CN102846704A - A Leonurus japonicus injection, its preparation method, and method for detecting total alkaloids - Google Patents
A Leonurus japonicus injection, its preparation method, and method for detecting total alkaloids Download PDFInfo
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Abstract
The invention provides a Leonurus japonicus injection and its preparation method. The injection contains Leonurus japonicus ultrafiltrate, pharmaceutically acceptable compound for adjusting osmotic pressure, and optionally water for injection. The Leonurus japonicus injection provided by the invention is a large-volume injection, has high effective component content in crude drug and stable physicochemical properties, and can be directly used for intravenous injection. The invention also provides a method for detecting total alkaloids in Leonurus japonicus injection. The method can simultaneously detect leonurine and stachydrine hydrochloride in Leonurus japonicus injection to better reflect the content of total alkaloids in Leonurus japonicus injection.
Description
Technical field
The invention belongs to the pharmaceutical technology field.Particularly, the present invention relates to a kind of Leonurus Heterophyllus injection for intravenous drip and preparation method thereof and detection method.
Background technology
Herba Leonuri is the fresh of labiate Herba Leonuri Leonurus Japonicus Houtt. or goes up dryly herb, begins to carry a Shennong's Herbal, classifies as top gradely, is conventional Chinese medicine.Herba Leonuri acrid in the mouth, little hardship, cold nature, people's pericardium, Liver Channel have eliminating blood stasis to promote regeneration of blood, the effect of promoting blood flow to regulate menstruation.In the treatment obstetrical and gynecological disease, doctor family claims that a Herba Leonuri is the blood system panacea to traditional medicine with Herba Leonuri and formulation application thereof.
The chemical constituent of Herba Leonuri mainly contains:
(1) alkaloids: Herba Leonuri contains total alkaloids, wherein leonurine (Leonurine), stachydrine (Stachydrine), Herba Leonuri pyridine and leonurinine etc.;
(2) flavonoid: 5,7,3 ', 4 ', 5 '-pentamethoxyl flavone, wogonin, apiolin-7-O-glucose bran glycosides, Dai, Quercetin, kaempferol and glycosides, rutin etc.;
(3) Diterpenes: prehispanolone, Herba Leonuri element, front Herba Leonuri second element;
(4) fatty acid: contain Fumaric acid, lauric acid, linolenic acid, oleic acid etc.;
(5) volatile oil;
(6) contain trace element: wherein contain Fe, Mn, Zn, Rb content is higher.
Modern study shows that the pharmacological action of Herba Leonuri has:
(1) to the effect of uterus, mammary gland: Herba Leonuri is China's regulating menstruation, hemostasis medicine commonly used among the people, has stronger uteri excitation effect, its effective ingredient is leonurine, to isolated uterine, all be excitation in body uterus and uterine vascular, uterine contraction is obviously strengthened, tensity increases, and the persistent period strengthens;
(2) to the effect of cardiovascular system: Herba Leonuri is to early stage ischemia, even has a better role near the cardiac muscle of ischemic necrosis.Being rich in the Herba Leonuri multiplely has the trace element of protective effect to cardiovascular system, and the cardiovascular diseases such as myocardial infarction, coronary heart diseases and angina pectoris are had certain preventive and therapeutic effect;
(3) to the effect of blood system: Herba Leonuri can significantly reduce packed cell volume, low part, low part, viscosity index (VI) and the erythrocyte aggregation index cut of whole blood reduction ratio viscosity cut of whole blood contrast viscosity, the anticoagulant effect, reduce blood and Plasma Viscosity, prevention and inhibition tiny blood vessels thrombosis;
(4) to immune effect: Herba Leonuri has the effect that enlivens Lymph microcirculation, can expand lymphatic vessel, strengthens vasculolymphatic contractility, promotes generation and the backflow of lymph fluid, constant to environment in the body recovery, and the raising of immunity is useful;
(5) to the effect of kidney: clinical research shows that Herba Leonuri has to be adjusted the systemic blood circulation, dispels the effect that the stasis of blood stagnates, eliminates edema, recovers renal function, can be used for treating acute and chronic oedema due to nephritis, and nutritional edema and the cause of disease be the edema of name not.Its aqueous solution effect is faint, is using aqueous solution while use in conjunction volatile oil that diuresis is strengthened.Zoopery shows that Herba Leonuri can be protected by the Acute Renal Failure due to the gentamycin;
(6) other effects: heavy dose of Herba Leonuri has the effect of certain inhibition dermatophytes, is usually used in clinically urticaria, itchy skin eruption, sore and toxic and treatment female skin pruritus, and oral administration and externally all can.Experimental results show that Herba Leonuri exerts the dermatophytess such as card Salmonella in various degree inhibitory action is all arranged to oidium schoenleinii, ulotrichy budlet spore tinea bacterium, epidermophyton rubrum, star.
Herba Leonuri is gynecological's panacea, determined curative effect, clinical alone more, such as Herba Leonuri paste, Herba Leonuri granule, Leonurus japonicus Houtt oral liquid, Yimucao capsule, YIMUCAO PIAN etc., but be general water extraction or aqueous extraction-alcohol precipitation technology, preparation is refining, and have that bitterness, dose are large, slow curative effect, unsettled shortcoming; Existing Leonurus Heterophyllus injection is because capacity is little, to contain biological effective components low, so therapeutic effect is undesirable; In addition, Leonurus Heterophyllus injection often needs Leonurus Heterophyllus injection is added glucose or sodium chloride injection dilution when clinical practice, use through intravenous drip again, because the liquid of getting before instiling mixes, brought some shortcomings, not only formality is loaded down with trivial details, and is subjected to easily the pollution of environment, apparatus, user produce infusion reaction as have a fever, feel sick, feel cold, tachycardia or excessively slow etc., section jeopardizes patient's life in the time of seriously.。
In addition, the detection of at present relevant motherwort formulation all is to detect single component, related to the stachydrine hydrochloride that adopts in the fingerprint chromatogram method mensuration Leonurus Heterophyllus injection such as CN1973855A, but the main effective ingredient of Herba Leonuri is leonurine and stachydrine, only stachydrine hydrochloride in the Leonurus Heterophyllus injection is measured the accurate content that can't comprehensively reflect crude drug effective ingredient in the injection.
Therefore, existing for novel Leonurus Heterophyllus injection, especially high-capacity injection at present is the demand of infusion solutions.In addition, also need to provide a kind of method that can accurately detect the crude drug effective ingredient of Leonurus Heterophyllus injection.
Summary of the invention
For the problems referred to above, one of purpose of the application provides a kind of novel jumbo Leonurus Heterophyllus injection.
Another object of the present invention provides the preparation method of this large capacity Leonurus Heterophyllus injection.
A further object of the present invention provides a kind of detection method that can detect the total alkaloids in the Leonurus Heterophyllus injection, the method detects leonurine and stachydrine hydrochloride simultaneously, thereby can be used for reflecting the content of large capacity Leonurus Heterophyllus injection Chinese medicine effective ingredient.
