CN103301161A - Method for preparing traditional Chinese medicine preparation by use of ground beetle extract and application of traditional Chinese medicine preparation - Google Patents
Method for preparing traditional Chinese medicine preparation by use of ground beetle extract and application of traditional Chinese medicine preparation Download PDFInfo
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Abstract
The invention relates to a method for preparing a traditional Chinese medicine preparation by use of a ground beetle extract combined with a modern technology according to records of traditional Chinese medicines. The method comprises the following steps: (1) preparing a ground beetle extract liquor; (2) performing ultrafiltration by an ultrafiltration membrane; (3) filtering by a nanofiltration membrane; (4) sterile-freezing and drying to prepare freeze-dried powder; (5) preparing capsules and powder-injection. The traditional Chinese medicine preparation prepared by the ground beetle extract according to the preparation method not only has the functions of removing blood stasis and promoting reunion of fractured bones verified by practice of traditional Chinese medicine, but also has the effects of improving purine metabolism, adjusting the function of the immune system, enhancing recovery of bone tissue and preventing tumor necrosis factors. In addition, the preparation further has the anti-thrombosis effect and the effect of dissolving thrombus, and has the delaying effect to atherosclerosis. The preparation is an ideal traditional Chinese medicine preparation for treating rheumatic pain, preventing tumor and improving the immunity.
Description
Technical field
The present invention relates to technical field of Chinese medicines, specifically, is a kind of method for preparing Chinese medicine preparation with Eupolyphaga seu steleophaga extract, and described Chinese medicine preparation with the Eupolyphaga seu steleophaga extract preparation can be applied to treat the fields such as rheumatic osteopathia, antitumor, enhancing body immunity.
Background technology
Rheumatic osteopathia belongs to arthromyodynia in theory of Chinese medical science, and numbness, namely numbness is the obstructed meaning of numbness resistance.QI and blood can not be unobstructed after human body flesh table grain suffers exopathogen invasion and attack, thereby the muscle BJM that causes etc. locates class illness such as misery, severe one numbness, joint stuffiness, all is called arthromyodynia.Arthromyodynia sees " Plain Questions numbness opinion " the earliest: " so-called fraud person ... reinfection is in the wet gas of wind and cold also ", wherein, rheumatism cold-evil is the main cause that causes arthromyodynia.Claim that in " Medical Treasures of the Golden Chamber " this disease is " severe and migratory arthralgia ".The wind heat disease of modern medicine, gout, rheumatic arthritis, rheumatoid arthritis, lumbar muscle strain etc. all belong to the category of arthromyodynia.The main pathogenesis of arthromyodynia is evil resistance venation, and QI and blood is popular unfavorable, and with muscle, arthralgia, movable unfavorable is primary symptom, but laid particular stress on by heresy, and the body negative and positive have inclined to one side Sheng; Therefore disease performance and inconsistent.Become migratory arthralgia such as windage yaw Sheng, arthralgia migration person; Wet partially Sheng, the heavy pain person in joint claim damp arthralgia; Cold partially Sheng, joint cold type of pain person claim arthralgia aggravated by cold; Heat is contained partially, burning in the joint, painful person is called pyretic arthralgia.The treatment of arthromyodynia, originally mainly with bloodstasis, disease is then joined the product of 5 bone and muscle strengthening, invigorating the liver and kidney for a long time.Theory of Chinese medical science thinks, the osteopathia that this card causes is because of insufficiency of vital energy and blood, and the Wei Bugu of battalion is due to rheumatism cold-evil whenever takes advantage of a weak point more.Excessive QI has inclined to one side Sheng, therefore be divided into above four large classes.Should note invigorating QI and blood or nourishing the liver and kidney in the treatment.Broken silt is reduced phlegm, insects are searched and pick all and can use with disease.Simultaneously, the lumbago that the causes of disease such as stagnation of cold-damp, damp and hot obstruct, qi depression to blood stasis cause in the traditional Chinese medical science category, the said rheumatism of modern medicine, rheumatoid disease, lumbar muscle strain, hyperosteogeny etc. all can be caused by arthromyodynia.
