CN1723888A - Medicine with function of increasing whole blood - Google Patents
Medicine with function of increasing whole blood Download PDFInfo
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- CN1723888A CN1723888A CN 200410040266 CN200410040266A CN1723888A CN 1723888 A CN1723888 A CN 1723888A CN 200410040266 CN200410040266 CN 200410040266 CN 200410040266 A CN200410040266 A CN 200410040266A CN 1723888 A CN1723888 A CN 1723888A
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Abstract
A medicine in the form of injection or orally taken forms for increasing the red cells, white cells, thrombocyte and hemoglobin in blood contains catechin and/or epicatechin and the pharmacologically acceptable auxiliary.
Description
Technical field
The present invention relates to a kind of medicine, specifically is a kind of medicine made from the active drug composition that is obtained by extraction separation in the natural drug or chemosynthesis mode with erythrocyte in the elevating blood, leukocyte, platelet, hemoglobin function.
Background technology
(tea polyphenols TP) is a kind of chemical compound composition with pharmacologically active to tea polyphenols, and its main pharmacologically active is for all to have tangible inhibition ability to various bacteria, fungus, yeast in present studies show that.A large amount of experiments show, TP is put bacterium to the mutans streptococcus in the oral cavity with sticking, to spirillum, Salmonella enteritidis, escherichia coli, bacillus pyocyaneus, shigella flexneri, Song Shi dysentery bacterium, Bacillus typhi, Salmonella paratyphi, yellow staphylococcus haemolyticus, golden yellow streptococcus in the gastrointestinal tract, and cause that funguses such as the Trichophyton of human body skin disease, novel stealthy ball yeast and tinea alba bacterium all have certain inhibitory action or lethal effect.Have certain mutation and antitumor, the effect of cancer cell metastasis.Have good effect for reducing blood fat, the rat heart muscle toxicity that rat myocardium from injury, amycin are caused has the effect of alleviating, and myocardial ischemia reperfusion injury (IRI) and cerebral ischemia reperfusion injury are had protective effect.TP also has the antiarrhythmic effect simultaneously.Have hepatoprotective effect, the effect that alleviates chemotherapeutic toxicity is arranged.(" Anhui Chinese Medicine College journal " 2002,21 (5): 57-60).
The main effective ingredient of tea polyphenols is catechin [Catechin, chemistry 2H-1-Benzopyran-3 by name, 5,7-triol, and 2-(3,4-dihydroxyphenyl)-3,4-dihydro-, (2R-trans-)] and epicatechin [EpiCatechin, chemistry 2H-1-Benzopyran-3 by name, 5,7-triol, and 2-(3,4-dihydroxyphe-nyl)-3,4-dihydro-, (2R-cis-)].Catechin compounds can derive from rosaceous plant Fructus Pruni Tomentosae (Prunus tomentosa) timber usually; The Ginkgoaceae Folium Ginkgo; The red nanmu of Lauraceae (Ginkgo biloba) heartwood; Beard section Fructus Elaeagni Angustifoliae (Etaeagnus angustifolia) peel of stem, branch; Section Japan produces bitter (Meliaazedarach var.japonica) bast; Apocynaceae plant Herba Apocyni veneti (Apocynum venetum) leaf; Leguminous plant catechu (Acacia catechu) heartwood; Babassu Semen Arecae (Areca catechu) endosperm; Salicaceae plant Hemerocallis citrina Baroni willow (Salix caprea).Show that by present existing research report the pharmacologically active of this composition can be embodied in aspects such as cardiovascular system, antioxidation, serum lipids and liver protectings.For example, people such as Xu Minhua are in " catechin is to the influence of Carnis Coturnicis japonicae experimental atherosclerosis " (" preclinical medicine and clinical " 2002,22 (5): reported once 447-450) that catechin had tangible blood lipid regulation, anti peroxidation of lipid, alleviates fatty liver, reduces fructosamine and resists atherosclerotic effect.
Lin Qinlu etc. are in " catechin is to the influence of animal serum SOD " (" Food Science " 2001,22 (11): report 53-56), catechin has the effect of obvious rising mouse serum SOD, and the catechin of different combining forms (free type, lipid build, micelle build) is through the influence difference of different administration approach (irritating stomach, intravenous injection) to SOD in serum, the effect of liposome, glue bundle body conjunction type catechin rising SOD in serum is better than free type (P＜0.05), and the intravenous injection effect is better than oral (P＜0.05).
People such as Ye Xiyun are at " catechin is to the protective effect of cell injury due to the LYSOLECITHIN SUNLECITHIN A choline " (" East China Normal University's journal (natural science edition) " 2001; 9 (3): report 97-101), catechin can cause the damage of VEC (vascular endothelial cell) by antioxidation approach opposing LPC (LYSOLECITHIN SUNLECITHIN A choline) and X/XO (xanthine/xanthine oxidase).
What the people such as goes to the lavatory in " catechin is to nephropathy rat nitric oxide and the uropoietic influence of albumen " (" Food Science " 2004; 24 (1): report 121-124), catechin and hormons are used has synergistic therapeutic action, can alleviate kidney damage.It may be by raising the content of kidney part and plasma nitric oxide (NO), reducing the drainage of nephropathy rat 24h urine protein, to delay the chronic progress of kidney pathology.
Liu Xiang newly waits the people at " catechin and epicatechin are to the influence of mouse serum lipid " (" Agricultural University Of Hunan's journal (natural science edition) " 2002; 28 (3): 232-233), reported entering the intravital catechin of white mice through different approaches and enter, but different types of catechin is to the report of various lipids influence researchs in the hyperlipidemia mouse serum through same approach.Its result of study shows, no matter be to adopt administration by gavage, or intravenous injection, handle through catechin and epicatechin, each test group of every white mice 1.5mg/d all has obvious reduction hyperlipidemia mouse serum TC (serum total cholesterol), TG (triglyceride), LDL-C (low-density lipoprotein cholesterol), the effect of rising HDL-C (HDL-C), and the catechin effect is better than epicatechin.