Be used for realizing that the technical scheme of above-mentioned purpose is as follows:
On the one hand, the invention provides a kind of Leonurus Heterophyllus injection, described injection comprises the chemical compound of Herba Leonuri ultrafiltrate, pharmaceutically acceptable adjusting osmotic pressure and water for injection randomly.
Wherein, in the Leonurus Heterophyllus injection of 250ml, the content that this injection comprises total alkaloids is 10-500mg;
Preferably, to comprise the content of total alkaloids be 20-250mg to described injection;
Further preferably, the described injection content that comprises total alkaloids is 40-125mg;
More preferably, to comprise the content of total alkaloids be 45-50mg to described injection.
In addition, in above-mentioned Leonurus Heterophyllus injection, according to " 2010 editions appendix IXR of Chinese Pharmacopoeia particulate matter inspection technique " first method, light blockage method inspection, the particulate matter greater than 10 μ m in every 50ml injection is no more than 20; Microgranule greater than 25 μ m is no more than 5.
In above-mentioned Leonurus Heterophyllus injection, the method preparation of described Herba Leonuri ultrafiltrate by comprising the steps: get Herba Leonuri, decoct with water, merging filtrate, centrifugal treating or process microporous filter membrane are processed, and carry out ultrafiltration;
Preferably, the method preparation of described Herba Leonuri ultrafiltrate by comprising the steps:
A, get Herba Leonuri, decoct with water 2～5 times, collecting decoction, 1500～5000 rev/mins of centrifugal treating 5～30 minutes or process through microporous filter membrane, the supernatant of getting after the processing is for subsequent use;
B, get the supernatant that step a obtains, adopting the molecular retention amount is that 10,000～100,000 daltonian ultrafilter membranes carry out ultrafiltration, pressure 0.05-0.25MPa, and ultrafiltrate is for subsequent use;
C, get the ultrafiltrate that step b obtains, add appropriate hydrochloric acid adjust pH to 4.0～7.0, leave standstill more than 8～24 hours, adopting the molecular retention amount is that 1000～50000 daltonian ultrafilter membranes carry out ultrafiltration, pressure 0.05-0.25MPa, and ultrafiltrate is for subsequent use.
The water that adds in step a is 8～12 times (weight ratios) of medical material; The microporous filter membrane aperture of using is 0.45 μ m.
The ultrafilter membrane that uses in step b and c is selected from respectively ceramic membrane, cellulose acetate membrane, poly (ether sulfone) film, polyamide membrane or poly-fluorine film.
And the chemical compound of the pharmaceutically acceptable adjusting osmotic pressure of employing is selected from one or more in glucose, sodium chloride, magnesium chloride, calcium chloride, sodium lactate, xylitol, sorbitol and/or the dextran;
Preferably, described chemical compound is selected from one or more in glucose, sodium chloride, calcium chloride, dextran and/or the sodium lactate.
Further preferably, the osmotic pressure of described Leonurus Heterophyllus injection oozes for waiting, and namely the plasma osmotic pressure with the people equates.
On the other hand, the present invention also provides a kind of preparation method of Leonurus Heterophyllus injection, and described preparation method may further comprise the steps:
Get Herba Leonuri, decoct with water, merging filtrate, centrifugal treating or process through microporous filter membrane is carried out ultrafiltration, and ultrafiltrate adds the chemical compound accent of pharmaceutically acceptable adjusting osmotic pressure etc. and oozes, and adds water for injection or concentrated, sterilization, fill.
Preferably, said method comprising the steps of:
A, get Herba Leonuri, decoct with water 2～5 times, collecting decoction, 1500～5000 rev/mins of centrifugal treating 5～30 minutes or process through microporous filter membrane, the supernatant of getting after the processing is for subsequent use;
B, get the supernatant that step a obtains, adopting the molecular retention amount is that 10,000～100,000 daltonian ultrafilter membranes carry out ultrafiltration, pressure 0.05-0.25MPa, and ultrafiltrate is for subsequent use;
C, get the ultrafiltrate that step b obtains, add appropriate hydrochloric acid adjust pH to 4.0～7.0, leave standstill more than 8～24 hours, adopting the molecular retention amount is that 1000～50,000 daltonian ultrafilter membranes carry out ultrafiltration, pressure 0.05-0.25MPa, and ultrafiltrate is for subsequent use;
D, get the chemical compound accent etc. that ultrafiltrate that step c obtains adds pharmaceutically acceptable adjusting osmotic pressure and ooze, add water for injection or concentrated, fill nitrogen, sterilization, fill.
Wherein, the water that adds in step a is 8～12 times (weight ratios) of medical material; The microporous filter membrane aperture of using is 0.45 μ m.
Ultrafilter membrane among described step b and the c is respectively preferably from ceramic membrane, cellulose acetate membrane, poly (ether sulfone) film, polyamide membrane or poly-fluorine film.
Preferably, the chemical compound of described pharmaceutically acceptable adjusting osmotic pressure is selected from one or more in glucose, sodium chloride, magnesium chloride, calcium chloride, sodium lactate, xylitol, sorbitol and/or the dextran;
Further preferably, described chemical compound is selected from one or more in glucose, sodium chloride, calcium chloride, dextran and/or the sodium lactate.
Another aspect, the present invention also provides the detection method of total alkaloids in a kind of Leonurus Heterophyllus injection, and described detection method comprises the content that detects leonurine and stachydrine hydrochloride in the described Leonurus Heterophyllus injection.
Preferably, described detection method is according to high-performance liquid chromatogram determination, and described chromatographic condition comprises:
Take octadecylsilane chemically bonded silica as filler; Take 0.1% phosphoric acid solution-acetonitrile-methanol (40: 30: 30) (volume ratio) as mobile phase, column temperature is 30 ℃, and the pH that wherein regulates 0.1% phosphoric acid solution with triethylamine is 4.0; The detection wavelength is 276nm; Flow velocity: 0.8mL/min; Sample size 20 μ l; Number of theoretical plate calculates by the stachydrine hydrochloride peak and is not less than 3000.
Below be detailed description of the present invention:
Hyperfiltration technique is the new branch that emerges of separation engineering, and it is that microcellular structure by the film surface carries out Selective Separation to material.When liquid mixture is flowed through the film surface under certain pressure, solvent and micromolecule solute see through film (title ultrafiltrate), and macromolecular substances then is trapped, and its concentration in stock solution is improved gradually, thereby realize large and small intermolecular separation, reach the purpose of purification medicinal liquid.
Because Chinese medicine ingredients is complicated, except keeping to greatest extent the effective ingredient, also must remove as much as possible impurity during preparation.Have macromolecular substances and many microgranules, submicron and the flocculates etc. such as a large amount of tannins, protein, starch, resin in decocting for Chinese herbal medicine liquid, they generally do not have a drug action, but affect the quality of product.The quality that adopts hyperfiltration technique to prepare Chinese medicine is better than traditional method.Its main feature owing to removed the impurity such as tannin, has obviously improved clarity and the stability of injection for 1.; 2. since when separating without phase transformation, thereby be conducive to keep biological activity and the physical and chemical stability of Chinese medicine, be easy to keep the effective ingredient in the original formulation; 3. technological process and with short production cycle is easy to operation; 4. ultrafiltration preparation active constituent content is high by 10%～100% than well-established law, thereby economizes in raw materials, and saves simultaneously a large amount of solvents.