Treatment arthromyodynia and lumbago (doctor trained in Western medicine is referred to as " rheumatic osteopathia ") mostly are with Western medicine and carry out symptomatic treatment at present.Therapeutic effect is obvious in a short time for the chemical classes medicine of Western medicine, still, can have side effects after the somebody takes sometimes, and treatment also is difficult to remove old complaint, can occur state of an illness bounce-back or recurrence in treatment or after the treatment.If with Chinese traditional treatment arthromyodynia and lumbago, mainly be to adopt Chinese medicine to treat.Theory of Chinese medical science thinks, the main pathogenesis of arthromyodynia is evil resistance venation, and QI and blood is popular unfavorable and cause muscle, arthralgia, movable unfavorable.Therefore Chinese medicine to the treatment more options of arthromyodynia by through the cave, the relevant approach of internal organs, the medicine that human body multisystem, many organs, too many levels are adjusted is treated, mainly be indirectly to treat arthromyodynia and rheumatic osteopathia by adjusting human organ function and body's immunity, thereby, the treatment of the traditional Chinese medical science is fast and obvious not as doctor trained in Western medicine, but the side effect of Chinese medicine is less, and the chemical reaction of taking the Chinese medicine descendant can be less.
In addition, the method for modern medicine (doctor trained in Western medicine) treatment tumor has varied, comprises the methods such as operative treatment, radiotherapy, chemotherapy and therapy of combining Chinese and Western medicine.The method that benign tumor can be selected to excise is treated, but, the side of excision can not reach the purpose of thorough treatment and control for malignant tumor, therefore, to carry out selectively chemotherapy behind the excision (malignant tumor), but chemotherapy also can damage people's normal cell and body tissue in killing and wounding the cancerous cell process.So, the method for therapy of combining Chinese and Western medicine tumor has appearred now.And Chinese medicine mainly is by carrying out on the basis that the mechanism of human body is regulated to the control of tumor, and little to the direct injury of human body, the toxic and side effects of medicine is little, and somebody and the disease that has are had obvious curative effect equally, is easy to be accepted by the patient.Chinese medicine thinks, the disease of tumor is drawn for qi stagnation blood stasis expectorant solidifying, and ancient medicine is blamed in empty, strongly fragrant, expectorant, the stasis of blood four ends more.The the underlying cause of Carcinogenesis is that sickened body had the causes of disease such as disorder of YIN and YANG, zang-fu disharmony before canceration, and multiple long-time stimulus with factors such as lightweight, diet causes the pathogen invasion, and the healthy energy virtual loss finally causes canceration.Therefore in history, the traditional Chinese medical science is being sought always and can treated arthromyodynia each sick method and Chinese medicine.
Eupolyphaga Seu Steleophaga has another name called Eupolyphaga Seu Steleophaga, eupolyphaga, is Corydiidae insecticide eupolyphoge sinensis
Eupolyphaga sinensis WalkerOr Ji eupolyphoge sinensis
Stelephaga plancyi (Boleny)Female worm all.On the books on Chinese medicine, Eupolyphaga Seu Steleophaga has embodied positive effect as the insects medical material in the rheumatic osteopathia that the treatment arthromyodynia causes.Modern medicine study also confirms, Eupolyphaga Seu Steleophaga contains the inorganic elementss such as the aminoacid of needed by human and potassium, calcium, magnesium, zinc, phosphorus.As if but the traditional method of the traditional Chinese medical science can not reach optimum state to Eupolyphaga Seu Steleophaga as the utilization of insects medical material.In the research to Chinese medicine, it is found that: utilize modern technologies can promote the development of Chinese medicine; Applicating modern times technology can reach a new high the therapeutic effect of some Chinese medicines.Eupolyphaga Seu Steleophaga is as one of traditional insects medical material of Chinese medicine, utilize modern technologies to its applied research at present or inadequate.
Summary of the invention
The object of the invention is to, the record according to " Eupolyphaga Seu Steleophaga can be treated the rheumatic osteopathia that arthromyodynia causes " on theory of Chinese medical science and the Chinese medicine provides a kind of method for preparing Chinese medicine preparation with Eupolyphaga seu steleophaga extract in conjunction with modern technologies; The second purpose of the present invention is, with the Chinese medicine preparation of preparation be applied to wider, comprise the scope for the treatment of rheumatic osteopathia, antitumor and enhancing body immunity.
