CN1210033C - Immunodepressive medicine and its preparing process and application - Google Patents
Immunodepressive medicine and its preparing process and application Download PDFInfo
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- CN1210033C CN1210033C CN 02110826 CN02110826A CN1210033C CN 1210033 C CN1210033 C CN 1210033C CN 02110826 CN02110826 CN 02110826 CN 02110826 A CN02110826 A CN 02110826A CN 1210033 C CN1210033 C CN 1210033C
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Abstract
The present invention belongs to the field of medicine production, which specifically relates to an immunosuppressive medicine using a monomer compound of demethylzeylasteral extracted and obtained form tripterygium wilfordii as an effective component, and a method for preparing the immunosuppressive medicine. The monomer compound of demethylzeylasteral of the present invention can be used as a medical raw material to be made into oral sustained-release agents, soft capsules, injection agents, ointments, etc. The immunosuppressive medicine of the present invention has the advantages of extremely little toxicity and side effect, wide sources, low production cost, low price and convenient application. The medicine of the present invention has obvious effect on rejection reaction resistance after the medicine is used for organ transplantation. Besides, the present invention can be used for treating clinical other autoimmune diseases, such as lupus erythematosus, etc.
Description
Technical field
The invention belongs to pharmaceutical field, being specifically related to a kind of is the immunosuppressive drug and preparation method thereof of effective ingredient and by its preparation anti-rejection and treat the application of some autoimmune disease after organ transplantation with the DEMETHYLZEYLASTERAL DZY T-96 TZ-93.
Background technology
Organ transplantation is the progress that 20th century surgery attracts people's attention most, comes true from illusion.By the end of the year 1997, patient surplus the whole world existing 610,000 person-time has accepted the almost various dissimilar organ transplantations of whole body such as the heart, lung, liver, kidney, pancreas, spleen, small intestinal, testis, cerebral tissue, surplus China existing 30,000 person-time, year transplant several 4000 surplus the people, and also have ascendant trend year by year.In addition, renal transplant patient's survival rate and life quality also improve greatly.The great achievement of all these organ transplantations directly has benefited from the exploitation of the immunosuppressive drug of medicine mediation, current except rare organ transplantation, as the organ transplantation between the monozygotic twins, the function of keeping institute's transplant organ is normal, and it is inevitable carrying out the medicine immunosuppressant therapy all the life.So new and effective, low toxicity, low price, using easily, immunosuppressant is the welcome that is subjected to doctor and patient deeply.The treatment of many autoimmune diseasees clinically in addition also mainly depends on the treatment of immunosuppressant.Though immunosuppressant therapy has experienced the development in a century, from the lonizing radiation that destroy all noble cellss, chemicals indiscriminately to a ciclosporin A that only suppresses to participate in immunoreactive cell, Prograf etc., make a lot of progress, but rejection is still the main cause of transplant organ loss of function, chronic rejection lacks effective Therapeutic Method and the big and expensive price of present most immunosuppressant toxic side effects, makes organ transplantation also not fully up to expectations.。
Existing research is separated to a kind of faint yellow crystalline material from the plant Radix Tripterygii Wilfordii, its molecular weight is 480, and fusing point is 260 ℃ (decomposition), and molecular formula is C
29H
36O
6Phenol demethyl triterpenoid, its English name is that (be called for short Dzy, T-96 TZ-93), names DEMETHYLZEYLASTERAL DZY T-96 TZ-93 to demethylzeylasteral.Its chemical structural formula is following represented:
Summary of the invention
The purpose of this invention is to provide a kind of DEMETHYLZEYLASTERAL DZY T-96 TZ-93 of from the Radix Tripterygii Wilfordii plant, extracting as crude drug, be used to prepare immunosuppressive drug.Further aim of the present invention is to provide a kind ofly to prepare DEMETHYLZEYLASTERAL DZY T-96 TZ-93 slow releasing capsule, soft capsule by the DEMETHYLZEYLASTERAL DZY T-96 TZ-93 crude drug, the preparation technology of injection and ointment.Medicine of the present invention is to be made of the excipient of DEMETHYLZEYLASTERAL DZY T-96 TZ-93 with solid or liquid, and solid used herein or liquid excipient are well known in the art, and is pharmaceutically acceptable excipient.DEMETHYLZEYLASTERAL DZY T-96 TZ-93 and its proportioning of excipient that medicine of the present invention contains effective dose are 1: 20-300.
