The specific embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Embodiment 1 pyrroloquinoline quinone PQQ is right
60The research of mice survival condition influence under the radiation of Co gamma-rays
1 test objective
Utilize mice
60Co gamma-ray irradiation model is observed the influence of pyrroloquinoline quinone to the mice survival condition.
2 experiment materials
2.1 given the test agent
Medicine name: pyrroloquinoline quinone, be called for short PQQ, be commercially available, or can be according to (microorganism journal .2007,47 (6): 982-986.) disclosed method preparation.
2.2 positive control drug
Medicine name: nilestriol (Nilestriol) is bought from Radiation Medical Science Inst., Chinese Academy of Military Medical Science (CN nine Room.
Batch number: 20081007.
Character: white powder.
2.3 laboratory animal
Adult KM mice, Military Medical Science Institute's Experimental Animal Center breeding.The mice body weight is 16.0~18.0g.Laboratory animal credit number: SCXK-(army) 2002-001.Laboratory animal is at the animal center SPF of Military Medical Science Institute experiment indoor feeding, the laboratory quality certification number: SYXK (army)-2002-016.10 mices of every cage are raised to be the mice feed prepared specially, freely drink water.The indoor temperature of zoopery remains on about 25 ℃, and relative humidity remains on 40-70%, illumination every day 12 hours.
3 experimental techniques
3.1 illuminate condition
60The Co gamma-rays, absorbed dose rate 236.05cGy/min.Exposure mice survival rate test absorbed dose are 8.0Gy, the whole body once irradiating.
3.2 group technology
Five groups are established in the test of mice survival rate: test is provided with dosage group (4mg/kg) among irradiation control group, positive drug control group (nilestriol 10mg/kg), PQQ low dose group (2mg/kg), the PQQ, PQQ high dose group (8mg/kg group).Be subjected to the situation of 8.0Gy roentgenization mice survival rate.Every group 20, male and female half and half.
3.3 medication
Each group is the oral administration gavage administration, and the administration time scheme sees the following form 1
Table 1 administration time calendar
3.4 observation index and date processing
Press such scheme irradiation and administration,
60After the Co8.0Gy dose irradiation, every day at the upper and lower noon is respectively observed mice 1 time, and record dead animal number and date of death, be 30d observing time.
Calculate surviving fraction PF (=processed group dead animal mean survival time/irradiation control group dead animal mean survival time).Enumeration data X
2Check, measurement data is carried out statistical procedures with the T method of inspection.Draw mice survival curve (Kaplan-Meier curve), carry out statistical analysis with sequential card side (Log rank test) method.
4 experimental results
4.1PQQ to being subjected to influence according to the mice survival condition
Be subjected to the situation of 8.0Gy roentgenization mice survival rate, in 30 days observation period, the irradiation control group animal only survives 2, survival rate is 10%, and PQQ 2mg/kg group survival rate is 35%, and PQQ 4mg/kg group survival rate is 60%, statistical significance is arranged, and animals survived quantity is more; The survival rate of positive drug group is 40%; But the survival rate of PQQ 8mg/kg group is 25% only, sees Table 2.
Table 2PQQ is to being subjected to the influence of 8.0Gy roentgenization mice survival rate
Annotate: compare " * " P<0.05 with irradiation control group; " * * " P<0.01; " * * * " P<0.001
The survival curve figure of raying mice (Kaplan-Meier curve), see Fig. 1, the survival curve of PQQ administration group and positive control treated animal is significantly higher than the radiation matched group, and wherein the sequential X 2 test of dosage group has statistical significance among positive drug, PQQ low dose group and the PQQ.
Add up each treated animal natural law of on average surviving, see Table 3.PQQ administration group 2mg/kg and the average survival of 4mg/kg group natural law are higher than irradiation control group, and wherein buck survival natural law is longer, and statistical significance is arranged.
The influence that table 3PQQ survives to the mice that is subjected to the 8.0Gy roentgenization
Annotate: compare " * " P<0.05 with irradiation control group; " * * " P<0.01; " * * * " P<0.001; Observing time is by 30d.
Statistics is respectively organized the dead animal natural law of on average surviving, and sees Table 4, with irradiation control group relatively, PQQ group dead animal prolonged survival period, surviving fraction PF is greater than 1.
The influence that table 4PQQ survives to the dead mice that is subjected to the 8.0Gy roentgenization
Annotate: compare " * " P<0.05 with irradiation control group; " * * " P<0.01; " * * * " P<0.001;
5 discuss and conclusion
The most of animal deads of radiation control animals, current deposit rate are 10% only, and the survival rate of being tried the thing treated animal is 25%-60%, is higher than the radiation matched group; The survival rate of positive drug treated animal is 40%.On average the survive observation index such as average survival natural law of natural law and dead animal of comprehensive irradiation animal dis motility rate, animal survival curve, each processed group are subjected to reagent PQQ that the survival condition of exposure animal is had stronger protective effect.