For achieving the above object, the technical solution used in the present invention is as follows.
A kind of method for preparing Chinese medicine preparation with Eupolyphaga seu steleophaga extract is characterized in that, comprises following steps:
(1) preparation Eupolyphaga Seu Steleophaga extracting solution
At ambient temperature, to take by weighing through the Eupolyphaga Seu Steleophaga concocted and deionized water (be the words of 10g through the Eupolyphaga Seu Steleophaga concocted to the ratio of 1:10 in mass ratio, deionized water is 100ml), then described Eupolyphaga Seu Steleophaga was put under the deionized water room temperature lixiviate 1 hour, ultrasonication is 40 minutes after the lixiviate, then use the gauze coarse filtration, obtain the Eupolyphaga Seu Steleophaga extracting solution;
(2) use the ultrafilter membrane ultrafiltration
The Eupolyphaga Seu Steleophaga extracting solution that step (1) obtains is at room temperature used the ultrafilter membrane ultrafiltration, and molecular cut off is greater than 30000, will through ultrafilter membrane, molecular weight saves backup less than 30000 ultrafiltrate;
(3) filter with NF membrane
The ultrafiltrate that step (2) is obtained at room temperature filters with NF membrane, and molecular cut off is got molecular weight for subsequent use greater than 300 concentrated solution greater than 300;
(4) molecular weight that step (3) is obtained is made lyophilized powder greater than the aseptic lyophilization of 300 concentrated solution;
(5) lyophilized powder that step (4) is obtained is made capsule and injectable powder, obtains target product---the Chinese medicine preparation made from Eupolyphaga seu steleophaga extract.
For realizing above-mentioned the second purpose, the technical solution used in the present invention is as follows.
The application of Chinese medicine preparation in treatment rheumatic osteopathia, treatment oncosis with the Eupolyphaga seu steleophaga extract preparation.
The application of Chinese medicine preparation in improving body immunity, balance the body cardiovascular system activity with the Eupolyphaga seu steleophaga extract preparation.
Chinese medicine preparation with the Eupolyphaga seu steleophaga extract preparation is being repaired the damaged cartilage cell, is being regulated the application in the purine metabolism.
Good effect of the present invention is:
(1) for prevention and the treatment of cold-dampness type arthromyodynia, tumor, makes a kind of new Chinese medicine preparation by modern means with Eupolyphaga seu steleophaga extract.
(2) from the situation of Chinese medicine record in conjunction with modern technologies analysis and various cell experiments, Chinese medicine preparation with the Eupolyphaga seu steleophaga extract preparation of the present invention not only has the removing blood stasis that Traditional Chinese Medicine facts have proved, the effect of reunion of fractured tendons and bones, and have the effect that improves purine metabolism, regulates function of immune system and enhancing osseous tissue rehabilitation and anti-tumor necrosis factor; In addition, cell experiment has verified that also Eupolyphaga seu steleophaga extract has antithrombotic to form and thrombolytic effect, has the effect that delays to atherosclerosis.
(3) the Chinese medicine preparation medicinal effects with the Eupolyphaga seu steleophaga extract preparation that adopts preparation method of the present invention to obtain is remarkable, have no side effect, taking or treat and use conveniently, is a kind of Chinese medicine preparation of comparatively desirable treatment rheumatic ostalgia, antitumor and raising immunity, can supply extensive use.
(4) preparation method of the present invention, preparation technology are simple, and easily operation is implemented.
Description of drawings
To be the present invention prepare the uv-spectrogram of Chinese medicine preparation of the method preparation of Chinese medicine preparation with Eupolyphaga seu steleophaga extract to Fig. 1.
The specific embodiment
Below continue to introduce the specific embodiment of the present invention and 5 pharmacological evaluation reports are provided.But enforcement of the present invention is not limited to following embodiment.
Prepare the method for Chinese medicine preparation with Eupolyphaga seu steleophaga extract, comprise following steps:
(1) preparation Eupolyphaga Seu Steleophaga extracting solution
At ambient temperature, the ratio of 1:10 takes by weighing Eupolyphaga Seu Steleophaga and the deionized water through concocting in mass ratio, the present embodiment is taken Eupolyphaga Seu Steleophaga, the 100ml deionized water of 10g through concocting, then described Eupolyphaga Seu Steleophaga is put into deionized water (room temperature) lixiviate 1 hour, ultrasonication is 40 minutes after the lixiviate, then use the gauze coarse filtration, obtain the Eupolyphaga Seu Steleophaga extracting solution.