Medicine of the present invention is prepared by following step:
1. the extraction of crude drug DEMETHYLZEYLASTERAL DZY T-96 TZ-93
Technical process comprises drying, the pulverizing of Radix Tripterygii Wilfordii raw material, the extraction of organic solvent, and Recovery of Organic Solvent is separated the evaluation of monomeric compound with silica gel.It is characterized in that drying and pulverize medical material, put in the pot that refluxes, add 1-3 ethyl acetate doubly, reflux 3 hours, repeat 2 times, get acetic acid ethyl acetate extract, reclaim solvent, get dry extract, yield is 8.25%, get the silica gel that extractum 5-10 doubly measures and carry out column chromatography, adopt the n-hexane/acetone gradient elution, obtain the faint yellow crystalline material of DEMETHYLZEYLASTERAL DZY T-96 TZ-93.
2. the preparation of DEMETHYLZEYLASTERAL DZY T-96 TZ-93 preparation
1) it is as follows to prepare the technology of DEMETHYLZEYLASTERAL DZY T-96 TZ-93 slow releasing capsule by the DEMETHYLZEYLASTERAL DZY T-96 TZ-93 crude drug:
With stearyl alcohol heating and melting in water-bath, add DEMETHYLZEYLASTERAL DZY T-96 TZ-93 and stir evenly, cold after, grind, add the hypromellose gel and make soft material, granulate, drying, granulate incapsulates promptly.
2) it is as follows to prepare the technology of DEMETHYLZEYLASTERAL DZY T-96 TZ-93 soft capsule by the DEMETHYLZEYLASTERAL DZY T-96 TZ-93 crude drug:
DEMETHYLZEYLASTERAL DZY T-96 TZ-93 is mixed with a small amount of vegetable oil, and it is miscible that the back of waiting to be uniformly dispersed adds the surplus vegetable oil, gets medicinal liquid.Other joins gelatin solution, wherein: 100 parts in gelatin, 55 parts of glycerol, 100 parts in water is standby, adopts dropping preparation method, makes the DEMETHYLZEYLASTERAL DZY T-96 TZ-93 soft capsule.
3) it is as follows to prepare the injection technology of DEMETHYLZEYLASTERAL DZY T-96 TZ-93 by the DEMETHYLZEYLASTERAL DZY T-96 TZ-93 crude drug:
DEMETHYLZEYLASTERAL DZY T-96 TZ-93 adds an amount of ethanol and propylene glycol grinds well, and gradation adds entry and mixes, filtering with microporous membrane, and in the packing glass ampule, sterilization, promptly.
4) it is as follows to prepare the ointment technology of DEMETHYLZEYLASTERAL DZY T-96 TZ-93 by the DEMETHYLZEYLASTERAL DZY T-96 TZ-93 crude drug:
Get monoglyceride, octadecanol and liquid Paraffin heating make fusion.Other removes the chloroform of 10 times of amounts of first demethylzelasteral and the methanol of equivalent, joins in the DEMETHYLZEYLASTERAL DZY T-96 TZ-93, makes dissolving, slowly adds in the above-mentioned fused medicinal liquid again, and temperature constantly stirs at 80 ℃, makes evenly.In addition ethyl hydroxybenzoate, dodecane base flow acid sodium, G ﹠ W are mixed the back and all dissolve when straight fire is heated to 90 ℃, when treating temperature to 80 ℃ and above-mentioned medicinal liquid do not stopping to stir under companion's the situation, slowly added, continue again not stop to stir the companion until cold one-tenth thick paste shape, add an amount of Ah azoles's anthracene, stir evenly promptly.
