is embedded, section, is given conventional
Hematoxylin eosin staining, micro- Microscopic observation.
Electron microscope specimen makes and materials：First the disconnected neck of mouse is put to death, mouse head is then cut, two is cut off along sagittal line
Partly it is put into tissue fixative solution and soaks two weeks.Then dissect again, take out small rathole fornices point, seen using SEM
Examine its conjunctiva of right eye epithelial cell and goblet cell.
3.0 statistical method：One-way analysis of variance, is analyzed with SPSS softwares.
3 experimental results
3.1Schirmer I, fluorescein sodium stained positive reaction experiment result
Shirmer I result of the test, corneal fluorescein dye of the normal group with model group, Chinese herbs group and positive controls
Color results contrast difference has significant (P<0.05), it is shown in Table 1.
The each group Schirmer I result of the tests of table 1, fluorescein sodium stained positive reaction experiment compare (average ± standard
Difference, each group n=10)
Compared with normal group, ▲ P<0.01.Compared with model group, ● P<0.01.Compared with positive controls, ■ P<
0.01。
Each group fluorescein sodium stained positive reaction experiment result is shown in Fig. 1~5, wherein, normal group cornea unstressed configuration element sodium dye
Color positive reaction；Model group corneal fluorescein sodium stained positive significant reaction, it is seen that green fluorescence material full of cornea 4 as
Limit；The plain sodium stained positive reaction of LBP-X treatment group cornea unstressed configuration, with normal group no significant difference, illustrates that treatment can be preferable
Recover cornea integrality in ground；Compared with model group, positive controls cornea Green reactive material is reduced, but not up to matrimony vine is more
Sugared treatment group's level.And LBP-X high dose group curative effect is better than low dose group.
3.2 observe lachrymal gland morphologic observation under an optical microscope
As a result Fig. 6~10 are seen, wherein,
Normal group：Lachrymal gland leaflet structure marshalling, lacrimal epithelial cell is high column.
Model group：It can be seen that lacrimal epithelial cell atrophy, it is seen that vacuole is merged.
LBP-X and positive drug control group：Lachrymal gland structure is shown no obvious abnormalities, and lumen of gland epithelium result is complete.
ESEM amplifies under 2000 times, ten visuals field, the average goblet cell number of each group conjunctiva and inflammatory cell number
Compared with normal group, ▲ P<0.01.Compared with model group, ● P<0.01.Compared with positive controls, ■ P<
0.01。
Pass through scanning electron microscopic observation (see Figure 11~15), it is possible to find normal group has a more intact goblet cell, and mould
Type group is almost without goblet cell, and with inflammatory cell infiltration.LBP-X group and positive controls eye drops group cup-shaped are thin
Born of the same parents are significantly more than model group, and inflammatory cell infiltration substantially mitigates, and wherein LBP-X group curative effect is more notable, and LBP-X is high
Dosage group curative effect is better than LBP-X low dose group, and drug effect dose proportional relation.
Atropic category medicine is subcutaneously injected in this experimental selection：Hydrochloric acid raceanisodamine makes mouse dry eye model, passes through
Competitive assays mAChR, so that causing lacrimal secretion to reduce produces dry eye condition.Tear is by slurries and glues
Liquid is constituted, and wherein serosity tear accounts for the 98% of full tear.This experimental result finds that LBP-X can substantially increase lacrimal secretion.
The attachment for being completely conducive to tear film of corneal epithelium, and the performance of the normal corneal physiological function of tear film also has weight
The effect wanted.As the modeling time increases, mouse cornea epithelium gradually loses neat closely cell arrangement result, cell level
It is disorderly, increase, cells of superficial layer destruction comes off, and loses support and the effect of stable tear film, will cause the unstable of tear film.Fluorescein
Sodium is not dyed to healthy corneal epithelial cell, and the cornea of dyeing damage is to penetrate into institute in matrix by the disperse of damaged cell gap
Cause.When Cell tracking is destroyed, the disperse of fluorescein sodium energy is penetrated into space between cells.Experiment shows LBP-X eye drops in treatment
Afterwards, ocular fluorescein sodium dyeing is significantly reduced, and with normal group no significant difference, shows that LBP-X eye drops can be protected ideally
The form and function of corneal epithelium, have recovered corneal epithelium, maintain the stability of tear film.
Tear film innermost layer is rete malpighii, and rete malpighii adheres to cornea microvillus, on microcreping wall, tear film is equably covered
In ocular, and eye surface tension force can be reduced.Mucus is mainly produced by Conjunctival Goblet Cells, and its main component is various molecular weight
Hydrophobicity anterior corneal surface can be converted into hydrophily by mucoprotein, mucoprotein, made tear spread in anterior corneal surface, formed smooth light
Plane is learned, to keeping the moistening of cornea and maintaining the stabilization of tear film to play an important role.Conjunctival Goblet Cells quantity subtracts during scheroma
Few, secreted the mucus quantity is not enough to form complete film in corneal epithelium, and prevents serosity tear from adsorbing well
, tear film stability reduction.Find that LBP-X eye drip liquid energy ideally recovers conjunctiva cup-shaped by ESEM in this research
Cell number.
Claims (2)
1. application of the LBP-X in the medicine for preparing treatment scheroma, it is characterised in that described medicine is LBP-X
The external preparation being made with the medically acceptable auxiliary material of conventional amount used, wherein the molecular weight of the LBP-X is more than 2000
Dalton and it is made by following methods：
(1) take matrimony vine to be extracted by solvent of pure water, then extract solution is used into molecular cut off for the ultrafiltration of 2000 dalton
Film ultrafiltration, collects the filtered fluid of liquid feeding side；
(2) enzymolysis deproteinized is carried out toward addition neutral proteinase in filtered fluid, then, go out enzyme, centrifugation, takes supernatant；
(3) by DEAE- cellulose chromatographies post on supernatant, and pillar, Ran Houkao are rinsed with 0.1M~1M phosphate buffer
Mas bright blue method detects albumen, discards the eluent that absorbance is more than 0.1；
(4) on-line checking is carried out to the eluent left to evaporate photodetector, collects each crest segment efflux, and with phenol sulphur
Acid system detects the polyoses content of each crest segment efflux, and retention absorbance is more than 0.1 efflux；
(5) by the efflux of retention, using the nanofiltration membrane that molecular cut off is 200 dalton, the filtering of tapping side is discarded
Liquid, remove phosphate-buffered salt, then, the filtered fluid of liquid feeding side is concentrated, vacuum freeze drying, obtain molecular weight for more than
The LBP-X of 2000 dalton.
2. application according to claim 1, it is characterized in that, described external preparation is eye drops or gel.
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| CN111419869A (en) * | 2019-01-09 | 2020-07-17 | 宁夏天仁枸杞生物科技股份有限公司 | Application of lycium barbarum polysaccharide in preparation of medicine for preventing and treating eye diseases |
| CN110051683B (en) * | 2019-05-06 | 2021-07-27 | 广州中医药大学(广州中医药研究院) | Application of rhizoma anemarrhenae polysaccharide in preparing medicine for treating xerophthalmia |
| CN114588250B (en) * | 2022-03-18 | 2024-01-09 | 宁夏杞肽科技有限公司 | Application of lycium barbarum glycopeptide in preparing medicine for preventing or treating xerophthalmia |
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| 枸杞多糖的分离、纯化、分析及免疫作用的研究;王玲等;《昆明医学院学报》;19950630;第16卷(第2期);29-32 * |
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