(2) use the ultrafilter membrane ultrafiltration
The Eupolyphaga Seu Steleophaga extracting solution that step (1) obtains is at room temperature used the ultrafilter membrane ultrafiltration, and molecular cut off will save backup through the ultrafiltrate (molecular weight is less than 30000) of ultrafilter membrane greater than 30000.
(3) filter with NF membrane
The ultrafiltrate that step (2) is obtained at room temperature filters with NF membrane, and molecular cut off is got molecular weight for subsequent use greater than 300 concentrated solution greater than 300.
(4) molecular weight that step (3) is obtained is made lyophilized powder greater than the aseptic lyophilization of 300 concentrated solution.
(5) lyophilized powder that step (4) is obtained is made capsule and injectable powder, obtains target product---the Chinese medicine preparation made from Eupolyphaga seu steleophaga extract.
Through the ultraviolet spectrophotometer analysis, the present invention has maximum absorption peak (referring to Fig. 1) with the Chinese medicine preparation that Eupolyphaga seu steleophaga extract prepares the method preparation of Chinese medicine preparation near 290nm.
Below introduce with the situation of method of the present invention with the Chinese medicine preparation experiment Analysis of Eupolyphaga seu steleophaga extract preparation, by 5 cell experiments and zoopery the function of described Chinese medicine preparation is analyzed, checked and verifies.
Pharmacological evaluation 1
To be used for the Chinese medicine preparation of Eupolyphaga seu steleophaga extract preparation the cell experiment of the impact (cardiac vascular activity) of production by human umbilical vessel endothelial cells NO, to understand the function of its blood circulation promoting and blood stasis dispelling.
(1) tested medicine: described Chinese medicine preparation with the Eupolyphaga seu steleophaga extract preparation is dissolved in DMEM(annotates: a kind of culture medium that contains each seed amino acid and the glucose) solution of gained.
(2) subject cell: Human umbilical vein endothelial cells.
(3) situation of cell proliferation after the described tested medicine of observation discharges the impact of NO and adds described tested medicine Human umbilical vein endothelial cells, can produce facilitation to the release of the NO of Human umbilical vein endothelial cells as evaluation criterion take described tested medicine, determine to promote vasodilation to reach the distribution of regulating blood pressure and blood flow, the formation of anti-tampon with the Chinese medicine preparation of Eupolyphaga seu steleophaga extract preparation.
(4) experimentation
Cultivate Human umbilical vein endothelial cells, with the DMEM culture medium Chinese medicine preparation of the present invention is dissolved, configures, described Human umbilical vein endothelial cells (adopted 800,400,200,100,50,25,12.5,6.25 in 48～72 hours through described tested drug treating
Eight concentration of/ml, a negative control medicine and a positive control drug, three multiple holes); Then get cell conditioned medium liquid, with the propagation of CCK8 kit detection cell; Detect the release of NO with the NO test kit.
Wherein, the computational methods of NO content are: NO content (
)
=(measuring pipe absorbance-blank tube absorbance)/(standard substance absorbance-blank tube absorbance) * standard substance concentration (100
Extension rate (1) before the) * sample test.
(5) experimental result
The Chinese medicine preparation made from Eupolyphaga seu steleophaga extract is on the impact (seeing Table 1) of production by human umbilical vessel endothelial cells NO:
The Chinese medicine preparation that table 1. is made with Eupolyphaga seu steleophaga extract is on the impact of production by human umbilical vessel endothelial cells NO
(6) result of pharmacological evaluation 1 shows
1. with the Chinese medicine preparation of Eupolyphaga seu steleophaga extract preparation 6.25
Demonstrate maximum proliferative amount under the/mL concentration and be higher than blank group, this explanation, the facilitation that under this concentration Human umbilical vein endothelial cells is increased is the strongest.
