CN104096014A - Traditional Chinese medicine effective parts combination for mobilization of marrow mesenchymal stem cells - Google Patents
Traditional Chinese medicine effective parts combination for mobilization of marrow mesenchymal stem cells Download PDFInfo
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Abstract
The invention belongs to the technical field of medicines, relates to application of a variety of traditional Chinese medicine mixture compounds, and particularly relates to a preparation method of a traditional Chinese medicine compound effective part preparation and pharmaceutical field use. The traditional Chinese medicine compound effective part is ginseng saponin, tanshinone II a, astragaloside, salvia miltiorrhiza phenanthrenequinone, compound aglycone, compound protein and total alkaloid, ginseng polysaccharide, compound polysaccharide, salvia miltiorrhiza phenolic acid B, hydroxy safflower yellow A, flavonoid glycoside, total volatile oil and other effective parts extracted from the preparation. By ''primary functional effective component parts'', ''primary and secondary functional effective component parts'', ''all effective component parts'' and other anti-myocardial ischemia series research, a best effective component part is determined, and the research shows that by the traditional Chinese medicine compound effective part combination, ischemic myocardium repair effect can be played by stimulation of value addition, migration, myocardial homing and other different links of vascular endothelial cells. Fig. 1 shows a 100 times chemical picture of immune cells 4 weeks after induction (A: blank control group; B:5-aza group alpha-Sarcomeric Actine; C: 5-aza group Desmin; D: the effective part formula group alpha-Sarcomeric Actine; E: the effective part formula group Desmin).
Description
Technical field
The invention belongs to medical technical field, relate to the purposes of plurality of Chinese mixing cpd, be specifically related to the preparation method of a kind of Chinese medicine compound effective site side and the purposes of field of medicaments.
Background technology
This effective ingredient in Chinese combination (this effective part group) is to use the ruling by law of " foster the motive, and promoting blood circulation " to treat effective prescription of obstruction of qi in the chest and cardialgia, is use Radix Ginseng, the Radix Astragali, Ramulus Cinnamomi reinforce functional activities of the heart, warm promoting blood circulation; The Radix Rehmanniae, Fructus Schisandrae Chinensis Yin-nourishing and body fluid promoting, and venting QI-blood; Radix Salviae Miltiorrhizae, Flos Carthami nourishing blood and promoting blood circulation, a surname's qualcomm meridian, assistant is with Rhizoma Alismatis promoting diuresis to eliminate damp pathogen, changes the turbid and nourishing YIN of its expectorant, the Fructus Jujubae coordinating the actions of various ingredients in a prescription that fills blood.All medicines share, and reach with promoting blood circulation, take good care of the effect of the motive.Through us, study and show for many years, this prescription all has significant clinical efficacy for ischemic heart desease, hyperlipemia, atherosclerosis etc.Can improve coronary circulation, increase Coronary microcirculation blood flow; Strengthen the antithrombotic of cardio-vascular endothelial cells, anticoagulant is active; Regulating lipid metabolism; There is antagonism myocardial ischemia reperfusion injury, prevent the murder by poisoning of oxygen-derived free radicals and the effects such as insulin sensitivity of increase body.
This effective ingredient in Chinese be from this prescription, extract ginsenoside, Tanshinone I Ia,, astragaloside, Radix Salviae Miltiorrhizae phenanthrenequione, compound recipe glycoside, compound recipe aglycon, compound recipe albumen, total alkaloids, ginseng polysaccharide, compound polyose,, the effective site such as the fragrant phenolic acid B of Radix Salviae Miltiorrhizae, S-A Hydroxysafflor yellow A, lignanoid, flavonoid glycoside, total volatile oil.By the series of studies to wherein " effective ingredient position plays a major role ", " main and less important effective site reciprocal action " and " the effective dose compatibility at each effective ingredient position " etc. resist myocardial ischemia, and then " the best effective ingredient position " determined." effective part group " is an effective class chemical substance of extracting from Chinese medicine compound, it than former side's enrichment effective ingredient.Show after deliberation, this effective ingredient in Chinese combination, can be by stimulating the different links such as vascular endothelial cell " increment ", " migration " and " cardiac muscle of going back to the nest " to play the effect of repairing ischemic myocardium.
