CN104352711B - The new application of Chinese medicine compound pharmaceutical composition - Google Patents
The new application of Chinese medicine compound pharmaceutical composition Download PDFInfo
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- CN104352711B CN104352711B CN201410549398.4A CN201410549398A CN104352711B CN 104352711 B CN104352711 B CN 104352711B CN 201410549398 A CN201410549398 A CN 201410549398A CN 104352711 B CN104352711 B CN 104352711B
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- A61K36/21—Amaranthaceae (Amaranth family), e.g. pigweed, rockwort or globe amaranth
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Abstract
The invention discloses the purposes containing the bulk drug that following weight matches in the medicine for improving Maternal-placental immune toler ance is prepared：18 30 parts of cultivated land, 9 12 parts of Fructus Corni, 8 16 parts of Chinese yam, 9 12 parts of the fruit of Chinese wolfberry, 8 16 parts of Semen Cuscutae, 8 16 parts of deer horn glue, 1 16 parts of radix cyathulae, 1 16 parts of tortoise-shell glue.Bushen Compound Zuogui Wan of the present invention can improve the Maternal-placental immune toler ance of parent, and it is good to be expected to Pathologic pregnancy, the potential applicability in clinical practices such as treatment Maternal-fetal immunity unbalance caused habitual abortion, PIH, IUGR.
Description
Technical field
The present invention relates to a kind of new application of Chinese medicine compound pharmaceutical composition, specifically improving Maternal-fetal immunity preparing
Purposes in the medicine of tolerance.
Background technology
Gestation is a complicated physiology course.In terms of immunology angle, fetus and its adjunct with paternal antigen
Similar half allogeneic, will be led to that mother's immune system carries out Immune discrimination and response to it；From the point of view of pregnancy outcome, and with
Transplanting is different, and parent has immune tolerance, i.e. Maternal-placental immune toler ance to fetus, fetus is ostracised.Maternal-placental immune toler ance
Particularly significant for successful pregnancy, once Maternal-fetal immunity is unbalance, by occurring, the pathologic such as habitual abortion, PIH, IUGR are pregnant
It is pregnent.
Traditionally it has been recognized that, there is MHC- I to be used as its surface marker on any cell membrane, MHC-I is reflected carefully
The gene in intracellular portion is formed.But progress, the mechanism of Maternal-placental immune toler ance are directly to contact female hemic and immune system
Fetus trophocyte, the antigens of MHC- I that reflection cell interior gene is formed are not expressed on surface but, but expression does not possess polymorphism
HLA-G.
HLA-G, human leucocyte antigen (HLA)-G (human leucocyte antigen-G), it has similar knot to M HC-I
Structure domain-functionalities, but it is seen with height homotype feature in different plant species, therefore parent NK cells are unable to recognition expression this are anti-
Former trophocyte, therefore do not possess lethality to fetal placenta.In addition, HLA-G also has immune chemotaxis, it is this to act on tire
Played a key effect in youngster's development.Identification to HLA-G causes maternal immunity cell to discharge specific chemical signal, these signals
Gestational period immune system is adjusted.Research is found.The time of fertilization in vitro, preovulatory soluble HLA-G are horizontal too low with early stage
Miscarry relevant；In addition, HLA-G expresses increase on the trophocyte of normal pregnancy, and in the placenta of preeclamptic patients then
Reduce or lack.In other words, HLA-G expressions be react body Maternal-placental immune toler ance situation important indicator, Ke Yitong
The change for crossing detection HLA-G expressions judges body Maternal-placental immune toler ance situation.
In addition, research is found, TNF (tumor necrosis factor, TNF) receptor superfamily Fas/
FasL, indoleamine 2,3- dioxygenases (indoleamine 2,3-dioxygenase, IDO), α ν β 3 are also in Maternal-placental immune toler ance
In there is key effect, their expression can react body Maternal-placental immune toler ance situation.
The content of the invention
The technical scheme is that provide the new application of Chinese medicine compound pharmaceutical composition.Specifically carried in preparation
Purposes in the medicine of high Maternal-placental immune toler ance.
