CN101766640B - Preparation for panaxoside-Rd aqueous solution of propylene glycol and new application thereof in inflammation resistance, immune suppression and organ transplant rejection resistance - Google Patents
Preparation for panaxoside-Rd aqueous solution of propylene glycol and new application thereof in inflammation resistance, immune suppression and organ transplant rejection resistance Download PDFInfo
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- A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
- A61K36/258—Panax (ginseng)
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- A61P9/00—Drugs for disorders of the cardiovascular system
Abstract
The invention relates to preparation for panaxoside-Rd aqueous solution of propylene glycol and new application thereof in inflammation resistance, immune suppression and organ transplant rejection resistance. The invention provides panaxoside-Rd solution and a preparation method thereof. The solution is formed by dissolving the panaxoside-Rd in propylene glycol aqueous solution containing 50 to 100 volume percent of propylene glycol, and the content of the panaxoside-Rd is 1 to 100 mg/ml, wherein the volume percentage of the propylene glycol is based on the total volume of the aqueous solution of propylene glycol. The invention further provides a medicinal composition containing the solution of the invention and a preparation method thereof, and application of the panaxoside-Rd or the panaxoside-Rd solution of the invention in preparing a medicament for inflammation resistance, immune suppression and/or organ transplant rejection resistance.
Description
Technical field
The present invention relates to technical field of medicine, more specifically, relate to panaxoside-Rd aqueous solution of propylene glycol, its preparation and preparation method and application with highly dissoluble and stability.
Background technology
Ginsenoside-Rd is a kind of monomeric compound that is isolated through multistep from the Chinese medicine panax araliaceae plant, belongs to protopanoxadiol type saponin.Its chinesization formal name used at school is 20-(S)-protopanoxadiol-3[O-β-D-Glucopyranose. (1 → 2)-β-D-pyranglucoside]-20-O-β-D-pyranglucoside, English chemistry 20 (S)-Protopanaxadiol 3-O-[β-D-glucopyranosyl (1 → 2)-β-D-glucopyranosyl by name]-20-O-β-glucopyranoside, chemical constitution is as follows:
The ginsenoside-Rd outward appearance is white to micro-yellow powder, and odorless is soluble in methanol, ethanol, propylene glycol, n-butyl alcohol and hot water; Be dissolved in acetone, chloroform and water.Main physicochemical constant is: 209～214 ℃ of fusing points, specific optical rotation [α] D22+21.16 (C=4.98, MeOH) absorbs λ in the METHANOL MEDIUM medium ultraviolet
Max=201nm.Typical concentrations is 20 volume % propylene glycol solutions of the ginsenoside-Rd of 10mg/ml, and its pH is 7.6～7.72.
Ginsenoside-Rd has the effect of specific inhibition receptor dependency calcium channel, knownly can be used for treating the acute ischemic cerebral apoplexy disease, and for example its specification is that the injection that 10mg/ml contains propylene glycol intends being used for the treatment of the acute ischemic cerebral apoplexy disease.This medicine is first class national new drug, has finished at present preclinical study, has entered clinical experimental stage after State Food and Drug Administration's approval.
Yet, find that in application original aqueous solution of propylene glycol with 20 volume % is preserved at low temperatures as the ginsenoside-Rd injection (ginsenoside-Rd content 10mg/ml) of solvent and easily separated out precipitation.Think that in the past storage temperature is too low so that ginsenoside-Rd dissolubility reduction in the aqueous solution of propylene glycol of 20 volume %.But find that in process of production ginsenoside-Rd can not form true solution (having opalescence to exist) at the aqueous solution of propylene glycol of 20 volume %.Owing to the granule that forms is minimum few again, naked eyes are difficult for discovering, and it detects clarity by the pharmacopeia method is qualified (measuring because being that dilution is rear).Yet the Tyndall phenomenon of this solution is positive, and the granule that has 1-100nm is described.The granule of this size can form opalescence, and at room temperature owing to Brownian movement, but stable suspersion can not form precipitation.Because this part granule that exists in this ginsenoside-Rd injection at low temperatures Brownian movement weakens, so that the easy gathering of granule is agglomerating, thereby mass penalty forms precipitation.Because the implicit granule of this kind is not done detection in the preparation processing process, make the Rd injection of different batches again, form at random the preparation of many different solubilities.This just may be so that the dissolubility of preparation be out of control.
This shows that existing ginsenoside-Rd injection is transferred postpone at the lower environment of temperature and separated out precipitation and can have influence on this product quality of the pharmaceutical preparations control and estimate, and makes this product in use have certain risk.
Therefore, the dissolubility and the stability that how to improve active substance in the pharmaceutical composition are this area problem demanding prompt solutions.And for ginsenoside-Rd solution, the raising of its dissolubility, stability promotes ginsenoside-Rd to become problem in the urgent need to address in the clinical available medicine especially.
In addition, because ginsenoside-Rd content is lower in original ginsenoside-Rd injection, it is used and only limits at present treat the acute ischemic cerebral apoplexy disease.Further improve the content of ginsenoside-Rd in the preparation and the stability of preparation, and the new purposes of exploitation novel formulation, be to promote in this area that ginsenoside-Rd becomes another problem in the urgent need to address in the clinical available medicine.
Summary of the invention
One of purpose of the present invention is to provide a kind of ginsenoside-Rd solution with high-dissolvability and stability and preparation method thereof.Another object of the present invention is to provide ginsenoside-Rd or Radix Ginseng of the present invention saponin-Rd solution effect and their purposes in the preparation related drugs in antiinflammatory, immunosuppressant or anti-organ-graft refection.
In a first aspect of the present invention, a kind of ginsenoside-Rd solution is provided, described solution is that ginsenoside-Rd is the solution that forms in the aqueous solution of propylene glycol of 50-100 volume % at contained propylene glycol, the content of described ginsenoside-Rd is 1～100mg/ml solution, wherein, the percent by volume of propylene glycol is take the cumulative volume of aqueous solution of propylene glycol as benchmark.
In a preference, described solution is the solution that ginsenoside-Rd forms in the aqueous solution of propylene glycol of 55～100 volume %, the aqueous solution of propylene glycol of preferred 55～80 volume %, the more preferably aqueous solution of propylene glycol of 55～75 volume %.
In an embodiment of the invention, described solution is true solution.
In a preference, after 5 days, Tyndall phenomenon is negative described ginsenoside-Rd solution in room temperature, cold preservation and freezing placement.After preferably described ginsenoside-Rd solution at room temperature being placed a moment or spend the night after being preserved 5-50 days, preferred 36 days under room temperature and 2～6 ℃ of conditions, after preserving 48 hours under-20 ℃ of conditions or after preserving 36 days under-20 ℃ of conditions, the Tyndall phenomenon of described ginsenoside-Rd solution is negative.
In an embodiment of the invention, the content of ginsenoside-Rd is 5～75mg/ml in the described solution.
In a preference, the content of ginsenoside-Rd is 10～75mg/ml in the described solution, preferred 20～75mg/ml, more preferably 50～75mg/ml.
In another preference, the purity of described ginsenoside-Rd is 90-100wt%, and preferred purity is 95-99.99wt%, and more preferably purity is 95-99.8wt%.
In an embodiment of the invention, described ginsenoside-Rd solution is selected from lower group: greater than the ginsenoside-Rd of 25mg/ml～75mg/ml at 60-80 volume %, the solution in the aqueous solution of propylene glycol of preferred 75 volume %; Greater than the ginsenoside-Rd of 10mg/ml～25mg/ml at 55-75 volume %, the solution in the aqueous solution of propylene glycol of preferred 60 volume %; Or the ginsenoside-Rd of 5～10mg/ml is at 55-75 volume %, the solution in the aqueous solution of propylene glycol of preferred 55 volume %.
In a preference, described ginsenoside-Rd solution is the solution that the 5-25mg/ml ginsenoside-Rd forms at 60 volume % aqueous solution of propylene glycol.
In another preference, described solution is≤solution that the 5mg/ml ginsenoside-Rd forms in 55～75 volume % aqueous solution of propylene glycol, or the solution that forms in 75 volume % aqueous solution of propylene glycol of 5～75mg/ml ginsenoside-Rd.
In another preference, described ginsenoside-Rd solution is external, for oral administration or injection preparation form, preferred intravenous injection liquid, intramuscular injection liquid, oral administration solution, spray form.
In a second aspect of the present invention, a kind of method for preparing Radix Ginseng saponin of the present invention-Rd solution is provided, described method comprises: (a) aqueous solution of propylene glycol with contained propylene glycol 〉=50 volume % dissolves ginsenoside-Rd; (b) dilution step (a) gained solution randomly, the solution take the content that obtains ginsenoside-Rd as 1～100mg/ml solution.
In a preference, used diluent is the aqueous solution of propylene glycol that water or propylene glycol concentration are lower than used aqueous solution of propylene glycol in the step (a) in the step (b).
In an embodiment of the invention, described method comprises: behind the aqueous solution of propylene glycol dissolving ginsenoside-Rd with 100 volume %, gained solution is diluted again.
In a third aspect of the present invention, a kind of pharmaceutical composition is provided, it comprises: ginsenoside-Rd solution of the present invention and pharmaceutically acceptable carrier.
In a preference, comprise 0.01～100wt% ginsenoside-Rd solution of the present invention in the described pharmaceutical composition, preferred 0.1～99.9wt%, more preferably 1-99wt%.
In another preference, comprise the ginsenoside-Rd solution of the present invention of 0.5-7.5wt% in the described pharmaceutical composition, more preferably 0.5-2.5wt%.
In another preference, the purity of described ginsenoside-Rd is 90-100wt%, and preferred purity is 95-99.99wt%, and more preferably purity is 95-99.8wt%.
In another preference, described pharmaceutical composition is external, for oral administration or injection preparation form, preferred intravenous injection liquid, intramuscular injection liquid, oral administration solution, spray form, more preferably described injection preparation form is that intravenous injection is used or the intramuscular injection dosage form, more preferred intravenous injection dosage form.
In another preference, described pharmaceutical composition also comprises other active substance, and described other active substance is selected from: anti-inflammatory agent, immunosuppressant, acute ischemic cerebral apoplexy therapeutic agent or anti-organ-graft refection's agent.
In a fourth aspect of the present invention, a kind of method for preparing the pharmaceutical composition that contains ginsenoside-Rd is provided, described method comprises: ginsenoside-Rd solution of the present invention is mixed with pharmaceutically acceptable carrier.
In a preference, described method comprises by method of the present invention and prepares ginsenoside-Rd solution, and gained solution is mixed with pharmaceutically acceptable carrier.
In another preference, described method also comprises with diluent dilutes ginsenoside-Rd solution of the present invention, preferably dilutes 1-10 doubly, more preferably dilutes 1-5 doubly, most preferably dilutes 1-2 doubly.Described diluent is: injection normal saline, glucose solution or G/NS.
In another preference, described pharmaceutical composition is external, for oral administration or injection preparation form, preferred intravenous injection liquid, intramuscular injection liquid, oral administration solution, spray, more preferably described injection preparation form is that intravenous injection is used or the intramuscular injection dosage form, more preferred intravenous injection dosage form.In another preference, described pharmaceutically acceptable carrier is solvent for injection, and is preferred: the buffering injection solution that injection normal saline, glucose solution or G/NS or other are commonly used clinically.
In a fifth aspect of the present invention, provide ginsenoside-Rd or ginsenoside-Rd solution of the present invention for the preparation of the purposes in antiinflammatory, immunosuppressant and/or anti-organ-graft refection's the medicine.