Summary of the invention
Object of the present invention provides a kind of preparation method of Chinese medicine compound effective site combination.This medicine can promote mesenchymal stem cells differentiation to become cardiac-like muscle cell with inducing bone mesenchymal differentiation of stem cells.
The technical problem of its less important solution: this effective ingredient in Chinese is combined in and promotes the Bone Marrow Mesenchymal Stem Cells Transplantation medical usage that Cardiocytes direction transforms after cardiac muscle.
Described mesenchymal stem cells MSCs directed differentiation is cardiac-like muscle cell.According to this effective ingredient in Chinese, be combined in the successful case of inducing bone mesenchymal stem cell directional differentiation, can utilize mesenchymal stem cells MSCs as the cell model of new screening treatment myocardial infarction disease medicament.
Another technical problem to be solved of the present invention is to provide and promotes the in-vitro directed cardiac-like muscle cell that is converted into of mesenchymal stem cells MSCs.For this reason, the technical solution used in the present invention is: it adopts a kind of effective effective ingredient in Chinese combination as short cell differential agent, and inducing bone mesenchymal stem cell directional is divided into cardiac-like muscle cell.
The present invention also provides: the cardiac-like muscle cell that mesenchymal stem cells MSCs becomes by this effective ingredient in Chinese combination directed differentiation is in vivo as the purposes of heart tissue engineering.With seed cell or the bio-artificial heart of this effective ingredient in Chinese combination induction differentiation, transplant, can avoid immunological rejection that heart transplantation occurs and heart donor few problem of originating.
The present invention also provides: a kind of effective ingredient in Chinese combinatorial association mesenchymal stem cells MSCs is the purposes in preparation treatment myocardial infarction disease medicament as short cell differential agent, the medicine that this effective ingredient in Chinese combination is prepared into treatment myocardial infarction disease, associating Bone Marrow Mesenchymal Stem Cells Transplantation is treated great heart disease.
Accompanying drawing explanation
The quadratic regression curve of Fig. 1 drug level and primary cellular defect rate and regression equation (the quadratic regression curve B drug level of A drug level and primary cellular defect rate and the quadratic regression equation of loss ratio)
100 times of (A: blank group of 4 weeks immunocytochemistry pictures after Fig. 2 induces; B:5-aza group α-Sarcomeric Actine; C:5-aza organizes Desmin; D: effective site formula group α-Sarcomeric Actine; E: effective site formula group Desmin)
the specific embodiment
The present invention is studied and is shown by embodiment, the combination of this effective ingredient in Chinese can promote mesenchymal stem cells MSCs in vitro directed differentiation be cardiac-like muscle cell.The present invention is described further in conjunction with the accompanying drawings and embodiments.
Embodiment 1: the clinical research of the Main Function composition of mesenchymal stem cells MSCs is mobilized in this effective ingredient in Chinese combination
(1) laboratory animal and modeling: select 120 of wistar healthy rats, body weight is at 200 ± 20g, male, according to < < new Chinese medicine, develop and declare the requirement of > > about " pharmacodynamic study for the treatment of chest arthralgia precordial pain syndrome Chinese medicine ", with reference to the modeling of " pituitrin brings out acute myocardial ischemia " method, press the dosage of 0.75u/kg, tail vein injection pituitrin, has pushed away in constant speed 10s.Select electrocardiogram J point (J point is the end of a period of QRS wave group and ST section junction) and rise, or more than ST section raises or force down 0.1mv, the first towering rear low flat or inversion person of T ripple; Within after fast injection pituitrin 30 seconds, occur that Rat Ecg S-T raises or after 2 minutes low the putting down of T ripple successfully indicate for modeling.
