CN103301161B - The method of Chinese medicine preparation and the application of this Chinese medicine preparation is prepared with Eupolyphaga seu steleophaga extract - Google Patents
The method of Chinese medicine preparation and the application of this Chinese medicine preparation is prepared with Eupolyphaga seu steleophaga extract Download PDFInfo
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Abstract
The present invention records according to Chinese medicine, the method preparing Chinese medicine preparation with Eupolyphaga seu steleophaga extract studied in conjunction with modern technologies, and its step comprises: (1) prepare Eupolyphaga Seu Steleophaga extracting solution; (2) use ultrafilter membrane ultrafiltration; (3) use nanofiltration membrane; (4) lyophilized powder is made in sterile cryo drying; (5) make capsule and injectable powder.That prepares by method of the present invention not only has with Chinese medicine preparation prepared by Eupolyphaga seu steleophaga extract the removing blood stasis that Traditional Chinese Medicine facts have proved, effect of reunion of fractured tendons and bones, and there is the effect improving purine metabolism, immunity moderation systemic-function and strengthen osseous tissue rehabilitation and anti-tumor necrosis factor; In addition also have antithrombus formation and thrombolytic effect, atherosclerosis being had to the effect delayed, is the Chinese medicine preparation of a kind of ideal treatment rheumatic ostalgia, antitumor and raising immunity.
Description
Technical field
The present invention relates to technical field of Chinese medicines, specifically, is a kind of method that Eupolyphaga seu steleophaga extract prepares Chinese medicine preparation, and Chinese medicine preparation prepared by described Eupolyphaga seu steleophaga extract can be applied to the fields such as treatment rheumatic osteopathia, antitumor, enhancing body immunity.
Background technology
Rheumatic osteopathia belongs to arthromyodynia in theory of Chinese medical science, and numbness, namely numbness and numbness hinder obstructed meaning.After human body flesh table grain is attacked by exopathogen, QI and blood can not be unobstructed, and the places such as the muscle BJM thus caused are painful, and class illness such as severe one numbness, joint stuffiness, are all called arthromyodynia.Arthromyodynia sees the earliest " Plain Questions numbness opinion ": " so-called fraud person ... reinfection is in the wet gas of wind and cold also ", wherein, rheumatism cold-evil is the main cause causing arthromyodynia.In " Medical Treasures of the Golden Chamber ", claim this disease to be " severe and migratory arthralgia ".The wind heat disease, gout, rheumatic arthritis, rheumatoid arthritis, lumbar muscle strain etc. of modern medicine 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 lays particular stress on by heresy, and body negative and positive have inclined Sheng; Therefore disease performance and inconsistent.As windage yaw Sheng, arthralgia migration person become migratory arthralgia; Wet Sheng, the heavy pain person in joint partially claim damp arthralgia; Cold Sheng, joint cold type of pain person partially claim arthralgia aggravated by cold; Heat is contained partially, burning in joint, pain person is called pyretic arthralgia.The treatment of arthromyodynia, originally mainly with bloodstasis, sick product of then joining 5 bone and muscle strengthening, invigorating the liver and kidney for a long time.Theory of Chinese medical science is thought, this demonstrate,proves the osteopathia caused is because of insufficiency of vital energy and blood, battalion Wei Bugu, caused by rheumatism cold-evil often takes advantage of a weak point more.Excessive QI has inclined Sheng, therefore is divided into above four large classes.Invigorating QI and blood or nourishing the liver and kidney should be noted in treatment.Broken silt is reduced phlegm, insects are searched to pick and all can use with disease.Meanwhile, the lumbago that in traditional Chinese medical science category, the cause of disease such as stagnation of cold-damp, damp and hot obstruct, qi depression to blood stasis causes, the said rheumatism of modern medicine, rheumatoid disease, lumbar muscle strain, hyperosteogeny etc. all can be caused by arthromyodynia.
