Summary of the invention
The present invention is on the basis of research ancient prescription, and abundant, the low toxin of performance Chinese medicine ingredients through screening and system test research, provides a kind of Radix Angelicae Sinensis, Radix Hedysari ultrafiltration membrane extract.
Another object of the present invention is to provide the method for Radix Angelicae Sinensis, Radix Hedysari ultrafiltration membrane extract.
A further object of the present invention is that Radix Angelicae Sinensis, Radix Hedysari ultrafiltration membrane extract are used for the application of radioprotective harm protection.
The objective of the invention is to be achieved through the following technical solutions:
A kind of Radix Angelicae Sinensis, Radix Hedysari ultrafiltration membrane extract is characterized in that Radix Angelicae Sinensis and Radix Hedysari component ratio are 1: 5~6: 6.
The method of Radix Angelicae Sinensis, Radix Hedysari ultrafiltration membrane extract the steps include:
Get Radix Angelicae Sinensis Radix Hedysari decoction pieces and decoct with water by a certain percentage secondary, add 6 times of water gagings of Radix Angelicae Sinensis and Radix Hedysari prepared slice quality for the first time and decoct 2h, add 4 times of water gagings of Radix Angelicae Sinensis and Radix Hedysari prepared slice quality for the second time and decoct 1h, merge the decocting liquid hold over night twice, filter decocting liquid, get filtrate; Filtrate is again through the ultrafilter membrane ultrafiltration, ultrafiltration pressure≤0.4Mpa, and temperature≤40 ℃, ultrafiltrate is spray-dried, makes dry powder.
Above-mentioned ultrafilter membrane is 10-2 ten thousand molecular weight ultrafilter membranes, and membrane material is polyether sulfone (PES), and 100,000 molecular weight membrane aperture are 0.05 μ m, and 50,000 molecular weight ultrafilter membrane apertures are 0.25 μ m, and 20,000 molecular weight ultrafilter membrane apertures are 0.01 μ m.
Radix Angelicae Sinensis, Radix Hedysari ultrafiltration membrane extract physicochemical property:
1. this product is pale yellow powder, and is soluble in water, is insoluble to ethanol, methanol.The easy moisture absorption, pH value 5-6.This product aqueous solution is aobvious purple under the 365nm uviol lamp.
2. this product aqueous solution adds 3 of 10% alpha-Naphthol alcoholic solution, after shaking up test tube is tilted, and adds concentrated sulphuric acid along test tube wall and purplish red colour circle occurs.
The beneficial effect of advantage of the present invention and generation:
1, the present invention is intended to bring into play Radix Angelicae Sinensis and has to enrich blood and invigorate blood circulation, menstruction regulating and pain relieving, function of relaxing bowel and Radix Hedysari invigorating QI to consolidate the body surface resistance, the diuresis poison holding, evacuation of pus, expelling pus and promoting granulation function and the two flavor advantages such as Chinese materia medica tocixities are low, and from the chemistry with pharmacology's index be that point of penetration is studied Different Proporations of Ingredients in Danggui Buxue Decoction and effectiveness thereof, form little compound recipe, its effect is mainly enriches blood and improves body's immunity, and immunity of organisms descends and hemogram problem on the low side after solving body and accepting irradiation.
2, adopt the ultrafilter membrane isolation technics to extract separation this compound recipe, farthest kept the active components such as polysaccharide, dispelled other useless compositions, improved the bioavailability of former medicine, kept to greatest extent former side's curative effect.
3, ultrafiltration membrane extract of the present invention adopts modern preparation technique to be prepared into capsule on the basis of extracting effective site, and take for user and provide convenience.
4, Radix Angelicae Sinensis, Radix Hedysari are large genuine medicinal materials of our province; aboundresources; the quality high-quality; animal experiment study of the present invention shows; Radix Angelicae Sinensis, Radix Hedysari ultrafiltration membrane extract have definite defencive function to radiation; this medicine is prepared into the health product that radiation had defencive function, can facilitates our province Chinese material medicine resource advantage to transform to economic advantages.