Herba Leonuri infusion solutions provided by the invention, adopted ultrafiltration technology in its preparation process, the large capacity Leonurus Heterophyllus injection of acquisition is made by Herba Leonuri ultrafiltrate, glucose or sodium chloride or magnesium chloride or calcium chloride or sodium lactate or xylitol or sorbitol or dextran (dextran that comprises big or middle or small-molecular weight) etc. and/or water for injection.
In the above-mentioned large capacity Leonurus Heterophyllus injection, in the 250ml injection, the content of total alkaloids is 10-500mg; Be preferably 20-250mg; 40-125mg more preferably; 45-50mg more preferably.
Particularly, large capacity Leonurus Heterophyllus injection of the present invention is prepared by the preparation method that comprises the steps:
The preparation of a, Herba Leonuri extracting solution: get Herba Leonuri, decoct with water 2～5 times, collecting decoction, centrifugal treating (1500～5000 rev/mins, 5～30 minutes) or process through microporous filter membrane, the clear liquor of getting after the processing is for subsequent use;
B, one-level ultrafiltration: getting above-mentioned supernatant, to adopt the molecular retention amount be that 10,000～100,000 daltonian ceramic membranes or cellulose acetate membrane or poly (ether sulfone) film or polyamide membrane or poly-fluorine film etc. carry out ultrafiltration, pressure 0.05-0.25MPa, and ultrafiltrate is for subsequent use;
C, two-stage ultrafiltering: get above-mentioned ultrafiltrate, add appropriate hydrochloric acid adjust pH to 4.0～7.0, leave standstill more than 8～24 hours, adopting the molecular retention amount is that 1000～50000 daltonian ceramic membranes or cellulose acetate membrane or poly (ether sulfone) film or polyamide membrane or poly-fluorine film etc. carry out ultrafiltration, pressure 0.05-0.25MPa, ultrafiltrate is for subsequent use;
D, get above-mentioned ultrafiltrate and add accent such as glucose or sodium chloride etc. and ooze, add water for injection or concentrate, fill nitrogen, sterilization filling gets the large capacity Leonurus Heterophyllus injection of finished product, it is to be made by Herba Leonuri ultrafiltrate, glucose or sodium chloride etc. and/or water for injection; In the 250ml injection, containing the crude drug effective ingredient is Herba Leonuri 2.5 grams～250 grams.
Product of the present invention by above method preparation is a kind of high-quality infusion solutions, and this infusion solutions physicochemical property is stable, and its clarity is high and microgranule is few.By sample thief 50ml, according to " 2010 editions appendix IXR of Chinese Pharmacopoeia particulate matter inspection technique " first method, the light blockage method inspection finds to contain in the 50ml test sample the above microgranule of 10 μ m≤20, contain the above microgranule of 25 μ m≤5, far below the existing standard of country.
In addition, product of the present invention also has following performance:
Protein: get this product 1ml, add 30% sulfosalicylic acid solution 1ml of new preparation, shake up, placed 5 minutes, do not occur muddy, up to specification.
Tannin: get this product 1ml, add the normal saline 5ml that contains 1% Ovum Gallus domesticus album of new preparation, placed 10 minutes, do not occur muddy or precipitation, up to specification.
Resin: get this product 5ml, put in the separatory funnel, add chloroform 10ml jolting and extract, divide and get chloroform liquid, put evaporate to dryness in the water-bath, residue adds glacial acetic acid 2ml makes dissolving, puts in the tool plug test tube, adds water 3ml, mixing was placed 30 minutes, precipitation do not occur, and is up to specification.
Oxalates: get this product 2ml, add 2～3 of 3% calcium chloride test solutions, placed 10 minutes, do not become turbid or precipitate, up to specification.
Heavy metal and harmful element: measure according to lead, cadmium, arsenic, hydrargyrum, copper algoscopy (an appendix IX of Chinese Pharmacopoeia version in 2010 B), the leaded 0.6mg that is less than of every 1L, cadmium is less than 0.15mg, and arsenic is less than 0.3mg, hydrargyrum is less than 0.1mg, copper is less than 20mg, and is all up to specification.
Residue on ignition: get this product 2ml, measure (an appendix IX of Chinese Pharmacopoeia version in 2010 J) in accordance with the law, 5 parts of meansigma methodss are 1.0% (g/ml), and are up to specification.
Total solid: precision is measured this product 10ml, puts in the evaporating dish that is dried to constant weight, and evaporate to dryness in the water-bath 105 ℃ of dryings 3 hours, in the dislocation exsiccator, cooled off 30 minutes, and rapid accurately weighed weight is calculated.Leave over residue and be 1.0%～3.0% (g/ml), up to specification.
Pyrogen: get this product, check (appendix XIIIA of Chinese Pharmacopoeia version in 2010) in accordance with the law, dosage is by the every 1kg injection of rabbit body weight 1.5ml, and is without heat source response, up to specification.
The present invention also provides the detection method of Leonurus Heterophyllus injection, and the method mainly comprises to be measured the total alkaloid content in the Leonurus Heterophyllus injection.Total alkaloids comprises leonurine and stachydrine hydrochloride, and main testing conditions is as follows:
Measure according to high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2010).
Chromatographic condition and system suitability:
Take octadecylsilane chemically bonded silica as filler; Take 0.1% phosphoric acid solution (regulating pH4.0 with triethylamine)-acetonitrile-methanol (40: 30: 30) as mobile phase, column temperature is 30 ℃; The detection wavelength is 276nm; Flow velocity: 0.8mL/min; Sample size 20 μ l.Number of theoretical plate calculates by the stachydrine hydrochloride peak should be not less than 3000.
The preparation of reference substance solution:
The reference substance stock solution: it is an amount of that precision takes by weighing the reference substance of leonurine and stachydrine hydrochloride, adds mobile phase solution dissolved dilution to becoming leonurine and stachydrine hydrochloride concentration to be respectively the mixed contrast solution of 30 μ g/mL and 150 μ g/mL.
Reference substance solution: precision is measured reference substance stock solution 5.0mL, puts in the 10mL measuring bottle, adds the mobile phase solution dilution to scale, shakes up.
The preparation of need testing solution: precision is measured this product 2.0ml, puts in the 10ml measuring bottle, adds the mobile phase solution dilution to scale, shakes up, and get final product.
Algoscopy: precision is drawn each 20 μ l of above-mentioned two kinds of solution respectively, and the injection liquid chromatography is measured, and calculates, and get final product.