People such as Li Jianxiang are at " catechin compounds causes the protective effect of rat chronic hepatic injury to carbon tetrachloride " (" industrial hygiene and occupation disease " 2003; 29 (1): 20-22) report, the catechin compounds administration is after 2 months, middle and high dosed administration group (100,200mg/kg) all can reduce the vigor of rat blood serum ALT due to the carbon tetrachloride chronic hepatic injury model and lipid peroxidation product MDA and the hepatic tissue content (p＜0.01 or p＜0.05) through proline, it is suitable through the effect and the bifendate of proline content that it reduces hepatic tissue.It is similar that the pathology of hepatic tissue improves situation two medicines, and anti-membrane lipid peroxidation is better than bifendate.Conclusion is that catechin compounds has the effect of protection chemical liver injury, the transaminase lowering vigor is arranged, alleviate the effect that hepatic pathology damages.
People such as Li Jianxiang are at " effect observation of catechin anti-hepatitis B virus " (" Chinese Journal of Preventive Medicine " 2001; 35 (6): report 404-407), catechin has anti-hepatitis virus, transaminase lowering activity, alleviates the hepatic pathology damaging action.
In present document, all do not see relevant catechin, epicatechin and tea polyphenols as yet to the pharmacological action of blood constituent function aspects and the research of influence, and with its report as the whole blood function medicament that is used to raise.
Summary of the invention
At above-mentioned situation, the present invention will provide a kind of with the active drug composition catechin, epicatechin or the tea polyphenols kind that obtain by extraction separation in the natural drug or chemosynthesis mode as the active drug composition, have the medicine of the whole blood function that comprises erythrocyte, leukocyte, platelet, hemoglobin in the elevating blood.
The present invention has the medicine of rising whole blood function, be with at least a in catechin or the epicatechin, the tea polyphenols that particularly contains catechin and epicatechin simultaneously is as the active drug composition, becomes the pharmaceutical preparation of injection-type or oral type with acceptable auxiliary element mutual group in the medicine.
When wherein said active drug composition adopts tea polyphenols, with gross weight content 〉=80% of wherein catechin and epicatechin for well.Acceptable auxiliary element in the said medicine, can comprise as auxiliary adding ingredient commonly used such as disintegrating agent commonly used in oral formulations, excipient, lubricant, binding agent, filler, and can handle by corresponding conventional oral formulations process, promptly may be made in the oral drugs of the solid preparation forms such as slow releasing agent, controlled release agent of tablet, pill, capsule or appropriate format; Mix with the surfactants of using always such as solubilizing agent, emulsifying agent, wetting agent, foaming or defoamer, diluent, antiseptic, stabilizing agent, correctives, thickening agent etc., handle by corresponding common process method, promptly may be made in the oral drugs of liquid preparation forms such as water preparation, syrup.In addition, acceptable auxiliary element in this medicine, can be appropriate solvent and/or the additives that in injectable drug, allow to use, and can be, promptly can be prepared into the injection agent medicine of muscle such as corresponding liquid drugs injection or powder pin or vein form by after the corresponding preparation PROCESS FOR TREATMENT.
Following correlation test result can know to show the above-mentioned active drug composition of the present invention catechin, epicatechin, the tea polyphenols role and influence to blood constituent.
Tea polyphenols in the trial drug by extracting in the Folium Camelliae sinensis, is the tea polyphenols commodity, and its labelled amount is a catechin total content 80%.
Catechin in the trial drug and epicatechin except that can or containing as Caulis Spatholobi etc. by Folium Camelliae sinensis extraction separation obtains in the plant of specific examples of such components, also can adopt the catechin and the epicatechin of existing commercial goods.
1. the active drug composition is to the test of compound anemia model mice peripheral hemogram influence
With 110 of male and female half and half Kunming mouses of body weight 18-22g, be divided into 11 groups at random by body weight, 10 every group.Except that the normal control group, all the other 10 groups of mice 4d and 7d injection acetylphenylhydrazine 50mg/kg, the 5th, 6,7d injection cyclophosphamide 40mg/kg, cause the anemia model.Normal control group and anemia model group intraperitoneal injection of saline every day 0.1ml/10g, the medicine group according to dosage every day 0.1ml/10g, continuous 9d.1h after the last administration, each organizes mice from the eye socket venous blood collection, measures hemogram, and the result is as shown in table 1.
The influence of table 1 pair composite model anemia mice hemogram (x ± s)
| Group | WBC(10 9/L) | RBC(10 12/L) | HGB(g/L) | PLT(10 9/L) |
| Matched group | 9.07±1.77 | 9.0±0.91 | 132.2±8.60 | 743.5±53.86 |
| Model group | 3.68±1.22 | 5.24±1.58 | 99.67±16.39 | 723.3±118.8 |
| Tea polyphenols (10mg/kg) | 3.71±1.24 | 5.36±1.11 | 104.5±10.94 | 757.2±120.4 |
| Tea polyphenols (20mg/kg) | 3.78±1.11 | 5.69±1.05 | 111.3±8.98 * | 839±218.4 |
| Tea polyphenols (40mg/kg) | 4.12±1.53 | 6.62±1.34 * | 115.3±17.64 * | 952.1±204.7 ** |
| Catechin (6mg/kg) | 3.61±1.04 | 5.30±1.16 | 100.5±10.84 | 756.9±120.3 |
| Catechin (12g/kg) | 3.58±1.11 | 5.64±1.09 | 111.9±8.99 * | 834±217.4 |
| Catechin (24mg/kg) | 4.02±1.51 | 6.60±1.27 * | 116.3±17.59 * | 949.1±239.7 ** |
| Epicatechin (6mg/kg) | 3.68±1.26 | 5.28±1.19 | 101.5±10.89 | 753.2±122.4 |
| Epicatechin (12mg/kg) | 3.75±1.15 | 5.59±1.05 | 111.7±8.78 * | 836±211.4 |
| Epicatechin (24mg/kg) | 4.05±1.50 | 6.56±1.19 * | 115.9±17.52 * | 948.1±214.7 ** |
Annotate: tea polyphenols group, catechin group, epicatechin group and model group compare,
*Expression P＜0.01,
*Expression P＜0.05.
The above results shows, give mice with the dosage lumbar injection tea polyphenols of 40mg/kg and the catechin and the epicatechin of 24mg/kg dosage, all can effectively resist reduction, the composite model anemia mice that significantly raises erythrocyte, content of hemoglobin and number of platelets owing to acetylphenylhydrazine and caused by cyclophosphamide mice peripheral hemogram.