Current treatment arthromyodynia and lumbago (doctor trained in Western medicine is referred to as " rheumatic osteopathia ") mostly are carries out symptomatic treatment with Western medicine.The chemical classes medicine of Western medicine in a short time therapeutic effect is obvious, but can have side effects after somebody takes sometimes, treatment is also difficult to remove old complaint, in the treatment or there will be state of an illness bounce-back or recurrence after treatment.If by Chinese traditional treatment arthromyodynia and lumbago, Chinese medicine is mainly adopted to treat.Theory of Chinese medical science is thought, 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 passes through through cave the treatment more options of arthromyodynia, the relevant approach of internal organs, the medicine that human body multisystem, multiple organ, too many levels adjust is treated, mainly indirectly treat arthromyodynia and rheumatic osteopathia by adjustment human organ function and body's immunity, thus, the treatment of the traditional Chinese medical science is fast not as doctor trained in Western medicine and obvious, but the side effect of Chinese medicine is less, the chemical reaction taking Chinese medicine descendant can be less.
In addition, the method that modern medicine (doctor trained in Western medicine) treats tumor has varied, comprises the methods such as operative treatment, radiotherapy, chemotherapy and therapy of combining Chinese and Western medicine.Benign tumor can select the method for excising to treat, but, the side of excision can not reach the object of thoroughly treatment and control for malignant tumor, therefore, chemotherapy to be carried out selectively after excision (malignant tumor), but chemotherapy also can damage the normal cell of people and body tissue while killing and wounding in cancerous cell process.So, there is now the method for therapy of combining Chinese and Western medicine tumor.And Chinese medicine to the control of tumor mainly by what the basis that regulates the mechanism of human body was carried out, little to the direct injury of human body, the toxic and side effects of medicine is little, has obvious curative effect equally to somebody and the disease that has, is easy to be accepted by patient.Chinese medicine is thought, the disease of tumor is cited as qi stagnation blood stasis expectorant and coagulates, and ancient medicine is blamed in empty, strongly fragrant, expectorant, the stasis of blood four end more.The most the underlying cause of Carcinogenesis is that sickened body had the cause of disease such as disorder of YIN and YANG, zang-fu disharmony before canceration, causes pathogen to be invaded with the long-time stimulus of the factor such as lightweight, diet again, and healthy energy virtual loss, finally causes canceration.Therefore in history, the traditional Chinese medical science is finding the method and Chinese medicine that can treat each disease of arthromyodynia always.
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 embodies positive effect as insects medical material in the rheumatic osteopathia that causes for the treatment of arthromyodynia.Modern medicine study also confirms, Eupolyphaga Seu Steleophaga contains the inorganic elements such as aminoacid and potassium, calcium, magnesium, zinc, phosphorus of needed by human.But the traditional method of the traditional Chinese medical science seems to reach optimum state to the utilization of Eupolyphaga Seu Steleophaga as insects medical material.It is found that in the research of Chinese medicine: utilize modern technologies can promote the development of Chinese medicine; The therapeutic effect of some Chinese medicines can reach a new high by applicating modern times technology.Eupolyphaga Seu Steleophaga, as one of traditional insects medical material of Chinese medicine, utilizes the applied research of modern technologies to it current or inadequate.
Summary of the invention
The object of the invention is to, according to the record of " Eupolyphaga Seu Steleophaga can treat the rheumatic osteopathia that arthromyodynia causes " in theory of Chinese medical science and Chinese medicine, provide a kind of Eupolyphaga seu steleophaga extract to prepare the method for Chinese medicine preparation in conjunction with modern technologies; The second object of the present invention is, the Chinese medicine preparation of preparation is applied to scope that is wider, that comprise treatment rheumatic osteopathia, antitumor and enhancing body immunity.
For achieving the above object, the technical solution used in the present invention is as follows.