The specific embodiment
Below in conjunction with embodiment, technical solution of the present invention is further described again:
Embodiment 1
The Chinese crude drug that the present invention adopts is from Minxian County, gansu Province, Radix Angelicae Sinensis available from production base, Minxian County, Radix Hedysari all available from medical material station, Minxian County.
Get Radix Angelicae Sinensis Radix Hedysari decoction pieces and decoct with water secondary in 1: 5 ratio, add 6 times of water gagings of Radix Angelicae Sinensis Radix Hedysari prepared slice quality for the first time and decoct 2h, add 4 times of water gagings of Radix Angelicae Sinensis Radix Hedysari prepared slice quality for the second time and decoct 1h, merge the decocting liquid hold over night twice, filter decocting liquid, get filtrate; Through 100,000 molecular weight (molecular cut off is 100,000) ultrafilter membrane---polyether sulfone (PES) membrane ultrafiltration, ultrafiltration pressure is 0.3Mpa to filtrate again, and temperature is 35 ℃, and ultrafiltrate is spray-dried, makes dry powder.
Embodiment 2
In order to show the present invention to the effect of radioprotective harm protection, the present invention has carried out superoxide dismutase (SOD) activity experiment, serum hemolysin experiment in leukocyte counts experiment, Quantity of DNA determination experiment, mouse Bone marrow cells micronucleus experiment, the blood to animal.
1. experiment material
1.1 animal
The KM mice, the SPF level, male, body weight 20 ± 2g is provided by Gansu college of traditional Chinese medicine Experimental Animal Center, the certification of fitness number: SCXK (sweet) 2004-0006, Experimental Establishment use certificate number: SYXK (sweet) 2004-0006.The animal sub-cage rearing, the food solid feed, feedstuff is provided by Beijing section Australia laboratory animal feed corporation,Ltd that pulls together, drinking water freely, 20~25 ℃ of room temperatures, relative humidity 45%~52%.
1.2 medicine and preparation
Radix Angelicae Sinensis Radix Hedysari ultrafiltration membrane extract is respectively Radix Angelicae Sinensis Radix Hedysari (1: 5) ultrafiltration membrane extract group, Radix Angelicae Sinensis Radix Hedysari (2: 4) ultrafiltration membrane extract group, Radix Angelicae Sinensis Radix Hedysari (3: 3) ultrafiltration membrane extract group, every gram crude drug closes extract xeraphium 0.109g (0.109g/g), provided by Gansu college of traditional Chinese medicine scientific experiment center, Radix Angelicae Sinensis decoction for tonifying blood every consumption per day 36g crude drug of being grown up, become body weight for humans press 60kg calculating, then people's a consumption per day is 0.6g crude drug/kg (0.0656g medicated powder/kg).According to the direct scaling method of the clinical kg body weight dosage of man and animal multiple, the dose,equivalent of getting mice is 5 times of (3g crude drug/kg of human body recommended amounts, 0.328 medicated powder/kg) as low dose of, 10 times of (6g/kg, 0.656 medicated powder/kg) as in dosage, 20 times (12g/kg, 1.312 medicated powder/kg) as heavy dose of, the amount of the administration of gavage is 0.2ml/10gbw.
1.3 key instrument and reagent
Full-automatic blood counting instrument (U.S. Abbott Laboratories, model: CD1200); Clinac (BJ Medical Equipment Research Institute, model BD-6M); Flow cytometer (U.S. Couter, model EPICS XL); Electronic balance (Beijing Sartorius, model: BL121SS); Binocular optical microscope (Japan, Olympus company); Image analysis system (Beijing mighty force new century Development Co., Ltd, model WL-9000); Centrifugal precipitation mechanism (Shanghai medical analytical instrument product); Ultraviolet spectrophotometer (day island proper capital, model: UV-2401PC).Hemolysin (CD1200 of U.S. Abbott Laboratories, lot number 6260312); Diluent (three and the armarium company limited, lot number: 0612001); Methanol (Tianjin moral grace chemical reagent company limited, lot number: 20070712); Calf serum (Hangzhou Sijiqing Biological Engineering Material Co., Ltd., lot number: 071005); Giemsa dye liquor (the pH6.8PBS preparation is provided by Gansu basic courses department of college of traditional Chinese medicine biology laboratory); The SOD test kit; Hank ' s liquid (Beijing ancient cooking vessel state biotechnology Co., Ltd, lot number: 87P00138); Pentobarbital sodium (Solution on Chemical Reagents in Shanghai company of Chinese Medicine group, lot number: 20030816); EDTA-K
2Anticoagulant (Tianjin section close europeanized reagent development centre, lot number: 20080218)
2. experimental technique
2.1 model preparation
Adopt clinac (BJ Medical Equipment Research Institute, model BD-6M) the disposable total irradiation mice of low energy X ray modeling, skin spacing 100cm, wherein leukocyte counts experiment, Quantity of DNA determination experiment, mouse Bone marrow cells micronucleus experiment, the setting radiation dose is 5.0Gy/min; It is 8.0Gy/min that superoxide dismutase in the blood (SOD) activity experiment is set radiation dose; It is 3.0Gy/min that radiation dose is set in the serum hemolysin experiment.
