CN106074585B - Ginsenoside Rb1 and Rd combination and its application in the drug of preparation treatment photoreceptor cell death related disease - Google Patents
Ginsenoside Rb1 and Rd combination and its application in the drug of preparation treatment photoreceptor cell death related disease Download PDFInfo
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Abstract
The present invention relates to the application of ginsenoside Rb1 and Rd combination in preparation treatment Retinal degeneration drug.The present invention uses the mouse model of retinal light damage, simulate the Common key pathology link in a variety of Retinal degeneration generating processes, ginsenoside Rb1 and Rd combination are studied to the intervention effect of retinal light damage mouse model, the result shows that the pharmaceutical composition can maintain outer nuclear layer form, it effectively prevent outer nuclear layer degeneration to damage, prevent the reduction of outer nuclear layer thickness, inhibit photoreceptor cell dead, antagonism retinal pigment epithelium and photoreceptor cell oxidative stress, effectively inhibit the inflammatory reaction of retinal damage related immune, significantly improve Retinal degeneration.Therefore ginsenoside Rb1 and Rd combination can be used for preparing a variety of Retinal degeneration drugs treated including age-related macular degeneration, retinal pigment degeneration, Stargardt disease, the cone-rod cell malnutrition, diabetic retinopathy etc..
Description
Technical field
The present invention relates to pharmaceutical technology fields, specifically, being related to ginsenoside Rb1 and Rd combination in preparation treatment with light
Receptor cell death, retinal pigment epithelium and photoreceptor cell oxidative stress are central pathological including age phase
Closing property macular degeneration, Stargardt disease, the cone-rod cell malnutrition, retinal pigment degeneration and diabetic retinopathy
The application in Retinal degeneration drug including change.
Background technique
Retina degenerative disease is one group on retinal light injury photoreceptor oxidative stress, death and retinal pigment
Chrotoplast oxidative stress is what central pathological changed, can cause the vision disorder even retinopathy of blindness comprising the age is related
Property macular degeneration, Stargardt disease, the cone-rod cell malnutrition, retinal pigment degeneration etc..In addition, photoreceptor is thin
Born of the same parents' excessive oxidation stress be the important early stage pathology link that diabetic retinopathy occurs., view dead for Retina
Generation of the treatment of retinal pigment epithelial cell and photoreceptor cell oxidative stress to above-mentioned associated retinal retrogression pathological changes
Effective intervention effect is played in development.
Age-related macular degeneration is most common the vision health even view of blinding of seriously endangering in world wide
One of the main Types of film retrogression pathological changes.China's epidemiological survey shows that population ages' Macular became in 50 years old or more
The illness rate nearly 15.5% of property.The illness rate that aging of population directly results in age-related macular degeneration increases year by year, structure
At serious social public health problem.The clinical manifestation of age-related macular degeneration is generally divided into " stemness " and " moist " two
Seed type.Dry age macular degeneration mostly occurs in 50 years old or more.Eyesight shows as slow progressive decline or metamorphopsia,
90% or more of the morbidity of age-related macular degeneration is accounted for, there is no effective therapeutic agent at present.The cone, rod cell nutrition are not
Good is one group of hereditary disease, is caused by gene mutations such as ABCA4, not with hypopsia, yctalopia, visual field diminution, photoreceptor function
Good is main feature, there is no effective therapeutic agent at present.It with inherent cause is leading progress that retinal pigment degeneration, which is a kind of,
The Retinal degeneration of property, dystrophic, being related to gene diversity or even same gene can show as in different patients
Different mutation.It is mainly shown as chronic progressive visual field missing, ablepsia, yctalopia, electroretinogram exception, vision disorder very
To blindness, effective therapeutic agent there is no at present.Diabetes are one of the great metabolic diseases that hyperglycemia causes.Cut-off 2012
September, about 300,000,000 4 thousand 7 million people suffers from diabetes in worldwide.Diabetes, which have become, to be only second to cardiovascular disease and swells
The third-largest non-communicable diseases of tumor.Diabetic retinopathy is one kind most commonly seen in diabetic complication, in glycosuria
The early stage of sick process may occur in which, be first factor for leading to blindness in worldwide.In diabetic retinopathy
Disease incidence gradually rises with the lengthening of diabetic duration, and person's disease incidence increases to 69%-90% within 10 years or more.The course of disease 15 years or more
Diabetic about 2% blind because of retinopathy, about 10% patient shows as serious vision impairment.Photoreceptor
The Modern medical therapy intervention of cell transition oxidative stress still has a large amount of blank.
