CN107441106B - The pharmaceutical composition of ginsenoside RG5 and RZ1 a kind of and the application in brain protection - Google Patents
The pharmaceutical composition of ginsenoside RG5 and RZ1 a kind of and the application in brain protection Download PDFInfo
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- CN107441106B CN107441106B CN201710809967.8A CN201710809967A CN107441106B CN 107441106 B CN107441106 B CN 107441106B CN 201710809967 A CN201710809967 A CN 201710809967A CN 107441106 B CN107441106 B CN 107441106B
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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Abstract
The active constituent of application the present invention relates to the pharmaceutical composition of ginsenoside RG5 and RZ1 a kind of and in brain protection, the pharmaceutical composition is ginsenoside RG5 and RZ1, can be used for preparing the drug of brain protection.The present invention provides a kind of pharmaceutical preparation, including ginsenoside RG5 and RZ1 pharmaceutical composition, also comprising pharmaceutically acceptable carrier, pharmaceutically acceptable dosage form is made, the pharmaceutically acceptable carrier includes one or more solids, semisolid or Auxiliary Liquid Material, and the pharmaceutically acceptable dosage form includes injection, tablet, capsule, granule, pill, syrup, powder, paste.In pharmaceutical composition provided by the invention, ginsenoside RG5 and RZ1 have the function of mutually promoting through blood-brain barrier, are significantly higher than the blood brain barrier transmissivity of ginsenoside RG5 or RZ1 monomer, and therefore, the two can be with drug combination.
Description
Technical field
It is protected the present invention relates to the pharmaceutical composition of pharmaceutical composition more particularly to a kind of ginsenoside RG5 and RZ1 and in brain
Application in shield.
Background technology
Known ginsenoside has multiple pharmacological effect, including brain protection and neuroprotection.Ginsenoside RG5 and RZ1
It is the rare saponin(e being dehydrated by ginsenoside RG3, isomer each other, it is C20-C22 along alkene to differ only in the former,
The latter is the anti-alkene of C20-C22 (seeing below formula).Applicants have found that ginsenoside RG5 and RZ1 has more than ginsenoside RG3
For excellent brain protection and neuroprotection.But ginsenoside RG5 and RZ1 molecular weight is larger, side chain there are one diglycosyl,
Blood-brain barrier percent of pass is low, and availability is low.
Blood-brain barrier (BBB) is mammalian central nervous system (CNS) distinctive defence organization, and the brain in brain tissue is micro-
Vascular endothelial cell is closely coupled, gapless between endothelial cell, and capillary outer surface is almost astroglia
It surrounds, this special construction forms the physiologic barrier of protection brain, and the physiologic barrier is in blocking pathogenic microorganism and toxicity production
Object, foreign particles include dye granule etc. from blood flow into brain tissue and cerebrospinal fluid in, with protect CNS from damage while,
It hinders most of chemical composition and enters CNS, or make the drug concentration into CNS very low, seriously affected drug effect.
In order to improve the concentration of ginsenoside RG5 and RZ1 in brain tissue, the BBB transmitances for improving the two are needed first.
Invention content
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of pharmaceutical compositions of ginsenoside RG5 and RZ1
Object and the application in brain protection, improve the blood brain barrier transmissivity of ginsenoside RG5 and RZ1.
To achieve the above object, the present invention provides following technical schemes：
A kind of pharmaceutical composition, active constituent are ginsenoside RG5 and RZ1.
Application of the aforementioned pharmaceutical compositions in preparing brain-protection drugs.
A kind of pharmaceutical preparation, including pharmacy is made also comprising pharmaceutically acceptable carrier in above-mentioned pharmaceutical composition
Upper acceptable dosage form.
Preferably, the pharmaceutically acceptable carrier includes one or more solids, semisolid or Auxiliary Liquid Material.
Preferably, the pharmaceutically acceptable dosage form includes injection, tablet, capsule, granule, pill, sugar
Starch agent, powder, paste.