In an embodiment of the invention, described medicine for antiinflammatory is used for the treatment of and is selected from lower group inflammatory diseases: rheumatic, rheumatoid arthritis, ankylosing spondylitis, rheumatic valvulitis, tuberculous meningitis, pleuritis or pericarditis; Describedly be used for the treatment of or auxiliary treatment is selected from lower group immune correlated disease: the inflammatory disease of the skin that the immunological rejection of autoimmune disease, organ transplantation postoperative, IV allergic reaction type are participated in for the immunosuppressant medicine.
In a preference, described autoimmune disease is selected from: rheumatic and rheumatoid arthritis, systemic lupus erythematosus (sle), myasthenia gravis, autoimmune hemolytic anemia or nephrotic syndrome or ulcerative colitis.
Description of drawings
Fig. 1 has illustrated ginsenoside-Rd solution to the dermatoplastic impact of allograft transplantation, is depicted as the photo of rear the 12nd day skin grafts of operation.Wherein, be respectively shown in each photo:
Figure 1A: Normal group (autotransplantation);
Figure 1B: model control group (55 volume % aqueous solution of propylene glycol, im, 0.2ml/100g body weight);
Fig. 1 C: positive controls (cyclosporin A injection, 5mg/kg body weight, im, 0.2ml/100g body weight);
Fig. 1 D:Rd low dose group (12.5mg Rd/kg body weight, im, 0.2ml/100g body weight, ginsenoside-Rd content 6.25mg/ml);
Dosage group among Fig. 1 E:Rd (25mg Rd/kg body weight, im, 0.2ml/100g body weight, ginsenoside-Rd content 12.5mg/ml);
Fig. 1 F:Rd high dose group (50mg Rd/kg body weight, im, 0.2ml/100g body weight, ginsenoside-Rd content 25mg/ml).
The specific embodiment
It is solvent that present ginsenoside-Rd injection adopts 20 volume % propylene glycol, and than under the low environment, the active medicine dissolubility reduces, and easily separates out precipitation in temperature, and impact is used and safety.And the clinical practice of existing ginsenoside-Rd also only limits to treat acute ischemic cerebral apoplexy disease etc.
In order to address these problems, the inventor conducts in-depth research, and find unexpectedly can be by adjusting propylene glycol concentration so that ginsenoside-Rd dissolve fully, thereby obtain the ginsenoside-Rd true solution, improve its dissolubility and stability.The inventor has also further studied the purposes of ginsenoside-Rd, and found that ginsenoside-Rd (ginsenoside-Rd true solution especially of the present invention) has good antiinflammatory and immunosuppressive action, thereby can be used for clinical treatment inflammatory diseases and immune correlated disease.On this basis, the inventor has finished the present invention.
Ginsenoside-Rd and aqueous solution of propylene glycol
Among the present invention, term " ginsenoside-Rd ", " active substance of the present invention " and " active medicine " are used interchangeably, and all refer to have the chemical compound of structural formula shown in the background technology part.
The ginsenoside-Rd that the present invention is used can be isolated through multistep in Chinese medicine Araliaceae (for example Radix Notoginseng), also can be synthetic by the method for chemosynthesis.In the present invention, preferably using purity is ginsenoside-Rd more than 90, and preferred purity is 95-99.99wt%, and more preferably purity is 95-99wt%.
Among the present invention, term " propylene glycol " refers to 1, the 2-propylene glycol, " aqueous solution of propylene glycol " refers to the double solvents that is comprised of propylene glycol and water, for example " aqueous solution of propylene glycol of 55 volume % " or " aqueous solution of propylene glycol of 55 volume % " all refer to cumulative volume take aqueous solution of propylene glycol as benchmark, and contained propylene glycol is the aqueous solution of propylene glycol of 55 volume %.In the present invention, " aqueous solution of propylene glycol of 100 volume % " refer to pure propylene glycol, and are wherein not moisture.
Ginsenoside-Rd true solution and preparation method thereof
In the present invention, term " true solution " refers to a kind of homodisperse system, and solute is distributed in the solvent and between the two equably with molecular size and exists without the interface.That the principal character of true solution shows as is transparent, scattering of light can not occur, diffusion velocity is fast, solute and solvent all can pass through semipermeable membrane.Under certain condition, the solute of true solution and solvent can automatically not be separated into biphase, are the thermodynamically metastable fixed system.
In the present invention, term " ginsenoside-Rd solution ", " ginsenoside-Rd solution of the present invention ", " ginsenoside-Rd true solution " or " solution of the present invention " are used interchangeably.In the present invention, if gained solution clear without Tyndall phenomenon, and had thermodynamic stability in rear 5 days in room temperature, cold preservation and freezing placement, then be judged to be " true solution ".
Existing aqueous solution of propylene glycol with 20 volume % is not true solution as the ginsenoside-Rd injection (ginsenoside-Rd content 10mg/ml) of solvent, and it has opalescence to exist.Although it is qualified that this solution detects clarity by the pharmacopeia method, its Tyndall phenomenon is positive, and the granule that has 1-100nm is described.Under the low temperature, this part particle's Brownian movement weakens, so that the easy gathering of granule is agglomerating, thereby mass penalty forms precipitation, thereby so that the dissolubility of preparation is out of control.
The present invention obtains the true solution of ginsenoside-Rd by the concentration that improves aqueous solution of propylene glycol, thereby so that described solution all has good thermodynamic stability after room temperature, cold preservation and freezing placement, and adopt dicyandiamide solution of the present invention can so that the concentration of ginsenoside-Rd in solution be improved, thereby be suitable for multiple new purposes.
Particularly, solvent for use is the aqueous solution of propylene glycol of contained propylene glycol 〉=50 volume % in the ginsenoside-Rd solution of the present invention, the aqueous solution of propylene glycol of preferred 55～100 volume %, the more preferably aqueous solution of propylene glycol of 55～80 volume %, the most preferably aqueous solution of propylene glycol of 55～75 volume %.As preparing injection with this solution, the concentration of propylene glycol is unsuitable too high, for example can be 55～60 volume %, preferred 60 volume %.
The content of ginsenoside-Rd can be 1～100mg/ml in ginsenoside-Rd solution of the present invention, preferred 5～75mg/ml, more preferably 10～75mg/ml, 20～75mg/ml or 50～75mg/ml.(for example in 20～75mg/ml) the situation, the aqueous solution of propylene glycol that preferably adopts high concentration is solvent, for example the aqueous solution of propylene glycol of 60～75 volume % obtaining higher ginsenoside-Rd content.
Preferred described ginsenoside-Rd solution is≤solution that the 5mg/ml ginsenoside-Rd forms in 55～75 volume % aqueous solution of propylene glycol in the present invention, the solution that 5～75mg/ml ginsenoside-Rd forms in 75 volume % aqueous solution of propylene glycol, or the more preferably solution that in 60 volume % aqueous solution of propylene glycol, forms of 5～25mg/ml ginsenoside-Rd.
In the ginsenoside-Rd true solution of the present invention, no matter the content of ginsenoside-Rd is higher than or is lower than existing aqueous solution of propylene glycol with 20 volume % as the ginsenoside-Rd injection (ginsenoside-Rd content 10mg/ml) of solvent, its stability all is better than existing injection.
Ginsenoside-Rd solution of the present invention is preserved the long period (for example 12 days, 14 days or 50 days) and preserve the short period (24h for example under freezing (20 ℃) condition under room temperature and cold preservation (2～6 ℃) condition, all can not occur Precipitation 48h) or have opalescence to produce, and after described solution is under freezing conditions preserved the long period (for example 36 or 60 days), place at room temperature after moments later jolting or the jolting that room temperature places behind the certain hour that solution recovers clarification and without opalescence.
Radix Ginseng saponin of the present invention-Rd solution can be prepared as follows: method A. directly dissolves the ginsenoside-Rd powder with the aqueous solution of propylene glycol of variable concentrations, to obtain required ginsenoside-Rd concentration; Or method B. is dissolved in ginsenoside-Rd first in the pure propylene glycol (i.e. 100% aqueous solution of propylene glycol), dilute so that the content of ginsenoside-Rd reaches 1～100mg/ml solution, and so that aqueous solution of propylene glycol reaches required concentration again.Method for optimizing B, used diluent is water or propylene glycol weak solution (being that propylene glycol concentration is lower than required propylene glycol final concentration) in the dilution.
Pharmaceutical composition
A kind of pharmaceutical composition is provided in the present invention, and it contains ginsenoside-Rd solution of the present invention and pharmaceutically acceptable carrier.Among the present invention, term " contains " the various compositions of expression and can be applied to together in mixture of the present invention or the compositions.Therefore, term " mainly by ... form " and " by ... composition " be included in during term " contains ".Among the present invention, term " pharmaceutically acceptable " composition refers to be applicable to people and/or animal and without excessive bad side reaction (such as toxicity, stimulation and allergy), the material of rational benefit/risk ratio is arranged namely.
Among the present invention, term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, itself does not induce the harmful antibody of individuality that produces accepting said composition, and after the administration or give do not have undue toxicity after the health product.In " Lei Mingdun pharmaceutical science " (Remington ' s Pharmaceutical Sciences, Mack Pub.Co., N.J.1991), can find discussing fully about pharmaceutically acceptable carrier.
In an embodiment of the invention, take the gross weight of described pharmaceutical composition as benchmark, comprise 0.01～100wt% ginsenoside-Rd solution of the present invention in the described pharmaceutical composition, preferred 0.1～99.9wt%, more preferably 1-99wt%.(wt% refers to ginsenoside-Rd solution shared percentage by weight in pharmaceutical composition herein, and unit of weight is gram).In an embodiment of the invention, comprise the ginsenoside-Rd solution of the present invention of 0.5-7.5g% (W/V) in the described pharmaceutical composition, preferred 0.5-7.5g% (W/V), more preferably 0.5-2.5g% (W/V).(unit wherein (W/V) refers to the grams of the ginsenoside-Rd solution that contains in every 100ml pharmaceutical composition).In another embodiment of the present invention, take the pharmaceutical composition cumulative volume as benchmark, the amount of contained ginsenoside-Rd is 5-100mg/ml in the described pharmaceutical composition, more preferably 5-75mg/ml, most preferably 5-25mg/ml.
Pharmaceutical composition of the present invention can be external, for oral administration or injection preparation form, preferred intravenous injection liquid, intramuscular injection liquid, oral administration solution, spray, more preferably described injection preparation form is that intravenous injection is used or the intramuscular injection dosage form, more preferred intravenous injection dosage form.
When the pharmaceutical composition (being the ginsenoside-Rd solution) of preparation liquid form of the present invention, can adopt following method: prepare ginsenoside-Rd solution of the present invention; Then with water for injection with ginsenoside-Rd solution dilution 0-10 doubly, preferred 0-5 times, more preferably 0-3 times, most preferably 0-2 times, and with gained diluent and solution solvent.Described solution can be injection, external preparation or Oral preparation.In an embodiment of the invention, described solution solvent is solvent for injection, and it is preferably injection normal saline, glucose solution or G/NS.
In another embodiment of the present invention, described pharmaceutical composition also comprise other active substance or can with these active substance couplings, described other active substance is selected from: anti-inflammatory agent, immunosuppressant, acute ischemic cerebral apoplexy therapeutic agent or anti-organ-graft refection's agent.