The invention provides containing the bulk drug that following weight matches prepare improve Maternal-placental immune toler ance medicine in
Purposes：Cultivated land 18-30 parts, Fructus Corni 9-12 parts, Chinese yam 8-16 parts, fruit of Chinese wolfberry 9-12 parts, Semen Cuscutae 8-16 parts, deer horn glue 8-16
Part, radix cyathulae 1-16 parts, tortoise-shell glue 1-16 parts.
The weight proportion of the bulk drug is as follows：Cultivated land is 24 parts, Fructus Corni is 9-12 parts, Chinese yam is 12 parts, the fruit of Chinese wolfberry
For 9-12 parts, Semen Cuscutae be 12 parts, deer horn glue is 12 parts, 9 parts of radix cyathulae, 12 parts of tortoise-shell glue.
The weight proportion of the bulk drug is as follows：Cultivated land is 24 parts, Fructus Corni is 12 parts, Chinese yam is 12 parts, the fruit of Chinese wolfberry is
12 parts, Semen Cuscutae be 12 parts, deer horn glue is 12 parts, 9 parts of radix cyathulae, 12 parts of tortoise-shell glue.
The medicine is using foregoing as active component, plus pharmaceutically acceptable auxiliary material or complementary composition
The preparation being prepared.
Described preparation is oral formulations.
Described oral formulations are granule, powder, pill, capsule, tablet or oral liquid.
Bushen Compound Zuogui Wan of the present invention can raise the expression of the genes of HLA-G, Fas/FasL, IDO and α ν β 3, show it
There is the Maternal-placental immune toler ance for improving parent, the medicine for improving Maternal-placental immune toler ance can be prepared into, be expected to treat
Pathologic pregnancy, the potential applicability in clinical practice such as habitual abortion, PIH, IUGR are good caused by Maternal-fetal immunity is unbalance.
Obviously, according to the above of the present invention, according to the ordinary technical knowledge and customary means of this area, do not departing from
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The embodiment of form by the following examples, the above of the present invention is remake further specifically
It is bright.But the scope that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to following example.It is all to be based on the above of the present invention
The technology realized belongs to the scope of the present invention.
Brief description of the drawings
Various gene transcript expression situations in Fig. 1 Serum Containing Zuogui Pill Substances group, positive controls and blank group；Ordinate
For 2△ct, wherein " * " withRepresent that two groups are compared P ＜ 0.05；
Various gene transcript expression situations in Fig. 2 Serum Containing Zuogui Pill Substances group, positive controls and blank group；Ordinate
For 2△ct, wherein " * " withRepresent that two groups are compared P ＜ 0.05；
Fig. 3 is each gene time gradient expression of Contained Serum of 5% concentration, and abscissa is gene, ordinate 2△ct, wherein " * " withRepresent that two groups are compared P ＜ 0.05；
The protein expression situations of α ν β 3 in Fig. 4 Serum Containing Zuogui Pill Substances group, positive controls and blank group；Ordinate is ng/
Mg prot, wherein " * " withRepresent that two groups are compared P ＜ 0.05；
HLA-G protein expression situations in Fig. 5 Serum Containing Zuogui Pill Substances group, positive controls and blank group；Ordinate is
Ng/mg prot, wherein " * " withRepresent that two groups are compared P ＜ 0.05；
The time gradient situation of the protein expressions of Contained Serum α ν β 3 of the concentration of Fig. 6 5%；Abscissa is the time, and ordinate is
Ng/mg prot, wherein " * " withRepresent that two groups are compared P ＜ 0.05；
The time gradient situation abscissa of the Contained Serum HLA-G protein expressions of the concentration of Fig. 7 5% is the time, and ordinate is
Ng/mg prot, wherein " * " withRepresent that two groups are compared P ＜ 0.05；
Embodiment
The preparation (Zuogui Wan) of 1 pharmaceutical composition of the present invention of embodiment
Weigh raw material：Prepared rhizome of rehmannia 200g, Semen Cuscutae 100g, root of bidentate achyranthes 75g, tortoise-shell glue 100g, deer horn glue 100g, Chinese yam
100g, Fructus Corni 100g, fruit of Chinese wolfberry 100g；
Preparation method：The taste of the above eight, in addition to deer horn glue, tortoise-shell glue, the Six-element such as remaining prepared rhizome of rehmannia is ground into fine powder, and sieving is mixed
It is even.Deer horn glue, tortoise-shell glue molten are taken, is mixed with above-mentioned fine powder.Add refined honey 10g and appropriate water per 100g powder, general ball, do
It is dry, produce；Or honey is not added with, only powder powder；Or powder is taken, add pharmaceutically acceptable auxiliary material or complementary composition system
It is standby into granule, tablet, capsule, oral liquid.