Application of the said medicine preparation in preparing brain-protection drugs.
Applications of the ginsenoside RG5 in preparing the drug for improving ginsenoside RZ1 blood brain barrier transmissivities.
Applications of the ginsenoside RZ1 in preparing the drug for improving ginsenoside RG5 blood brain barrier transmissivities.
Advantages of the present invention：
In pharmaceutical composition provided by the invention, ginsenoside RG5 and RZ1 have the work mutually promoted through blood-brain barrier
With being significantly higher than the blood brain barrier transmissivity of ginsenoside RG5 or RZ1 monomer, therefore, the two can be with drug combination.
Description of the drawings
Fig. 1 is the chemical structural formula of ginsenoside RG5 and RZ1；
Fig. 2 is the bEnd.3 cellular morphologies observed under inverted microscope；
Fig. 3 is the drug transmitance (%) of each group ginsenoside RG5 and/or RZ1.
Specific implementation mode
Embodiment 1：Ginsenoside RG5, RZ1 collaboration promotes to penetrate blood-brain barrier
One, experimental method
1, the culture of mouse bEnd.3 cells with freeze
By mouse bEnd.3 cells (Shanghai Cell Bank of the Chinese Academy of Sciences offer) with DMEM high glucose mediums (containing 15%FBS,
1%100U/mL blueness-streptomysin) 37 DEG C, 95% humidity, 5%CO2Culture, changes liquid, waits for that cell exists every other day in constant incubator
It is grown in culture bottle to when about 85% fusion and is digested with 0.25% pancreatin, and press 1:2 ratios pass on.Frozen stock solution (10% is prepared in advance
DMSO and 90%FBS mixed liquors), preserve precooling in 40 DEG C of refrigerators.Logarithmic growth phase cell discards the culture in culture bottle
The PBS of base, pre-temperature is washed 3 times, and 0.25% trypsin digestion cell is observed under the microscope, is added when cell is slightly variable circle new
The culture medium of fresh pre-temperature terminates digestion, dispels cell, cell suspending liquid is centrifuged, discard culture medium, the frozen stock solution of precooling is added
Enter in cell precipitation and suspension cell, every cryopreservation tube are added lmL frozen stock solutions, are marked after sealing, freeze falling temperature gradient again：4
DEG C, 2h；20 DEG C, 2h；80 DEG C, 2h；It is stored in liquid nitrogen container after freezing.
2, the cytotoxicity of mtt assay detection ginsenoside RG5, RZ1
Precision weighs ginsenoside RG5, RZ1 (self-control, purity are more than 95%, and chemical structural formula is shown in Fig. 1) respectively, molten respectively
In DMSO, it is configured to the mother liquor of a concentration of 10mmol/L.Precision measures appropriate mother liquor and plasma-free DMEM medium progress is added
Gradient dilution respectively obtains RG5, RZ1 solution of final concentration of 0,0.005,0.05,0.5,5,50,500 μm of ol/L, wherein respectively
DMSO volume fractions < 0.1% in solution.
All drug solutions are all made of 0.22 μm of miillpore filter degerming.
Single cell suspension is made in the bEnd.3 cells of logarithmic growth phase, the digestion of 0.25% pancreatin, and adjustment cell count is about
It is 2 × 104A/mL is inoculated in 96 orifice plates, before orifice plate use PBS rinses 2 times and being put into 37 DEG C of incubators be saturated
30min.100 μ L cell suspensions are added per hole, when inoculation otherwise stop mixing to prevent cell precipitation.6 multiple holes of every group of setting, edge
Hole is filled using PBS, reduces the evaporation of culture medium to the greatest extent, and 96 orifice plates completed are placed in incubator.After cell is adherent, into
Row synchronization process.After synchronization, the drug that respective concentration is added continues to be incubated for 24 hours.After pharmaceutical intervention, add per hole
Enter 10 μ L MTT cultures 4h.Crystallization can be formed after 4h, carefully siphon away supernatant, and 150 μ L DMSO of addition set shaking table low speed and shake
10min makes crystallization dissolve.Absorbance (A) value is measured at 490nm using multi-function microplate reader, calculates cell survival rate：Cell
Survival rate=A experiments/A controls.