Antiinflammatory action
Inflammation is to have the defense reaction that the biological tissue of vascular system occurs damage factor, symptom or the complication of numerous disease, because virulence factor is different and physical function difference, inflammation is not quite similar serious inflammatory reaction even life-threatening to the infringement of tissue and body.Anti-inflammatory agent is as the second largest class medicine of clinical application, consumption is only second to anti-infective, chemosynthesis anti-inflammatory agent commonly used mainly contains adrenocortical hormones and nonsteroidal antiinflammatory drug, it is found that in prolonged application there are many untoward reaction in this AID, this is restricted the clinical practice of chemosynthesis anti-inflammatory agent, and people more and more pay attention to the less anti-inflammatory drug of exploitation toxic and side effects from Chinese medicine.
The discovered in recent years Radix Notoginseng total arasaponins has obvious antagonism to various acute exudative inflammation model, ginsenoside-Rd is separation and Extraction activated monomer out from Radix Notoginseng total arasaponins, studies its anti-inflammatory activity and is intended to provide theoretical foundation for excavating the effective antiinflammatory medicine.This experiment has adopted the swollen model of rat and mice chondrus ocellatus Holmes colloidality foot that the antiinflammatory performance of Radix Ginseng saponin of the present invention-Rd solution is studied.
This paper result of study shows that iv and im ginsenoside-Rd have remarkable inhibitory action to rat and sufficient the swelling of mice chondrus ocellatus Holmes colloidality, point out this medicine that the acute inflammation process of oozing out is had obvious inhibitory action, illustrate that Rd has good antiinflammatory action.The mechanisms of anti-inflammatory of Rd may be relevant with the release of inflammation-inhibiting medium and inflammatory cytokine, about the mechanisms of anti-inflammatory of Rd, remains further to be furtherd investigate.
With reference to this experimental result, ginsenoside-Rd antiinflammatory biological activity is clear and definite, along with the further investigation of its pharmacological action and mechanism of action thereof, ginsenoside-Rd can be used as anti-inflammatory agent and tries out clinically in the treatment of the inflammation diseases such as rheumatic, rheumatoid arthritis, ankylosing spondylitis, rheumatic valvulitis, tuberculous meningitis, pleuritis, pericarditis.
Immunosuppressive action
Delayed hypersensitivity is a kind of cell immune response of the T of dependence cell, and its principal character is that the sensitization body the struvite pathology damage of delayed occurs at antigen attack position.Adopted 2 among the present invention, 4-dinitrochlorobenzene (DNCB) brings out the part that makes mice and produces delayed allergy, be about to be applied in the mice abdominal skin after the low molecule hapten DNCB dilution of chemistry, be combined into complete antigen with skin protein, stimulate thus the T lymphopoiesis to become primed lymphocyte, after 4 days DNCB is applied in mice ear and attacks, make the local delayed allergy that produces.
Immunosuppressive action for research ginsenoside-Rd solution, the inventor has taked multiple dosage regimen in experiment, wherein take the IV allergic reaction type induce phase (sensitization phase) as main omnidistance administration and antigen attack after the effect of the delayed hypersensitivity two kinds of dosage regimens of administration that (excite phase) mutually, confirm that all ginsenoside-Rd has remarkable inhibitory action to DNCB induced mice delayed hypersensitivity, and excite front single to give Rd to the not impact of mice delayed hypersensitivity.Owing to induce take immunoreation as main, effect is take inflammatory reaction as main, ginsenoside-Rd all can obviously suppress the IV allergic reaction type in this two-phase administration, point out this medicine may have the dual function of immunosuppressant and antiinflammatory, and our existing experiment confirm ginsenoside-Rd has significant antiinflammatory action, and is consistent with this experimental result.
Its morbidity of atopic dermatitis, contact or the skin allergic diseases such as allergic dermatitis, eczema is except having the I allergic reaction type participates in clinically, and is also normal relevant with the IV allergic reaction type, and the performance of IV allergic reaction type often is active chronic inflammation.Experimental result shows, ginsenoside-Rd has the effect of significant antiinflammatory and anti-IV type allergy, can suppress generation and the development of delayed hypersensitivity, and can more promptly improve the skin inflammatory symptoms, this may treat clinically the inflammatory disease of the skin that the IV allergic reaction type participates in for it theoretical foundation is provided.
Organ transplantation is the whole effective and necessary treatment means of saving patients ' lives latter stage of Organ Failure, postoperative patient need be taken immunosuppressant all the life, immunosuppressant poor specificity take ciclosporin A and tacrolimus (FK506) as representative, toxicity are high at present, can cause the toxic and side effects of the internal organs such as tumor, severe infections and liver, kidney, the immunosuppressant of research high-efficiency low-toxicity is the emphasis of current medical research.Be the immunosuppressive action of research ginsenoside-Rd, we have studied the impact of ginsenoside-Rd on human T lymphocyte's propagation, and discovery can obviously suppress human T lymphocyte's propagation that ConA causes, and is concentration dependent.Allograft transplantation skin transplantation model has also been adopted in this experiment, this is the organ transplantation classical model, the impact of skin grafts mean survival time is investigated the anti-immunologic rejection effect of ginsenoside-Rd by observing ginsenoside-Rd, the result shows that ginsenoside-Rd can effectively prolong the time-to-live of skin grafts, has immunosuppressive action.
Thus, the inventor has proved that ginsenoside-Rd can be used as clinically immunosuppressant and is used for the various autoimmune disease, such as rheumatic and rheumatoid arthritis, systemic lupus erythematosus (sle), myasthenia gravis, autoimmune hemolytic anemia, nephrotic syndrome, ulcerative colitis etc., also can be used for the clinically immunological rejection of organ transplantation postoperative, but its immunosuppressive action is weaker than CsA, point out it clinically mainly as the adjuvant drug of other immunosuppressant, reduce untoward reaction by the consumption that reduces other immunosuppressant after share.
Advantage of the present invention
Concentration by adjusting the solvent aqueous solution of propylene glycol so that ginsenoside-Rd dissolve fully, thereby obtain the true solution of ginsenoside-Rd, improve its dissolubility and stability;
2. can improve in the injection activity substance content or reduce the injecting drug use volume;
3. found the new purposes of ginsenoside-Rd, namely ginsenoside-Rd is used for the new purposes that antiinflammatory, immunosuppressant and anti-organ transplantation are got rid of, thereby can be used for clinical treatment inflammatory diseases and immune correlated disease.
Those of ordinary skills also can realize other advantage of the present invention by description of the invention, and are low, easy to prepare etc. such as cost.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to normal condition or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise percentage ratio and umber calculate by weight.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that better implementation method described in the literary composition and material only present a demonstration.
The formulating of the low concentration ginsenoside-Rd solution of embodiment 1. propylene glycol hydrotropies
Purpose: adjust propylene glycol concentration so that ginsenoside-Rd can dissolve fully, in the hope of the acquisition true solution, thereby guarantee clinical drug safety.
Method: select different volumes than (20 volume %, 30 volume %, 40 volume %, 50 volume %, 55 volume %, 60 volume %, 75 volume % and 100 volume %) aqueous solution of propylene glycol as solvent, content with 10mg/ml and 5mg/ml adds ginsenoside-Rd, determines the propylene glycol concentration that ginsenoside-Rd is fully dissolved.After obtaining true solution, under freezing or refrigerated condition, investigate the dissolved state of medicinal liquid.Useful formulations with adding in the 250ml normal saline mixing after the water for injection dilution or directly using normal saline dilution, is investigated the dissolved state in the drug dilution process.
One, to dissolving ginsenoside-Rd fully and obtaining the selection of the propylene glycol concentration of true solution
1. material
The ginsenoside-Rd pressed powder (is provided lot number: 050815) by GuangDong TaiHe Biology Pharmacy Co., Ltd;
Propylene glycol (Beijing Chemical Plant produces for 1,2-PD, analytical pure).
2. method
Get respectively the ginsenoside-Rd pressed powder, be dissolved in 10mg/ml in the aqueous solution of propylene glycol of variable concentrations.Propylene glycol concentration is 20 volume %, 30 volume %, 40 volume %, 50 volume %, 60 volume %, 75 volume % or 100 volume %.The compound method of sample has 2 kinds: the A method: the aqueous solution of propylene glycol with variable concentrations directly dissolves the ginsenoside-Rd pressed powder; The B method: first the ginsenoside-Rd pressed powder is dissolved in the pure propylene glycol, it is quantitative to add water again, makes propylene glycol reach desired concn.Place cold preservation under the low temperature (2-6 ℃) to preserve gained solution, observe solution state, detect its Tyndall phenomenon, be about to sample and place the darkroom, during with a strong beam irradiating liquids, can form the colloid that is of light " path ".The phenomenon of the bright path of this formation is Tyndall phenomenon, and true solution does not have Tyndall phenomenon.The Tyndall phenomenon feminine gender is then thought favorable solubility, can judge that it is true solution.The results are shown in Table 1-1:
The selection of table 1-1. propylene glycol concentration (V/V volume ratio)
*The expression sample represents sample A method preparation with B method preparation, #
3. results and analysis:
All samples is placed under the low temperature (2 ℃～6 ℃) and preserves.Behind the 12h, propylene glycol concentration is that the sample of 20 volume % and 30 volume % all has Precipitation.When placing 1 day, propylene glycol concentration is that the sample of 40 volume % also has precipitation to generate.In the time of can tentatively inferring propylene glycol concentration≤40 volume %, can not dissolve ginsenoside-Rd fully.In the time of the 9th day, No. 8 (propylene glycol concentration is that 50 volume % have faint opalescence to the sample of A method configuration, and Tyndall phenomenon is positive.When low temperature was placed 14 days, the sample of the Tyndall phenomenon that occurs had precipitation or white particle to separate out.
And same time point, with propylene glycol No. 3 samples identical with ginsenoside-Rd concentration of B method preparation, dissolubility is better.Can find out, when the propylene glycol concentration of sample 〉=50 volume %, can dissolve preferably ginsenoside-Rd (10mg/ml), obtain true solution.The dissolubility that ginsenoside-Rd is described is relevant with propylene glycol concentration.Therefore, the concentration of adjustment solvent propylene glycol is to make ginsenoside-Rd fully dissolve a vital step.
Simultaneously, in process for preparation, be better than possibly directly using first aqueous solution of propylene glycol dissolving ginsenoside-Rd (A method) the gained sample of variable concentrations with the sample that adds again quantitative water (B method) preparation behind the pure propylene glycol dissolving Rd.Infer that thus propylene glycol solution increases the dissolubility of ginsenoside-Rd during the preparation of B method.
Two, the solution state under ginsenoside-Rd true solution cold preservation (2 ℃～6 ℃) or freezing (20 ℃) condition is investigated
As mentioned above, at room temperature obtain the ginsenoside-Rd true solution, under low temperature (2 ℃～6 ℃ and-20 ℃) condition, preserve, observe its solution state, and detect Tyndall phenomenon.Get the ginsenoside-Rd pressed powder, be dissolved in 10mg/ml and 5mg/ml in the propylene glycol of concentration 〉=50 volume % (preparation of B method).At room temperature solution clarification of the sample that makes, Tyndall phenomenon all is negative.Respectively sample is placed under the environment of 2 ℃～6 ℃ and-20 ℃ and preserves.Observe its solution state, detect its Tyndall phenomenon.The results are shown in Table 1-2,1-3:
Propylene glycol true solution solution state under refrigerated condition of table 1-2. ginsenoside-Rd is investigated the result
The propylene glycol true solution of table 1-3. ginsenoside-Rd under freezing conditions solution state is investigated the result
Preserve 36d under the table 1-4. freezing conditions, sample solution state after room temperature is placed a moment and spent the night is investigated the result
Results and analysis
With the sample of B method preparation, at room temperature, when propylene glycol concentration was between 50～75 volume %, ginsenoside-Rd added with 5mg/ml and 10mg/ml content, the solution clarification, and Tyndall phenomenon is negative.