The present invention has carried out drug effect and toxicological experiment to the crude drug DEMETHYLZEYLASTERAL DZY T-96 TZ-93, and the result is as follows:
(1) drug effect result
1, DEMETHYLZEYLASTERAL DZY T-96 TZ-93 external to the immunosuppressant experimentation
1), DEMETHYLZEYLASTERAL DZY T-96 TZ-93 inhibitory action that splenocyte is transformed under mitogenstimulated
Get the BALB/c mouse spleen and make splenocyte suspension, in containing the RPMI-1640 culture fluid of 10% calf serum, hatch, the DEMETHYLZEYLASTERAL DZY T-96 TZ-93, cyclosporine A (CsA) and the Tripterygium glycosides (TII) that add variable concentrations, respectively under ConA and the primary stimuli of PHA mitosis, use the MTT method and measure the conversion situation of splenocyte, understand the inhibition degree that DEMETHYLZEYLASTERAL DZY T-96 TZ-93 transforms splenocyte.
Result: the inhibitory action that splenocyte is transformed: adding concentration when being 0.125,0.25 μ g/ml DEMETHYLZEYLASTERAL DZY T-96 TZ-93, no matter be that ConA or PHA stimulate, its OD value and suitable (P＞0.05) that do not add DEMETHYLZEYLASTERAL DZY T-96 TZ-93, and be in the DEMETHYLZEYLASTERAL DZY T-96 TZ-93 of 0.5 μ g/ml and 1.0 μ g/ml in concentration, the OD value obviously descends, with do not add DEMETHYLZEYLASTERAL DZY T-96 TZ-93 significant difference (P＜0.001) relatively arranged, show that DEMETHYLZEYLASTERAL DZY T-96 TZ-93 transforms the inductive splenocyte of ConA, PHA the obvious suppression effect is arranged.Wherein add the OD value littler (p＜0.05) of the OD value of 0.5 μ g/ml DEMETHYLZEYLASTERAL DZY T-96 TZ-93 than the TII that adds 1 μ g/ml, OD value zero difference (p＞0.05) during with the CsA that adds 1 μ g/ml, the inhibitory action that DEMETHYLZEYLASTERAL DZY T-96 TZ-93 is described is stronger than TII, and suitable with the inhibitory action of CsA when reaching finite concentration.
2), DEMETHYLZEYLASTERAL DZY T-96 TZ-93 inhibition test that mixed lymphocytes is cultivated:
Get the C57BL/6 mouse boosting cell and mix with the BALB/c mouse splenocyte of deactivation, hatch in the RPMI-1640 culture fluid, and add DEMETHYLZEYLASTERAL DZY T-96 TZ-93, CsA or the TII of variable concentrations respectively, 4h adds 1 μ ci before stopping cultivation
3H-TdR on glass fiber filter paper, measures the dpm value with cell harvesting on β liquid scintillation counter, observe splenocyte and transform situation, understands the inhibition degree that DEMETHYLZEYLASTERAL DZY T-96 TZ-93, TII and CsA transform splenocyte.
Result: mix the inhibitory action that splenocyte transforms: similar to the experimental study that the mitogenstimulated splenocyte transforms, DEMETHYLZEYLASTERAL DZY T-96 TZ-93, TII or CsA that this test adds variable concentrations show that all conversion has inhibitory action to splenocyte, and the variable concentrations inhibition strength differs.The DEMETHYLZEYLASTERAL DZY T-96 TZ-93 that adds 1 μ g/ml concentration is strong especially to mixing splenocyte conversion inhibitory action.
3), DEMETHYLZEYLASTERAL DZY T-96 TZ-93 is to inducing the inhibiting test of IL-2.
Get the BALB/c mouse spleen and make splenocyte suspension, hatch in the RPMI-1640 culture fluid, not adding or add ConA stimulates observation of cell to generate IL-2, and adds DEMETHYLZEYLASTERAL DZY T-96 TZ-93, TII or the CsA of variable concentrations, and getting supernatant is sample to be tested.Cultivate in the RPMI-1640 culture fluid with CTLL-2 cell that relies on IL-2 and the sample to be tested that has prepared,, calculate the IL-2 activity of sample with the rIL-2 result of standard with the conversion of MTT method mensuration CTLL-2.