3, prepared by BBB in vitro models
Using the in-vitro simulated normal condition BBB of monolayer endothelial cell.In the thin of the small indoor inoculation Suitable Densities of Transwell
Born of the same parents, in 5%CO2, in 37 DEG C of incubators culture to cell fusion state.It is every using cell potential instrument from the 2nd day after inoculation
It measures the membrane potential (TEER) of cell, METHOD FOR CONTINUOUS DETERMINATION 10d, when continuous five days resistance values measured are close to constant, and locates
In 80-140 Ω cm2When show cell tight and complete, it is believed that BBB is basically formed.TEER=(Ω experiment-Ω blank) × S,
Ω is cross-film resistivity measurements, and S is that (12 hole plate counterdie areas are 1.1cm to plate counterdie area2)。
4, drug is detected through the permeability of BBB external models
Experiment is divided into control group, solvent group, RG5 groups, RZ1 groups, RG5+RZ1 groups.Control group is respectively to supply pool and reception
Serum free medium 0.5mL and 1.5mL are added in pond.The free serum culture containing 0.1%DMSO is added in solvent group in supply pool
Serum free medium 1.5mL is added in base 0.5mL in reception tank.(50 μm of ol/ containing RG5 are added in administration group into supply pool respectively
L serum-free is added in reception tank by), the serum free medium 0.5mL of RZ1 (50 μm of ol/L), RG5+RZ1 (50+50 μm of ol/L)
Culture medium 1.5mL.Before dosing and dosing measures the TEER of each group respectively afterwards for 24 hours.And take afterwards for 24 hours in supply pool in dosing,
The culture medium in outside measures the concentration of contained RG5, RZ1 in culture medium.With the amount of reception tank RG5, RZ1 divided by the total amount of dosing
Calculate drug transmitance：Transmitance=reception tank medication amount/supply pool medication amount.
5, statistical procedures
Multisample mean compares using variance analysis, and two sample averages compare to be examined using t, counts soft using SPSS22.0
Part is handled, and each group of data is indicated with mean value ± deviation.
Two, experimental result
1, morphological observation
With 0.25% trypsin digestion cell, it is inoculated in 25cm2In culture bottle, most cells adherent growth changes liquid every other day,
About 2-3d fusions are in blocks when cell state is good, pass on 1 time, observed under inverted microscope, and bEnd.3 cells are not in when growth
Regular shape has pseudopodium stretching, extension, adherent growth, such as Fig. 2 (× 200).
2, influences of ginsenoside RG5, the RZ1 to bEnd.3 cell survival rates
After ginsenoside RG5, RZ1 handle bEnd.3 cells for 24 hours respectively, solvent does not influence the survival of cell.With compare
Group is compared, and for ginsenoside RG5, RZ1 concentration in 0.005-50 μm of ol/L, cell survival rate is more than 85%, when concentration is high again just
Cell survival rate can be significantly reduced, therefore, a concentration of 50 μm of ol/L of ginsenoside RG5, RZ1 is selected to carry out follow-up study.
3, ginsenoside RG5, RZ1 collaborations promote the influence through BBB external model permeabilities
It is substantially change (P > 0.05) the experimental results showed that being had no with TEER after administration for 24 hours before each group administration.Using this reality
The method for testing foundation early period detects RG5, RZ1 concentration in culture medium, and compared with RG5 groups, the RG5 transmitances of RG5+RZ1 groups are notable
It improves (P < 0.05)；Compared with RZ1 groups, the RZ1 transmitances of RG5+RZ1 groups significantly improve (P < 0.05).