Eupolyphaga seu steleophaga extract prepares a method for Chinese medicine preparation, it is characterized in that, comprises following steps:
(1) Eupolyphaga Seu Steleophaga extracting solution is prepared
At ambient temperature, in mass ratio the ratio of 1:10 take through process of preparing Chinese medicine Eupolyphaga Seu Steleophaga and deionized water (through process of preparing Chinese medicine Eupolyphaga Seu Steleophaga be the words of 10g, deionized water is 100ml), then described Eupolyphaga Seu Steleophaga to be put under deionized water room temperature lixiviate 1 hour, ultrasonication 40 minutes after lixiviate, then use gauze coarse filtration, obtain Eupolyphaga Seu Steleophaga extracting solution;
(2) ultrafilter membrane ultrafiltration is used
The Eupolyphaga Seu Steleophaga extracting solution that step (1) obtains at room temperature is used ultrafilter membrane ultrafiltration, and molecular cut off is greater than 30000, by through ultrafilter membrane, ultrafiltrate that molecular weight is less than 30000 saves backup;
(3) nanofiltration membrane is used
The ultrafiltrate that step (2) obtains at room temperature is used nanofiltration membrane, and molecular cut off is greater than 300, get molecular weight be greater than 300 concentrated solution for subsequent use;
(4) molecular weight that step (3) obtains is greater than the concentrated solution sterile cryo drying of 300, makes lyophilized powder;
(5) lyophilized powder that step (4) obtains is made capsule and injectable powder, obtain target product---the Chinese medicine preparation made with Eupolyphaga seu steleophaga extract.
For realizing above-mentioned second object, the technical solution used in the present invention is as follows.
The application of the Chinese medicine preparation prepared with Eupolyphaga seu steleophaga extract in treatment rheumatic osteopathia, treatment oncosis.
The Chinese medicine preparation prepared with Eupolyphaga seu steleophaga extract is improving the application in body immunity, adjustment cardiovascular system of human body activity.
The Chinese medicine preparation prepared with Eupolyphaga seu steleophaga extract is repairing the application in damaged cartilage cell, adjustment purine metabolism.
Good effect of the present invention is:
(1) for prevention and the treatment of cold-dampness type arthromyodynia, tumor, a kind of new Chinese medicine preparation is made by modern means Eupolyphaga seu steleophaga extract.
(2) situation in conjunction with modern technologies analysis and various cell experiment is recorded from Chinese medicine, Chinese medicine preparation prepared by Eupolyphaga seu steleophaga extract of the present invention not only has the removing blood stasis that Traditional Chinese Medicine facts have proved, effect of reunion of fractured tendons and bones, and there is the effect improving purine metabolism, immunity moderation systemic-function and strengthen osseous tissue rehabilitation and anti-tumor necrosis factor; In addition, cell experiment also demonstrates Eupolyphaga seu steleophaga extract antithrombus formation and thrombolytic effect, atherosclerosis is had to the effect delayed.
(3) the Chinese medicine preparation medicinal effects prepared with Eupolyphaga seu steleophaga extract adopting preparation method of the present invention to obtain is remarkable, have no side effect, take or treatment use convenience, be the Chinese medicine preparation of a kind of ideal treatment rheumatic ostalgia, antitumor and raising immunity, can extensive use be supplied.
(4) preparation method of the present invention, preparation technology simply, easily operate enforcement.
Accompanying drawing explanation
Fig. 1 is the uv-spectrogram of Chinese medicine preparation prepared by method that the present invention's Eupolyphaga seu steleophaga extract prepares Chinese medicine preparation.
Detailed description of the invention
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) Eupolyphaga Seu Steleophaga extracting solution is prepared
At ambient temperature, in mass ratio the ratio of 1:10 take through process of preparing Chinese medicine Eupolyphaga Seu Steleophaga and deionized water, the present embodiment takes 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 40 minutes after lixiviate, then use gauze coarse filtration, obtain Eupolyphaga Seu Steleophaga extracting solution.
(2) ultrafilter membrane ultrafiltration is used
The Eupolyphaga Seu Steleophaga extracting solution that step (1) obtains at room temperature is used ultrafilter membrane ultrafiltration, and molecular cut off is greater than 30000, is saved backup by the ultrafiltrate (molecular weight is less than 30000) through ultrafilter membrane.