The result: DEMETHYLZEYLASTERAL DZY T-96 TZ-93 generates the inhibitory action of IL-2 to lymphocyte: in to lymphocytic cultivation, not adding ConA stimulates, and sophisticated lymphocyte also generates and discharges IL-2, and its burst size is lower.During unconstrained medicine, at 12 hours, 24 hours and 36 hours incubation times, the burst size of IL-2 is respectively 7.50 ± 0.26,2.43 ± 0.12 and 2.37 ± 0.007u/ml, wherein with other two times group notable difference (p＜0.001) was arranged relatively in 12 hours, the IL-2 activity that shows lymphocyte release is the highest to cultivate 12 hours, DEMETHYLZEYLASTERAL DZY T-96 TZ-93, TII and CsA do not have influence to the IL-2 that mature lymphocyte discharges, and the confirmation DEMETHYLZEYLASTERAL DZY T-96 TZ-93 does not have direct killer cell effect.In the lymphocyte of cultivating, after adding mitogen ConA, the activity of IL-2 significantly increases, in unconstrained dose sample, it is respectively 48.75 ± 3.14,45.33 ± 2.24 and 27.37 ± 1.16U/ml cultivating in 12 hours, 24 hours and 36 hours the IL-2 amount, and 36 hours was low (p＜0.05) than 12 hours.After adding the DEMETHYLZEYLASTERAL DZY T-96 TZ-93 medicine, cultivate when DEMETHYLZEYLASTERAL DZY T-96 TZ-93 concentration was 1 μ g/ml in 12 hours, the inhibition of IL-2 very remarkable (12.36 ± 2.39U/ml), (48.75 ± 3.14U/ml) relatively have marked difference (p＜0.001) with check sample, TII sample (43.17 ± 2.65U/ml) comparisons with 1 μ g/ml concentration, IL-2 is starkly lower than the TII sample in the DEMETHYLZEYLASTERAL DZY T-96 TZ-93 sample, and the prompting DEMETHYLZEYLASTERAL DZY T-96 TZ-93 is than the inhibitory action strong (p＜0.001) of TII; Compare with the CsA inhibitory action of each concentration, the inhibitory action of DEMETHYLZEYLASTERAL DZY T-96 TZ-93 is strong not as good as the CsA effect, the IL-2 activity of CsA when 0.25,0.5,1 μ g/ml is 10.17 ± 1.59,8.70 ± 0.46 respectively, 5.37 ± 0.70u/ml, all have significance to suppress, the growing amount of its IL-2 is all low than the DEMETHYLZEYLASTERAL DZY T-96 TZ-93 of 1 μ g/ml concentration.What be worth proposition is to compare with CsA and check sample, the IL-2 activity should consume gradually with the prolongation of 12 hours, 24 hours, 36 hour time and reduce, but in being arranged, cultivated 12 hours to 36 hours by the sample of DEMETHYLZEYLASTERAL DZY T-96 TZ-93, the IL-2 activity is not seen the trend of attenuating, the prompting DEMETHYLZEYLASTERAL DZY T-96 TZ-93 is except that having the IL-2 of inhibition generation, suppress the effect of IL-2 receptor active in addition, and reduce the consumption of IL-2.
4), DEMETHYLZEYLASTERAL DZY T-96 TZ-93 promotes the inhibitory action of CTLL-2 cell proliferation to add DEMETHYLZEYLASTERAL DZY T-96 TZ-93, TII, the CsA of variable concentrations in the CTLL-2 cell suspension that relies on IL-2 to recombinant il-2, and add rIL-2 CTLL-2 is transformed, measure the OD value with the MTT method, understand the inhibitory action of DEMETHYLZEYLASTERAL DZY T-96 TZ-93 the short CTLL-2 cell transformation of rIL-2.
Result: in the CTLL-2 cell of the dependence IL-2 that cultivates, add rIL-2, blank OD value is 0.154 ± 0.013, after the DEMETHYLZEYLASTERAL DZY T-96 TZ-93 that adds 0.5 μ g/ml and 1 μ g/ml concentration, its OD value obviously descends, be respectively 0.024 ± 0.001 and 0.014 ± 0.003, the prompting DEMETHYLZEYLASTERAL DZY T-96 TZ-93 has the effect (p＜0.001) that suppresses the IL-2 receptor active.In the TII of 0.5 μ g/ml and 1 μ g/ml concentration group, its OD value and matched group more also have small size decline (P＜0.05 and P＜0.01), but strong not as good as DEMETHYLZEYLASTERAL DZY T-96 TZ-93.The CsA OD value of each concentration all with dummy quite (p＞0.05), show that CsA does not have influence to the IL-2 receptor active.