Conclusion: PQQ is right
60The death of Co8.0Gy irradiation induced mice has protective effect, and the protective effect when dosage is 4mg/kg is the strongest.
Embodiment 2 pyrroloquinoline quinone are to the influence of Co radiation gamma mouse bone marrow cells hematopoietic function
1 test objective
Utilize mice
60Co gamma-ray irradiation model is observed the influence of pyrroloquinoline quinone to mouse hemopoietic system.
2 experiment materials
With embodiment 1
2.3 laboratory animal
With embodiment 1
3 experimental techniques
3.1 illuminate condition
60The Co gamma-rays, absorbed dose rate 236.05cGy/min.Exposure mice survival rate test absorbed dose are 4.0Gy.The result of embodiment 1 shows, 8.0Gy be fatal dose, 4.0Gy is a sublethal dose, because will study the therapeutical effect of PQQ to the bone marrow hematogenesis radiation injury, if adopt fatal dose can cause mice dead in experimentation, thereby influence the analysis of experimental result.Therefore present embodiment adopts sublethal dose whole body once irradiating.
3.2 group technology
Three groups are established in the test of mice survival rate: irradiation control group, positive drug control group (nilestriol 10mg/kg), PQQ organize (4mg/kg).Every group 20, male and female half and half.
3.3 medication
Each group is the oral administration gavage administration, and the administration time scheme sees the following form 5.
Table 5 administration time calendar
3.4 detect index and method
Press such scheme irradiation and administration,
60After the Co 4.0Gy dose irradiation, the blood sampling of pre-irradiation afterbody, after each leading indicator irradiation of mensuration hematology, afterbody blood sampling in per 3 days is once measured hematology's leading indicator, and the observation period is limited to 21 days.After the last blood sampling, put to death animal, measure the dna content and the cell quantity of femur bone marrow.
The hematological indices detection method: cytometry is measured with Sysmex F820 blood analyser, and used main agents comprises: hemolysin-SYS-II, No. the 1400019th, Shandong Ji food medicine prison tool (standard) word 2007, Shandong STAC Medical Science And Technology Co., Ltd.; Diluent-SYS, No. the 1400018th, Shandong Ji food medicine prison tool (standard) word 2007, Shandong STAC Medical Science And Technology Co., Ltd..
The reticulocyte count method is that afterbody is taken a blood sample the adding of 50 μ l blood with the yellow cresyl blue dyeing of volume, hatches 30min for 37 ℃, smear, microscopically counting.
3.5 observation index and date processing
Data are represented with mean ± standard deviation, enumeration data X
2Check, measurement data is carried out statistical procedures with the T method of inspection.
4 experimental results
4.1 the variation of leucocyte content
Mice was subjected to roentgenization 21 days, the leukocyte Changing Pattern: drop to minimum according to the 3rd day leukocyte in back, return to substantially in the time of 21 days near normal (referring to table 6 and table 7).Since in the blood before the WBC medicine and behind the medicine fluctuation of detected value bigger, mainly by its with medicine before the relative value that compares judge the influence that is subjected to the reagent thing, the practical measurement value is through for referencial use.Played administration group and the rising of positive group leukocyte obviously (P<0.05, relative value) in 12 days, prompting is subjected to the reagent thing can promote the recovery of the peripheral blood leucocyte of acute radiation sickness mice.
Table 6 is subjected to shine 21 days leukocyte changing values of mice, and (WBC 10
9/ L)
Table 7 is subjected to according to 21 days leukocyte relative changing values (WBC) of mice
Annotate: compare " * " P<0.05 with irradiation control group; " * * " P<0.01
4.2 the variation of erythrocyte content
Mice was subjected to roentgenization 21 days, the change of red blood cell rule: dropped to minimum on the 6th day according to the back, return to normal (see Table 8 and table 9) in the time of 21 days.It is bigger that each group is measured numerical fluctuations, do not see evident difference.
Table 8 is subjected to shine 21 days change of red blood cell values of mice, and (RBC 10
12/ L)
Table 9 is subjected to according to 21 days erythrocyte relative changing values (RBC) of mice
Annotate: compare " * " P<0.05 with irradiation control group; " * * " P<0.01
4.3 the variation of content of hemoglobin
Mice was subjected to roentgenization 21 days, the hemoglobin Changing Pattern: bigger according to back fluctuation, return to substantially in the time of 21 days near normal (see Table 10 and table 11).It is bigger that each group is measured numerical fluctuations, do not see evident difference.