2.2 animal grouping
2.21 the leukocyte counts experiment is divided into four groups at random, 15/group, be respectively model group, model group+Radix Angelicae Sinensis Radix Hedysari (1: 5) group, model group+Radix Angelicae Sinensis Radix Hedysari (2: 4) group, model group+Radix Angelicae Sinensis Radix Hedysari (3: 3) group (do not establish blank group, with the index before every group of radiation as blank).
2.22 superoxide dismutase (SOD) activity experiment, serum hemolysin experiment all are divided into five groups at random in Quantity of DNA determination experiment, mouse Bone marrow cells micronucleus experiment, the blood, 15/group, model group+Radix Angelicae Sinensis Radix Hedysari (1: 5) group, model group+Radix Angelicae Sinensis Radix Hedysari (2: 4) group, model group+Radix Angelicae Sinensis Radix Hedysari (3: 3) group
Blank group is only given normal saline, a model group not administration of modeling, and the medicine group gavages the corresponding dosage medicine every day, and concrete administration time is seen concrete experimental technique.
2.3 experimental result statistical method
Measurement data, adopt variance analysis, carry out first homogeneity test of variance according to the program of variance analysis before the variance analysis, the data of abnormal or heterogeneity of variance are carried out suitable variable conversion, after satisfying the neat requirement of normal state or variance, add up with the data after the conversion; If do not reach yet normal state or the neat purpose of variance after the variable conversion, use rank test instead and add up.For enumeration data, adopt X
2Check.
2.4 experimental procedure and result
2.4.1 peripheral leukocytes counting (comprising body weight change, relevant organ index and pathology infringement)
Except a model group not administration of modeling, the continuous gastric infusion of other administration group 30 days continued oral administration gavage to the 45 days after the irradiation.Adopted peripheral blood 20 μ L in rear the 3rd day, the 7th day and the 14th day four times respectively at pre-irradiation, irradiation, add diluent (three and the armarium company limited, lot number: 0612001) to 40ul, with full-automatic blood cell analysis instrument (U.S. Abbott Laboratories, model: CD1200) detect routine blood test, white mice is put to death in the rear cervical vertebra dislocation of weighing last day, gets spleen, thymus is weighed and calculate organ index, and experimental result sees Table 1 and table 2.
Table 1 is on the impact of linear accelerator low energy X ray irradiation murine interleukin (x ± SD)
Annotate: compare with model group
※P<0.05
※ ※P<0.01
※ ※ ※P<0.001
Table 2 is on the impact of linear accelerator low energy X ray irradiation mouse liver, thymus and index and spleen index (x ± SD)
Annotate: compare with model group
※P<0.05
※ ※P<0.01
※ ※ ※P<0.001
As can be seen from Table 1: 3d, 7d, 14d after the irradiation, the numeration of leukocyte of radiation model control group and pre-irradiation compare, and difference all has statistical significance (P<0.001), and the establishment of radiation damage model is described.3d after the irradiation, 7d, each ratio group numeration of leukocyte of the 14th Radix Angelicae Sinensis Radix Hedysari is apparently higher than the radiation model control group, and wherein Radix Angelicae Sinensis Radix Hedysari (1: 5) group compares with model group, and statistical significance is (P<0.001) significantly; 7d, 14d Radix Angelicae Sinensis Radix Hedysari (2: 4) group more also has significantly to difference with model group; 7d, 14d with blank group more also has statistical significance (P<0.05) at 3: 3.3d relatively has the trend of increasing behind the 7th day and each leukocytic quantity of ratio group mouse peripheral blood of 14d tested material and irradiation behind the irradiation, and the peripheral blood leucocyte that the prompting tested material causes radiation reduces protective action, and can promote its quantity to recover.