(2) experimental drug
1. tested medicine: medicine former side consist of by a certain percentage Radix Ginseng, the Radix Astragali, Ramulus Cinnamomi, the Radix Rehmanniae, Fructus Schisandrae Chinensis, Radix Salviae Miltiorrhizae, Flos Carthami, Rhizoma Alismatis, Fructus Jujubae etc., medical material is purchased from attached Rui Kang hospital of Guangxi traditional Chinese medicine university, and kind Jun Jing Guangxi traditional Chinese medicine university Chinese medicine preparation expert testimony is approved.After attached Rui Kang hospital of Guangxi traditional Chinese medicine university Chinese medicine ethnic drug research and development base decocting, press steam distillation, water alcohol method, the methods such as cationic resin absorption method are prepared into and comprise ginsenoside, ginseng polysaccharide, tanshinone ⅡA, Radix Salviae Miltiorrhizae phenanthrenequione, compound recipe glycoside, compound recipe aglycon, compound polyose, compound recipe albumen, total alkaloids and 10 effective ingredient positions of total volatile oil, make respectively effective ingredient position oral liquid.
2. contrast medicine: normal saline.
(3) grouping experiment
Get above-mentioned modeling rat, be divided at random 15 groups, every group 8, comprise ginsenoside, Tanshinone I Ia,, astragaloside, Radix Salviae Miltiorrhizae phenanthrenequione, compound recipe glycoside, compound recipe aglycon, compound recipe albumen, total alkaloids, ginseng polysaccharide, compound polyose,, the fragrant phenolic acid B of Radix Salviae Miltiorrhizae, S-A Hydroxysafflor yellow A, lignanoid, flavonoid glycoside, total volatile oil, blank model group and normal healthy controls group.Wherein 15 effective site groups all at feed standard feed simultaneously, gavage corresponding effective site oral liquid, each 4 milliliters; Model control group, normal healthy controls group award the normal saline that gavages equivalent.Every day 1 time, continuous 10 days, in the 11st day, start to detect peripheral blood CD34
+cell.
(3) dosage: definite LD50 or the maximum tolerated dose of each effective ingredient acute toxicity test of in earlier stage doing by this problem determined dosage, with distilled water diluting to 4ml, each group of effective components gavages corresponding effective ingredient oral liquid, and normal healthy controls group and blank model group give 4ml distilled water.
(4) blood CD34
+cell detection
Phlebotomize, 100 μ L add 10 μ L CD34
+fITC antibody, hatches 30min in 4 ℃ of camera bellows; Negative control adds 10 μ L PBS liquid, in 4 ℃ of camera bellows, hatches 30min.Every pipe adds 2mL erythrocyte cracked liquid fully to mix, lucifuge reaction 10min under room temperature, the centrifugal 5min of 1500 * g.Abandon supernatant, add 2mL PBS liquid, mix, the centrifugal 5min of 500 * g.Come again.Abandon supernatant, add 0.5mL PBS to mix, use FACS calibur flow cytometer (U.S. company BD) to analyze peripheral blood CD34
+cell rate.With peripheral white blood cell, be multiplied by CD34
+cell rate draws peripheral blood CD34
+cell number.
As shown in Table 1: the 1. blood CD34 of model control group
+cell number and the comparison of normal healthy controls group, difference has significant, P < 0.01; 2. ginsenoside's group all has significant statistical significance with model group comparison (P < 0.01), salvianolic acid B group and model group comparison (P < 0.05), astragaloside group and model group (P < 0.01), flavonoid glycoside group and model group comparison (P < 0.05), total volatile oil group and model group comparison (P < 0.01).Therefore confirm that these five kinds of compositions are the main component playing a role.
Table 1 is respectively organized blood CD34
+comparison
| Group | CD34 +(*10 6/L) |
| Ginsenoside's group | 8.91±1.56 **ΔΔ |
| Tanshinone I Ia group | 8.01±2.13 ** |
| Astragaloside group | 9.11±2.02 **ΔΔ |
| Radix Salviae Miltiorrhizae phenanthrenequione group | 7.69±2.38 ** |
| Compound recipe glycoside group | 7.33±1.89 ** |
| Compound recipe aglycon group | 6.69±1.88 ** |
| Compound recipe protein groups | 7.05±2.21 ** |
| Total alkaloids group | 7.01±2.33 ** |
| Ginseng polysaccharide's group | 7.85±2.11 ** |
| Compound polyose group | 6.78±1.32 ** |
| The fragrant phenolic acid B group of Radix Salviae Miltiorrhizae | 8.67±2.65 **ΔΔ |
| S-A Hydroxysafflor yellow A group | 8.03±2.48 ** |
[0026]?