(3) nanofiltration membrane is used
The ultrafiltrate that step (2) obtains at room temperature is used nanofiltration membrane, and molecular cut off is greater than 300, get molecular weight be greater than 300 concentrated solution for subsequent use.
(4) molecular weight that step (3) obtains is greater than the concentrated solution sterile cryo drying of 300, makes lyophilized powder.
(5) lyophilized powder that step (4) obtains is made capsule and injectable powder, obtain target product---the Chinese medicine preparation made with Eupolyphaga seu steleophaga extract.
Through ultraviolet spectrophotometer analysis, Chinese medicine preparation prepared by the method that the present invention's Eupolyphaga seu steleophaga extract prepares Chinese medicine preparation has maximum absorption peak (see Fig. 1) near 290nm.
Below introduce the situation to the Chinese medicine preparation experiment Analysis prepared with method Eupolyphaga seu steleophaga extract of the present invention, by 5 cell experiments and zoopery, the function to described Chinese medicine preparation is analyzed, checks and is verified.
pharmacological evaluation 1
The Chinese medicine preparation prepared with Eupolyphaga seu steleophaga extract is used for 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) test medicine: Chinese medicine preparation prepared by described Eupolyphaga seu steleophaga extract is dissolved in DMEM(note: a kind of culture medium containing each seed amino acid and glucose) solution of gained.
(2) subject cell: Human umbilical vein endothelial cells.
(3) described test medicine is observed on the impact of Human umbilical vein endothelial cells release NO and the situation of cell proliferation after adding described test medicine, facilitation can be produced for evaluation criterion to the release of the NO of Human umbilical vein endothelial cells with described test medicine, determine that can the Chinese medicine preparation prepared with Eupolyphaga seu steleophaga extract promote that vasodilation is to reach the distribution regulating blood pressure and blood flow, the formation of anti-tampon.
(4) experimentation
Cultivate Human umbilical vein endothelial cells, dissolve by DMEM culture medium, configure Chinese medicine preparation of the present invention, described Human umbilical vein endothelial cells (adopts 800,400,200,100,50,25,12.5,6.25 through described test medicine process 48 ~ 72 hours
/ ml eight concentration, a negative control medicine and a positive control drug, three wells); Then cell conditioned medium liquid is got, by the propagation of CCK8 kit detection cell; The release of NO is detected with 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 concentration (100
extension rate (1) before) × sample test.
(5) experimental result
The Chinese medicine preparation made with Eupolyphaga seu steleophaga extract is on the impact (see 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. the Chinese medicine preparation prepared with Eupolyphaga seu steleophaga extract is 6.25
demonstrate maximum proliferative amount under/mL concentration and higher than blank group, this illustrates, the facilitation increased Human umbilical vein endothelial cells under this concentration is the strongest.
2., in the result of NO secretion, there is with Chinese medicine preparation prepared by Eupolyphaga seu steleophaga extract the effect of stronger promotion production by human umbilical vessel endothelial cells NO.The NO produced due to vascular endothelial cell is passed to vascular smooth muscle cell rapidly by cell membrane, makes smooth muscle loosening. and arteries is expanded, thus regulates blood pressure and blood distribution.Endogenous NO regulates vascular endothelial growth, triggers vaso-active substance, promotes angiogenic growth and regeneration.The NO that vascular endothelial cell produces all has the effect keeping vascular endothelial cell integrity under physiology, pathologic condition.NO can anticoagulant and adhesion, reduces leukocytic gathering, the formation of anti-tampon; Can also lymphopoiesis be suppressed, 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 content of NO increases Chinese medicine preparation prepared by explanation Eupolyphaga seu steleophaga extract improving in Cardiovascular and serves useful effect in pharmacological evaluation 1.The concept of the traditional Chinese medical science is the effect playing blood circulation promoting and blood stasis dispelling.
pharmacological evaluation 2
The cell experiment of purine metabolism inhibit activities: Chinese medicine preparation prepared by understanding Eupolyphaga seu steleophaga extract is to the inhibitory action of xanthine oxidase.