The preparation of 2 pharmaceutical composition of the present invention of embodiment
Weigh raw material：Prepared rhizome of rehmannia 18g, Semen Cuscutae 8g, root of bidentate achyranthes 1g, tortoise-shell glue 1g, deer horn glue 8g, Chinese yam 8g, Fructus Corni 9g,
Fruit of Chinese wolfberry 9g；
Prepared according to the method for embodiment 1.
The preparation of 3 pharmaceutical composition of the present invention of embodiment
Weigh raw material：Prepared rhizome of rehmannia 30g, Semen Cuscutae 12g, root of bidentate achyranthes 16g, tortoise-shell glue 16g, deer horn glue 16g, Chinese yam 16g, mountain Zhu
Cornel 12g, fruit of Chinese wolfberry 16g；
Prepared according to the method for embodiment 1.
Above-mentioned raw materials can use crude drug in whole, can also use the processed product of crude drug in whole.
It is by effect experiment and clinical trial certificate beneficial effects of the present invention below.
1 Bushen Compound of the present invention of experimental example raises the expression of Maternal-placental immune toler ance related gene
1st, experiment material
1 material
1.1 Specimen origin samples from October, 2013 in April, 2014 Hospital Affiliated To Chengdu Traditional Chinese Medicine Univ pregnant 5~
The pregnant early stage chorionic villi of the normal pregnant women of 10 weeks row artificial abortions.
1.2 laboratory apparatus cell culture incubators (Thermo companies, 3111 types), electric heating constant-temperature water-bath tank (Shanghai Medical seven
Factory), inverted microscope (Leica companies, DMI3000B types), ELIASA (Thermo companies, Multiskan spectrum), determine
Measure PCR instrument (Bio-Rad companies, CFX96), interior cut tissue homogenizer (ART-MICCRA), UV detector
(BECKMAN)。
1.3 reagents and primer RPMI1640 nutrient solutions, hyclone, NTx enzyme, EDTA, it is dual anti-(Gibco-BRL,
MD, USA)；Absolute ethyl alcohol, isopropanol, chloroform (Chengdu Ke Long chemical reagents factory)；Diagnostic Kit for Human
leukocyte antigen G(HLA-G)(ELISA)(Sichuan xinchuang Biotech co.,CTD)；Human
Total Integrinανβ3ELISA(R&D Systems,Catalog No.DYC30502,USA)；
Reagents (Macherey-Nagel, 15596-026)；IScript cDNA Synthesis Kit (BIO-RAD, Lot：
170-8891)；Q-PCR assay kits (Roche companies, FastStart Universal SYBR Green Master
(ROX))；Primer (Shanghai Jierui Biology Engineering Co., Ltd, Invitrogen trade Co., Ltd) (table 1 is primer sequence) etc..