The invention provides a kind of novel Leonurus Heterophyllus injection and preparation method thereof, and the detection method of total alkaloids in this Leonurus Heterophyllus injection.Compared with prior art, product provided by the invention has the following advantages:
The first, Leonurus Heterophyllus injection provided by the invention is large capacity Leonurus Heterophyllus injection, can be directly used in intravenous drip, improves clinical practice level and the safety of Herba Leonuri, makes things convenient for patient to use, and has overcome the shortcoming of existing product;
The second, the preparation method of Leonurus Heterophyllus injection of the present invention combines hyperfiltration technique, play the effects such as remove impurity, depyrogenation, degerming by ultrafiltration, technical process is simple, without phase transformation, energy-conservation, efficient, non-secondary pollution, need not high temperature or other chemical methodes, and good separating effect, the quality, stability and the safety that have improved Leonurus Heterophyllus injection;
Three, the detection method of at present relevant motherwort formulation all is to detect single component, therefore can not reflect accurately, all sidedly the content of existing motherwort formulation effective ingredient.And the detection method of Leonurus Heterophyllus injection provided by the invention adopts high-efficient liquid phase chromatogram technology, the testing conditions that wherein adopts can detect two alkaloid components (leonurine and stachydrine hydrochloride) in the Leonurus Heterophyllus injection simultaneously, especially under testing conditions of the present invention, the appearance time at these two peaks is moderate, separate good, simultaneously with sample in other impurity peaks all separate well, thereby testing conditions of the present invention can detect the content of the total alkaloids (in leonurine and stachydrine hydrochloride sum) in the motherwort formulation (such as infusion solutions), can be used for the better quality of control motherwort formulation, have easy, accurately reach reliable characteristics.
Description of drawings
Below, describe by reference to the accompanying drawings embodiments of the invention in detail:
Fig. 1 is respectively the HPLC spectrogram of leonurine and stachydrine hydrochloride reference substance, and its left figure is leonurine, and right figure is stachydrine hydrochloride.
Fig. 2 is the HPLC spectrogram of large capacity Leonurus Heterophyllus injection sample of the present invention, and wherein 1 is leonurine, and 2 is stachydrine hydrochloride.
The specific embodiment
Below in conjunction with the specific embodiment the present invention is further described in detail, the embodiment that provides is only in order to illustrate the present invention, rather than in order to limit the scope of the invention.
Experimental technique among the following embodiment if no special instructions, is conventional method.Used test material among the following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.
Embodiment 1: the preparation of large capacity Leonurus Heterophyllus injection
The preparation of a, Herba Leonuri extracting solution and processing: get Herba Leonuri 1kg, add water 10kg at every turn and decoct 3 times, collecting decoction, centrifugal treating 30 minutes (1500 rev/mins), it is for subsequent use to get supernatant;
B, one-level ultrafiltration: getting above-mentioned supernatant employing molecular retention amount is that 10,000 daltonian poly (ether sulfone) films carry out ultrafiltration, pressure 0.05MPa, filtrate for later use;
C, two-stage ultrafiltering: get above-mentioned filtrate, add an amount of 1mol/L hydrochloric acid adjust pH to 5.0, left standstill 8 hours, adopting the molecular retention amount is that 3000 daltonian poly (ether sulfone) films carry out ultrafiltration, pressure 0.2MPa, and ultrafiltrate is for subsequent use;
D, get above-mentioned ultrafiltrate and add glucose accent etc. and ooze, adding water for injection, to make cumulative volume be 100L again, fill, the large capacity Leonurus Heterophyllus injection of finished product of sterilizing to get.
Adopt the HPLC method that provides among the embodiment 9 to measure, total alkaloids is in the total amount of leonurine and stachydrine hydrochloride, and it is 0.05mg that every 1ml contains total alkaloids; Total alkaloids is 10mg in every 250ml sample.
This infusion solutions physicochemical property is stable, and its clarity is high and microgranule is few, the preparation Performance Detection:
Particulate matter---sample thief 50ml, according to " 2010 editions appendix IXR of Chinese Pharmacopoeia particulate matter inspection technique " first method, light blockage method inspection, particulate matter 〉=10 μ m :≤20; 〉=25 μ m :≤5.
Protein---get this product 1ml, add 30% sulfosalicylic acid solution 1ml of new preparation, shake up, placed 5 minutes, do not occur muddy, up to specification.
Tannin---get this product 1ml, add the normal saline 5ml that contains 1% Ovum Gallus domesticus album of new preparation, placed 10 minutes, do not occur muddy or precipitation, up to specification.
Resin---get this product 5ml, put in the separatory funnel, add chloroform 10ml jolting and extract, divide and get chloroform liquid, put evaporate to dryness in the water-bath, residue adds glacial acetic acid 2ml makes dissolving, puts in the tool plug test tube, adds water 3ml, mixing was placed 30 minutes, precipitation do not occur, and is up to specification.
Oxalates---get this product 2ml, add 2～3 of 3% calcium chloride test solutions, placed 10 minutes, do not become turbid or precipitate, up to specification.
Embodiment 2: the preparation of large capacity Leonurus Heterophyllus injection
The preparation of a, Herba Leonuri extracting solution and processing: get Herba Leonuri 1kg, add water 8kg at every turn, decoct 3 times, collecting decoction, centrifugal treating 15 minutes (3000 rev/mins), it is for subsequent use to get supernatant;
It is that 10,000 daltonian ceramic membranes carry out ultrafiltration, pressure 0.1MPa, filtrate for later use that above-mentioned supernatant employing molecular retention amount is got in b, one-level ultrafiltration;
C, two-stage ultrafiltering are got above-mentioned filtrate, add appropriate hydrochloric acid adjust pH to 5.5, leave standstill 10 hours, and adopting the molecular retention amount is that 1000 daltonian ceramic membranes carry out ultrafiltration, pressure 0.1MPa, and ultrafiltrate is for subsequent use;
D, get above-mentioned ultrafiltrate and add sodium chloride accent etc. and ooze, adding water for injection, to make cumulative volume be 50L again, fill, the large capacity Leonurus Heterophyllus injection of finished product of sterilizing to get.
Adopt the HPLC method that provides among the embodiment 9 to measure, it is 0.08mg that every 1ml contains total alkaloids; Total alkaloid content is 20mg in every 250ml sample.
This infusion solutions physicochemical property is stable, and its clarity is high and microgranule is few, the preparation Performance Detection:
Particulate matter---sample thief 50ml, according to " 2010 editions appendix IXR of Chinese Pharmacopoeia particulate matter inspection technique " first method, light blockage method inspection, particulate matter 〉=10 μ m :≤20; 〉=25 μ m :≤5.
Protein---get this product 1ml, add 30% sulfosalicylic acid solution 1ml of new preparation, shake up, placed 5 minutes, do not occur muddy, up to specification.
Tannin---get this product 1ml, add the normal saline 5ml that contains 1% Ovum Gallus domesticus album of new preparation, placed 10 minutes, do not occur muddy or precipitation, up to specification.