(1) test medicine: the Chinese medicine preparation prepared with Eupolyphaga seu steleophaga extract is dissolved in PBS(phosphate buffer) solution that obtains.
(2) experimental technique
Xanthine oxidase (Xanthine Oxidase claims simple XOD) is a kind of important enzyme in nucleic acid in vivo metabolism, and energy catalysis xanthine and hypoxanthine oxidation generate uric acid, produce peroxide radical simultaneously.Therefore, suppress the activity of xanthine oxidase, both can reduce the formation of uric acid in body, the tissue injury that free radical causes can be reduced again.
Carry out the mensuration of described test medicine to the suppression ratio of xanthine oxidase.Xanthine concentration is 100mg/L, and xanthine oxidase concentration is 50U/L, and the solution concentration of the Chinese medicine preparation prepared with Eupolyphaga seu steleophaga extract that pharmacological evaluation 2 configures is 10,50,90,150,300,450
/ mL.2.0ml xanthine solution is added in 10mL plastic centrifuge tube, 25 DEG C of pre-temperature 10min, add PBS buffer solution 0.1ml, add the xanthine oxidase solution 2.0ml through 25 DEG C of pre-temperature again, measure the absorbance of 290nm place different time points, within every 30 seconds, measure 1 time, METHOD FOR CONTINUOUS DETERMINATION 450 seconds, by time and absorbance data by MATLAB process, calculate enzyme activity VXOD.
In 10mL plastic centrifuge tube, add 2.0mL xanthine solution, 25 DEG C of pre-temperature 10min, add PBS buffer solution 2.1mL, every 30s measures the absorbance at 290nm place, and METHOD FOR CONTINUOUS DETERMINATION 450s, calculates blank
.
2.0mL substrate solution is added in 10mL plastic centrifuge tube, 25 DEG C of pre-temperature 10min, add testing sample 0.1mL, add the xanthine oxidase solution 2.0mL through 25 DEG C of pre-temperature again, every 30s measures the absorbance of 290nm, METHOD FOR CONTINUOUS DETERMINATION 450s, calculates the maximum reaction rate Vs of xanthine enzyme under sample effect.
The computational methods of its suppression ratio are: suppression ratio
(2).
(3) experimental result
The Chinese medicine preparation prepared with Eupolyphaga seu steleophaga extract is to the suppression ratio (see table 2) of xanthine enzyme:
The Chinese medicine preparation that table 2. is prepared with Eupolyphaga seu steleophaga extract is to the suppression ratio of xanthine enzyme
。
Through 3 repeated trials, the result of table 2 is shown as: be 77.04% to the maximal percentage inhibition of xanthine oxidase, and the suppression ratio of other groups more parallel is maximum.
(4) result of pharmacological evaluation 2 shows
The Chinese medicine preparation prepared with Eupolyphaga seu steleophaga extract can suppress the metabolism of xanthine oxidase, reduces the generation of uric acid, also has the mediation effect of Chinese medicine, invigorating the spleen and replenishing QI simultaneously, and emergency is eased pain.
Pharmacological evaluation 2 demonstrates on the basis of modern medicine: the pain of the Chinese medicine preparation prepared with Eupolyphaga seu steleophaga extract by regulating xanthine metabolism to alleviate rheumatism arthritis.
pharmacological evaluation 3
Zoopery to rheumatic osteopathia therapeutical effect: Chinese medicine preparation prepared by understanding Eupolyphaga seu steleophaga extract is to IL-1
the impact of the rat cartilage cell propagation of induction.
(1) test medicine: the Chinese medicine preparation prepared with Eupolyphaga seu steleophaga extract.