Table 2 is the result show, all decline (P<0.001) of the blank group of the Thymus and spleen index of mice behind the irradiation, wherein Radix Angelicae Sinensis Radix Hedysari (1: 5) group can improve the index of two internal organs comparatively significantly, relatively has significant difference (P<0.05) with model group.
2.4.2 the impact on Quantity of DNA
Except a model group not administration of modeling, blank group give with normal saline outside, the continuous gastric infusion of other administration group 30 days, continued oral administration gavage to the 33 days after the irradiation, about 2h after i.e. administration in the 33rd day in the 3rd day after the irradiation, white mice is put to death in the cervical vertebra dislocation, separate femur, draw Hank ' s liquid (Beijing ancient cooking vessel state biotechnology Co., Ltd, the lot number: 87P00138), go out the whole medullary cells in the femur of certain volume with 1mL syringe (No. 5 syringe needles); At last, allow cell suspension pass through the syringe of No. 4 syringe needles, cell is fully disperseed in suspension.Detect dna content (iodate pyridine method) with flow cytometer (U.S. Couter, model EPICS XL), the results are shown in Table 3.
Table 3 is on the impact of linear accelerator low energy X ray irradiation bone marrow cells in mice dna content (x ± SD)
Annotate: compare with model group
※P<0.05
※ ※P<0.01
※ ※ ※P<0.001;
Learn from table 3: the blank group of bone marrow cells in mice dna content all descends (P<0.001) behind the irradiation, and wherein Radix Angelicae Sinensis Radix Hedysari (1: 5) can improve its content comparatively significantly, relatively has significant difference (P<0.05) with model group.
2.4.3 the impact of mouse Bone marrow cells micronucleus number
Except a model group not administration of modeling, blank group give with normal saline outside, the continuous gastric infusion of other administration group 30 days, continued oral administration gavage to the 33 days after the irradiation, about 2h after i.e. administration in the 33rd day in the 3rd day after the irradiation, white mice is put to death in the cervical vertebra dislocation, gets breastbone or femur, extrude the calf serum mixing of bone marrow fluid and slide one end, routine smear with mosquito forceps.Or wash marrow cavity of femur with calf serum and make the cell suspension smear, behind the smear natural drying, put into methanol (Tianjin moral grace chemical reagent company limited, lot number: fixing 5-10min 20070712), put into Giemsa and use liquid (Giemsa dye liquor, pH6.8PBS preparation, provided by Gansu basic courses department of college of traditional Chinese medicine biology laboratory), dyeing 10-15min with phosphate buffer or distilled water flushing, dries immediately.Microscopy, micronucleus cell number in 1000 polychromatic erythrocytes of every animal counting, and calculate micronuclear rates, the results are shown in Table 4.
Table 4 is on the impact of linear accelerator low energy X ray irradiation mouse bone marrow cells Other nucleated cells differential count (n=15, x ± SD)
Annotate: compare with model group
※P<0.05
※ ※P<0.01
※ ※ ※P<0.001;
As can be seen from Table 4: rear the 3rd day of irradiation, the mouse bone marrow cells number of nucleated cells obviously increases (P<0.001), wherein Radix Angelicae Sinensis Radix Hedysari (1: 5) group, Radix Angelicae Sinensis Radix Hedysari (2: 4) group, Radix Angelicae Sinensis Radix Hedysari (3: 3) group all can be resisted this effect, more all has statistical significance ((P<0.001 with model group, P<0.01, P<0.01).