2 methods
2.1 chorionic villi culture operation consents after materials, wash the operating theater instruments autoclaving such as negative-pressure suction bottle with PBS
Chorionic villi is put into RPMI1640 nutrient solution (mycillins immediately：In 100U/ml).It is miscellaneous that blood clot etc. is removed under the microscope
Matter, selects the part that fine hair is short, branch is more, has microvillus, PBS, and rotten shape is cut into horizontalization ware, adds the type glue of 2mg/ml I
Protoenzyme (0.02%EDTA) water-bath digests 10-20min, is terminated and disappeared with isometric RPMI1640 nutrient solutions (10% hyclone)
Change, 1000rpm/min, centrifuge 3min, abandon supernatant.So rinsing 1~2 time, adds a small amount of complete culture solution, histocyte is made
Suspension, it is inoculated in by 5 pieces/cm2 concentration in 24 orifice plates, 37 DEG C, 5%CO2 cultures, supplies nutrient solution after 24h, hereafter changed per 3d
Liquid once, per microexamination under clear water surface.
The preparation of 2.2 Serum Containing Zuogui Pill Substances
By 10 body weight are homogeneous, the rat of male and female half and half is giving Zuogui Wan to gavage, dosage is 5.0g/kg, administration
Capacity is 20ml/kg.30min, 60min, 90min eyeground vein clump take blood after administration, and blood stands 2h, 3000rpm from
Heart 10min, different time sections serum is separated, the serum of 3 periods is mixed, high performance liquid chromatography (High
Performance Liquid Chromatography, HPLC) serum is analyzed, filtration sterilization is standby.
1. tissue cultures carry out grouping experiment to 2.3 Serum Containing Zuogui Pill Substances processing chorionic villi after 5 days, i.e., concentration be 2%,
5%th, 10% Serum Containing Zuogui Pill Substances group, positive controls (control groups, i.e. progesterone：1000ng/ml) and blank group
(blank groups, i.e., the RPMI1640 culture mediums without FBS), carry out continuous processing 72h；3. detection obtains the Contained Serum of high expression
Group, carries out the screening of time gradient (24h, 48h, 72h), and detection method is the same.Above experiment packet is all provided with 3 multiple holes and repetition
Experiment 2 times.
Each group supernatant is abandoned in the detection of related gene expression in 2.4 chorionic villis, the total serum IgE of relevant hole tissue is extracted, through purple
Outer spectrophotometer quantifies and detects its purity (A260/A280 is between 1.8~2.0), synthesizes cDNA immediately, finally with
CDNA is template, using Sybr Green Qpcr reagents carry out real-time fluorescence quantitative PCR analysis with survey endometrium receptivity and
The transcriptional expression of Maternal-placental immune toler ance related gene is horizontal.Detection gene is α ν β 3, HLA-G, Fas/FasL, IDO, reference gene
For people β-Actin.Designed for the detection primer of said gene using software PrimerExpress3.0, primer information such as table 1.
The detection of α ν β 3 and HLA-G expression of gene protein collects each group chorionic villi with PBS in 2.5 chorionic villis, adds
1% protease inhibitors, after being homogenized using tissue homogenizer, 1000rpm/min, 10min is centrifuged, draw supernatant, used
α ν β 3, HLA-G protein expressions in ELISA method detection each group supernatant, in strict accordance with α ν β 3, the experiment of HLA-G kit specifications
Step is carried out, and OD values are read at 450nm with full-automatic ELIASA, obtain α ν β 3, HLA-G concentration；Biuret rule according to
Total protein measure kit specification operation, ELIASA detect its total protein concentration at 562nm.Calculate α ν β 3, HLA-G albumen
Ratio with organizing total protein, so obtains α ν β 3, HLA-G actual protein content.
2.6 data analysis experimental datas are analyzed using the statistical softwares of SPSS 17.0, as a result withRepresent, respectively
Two sample averages compare using independent samples t test between group, and P ＜ 0.05 are then with statistical significance.
Table 1 is for different endometrium receptivities and the Qpcr primers of Maternal-placental immune toler ance related gene
3 experimental results
3.1 Serum Containing Zuogui Pill Substances are to chorionic villi α ν β 3, the influence of HLA-G, Fas/FasL and IDO gene transcript expression
From Fig. 1-Fig. 2, compared with blank group, the Serum Containing Zuogui Pill Substances of the present invention of various concentration significantly improve α
ν, β 3, HLA-G, Fas, FasL and IDO gene transcriptional expression (P ＜ 0.05) wherein 5% concentration Contained Serum it is the most notable.