Above-mentioned experimental result confirms: DEMETHYLZEYLASTERAL DZY T-96 TZ-93 has clear and definite inhibitory action to the conversion of mitogen ConA or PHA and allosome MHC antigenic stimulus mouse boosting cell, it is stronger than the effect of TII that DEMETHYLZEYLASTERAL DZY T-96 TZ-93 suppresses immunoreactive effect, when reaching finite concentration, its inhibiting intensity even suitable with CsA.DEMETHYLZEYLASTERAL DZY T-96 TZ-93 not only has the effect that splenocyte generated or discharged IL-2 that suppresses, and also has the inhibitory action to the IL-2 receptor active.
2, DEMETHYLZEYLASTERAL DZY T-96 TZ-93 is to the experimentation of rat original position renal transplantation model
1), sets up rat original position renal transplantation study model
Adopt the 250-300g body weight, male Wistar and SD rat are as confession, the receptor Mus of renal transplantation.Expose the left kidney of donor rat and under operating microscope, dissect free left kidney arteriovenous, ureter epimere, the renovascular ventral aorta up and down in a blocking-up left side, through the kidney preserving liquid 8ml of ventral aorta, cut left kidney and repair left kidney blood vessel and ureter 0～4 ℃ of left renal perfusion.Fully expose the left kidney of receptor rat, free left kidney and blood vessel, ureter epimere under operating microscope, near ventral aorta blocking-up renal artery, vein, cross-section arteriovenous and ureter epimere excise left kidney, and the flushing of stump blood vessel heparin saline is interrupted end to end anastomosis respectively for renal artery and receptor renal artery, two fixed points end end are continuously sewed up renal veins, open blood vessel sees that kidney blood supplies well, color is ruddy, ureter stream urine, and ureter and ureter epimere interrupted suture excise right kidney.Art finishes the rat insulation up to reviving.The result: the success rate of the kidney of rats transplantation model of setting up through this method is higher, can reach 80% success rate, and the successful rat excretory urography of performing the operation sees that transplanted kidney function is good, drains unobstructedly, does not have obvious urinary tract obstruction.Blood BUN, Cr check normal range.Compare with BUN, the Cr of self before the art, t checks P＞0.05, and difference does not have significance, and the prompting renal function is good.The 3 days transplanted kidney of performing the operation cut does pathological observation, and nephridial tissue has a small amount of lymphocytic infiltration under the mirror, and glomerule and renal tubules are intact.Conclusion: under operating microscope, set up left kidney original position kidney of rats transplantation model, can reach the normal requirement of renal function.
2), DEMETHYLZEYLASTERAL DZY T-96 TZ-93 and TII suppress the comparative study of rat implantation kidney repulsive interaction
Male Wistar, SD rat are confession, receptor, and 70 of row renal transplantation rats are divided into 7 groups at random, and 8 every group, one is a matched group.Every group has 2 preparations to put to death in the time of 14 days in addition.Matched group every Mus normal saline every day 4ml irritates stomach, three of DEMETHYLZEYLASTERAL DZY T-96 TZ-93 medications are organized every day respectively every Mus and are irritated the DEMETHYLZEYLASTERAL DZY T-96 TZ-93 medicinal liquid that 4ml includes 5mg/kg, 10mg/kg, 20mg/kg, TII divides three groups, and every Mus filling every day stomach 4ml includes 20mg/kg, 40mg/kg and 60mg/kg medicinal liquid respectively.Irritate the stomach time and be every day to dead or 14 days.Observe activities in rats and feed situation, before the medication and medication respectively got blood in 14 days and once check hemoglobin, liver function situation, medication was put to death 1 or get intestinal, liver, the capable histopathologic examination of kidney when dead in 14 days.The survival of rats natural law respectively organized in record respectively.