2.4.4 the impact that centering superoxide dismutase (SOD) is active
Except a model group not administration of modeling, blank group give with normal saline outside, the continuous gastric infusion of other administration group 30 days, continued oral administration gavage to the 37 days after the irradiation, after irradiation the 7th day, with ultraviolet spectrophotometer (day island proper capital, model: UV-2401PC) carry out SOD and detect, the results are shown in Table 5.
Table 5 is on the impact of SOD in the linear accelerator low energy X ray irradiation mice serum (x ± SD)
Annotate: compare with model group
※P<0.05
※ ※P<0.01
※ ※ ※P<0.001;
As can be seen from Table 5: rear the 7th day of irradiation, SOD content obviously descend (P<0.001) in the mice serum, wherein Radix Angelicae Sinensis Radix Hedysari (1: 5) group, Radix Angelicae Sinensis Radix Hedysari (2: 4) group all can be resisted this effect, more all has statistical significance ((P<0.01, P<0.05) with model group.
2.4.5 the impact (Hemagglutination Method) on serum hemolysin
Except a model group not administration of modeling, blank group give with normal saline outside, the continuous gastric infusion of other administration group 30 days continued oral administration gavage to the 45 days after the irradiation, after irradiation the 15th day, carried out the mensuration of serum hemolysin.
Immune animal and serum separate: get Sanguis caprae seu ovis, immune animal and serum separate gets sheeps blood erythrocyte (SRBC), and with normal saline washing 3 times, each centrifugal 2000r/min is 10min altogether.Hematocrit SRBC is made into the cell suspension of 2% (v/v) with normal saline, and every Mus lumbar injection 0.2mL carries out immunity.After 5 days, extract eyeball and get blood in centrifuge tube, place about 1h, solidification blood and tube wall are peeled off, serum is fully separated out, the centrifugal 10min of 2000r/min collects serum.
Agglutination with normal saline with the serum doubling dilution, the dilution serum of difference is placed respectively in the Microhemagglutination brassboard, every hole 100 μ L, the SRBC suspension that adds again 100 μ L 0.5% (v/v), mixing, pack into and add a cover in the moistening square position, in 37 ℃ of incubation 3h, observe the hemagglutination degree.
The serum agglutination degree generally is divided into 5 grades of (0-IV) records, is calculated as follows the antibody product,
Antibody horizontal=(S1+2S2+3S3 ... nSn)
In the formula 1,2,3 ... n represents two-fold dilution's index, and S represents the rank of coagulation degree, and the antibody product is larger, and the expression serum antibody is higher.
0 grade of erythrocyte all sinks, and concentrates on the bottom, hole and forms fine and close round point shape, and liquid is fair-skinned clearly all around.
The most of heavy collection of I level erythrocyte becomes the round spot shape at the bottom of the hole, the erythrocyte of a small amount of coagulation is arranged all around.
The erythrocyte of II level coagulation forms thin layer at the bottom of the hole, a loose red point can be obviously seen at the center.
The even shakedown of erythrocyte of III level coagulation becomes skim at the bottom of being dispersed in the hole, and a small red dot mays be seen indistinctly at the center.
The even shakedown of erythrocyte of IV level coagulation becomes skim at the bottom of being dispersed in the hole, and grumeleuse becomes the convolution shape sometimes.
Experimental result sees Table 6.
Table 6 is on the impact of linear accelerator low energy X ray irradiation mice serum hemolysin (x ± SD)
Annotate: compare with model group
※P<0.05
※ ※P<0.01
※ ※ ※P<0.001;
Compare with the blank group
◇P<0.05
◇ ◇P<0.01
◇ ◇ ◇P<0.001.
As can be seen from Table 6: rear the 15th day of irradiation, mice serum hemolytic antibody level obviously descend (P<0.01), wherein Radix Angelicae Sinensis Radix Hedysari (1: 5) group, Radix Angelicae Sinensis Radix Hedysari (2: 4) group, Radix Angelicae Sinensis Radix Hedysari (3: 3) group all can be resisted this effect, more all have statistical significance ((P<0.0001) with model group.