(2) animal subject: SD rat, 6,7 ages in days, male and female half and half; SD rat is purchased from Shanghai western 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 to put to death, being soaked in alcohol content is take out after several minutes in the ethanol of 95%, take out femoral head, shoulder joint and knee cartilage tissue, use PBS(phosphate buffer) rinse for several times, add a small amount of PBS to shred, with the II Collagenase Type 37 DEG C digestion of 0.2%, pipette supernatant and cultivate in culture dish as individual cells suspension inoculation, change liquid every other day.Secondary Culture after cell covers with substantially, when reaching 2nd generation, adjustment 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 by culture hole after inoculating 24 h
matched group, given the test agent group, positive controls, the multiple hole of every sample 3, change corresponding culture fluid respectively:
1. blank group: change DMEM (containing 5% FBS, 0.02% tween 80);
2. model group: change DMEM (containing 10
the IL-1 β of/L, 0.02% tween 80 and 5% FBS);
3. test medicine and positive controls: change DMEM (containing 10
the IL-1 of/L
, 0.02% tween 80,5% FBS and each acute drug or positive drug);
Each group is put into incubator continuation cultivation 48h and be used for 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 measures the optical density value (OD value) in every hole, and is converted into the rate of increase (formula 1), analyze given the test agent proliferation under within 3 hours after administration, being placed in microplate reader 450 nm wavelength.
The rate of increase (%)=
(3).
(4) experimental result
Test medicine is to IL-1
the proliferative effect (see table 2) of the rat osteoarthritis chondrocytes of induction:
Table 3. test medicine is to IL-1
the proliferative effect (O.D.) of the rat osteoarthritis chondrocytes of induction
；
In table 3, * represents P<0.05, has significant difference; * represents P<0.01, has significant difference.
(4) result of pharmacological evaluation 3 shows
From table 3, model group is adding IL-1
after, OD value compared with normal group obviously reduces, and cell quantity significantly reduces, and this phenomenon illustrates IL-1
there is presenility in existing effect inducing cell, the success of rat bone articular degeneration model construction.Meanwhile, test medicine group is at (0.08-9.77
/ ml) all demonstrate obvious cel l proliferation in concentration range, and in dose dependent, and be 9.77 in concentration
during/mL, effect is the most obvious: the rate of increase is 113.071%, reaches its maximum drug effect.It can thus be appreciated that the Chinese medicine preparation prepared with Eupolyphaga seu steleophaga extract effectively can treat osteoarthritis, the injury that the propagation of chondrocyte causes joint to alleviate degenerative osteoarthritis effectively can be promoted.
pharmacological evaluation 4
Zoopery on immunoregulation effect: Chinese medicine preparation prepared by understanding Eupolyphaga seu steleophaga extract is on the impact of mouse spleen lymphocyte immunologic function.
(1) test medicine: the solution Chinese medicine preparation prepared with Eupolyphaga seu steleophaga extract being dissolved in DMEM gained.
(2) animal subject: C57 mice, 10,6-8 week age, male and female half and half; C57 mice is purchased from Laboratory Animal Resource center, Shanghai (credit number: SCXK (Shanghai) 2008-0016).
(3) experimental technique
Break test mice neck, asepticly gets spleen, prepares splenocyte suspension after grinding with filter screen, ensures primary cell activity more than 95%, adjustment cell concentration to 3 × 10
6individual/mL; Add ConA and induce spleen lymphocyte proliferation, through test medicine, (concentration is (3.125,6.25,12.5 25,50,100,200,400 to cell
– 1eight concentration, 3 hole parallel laboratory tests) process, measure IFN-in cells and supernatant by ELISA method after cultivating 72h
content.
(4) experimental result
1. test medicine is to mouse boosting cell IFN-
the impact (see table 4) of secretion
Table 4. test medicine is to mouse boosting cell IFN-
the impact (O.D.) of secretion
；
In table 4, * represents P<0.05, has significant difference; * represents P<0.01, has significant difference.