The result: DEMETHYLZEYLASTERAL DZY T-96 TZ-93 medication 5mg/kg rat mean survival time is 7.1 ± 0.65 days, and with matched group comparison in 6.9 ± 0.64 days there was no significant difference (P＞0.05), prompting DEMETHYLZEYLASTERAL DZY T-96 TZ-93 5mg/kg repels the unrestraint effect to the rat implantation kidney; When DEMETHYLZEYLASTERAL DZY T-96 TZ-93 is used 10mg/kg and 20mg/kg, its mean survival time (MST), brought up to 14.7 ± 1.04 days and 18.0 ± 1.51 days, with matched group comparing difference remarkable (p＜0.001), show that survival has the prolongation effect to these two dosage DEMETHYLZEYLASTERAL DZY T-96 TZ-93 to the rat implantation kidney.Relatively the 20mg/kg group is long than 10mg/kg group survival period between its two groups.Compare with TII medication group, the survival period of DEMETHYLZEYLASTERAL DZY T-96 TZ-93 10mg/kg survival period and TII 40mg/kg and 60mg/kg is (p＞0.05) quite, but the survival period of DEMETHYLZEYLASTERAL DZY T-96 TZ-93 20mg/kg is all grown (p＜0.01) for any one group than TII.Dirty typical case and the serious acute rejection of showing as of the dead Ren Mus of histopathological examination matched group, DEMETHYLZEYLASTERAL DZY T-96 TZ-93 5mg/kg group changes similar to matched group to the transplanted kidney of TII 20mg/kg group.DEMETHYLZEYLASTERAL DZY T-96 TZ-93 10mg/kg group and TII 40mg/kg and 60mg/kg group, saw more lymphocyte at 14 days in the transplanted kidney tissue, existing rejection occurs, rarely seen a small amount of lymphocytic infiltration in the nephridial tissue when having only DEMETHYLZEYLASTERAL DZY T-96 TZ-93 20mg/kg to organize 14 days, not seeing has tangible rejection sign.Small intestinal regulating liver-QI tissue examination no abnormality seen.There is not significance (p＞0.05) with 14 days hemoglobin of medication, ALT, TP inspection difference before the medication.
Conclusion: the DEMETHYLZEYLASTERAL DZY T-96 TZ-93 administration has definite inhibitory action to the repulsion of rat implantation kidney, its action intensity and dosage are proportionate., 10mg/kg and 20mg/kg medication all survivals of energy significant prolongation transplanted kidney in 14 days, but it is even more ideal with the 20mg/kg group, compare with TII medication group, DEMETHYLZEYLASTERAL DZY T-96 TZ-93 20mg/kg effect is eager to excel.DEMETHYLZEYLASTERAL DZY T-96 TZ-93 20mg/kg administration still was unlikely to rat is caused toxic side effects in 14 days.
3), DEMETHYLZEYLASTERAL DZY T-96 TZ-93 and CsA suppress the comparative study of rat implantation kidney repulsive interaction
With male Wistar, SD rat is confession, receptor, and 80 of row renal transplantation rats are divided into 8 groups, 8 every group at random.Every group has 2 preparations to put to death in the time of 14 days in addition.Each group gives 4ml respectively and contains Pred 10mg/kg, DEMETHYLZEYLASTERAL DZY T-96 TZ-93 10mg/kg, DEMETHYLZEYLASTERAL DZY T-96 TZ-93 20mg/kg, DEMETHYLZEYLASTERAL DZY T-96 TZ-93 10mg/kg+Pred 10mg/kg, CsA 5mg/kg, CsA10mg/kg and CsA 10mg/kg+Pred 10mg/kg medicinal liquid, and every Mus is irritated stomach every day once to dead or 14 days.Simultaneously observed and recorded is respectively organized the existence natural law of rat, get dead Mus or put to death the preparation rat in 14 days and get small intestinal, liver, kidney etc. and do the tissue pathological slice inspection, and before the medication and hemoglobin, ATL, TP 14 days the time check.