Ginsenoside Rb1's compound English is abbreviated as Ginsenoside Rb1, and Chinese chemical name is referred to as ginsenoside
Rb1, chemical structure are shown in formula I.Ginsenoside Rd's compound English is abbreviated as Ginsenoside Rd, Chinese chemical name
Referred to as ginsenoside Rd, chemical structural formula is as shown in Formula II.
Photoreceptor cell is to refer to experience light stimulus, and generate the sensory cell that nerve impulse is transmitted to nervous centralis,
It is passed in brain processes and plays an extremely important role through vision system in external information, photoreceptor cell includes that retinal rod is thin
Born of the same parents and cone cell, wherein rod cell department noctovision, cone cell department photopic vision and epicritic vision.Photoreceptor cell apoptosis
Research confirm that photoreceptor cell is very fragile, vulnerable to the damage of environment and light source, it be central nervous system nerve it is thin
Born of the same parents, once it is impaired, it cannot regenerate.Chinese patent 201210228744X discloses naringenin and is preparing anti-retinal photoreceptor
Application in the drug of Apoptosis, compound naringenin there is protection to make the photochemical damage of retinal light injury photoreceptor
With, and it is resistant to the apoptosis of retinal light injury photoreceptor, it can be used for preparing various resisting apoptosis of a retinal photoreceptor cell drugs.
It is treating however, existing literature has not been reported ginsenoside Rb1 and Rd combination with photoreceptor cell on dead, retinal pigment
Skin and photoreceptor cell oxidative stress are the disease of central pathological, including age-related macular degeneration, retinal pigment become
In terms of the Retinal degenerations such as property, Stargardt disease, the cone-rod cell malnutrition, diabetic retinopathy
Pharmacological activity.
Summary of the invention
The purpose of the present invention is aiming at the shortcomings in the prior art, provide it is a kind of prevention and/or treatment retina degeneration
The pharmaceutical composition of lesion.
Another purpose of the invention is to provide the purposes of pharmaceutical composition as described above.
To achieve the above object, the technical solution adopted by the present invention is that:
A kind of pharmaceutical composition prevented and/or treat Retinal degeneration, the drug is by following bulk drugs system
It is standby to form: ginsenoside Rb1, ginsenoside Rd, the ginsenoside Rb1, ginsenoside Rd structural formula respectively such as Formulas I and formula
Shown in II.
The ginsenoside Rb1 and ginsenoside Rd's mass ratio are 26:5-10 as a preferred implementation manner,；
The ginsenoside Rb1 and ginsenoside Rd's mass ratio are 26:9 as a preferred implementation manner,.
Described pharmaceutical composition includes the pharmaceutical composition of therapeutically effective amount as described above as a preferred implementation manner,
Object and its pharmaceutically acceptable excipient, carrier or diluent.
The pharmaceutically acceptable carrier includes the additives of ophthalmically acceptable solvent, injection as a preferred implementation manner,
With additives, tablet additives, surfactant and stabilizer；The ginsenoside Rb1 and Rd group are combined into the medicine group
Close the main active of object.
In the present invention, the ginsenoside Rb1 and Rd pharmaceutical composition can be used alone or as active constituent and its
He is used in mixed way conventional auxiliary material.
Described pharmaceutical composition further includes the drug of other treatment eye disease as a preferred implementation manner,.
To realize above-mentioned second purpose, the technical solution adopted by the present invention is that:
In a first aspect, providing drug as described above for treating and/or preventing Retinal degeneration, the view
Film retrogression pathological changes are that or have retinal pigment epithelium or light sensation using retinal light injury photoreceptor death as central pathological
The oxidative stress of receiver cell participates in.
The Retinal degeneration is selected from as a preferred implementation manner: age-related macular degeneration,
Stargardt disease, the cone-rod cell malnutrition, retinal pigment degeneration and diabetic retinopathy.