Resin---get this product 5ml, put in the separatory funnel, add chloroform 10ml jolting and extract, divide and get chloroform liquid, put evaporate to dryness in the water-bath, residue adds glacial acetic acid 2ml makes dissolving, puts in the tool plug test tube, adds water 3ml, mixing was placed 30 minutes, precipitation do not occur, and is up to specification.
Oxalates---get this product 2ml, add 2～3 of 3% calcium chloride test solutions, placed 10 minutes, do not become turbid or precipitate, up to specification.
Embodiment 3: the preparation of large capacity Leonurus Heterophyllus injection
The preparation of a, Herba Leonuri extracting solution and processing: get Herba Leonuri 1kg, add water 10kg at every turn, decoct twice, collecting decoction, centrifugal treating 15 minutes (5000 rev/mins), it is for subsequent use to get supernatant;
B, one-level ultrafiltration: getting above-mentioned supernatant employing molecular retention amount is that 60,000 daltonian cellulose acetate membrane are carried out ultrafiltration, pressure 0.08MPa, filtrate for later use;
C, two-stage ultrafiltering: get above-mentioned filtrate, add appropriate hydrochloric acid adjust pH to 6.0, left standstill 12 hours, adopting the molecular retention amount is that 10,000 daltonian cellulose acetate membrane are carried out ultrafiltration, pressure 0.12MPa, and ultrafiltrate is for subsequent use;
D, get above-mentioned ultrafiltrate and add calcium chloride accent etc. and ooze, adding water for injection, to make cumulative volume be 25L again, fill, the large capacity Leonurus Heterophyllus injection of finished product of sterilizing to get.
Adopt the HPLC method that provides among the embodiment 9 to measure, it is 0.16mg that every 1ml contains total alkaloids; Total alkaloid content is 40mg in every 250ml sample.
This infusion solutions physicochemical property is stable, and its clarity is high and microgranule is few, the preparation Performance Detection:
Particulate matter---sample thief 50ml, according to " 2010 editions appendix IXR of Chinese Pharmacopoeia particulate matter inspection technique " first method, light blockage method inspection, particulate matter 〉=10 μ m :≤20; 〉=25 μ m :≤5.
Protein---get this product 1ml, add 30% sulfosalicylic acid solution 1ml of new preparation, shake up, placed 5 minutes, do not occur muddy, up to specification.
Tannin---get this product 1ml, add the normal saline 5ml that contains 1% Ovum Gallus domesticus album of new preparation, placed 10 minutes, do not occur muddy or precipitation, up to specification.
Resin---get this product 5ml, put in the separatory funnel, add chloroform 10ml jolting and extract, divide and get chloroform liquid, put evaporate to dryness in the water-bath, residue adds glacial acetic acid 2ml makes dissolving, puts in the tool plug test tube, adds water 3ml, mixing was placed 30 minutes, precipitation do not occur, and is up to specification.
Oxalates---get this product 2ml, add 2～3 of 3% calcium chloride test solutions, placed 10 minutes, do not become turbid or precipitate, up to specification.
Embodiment 4: the preparation of large capacity Leonurus Heterophyllus injection
The preparation of a, Herba Leonuri extracting solution and processing: get Herba Leonuri 1kg, add 12 times of water gagings at every turn, decoct 3 times, collecting decoction, centrifugal treating 20 minutes (3000 rev/mins), it is for subsequent use to get supernatant;
B, one-level ultrafiltration: getting above-mentioned supernatant employing molecular retention amount is that 10,000 daltonian poly-fluorine films carry out ultrafiltration, pressure 0.15MPa, filtrate for later use;
C, two-stage ultrafiltering: get above-mentioned filtrate, add appropriate hydrochloric acid adjust pH to 5.5, left standstill 8 hours, adopting the molecular retention amount is that 5000 daltonian poly-fluorine films carry out ultrafiltration, pressure 0.15MPa, and ultrafiltrate is for subsequent use;
D, get above-mentioned ultrafiltrate and add dextran accent etc. and ooze, adding water for injection, to make cumulative volume be 25L again, fill, the large capacity Leonurus Heterophyllus injection of finished product of sterilizing to get.
Adopt the HPLC method that provides among the embodiment 9 to measure, it is 0.18mg that every 1ml contains total alkaloids; Total alkaloid content is 45mg in every 250ml sample.
This infusion solutions physicochemical property is stable, and its clarity is high and microgranule is few, the preparation Performance Detection:
Particulate matter---sample thief 50ml, according to " 2010 editions appendix IXR of Chinese Pharmacopoeia particulate matter inspection technique " first method, light blockage method inspection, particulate matter 〉=10 μ m :≤20; 〉=25 μ m :≤5.
Protein---get this product 1ml, add 30% sulfosalicylic acid solution 1ml of new preparation, shake up, placed 5 minutes, do not occur muddy, up to specification.
Tannin---get this product 1ml, add the normal saline 5ml that contains 1% Ovum Gallus domesticus album of new preparation, placed 10 minutes, do not occur muddy or precipitation, up to specification.
Resin---get this product 5ml, put in the separatory funnel, add chloroform 10ml jolting and extract, divide and get chloroform liquid, put evaporate to dryness in the water-bath, residue adds glacial acetic acid 2ml makes dissolving, puts in the tool plug test tube, adds water 3ml, mixing was placed 30 minutes, precipitation do not occur, and is up to specification.
Oxalates---get this product 2ml, add 2～3 of 3% calcium chloride test solutions, placed 10 minutes, do not become turbid or precipitate, up to specification.
Embodiment 5: the preparation of large capacity Leonurus Heterophyllus injection
The preparation of a, Herba Leonuri extracting solution and processing: get Herba Leonuri 1kg, add 10 times of water gagings at every turn, decoct 3 times, collecting decoction, centrifugal treating 20 minutes (4000 rev/mins), it is for subsequent use to get supernatant;
B, one-level ultrafiltration: getting above-mentioned supernatant employing molecular retention amount is that 10,000 daltonian polyamide membrane are carried out ultrafiltration, pressure 0.2MPa, filtrate for later use;
C, two-stage ultrafiltering: get above-mentioned filtrate, add appropriate hydrochloric acid adjust pH to 6.0, left standstill 8 hours, adopting the molecular retention amount is that 3000 daltonian polyamide membrane are carried out ultrafiltration, pressure 0.2MPa, and ultrafiltrate is for subsequent use;
D, get above-mentioned ultrafiltrate and add sodium lactate accent etc. and ooze, adding water for injection, to make cumulative volume be 25L again, fill, the large capacity Leonurus Heterophyllus injection of finished product of sterilizing to get.
Adopt the HPLC method that provides among the embodiment 9 to measure, it is 0.2mg that every 1ml contains total alkaloids; Total alkaloid content is 50mg in every 250ml sample.
This infusion solutions physicochemical property is stable, and its clarity is high and microgranule is few, the preparation Performance Detection:
Particulate matter---sample thief 50ml, according to " 2010 editions appendix IXR of Chinese Pharmacopoeia particulate matter inspection technique " first method, light blockage method inspection, particulate matter 〉=10 μ m :≤20; 〉=25 μ m :≤5.