As shown in table 4, described test medicine is 3.125 ~ 400
all can to the IFN-of mouse spleen lymphocyte in/ml concentration range
secretion level has Pasitive Regulation Effect of Genseng, and in obvious dose dependent.Wherein, when concentration is 400
during/ml, facilitation reaches the strongest.Chinese medicine preparation adjustable dynamic immune response in high concentration situation prepared by this result display Eupolyphaga seu steleophaga extract, regulates lymphocytic Proliferation and differentiation to reach the object of effecting a permanent cure with enhancing human body immunity effect.Meanwhile, the state (see table 5) of cell has been investigated in this experiment with CCK-8 test
Table 5. test medicine is on the impact (O.D.) of mouse spleen cell proliferation
；
In table 5, * represents P<0.05, has significant difference; * represents P<0.01, has significant difference.
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: Chinese medicine preparation prepared by understanding Eupolyphaga seu steleophaga extract is on mice Ana-1 macrophage immunity function, antineoplastic impact.
(1) test medicine: the solution Chinese medicine preparation prepared with Eupolyphaga seu steleophaga extract being dissolved in DMEM gained.
(2) subject cell: Ana-1 mouse macrophage.
(3) experimental technique
1. by Ana-1 mouse macrophage at 37 DEG C, 5%CO
2under condition, with containing 10% calf serum, penicillin (1 × 10
5uL
-1) and streptomycin (100 mgL
-1) DMEM culture fluid carry out Secondary Culture.Experiment cell is all in exponential phase.
2. get the cell concentration of needs, add the fresh complete medium re-suspended cell needing 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 DEG C, 5% CO
2cultivate.
3. before dosing, discard old culture fluid, wash 3 times with fresh medium, add fresh drug incubation 30min to be measured, with enzyme-linked method working sample IL-1
, IL-6 and TNF-
content, often organizes repetition 3 times, adds CCK8 reagent simultaneously, detects cell viability state.
(4) experimental result
1. test medicine is to IL-1
the proliferative effect (see table 6) of cell
Table 6. test medicine is to IL-1
the proliferative effect (OD value/A) of cell
；
In table 6, * represents P<0.05, has significant difference; * represents P<0.01, has significant difference.
As shown in table 6, described test medicine is 50 ~ 400
the IL-1 of mouse spleen lymphocyte all significantly can be strengthened in/ml concentration range
secretion level is obvious dose dependent in high concentration range, especially when concentration is 400
during/ml, facilitation is the most outstanding.IL-1
it is the most important cytokine stimulating T cell, B cell and NK cell activation, propagation and differentiation, produce primarily of macrophage, it has biologic activity very widely, and the inflammation and immune defence reaction, the hemopoietic function of bone marrow that participate in body regulate and almost comprise the autologous balancing of all tracts.
2. test medicine intervenes the impact (see table 7) that Ana-1 cell produces IL-6
Table 7. test medicine intervenes the impact (OD value/A) that Ana-1 cell produces IL-6
；
In table 7, * represents P<0.05, has significant difference; * represents P<0.01, has significant difference.
As shown in table 7, described test medicine is 3.125 ~ 400
all significantly can strengthen the IL-6 secretion level of mouse spleen lymphocyte in/ml concentration range, be obvious dose dependent in high concentration range, especially when concentration is 200
during/ml, facilitation is the most outstanding.The T cell that IL-6 can activate and the lymphokine that fibroblast produces, can make B cell precursor become the cell producing antibody; The growth and differ entiation of original bone marrow-derived cells is worked in coordination with, promoted to energy and colony stimulating factor, strengthens the cracking function of natural killer cell.Therefore visible, with Chinese medicine preparation prepared by Eupolyphaga seu steleophaga extract, there is transfer immune response, drive the propagation of original bone marrow-derived cells and NK cell and effect of effect.
3. test medicine produces TNF-to Ana-1 cell
impact (see table 8)
Table 8. test medicine produces TNF-to Ana-1 cell
impact (OD value/A)
；
In table 8, * represents P<0.05, has significant difference; * represents P<0.01, has significant difference.