With the sample of B method preparation, investigate result (table 1-2) during cold preservation, all samples is when preserving for 2 ℃, and the content of ginsenoside-Rd is that the sample of 5mg/ml and 10mg/ml does not have Precipitation in 2d, and Tyndall phenomenon is negative.And place 12d, the visible faint opalescence of No. 5 samples (propylene glycol concentration=50 volume %, the content of ginsenoside-Rd are 10mg/ ml) at 2 ℃.During 2 ℃ of placements (50d) more of a specified duration, No. 5 sample adularescent granule is separated out.
Sample with the preparation of B method, under freezing conditions investigate (table 1-3), the result works as all samples and is placed on 24h under-20 ℃ of environment, ginsenoside-Rd content is that 10mg/ml, propylene glycol concentration are that the sample Tyndall phenomenon of 50～60 volume % is positive, and propylene glycol concentration is the sample clarification of 75 volume % and without opalescence.And also visible faint opalescence of No. 1 sample (propylene glycol concentration is 50 volume %, and ginsenoside-Rd content is 5mg/ml).All the other sample solution clarifications, Tyndall phenomenon is negative.But place some days (36d), propylene glycol concentration≤60 volume %, ginsenoside-Rd content is that the sample of 10mg/ml or 5mg/ml has Precipitation or produces muddy.And propylene glycol concentration is 75 volume %, and ginsenoside-Rd content is that the sample of 10mg/ml or 5mg/ml is still clarified and without opalescence.Preserved 36 days sample under the freezing conditions in the lower placement of room temperature (18 ℃) for a moment, ginsenoside-Rd is that (propylene glycol concentration is that 50～60 volume % and ginsenoside-Rd are that (propylene glycol concentration is that 60 volume % precipitation can be dissolved rapidly by jolting, again the clarification of the sample solution after the dissolving and without opalescence for the sample of 10mg/ml for the sample of 5mg/ml.And propylene glycol concentration is 50 volume % and 55 volume %, and ginsenoside-Rd is can not at once dissolve after the sample jolting of 10mg/ml, need spend the night to place and could clarify and without opalescence (showing 1-4).
The result shows, when the content of ginsenoside-Rd is 5mg/ml, propylene glycol concentration 〉=55 volume %, no matter under room temperature, cold preservation (2～6 ℃) or freezing (20 ℃) condition, preserve the short period, the situations such as Precipitation or opalescence (Tyndall phenomenon is positive) can not appear, but freezing (20 ℃) the lower holding time of condition is than long time (36 days), propylene glycol concentration is that opalescence or muddiness appear in the sample of 55 volume % and 60 volume %, and jolting can make solution again clarify and without opalescence after room temperature was placed.Can judging these two kinds, to contain ginsenoside-Rd be that the sample of 5mg/ml is clinical useful formulations.
Although and sample solution state under room temperature and cold preservation (2～6 ℃) environment of propylene glycol concentration to be the content of 55 volume %, ginsenoside-Rd be 10mg/ml is stable, the muddiness that produces when preserving 36 days under freezing (20 ℃) condition needs room temperature to place to spend the night could recover to clarify.Therefore, this type of solution can be preserved under refrigerated condition, but does not advise under freezing conditions preserving.
It should be noted that in addition the propylene glycol concentration that can add in the injection is generally 20～60 volume %, is more suitable for the requirement of production technology.Propylene glycol concentration is 75 volume %, the content of ginsenoside-Rd is that the sample of 5mg/ml and 10mg/ml all is true solution under room temperature, cold preservation or freezing conditions, but liquid viscosity increases during owing to the propylene glycol excessive concentration, producting process difficulty increases, can fall low viscous material by interpolation and make injection, select falling low viscous material according to this area general knowledge.
Three, dilution test
1. the impact of dilution process
One-step method dilution: ginsenoside-Rd solution is not added water and directly injects the 250ml normal saline;
The two step method dilution: sample thief 1ml (containing ginsenoside-Rd 10mg/ml) or 2ml (containing ginsenoside 5mg/ml) mix with isopyknic water, in the 250ml normal saline that reinjects.
Observe solution state, relatively the superiority-inferiority of the method for ginsenoside-Rd injection is prepared in two step method dilution or one-step method dilution, the results are shown in Table 1-5:
The comparison of table 1-5. one-step method dilution or two step method dilution
Propylene glycol concentration is 50～75 volume %, when ginsenoside's solubility is 5-10mg/ml, each preparation with muddiness can not appear after the water for injection equal-volume mixes.Ginsenoside-Rd content directly injects the 250ml normal saline at 10mg/ml, propylene glycol concentration at the sample of 50～75 volume % muddiness can occur.Ginsenoside-Rd content is at 5mg/ml, and propylene glycol concentration is directly injected the 250ml normal saline at the sample of 55～75 volume % also can be muddy.And it is muddy to select two step of water for injection dilution method that medicine is formed in the 250ml normal saline, and therefore repeatability better be more suitable in clinical practice.
2. the dilution amount of water is on the impact of middle sample dissolved state
During two step method dilution ginsenoside-Rd propylene glycol solution, too much such as amount of water, might cause medicine to be separated out owing to propylene glycol concentration reduces.Thereby during clinical dilution medicine, to amount of water individual span of control should be arranged.This experiment two step method dilute sample changes amount of water, the minimum water consumption when investigation sample and water mixedly appear muddiness.The results are shown in Table 1-6:
Table 1-6. amount of water is on the impact of middle sample dissolved state
During two step method dilution ginsenoside-Rd propylene glycol solution, when amount of water is too much, can cause that the solution Chinese medicine separates out, and so that occur serious muddy in the meso sample.And when injection Chinese medicine content increases, but amount of water is less.Reference table 1-6 result judges, adopts the ginsenoside-Rd propylene glycol solution to add water two-fold dilution's way, and the amount of water in the time of can guaranteeing drug dilution is away from causing muddy amount of water, and can be owing to clinical manipulation is forbidden to cause behind the drug dilution muddy.Therefore, in two step dilution methods, the consumption of control water for injection is a step of most critical in the dilution.
Four, about the discussion of embodiment 1
Original ginsenoside-Rd injection selects 20 volume % propylene glycol can not well make the ginsenoside-Rd pressed powder fully dissolve (referring to the background technology part) as solvent.Solution clarification under the room temperature, but Tyndall phenomenon is positive.
The inventor finds can obtain true solution by adjusting propylene glycol concentration by experiment unexpectedly.When propylene glycol concentration 〉=50 volume %, ginsenoside-Rd content is that the sample of 5mg/ml is preserved the short period and can be kept stable dissolved state under room temperature, cold preservation (2～6 ℃) and freezing (20 ℃) condition, Precipitation can not occur or opalescence is arranged and causes the underproof phenomenon of preparation.In 250ml normal saline that a times water for injection dilution is reinjected, muddiness can not appear, can supply clinical practice.Although this sample freezing (20 ℃) the lower holding time of condition than long time (36d), muddiness can occur or separate out precipitation, (18 ℃) can make it again dissolve by jolting for a moment fast under the room temperature but be placed on, again the sample solution clarification after the dissolving and without opalescence.Therefore, can select propylene glycol concentration is that the sample of 5mg/ml is as medicinal for 〉=55 volume %, ginsenoside-Rd content.These samples solution dissolved state under room temperature and refrigerated condition is good and without opalescence.Can occur precipitation in freezing (20 ℃) the lower holding time of condition than long time, but place under the room temperature after a moment, the jolting rapidly dissolvablely, again liquid clarification and without opalescence after the dissolving is true solution still, can be used as the clinical preparation of using.
Select the propylene glycol solution of higher concentration as solvent, can solve the problem that ginsenoside-Rd fully dissolves final acquisition true solution.If reduce simultaneously the content of unit volume ginsenoside-Rd to 5mg/ml, can guarantee also that then sample can not separate out precipitation yet or opalescence occur under-20 ℃ of conditions.
Investigate further and find: with pure propylene glycol dissolving ginsenoside-Rd preparating liquid, be better than the method with aqueous solution of propylene glycol dissolving ginsenoside-Rd.But when dissolved substance, the medicine of make suring dissolves the formation true solution fully, should add the survey opalescence.
When dilution, selected water for injection but not the original special-purpose diluent that uses in pharmaceutical factory.Reduce to a certain extent concentration and the consumption of propylene glycol, also reduced re-injecting the possibility that occurs muddiness in the 250ml normal saline, greatly increased convenience and the safety of clinical application.The preferred two step dilution methods that adopt dilute, and the injection water yield that adds is too much unsuitable.
The adjustment of the concentration by propylene glycol has solved the deliquescent problem of ginsenoside-Rd.When the concentration of propylene glycol 〉=50 volume %, unit volume dissolving 5mg or 10mg ginsenoside-Rd can obtain true solution under the room temperature, without opalescence, filter, packing is all very convenient.Cold preservation (2～6 ℃) or freezing (20 ℃) short period, ginsenoside-Rd content does not have Precipitation at the sample of concentration 55～75 volume % of 5mg/ml, propylene glycol.
Recommend the clinical practice sample: ginsenoside-Rd 5mg/ml, every 2ml, propylene glycol concentration 55%～75 volume %.But be that contained propylene glycol is just in time for 1.1ml in the 55 volume %2ml medicinal liquids in propylene glycol concentration, the Propanediol of giving to the people during with former clinical trial equates, does not have the problem of reappraising safety.
Embodiment 2. high concentration propylene glycol are on the deliquescent impact of high concentration ginsenoside-Rd
Purpose: increase the concentration of propylene glycol, increase successively the content of ginsenoside-Rd, investigate the concentration of propylene glycol to the deliquescent impact of ginsenoside-Rd.
Method: select the propylene glycol of concentration 〉=50 volume %, add ginsenoside-Rd [first with adding again quantitative water (B method) behind the pure propylene glycol dissolving ginsenoside-Rd powder] with different content (20mg/ml, 25mg/ml, 50 mg/ml, 75mg/ml) respectively.At room temperature placed some days, the sample that solution state is good places refrigerator cold-storage or freezing preservation, observes solution state, detects its Tyndall phenomenon.
1. material (with embodiment 1)
2. method
Select the propylene glycol of concentration 〉=50 volume %, add with different content (20mg/ml, 50mg/ml and 75mg/ml) respectively and the dissolving ginsenoside-Rd.Sample is placed in room temperature, observed solution state, detect Tyndall phenomenon.The results are shown in Table 2-1.Be that the sample of 60 volume % and 75 volume % places refrigerator cold-storage or-20 ℃ to take out room temperature preservation after freezing some hours again with propylene glycol concentration, observe solution state, detect Tyndall phenomenon.The results are shown in Table 2-2, table 2-3.
Propylene glycol concentration is on the deliquescent impact of ginsenoside-Rd under the table 2-1. room temperature
*Ginsenoside-Rd. room temperature
*60% propylene glycol, ginsenoside-Rd content are that the sample of 20mg/ml and 25mg/ml was at room temperature preserved 60 days, and solution is clarified without opalescence
The good sample of solution state under the table 2-2. room temperature, solution state and Tyndall phenomenon when refrigerator cold-storage (2 ℃) 25d
*60% propylene glycol, ginsenoside-Rd content are to preserve 60 days under the sample cold preservation of 20mg/ml and 25mg/ml, and solution is clarified without opalescence
Under the freezing preservation condition of table 2-3., the deliquescent relation of propylene glycol concentration and ginsenoside-Rd and the research of redissolving
The clarification of "-" expression solution is without opalescence
*60% propylene glycol, ginsenoside-Rd content be the freezing lower preservation of the sample of 20mg/ml and 25mg/ml after 60 days solution muddy, but under the replacement room temperature behind the 10min solution clarification without opalescence
3. results and analysis
Place in room temperature, concentration is the ginsenoside-Rd that the propylene glycol of 50 volume % can not dissolve 20～75mg/ml.Concentration is that the propylene glycol solution of 60 volume % and 75 volume % can dissolve relatively large ginsenoside-Rd, and the solution clarification is without opalescence (table 2-1).