Protein---get this product 1ml, add 30% sulfosalicylic acid solution 1ml of new preparation, shake up, placed 5 minutes, do not occur muddy, up to specification.
Tannin---get this product 1ml, add the normal saline 5ml that contains 1% Ovum Gallus domesticus album of new preparation, placed 10 minutes, do not occur muddy or precipitation, up to specification.
Resin---get this product 5ml, put in the separatory funnel, add chloroform 10ml jolting and extract, divide and get chloroform liquid, put evaporate to dryness in the water-bath, residue adds glacial acetic acid 2ml makes dissolving, puts in the tool plug test tube, adds water 3ml, mixing was placed 30 minutes, precipitation do not occur, and is up to specification.
Oxalates---get this product 2ml, add 2～3 of 3% calcium chloride test solutions, placed 10 minutes, do not become turbid or precipitate, up to specification.
Embodiment 6: the preparation of large capacity Leonurus Heterophyllus injection
The preparation of a, Herba Leonuri extracting solution and processing: get Herba Leonuri 1kg, add 10 times of water gagings at every turn, decoct 3 times, collecting decoction is crossed microporous filter membrane (0.45 μ m) pretreatment, gets filtrate for later use;
B, one-level ultrafiltration: getting above-mentioned supernatant employing molecular retention amount is that 10,000 daltonian poly (ether sulfone) films carry out ultrafiltration, pressure 0.1MPa, filtrate for later use;
C, two-stage ultrafiltering: get above-mentioned filtrate, add an amount of 1mol/L hydrochloric acid adjust pH to 5.0, left standstill 8 hours, adopting the molecular retention amount is that 3000 daltonian poly (ether sulfone) films carry out ultrafiltration, pressure 0.1MPa, and ultrafiltrate is for subsequent use;
D, get above-mentioned ultrafiltrate and add glucose accent etc. and ooze, being concentrated into cumulative volume is 10L, fill, the large capacity Leonurus Heterophyllus injection of finished product of sterilizing to get.
Adopt the HPLC method that provides among the embodiment 9 to measure, it is 0.5mg that every 1ml contains total alkaloids; Total alkaloid content is 125mg in every 250ml sample.
This infusion solutions physicochemical property is stable, and its clarity is high and microgranule is few, the preparation Performance Detection:
Particulate matter---sample thief 50ml, according to " 2010 editions appendix IXR of Chinese Pharmacopoeia particulate matter inspection technique " first method, light blockage method inspection, particulate matter 〉=10 μ m :≤20; 〉=25 μ m :≤5.
Protein---get this product 1ml, add 30% sulfosalicylic acid solution 1ml of new preparation, shake up, placed 5 minutes, do not occur muddy, up to specification.
Tannin---get this product 1ml, add the normal saline 5ml that contains 1% Ovum Gallus domesticus album of new preparation, placed 10 minutes, do not occur muddy or precipitation, up to specification.
Resin---get this product 5ml, put in the separatory funnel, add chloroform 10ml jolting and extract, divide and get chloroform liquid, put evaporate to dryness in the water-bath, residue adds glacial acetic acid 2ml makes dissolving, puts in the tool plug test tube, adds water 3ml, mixing was placed 30 minutes, precipitation do not occur, and is up to specification.
Oxalates---get this product 2ml, add 2～3 of 3% calcium chloride test solutions, placed 10 minutes, do not become turbid or precipitate, up to specification.
Embodiment 7: the preparation of large capacity Leonurus Heterophyllus injection
The preparation of a, Herba Leonuri extracting solution and processing: get Herba Leonuri 1kg, add 10 times of water gagings at every turn, decoct 2 times, collecting decoction, centrifugal treating 15 minutes (4000 rev/mins), it is for subsequent use to get supernatant;
B, one-level ultrafiltration: getting above-mentioned supernatant employing molecular retention amount is that 10,000 daltonian polyamide membrane are carried out ultrafiltration, pressure 0.1MPa, filtrate for later use;
C, two-stage ultrafiltering: get above-mentioned filtrate, add appropriate hydrochloric acid adjust pH to 6.5, left standstill 8 hours, adopting the molecular retention amount is that 3000 daltonian polyamide membrane are carried out ultrafiltration, pressure 0.2MPa, and ultrafiltrate is for subsequent use;
D, get above-mentioned ultrafiltrate and add sodium lactate accent etc. and ooze, being concentrated into cumulative volume is 5L, fill, the large capacity Leonurus Heterophyllus injection of finished product of sterilizing to get.
Adopt the HPLC method that provides among the embodiment 9 to measure, it is 1.0mg that every 1ml contains total alkaloids; Total alkaloid content is 250mg in every 250ml sample.
This infusion solutions physicochemical property is stable, and its clarity is high and microgranule is few, the preparation Performance Detection:
Particulate matter---sample thief 50ml, according to " 2010 editions appendix IXR of Chinese Pharmacopoeia particulate matter inspection technique " first method, light blockage method inspection, particulate matter 〉=10 μ m :≤20; 〉=25 μ m :≤5.
Protein---get this product 1ml, add 30% sulfosalicylic acid solution 1ml of new preparation, shake up, placed 5 minutes, do not occur muddy, up to specification.
Tannin---get this product 1ml, add the normal saline 5ml that contains 1% Ovum Gallus domesticus album of new preparation, placed 10 minutes, do not occur muddy or precipitation, up to specification.
Resin---get this product 5ml, put in the separatory funnel, add chloroform 10ml jolting and extract, divide and get chloroform liquid, put evaporate to dryness in the water-bath, residue adds glacial acetic acid 2ml makes dissolving, puts in the tool plug test tube, adds water 3ml, mixing was placed 30 minutes, precipitation do not occur, and is up to specification.
Oxalates---get this product 2ml, add 2～3 of 3% calcium chloride test solutions, placed 10 minutes, do not become turbid or precipitate, up to specification.
Embodiment 8: the preparation of large capacity Leonurus Heterophyllus injection
The preparation of a, Herba Leonuri extracting solution and processing: get Herba Leonuri 1kg, add water 10kg at every turn and decoct 3 times, collecting decoction, centrifugal treating 30 minutes (2500 rev/mins), it is for subsequent use to get supernatant;
B, one-level ultrafiltration: getting above-mentioned supernatant employing molecular retention amount is that 10,000 daltonian poly (ether sulfone) films carry out ultrafiltration, pressure 0.2MPa, filtrate for later use;
C, two-stage ultrafiltering: get above-mentioned filtrate, add an amount of 1mol/L hydrochloric acid adjust pH to 5.5, left standstill 8 hours, adopting the molecular retention amount is that 3000 daltonian poly (ether sulfone) films carry out ultrafiltration, pressure 0.2MPa, and ultrafiltrate is for subsequent use;
D, get above-mentioned ultrafiltrate and add glucose accent etc. and ooze, being concentrated into cumulative volume is 2.5L, fill, the large capacity Leonurus Heterophyllus injection of finished product of sterilizing to get.