2. the active drug composition is to the test of caused by cyclophosphamide anemia model mice peripheral hemogram influence
With 110 of male and female half and half Kunming mouses of body weight 18-22g, be divided into 11 groups at random by body weight, every group 10, except that the normal control group, all the other 10 groups of mices the 5th, 6,7 days subcutaneous injection cyclophosphamide (CY) 40mg/kg, normal control group and anemia model group intraperitoneal injection of saline every day 0.1ml/10g, the medicine group according to dosage every day 0.1ml/10g, continuous 9d.1h after the last administration, each organizes mice from the eye socket venous blood collection, measures hemogram, and the result is as shown in table 2.
Table 2 result shows, all can significantly the raise reduction of mice peripheral blood leucocyte of caused by cyclophosphamide of tea polyphenols, catechin, epicatechin, and tea polyphenols is under the dosage of 40mg/kg, the reduction to the mice peripheral blood leucocyte of caused by cyclophosphamide under the dosage of 24mg/kg of catechin and epicatechin has good antagonism, and has compared significant difference with model group.
Active drug composition element to the test of hemorrhagic anemia mouse red blood cell number, hemoglobin influence with 110 of male and female half and half Kunming mouses of body weight 18-22g, be divided into 11 groups at random by body weight, every group 10, except that the normal control group, all the other 10 groups of mices from eye socket blood-letting 0.5ml/ are only caused the hemorrhagic anemia model.Normal control group and anemia model group intraperitoneal injection of saline every day 0.1ml/10g, the medicine group according to dosage every day 0.1ml/10g, continuous 7d.1h after the last administration, each organizes mice from the eye socket venous blood collection, measures RBC number, hemoglobin, and the result is as shown in table 3.
The influence of table 2 pair Cy anemia mice hemogram (x ± s)
| Group | WBC(10 9/L) | RBC(10 12/L) | HGB(g/L) | PLT(10 9/L) |
| Matched group | 9.27±2.07 | 10.93±0.91 | 132.20±8.60 | 743.5±53.86 |
| Model group | 1.53±0.38 | 8.27±2.79 * | 122.33±6.00 | 822.89±140.62 |
| Tea polyphenols (10mg/kg) | 1.83±0.99 | 9.53±1.38 | 128.53±25.38 | 599.98±189.28 |
| Tea polyphenols (20mg/kg) | 2.20±0.95 | 10.27±1.57 | 135.53±22.08 | 689.65±264.05 |
| Tea polyphenols (40mg/kg) | 2.28±0.82 * | 11.00±1.74 * | 141.22±21.56 * | 707.78±259.03 |
| Catechin (10mg/kg) | 1.80±0.89 | 9.47±1.36 | 127.83±25.58 | 589.98±191.28 |
| Catechin (20mg/kg) | 2.15±1.05 | 10.07±1.59 | 130.53±22.00 | 685.65±259.07 |
| Catechin (40mg/kg) | 2.18±0.79 * | 10.59±1.70 * | 137.22±21.16 * | 700.78±256.03 |
| Epicatechin (6mg/kg) | 1.73±0.79 | 9.45±1.35 | 126.53±25.18 | 590.97±188.28 |
| Epicatechin (12mg/kg) | 2.10±0.94 | 9.99±1.37 | 129.73±21.08 | 684.65±267.05 |
| Epicatechin (24mg/kg) | 2.19±0.92 * | 10.50±1.54 * | 134.23±18.56 * | 701.78±268.03 |
Annotate: tea polyphenols group, catechin group, epicatechin group and model group compare,
*Expression P＜0.01,
*Expression P＜0.05.
The influence of table 3 pair hemorrhagic anemia mouse red blood cell number, hemoglobin (x ± s)
| Group | Number of animals | Dosage mg/kg | RBC(10 12/L) | HGB(g/L) |
| Matched group | 10 | 0 | 8.40±0.61 | 118.4±9.7 |
| The anemia model group | 10 | 0 | 6.34±0.90 | 83.0±14.2 |
| Tea polyphenols | 10 | 20 | 6.23±0.61 | 82.0±8.4 |
| Tea polyphenols | 10 | 40 | 7.14±0.57 * | 94.3±9.8 |
| Tea polyphenols | 10 | 80 | 7.87±0.56 ** | 100.7±7.6 ** |
| Catechin | 10 | 12 | 6.19±0.51 | 80.0±8.0 |
| Catechin | 10 | 24 | 7.00±0.47 * | 90.3±9.9 |
| Catechin | 10 | 48 | 7.57±0.46 ** | 97.0±7.5 ** |
| Epicatechin | 10 | 12 | 6.18±0.71 | 79.0±8.4 |
| Epicatechin | 10 | 24 | 7.06±0.60 * | 91.3±9.0 |
| Epicatechin | 10 | 48 | 7.33±0.66 ** | 96.7±6.6 ** |
Annotate: tea polyphenols group, catechin group, epicatechin group and model group compare,
*Expression P＜0.01,
*Expression P＜0.05.
Table 3 result shows, tea polyphenols, catechin, epicatechin can significantly raise hemorrhagic anemia mouse red blood cell number, hemoglobin number have good dose-effect dependence, and tea polyphenols has been compared utmost point significant difference at the dosage of 80mg/kg with model group.Catechin has been compared utmost point significant difference with epicatechin at the dosage of 48mg/kg with model group.