Sample-20 ℃ muddy or freeze after freezing some hours, take out room temperature and place, the solution clarification is without opalescence (table 2-3).This with before experiment in the sample (propylene glycol concentration is 75 volume %, ginsenoside-Rd content 10mg/ml) prepared be consistent in refrigerator-20 ℃ solution clarification in freezing 36 days without the result of opalescence.As seen the propylene glycol of 75 volume % can than a small amount of ginsenoside-Rd of good dissolving, be stablized without opalescence at the cryopreservation solution state.And the propylene glycol dissolving more amount ginsenoside-Rd of working as 75 volume %, in refrigerator-20 ℃ freezing preservation, ginsenoside-Rd content is higher, easier freezing.Dissolving 75mg/ml ginsenoside-Rd was put in refrigerator-20 ℃ freezing preservation more than one month in the propylene glycol of 75 volume %, again place under the room temperature and can recover clarification a moment and without opalescence, can infer that concentration is that 75% propylene glycol is preferably solvent of dissolving ginsenoside-Rd.With this ginsenoside-Rd liquid preparation stable in properties of preparing.
Be that 60 volume %, ginsenoside-Rd content are that the sample of 20mg/ml and 25mg/ml places respectively under room temperature, cold preservation, the freezing conditions and preserved 60 days to propylene glycol concentration in addition, find that its solution under room temperature and cold storage environment is all clarified and without opalescence, although and the sample of freezing preservation occurs muddy but place and place behind the 10min solubilized under the room temperature and without opalescence, can clinical practice.
4. discuss
Experiment by embodiment 1 has recognized that: propylene glycol concentration 〉=55 volume %, and ginsenoside-Rd content is preserved under cold preservation or freezing conditions at the sample of 5mg/ml, and solution state is good and without opalescence.In order to investigate propylene glycol concentration to the deliquescent impact of ginsenoside-Rd, we have carried out the experiment of embodiment 2.
In this experiment, increase propylene glycol concentration and increase the content of ginsenoside-Rd, at room temperature place and observe solution state.As propylene glycol concentration 〉=55 volume %, when preferred concentration is 60 volume % and 75 volume %, can dissolve relatively large ginsenoside-Rd, and the solution clarification is without opalescence.Propylene glycol concentration is the sample solution clarification of 75 volume % and without opalescence.With sample place refrigerator-20 ℃ freezing some hours muddy or freeze after, take out room temperature and place, solution clarification and without opalescence.Be that 75 volume %, ginsenoside-Rd content are freezing long period holding time of sample of 75mg/ml to propylene glycol concentration, sample freezes.Placing under the room temperature a moment is solubilized and without opalescence.And propylene glycol concentration is 75 volume %, ginsenoside-Rd content be 10mg/ml sample-20 ℃ of freezing preservations more than one month solution state still well without opalescence.Be that 60 volume %, ginsenoside-Rd content are that the sample of 20mg/ml and 25mg/ml places respectively under room temperature, cold preservation, the freezing conditions and preserved 60 days to propylene glycol concentration, find that its solution under room temperature and cold storage environment is all clarified and without opalescence, although and the sample of freezing preservation occurs muddy but place and place behind the 10min solubilized under the room temperature and without opalescence, can clinical practice.Judge thus: concentration is the dissolubility that the propylene glycol of 60～75 volume % can increase ginsenoside-Rd preferably, obtains at room temperature and low temperature (2 ℃) stored refrigerated very stable solution all.It is some day that this sample is under freezing conditions preserved, and at room temperature places at once to recover clarification and without opalescence.
The conclusion that is drawn by above-mentioned experimental result is: increase the dissolubility that propylene glycol concentration can increase ginsenoside-Rd preferably.The dissolubility that just can obtain required true solution and increase ginsenoside-Rd by increasing propylene glycol concentration, the gained sample is preserved stable in properties at room temperature or freezing.
Embodiment 3. ginsenoside-Rds are on the impact of inflammation
This experiment adopts carrageenin as the acute inflammation derivant, it is injected in mice and rat paw are subcutaneous to bring out rat and the swollen model of mice foot is observed ginsenoside-Rd to the impact of inflammation.
1. animal
120 of Kunming mouses, male and female half and half, the cleaning level, body weight 18～22g, available from Lanzhou University's Experimental Animal Center, credit number: SCXK (sweet) 20050007; 96 of Wistar rats, male and female half and half, the SPF level, body weight 180～220g, available from Gansu college of traditional Chinese medicine scientific experiment center, credit number: SCXK (sweet) 20040006.
2. medicine
2.1 ginsenoside-Rd: batch number is provided by GuangDong TaiHe Biology Pharmacy Co., Ltd: 980303, be mixed with before use the solution of respective concentration in one day with the dissolving of 55% propylene glycol.
2.2 dexamethasone sodium phosphate injection: Tianjin Pharmaceutical Jiaozuo Co., Ltd. produces, batch number: 08020111, become respective concentration with normal saline dilution before use.
2.3 carrageenin: Wako Pure Chemical Industries, Ltd. produces, batch number: 037-09692, and 24h is mixed with the solution that concentration is 1% (W/V) with normal saline before use, 4 ℃ of Refrigerator stores.
2.4 0.9% sodium chloride injection: Gansu Fuzheng Pharmaceutical Sci ﹠ Tech Co., Ltd. produces, batch number: 200709100102.
3. instrument
Electronic balance, model: FA1604S is made by Shanghai balance equipment factory;
The toes capacity measurer, model: YLS-7B is made by Shandong Academy of Medical Sciences equipment station.
4. method
4.1 intravenous injection (iv) various dose ginsenoside-Rd on Carrageenan causes the swollen impact of mice foot
60 of Kunming mouses, male and female half and half, body weight 18～22g is divided into 6 groups at random, and 10 every group, grouping and administration situation are as follows: first group: model control group, 55 volume % propylene glycol, iv, 0.1ml/10g body weight; Second group: positive controls, dexamethasone sodium phosphate, iv, 2mg dexamethasone sodium phosphate/kg body weight, 0.1ml injection/10g body weight; The 3rd group: Rd low dose group, iv, 4mg Rd/kg body weight, 0.1ml Rd injection/10g body weight; The 4th group: dosage group among the Rd, iv, 20mg Rd/kg body weight, 0.1ml Rd injection/10g body weight; The 5th group: Rd high dose group, iv, 100mg Rd/kg body weight, 0.1ml Rd injection/10g body weight; The 6th group: im Rd high dose group, 100mg Rd/kg body weight, 0.1ml Rd injection/10g body weight.
Each group causes inflammation at every group of every Mus left hind foot plantar subcutaneous injection 1% (W/V) carrageenin respectively to 1h behind the relative medicine, and 50 μ l/ only.Cause scorching rear 4h and cut left and right sides metapedes along the ankle joint, accurately weigh.Adopt following formula to calculate swelling and the inhibitory rate of intumesce of each group:
Swelling=left heavy sensation in the foot-right heavy sensation in the foot,
Statistical procedures uses Excel-2003 to add up, and each is organized data and is measurement data, represents with x ± s, and the significance of group difference is checked with t.
4.2 intramuscular injection (im) various dose Rd on Carrageenan causes the swollen impact of mice foot
60 of Kunming mouses, male and female half and half, body weight 18～22g is divided into 6 groups at random, and 10 every group, grouping and administration situation are as follows: first group: model control group, 55 volume % propylene glycol, im, 0.1ml/10g body weight; Second group: positive controls, dexamethasone sodium phosphate, im, 2mg dexamethasone sodium phosphate/kg body weight, 0.1ml injection/10g body weight; The 3rd group: Rd 12.5mg/kg body weight group, im, 0.1ml Rd injection/10g body weight; The 4th group: Rd25mg/kg body weight group, im, 0.1ml Rd injection/10g body weight; The 5th group: Rd 50mg/kg body weight group, im, 0.1ml Rd injection/10g body weight; The 6th group: Rd 100mg/kg body weight group, im, 0.1ml Rd injection/10g body weight.
Each organizes 1h behind the im relative medicine, causes inflammation at every group of every Mus right hind foot plantar subcutaneous injection 1% (W/V) carrageenin respectively, and 50 μ l/ only.Cause scorching rear 4h and cut left and right sides metapedes along the ankle joint, accurately weigh.Adopt the formula in 4.1 to calculate swelling and the inhibitory rate of intumesce of each group, and in 4.1, carry out statistical procedures.
4.3 intramuscular injection (im) various dose Rd on Carrageenan causes the swollen impact of rat foot
96 of Wistar rats, 180～220g, male and female half and half are divided into 6 groups at random, and 16 every group, grouping and administration situation are as follows: first group: Normal group; Second group: model control group, 55 volume % propylene glycol, im, 0.2ml/100g body weight; The 3rd group: positive controls, dexamethasone sodium phosphate, 1mg/kg body weight, 0.2ml injection/100g body weight, im; The 4th group: Rd low dose group, im, 12.5mg Rd/kg body weight, 0.2ml Rd injection/100g body weight; The 5th group: dosage group among the Rd, im, 25mg Rd/kg body weight, 0.2ml Rd injection/100g body weight; The 6th group: Rd high dose group, im, 50mg Rd/kg body weight, 0.2ml Rd injection/100g body weight.
Make a mark line in rat left hind ankle with marking pen before the administration, and measure left sufficient normal foot sole of the foot volume (mark line is following), double, ask its meansigma methods as basic volume.Each organizes 1h behind the im relative medicine, cause inflammation at each Mus left hind foot plantar subcutaneous injection 1% carrageenin respectively, 0.1ml/ only, Normal group subcutaneous injection normal saline, measure respectively and cause scorching rear 1h, 2h, 4h, the left whole sole of the foot volume of 6h (measuring by mark line before the administration) calculates swelling and inhibitory rate of intumesce.
The scorching front foot sole of the foot volume of the scorching metapedes sole of the foot volume of swelling=cause-cause
Statistical procedures uses Excel-2003 to add up, and each is organized data and is measurement data, represents with x ± s, and the significance of group difference is checked with t.
5. result
5.1 intravenous injection various dose ginsenoside-Rd on Carrageenan causes the swollen impact of mice foot
Intravenous injection Radix Ginseng saponin-Rd on Carrageenan causes the sufficient swollen remarkable inhibitory action that has of mice, and dose-effect relationship is arranged, can obviously alleviate the symptom such as red, swollen of carrageenin induced mice metapedes, compare with the model control group paw swelling, during dexamethasone 2mg/kg body weight iv, P＜0.01 (inhibitory rate of intumesce is 43.09%), during ginsenoside-Rd 4mg/kg body weight iv, P＞0.05 (inhibitory rate of intumesce is 18.26%), during ginsenoside-Rd 20mg/kg body weight iv, P＜0.05 (inhibitory rate of intumesce is 19.10%); During ginsenoside-Rd 100mg/kg body weight iv, P＜0.01 (inhibitory rate of intumesce is 43.76%), during ginsenoside-Rd 100mg/kg body weight im, P＜0.01 (inhibitory rate of intumesce is 49.40%), antiphlogistic effects is remarkable, and experimental result is shown in table 3-1.