| Lignanoid's group | 6.43±1.48 ** |
| Flavonoid glycoside group | 8.33±2.18 **ΔΔ |
| Total volatile oil group | 9.21±2.10 **ΔΔ |
| Model control group | 5.89±2.28 **ΔΔ |
| Normal healthy controls group group | 2.17±0.75 |
Note: with the comparison of health group,
*p < 0.05,
*p < 0.01; With model control group comparison,
Δp < 0.05,
Δ Δp < 0.01.
This effective ingredient in Chinese of embodiment 2 is mobilized the research of the effective dose compatibility of mesenchymal stem cells MSCs
More than research shows that ginsenoside, salvianolic acid B, astragaloside, flavonoid glycoside, 5 kinds of compositions of total volatile oil have obvious mobilization mesenchymal stem cells MSCs to the effect in peripheral blood.This experiment will be observed above-mentioned 5 effective ingredient portions effect that position does not form prescription mobilization mesenchymal stem cells MSCs with dosage level, thereby defines the optimal dose of effective constituent position side.
(1) experimental drug
Tested medicine: 5 kinds of effective ingredient positions of effective site side form 3 various dose levels of 4 factors, in Table 2.
5 kinds of effective ingredient position L9 (3 of table 2
4) orthogonal table test arrangement
Note: except the unit of total volatile oil is that ml, all the other composition units are g.
(2) animal model: with experiment 1
(3) grouping experiment: get 72 of above-mentioned rats, be divided at random 9 groups, 8 every group, by L9 (3
4) orthogonal trial test (as table 3).9 groups all at feed standard feed simultaneously, gavages corresponding effective site oral liquid, each 4 milliliters; Every day 1 time, continuous 10 days, in the 11st day, start test.
[L9 (3 for the effective dose compatibility test orthogonal design at each effective ingredient position of table 3
4)]
(4) blood CD34
+cell detection is with experiment 1
Table 4 be variant horizontal dosage effective site side to Rat Ecg J point displacement Orthogonal experiment results, adopt the analysis of intuitive analysis method to show, improving aspect electrocardiogram, it is good compared with 1,2 levels that ginsenoside gets 3 levels; It is good compared with 1,2 levels that tanshinone ⅡA is got 3 levels; It is good compared with 1,3 levels that ginseng polysaccharide gets 2 levels; It is good compared with 2,3 levels that flavonoid glycoside and total volatile oil are got 1 level.
Table 4 is respectively tested peripheral blood CD34
+cell counting Orthogonal experiment results (* 10
6/ L)
Embodiment 3: this effective ingredient in Chinese formula promote mesenchymal stem cells MSCs in vitro directed differentiation be cardiac-like muscle cell.
(1) cultivation of mesenchymal stem cells MSCs (MSCs):
Get rat tibia and femur bone marrow, with after percoll density gradient centrifugation, middle tunica albuginea confluent monolayer cells is (containing the L-DMEM culture medium of 10% hyclone) in conditioned medium, by after cell counting with 10
9/ ml density is inoculated in 50cm
2in culture bottle, when cell reaches 80%, the cultivation of going down to posterity.