Second aspect provides purposes of the pharmaceutical composition as described above in pharmacy or reagent, the drug or reagent
For protecting form, the structure or function of retina.
The third aspect provides purposes of the pharmaceutical composition as described above in pharmacy or reagent, the drug or reagent
For inhibiting retinal light damage.
Fourth aspect provides purposes of the pharmaceutical composition as described above in pharmacy or reagent, the drug or reagent
For:
(1) for preventing or treating retinal light injury photoreceptor death；
(2) it is used to preventing or treating retinal pigment epithelium or photoreceptor cell excessive oxidation stress；
(3) for maintaining outer nuclear layer form；
(4) for preventing the reduction of outer nuclear layer thickness；
(5) for inhibiting retinal damage related immune inflammatory reaction.
Described pharmaceutical composition is pharmaceutically acceptable as single component or with other as a preferred implementation manner,
Ingredient constitutes composition to pharmacy or reagent.
In the present invention, described pharmaceutical composition ginsenoside Rb1 and Rd are that inventor passes through greatly in numerous ginsenosides
Obtained from measuring experiment screening, the combination of ginsenoside Rb1 and Rd have the advantages that good drug efficacy, dosage are small.
The invention has the advantages that:
The present invention uses the mouse model of retinal light damage, simulates in a variety of Retinal degeneration generating processes
Common pathology link, i.e. photoreceptor cell death, retinal pigment epithelium and photoreceptor cell oxidative stress, to people
Join saponin(e Rb1 and Rd and combines anti-light receptor cell death, anti-retinal pigment epithelium oxidative stress, anti-retinal damage
The effect that related immune inflammatory reaction, prevention and treatment Retinal degeneration occur is studied, the results showed that ginsenoside Rb1
Photoreceptor cell mortality, retinal pigment epithelium and the photoreceptor cell oxygen induced light injury is combined with Rd
Change stress, retinal light injury photoreceptor degenerative change and retinal damage related immune inflammatory reaction there is significant inhibit
Effect, can significantly treat and improve Retinal degeneration, therefore can be used for preparing treatment includes that age-related macular becomes
Including property, retinal pigment degeneration, Stargardt disease, the cone-rod cell malnutrition, diabetic retinopathy etc.
A variety of Retinal degeneration drugs.
Detailed description of the invention
Attached drawing 1 is the OCT qualification result of ginsenoside Rb1 and Rd combined therapy to photoreceptor cell protective effect.Its
Middle normal control mice receives 50 μ l30%DMSO solvent controls, and light injury model mice receives 50 μ l30%DMSO solvents pair
According to ginsenoside Rb1 and Rd group are combined mouse and receive 50 μ l ginsenoside Rb1s and Rd combined therapy, ginsenoside Rb1 and Rd agent
Amount is respectively 65mg/kg and 22.5mg/kg weight, and all treatments are all made of intraperitoneal injection, in addition to normal control mice, control
30 minutes each group mouse receive white light after treatment, and illumination condition 10,000Lux continues 30 minutes.7 days after illumination, mouse
Mydriasis after the anesthesia of 1% yellow Jackets respectively, carries out retinal structure imaging analysis using OCT.No light: normal control,
Do not receive illumination；Light_vehicle: light loss wound model；Light_Rb1&Rd: ginsenoside Rb1 and Rd combined therapy and light
According to group.
Attached drawing 2 is the quantitative analysis of ginsenoside Rb1 and Rd combined therapy to retina protective effect, and OCT analysis is completed
Afterwards, quantitative analysis has been carried out to ONL thickness.
Attached drawing 3 is the histopathological study of ginsenoside Rb1 and Rd combined therapy to retina protective effect.It is all small
Mouse was put to death after OCT image in 7 days, and solution takes eyeball, and 4% paraformaldehyde is fixed, and carried out paraffin embedding and slice.The stone of 4 μ m-thicks
Wax slice carries out H&E dyeing.The result of H&E dyeing carries out micro- sem observation and takes pictures and analyze.