Table 3-1 intravenous injection various dose ginsenoside-Rd on Carrageenan causes the swollen impact (x ± s, n=10) of mice foot
Annotate: compare with model control group,
*P＜0.05,
*P＜0.01
5.2 intramuscular injection various dose ginsenoside-Rd on Carrageenan causes the swollen impact of mice foot
Compare with model control group, the intramuscular injection ginsenoside-Rd can obviously alleviate the red of carrageenin induced mice metapedes, the symptom such as swollen, the mice foot that can significantly suppress due to the carrageenin is swollen, and dose-effect relationship is arranged, compare with the model control group paw swelling, during dexamethasone 2mg/kg body weight im, P＜0.01 (inhibitory rate of intumesce is 35.43%), when ginsenoside-Rd is 12.5mg/kg body weight im, P＞0.05 (inhibitory rate of intumesce is 15.80%), ginsenoside-Rd is 25,50, during 100mg/kg body weight im, all＜0.01 (inhibitory rate of intumesce is respectively 44.47% to P, 53.80%, 58.33%), antiphlogistic effects is remarkable, and experimental result is shown in table 3-2.
Table 3-2 intramuscular injection various dose ginsenoside-Rd on Carrageenan causes the swollen impact (x ± s, n=10) of mice foot
Annotate: compare with model control group,
*P＜0.01
5.3 intramuscular injection various dose ginsenoside-Rd on Carrageenan causes the swollen impact of rat foot
The ginsenoside-Rd on Carrageenan causes sufficient the swelling of rat remarkable inhibitory action, and dose dependent is arranged.Model control group and Normal group relatively cause scorching rear each time point P all＜0.01, show the modeling success; Compare with the model control group paw swelling, dexamethasone is causing scorching rear each time point P all＜0.01, and the swollen suppression ratio of foot is respectively 59.30%, 62.82%, 65.63% and 78.70% at each time point; Each administration group except the ginsenoside-Rd low dose group cause scorching after 4hP＜0.05, all the other each groups at each time point P all＜0.01, the swollen suppression ratio of ginsenoside-Rd 12.5mg/kg body weight group foot is respectively 62.79% at each time point, 42.31%, 40.63% and 47.22%, the swollen suppression ratio of ginsenoside-Rd 25mg/kg body weight group foot is respectively 67.44% at each time point, 46.15%, 40.63% and 74.07%, the swollen suppression ratio of ginsenoside-Rd 50mg/kg body weight group foot is respectively 68.60% at each time point, 58.97%, 46.88% and 75.00%, antiphlogistic effects is remarkable, and experimental result is such as table 3-3, shown in the table 3-4:
Table 3-3 im various dose ginsenoside-Rd on Carrageenan causes the impact (x ± s, n=16) of scorching rear different time points rat paw edema degree
Annotate: compare with Normal group,
*P＜0.01; Compare with model control group,
▲P＜0.05,
▲ ▲P＜0.01
Table 3-4 im various dose ginsenoside-Rd causes the swollen suppression ratio of foot of scorching rear different time points at carrageenin
It is swollen that the result shows that vein and intramuscular injection ginsenoside-Rd all can significantly suppress rat and mice foot that carrageenin causes, paw swelling is obviously descended, and dose-effect relationship is arranged, and inhibitory rate of intumesce also is obvious dose dependent.Compare with the model control group paw swelling, mice is when iv (intravenous injection) dexamethasone 2mg/kg body weight, P＜0.01 (inhibitory rate of intumesce is 43.09%), during iv ginsenoside-Rd 4mg/kg body weight, P＞0.05 (inhibitory rate of intumesce is 18.26%), during iv ginsenoside-Rd 20mg/kg body weight, P＜0.05 (inhibitory rate of intumesce is 19.10%); During iv ginsenoside-Rd 100mg/kg body weight, P＜0.01 (inhibitory rate of intumesce is 43.76%); Mice is when im dexamethasone 2mg/kg body weight, P＜0.01 (inhibitory rate of intumesce is 35.43%), during im ginsenoside-Rd 12.5mg/kg body weight, P＞0.05 (inhibitory rate of intumesce is 15.80%), when im ginsenoside-Rd 25,50,100mg/kg body weight, P all＜0.01 (inhibitory rate of intumesce is respectively 44.47%, 53.80%, 58.33%); Rat is when im dexamethasone 1mg/kg body weight, causing scorching rear each time point P all＜0.01, the swollen suppression ratio of foot is respectively 59.30% at each time point, 62.82%, 65.63% and 78.70 %, im ginsenoside-Rd 12.5,25, during the 50mg/kg body weight, except ginsenoside-Rd 12.5mg/kg body weight dosage group cause scorching after 4h P＜0.05, all the other each dosage groups at each time point P all＜0.01, the swollen suppression ratio of ginsenoside-Rd 12.5mg/kg body weight group foot is respectively 62.79% at each time point, 42.31%, 40.63% and 47.22%, the swollen suppression ratio of ginsenoside-Rd 25mg/kg body weight group foot is respectively 67.44% at each time point, 46.15%, 40.63% and 74.07%, the swollen suppression ratio of ginsenoside-Rd 50mg/kg body weight group foot is respectively 68.60% at each time point, 58.97%, 46.88% and 75.00%.
6. conclusion
Ginsenoside-Rd all has significant antiinflammatory action when vein and intramuscular injection, its powerful anti-inflammatory activity is pointed out it to can be used as anti-inflammatory agent to have clinically extensive use, can try out in the treatment of the inflammation diseases such as rheumatic, rheumatoid arthritis, ankylosing spondylitis, rheumatic valvulitis, tuberculous meningitis, pleuritis, pericarditis.
Ginsenoside-Rd solution of the present invention and ginsenoside-Rd solution phase ratio of the prior art have higher stability and dissolubility, can contain the ginsenoside-Rd of higher concentration, therefore are more suitable for for clinical anti-inflammatory treatment.
The immunosuppressive action of embodiment 4. ginsenoside-Rds
The immunosuppressive action of ginsenoside-Rd is observed in the impact of survival of grafted skin time after human T lymphocyte's propagation that this experiment is induced mice delayed hypersensitivity (DTH), ConA by the research ginsenoside-Rd and the allograft transplantation skin transplantation.
One, ginsenoside-Rd (Rd) is on the impact of mice delayed hypersensitivity (DTH)
1 material
1.1 animal
202 of Kunming mouses, the SPF level, body weight 18～22g, male and female half and half, available from Gansu college of traditional Chinese medicine scientific experiment center, credit number: SCXK (sweet) 20040006.
1.2 medicine
Ginsenoside-Rd is provided by GuangDong TaiHe Biology Pharmacy Co., Ltd, lot number: 050815, and the administration proxima luce (prox. luc) is made into respective concentration with the dissolving of 55% propylene glycol; Dexamethasone sodium phosphate injection, Tianjin Pharmaceutical Jiaozuo Co., Ltd. produces, lot number: 08020111, before use with normal saline dilution to desired concn; DNFB (DNCB), upper marine Qin chemical reagent company limited is produced, lot number: 20080311, use before use acetone-Oleum Sesami (V
Acetone: V
Oleum SesamiBe 1: 1) be made into 1% (1g DNCB/100ml acetone-Oleum Sesami) concentration; Sodium sulfide (Na
2S), the Chengdu chemical reagent factory is produced, lot number: 910923-1.
1.3 the instrument electronic balance, model: FA1604S, balance equipment factory in Shanghai makes.
2. method
2.1 excite front single-dose on the impact of mice delayed hypersensitivity
84 of Kunming mouses, body weight 18～22g, male and female half and half are divided into 6 groups at random by body weight, and 14 every group, grouping and administration situation are as follows: first group: Normal group; Second group: model control group, 55 volume % propylene glycol solutions, im, 0.1ml/10g body weight; The 3rd group: positive controls, dexamethasone sodium phosphate, 2mg/kg body weight, im, 0.1ml injection/10g body weight; The 4th group: Rd low dose group, 25mg Rd/kg body weight, im, 0.1ml Rd injection/10g body weight; The 5th group: dosage group among the Rd, 50mg Rd/kg body weight, im, 0.1ml Rd injection/10g body weight; The 6th group: Rd high dose group, 100mg Rd/kg body weight, im, 0.1ml Rd injection/10g body weight.
Use 10%Na
2S (W/V) is with the about 3cm of mouse part skin unhairing
2The zone, only measure with 50 μ l/ in depilation zone and smear 1%DNCB sensitization next day, and the next day, strengthen again to smear with method.Respectively organized the intramuscular injection relative medicine on the 4th day after the sensitization, and 1h evenly smears 1%DNCB at the left ear tow sides of mice after administration, 10 μ l/ only attack.The dislocation of mice cervical vertebra is put to death behind the 24h, cuts two ears along the auricle baseline, lays auricle at the same position of two ears respectively with the 8mm borer, accurately weighs.As swelling, take off mouse thymus simultaneously and spleen is accurately weighed with the difference of left and right sides auricle weight, and thymus recast heavy take the spleen of every 10g body weight is as index and spleen index and thymus index respectively.
2.2 not simultaneously the phase multiple dosing on the impact of delayed hypersensitivity
118 of Kunming mouses, body weight 18～22g, male and female half and half are divided into 10 groups at random by body weight, and every group 11-12, grouping and administration situation are as follows: first group: Normal group; Second group: model control group, 55 volume % propylene glycol solutions, im, 0.1ml/10g body weight; The 3rd group: the omnidistance administration group of positive control, dexamethasone sodium phosphate, 2mg/kg body weight, im, 0.1ml injection/10g body weight; The 4th group: the omnidistance administration group of Rd low dosage, 25mg Rd/kg body weight, im, 0.1ml Rd injection/10g body weight; The 5th group: the omnidistance administration group of dosage among the Rd, 50mg Rd/kg body weight, im, 0.1ml Rd injection/10g body weight; The 6th group: the omnidistance administration group of Rd high dose, 100mg Rd/kg body weight, im, 0.1ml Rd injection/10g body weight; The 7th group: positive control excites phase administration group, dexamethasone sodium phosphate, 2mg/kg body weight, im, 0.1ml injection/10g body weight; The 8th group: the Rd low dosage excites phase administration group, 25mg Rd/kg body weight, im, 0.1ml Rd injection/10g body weight; The 9th group: dosage excites phase administration group among the Rd, 50mg Rd/kg body weight, im, 0.1ml Rd injection/10g body weight; The tenth group: the Rd high dose excites phase administration group, 100mg Rd/kg body weight, im, 0.1ml Rd injection/10g body weight.
Omnidistance administration group (the second～six group) experimental technique is as follows: mice by the body weight random packet after intramuscular injection give relative medicine, once a day, continuous 7 days, dosage and method were as implied above.For the first time used 10%Na the same day after the administration
2S is with the about 3cm of skin of abdomen unhairing
2The zone, be applied in mice depilation zone with sensitization with 1%DNCB 50 μ l/ Mus next day behind administration 1h, and the next day, again smears with method and to strengthen.1h after the administration in the 7th day evenly smears at the left ear tow sides of mice with 1%DNCB10 μ l/ Mus and to attack, and the dislocation of mice cervical vertebra is put to death behind the 24h, cuts two ears along the auricle baseline, lays auricle in two ear same area respectively with the 8mm borer, accurately weighs.As swelling, take off mouse thymus simultaneously and spleen is accurately weighed with the difference of left and right sides auricle weight, and thymus recast heavy take the spleen of every 10g body weight is as index and spleen index and thymus index respectively.Wherein, first group not sensitization only attack.