As seen from Figure 3, influence of the Serum Containing Zuogui Pill Substances of the present invention to β 3, HLA-G, Fas, FasL genetic transcription is in the time
Dependence influences, and for the influence to α ν and IDO genes then without existing time dependence, it can significantly raise α ν bases in 24h and 72h
The transcriptional expression of cause, the then unobvious in 48h, and the influence to the transcriptional expression of IDO genes is maximum in 48h.
Influence of 3.2 Serum Containing Zuogui Pill Substances to α ν β 3, HLA-G protein expressions
From Fig. 4-Fig. 5, compared with blank group, the Serum Containing Zuogui Pill Substances of the present invention of various concentration significantly improve α ν
β 3 and HLA-G protein expressions (P ＜ 0.05), in the Contained Serum of 5% concentration significantly, HLA-G is then dense in 2% by wherein α ν β 3
The Contained Serum of degree is significantly (the equal ＜ 0.05 of P).
Can be seen by Fig. 6-Fig. 7, Serum Containing Zuogui Pill Substances of the present invention to HLA-G albumen, the influence of the expression of the albumen of α ν β 3,
With the extension of time, HLA-G albumen, the expression of the albumen of α ν β 3 first increase, it is rear to reduce, then raise, wherein, the highest in 72h.
Experimental result illustrates that medicine Zuogui Wan of the present invention can raise the table of the genes of HLA-G, Fas/FasL, IDO and α ν β 3
Reach, it is possible to increase the Maternal-placental immune toler ance of parent.
To sum up, Bushen Compound Zuogui Wan of the present invention can raise the expression of the genes of HLA-G, Fas/FasL, IDO and α ν β 3,
Show its Maternal-placental immune toler ance for having the function that to improve parent, can prepare as the medicine of Maternal-placental immune toler ance is improved,
It is good to be expected to Pathologic pregnancy, the potential applicability in clinical practices such as treatment Maternal-fetal immunity unbalance caused habitual abortion, PIH, IUGR
It is good.
Claims (5)
1. by purposes of the bulk drug that following weight proportioning forms in the medicine for improving Maternal-placental immune toler ance is prepared：
Cultivated land is 24 parts, Fructus Corni is 9-12 parts, Chinese yam is 12 parts, the fruit of Chinese wolfberry is 9-12 parts, Semen Cuscutae is 12 parts, deer horn
Glue be 12 parts, 9 parts of radix cyathulae, 12 parts of tortoise-shell glue.
2. the purposes according to claim 1, it is characterised in that：The weight proportion of the bulk drug is as follows：Cultivated land is 24
Part, Fructus Corni are 12 parts, Chinese yam is 12 parts, the fruit of Chinese wolfberry is 12 parts, Semen Cuscutae is 12 parts, deer horn glue is 12 parts, radix cyathulae
9 parts, 12 parts of tortoise-shell glue.
3. the purposes according to claim 1 or 2, it is characterised in that：The medicine is with the raw material of claim 1 or 2
The powder of medicine is active component, the preparation being prepared plus pharmaceutically acceptable auxiliary material or complementary composition.
4. the purposes according to claim 3, it is characterised in that：Described preparation is oral formulations.
5. the purposes according to claim 4, it is characterised in that：Described oral formulations are granule, powder, pill, glue
Wafer, tablet or oral liquid.
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| KR20040108512A (en) * | 2003-06-17 | 2004-12-24 | 주식회사 닥터즈메디코아 | Extract of herbal mixture and composition for the impotence improvement comprising the same |
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| CN103479690A (en) * | 2011-08-23 | 2014-01-01 | 成都中医药大学 | New application of traditional Chinese medicine compound medicinal composition |
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| 育阴灵冲剂对自然流产模型母胎界面Th1/Th2型细胞因子及妊娠结局的影响;韩延华等;《世界中西医结合杂志》;20101231;第5卷(第5期);第391-393页，尤其是第391页摘要结论部分，第392页左栏第3段 * |
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