(2) the maximal non-toxic concentration determination (mtt assay) of medicine
It is 66.5g/L that compatibility of drugs well makes final concentration later, and 0.22 μ m membrane filtration degerming, by 2
-ndoubling dilution (n is 1～9), is diluted to it respectively with cell culture fluid: 50g/L (2
-1), 25g/L (2
-2), 12.5g/L (2
-3), 6.25g/L (2
-4), 3.12g/L (2
-5), 1.56g/L (2
-6), 0.78g/L (2
-7), 0.39g/L (2
-8), 0.19g/L (2
-9) totally 9 dilution factors.Sonic oscillation, makes it to dissolve, and pH is between 7.2～7.4.Take the logarithm the 4th generation MSCs of trophophase, after 0.25% trypsinization, 1000r/min, 5min are centrifugal, are mixed with cell suspension, adjust concentration to 1 * 10 under microscope after cell counting
4/ mL, adds 96 orifice plates, every hole 0.2mL, 37 ℃, saturated humidity, the CO that is 0.05 containing volume fraction
2in incubator, inducing culture liquid is hatched after 24h, removes culture fluid and not adherent cell.It is 50g/L, 25g/L, 12.5g/L, 6.25g/L that experimental group adds respectively final concentration to contain effective site formula, each 20 μ L of the inducing culture liquid of 3.12g/L, 1.56g/L, 0.78g/L, 0.39g/L, 0.19g/L, then the hyclone culture fluid 180 μ L that add volume fraction 10%, cell control well does not add medicine, only add 200 μ L cell culture fluids, and establish blank.Each concentration is established 4 multiple holes.At 37 ℃, saturated humidity, the CO that is 0.05 containing volume fraction
2in incubator, cultivate and cultivate respectively after 24h, 48h, 72h, add the MTT20 μ L of 5mg/mL, hatch 4h for 37 ℃, abandon supernatant, every hole adds 150 μ L DMSO, is positioned over shake well in water-bath constant temperature oscillator, make dissolving crystallized, upper microplate reader detects absorbance A value at 570nm place.With statistics software SPSS13.0, surveyed A value is carried out to significance test, finally get the meansigma methods of 4 parallel holes, calculate cytoclasis percentage rate.Destructive rate (%)=[(A
groups of cells-A
administration group)/(A
groups of cells-A
blank group)] * 100%.Choose cell-damaging rate and be less than the non-toxic that 5% drug level is this medicine.
By known in table 5, when absorbance and the cell matched group there was no significant difference (P > 0.05) of drug level during lower than 1.56g/L, illustrate that medicine has no obvious cytotoxicity to MSCs in this concentration range; And along with the reduction of liquor strength, absorbance increases, and living cell rate improves.According to the relation between drug level and loss ratio, set up binary regression equation and determine that the maximal non-toxic concentration of medicine is 1.23% (accompanying drawing 1).
The toxicity test result of table 5 effective site formula to MSCs cell
d (λ=570nm)
*compare P < 0.05 with cell matched group;
*compare P < 0.01 with cell matched group
(3) pharmaceutical intervention:
Gather in the crops after the 4th generation MSCs, by 1 * 10
4/ ml, adds 24 well culture plates, after cell 100% merges, with DK liquid, washes cell, washes altogether 3 times; Add afterwards inducing culture.
Experiment is divided into four groups, and first group is observed drug group: in rat culture fluid, add 1.23% this effective ingredient in Chinese formula; Second group is 5-aza positive controls: in rat BMSCs culture fluid, add 10 μ mol/L5-aza; The 3rd group of negative matched group: add in rat BMSCs culture fluid and first group of equivalent DMEM.The 4th group is Normal group: in rat BMSCs culture fluid, add containing 10% hyclone DMEM.All put in incubator for 4 groups and hatch after 24h, remove the culture fluid of each group, with DMEM culture medium washing 2 times, then continue to cultivate by above-mentioned condition of culture, every 3d changes liquid 1 time.After induction differentiation 28d, take out and respectively organize BMSCs respectively, do the evaluation of cardiac-like muscle cell.
The SABC of induction MSCs identifies, do not induce in blank group of MSCs and do not find Desmin and α-Sarcomeric Actine positive cell.In 5-aza group and effective ingredient in Chinese formula group, the equal visible weak positive immune complex of Desmin immunohistochemical staining, is colony sample and distributes, filamentary structure as seen in kytoplasm, and positive cell is dispersed in distribution, is fusiformis and fibroblast sample form (accompanying drawing 2).In its OD value comparison in two groups of filling a prescription of 5-aza group and effective site, Desmin statistics there was no significant difference, α-Sarcomeric Actine5-aza group is lower than effective site formula group (P < 0.05 table 6).