2. in the result of NO secretion, has the effect of stronger promotion production by human umbilical vessel endothelial cells NO with the Chinese medicine preparation of Eupolyphaga seu steleophaga extract preparation.Because the NO that vascular endothelial cell produces is passed to rapidly vascular smooth muscle cell by cell membrane, makes smooth muscle loosening. the arteries expansion, thus regulate blood pressure and blood distribution.Endogenous NO is regulated vascular endothelial growth, triggers vaso-active substance, promotes angiogenic growth and regeneration.The NO that vascular endothelial cell produces all has the effect that keeps the vascular endothelial cell integrity under physiology, pathologic condition.NO can anticoagulant and adhesion, reduces leukocytic gathering, the formation of anti-tampon; Can also suppress lymphopoiesis, suppress the oxidation product of macrophage, suppress the activity of mastocyte; When chronic infection and inflammation, the N0 that activating macrophage, leukocyte produce participates in the effect of kill bacteria, virus, parasite, fungus, tumor cell.Therefore the increased content of NO explanation has been played useful effect aspect the Cardiovascular with the Chinese medicine preparation of Eupolyphaga seu steleophaga extract preparation improving in the pharmacological evaluation 1.The concept of the traditional Chinese medical science is the effect of playing blood circulation promoting and blood stasis dispelling.
Pharmacological evaluation 2
Purine metabolism suppresses active cell experiment: understand with the Chinese medicine preparation of the Eupolyphaga seu steleophaga extract preparation inhibitory action to xanthine oxidase.
(1) solution that tested medicine: will be dissolved in the PBS(phosphate buffer with the Chinese medicine preparation of Eupolyphaga seu steleophaga extract preparation) obtains.
(2) experimental technique
Xanthine oxidase (Xanthine Oxidase claims simple XOD) is a kind of important enzyme in the nucleic acid in vivo metabolism, and energy catalysis xanthine and hypoxanthine oxidation generate uric acid, produce simultaneously peroxide radical.Therefore, suppress the activity of xanthine oxidase, both can reduce the formation of uric acid in the body, can reduce the tissue injury that free radical causes again.
Carry out described tested medicine to the mensuration of the suppression ratio of xanthine oxidase.Xanthine concentration is 100mg/L, and xanthine oxidase concentration is 50U/L, and the solution concentration with the Chinese medicine preparation of Eupolyphaga seu steleophaga extract preparation that pharmacological evaluation 2 configures is 10,50,90,150,300,450
/ mL.In the 10mL plastic centrifuge tube, add 2.0ml xanthine solution, 25 ℃ of pre-temperature 10min, add PBS buffer solution 0.1ml, add again the xanthine oxidase solution 2.0ml through 25 ℃ of pre-temperature, measure the absorbance of 290nm place different time points, measured METHOD FOR CONTINUOUS DETERMINATION 450 seconds 1 time in per 30 seconds, time and absorbance data are processed by MATLAB, calculate enzyme activity VXOD.
Add 2.0mL xanthine solution in the 10mL plastic centrifuge tube, 25 ℃ of pre-temperature 10min add PBS buffer solution 2.1mL, and every 30s measures the absorbance at 290nm place, and METHOD FOR CONTINUOUS DETERMINATION 450s calculates blank
In the 10mL plastic centrifuge tube, add the 2.0mL substrate solution, 25 ℃ of pre-temperature 10min, add testing sample 0.1mL, add again the xanthine oxidase solution 2.0mL through 25 ℃ of pre-temperature, every 30s measures the absorbance of 290nm, METHOD FOR CONTINUOUS DETERMINATION 450s calculates the maximum reaction rate Vs of xanthine enzyme under the sample effect.
The computational methods of its suppression ratio are: suppression ratio
(2).
(3) experimental result
With the Chinese medicine preparation of Eupolyphaga seu steleophaga extract preparation to xanthine enzymeinhibition rate (seeing Table 2):
Table 2. with the Chinese medicine preparation of Eupolyphaga seu steleophaga extract preparation to xanthine enzymeinhibition rate
Through 3 repeated trials, the result of table 2 is shown as: the maximal percentage inhibition to xanthine oxidase is 77.04%, and the suppression ratio of more parallel other groups is maximum.
(4) result of pharmacological evaluation 2 shows
Can suppress the metabolism of xanthine oxidase with the Chinese medicine preparation of Eupolyphaga seu steleophaga extract preparation, reduce the generation of uric acid, also have simultaneously the mediation effect of Chinese medicine, invigorating the spleen and replenishing QI, emergency analgesia.