Attached drawing 4 is the immunohistochemistry research of ginsenoside Rb1 and Rd combined therapy to retina protective effect.It is all
Mouse was put to death after OCT image in 7 days, and solution takes eyeball, and 4% paraformaldehyde is fixed, and carried out paraffin embedding and slice.Take 4 μ m-thicks
Paraffin section de-waxing processing after carry out Rhodopsin, opsin M and DAPI immunostaining, respectively to rod cell acromere, view
It bores cellular matrix sheath and nucleus progress is immune labeled.Immunohistochemistry results carry out micro- sem observation and take pictures and analyze.
Attached drawing 5 is that ginsenoside Rb1 and Rd combine anti-retinal pigment epithelium and photoreceptor cell oxidative stress
Research.After illumination superoxide anion fluorescence probe DHE is injected intraperitoneally in 1 day in each group mouse respectively, puts to death after 2 hours,
Solution takes eyeball, separates and reject cornea and crystalline lens, remaining part containing retina carries out 4% paraformaldehyde and fixes, and carries out ice
Freeze embedding and slice, take the frozen section of 12 μ m-thicks to the red fluorescence result of each layer of retina carry out micro- sem observation take pictures and
Analysis.
Attached drawing 6 is the research for the activation hyperplasia that ginsenoside Rb1 and Rd combine anti-retina immune inflammation cell.Each group is small
Mouse is put to death for 3 days after illumination, and solution takes eyeball respectively, separates and reject cornea and crystalline lens, remaining part containing retina carries out
4% paraformaldehyde is fixed, and frost embedding and slice are carried out, and takes the frozen section of 12 μ m-thicks to carry out GFAP, Iba1 and DAPI immune
Dyeing respectively carries out M ü ller spongiocyte, microglia and nucleus immune labeled.Immunohistochemistry results into
The micro- sem observation of row is taken pictures and is analyzed.
Specific embodiment
The invention will be further elucidated with reference to specific embodiments.It should be understood that these embodiments are merely to illustrate this hair
It is bright rather than limit the scope of the invention.In addition, it should also be understood that, after having read the content of the invention recorded, art technology
Personnel can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Fixed range.
Term used herein " acceptable " refers to a prescription component or active constituent to the health of general treatment target
Inexcessive adverse effect.
Term used herein " treatment ", including mitigation, inhibition or the symptom or situation that improve disease；Inhibit complication
It generates；Improve or prevent potential metabolic syndrome；Inhibit the generation of disease or symptom, such as controls the development of disease or situation；Subtract
Light disease or symptom；Disease or symptom is set to decline；Mitigate the complication as caused by disease or symptom, or prevention or treatment by disease
Or sign caused by symptom.As used herein, a certain compound or pharmaceutical composition after administration, can make a certain disease, symptom
Or situation is improved, espespecially its severity is improved, delayed onset, slows down disease progression, or reduce the state of an illness duration.
No matter fix administration or interim administration, be administered continuously or interrupted continuous administration, can be attributed to or the situation related with administration.
Term used herein " pharmaceutically acceptable " herein refers to a kind of substance, such as carrier or dilution, will not make chemical combination
The bioactivity or property of object disappear, and relative nontoxic e.g. gives individual something, will not cause undesired biotic influence
Or it interacts in harmful manner with any component that it contains.
Embodiment
In the present embodiment, pass through retina eyeground tomoscan OCT technology, pathology and the immuning tissue of Noninvasive
Research means specify ginsenoside Rb1 and Rd combination to the intervention effect of retinal light damage mouse model.Research knot
Fruit shows that ginsenoside Rb1 and Rd combination can effective prevention photoreceptor cell be dead and the generation of Retinal degeneration,
Significant protective effect is played to the structure of retina.
One, method
1. drug
Ginsenoside Rb1 and ginsenoside Rd.Ginsenoside Rb1 is purchased from Shanghai Yuan Ye Biotechnology Co., Ltd,
41753-43-9, Lot#H02J6X4, purity >=98%.Ginsenoside Rd is purchased from Shanghai Yuan Ye Biotechnology Co., Ltd,
52705-93-8, Lot#H02J6X2, purity >=98%.