4. the active drug composition is to the test normal and influence of anemia mice medullary cell ability
Get Kunming mouse, be divided into normal group, model group, tea polyphenols group, catechin group, epicatechin group, tea polyphenols model group, catechin model group, epicatechin model group at random.Tea polyphenols group and tea polyphenols model group lumbar injection 40mg/kg, 0.1ml/10g, catechin group, epicatechin group, catechin model group, epicatechin model group lumbar injection 24mg/kg, 0.1ml/10g, normal control group and anemia model group intraperitoneal injection of saline every day 0.1ml/10g, every day 1 time, 15d continuously.In the 8th, 11 day model group and administration group mouse subcutaneous injection acetylphenylhydrazine 50mgkg
-1, from the 9th day pattern drawing group and administration group mouse peritoneal injection cyclophosphamide 50mgkg-1,3d continuously, the normal group mice such as injects simultaneously at capacity NS.The dislocation of mice cervical vertebra is put to death behind the last administration 1.5h, the aseptic bilateral femur of getting, inhale RPMI-1640 culture fluid flushing medullary cavity with No. 6 syringe needles, mix bone marrow cell suspension and cross 4 syringe needles, form single cell suspension and transfer cell number to 2 * 10 with the RPMI-1640 culture fluid system that contains 10% calf serum
5/ mL gets 200 μ l and adds 96 porocyte culture plates, establishes 3 multiple holes, at 37 ℃, 5%CO
2Cultivate 7d down with complete damp condition, the every hole of 4h discards culture supernatant 100 μ L before cultivating end, adding concentration is the MTT 10 μ L of 5mg/mL, cultivate every hole, end back and add 10%SDS (with the HCl configuration of 0.01M) 100 μ L, the vibration mixing, cultivate 4h down for 37 ℃, read absorbance at the 570nm place with microplate reader, the result is as shown in table 4.
The influence that table 4 pair is normal and the anemia mice bone marrow nucleated cell is bred (x ± s, n=6)
| Group | OD value 570nm |
| Normal group | 0.114±0.009 |
| The tea polyphenols group | 0.131±0.009 ** |
| The catechin group | 0.129±0.008 ** |
| The epicatechin group | 0.128±0.009 ** |
| Model group | 0.072±0.0178 |
| The tea polyphenols model group | 0.101±0.0120 ** |
| The catechin model group | 0.99±0.0119 ** |
| The epicatechin model group | 0.97±0.0120 * |
Annotate: tea polyphenols group, catechin group, epicatechin group and normal group compare, and tea polyphenols model group, catechin model group, epicatechin model group and model group compare,
*Expression P＜0.01,
*Expression P＜0.05.
Table 4 the result show, the normal matched group of bone marrow depression after the moulding-hemolytic anemia bone marrow cells in mice multiplication capacity has descended 34.78%, tea polyphenols, catechin, epicatechin can not only effectively improve the proliferation of bone marrow cells ability (P＜0.01) of normal mouse, and the reduction that can resist anemia mice proliferation of bone marrow cells ability.
5. induce the influence test of splenocyte, peritoneal macrophage, lung and the skeletal muscle conditioned medium of preparation through the active drug composition to normal and anemia mice bone marrow nucleated cell propagation
5.1 the preparation of splenocyte, peritoneal macrophage, lung and skeletal muscle conditioned medium:
Mice is divided four groups, and 6 every group, every lumbar injection tea polyphenols, catechin, epicatechin 0.1ml/10g or equivalent normal saline, every day 1 time, totally 15 days, mice was put to death in cervical vertebra dislocation in 1.5 hours behind the last medicine.
5.1.1 splenocyte conditioned medium (SCM) preparation:
The aseptic mice spleen of getting with the RPMI-1640 washing, shreds at 200 order steel wires, gently grinds, filters, 0.83%Tris-NH with sterilization glass inject cores
4Cl destroys erythrocyte, and ice bath left standstill 10 minutes, and 1500r/min is centrifugal, removes supernatant, uses twice of RPMI-1640 washed cell.Greater than 95%, make 1 * 10 with RPMI-1640 through determination of trypan blue staining cell motility rate again
7/ ml splenocyte suspension adds ConA 5 μ g/ml, is sub-packed in Tissue Culture Flask, at 5%CO
237 ℃ of incubations are 2 days in the moist atmosphere.Centrifugal results supernatant is SCM.The packing of penicillin bottle ,-20 ℃ of preservations are standby.
5.1.2 lung conditioned medium (LCM) preparation:
The aseptic bilateral lung of getting, with RPMI-1640 liquid flush away blood, remove connective tissue, in RPMI-164, mix the fritter that shreds into 3-5mm, adding the 2ml culture fluid by every mouse lung calculates, add the RPMI-1640 culture fluid that contains 10% calf serum and make the lung tissue suspension, be sub-packed in Tissue Culture Flask, at 5%CO
237 ℃ of incubations are 7 days in the moist atmosphere.The screen filtration piece of tissue, centrifugal results supernatant, 0.22 μ m filtering with microporous membrane, the packing of penicillin bottle ,-20 ℃ of preservations are standby.
5.1.3 peritoneal macrophage culture fluid (PM φ CM) preparation:
Mice is put to death in cervical vertebra dislocation, with 75% alcohol-pickled after, fully wash out abdominal cavity cell with 5ml D-Hank ' the s liquid of pre-cooling, the centrifugal 5min of 1500rpm/min uses the RPMI-1640 washed twice, accent cell to 2 * 10
6/ ml adds adherent 2h in 24 orifice plates, abandons culture fluid, and washed cell adds LPS and the culture fluid of 10 μ g/ml, and 37 ℃, 5%CO
2Incubation 48h in the moist atmosphere collects supernatant ,-20 ℃ of preservations.
5.1.4 skeletal muscle conditioned medium (SMCM) preparation:
The aseptic femoral muscle of getting, RMPI1640 washing is cut into the fritter of 3-5mm, soak 2h in 1640 after, add 0.3g in each cell bottle and handle back muscle, and additional culture fluid (RMPI-1640 that contains 10% calf serum) 3ml.37 ℃, 5%CO
2Saturated humidity was cultivated 7 days.The screen filtration piece of tissue, centrifugal results supernatant, 0.22 μ m filtering with microporous membrane, the packing of penicillin bottle ,-20 ℃ of preservations are standby.
5.1.5CSFs determination of activity:
Asepticly get bilateral femur normal and anemia mice (modeling method is with 2.1), inhale culture fluid flushing medullary cavity with No. 6 syringe needles, the mixing bone marrow cell suspension is crossed 4 syringe needles, and the formation single cell suspension makes 2 * 10 with the RPMI-1640 culture fluid
6/ ml nucleated cell suspension is as measuring the active reacting cells of CSFs.Get the SCM or the LCM of-20 ℃ of preservations, make 1/4,1/8 and 1/32 serial dilution respectively, measure its CSFs activity with medullary cell.Promptly in the flat Tissue Culture Plate in 96 holes (U.S. Falcon 3072), every hole adds 2 * 10
5The SCM or the LCM of/0.1ml bone marrow cell suspension and 0.1ml 1/4,1/8,1/32 dilution, control wells adds the equivalent culture fluid.At 5%CO
2Cultivated MTT colorimetric method for determining result 7 days for 37 ℃ in the moist atmosphere.