Pharmacological evaluation 2 has proved on the basis of modern medicine: the pain that can alleviate by regulating the xanthine metabolism rheumatism arthritis with the Chinese medicine preparation of Eupolyphaga seu steleophaga extract preparation.
Pharmacological evaluation 3
Zoopery to the rheumatic osteopathia therapeutical effect: understand Chinese medicine preparation with the Eupolyphaga seu steleophaga extract preparation to IL-1
The impact of the rat cartilage cell propagation of inducing.
(1) tested medicine: with the Chinese medicine preparation of Eupolyphaga seu steleophaga extract preparation.
(2) animal subject: SD rat, 6,7 ages in days, male and female half and half; The SD rat is available from west, Shanghai pul-Bi Kai laboratory animal company limited (credit number: SCXK (Shanghai) 2008-0016).
(3) experimental technique
7 age in days SD rats are taken off neck puts to death, be soaked in alcohol content and be in 95% the ethanol and take out after several minutes, take out femoral head, shoulder joint and knee cartilage tissue, use the PBS(phosphate buffer) flushing is for several times, adding a small amount of PBS shreds, 37 ℃ of digestion of II Collagenase Type with 0.2% pipette supernatant and cultivate in culture dish as the individual cells suspension inoculation, change every other day liquid.The cultivation of going down to posterity after cell covers with substantially, when reaching 2nd generation, adjusting cell concentration is 1 * 10
7/ L is inoculated in 96 well culture plates for experiment.Every hole inoculation 200
Cell suspension is divided into blank group, IL-1 with culture hole after inoculating 24 h
Matched group, given the test agent group, positive controls, every sample 3 multiple holes, change respectively corresponding culture fluid:
1. blank group: change DMEM (containing 5% FBS, 0.02% tween 80);
2. model group: change DMEM and (contain 10
The IL-1 β of/L, 0.02% tween 80 and 5% FBS);
3. tested medicine and positive controls: change DMEM and (contain 10
The IL-1 of/L
, 0.02% tween 80,5% FBS and each concentration medicine or positive drug);
Each group is put into incubator continue to cultivate 48h for the cell proliferation detection.Remove original cell culture medium, every hole adds 100
Full culture medium and 10
CCK8 solution is resuspended, continues to hatch, and in placing the optical density value (OD value) of measuring every hole under the microplate reader 450 nm wavelength after the administration in 3 hours, and is converted into the rate of increase (formula 1), and the given the test agent proliferation is analyzed.
The rate of increase (%)=
(3).
(4) experimental result
Tested medicine is to IL-1
The propagation impact (seeing Table 2) of the rat osteoarthritis chondrocytes of inducing:
The tested medicine of table 3. is to IL-1
The propagation impact (O.D.) of the rat osteoarthritis chondrocytes of inducing
In the table 3, * represents P＜0.05, and significant difference is arranged; * represents P＜0.01, and significant difference is arranged.
(4) result of pharmacological evaluation 3 shows
By as seen from Table 3, model group is adding IL-1
After, OD value compared with normal group obviously reduces, and cell quantity significantly reduces, this phenomenon explanation IL-1
Presenility occured in existing effect inducing cell, the success of rat bone articular degeneration model construction.Simultaneously, tested medicine group is at (0.08-9.77
/ ml) all demonstrate obvious cel l proliferation in the concentration range, and be dose dependent, and be 9.77 in concentration
Effect is the most obvious during/mL: the rate of increase is 113.071%, reaches its maximum drug effect.Hence one can see that, can effectively treat osteoarthritis with the Chinese medicine preparation of Eupolyphaga seu steleophaga extract preparation, can effectively promote the injury that the propagation of chondrocyte causes the joint to alleviate degenerative osteoarthritis.
Pharmacological evaluation 4
Zoopery on immunoregulation effect: understand with the impact on the mouse spleen lymphocyte immunologic function of the Chinese medicine preparation of Eupolyphaga seu steleophaga extract preparation.
(1) tested medicine: will be dissolved in the Chinese medicine preparation of Eupolyphaga seu steleophaga extract preparation the solution of DMEM gained.