Excite phase administration group (the seven～ten group) experimental technique as follows: to use 10%Na
2S is with the about 3cm of mouse part skin unhairing
2The zone, only measure with 50 μ l/ in depilation zone and smear 1%DNCB with sensitization next day, and the next day, strengthen again to smear with method.Evenly smeared 1%DNCB at the left ear tow sides of mice on the 4th day after the sensitization, 10 μ l/ only attack, and immediately administration 1 time after attacking, 9 o'clock evenings of the same day are administered once again, the rear 1h that is administered once again at 12 o'clock at noon next day puts to death the dislocation of mice cervical vertebra, cut two ears along the auricle baseline, lay auricle at the same position of two ears respectively with the 8mm borer, accurately weigh.As swelling, take off mouse thymus simultaneously and spleen is accurately weighed with the difference of left and right sides auricle weight, and thymus recast heavy take the spleen of every 10g body weight is as index and spleen index and thymus index respectively.Statistical procedures uses Excel-2003 to add up, and each is organized data and is measurement data, represents with x ± s, and the significance of group difference is checked with t.
3. result
3.1 excite front single-dose on the impact of mice delayed hypersensitivity
Model control group and Normal group compare, and ear swelling degree P＜0.01 illustrates the modeling success.Positive controls and model control group compare, ear swelling degree and thymus index P＜0.05, and index and spleen index P＜0.01 illustrates that dexamethasone single-dose before exciting has the effect of remarkable inhibition delayed hypersensitivity; But the basic, normal, high dosage group of Rd and model control group are relatively, and ear swelling degree, index and spleen index, thymus index P all＞0.05 illustrate that Rd single-dose before exciting does not affect delayed hypersensitivity, and experimental result is shown in table 4-1.
Table 4-1 excites front single-dose DNCB to be caused the impact (x ± s, n=14) of mice delayed hypersensitivity
Annotate: compare with Normal group,
*P＜0.01; Compare with model control group,
▲P＜0.05,
▲ ▲P＜0.01
3.2 not simultaneously the phase multiple dosing on the impact of delayed hypersensitivity
Model control group and Normal group compare, and ear swelling degree P＜0.01 illustrates the modeling success.The omnidistance administration group of positive control and model control group are relatively, ear swelling degree, thymus index and index and spleen index P are all＜0.01, inhibitory rate of intumesce is 60.86%, explanatorily fills in Mi Songjing and crosses and comprise and induce mutually and excite mutually omnidistance administration can significantly suppress the delayed hypersensitivity of mice; Compare with model control group, Rd is low, high dose group ear swelling degree P equal＜and 0.05, dosage group P＜0.01 among the Rd, except Rd high dose group thymus index P＜0.01, all the other each Rd group thymus indexs and index and spleen index P are all＞0.05, the basic, normal, high dosage group of Rd inhibitory rate of intumesce is respectively 39.96%, 52.31%, 42.98%, explanation before sensitization after excite in the situation of omnidistance administration, each dosage ginsenoside-Rd all has remarkable inhibitory action to DNCB induced mice delayed hypersensitivity, and the inhibitory action of middle dosage Rd is the most remarkable.
Positive control excites phase administration group and model control group relatively, ear swelling degree, thymus index and index and spleen index P are all＜0.01, inhibitory rate of intumesce is 70.19%, illustrates that dexamethasone is exciting phase (effect phase) multiple dosing also can significantly suppress the delayed hypersensitivity of mice; Compare with model control group, low dosage Rd ear swelling degree P＞0.05 (thymus index P＞0.05, index and spleen index P＜0.05), middle dosage Rd ear swelling degree P＜0.05 (thymus index P＞0.05, index and spleen index P＜0.01), high dose Rd ear swelling degree P＜0.01 (thymus index P＜0.01, index and spleen index P＜0.05), Rd is low, in, the high dose group inhibitory rate of intumesce is respectively 31.06%, 47.69%, 68.38%, in the situation that excites phase (effect phase) multiple dosing of explanation delayed hypersensitivity after antigen is attacked, in, the Rd of high dose has obvious inhibitory action to DNCB induced mice delayed hypersensitivity, and dose-effect relationship is arranged, and experimental result is shown in table 4-2.
Table 4-2 not simultaneously the phase multiple dosing DNCB is caused the impact (x ± s, n=11～12) of mice delayed hypersensitivity
Annotate: compare with Normal group,
*P＜0.05,
*P＜0.01; It is relatively thin with the model contrast,
▲P＜0.05,
▲ ▲P＜0.01
Two, ginsenoside-Rd is on the impact of human T lymphocyte's propagation
1. material
Ginsenoside-Rd is provided by GuangDong TaiHe Biology Pharmacy Co., Ltd, lot number: 980303.Hank ' s liquid, NaCl 136mM, KCl 5.36mM, KH
2PO
40.44mM, Na
2HPO
40.37mM, glucose 5mM, NaHCO
34.16mM, pH7.2.PBS buffer: NaCl 136mM, Na
2HPO
49.74mM, KH
2PO
41.47mM, KCl 2.68mM, pH 7.2.
2. method
2.1 become human peripheral blood T lymphocyte to separate
In the aseptic blood of getting on the experiment same day, anticoagulant heparin adds equal-volume Hank ' s liquid, draw diluted blood with suction pipe and gently place on the lymphocyte separation medium aspect, diluted blood and lymphocyte separation medium volume ratio are 4: 3, centrifugal 25 minutes of room temperature, 2000 rev/mins, draw white buffy coat with capillary pipette, add in another centrifuge tube, with 4 times of volume Hank ' s liquid dilution mixings, centrifugal 10 minutes, 1200 rev/mins, abandon supernatant, the PBS buffer is washed twice.With RPMI RPMI-1640 suspension cell (cell number 10
7Individual/ml), cell suspension is hatched 45min on 37 ℃ on aseptic nylon hair post, collects eluent, with RPMI RPMI-1640 suspension cell (cell number 4 * 10
8Individual/L), fluidic cell instrument system of fluorescence analysis detects T lymphocyte number＞95%.
2.2T lymphocyte proliferation assay
It is former as stimulating take ConA,
3H-TdR mixes method and measures the T lymphopoiesis.The preliminary experiment result shows that the moderate irritaiting concentration of ConA is 5 μ g/ml, so this concentration (for the reaction final concentration, producing approximately 87% multiplication effect) is adopted in this research.Cell suspension is added in 96 orifice plates, and (cell number is 2 * 10 to every hole 100 μ l
5Individual/hole), the grouping situation is as follows: the blank group: 200 μ l RPMI 1640; Negative control group: 100 μ l cell suspension+100 μ l RPMI 1640; ConA matched group: 100 μ l cell suspension+20 μ l ConA+80 μ l RPMI 1640; Rd experimental group: 100 μ l cell suspension+20 μ l ConA+20 μ l medicine+60 μ l RPMI 1640.
Establish three multiple holes for every group, at 37 ℃, 5%CO
2Hatched in the incubator 72 hours, after cell culture finished, the Trypan Blue viable count showed viable count 〉=98%.16h adds before the cell harvesting
3H-TdR (1.85 * 10
4The Bq/ hole), cultivate and finish the rear bull cell harvesting instrument collection culture of using on 9999 type glass fiber filters, 10% trichloroacetic acid is fixed, behind 95% alcohol flushing, 60 ℃ of oven dry, put into scintillation vial, add scintillation solution (composition: PPO 7g, POPOP0.5g, dimethylbenzene 1000ml) at liquid scintillation instrument (FJ2101 type liquid scintillation counter, state-run 262 factories, the instrument counting efficiency is 55.8%) upper counting cpm (umber of pulse/minute), at the bottom of the master copy＜50cpm.Medicine represents with suppression ratio the inhibitory action of T cell proliferation:
3. result
We adopt ConA as mitogen, can make the human T lymphocyte produce had significant proliferation, and this breeder reaction needs Ca
2+Interior stream participates in.Experimental result shows, ginsenoside-Rd can obviously suppress human T lymphocyte's propagation that ConA causes and be concentration dependent, cpm compares with positive controls, Rd is P＞0.05 (proliferation inhibition rate is 12.4%) when 5 μ M, P＜0.05 when 10 μ M (proliferation inhibition rate is 34.3%), when 20,40,80 μ M P all＜0.01 (proliferation inhibition rate is respectively 56.4%, 81.9% and 92.7%).And another non-voltage-dependent Ca
2+Channel blocker SK﹠amp; F96365 can significantly suppress human T lymphocyte's propagation equally, and is concentration dependent, and cpm compares with positive controls, SK﹠amp; F96365 is P＜0.05 (proliferation inhibition rate is 15.6%) when 1 μ M, when 3,10,30 μ M P all＜0.01 (proliferation inhibition rate is respectively 37.4%, 53.1% and 78.6%), the results are shown in Table 4-3.The results suggest ginsenoside-Rd can obviously suppress human T lymphocyte's propagation.
Table 4-3:SK﹠amp; F96365 and ginsenoside-Rd cause ConA
3The effect that H-TdR mixes the human T lymphocyte (x ± s.n=5)
Annotate: compare with positive controls,
*P＜0.05,
*P＜0.01
Three, ginsenoside-Rd is on the dermatoplastic impact of allograft transplantation
1. experiment material
1.1 animal
82 of receptor Wistar rats, SPF level, ♂, body weight 193～230g; 10 of donor SD rats, SPF level, ♂, body weight 218～263g.Available from Shanghai Slac Experimental Animal Co., Ltd., credit number: SCXK (Shanghai) 2007-0005.
1.2 medicine
Ginsenoside-Rd: lot number is provided by GuangDong TaiHe Biology Pharmacy Co., Ltd: 050815, dissolve the solution that is mixed with respective concentration with 55% propylene glycol before use.Cyclosporin A injection (CsA), Switzerland Novartis product, lot number: S0016A is diluted to desired concn with physiological saline solution before use.Pentobarbital sodium: the chemical experimental factory, Foshan City, lot number: 860901, with the physiological saline solution dilution, concentration is 0.4% before use.800,000 unit penicillin sodiums: HARBIN PHARMACEUTICAL GROUP CO., LTD. General Pharm. Factory production, lot number: A 080502713.Be 80,000 units/ml with normal saline dilution before use.
1.3 equipment
The routine operation apparatus, preoperative disinfection sterilization: mosquito forceps, curved scissors, staight scissors, eye scissors, ophthalmic tweezers, tissue clamps, needle holder, suture needle, stitching thread, gauze, adhesive plaster, swab stick, culture dish.
2. method
2.1 animal grouping and administration
82 of receptor Wistar rats, ♂, body weight 193～230g is divided into 6 groups at random by body weight before the skin transplantation, and 13～14 every group, grouping and administration situation are as follows: first group: Normal group (autotransplantation group); Second group: model control group, 55 volume % propylene glycol, im, 0.2ml/100g body weight; The 3rd group: positive controls, CsA, 5mg/kg body weight, im, 0.2ml injection/100g body weight; The 4th group: Rd low dose group, 12.5mg Rd/kg body weight, im, 0.2ml Rd injection/100g body weight; The 5th group: dosage group among the Rd, 25mg Rd/kg body weight, im, 0.2mlRd injection/100g body weight; The 6th group: Rd high dose group, 50mg Rd/kg body weight, im, 0.2ml Rd injection/100g body weight.
Each organizes front 3 days of Rhizoma Atractylodis Macrocephalae beginning administration (administration the previous day of positive controls Rhizoma Atractylodis Macrocephalae), and once a day, to postoperative the 15th day, dosage and method were as implied above.