Experimental result is shown in Table 1 and Fig. 3.
The drug transmitance (%) of table 1 each group ginsenoside RG5 and/or RZ1
| RG5 transmitances (%) | RZ1 transmitances (%) | |
| RG5 groups | 39.42±6.26 | / |
| RZ1 groups | / | 43.58±5.95 |
| RG5+RZ1 groups | 92.17±5.63 | 98.43±6.08 |
Embodiment 2：Brain protects ginsenoside injection
Active constituent is ginsenoside RG5 and RZ1, is prepared with sterile water and surfactant.
Embodiment 3：Brain protects ginsenoside tablet
Active constituent is ginsenoside RG5 and RZ1, and tablet is made with one or more solids, semisolid or Auxiliary Liquid Material.
Embodiment 4：Brain protects ginsenoside capsule
Active constituent is ginsenoside RG5 and RZ1, and capsule is made with one or more solids, semisolid or Auxiliary Liquid Material
Agent.
Embodiment 5：Brain protects ginsenoside granule
Active constituent is ginsenoside RG5 and RZ1, and particle is made with one or more solids, semisolid or Auxiliary Liquid Material
Agent.
Embodiment 6：Brain protects ginsenoside pill
Active constituent is ginsenoside RG5 and RZ1, and pill is made with one or more solids, semisolid or Auxiliary Liquid Material.
Embodiment 7：Brain protects ginsenoside syrup
Active constituent is ginsenoside RG5 and RZ1, and syrup is made with one or more solids, semisolid or Auxiliary Liquid Material
Agent.
Embodiment 8：Brain protects ginsenoside powder
Active constituent is ginsenoside RG5 and RZ1, and sugar, which is made, with one or more solids, semisolid or Auxiliary Liquid Material dissipates
Agent.
Embodiment 9：Brain protects ginsenoside paste
Active constituent is ginsenoside RG5 and RZ1, and paste is made with one or more solids, semisolid or Auxiliary Liquid Material.
Above-described embodiment proves that ginsenoside RG5 and RZ1 have the function of mutually promoting through blood-brain barrier, significantly high
In the blood brain barrier transmissivity of ginsenoside RG5 or RZ1 monomer, therefore, the two can be prepared into composition drug combination.
It will be appreciated by those skilled in the art that above-mentioned specific implementation mode is only used for explaining the present invention, protection of the invention
Range is not limited to above-mentioned specific implementation mode.
Claims (2)
1. applications of the ginsenoside RG5 in preparing the drug for improving ginsenoside RZ1 blood brain barrier transmissivities
2. applications of the ginsenoside RZ1 in preparing the drug for improving ginsenoside RG5 blood brain barrier transmissivities.
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Effective date of registration: 20180711 Address after: 211505 D1 building, 9 Chuang Chuang Road, Zhongshan science and Technology Park, Liuhe District, Nanjing, Jiangsu. Applicant after: Nanjing qiannianjian stem cell gene engineering Co Ltd Address before: 810003 Chengbei Biological Park, Xining, Qinghai, No. 7, No. three road. Applicant before: Qinghai colorful flower Biotechnology Co., Ltd. |
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Inventor after: Niu Min Inventor after: Wang Hongming Inventor after: Zhang Dandan Inventor after: Yang Yong Inventor after: Peng Yuze Inventor before: Zhang Dandan Inventor before: Yang Yong Inventor before: Peng Yuze |
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Effective date of registration: 20180928 Address after: 710000 Shaanxi, Xi'an, new town, 102 longevity street, 21 floor, 3 doors, 1 floors 1. Applicant after: Niu Min Address before: 211505 D1 building, 9 Chuang Chuang Road, Zhongshan science and Technology Park, Liuhe District, Nanjing, Jiangsu. Applicant before: Nanjing qiannianjian stem cell gene engineering Co Ltd |
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