Adopt the HPLC method that provides among the embodiment 9 to measure, total alkaloids is in the total amount of leonurine and stachydrine hydrochloride, and it is 2.0mg that every 1ml contains total alkaloids; Total alkaloids is 500mg in every 250ml sample.
This infusion solutions physicochemical property is stable, and its clarity is high and microgranule is few, the preparation Performance Detection:
Particulate matter---sample thief 50ml, according to " 2010 editions appendix IXR of Chinese Pharmacopoeia particulate matter inspection technique " first method, light blockage method inspection, particulate matter 〉=10 μ m :≤20; 〉=25 μ m :≤5.
Protein---get this product 1ml, add 30% sulfosalicylic acid solution 1ml of new preparation, shake up, placed 5 minutes, do not occur muddy, up to specification.
Tannin---get this product 1ml, add the normal saline 5ml that contains 1% Ovum Gallus domesticus album of new preparation, placed 10 minutes, do not occur muddy or precipitation, up to specification.
Resin---get this product 5ml, put in the separatory funnel, add chloroform 10ml jolting and extract, divide and get chloroform liquid, put evaporate to dryness in the water-bath, residue adds glacial acetic acid 2ml makes dissolving, puts in the tool plug test tube, adds water 3ml, mixing was placed 30 minutes, precipitation do not occur, and is up to specification.
Oxalates---get this product 2ml, add 2～3 of 3% calcium chloride test solutions, placed 10 minutes, do not become turbid or precipitate, up to specification.
Embodiment 9: the HPLC of large capacity Leonurus Heterophyllus injection total alkaloids detects
1 experimental apparatus and reagent
1.1 instrument
Agilent 1100 high performance liquid chromatographs (Agilent Chemstation work station);
Chromatographic column: Sunfire C18 (250mm * 4.6mm, 5 μ m);
Kromasil C18(250mm×4.6mm，5μm)；
Kromasil C18(150mm×4.6mm，5μm)；
Aichrom Bond-AQ-Cl8(4.6mm×250mm，5μm)；
1.2 reagent
Sample is provided by the thousand standing grain pharmaceutcal corporation, Ltd medicine centers of grinding, Xi'an.
Reference substance leonurine (Anhui nova institute of materia medica, purity 99.0%)
Stachydrine hydrochloride (Chinese pharmaceutical biological product is checked institute, and lot number: 712-0703 is for assay)
Reagent methanol, acetonitrile etc. are chromatographically pure, and water is the self-control ultra-pure water, and other reagent is analytical pure
2 chromatographic conditions
In the preliminary experiment process, select respectively Sunfire C18 (250mm * 4.6mm, 5 μ m), KromasilC18 (250mm * 4.6mm, 5 μ m), Kromasil C18 (150mm * 4.6mm, 5 μ m) and Aichrom Bond-AQ-Cl8 (4.6mm * 250mm, 5 μ m) four kinds of different chromatographic columns are studied, relatively good from the separating degree of the peak shape of chromatographic peak and separating effect Sunfire C18 (250mm * 4.6mm, 5 μ m) post.
In the test, once investigated multiple flow phase system (such as methanol-buffer; Acetonitrile-buffer etc.) impact that leonurine is separated with stachydrine hydrochloride, simultaneously the pH, flow velocity and the column temperature that detect wavelength, buffer are investigated, the result is at mobile phase 0.1% phosphoric acid solution (regulating pH4.0 with triethylamine)-acetonitrile-methanol (40: 30: 30) (volume ratio); Flow velocity: 0.8mL/min; Column temperature: leonurine and stachydrine hydrochloride appearance time are moderate in the time of 30 ℃, separate good, simultaneously with sample in other impurity peaks all separate well.The chromatographic condition of research is as follows:
Detect wavelength: 276nm;
Column temperature: 30 ℃;
Mobile phase: 0.1% phosphoric acid solution (regulating pH4.0 with triethylamine)-acetonitrile-methanol (40: 30: 30) (volume ratio); Flow velocity: 0.8mL/min;
The evaluation of 3 chromatographic conditions
By different experiments personnel, chromatographic system and object of study chromatographic condition and system suitability are investigated, the result is as follows:
A. separating degree: leonurine, stachydrine hydrochloride reach good separating with other impurity peaks in the sample, separating degree R＞1.5 (chromatogram of leonurine and stachydrine hydrochloride reference substance is seen Fig. 1, and the chromatogram of Leonurus Heterophyllus injection sample of the present invention is seen Fig. 2).
B. number of theoretical plate: in the stachydrine hydrochloride peak, number of theoretical plate is not less than 3000 in reference substance and the sample.
4 conclusions
Take 0.1% phosphoric acid solution (regulating pH4.0 with triethylamine)-acetonitrile-methanol (40: 30: 30) as mobile phase; Flow velocity is 0.8mL/mm; The detection wavelength is 276nm; Column temperature is 30 ℃.Number of theoretical plate is not less than 3000 chromatographic condition by the stachydrine hydrochloride peak can satisfy testing requirement.
5 methodological studies
Detection method of the present invention has been carried out methodological study, specific as follows:
5.1 the preparation of reference substance solution
It is an amount of that precision takes by weighing the reference substance of leonurine, stachydrine hydrochloride, adds the reference substance solution that the dissolving of mobile phase solution is made into respectively 0.5mg/mL, 1mg/mL.
5.2 the preparation of need testing solution
Because sample is bulk capacity injection, strict control is arranged on clarity and microgranule, and keep the physical presence state of each material in the injection as far as possible, so the method for sample introduction after adopting sample directly to dilute.
Precision is measured product 2ml of the present invention, puts in the 10ml measuring bottle, adds the mobile phase solution dilution to scale, shakes up, and get final product.
5.3 the investigation of linear relationship
The accurate absorption in leonurine reference substance solution 0.5mL, 1.0mL, 2.0mL, 4.0mL, 5.0mL to the 10mL volumetric flask used methanol constant volume, shakes up, and be for subsequent use.The accurate absorption in stachydrine hydrochloride reference substance solution 1.0mL, 2.0mL, 4.0mL, 5.0mL, 10.0mL to the 10mL volumetric flask used the mobile phase standardize solution, shakes up, and be for subsequent use.Draw each concentration solution 20 μ L and inject high performance liquid chromatograph.Record respectively the peak area of leonurine, stachydrine hydrochloride, set up take component content as abscissa, peak area is the standard curve equation of vertical coordinate, the results are shown in Table 1.
The linear relationship of two kinds of reference substances of table 1 Herba Leonuri
| Composition | Equation | R | The range of linearity (μ g) |
| Leonurine | Y＝1.21×10 8X-1.92×10 5 | 0.9998 | 2.50～25.0 |
| Stachydrine hydrochloride | Y＝1.24×10 9X-2.96×10 5 | 0.9996 | 10.0～100.0 |
5.4 precision test
To the need testing solution of same lot number sample, continuous sample introduction 5 pins, the peak area of calculating leonurine and stachydrine hydrochloride the results are shown in Table 2.