5.2SCM to influence normal and anemia mice bone marrow nucleated cell propagation
Induce the SCM of preparation in tea polyphenols, catechin, epicatechin body, no matter the propagation to the bone marrow nucleated cell of normal or anemia Mus all has obvious facilitation (table 5).Dilution factor 1: 8 time the proliferation function of the medullary cell of normal mouse has been compared utmost point significant difference with matched group, the proliferation function of the medullary cell of model mice has been compared utmost point significant difference and 1: 32 (p＜0.01) with model group dilution factor 1: 32 time.The result is as shown in table 5
The influence of CSFs vigor in the table 5 pair mice spleen conditioned medium (SCM) (x ± s, n=6)
| Group | OD value 570nm | ||
| Dilution factor 1: 4 | Dilution factor 1: 8 | Dilution factor 1: 32 | |
| Normal group | 0.099±0.004 | 0.123±0.009 | 0.098±0.003 |
| Tea Polyphenols group catechin group epicatechin group model group Tea Polyphenols model group catechin model group epicatechin model group | 0.106±0.003 ** 0.104±0.004 * 0.103±0.003 * 0.082±0.010 0.098±0.002 * 0.094±0.002 * 0.093±0.002 * | 0.145±0.007 ** 0.138±0.008 ** 0.139±0.009 ** 0.092±0.009 0.102±0.005 * 0.101±0.005 * 0.100±0.003 * | 0.106±0.005 * 0.105±0.006 * 0.104±0.006 * 0.085±0.009 O.101±0.005 ** 0.099±0.004 ** 0.099±0.005 ** |
Annotate: tea polyphenols group, catechin group, epicatechin group and normal group compare, and tea polyphenols model group, catechin model group, epicatechin model group and model group compare,
*Expression P＜0.01,
*Expression P＜0.05.
5.3LCM to influence normal and anemia mice bone marrow nucleated cell propagation
In tea polyphenols, catechin, epicatechin body, induce the LCM of preparation, propagation to the bone marrow nucleated cell of normal mice does not have obvious influence, and the propagation of the bone marrow nucleated cell of anemia Mus all there is obvious facilitation, and at 1/32 dilution factor the strongest facilitation is arranged, improved 34.12%, 31.76%, 31.76% than model group respectively.The result is as shown in table 6.
Table 6 catechin to the influence of CSFs vigor in the mouse lung conditioned medium (LCM) (x ± s, n=6)
| Group | OD value 570nm | ||
| Dilution factor 1: 0 | Dilution factor 1: 8 | Dilution factor 1: 32 | |
| The normal control group | 0.162±0.025 | 0.186±0.014 | 0.090±0.047 |
| Tea Polyphenols group catechin control group epicatechin group model group Tea Polyphenols model group catechin model group epicatechin model group | 0.170±0.057 0.168±0.059 0.165±0.046 0.100±0.020 0.130±0.021 * 0.128±0.020 * 0.125±0.019 * | 0.140±0.016 0.137±0.015 0.135±0.014 0.098±0.016 0.119±0.011 * 0.117±0.013 * 0.117±0.014 * | 0.128±0.012 0.125±0.009 O.122±0.008 0.085±0.015 0.114±0.019 ** 0.112±0.015 ** 0.112±0.016 ** |
Annotate: tea polyphenols group, catechin group, epicatechin group and normal group compare, and tea polyphenols model group, catechin model group, epicatechin model group and model group compare,
*Expression P＜0.01,
*Expression P＜0.05.
5.4PM φ CM is to influence normal and the little marrow nucleated cell propagation of anemia
In tea polyphenols, catechin, epicatechin body, induce the PM φ CM of preparation, propagation to the bone marrow nucleated cell of anemia Mus all has utmost point obvious facilitation, but the propagation to the bone marrow nucleated cell of normal mice only has a significant effect under 1/8 dilution factor, and utmost point significant difference (P＜0.01) is arranged.The result is as shown in table 7.
5.5SMCM to influence normal and anemia mice bone marrow nucleated cell propagation
The SMCM that induces preparation in tea polyphenols, catechin, epicatechin body propagation to the bone marrow nucleated cell of anemia Mus under 1/32 dilution factor has facilitation, and the propagation of the bone marrow nucleated cell of normal mice is not had obvious influence.The result is as shown in table 8.
In the table 7 pair Turnover of Mouse Peritoneal Macrophages culture fluid conditioned medium (PM φ CM)
The influence of CSFs vigor (x ± s, n=6)
| Group | OD value 570nm (x+s) | ||
| Dilution factor 1: 4 | Dilution factor 1: 8 | Dilution factor 1: 32 | |
| The normal control group | 0.128±0.009 | 0.126±0.005 | 0.125±0.004 |
| Tea Polyphenols control group catechin control group epicatechin control group model group Tea Polyphenols model group catechin model group epicatechin model group | 0.125±0.046 0.122±0.036 0.120±0.040 0.099±0.014 0.123±0.011 ** 0.127±0.018 ** 0.125±0.017 ** | 0.138±0.005 ** 0.135±0.004 ** 0.136±0.005 ** 0.092±0.014 0.117±0.014 ** 0.119±0.016 ** 0.220±0.018 ** | 0.130±0.010 0.125±0.009 0.124±0.007 0.10l±0.018 0.132±0.016 ** 0.130±0.014 ** 0.131±0.017 ** |
Annotate: tea polyphenols group, catechin group, epicatechin group and normal group compare, and tea polyphenols model group, catechin model group, epicatechin model group and model group compare,
*Expression P＜0.01,
*Expression P＜0.05.