(2) animal subject: C57 mice, 10, age in 6-8 week, male and female half and half; The C57 mice is available from Shanghai laboratory animal resource center (credit number: SCXK (Shanghai) 2008-0016).
(3) experimental technique
With the disconnected neck of tested mice, the aseptic spleen of getting grinds rear preparation splenocyte suspension with filter screen, guarantees the primary cell activity more than 95%, adjustment cell concentration to 3 * 10
6Individual/mL; Add ConA and induce spleen lymphocyte proliferation, (concentration is (3.125,6.25,12.5 25,50,100,200,400 to cell through tested medicine
– 1Eight concentration, 3 hole parallel laboratory tests) process, measure IFN-in the cells and supernatant with the ELISA method behind the cultivation 72h
Content.
(4) experimental result
1. tested medicine is to mouse boosting cell IFN-
The impact (seeing Table 4) of secretion
The tested medicine of table 4. is to mouse boosting cell IFN-
The impact (O.D.) of secretion
；
In the table 4, * represents P＜0.05, and significant difference is arranged; * represents P＜0.01, and significant difference is arranged.
As shown in table 4, described tested medicine is 3.125～400
All can be to the IFN-of mouse spleen lymphocyte in the/ml concentration range
Secretion level has Pasitive Regulation Effect of Genseng, and is obvious dose dependent.Wherein, when concentration 400
During/ml, it is the strongest that facilitation reaches.This result shows with Chinese medicine preparation adjustable motivation body immunne response in the high concentration situation of Eupolyphaga seu steleophaga extract preparation, regulates lymphocytic Proliferation and differentiation and reach the purpose of effecting a permanent cure with the enhancing human body immunity effect.Simultaneously, the state (seeing Table 5) of cell has been investigated in this experiment with the CCK-8 test
The tested medicine of table 5. is on the impact (O.D.) of mouse boosting cell propagation
In the table 5, * represents P＜0.05, and significant difference is arranged; * represents P＜0.01, and significant difference is arranged.
As can be known from the results of Table 5, Growth of Cells is good, and without mortality, cell state is normal, shows that this experimental data is reliable.
Pharmacological evaluation 5
Cell experiment on Ana-1 macrophage immunity function: the Chinese medicine preparation of understanding with the Eupolyphaga seu steleophaga extract preparation affects mice Ana-1 macrophage immunity function, antineoplastic.
(1) tested medicine: will be dissolved in the Chinese medicine preparation of Eupolyphaga seu steleophaga extract preparation the solution of DMEM gained.
(2) subject cell: Ana-1 mouse macrophage.
(3) experimental technique
1. with the Ana-1 mouse macrophage at 37 ℃, 5%CO
2Under the condition, with containing 10% calf serum, penicillin (1 * 10
5UL
-1) and streptomycin (100 mgL
-1) the cultivation of going down to posterity of DMEM culture fluid.Experiment all is in exponential phase with cell.
2. get the cell concentration that needs, add the fresh complete medium re-suspended cell that needs volume, inoculate in 96 orifice plates blank group and drug treating group to be measured (3.125,6.25,12.525,50,100,200
ML
-1), in 37 ℃, 5% CO
2Cultivate.
3. before dosing, discard old culture fluid, with fresh medium washing 3 times, add fresh drug incubation 30min to be measured, with enzyme-linked method working sample IL-1
, IL-6 and TNF-
Content, every group is repeated 3 times, adds simultaneously CCK8 reagent, detects the cell viability state.
(4) experimental result
1. tested medicine is to IL-1
The propagation impact (seeing Table 6) of cell
The tested medicine of table 6. is to IL-1
The propagation impact of cell (the OD value/A)
In the table 6, * represents P＜0.05, and significant difference is arranged; * represents P＜0.01, and significant difference is arranged.
As shown in table 6, described tested medicine is 50～400
All can significantly strengthen the IL-1 of mouse spleen lymphocyte in the/ml concentration range
Secretion level is obvious dose dependent in high concentration range, especially works as concentration 400
During/ml, facilitation is the most outstanding.IL-1
It is the most important cytokine that stimulates T cell, B cell and NK cell activation, propagation and differentiation, mainly produced by macrophage, it has very widely biologic activity, participate in the inflammation of body and immune defence reaction, hemopoietic function of bone marrow regulate and almost comprise all tracts from the body balancing.