Result: single time-to-live, notable difference (p＜0.01) is more all arranged with matched group with the equal energy of two dosage groups of DEMETHYLZEYLASTERAL DZY T-96 TZ-93 (10mg/kg and 20mg/kg) significant prolongation rat.Wherein the 10mg/kg group time-to-live uses the 5mg/kg dosage group of CsA suitable with list, and difference does not have significance (p＞0.05).The single repulsion with prednisone 10mg/kg prevention rat implantation kidney almost do not have effect, and its mean survival time (MST) and matched group comparison difference do not have significance meaning (p＞0.05).But DEMETHYLZEYLASTERAL DZY T-96 TZ-93 10mg/kg and Pred 10mg/kg share can prolong rats life cycle to 31.75 ± 6.48 day.The transplanted kidney of medication in the time of 14 days is in DEMETHYLZEYLASTERAL DZY T-96 TZ-93 20mg/kg, CsA10mg/kg group and two drug combination groups, and the nephropathy reason is checked no obvious pathological changes, and existing repulsion sign such as the lymphocytic infiltration in various degree of other groups.Each group is checked no abnormal in 14 days hemoglobin of medication, ALT, TP, get liver in the time of 14 days, small intestinal is done also no abnormality seen of pathologic finding.
Conclusion: confirm that DEMETHYLZEYLASTERAL DZY T-96 TZ-93 has stronger immunosuppressive action, use 10mg/kg or 20mg/kg time-to-live of prolong rats transplanted kidney significantly separately, the time-to-live strengthens with dosage within the specific limits and prolongs.Use Pred 10mg/kg separately the renal transplantation survival of rats phase is not had the prolongation effect, but 10mg/kg share with DEMETHYLZEYLASTERAL DZY T-96 TZ-93, but can prolong the survival of rats time of singly using with the DEMETHYLZEYLASTERAL DZY T-96 TZ-93 of dosage exponentially, use the DEMETHYLZEYLASTERAL DZY T-96 TZ-93 of suitable dosage at short notice, visible toxic side effects can not occur.
(2), toxicological experiment result
1, acute toxicity test:
Getting male and female mice body weight is 18～22g, each 30, be divided into 6 groups at random, each 5 of every group of male and female mices, each group once irritates stomach for every mice DEMETHYLZEYLASTERAL DZY T-96 TZ-93 by 192mg/kg, 240mg/kg, 300mg/kg, 375mg/kg and 468mg/kg respectively, and control group administered physiological saline, volume are 0.8-0.9ml, observe the dead mouse situation, ask LD
50Value, and taking internal organ pathologic finding.
Result: the LD of DEMETHYLZEYLASTERAL DZY T-96 TZ-93
50Be 286.135mg/kg.LD
5095% fiducial limit 283.92-288.39mg/kg.The main toxicity organ of DEMETHYLZEYLASTERAL DZY T-96 TZ-93 is the intestinal regulating liver-QI.
2, subacute toxicity test:
Get 40 of mices, be divided into 4 groups at random, 10 every group, male and female half and half.Give each group respectively by normal saline, DEMETHYLZEYLASTERAL DZY T-96 TZ-93 10mg, 20mg and 80mg/kg, irritate stomach once every day for every mice, totally 14 days, volume was 0.8ml, observed the pathologic finding of mice activity, diet, body weight, routine blood test, blood biochemistry and internal organs.
The result: during DEMETHYLZEYLASTERAL DZY T-96 TZ-93 80mg/kg every day administration, just begin to cause the mice anorexia, weight loss may reduce relevant with feed.80mg/kg group medication 14 days, hemoglobin and WBC have certain decline, and liver function test and pathologic finding prompting 80mg/kg have slight toxic action to liver, mucous membrane of small intestine fine hair mild swelling, a small amount of inflammatory cell infiltration in the lamina propria, and 10mg and 20mg/kg group do not see that above-mentioned pathological changes is arranged.
The main component of mentioning in toxicological study proof DEMETHYLZEYLASTERAL DZY T-96 TZ-93 and the present Radix Tripterygii Wilfordii is compared, as the oral LD of Tripterygium glycosides (TII) mice
50Be 159.70mg/kg, lumbar injection is 93.99mg/kg; The LD of triptolide (Radix Tripterygii Wilfordii lactone alcohol) mouse peritoneal injection
50Be 0.9mg/kg; Tripterine is 10.9mg/kg; And the oral LD of the mice of DEMETHYLZEYLASTERAL DZY T-96 TZ-93
50Be 286.135mg/kg, toxicity is starkly lower than these chemical compounds.