2. animal model
It is tested using 4-6 week old female Balb/c mouse (Si Laike, Shanghai).Mouse is randomly divided into normal control
Group, light injury model control group, ginsenoside Rb1 and Rd combined treatment, ginsenoside Rb and Rd dosage be respectively 65mg/kg and
22.5mg/kg weight is injected intraperitoneally for 30 minutes before illumination.Light stimulation will be set using the cold fluorescent lamp of disperse white, illumination condition
10,000Lux are set to, continues 30 minutes.After illumination 7 days, retinal morphology analysis is carried out respectively.
3. retinal structure is analyzed
The 7th day after drug therapy and illumination, 1% yellow Jackets anesthetized mice, after Tropicamide mydriasis, using toy
OCT (Phoenix Research Labs) is observed and is compared to each group Mouse Retina form respectively.
The analysis of 4.ONL thickness measurement
After OCT image, from ONH (the optic papilla), to inside and outside retina ONL thickness with 500 μm be between
Away from measuring, quantitative analysis is carried out to the effect of each group retinal structure protection.
5. retinal tissue pathology
7 days after OCT image, mouse is put to death, eyeball is taken, is fixed with 4% paraformaldehyde.Fixed eyeball tissue carries out stone
Wax embedding and slicing treatment.Paraffin section is with a thickness of 4 μm, for further H&E dyeing and immunohistochemical staining, including
Rhodopsin marks cone cell, opsin M to mark rod cell, and DAPI carries out nuclear marker.3 days after illumination, put to death
Mouse takes eyeball, rejects cornea and crystalline lens under microscope, the eyeball tissue containing retina is fixed with 4% paraformaldehyde.Gu
Fixed eyeball tissue carries out frozen section processing.Frozen tissue section is used for further immunohistochemistry with a thickness of 12 μm
Dyeing, including GFAP label M ü ller spongiocyte, Iba1 mark microglia, and DAPI carries out nuclear marker.
6. in-situ oxidation stress level is analyzed
1 day after drug therapy and illumination, superoxide anion fluorescence probe DHE is injected intraperitoneally in each group mouse respectively, and 2 is small
When put to death mouse, take eyeball, separate and reject cornea and crystalline lens, will contain retina part carry out 4% paraformaldehyde fix.
Fixed eyeball tissue carries out frozen section processing, and frozen tissue section is each for further analyzing retina with a thickness of 12 μm
The red fluorescence of layer.
7. statistical analysis
Data representation is means ± S.E, and data analysis uses indepedent sample t test method.p<0.05
It is defined as statistically-significant difference.
Two, result
It is seriously damaged 1. white light induces Mouse Retina
7 days after white light, retinal structure is imaged using OCT.As shown in Figure 1, white light induces seriously
With photoreceptor cell damage be main pathological manifestations retina degenerative change, be mainly shown as after white light 7 days
ONL is seriously damaged.
2. ginsenoside Rb1 and Rd combination are to retina protective effect
30 minutes before white light, mouse receives solvent or ginsenoside Rb1 and Rd and is treated in combination, ginsenoside Rb1 and
Rd dosage is respectively 65mg/kg and 22.5mg/kg weight, and volume is 50 μ l, intraperitoneal injection.7 days after illumination, carried out using OCT
Retinal structure analysis.As shown in Figure 1, the hair for significantly suppressing retinal light damage is treated in combination in ginsenoside Rb1 and Rd
It is raw, it is mainly shown as that ONL form remains intact, the quantitative analytical data (Fig. 2) of ONL thickness is shown and does not receive illumination
Normal mouse retina compares, and illumination causes retina ONL thickness seriously to reduce (p < 0.05 *), and ginsenoside Rb1 and Rd
It is treated in combination and significant protection, the close normal mouse view for not receiving illumination of respective ONL thickness is played to retina ONL
Film ONL thickness (* light injury model group compared with Normal group, p < 0.05；# ginsenoside Rb1 and Rd combination and light injury mould
Type group compares, p < 0.05).Histopathological study result (Fig. 3) shows each layer knot of the normal mouse retina for not receiving illumination
Structure is complete, and light injury mouse shows as ONL and seriously reduces, and the retina of ginsenoside Rb1 and Rd combined therapy group mouse is each
Layer structure is similar to light group is not received.Further immunohistochemistry research result (Fig. 4) shows the ONL of DAPI label