As shown in table 8, described test medicine is 3.125 ~ 400
all can to the TNF-of mouse spleen lymphocyte in/ml concentration range
secretion level has Pasitive Regulation Effect of Genseng, and in obvious dose dependent, wherein, when concentration is 200
during/ml, facilitation reaches the strongest.TNF-
be a kind of can direct killing tumor cell and to the cytokine of normal cell without overt toxicity, be one of bioactie agent that found up to now direct killing function of tumor is the strongest, its participates in immune response and inflammatory reaction.Therefore visible, the Chinese medicine preparation prepared with Eupolyphaga seu steleophaga extract is adjustable dynamic immune response, enhancing human body immunity power in high concentration situation, can more effectively treat tumor and rheumatic osteopathia.
Claims (1)
1., with the application of Chinese medicine preparation in preparation suppression xanthine oxidase metabolic drug prepared by Eupolyphaga seu steleophaga extract, the preparation process of described Chinese medicine preparation is:
(1) Eupolyphaga Seu Steleophaga extracting solution is prepared
At ambient temperature, the ratio of 1:10 takes Eupolyphaga Seu Steleophaga through concocting and deionized water in mass ratio, then described Eupolyphaga Seu Steleophaga to be put under deionized water room temperature lixiviate 1 hour, ultrasonication 40 minutes after lixiviate, then use gauze coarse filtration, obtain Eupolyphaga Seu Steleophaga extracting solution;
(2) ultrafilter membrane ultrafiltration is used
The Eupolyphaga Seu Steleophaga extracting solution that step (1) obtains at room temperature is used ultrafilter membrane ultrafiltration, and molecular cut off is greater than 30000, by through ultrafilter membrane, ultrafiltrate that molecular weight is less than 30000 saves backup;
(3) nanofiltration membrane is used
The ultrafiltrate that step (2) obtains at room temperature is used nanofiltration membrane, and molecular cut off is greater than 300, get molecular weight be greater than 300 concentrated solution for subsequent use;
(4) molecular weight that step (3) obtains is greater than the concentrated solution sterile cryo drying of 300, makes lyophilized powder;
(5) lyophilized powder that step (4) obtains is made capsule and injectable powder, obtain target product---the Chinese medicine preparation made with Eupolyphaga seu steleophaga extract.
Priority Applications (1)
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|---|---|---|---|
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| CN1965897A (en) * | 2006-11-24 | 2007-05-23 | 江苏天照药业有限公司 | Chinese medicinal composition for resisting hepatic fibrosis and method for preparing same |
| EP2324841A1 (en) * | 2008-07-29 | 2011-05-25 | Hebei Yiling Medicine Research Institute Co., Ltd. | Use of a traditional chinese medicinal composition for preparing medicine for promoting bone marrow-derived mesenchymal stem cell survival in vivo and cardiac muscle differentiation |
| CN102188481A (en) * | 2010-03-16 | 2011-09-21 | 博安兄弟制药（中国）有限公司 | Preparation method of particles for supplementing qi and activating blood circulation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1965897A (en) * | 2006-11-24 | 2007-05-23 | 江苏天照药业有限公司 | Chinese medicinal composition for resisting hepatic fibrosis and method for preparing same |
| EP2324841A1 (en) * | 2008-07-29 | 2011-05-25 | Hebei Yiling Medicine Research Institute Co., Ltd. | Use of a traditional chinese medicinal composition for preparing medicine for promoting bone marrow-derived mesenchymal stem cell survival in vivo and cardiac muscle differentiation |
| CN102188481A (en) * | 2010-03-16 | 2011-09-21 | 博安兄弟制药（中国）有限公司 | Preparation method of particles for supplementing qi and activating blood circulation |
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| 土鳖虫最佳提取部位及有效分子量确定;殷书梅等;《中药材》;20020228;第25卷(第2期);第126页右栏第1段 * |
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