2.2 skin transplantation method
Autotransplantation group (from body skin graft Hui Zhi): step is as follows: 1) anesthesia: 0.4% (W/V) pentobarbital sodium solution, 1ml/100g body weight lumbar injection.2) Skin autografts: autotransplantation group rat back graft area cropping, iodophor disinfection 3 times, ethanol takes off the skin graft of cutting the about 1cm * 1cm of a block size behind the iodine 2 times in 1cm place, the other right side of spinal column, put into the culture dish that fills physiological saline solution and prune subcutaneous tissue to corium, Hui Zhi was in wound surface after normal saline flushing was clean, with ophthalmic tweezers skin graft is paved and to be made it be close to plant bed, sterile gauze sucks normal saline and oozes out blood.Carry out four angle seams with the seam sword-shaped needle with No. 0 line of not damaged and close, skin graft is fixed on the wound surface.3) wrapping: wound with iodophor disinfection after, be covered with the sterile gauze that scribbles white vaseline, adhesive tape ring is around pressure dressing.The autotransplantation group is set, causes the possibility of skin graft necrosis to get rid of the factors such as operation technique failure, the postoperative skin graft infects and move.Postoperative rat sub-cage rearing is freely ingested, is drunk water.The penicillin 0.4ml infection of every Mus intramuscular injection of while 80,000 units/ml, once a day, continuous 5 days.
Heterogenous skin is transplanted and is adopted the back of the body-butt skin grafting, and step is as follows: 1) anesthesia: 0.4% (W/V) pentobarbital sodium solution, 1ml/100g body weight lumbar injection; 2) prepare for skin: the skin graft of cutting the about 1cm * 1cm of size behind the iodine 2 times is taken off in the dermatotome cropping of donor SD donor rat, iodophor disinfection 3 times, ethanol, puts into the culture dish that fills physiological saline solution and prunes subcutaneous tissue to corium, and normal saline flushing is clean rear for subsequent use; 3) allosome grafts: after the receptor Wistar rat back graft area cropping sterilization, cut a skin graft similar with supplying the skin size in 1cm place, the other right side of spinal column, good being affixed on the receptor plant bed for skin will be pruned in advance, with ophthalmic tweezers skin graft is paved and to be made it be close to plant bed, sterile gauze sucks normal saline and oozes out blood.Carry out four angle seams with the seam sword-shaped needle with No. 0 line of not damaged and close, skin graft is fixed on the plant bed; 4) wrapping: wound with iodophor disinfection after, be covered with the sterile gauze that scribbles white vaseline, adhesive tape ring is around pressure dressing.Postoperative rat sub-cage rearing is freely ingested, is drunk water.The penicillin 0.4ml infection of every Mus intramuscular injection of while 80,000 units/ml, once a day, continuous 5 days.
Postoperative the 7th day beginning every day unpacks to each group rat and observes the skin graft growing state and take pictures, if any skin graft displacement, pile up or come off and then think operative failure (operative failure rate can not greater than 10%), do not charge to experimental data, all the other record the survival of grafted skin time, statistical result represents with skin graft mean survival time (mean survival time, MST)
Skin grafts repels the definite of time (time-to-live): if skin-grafting is healed with host's back end section, skin graft is soft, and color and luster is consistent with surrounding skin, and inflammation thinks then that with congested not obvious rejection does not occur survival of grafted skin; If skin graft blackout, stiff incrustation or the area that comes off reach 50% and think that rejection has occured skin graft.
The experimental result statistical procedures uses Excel-2003 to add up, and each is organized data and is measurement data, represents with x ± s, and the significance of group difference is checked with t-.
3. result
The skin graft mean survival time of model control group is (11.67 ± 1.97) d, the skin graft mean survival time of CsA is (22.18 ± 1.17) d, and the skin graft mean survival time of the basic, normal, high dosage group of Rd is respectively (14.17 ± 2.69) d, (15.60 ± 0.52) d and (15.10 ± 0.99) d.MST compares with model control group, CsA organizes P＜0.01, Rd low dose group P＜0.05, the middle and high dosage group of Rd P all＜0.01, immunological rejection after each dosage Rd all can suppress the allograft transplantation skin transplantation is described, prolong the survival of grafted skin time, but its immunosuppressive action intensity is weaker than CsA, the results are shown in Table 4-4 and Fig. 1.
Table 4-4: different dosing group rat implantation survival of grafted skin time (x ± s, n=10～12)
Annotate: compare with model control group,
▲P＜0.05,
▲ ▲P＜0.01.
Morphologic observation is shown in the 12nd day skin grafts taken a picture after the operation of Fig. 1: first group: the Normal group skin-grafting is good with the plant bed healing, and skin graft is soft, and color and luster is consistent with surrounding skin, pinkiness; Second group: the skin-grafting generation rejection of model control group, skin graft blackout, stiff, incrustation; The 3rd group: the skin-grafting of positive controls and plant bed healing are good, and skin graft is soft, and color and luster is consistent with surrounding skin, and rejection does not occur pinkiness; The 4th group: the skin-grafting of Rd low dose group and plant bed healing are good, and skin graft is soft, and is slightly rubescent, local inflammation arranged, but rejection does not occur; The 5th group: the skin-grafting of dosage group and plant bed heal well among the Rd, and skin graft is soft, and color and luster is consistent with surrounding skin, and rejection does not occur pinkiness; The 6th group: the skin-grafting of Rd high dose group and plant bed healing are good, and skin graft is soft, and color and luster is consistent with surrounding skin, and rejection does not occur pinkiness.
4. conclusion
Ginsenoside-Rd can suppress the mice delayed hypersensitivity, human T lymphocyte's propagation that ConA induces and the immunological rejection after the allograft transplantation skin transplantation, illustrate that it has immunosuppressive action, may be used for the various autoimmune disease as immunosuppressant clinically, such as rheumatic and rheumatoid arthritis, systemic lupus erythematosus (sle), myasthenia gravis, autoimmune hemolytic anemia, nephrotic syndrome, ulcerative colitis etc., also can be used for the clinically immunological rejection of organ transplantation postoperative, but this experimental result shows the immunosuppressive action of each dosage Rd and all is weaker than CsA, point out its adjuvant drug that can be used as clinically transplant rejection, reduce untoward reaction by the consumption that reduces other immunosuppressant after share.In addition, because ginsenoside-Rd has the effect of significant antiinflammatory and anti-IV type allergy, can suppress generation and the development of delayed hypersensitivity, and can more promptly improve the skin inflammatory symptoms, point out it may be used for the treatment of the inflammatory disease of the skin that has the IV allergic reaction type to participate in clinically.
And ginsenoside-Rd solution phase of the present invention has higher stability and dissolubility for ginsenoside-Rd solution of the prior art, can contain the ginsenoside-Rd of higher concentration, therefore is more suitable for for clinical immunosuppressant.
All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (12)
1. ginsenoside-Rd solution, it is characterized in that, described solution is that ginsenoside-Rd is the solution that forms in the aqueous solution of propylene glycol of 55-80 volume % at contained propylene glycol, the content of described ginsenoside-Rd is 5-75mg/ml solution, wherein, the percent by volume of propylene glycol is take the cumulative volume of aqueous solution of propylene glycol as benchmark.
2. solution as claimed in claim 1 is characterized in that, described solution is true solution.
3. ginsenoside-Rd solution as claimed in claim 1 is characterized in that, described ginsenoside-Rd solution is selected from lower group: greater than the ginsenoside-Rd of the 25mg/ml solution of ginsenoside-Rd in the aqueous solution of propylene glycol of 60-80 volume % to 75mg/ml; Greater than the ginsenoside-Rd of the 10mg/ml solution of ginsenoside-Rd in the aqueous solution of propylene glycol of 55-75 volume % to 25mg/ml; Or the solution of the ginsenoside-Rd of 5～10mg/ml in the aqueous solution of propylene glycol of 55-75 volume %.
4. ginsenoside-Rd solution as claimed in claim 3 is characterized in that, described ginsenoside-Rd solution is greater than the ginsenoside-Rd of the 25mg/ml solution of ginsenoside-Rd in the aqueous solution of propylene glycol of 75 volume % to 75mg/ml.
5. ginsenoside-Rd solution as claimed in claim 3 is characterized in that, described ginsenoside-Rd solution is greater than the ginsenoside-Rd of the 10mg/ml solution of ginsenoside-Rd in the aqueous solution of propylene glycol of 60 volume % to 25mg/ml.
6. ginsenoside-Rd solution as claimed in claim 3 is characterized in that, the solution of ginsenoside-Rd in the aqueous solution of propylene glycol of 55 volume % that described ginsenoside-Rd solution is 5～10mg/ml.
7. method for preparing the described ginsenoside-Rd solution of claim 1, described method comprises:
(a) aqueous solution of propylene glycol with contained propylene glycol 〉=50 volume % dissolves ginsenoside-Rd;
(b) dilution step (a) gained solution randomly, the solution take the content that obtains ginsenoside-Rd as 5-75mg/ml solution.
8. method as claimed in claim 7 is characterized in that, described method comprises: behind the aqueous solution of propylene glycol dissolving ginsenoside-Rd with 100 volume %, gained solution is diluted again.
9. pharmaceutical composition, it comprises: each described ginsenoside-Rd solution among the claim 1-6; With pharmaceutically acceptable carrier.
10. method for preparing the pharmaceutical composition that contains ginsenoside-Rd, described method comprises: each described ginsenoside-Rd solution among the claim 1-6 is mixed with pharmaceutically acceptable carrier.
11. the described ginsenoside-Rd solution of claim 1 is for the preparation of the purposes in antiinflammatory, immunosuppressant and/or anti-organ-graft refection's the medicine.
12. purposes as claimed in claim 11, it is characterized in that described medicine for antiinflammatory is used for the treatment of and is selected from lower group inflammatory diseases: rheumatic arthritis, rheumatoid arthritis, ankylosing spondylitis, rheumatic valvulitis, tuberculous meningitis, pleuritis or pericarditis; Describedly be used for the treatment of or auxiliary treatment is selected from lower group immune correlated disease: the immunological rejection of autoimmune disease, organ transplantation postoperative or the inflammatory disease of the skin that type Ⅳ allergy is participated in for the immunosuppressant medicine.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008102081192A CN101766640B (en) | 2008-12-29 | 2008-12-29 | Preparation for panaxoside-Rd aqueous solution of propylene glycol and new application thereof in inflammation resistance, immune suppression and organ transplant rejection resistance |
| PCT/CN2009/070419 WO2010078730A1 (en) | 2008-12-29 | 2009-02-13 | Preparation of ginsenoside-rd propylene glycol aqueous solution and new use thereof in anti-inflammation, immunosuppression and anti organ-graft rejection |
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| CN2008102081192A CN101766640B (en) | 2008-12-29 | 2008-12-29 | Preparation for panaxoside-Rd aqueous solution of propylene glycol and new application thereof in inflammation resistance, immune suppression and organ transplant rejection resistance |
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| CN102716487B (en) * | 2011-03-29 | 2016-03-09 | 兰州大学 | Ginsenoside-Rd Transdermal absorption penetration enhancer and application thereof |
| CN102743402B (en) * | 2012-07-15 | 2014-06-04 | 浙江大学 | Application of panaxadiol saponins fraction in preparing medicine for preventing dermatitis and scar |
| CN107737134B (en) * | 2017-12-04 | 2018-10-23 | 吉林大学 | Application of the Panasenoside in anaphylactia |
| CN116459267A (en) * | 2022-01-11 | 2023-07-21 | 大连富生天然药物开发有限公司 | Anti-inflammatory application of ginsenoside Rd |
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| CN100502880C (en) * | 2004-11-09 | 2009-06-24 | 成都华神集团股份有限公司制药厂 | Notoginseng glycol-saponin composition and its preparation method and use |
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