Table 8 catechin to the influence of CSFs vigor in the mice flesh conditioned medium (SMCM) (x ± s, n=6)
| Group | OD value 570nm | ||
| Dilution factor 1: 4 | Dilution factor 1: 8 | Dilution factor 1: 32 | |
| The normal control group | 0.174±0.038 | 0.132±0.014 | 0.119±0.011 |
| Tea Polyphenols control group catechin control group epicatechin control group model group Tea Polyphenols model group catechin model group epicatechin model group | 0.197±0.020 0.190±0.019 0.189±0.021 0.104±0.027 0.136±0.025 0.131±0.018 0.136±0.027 | 0.142±0.010 0.137±0.014 0.139±0.016 0.112±0.004 0.120±0.009 0.118±0.007 0.114±0.006 | 0.137±0.022 0.132±0.018 0.130±0.022 0.096±0.010 0.168±0.005 * 0.167±0.009 * 0.168±0.008 * |
Annotate: tea polyphenols group, catechin group, epicatechin group and normal group compare, and tea polyphenols model group, catechin model group, epicatechin model group and model group compare,
*Expression P＜0.01,
*Expression P＜0.05.
6. the active drug composition produces the influence test of IL-1 to Turnover of Mouse Peritoneal Macrophages
Get kunming mice male and female half and half, be divided into eight groups at random: blank group, model group, tea polyphenols matched group, catechin matched group, epicatechin matched group, tea polyphenols model group, catechin model group, epicatechin model group by body weight.Blank group and model group mice are pressed the 0.1ml/10g body weight with the normal saline lumbar injection every day; The tea polyphenols matched group, the catechin matched group, the epicatechin matched group, the tea polyphenols model group, the catechin model group, epicatechin model group mice is pressed the 0.1ml/10g body weight respectively with tea polyphenols, catechin, epicatechin lumbar injection every day, continues 10 days.In the time of the 5th, 6,7 day, blank group and tea polyphenols matched group, the catechin matched group, the epicatechin control group mice is pressed the 0.1ml/10g body weight with the normal saline subcutaneous injection; Model group and tea polyphenols model group, the catechin model group, epicatechin model group mice by body weight O.1ml/10g with the cyclophosphamide subcutaneous injection.Above mice was put to death standby in the 11st day.
The preparation of IL-1 sample: the mice cervical vertebra takes off neck and puts to death, 75% soak with ethanol 5 minutes, D-Hank ' s liquid 5ml with pre-cooling is injected into mouse peritoneal, gently rub tens of times, fully wash out abdominal cavity cell, centrifugal 10 minutes of 2000rpm with RPMI-1640 culture fluid washed twice, transfers cell number to 2 * 10 with the RPMI-1640 that contains 5% calf serum
6/ ml added in the 24 porocyte culture plates adherent 2 hours.Abandon supernatant, washed cell is removed NAC.The LPS and the RPMI-1640 culture fluid that contains 10% calf serum that add 10 μ g/ml, at 37 ℃, 5%CO
2Incubation is 48 hours in the moist atmosphere; Collect supernatant ,-20 ℃ of preservations are the IL-1 testing sample.
The detection of IL-1 sample: with C
57The purebred mouse chest cell of BL/6J is as the target cell that detects IL-1.Mice is put to death in the cervical vertebra dislocation, and the aseptic thymus of getting pushed 200 order steel wires with sterilization glass syringe core, centrifugal 10 minutes of 2000rpm, and washed twice, trypan blue dyeing, the meter viable count transfers to 1 * 10 with the RPMI-1640 culture fluid that contains 10% calf serum
6/ ml adds 96 porocyte culture plates with cell suspension, every hole 100 μ l (1 * 10
5Individual cell).With 1/4,1/8,1/32 dilution proportion IL-1 testing sample, every hole adds 50 μ l diluents with the RPMI-1640 culture fluid, and each dilution factor is established 3 multiple holes.Every hole add sub-doses mitogen ConA to final concentration be 2 μ g/ml.Every hole total capacity is 200 μ l.At 37 ℃, 5%CO
2Cultivated 48 hours in the moist atmosphere.With MTT colorimetric method for determining result, as shown in table 9.
The influence that table 9 pair Turnover of Mouse Peritoneal Macrophages produces IL-1
| Group | 1/4 thinner ratio OD 570Value | 1/8 thinner ratio OD 570Value | 1/32 thinner ratio OD 570Value |
| Blank group | 0.108±0.009 | 0.111±0.009 | 0.125±0.005 |
| The tea polyphenols matched group | 0.125±0.009 ** | 0.125±0.005 ** | 0.131±0.009 |
| The catechin matched group | 0.126±0.010 ** | 0.126±0.008 ** | 0.129±0.005 |
| Epicatechin matched group model administration group tea polyphenols model group catechin model group epicatechin model group | 0.120±0.005 ** 0.108±0.016 0.113±0.013 0.111±0.011 0.112±0.009 | 0.122±0.006 ** 0.112±0.007 0.119±0.010 0.117±0.008 0.115±0.007 | 0.126±0.004 0.123±0.035 0.150±0.029 0.152±0.022 0.156±0.030 |
Annotate: tea polyphenols group, catechin group, epicatechin group and normal group compare, and tea polyphenols model group, catechin model group, epicatechin model group and model group compare,
*Expression P＜0.01,
*Expression P＜0.05.
Can find out from above result: behind normal and model mice lumbar injection tea polyphenols, catechin, the epicatechin medicinal liquid, the ability that peritoneal macrophage produces IL-1 improves, and wherein 1/4 has compared utmost point significant difference with the blank administration group of 1/8 thinner ratio with the blank group.
7. the active drug composition produces the influence test of IL-2 to mouse spleen lymphocyte
Get kunming mice male and female half and half, be divided into eight groups at random: blank group, model group, tea polyphenols matched group, catechin matched group, epicatechin matched group, tea polyphenols model group, catechin model group, epicatechin model group by body weight.Blank group and model group mice are pressed the 0.1ml/10g body weight with the normal saline lumbar injection every day; The tea polyphenols matched group, the catechin matched group, the epicatechin matched group, the tea polyphenols model group, the catechin model group, epicatechin model group mice is pressed the 0.1ml/10g body weight respectively with tea polyphenols, catechin, epicatechin lumbar injection every day, continues 10 days.In the time of the 5th, 6,7 day, blank group and tea polyphenols matched group, the catechin matched group, the epicatechin control group mice is pressed the 0.1ml/10g body weight with the normal saline subcutaneous injection; Model group and tea polyphenols model group, the catechin model group, epicatechin model group mice is pressed the 0.1ml/10g body weight with the cyclophosphamide subcutaneous injection.Above mice was put to death standby in the 11st day.