Embodiment 1
With drying and pulverize medical material, put in the pot that refluxes, add 1.5 times ethyl acetate, reflux 3 hours, repeat 2 times, get acetic acid ethyl acetate extract, reclaim solvent, get dry extract, yield is 8.25%, the silica gel of getting 7.5 times of amounts of extractum carries out column chromatography, adopts the n-hexane/acetone gradient elution, obtains the faint yellow crystalline material of DEMETHYLZEYLASTERAL DZY T-96 TZ-93.
The preparation of embodiment 2. DEMETHYLZEYLASTERAL DZY T-96 TZ-93 slow releasing capsule
With stearyl alcohol heating and melting in water-bath, add DEMETHYLZEYLASTERAL DZY T-96 TZ-93 5mg and stir evenly, cold after, grind, add the hypromellose gel and make soft material, the wet grain of 18 mesh sieve systems, 60 ℃ of dryings, granulate incapsulates promptly, be total to 250 of capsules.
Embodiment 3. preparation DEMETHYLZEYLASTERAL DZY T-96 TZ-93 soft capsules
Earlier DEMETHYLZEYLASTERAL DZY T-96 TZ-93 5mg is mixed with the 25g vegetable oil, pulverize with colloid mill, it is miscible that the back of waiting to be uniformly dispersed adds the surplus vegetable oil, gets medicinal liquid 200ml.Other joins gelatin solution (100 parts in gelatin, 55 parts of glycerol, 100 parts in water) standby, under the condition of 23 ± 2 ℃ of room temperatures, relative humidity 40%, medicinal liquid and gelatin solution make 500 DEMETHYLZEYLASTERAL DZY T-96 TZ-93 soft capsules altogether with rotating rolling capsule machine automatically, and when 28 ± 2 ℃ of relative humiditys 40% capsule are got in dry 20 hours.
The injection of embodiment 4. preparation DEMETHYLZEYLASTERAL DZY T-96 TZ-93
Remove first demethylzelasteral 400mg and add 40ml ethanol and 160ml propylene glycol, grind well, make dissolving, gradation adds entry to 400ml again, shakes up, and filtering with microporous membrane in the packing 2ml glass ampule, is sterilized, promptly according to a conventional method.
The ointment of embodiment 5. preparation DEMETHYLZEYLASTERAL DZY T-96 TZ-93
Get monoglyceride 13g, octadecanol 25g and liquid Paraffin 38ml heating make fusion, and other removes first demethylzelasteral 0.38g and adds the chloroform of 10 times of amounts and the methanol of equivalent, make dissolving, slowly add in the above-mentioned fused medicinal liquid again, temperature remains on about 80 ℃, constantly stir, make evenly.In addition ethyl hydroxybenzoate 0.25g, sodium lauryl sulphate 0.63g, glycerol 25g and water 150ml being mixed the back all dissolves when straight fire is heated to 90 ℃, when treating temperature to 80 ℃ and above-mentioned medicinal liquid under not stopping to stir, slowly added, continue again not stop to stir until cold one-tenth thick paste shape, add an amount of Ah azoles's anthracene, stir evenly promptly.
Immunosuppressive drug toxic side effects of the present invention is few, wide material sources, production cost are low, low price, uses conveniently, also can be used for treating clinical other autoimmune diseasees such as lupus erythematosus etc.
Claims (1)
1, the application of DEMETHYLZEYLASTERAL DZY T-96 TZ-93 in the medicine that preparation inhibition transplant organ repels.
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| CN102532239A (en) * | 2010-12-24 | 2012-07-04 | 苏州宝泽堂医药科技有限公司 | Method for extracting demethylzeylasteral from thunder god vine |
| CN104749009B (en) | 2015-03-30 | 2018-05-04 | 上海云泽生物科技有限公司 | immunosuppressant drug extraction reagent for immunoassay |
| CN112237584B (en) * | 2020-08-21 | 2021-10-01 | 中国医学科学院医药生物技术研究所 | Use of compounds for the prophylaxis and/or treatment of diseases caused by coronavirus infection |
| CN112294825A (en) * | 2020-09-27 | 2021-02-02 | 天津国际生物医药联合研究院 | Potential application of demethylzelarwood aldehyde in resisting mycobacterium tuberculosis infection |
| CN113827599A (en) * | 2021-09-23 | 2021-12-24 | 天津国际生物医药联合研究院 | Potential application of demethylzelaronal in resisting dengue virus infection |
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