In light injury Mouse Retina serious loss, compared with normal control, the rarely seen remnants expression of Rhodopsin, opsin M (* p <
0.05).Ginsenoside Rb1 and Rd combined therapy group Rhodopsin, opsin M expression pattern are similar to Normal group, ONL
Have no obvious damage (Rhodopsin: red, opsin M: red, DAPI: blue).It is visited using superoxide anion silver light
Needle DHE carries out the analysis of in-situ oxidation stress level, as shown in figure 5, not receiving each layer of normal control mice retina of illumination not
See red fluorescent, the retinal pigment epithelium (RPE) and ONL of light injury Mouse Retina be visible significantly increase it is red
Color fluorescence signal, ginsenoside Rb1 and Rd combined therapy group mouse are only in the visible faint red fluorescent of RPE.GFAP mark
Note M ü ller spongiocyte is mainly expressed in the normal control mice of non-illumination in nerve fibre layer (Fig. 6), light loss wound model
Mouse Retina then shows as ONL, IPL and INL and has obvious GFAP and expresses, ginsenoside Rb1 and Rd combined therapy group
GFAP expression pattern is similar to Normal group；The microglia of Iba1 label is mainly expressed in normal mouse retina
The retina of IPL (Fig. 6) light loss wound model is shown in that a large amount of positive Iba1 is expressed in ONL, OPL, and ginsenoside Rb1 and Rd group
The Iba1 expression pattern for closing treatment group is similar to Normal group.
In conclusion by using OCT iconography and retinal histopathology means to retinal light damage model
Research prompt ginsenoside Rb1 and Rd combination are to the death of retinal light injury photoreceptor, retinal pigment epithelium and light
Retinal degeneration caused by permissive cell oxidative stress, the inflammatory reaction of retinal damage related immune extremely has
There is significant preventive and therapeutic effect.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, under the premise of not departing from the method for the present invention, can also make several improvement and supplement, these are improved and supplement also should be regarded as
Protection scope of the present invention.
Claims (6)
1. purposes of the combination of ginsenoside Rb1 and Rd in pharmacy, which is characterized in that the drug is for treating and/or in advance
Anti- Retinal degeneration, the Retinal degeneration be using retinal light injury photoreceptor death as central pathological,
Or there is the participation of the oxidative stress of retinal pigment epithelium or photoreceptor cell；The combination of the ginsenoside Rb1 and Rd
As single active ingredient.
2. purposes according to claim 1, which is characterized in that the Retinal degeneration is selected from: age related
Macular degeneration, Stargardt disease, the cone-rod cell malnutrition, retinal pigment degeneration.
3. purposes of the combination of ginsenoside Rb1 and Rd in pharmacy or reagent, which is characterized in that the drug or reagent are used for
Protect form, the structure or function of retina；The combination of the ginsenoside Rb1 and Rd are as single active ingredient.
4. purposes of the combination of ginsenoside Rb1 and Rd in pharmacy or reagent, which is characterized in that the drug or reagent are used for
Inhibit retinal light damage；The combination of the ginsenoside Rb1 and Rd are as single active ingredient.
5. purposes of the combination of ginsenoside Rb1 and Rd in pharmacy or reagent, which is characterized in that the ginsenoside Rb1 and
The combination of Rd is used for as single active ingredient, the drug or reagent:
(1) for preventing or treating retinal light injury photoreceptor death；
(2) it is used to preventing or treating retinal pigment epithelium or photoreceptor cell excessive oxidation stress；
(3) for maintaining outer nuclear layer form；
(4) for preventing the reduction of outer nuclear layer thickness；Or
(5) for inhibiting retinal damage related immune inflammatory reaction.
6. -5 any purposes according to claim 1, which is characterized in that the combination conduct of the ginsenoside Rb1 and Rd
Single component constitutes composition to pharmacy or reagent with other pharmaceutically acceptable ingredients.
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| 血塞通对糖尿病视网膜病变患者眼底微循环及血流动力学的影响;段元青等;《山东医药》;20140606;第54卷(第21期);第63-65页，参见摘要 * |
| 超滤工艺对血塞通注射液中有效成分的影响研究;雄海伟等;《中药材》;20090331;第32卷(第3期);参见全文 * |
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