In sum, after body was subject to radiation, ray can directly act on marrow hemopoietic stem cells, caused the inmature hematopoietic cell of bone marrow impaired, and the cell proliferation splitting ability descends, and interrupt in mature cell regeneration source in the peripheral blood.The peripheral blood leucocyte life-span is short, upgrades soon, if the marrow hemopoietic function is suppressed, eukocyte can not in time obtain upgrading, and then quantity of leucocyte descends rapidly.Therefore, leukocyte is direct, the most classical index of reflection radiation damage.Data shows, the variation of leukocyte count has phasic property when obvious behind the irradiation, phase and temporary transient equal when the ging up when initial stage that shows as descends.The descend time cause minimum at initial stage is to occur in 3~13d behind the irradiation behind the mice irradiation, and the time started of phase is 14d during temporary rise.3d behind the irradiation, the 7th day and three time points of 14d have been selected in this experiment, observe tested material on irradiation after the impact of mouse blood system.3d observes the Radiation on Mouse peripheral blood leucocyte and has produced appreciable impact behind irradiation, and quantity of leucocyte reduces in large quantities, illustrates that irradiation has caused certain damage to the mice body.3d relatively has the trend of increasing behind the 7th day and the leukocytic quantity of the basic, normal, high dosage group of 14d tested material mouse peripheral blood and irradiation behind the irradiation, the peripheral blood leucocyte that the prompting tested material causes radiation reduces protective action, and can promote its quantity to recover.
Effects of ionizing radiation can directly act on DNA, protein and enzyme behind body, cause ionization and chemical bond rupture, and molecule degeneration and cellularity are destroyed, and micronucleus occurs change; The free radical that radiation-actuate is a large amount of causes the peroxidization of whole body.Superoxide dismutase SOD is the defence body because oxygen metabolism is accepted the main protective enzyme of the superoxide radical infringement that electron deficiency forms, and is relevant with the protection of aging, tumor, inflammation, immune disease, cardiovascular disease and the radiation of body.SOD has very important effect to the balance between the oxidative and anti-oxidative, can remove the peroxidating anion, Cell protection in radiation injury.The vigor remote-effects of SOD are removed the ability of free radical, so can be used as an objective biochemical indicator of Antiradiation injury.This description of test tested material can enhanced rad after superoxide dismutase (SOD) activity of mice body, thereby verified that also Radix Angelicae Sinensis, Hedysarum polybotrys can repair radiation to the damage of antioxidant system.
The radiation meeting makes body that complicated reaction occur, thereby produces in vivo a large amount of endogenous free radicals, causes peroxidization, can cause certain damage to the immune system of body.Humoral immunization is antibody-mediated by the bone-marrow-derived lymphocyte generation, after exotic antigen enters body, in peripheral lymphoid tissue's (spleen, lymph node etc.) with the specific b lymphocyte on membrane receptor (mLg) combination, thereby beginning bone-marrow-derived lymphocyte activation stimulates dissimilar antibody generations.SRBC is combined with the B cell surface receptor after entering body, and the bone-marrow-derived lymphocyte of activation further breaks up and secretes anti-SRBC antibody, i.e. hemolysin.So the content of serum hemolysin has reflected bone-marrow-derived lymphocyte and has produced the ability of antibody, also can be used as the objective indicator of concentrated expression and evaluation body's immunity, and then becomes the biochemical indicator of Antiradiation injury.Originally experimental results show that Radix Angelicae Sinensis Radix Hedysari ultrafiltration membrane extract has protective effect to immune system injury.The Radix Hedysari ultrafiltration membrane extract can be by keeping and improve function after Immune Organs of Body is subject to radiation damage effectively, thereby improve the radiation-resistant ability of body.So one of Radix Angelicae Sinensis Radix Hedysari ultrafiltration membrane extract radiation resistance mechanism may be relevant with its enhancing immune organ function.
3. the result judges
In five experiments that superoxide dismutase (SOD) is active in above peripheral blood leucocyte, spleen and thymus index, femur bone marrow cell DNA content, bone marrow cell micronucleus number, the blood, serum hemolysin affects; any two experimental results are positive, can judge that this extract has assistant protection function to radiation hazradial bundle.The real data aspect several such as blood system, antioxidant system and immune system all demonstrate Radix Angelicae Sinensis Radix Hedysari (1: 5) ultrafiltration membrane extract to irradiation after the radiation damage of mice certain protection and restitution are arranged.