The preparation of IL-2 sample: 75% soak with ethanol 5 minutes, the aseptic spleen of getting are put to death in the dislocation of experiment mice cervical vertebra, with the washing of RPMI-1640 culture fluid, on 200 order steel wires, shred, gently grind, filter with sterilization glass inject cores, make splenocyte suspension respectively, 0.83%Tris-NH
4Cl destroys erythrocyte, and ice bath left standstill 10 minutes, centrifugal 10 minutes of 2000rpm, with RPMI-1640 culture fluid washed cell twice, through determination of trypan blue staining cell motility rate greater than 95%.Make 1 * 10 with the RPMI-1640 culture fluid that contains 10% hyclone again
7/ ml splenocyte suspension adds ConA5 μ g/ml, is incubated at 24 porocyte culture plates, at 5%CO
237 ℃ of incubations are 24 hours in the moist atmosphere; Centrifugal collection supernatant ,-20 ℃ of preservations are the IL-2 testing sample.
IL-2 detects the preparation of cell: 75% soak with ethanol 5 minutes, the aseptic spleen of getting are put to death in the dislocation of BalB/C mice cervical vertebra, with the washing of RPMI-1640 culture fluid, on 200 order steel wires, shred, gently grind, filter with sterilization glass inject cores, make splenocyte suspension, 0.83%Tris-NH
4Cl destroys erythrocyte, and ice bath left standstill 10 minutes, centrifugal 10 minutes of 2000rpm, with RPMI-1640 culture fluid washed cell twice, through determination of trypan blue staining cell motility rate greater than 95%.Make 5 * 10 with the RPMI-1640 culture fluid that contains 10% hyclone again
6/ ml splenocyte suspension adds ConA2.5 μ g/ml, puts in the Tissue Culture Flask of 25ml incubation 96 hours.(Hank ' the s liquid of α-mm) washes twice to cell, and the RPMI-1640 culture fluid that contains 10% hyclone is washed once, and regulating cell concentration is 1 * 10 with 10mg/ml Alpha-Methyl mannoside
6/ ml is as the reaction target cell of measuring IL-2.
The detection of IL-2 sample: that gets-20 ℃ of preservations respectively organizes the IL-2 supernatant, all does 1/4,1/8 and 1/32 dilution, measures its IL-2 level with the activatory mouse spleen lymphocyte of ConA respectively.Promptly in 96 porocyte culture plates, every hole adds reacting cells 0.1ml (1 * 10
5Individual) and the dilution of equivalent after the IL-2 supernatant, every sample is all established 3 multiple holes.Every hole adds Alpha-Methyl mannoside (the 20 μ l of α-mm) of 100mg/ml.At 37 ℃, 5%CO
2Incubation 48h in the moist atmosphere.With MTT colorimetric method for determining result, as shown in table 10.
Can find out from above result: behind normal and model mice lumbar injection tea polyphenols, catechin, the epicatechin medicinal liquid, the ability of splenocyte generation IL-2 is greatly improved.Comprehensive each thinner ratio data show, may be when 1/8 thinner ratio the IL-2 sample ability of best stimulation target cell propagation is arranged.
8. the active drug composition produces the influence test of IL-3 to mouse spleen lymphocyte
Get kunming mice male and female half and half, be divided into eight groups at random: blank group, model group, tea polyphenols matched group, catechin matched group, epicatechin matched group, tea polyphenols model group, catechin model group, epicatechin model group by body weight.Blank group and model group mice are pressed the 0.1ml/10g body weight with the normal saline lumbar injection every day; The tea polyphenols matched group, the catechin matched group, the epicatechin matched group, the tea polyphenols model group, the catechin model group, epicatechin model group mice is pressed the 0.1ml/10g body weight respectively with tea polyphenols, catechin, epicatechin lumbar injection every day, continues 10 days.In the time of the 5th, 6,7 day, blank group and tea polyphenols matched group, the catechin matched group, the epicatechin control group mice is pressed the 0.1ml/10g body weight with the normal saline subcutaneous injection; Model group and tea polyphenols model group, the catechin model group, epicatechin model group mice is pressed the 0.1ml/10g body weight with the cyclophosphamide subcutaneous injection.Above mice was put to death standby in the 11st day.
The influence that table 10 pair mouse spleen lymphocyte produces IL-2
| Group | 1/4 thinner ratio OD 570Value | 1/8 thinner ratio OD 570Value | 1/32 thinner ratio OD 570Value |
| Blank group | 0.110±0.010 | 0.103±0.005 | 0.084±0.004 |
| The tea polyphenols matched group | 0.123±0.011 | 0.113±0.003 ** | 0.092±0.005 ** |
| The catechin matched group | 0.120±0.007 | 0.110±0.003 ** | 0.091±0.004 ** |
| The epicatechin matched group | 0.119±0.008 | 0.111±0.003 ** | 0.091±0.003 ** |
| Model group | 0.101±0.007 | 0.087±0.021 | 0.079±0.012 |
| Tea polyphenols model group catechin model group epicatechin model group | 0.115±0.009 * 0.111±0.008 * 0.112±0.009 * | 0.115±0.017 * 0.113±0.019 * 0.111±0.015 * | 0.091±0.009 0.088±0.013 0.086±0.010 |
Annotate: tea polyphenols group, catechin group, epicatechin group and normal group compare, and tea polyphenols model group, catechin model group, epicatechin model group and model group compare,
*Expression P＜0.01,
*Expression P＜0.05.
The preparation of IL-3 sample: 75% soak with ethanol 5 minutes, the aseptic spleen of getting are put to death in the dislocation of experiment mice cervical vertebra, with the washing of RPMI-1640 culture fluid, on 200 order steel wires, shred, gently grind, filter with sterilization glass inject cores, make splenocyte suspension respectively, 0.83%Tris-NH
4Cl destroys erythrocyte, and ice bath left standstill 10 minutes, centrifugal 10 minutes of 2000rpm, with RPMI-1640 culture fluid washed cell twice, through determination of trypan blue staining cell motility rate greater than 95%.Make 1 * 10 with the RPMI-1640 culture fluid that contains 10% hyclone again
7/ ml splenocyte suspension adds ConA5 μ g/ml, is incubated at 24 porocyte culture plates, at 5%CO
237 ℃ of incubations are 72 hours in the moist atmosphere; Centrifugal collection supernatant ,-20 ℃ of preservations are the IL-3 testing sample.