2. tested pharmaceutical intervention Ana-1 cell produces the impact (seeing Table 7) of IL-6
The impact of the tested pharmaceutical intervention Ana-1 cell generation of table 7. IL-6 (the OD value/A)
In the table 7, * represents P＜0.05, and significant difference is arranged; * represents P＜0.01, and significant difference is arranged.
As shown in table 7, described tested medicine is 3.125～400
All can significantly strengthen the IL-6 secretion level of mouse spleen lymphocyte in the/ml concentration range, be obvious dose dependent in high concentration range, especially work as concentration 200
Facilitation is the most outstanding during/ml.The lymphokine that the T cell that IL-6 can activate and fibroblast produce can make B cell precursor become the cell that produces antibody; Energy and colony stimulating factor are collaborative, growth and the differentiation of the original bone marrow-derived cells of promotion, strengthen the cracking function of natural killer cell.Therefore as seen, the Chinese medicine preparation for preparing with Eupolyphaga seu steleophaga extract has the transfer immune response, drives the propagation of original bone marrow-derived cells and NK cell and the effect of effect.
3. tested medicine produces TNF-to the Ana-1 cell
Impact (seeing Table 8)
The tested medicine of table 8. produces TNF-to the Ana-1 cell
Impact (OD value/A)
In the table 8, * represents P＜0.05, and significant difference is arranged; * represents P＜0.01, and significant difference is arranged.
As shown in table 8, described tested medicine is 3.125～400
All can be to the TNF-of mouse spleen lymphocyte in the/ml concentration range
Secretion level has Pasitive Regulation Effect of Genseng, and is obvious dose dependent, wherein, when concentration 200
Facilitation reaches the strongest during/ml.TNF-
Be a kind of can the direct killing tumor cell and to the cytokine of normal cell without overt toxicity, be one of the strongest bioactie agent of the direct killing function of tumor found up to now, its participates in immune response and inflammatory reaction.Therefore as seen, with Chinese medicine preparation adjustable motivation body immunne response, enhancing human body immunity power in the high concentration situation of Eupolyphaga seu steleophaga extract preparation, can more effectively treat tumor and rheumatic osteopathia.
Claims (4)
1. a method for preparing Chinese medicine preparation with Eupolyphaga seu steleophaga extract is characterized in that, comprises following steps:
(1) preparation Eupolyphaga Seu Steleophaga extracting solution
At ambient temperature, the ratio of 1:10 takes by weighing Eupolyphaga Seu Steleophaga and the deionized water through concocting in mass ratio, then described Eupolyphaga Seu Steleophaga is put under the deionized water room temperature lixiviate 1 hour, and ultrasonication is 40 minutes after the lixiviate, then use the gauze coarse filtration, obtain the Eupolyphaga Seu Steleophaga extracting solution;
(2) use the ultrafilter membrane ultrafiltration
The Eupolyphaga Seu Steleophaga extracting solution that step (1) obtains is at room temperature used the ultrafilter membrane ultrafiltration, and molecular cut off is greater than 30000, will through ultrafilter membrane, molecular weight saves backup less than 30000 ultrafiltrate;
(3) filter with NF membrane
The ultrafiltrate that step (2) is obtained at room temperature filters with NF membrane, and molecular cut off is got molecular weight for subsequent use greater than 300 concentrated solution greater than 300;
(4) molecular weight that step (3) is obtained is made lyophilized powder greater than the aseptic lyophilization of 300 concentrated solution;
(5) lyophilized powder that step (4) is obtained is made capsule and injectable powder, obtains target product---the Chinese medicine preparation made from Eupolyphaga seu steleophaga extract.
2. treating rheumatic osteopathia, treating the application in the oncosis with the Chinese medicine preparation with the Eupolyphaga seu steleophaga extract preparation of preparation method preparation claimed in claim 1.
3. the application of Chinese medicine preparation in improving body immunity, balance the body cardiovascular system activity with the Eupolyphaga seu steleophaga extract preparation for preparing with preparation method claimed in claim 1.
4. repairing the damaged cartilage cell, regulating the application in the purine metabolism with the Chinese medicine preparation with the Eupolyphaga seu steleophaga extract preparation of preparation method preparation claimed in claim 1.
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