Table 10 is subjected to according to 21 days hemoglobin changing values (HGB g/L) of mice
Table 11 is subjected to according to 21 days hemoglobin relative changing values (HGB) of mice
4.4 the variation of platelet content
Mice was subjected to roentgenization 21 days, the platelet Changing Pattern: dropped to minimum on the 6th to 9 day according to the back, return to normal (table 12 and table 13) in the time of 21 days.And each group does not have evident difference.
Table 12 is subjected to shine 21 days platelet changing values of mice, and (PLT 10
9/ L)
Table 13 is subjected to according to 21 days platelet relative changing values (PLT) of mice
4.5 the variation of reticulocyte content
Mice was subjected to roentgenization 21 days, the Reticulocyte Changing Pattern: the photograph back was increased to the highest on the 3rd day, returned to substantially in the time of 21 days near normal (table 14).And administration group and positive group obviously reduce (P<0.05) after 9 days, show the recovery that is subjected to the reagent thing can promote the peripheral blood Reticulocyte of acute radiation sickness mice.
Table 14 is subjected to according to 21 days reticulocyte changing values (‰) of mice
Annotate: compare " * " P<0.05 with irradiation control group; " * * " P<0.01
4.6 the variation of medullary cell quantity and bone marrow dna content
Mice was subjected to roentgenization 21 days, bone marrow cell changes: the bone marrow cell of administration group and positive drug group is apparently higher than irradiation control group (P<0.05), and the bone marrow cell of administration group is also apparently higher than the positive drug group, show the recovery that is subjected to the reagent thing can promote the medullary cell of acute radiation sickness mice, and recovery capability is better than positive drug (table 15).
Mice was subjected to roentgenization 21 days, and medullary cell DNA changes: the bone marrow DNA of administration group and positive group shows to be subjected to the reagent thing to recover to have certain facilitation (table 16) for the bone marrow DNA that promotes the acute radiation sickness mice a little more than irradiation control group.
Table 16 is subjected to according to 21 days bone marrow cells of mice and bone marrow DNA numerical value
Annotate: compare " * " P<0.05 with irradiation control group; " * * * " P<0.001
4.8 the measurement of relevant organ weights
Mice was subjected to roentgenization 21 days, and each organizes body weight does not have significant difference, and the spleen weight of PQQ group and positive drug group is light than matched group, but organ coefficient does not have marked difference.Dissect the back spleen tuberosity is observed, do not see that each group has significant change (table 17 and table 18).
Table 17 is subjected to according to 21 days body weight of mice and organ weights
Table 18 is subjected to according to 21 days body weight of mice and organ weights coefficient
7 conclusions
To being subjected to
60After the administration of Co irradiation mouse model, be subjected to reagent thing PQQ certain facilitation to be arranged, promote the elimination of damage erythroid hematopoiesis factor, help the recovery of Reticulocyte ratio for leukocytic recovery.The bone marrow cell that is tried thing group and positive drug group is higher than matched group, and the bone marrow dna content also has the tendency that increases.As seen tried the recovery Effects that thing has certain promotion bone marrow hematogenesis function.
Embodiment 3 pyrroloquinoline quinone solutions
Prescription: pyrroloquinoline quinone 40mg
0.9% normal saline 100mL
Take by weighing 40mg pyrroloquinoline quinone powder, add the 100mL normal saline, stirring is dissolved powder fully, makes the pyrroloquinoline quinone aqueous solution preparation of 0.4mg/mL.
Embodiment 4 pyrroloquinoline quinone capsules
Prescription: pyrroloquinoline quinone 500mg
Starch 10g
10% starch slurry is an amount of
Make 500 capsules altogether, contain PQQ1mg in each capsule.
Earlier PQQ is mixed with starch, pulverize with colloid mill.With 10% starch slurry system soft material, soft material is crossed 20 mesh sieve system wet granulars, in 60~70 ℃ of oven dry, dried granule is with 20 mesh sieve granulate with wet granular.The capsule board that the preparation of capsule adopts lucite to make is filled.Earlier capsule medicated cap, capsule status are opened, the capsule body inserts in the capsule board hole, regulates the levels distance, makes the capsule mouth equal with the plate face.Granule is laid on the plate face, vibrates capsule board gently, make particles filled even.After filling up each capsule, with the unnecessary granule cleaning of plate face, jack-up capsule body, fit capsule medicated cap takes out capsule, promptly.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.