The detection of IL-3 sample: the dislocation of BalB/C mice cervical vertebra is put to death, 75% soak with ethanol 5 minutes, the aseptic bilateral femur of getting, RPMI-1640 culture fluid flushing medullary cavity with No. 6 syringe needle suction pre-coolings, mix bone marrow cell suspension and cross 4 syringe needles, form single cell suspension, transfer cell number to 2 * 10 with the RPMI-1640 culture fluid that contains 10% calf serum
6Individual/ml.That gets-20 ℃ of preservations respectively organizes the IL-3 supernatant, all does 1/4,1/8 and 1/32 dilution.In 96 porocyte culture plates, every hole adds reacting cells 0.1ml (2 * 10
5Individual) and the dilution of equivalent after the IL-3 supernatant, every sample is all established 3 multiple holes.At 37 ℃, 5%CO
2Incubation 7d in the moist atmosphere, with MTT colorimetric method for determining result, as shown in table 11.
Can find out from above result: normal and model mice is behind lumbar injection tea polyphenols, catechin, epicatechin medicinal liquid, and the ability of splenocyte generation IL-3 has the raising of significance.During 1/4 and 1/8 thinner ratio, blank administration group has been compared utmost point significant difference with the blank group, and model administration group has been compared significant difference with model group.During 1/32 thinner ratio, all there are significant difference in blank administration group and blank group between model administration group and the model group.
Table 11 active drug composition catechin produces the influence of IL-3 to mouse spleen lymphocyte
| Group | 1/4 thinner ratio OD 570Value | 1/8 thinner ratio OD 570Value | 1/32 thinner ratio OD 570Value |
| Blank group | 0.097±0.004 | 0.122±0.011 | 0.096±0.002 |
| The tea polyphenols matched group | 0.105±0.002 ** | 0.141±0.009 ** | 0.102±0.006 * |
| The catechin matched group | 0.104±0.002 ** | 0.142±0.010 ** | 0.101±0.005 * |
| The epicatechin matched group | 0.003±0.002 ** | 0.143±0.012 ** | 0.099±0.003 * |
| Model group | 0.082±0.013 | 0.095±0.017 | 0.085±0.013 |
| Tea polyphenols model group catechin model group epicatechin model group | 0.099±0.012 * 0.100±0.015 * 0.101±0.014 * | 0.121±0.019 * 0.119±0.017 * 0.120±0.016 * | 0.102±0.014 * 0.105±0.016 * 0.105±0.015 * |
Annotate: tea polyphenols group, catechin group, epicatechin group and normal group compare, and tea polyphenols model group, catechin model group, epicatechin model group and model group compare,
*Expression P＜0.01,
*Expression P＜0.05.
Below example by the specific embodiment again foregoing of the present invention is described in further detail.But this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following example.Do not breaking away under the above-mentioned technological thought situation of the present invention, various replacements or change according to ordinary skill knowledge and customary means are made include within the scope of the invention.
The specific embodiment
Embodiment 1 oral liquid formulations
Get 10 kilograms of tea polyphenols, 100 kilograms of dissolvings of water add sucrose and sodium benzoate (or other sweeting agents of allowing), filter, and fill, promptly.
Embodiment 2 granules
Get 10 kilograms of tea polyphenols, 5 kilograms of sucrose, 5 kilograms of starch (or several correctivess of other adhesive of allowing), one-step-granulating method is granulated, granulate, packing, promptly.
Embodiment 3 tablets
Get 10 kilograms of tea polyphenols, 5 kilograms of sucrose, 5 kilograms of starch (or several correctivess of other adhesive of allowing), one-step-granulating method is granulated (or additive method granulation), drying, granulate adds magnesium stearate (or other available lubricants), the tabletting packing, promptly.
The tea polyphenol raw materials of above oral formulations can be 1～50 kilogram.
Embodiment 4 injections
Get 10 kilograms of tea polyphenols, with 100 kilograms of dissolvings of water for injection, add 0.9 kilogram in sodium chloride, add 0.01～1% the active carbon of solution total amount or the additive method of pharmaceuticals industry regulation and remove pyrogen, in 70～90 ℃ of insulations 10～60 minutes, filter, transfer pH4～9 with the additive method of hydrochloric acid, Chinese holly hydrochloric acid, sodium hydroxide (potassium) and pharmaceuticals industry regulation.In order to prevent this product oxidation stain, can add antioxidant in this product, as sodium pyrosulfite, sodium sulfite or other pharmaceuticals industries regulation antioxidant.Embedding, sealing by fusing, sterilization, promptly.
Claims (5)
1. the medicine that has rising whole blood function, it is characterized in that with at least a in catechin or the epicatechin be the active drug composition, form jointly with acceptable auxiliary element in the medicine.
2. the medicine with rising whole blood function as claimed in claim 1 is characterized in that said active drug composition is for containing the tea polyphenols of catechin and epicatechin simultaneously.
3. the medicine with rising whole blood function as claimed in claim 2 is characterized in that the catechin in the said active drug composition tea polyphenols and gross weight content 〉=80% of epicatechin.
4. as the described medicine of one of claim 1 to 3, it is characterized in that said medicine is the injection-type preparation with rising whole blood function.
5. as the described medicine of one of claim 1 to 3, it is characterized in that said medicine is the oral type preparation with rising whole blood function.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102688229A (en) * | 2011-03-22 | 2012-09-26 | 中国医学科学院药物研究所 | Application of epicatechin as drug for raising leucocytes |
| CN101549005B (en) * | 2008-03-31 | 2013-06-12 | 中国医学科学院药物研究所 | Application of Acacia catechu (L.) wild extract to medicines capable of improving white blood cells |
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2004
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101549005B (en) * | 2008-03-31 | 2013-06-12 | 中国医学科学院药物研究所 | Application of Acacia catechu (L.) wild extract to medicines capable of improving white blood cells |
| CN102688229A (en) * | 2011-03-22 | 2012-09-26 | 中国医学科学院药物研究所 | Application of epicatechin as drug for raising leucocytes |
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