The Desmin of table 6 MSCs and the comparison of α-Sarcomeric Actine immunocytochemistry OD mean
*compare P < 0.05 with 5-aza group group
2.6RT-PCR detected the expression 5-aza group, the induction of effective site formula group of the MSCs cardiac muscle specific factor after induction after MSCs14 days, and GATA-4mRNA and cTnI mRNA have low intensive expression, 28 days subsequently expression intensity significantly promote.And GATA-4mRNA has no positive band appearance in blank group; The positive band of cTnI mRNA a little less than.Semi-quantitative analysis 5-aza group, effective site formula group relative optical density value have statistical significance (P < 0.05, table 7) with blank group comparing difference.
The relative optical density value of the RT-PCR product of table 7 GATA-4 and cTnI mRNA
Compare * P < 0.05, * * P < 0.01 with blank group; Compare ##P < 0.01 with 5-aza group
Conclusion:
This effective ingredient in Chinese is combined in the external BMSCs Cardiocytes of can inducing to be broken up.
Claims (7)
1. a Chinese medicine compound effective site combination is containing ginsenoside's (3.53g crude drug/kg body weight), salvianolic acid B (2.12g crude drug/kg body weight), astragaloside (6.58g crude drug/kg body weight), flavonoid glycoside (1.87g crude drug/kg body weight), total volatile oil (34.35ml crude drug/kg body weight).
2. this effective ingredient in Chinese compositions, process as claimed in claim 1, is characterized in that: the preparation method of described a kind of effective ingredient in Chinese combination.
3. an effective ingredient in Chinese combined therapy and/or the medicine of prevention angiopathy are, the medicine of promotion mesenchymal stem cells differentiation becomes the medicinal usage of cardiac-like muscle cell with inducing bone mesenchymal differentiation of stem cells.
4. this effective ingredient in Chinese as claimed in claim 3 is combined in the medical usage that promotes that the external Cardiocytes direction of mesenchymal stem cells MSCs transforms, and it is characterized in that: described mesenchymal stem cells MSCs directed differentiation is cardiac-like muscle cell.
5. this effective ingredient in Chinese as described in right 3 requirements is combined in the medical usage of mobilizing into mesenchymal stem cells MSCs to peripheral blood, it is characterized in that: CD34 in this effective ingredient in Chinese combination energy peripheral blood
+cell number increases.
6. this effective ingredient in Chinese according to claim 3 is combined in and promotes the go back to the nest medical usage of cardiac muscular tissue of Bone Marrow Mesenchymal Stem Cells Transplantation after cardiac muscle, it is characterized in that: this effective ingredient in Chinese combination has promote the go back to the nest effect of cardiac muscular tissue of Bone Marrow Mesenchymal Stem Cells Transplantation after cardiac muscle.
7. this effective ingredient in Chinese combinatorial association Bone Marrow Mesenchymal Stem Cells Transplantation is treated the purposes in myocardial infarction heart blood silt disease medicament as short cell differential agent in preparation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3777866A4 (en) * | 2018-04-04 | 2022-01-05 | Tasly Pharmaceutical Group Co., Ltd. | Pharmaceutical composition and application thereof |
| CN116585301A (en) * | 2023-07-18 | 2023-08-15 | 青岛瑞源细胞生物科技开发有限公司 | Preparation for improving stem cell therapeutic capacity |
-
2013
- 2013-04-10 CN CN201310122465.XA patent/CN104096014A/en active Pending
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| EP3777866A4 (en) * | 2018-04-04 | 2022-01-05 | Tasly Pharmaceutical Group Co., Ltd. | Pharmaceutical composition and application thereof |
| US11491173B2 (en) | 2018-04-04 | 2022-11-08 | Tasly Pharmaceutical Group Co., Ltd. | Pharmaceutical composition and application thereof |
| CN116585301A (en) * | 2023-07-18 | 2023-08-15 | 青岛瑞源细胞生物科技开发有限公司 | Preparation for improving stem cell therapeutic capacity |
| CN116585301B (en) * | 2023-07-18 | 2023-09-22 | 青岛瑞源细胞生物科技开发有限公司 | Preparation for improving stem cell therapeutic capacity |
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