Table 2 Precision Experiment result
5.5 replica test
Get same lot number sample, totally 5 parts, get need testing solution according to " 5.2 " below legal system respectively, to measure, the content with standard curve method mensuration leonurine and stachydrine hydrochloride the results are shown in Table 3.
Table 3 repeated experiment result
5.6 stability test
The same need testing solution of accurate absorption, measure respectively at 0,2,4,8 and 24 hour sample introduction after the preparation, calculate the peak area of leonurine and stachydrine hydrochloride, RSD is respectively 2.24% and 2.01%, shows that leonurine and stachydrine hydrochloride are basicly stable at 24 hours.The results are shown in Table 4.
Table 4 stability test result
5.7 average recovery test
Precision is measured the large capacity Leonurus Heterophyllus injection sample 10mL of known content, totally 5 parts, add respectively a certain amount of leonurine and stachydrine hydrochloride reference substance, prepare 5 parts in sample by the need testing solution preparation method, sample size 20 μ L, measure each component content with standard curve method, the results are shown in Table 5, table 6.
Table 5 leonurine response rate experimental result
Table 6 stachydrine hydrochloride response rate experimental result
| Contain in the sample | Addition | The amount of recording | The response rate | Meansigma methods | RSD(％) |
| Amount (mg) | (mg) | (mg) | (％) | (％) | |
| 0.1036 | 0.10 | 0.2019 | 98.33 | ||
| 0.1021 | 0.10 | 0.2008 | 98.67 | ||
| 0.1027 | 0.10 | 0.2002 | 97.50 | 97.25 | 1.02 |
| 0.1013 | 0.10 | 0.2001 | 98.83 | ||
| 0.1026 | 0.10 | 0.1988 | 96.17 |
5.810 the assay of batch sample
By detection method of the present invention, measure 10 batch samples, it the results are shown in Table 7.
The large capacity Leonurus Heterophyllus injection assay result (mg/ml) of table 7
| Batch | Leonurine | Stachydrine hydrochloride | Total alkaloids |
| 1 | 0.051 | 0.124 | 0.175 |
| 2 | 0.052 | 0.121 | 0.171 |
| 3 | 0.050 | 0.122 | 0.172 |
| 4 | 0.051 | 0.126 | 0.177 |
| 5 | 0.051 | 0.125 | 0.176 |
| 6 | 0.053 | 0.124 | 0.177 |
| 7 | 0.050 | 0.121 | 0.171 |
| 8 | 0.050 | 0.124 | 0.174 |
| 9 | 0.055 | 0.123 | 0.178 |
| 10 | 0.052 | 0.124 | 0.177 |
According to the measurement result of 10 batch samples as seen, the assay limit of this product is that every 1ml contains total alkaloids and is no less than 0.15mg in leonurine and stachydrine hydrochloride sum.
Embodiment 10: the comparison of Leonurus Heterophyllus injection detection method of the present invention and prior art
CN1973855A relates to a kind of motherwort injection, wherein discloses employing HPLC method and has determined Herba Leonuri finger printing standard, the content of the total alkaloids in the stachydrine hydrochloride cubage motherwort injection that specifically obtains with mensuration.
The existing ministry standard method that detects at present Leonurus Heterophyllus injection is the content by the spectrophotometry stachydrine hydrochloride, specifically adopts following method:
The preparation precision of reference substance solution takes by weighing the stachydrine hydrochloride reference substance 25mg that is dried to constant weight at 105 ℃, puts in the 25ml measuring bottle, with the 0.1mol/L dissolve with hydrochloric acid solution and be diluted to scale, shakes up, and namely gets (the hydrochloric stachydrine 1mg of every 1ml).
The preparation precision of need testing solution is measured this product 5ml, puts in the 100ml measuring bottle, is diluted to scale with the 0.1mol/L hydrochloric acid solution, shakes up, and get final product.
Algoscopy respectively precision is measured reference substance solution, need testing solution, each 10ml of 0.1mol/L hydrochloric acid solution, place respectively three 25ml measuring bottles, each accurate 2% chromic thiocyanate ammonium salt solution 3ml that adds new system shakes up, and adds the 0.1mol/L hydrochloric acid solution to scale, shake up, put in the ice bath and placed 1 hour, filter, discard just filtrate, get subsequent filtrate, take the 0.1mol/L hydrochloric acid solution as blank.According to spectrophotography (Chinese Pharmacopoeia appendix V B), measure respectively trap at the wavelength place of 525nm, deduct respectively the trap of reference substance and test sample with the trap of blank reagent, calculate, and get final product.
Every contains total alkaloids with stachydrine hydrochloride (C7H13NO2HCl) calculating, should be 18～22mg.
The existing ministry standard method that the inventor adopts total alkaloids assay method among detection method of the present invention, the CN1973855A and Leonurus Heterophyllus injection is carried out Determination of Total Alkaloid relatively to the Leonurus Heterophyllus injection sample of embodiment 4 preparations, the results are shown in Table 8.
Three kinds of detection methods of table 8 are carried out the Determination of Total Alkaloid result relatively to same sample
Testing result by three kinds of methods relatively, the inventor finds that the result that detection method of the present invention and working standard method obtain is more approaching, the result is more accurate, obviously is better than the detection method of CN1973855A.In addition, the method complex operation of existing ministry standard adopts the precision of spectrophotometry also to be inferior to the detection method that the present invention adopts high performance liquid chromatography.
Claims (10)
1. a Leonurus Heterophyllus injection is characterized in that, described injection comprises the chemical compound of Herba Leonuri ultrafiltrate, pharmaceutically acceptable adjusting osmotic pressure and water for injection randomly.
2. Leonurus Heterophyllus injection according to claim 1 is characterized in that, in the 250ml injection, the content that described injection comprises total alkaloids is 10-500mg;
Preferably, to comprise the content of total alkaloids be 20～250mg to described injection;
Further preferably, the described injection content that comprises total alkaloids is 40～125mg;
More preferably, to comprise the content of total alkaloids be 45～50mg to described injection.
3. Leonurus Heterophyllus injection according to claim 1 and 2 is characterized in that, the particulate matter greater than 10 μ m among the every 50ml of described Leonurus Heterophyllus injection is no more than 20; Microgranule greater than 25 μ m is no more than 5.
4. each described Leonurus Heterophyllus injection in 3 according to claim 1 is characterized in that the method preparation of described Herba Leonuri ultrafiltrate by comprising the steps:
Get Herba Leonuri, decoct with water, merging filtrate, centrifugal treating or process microporous filter membrane are processed, and carry out ultrafiltration;
Preferably, the method preparation of described Herba Leonuri ultrafiltrate by comprising the steps:
A, get Herba Leonuri, decoct with water 2～5 times, collecting decoction, 1500～5000 rev/mins of centrifugal treating 5～30 minutes or process through microporous filter membrane, the supernatant of getting after the processing is for subsequent use;
B, get the supernatant that step a obtains, adopting the molecular retention amount is that 10,000～100,000 daltonian ultrafilter membranes carry out ultrafiltration, pressure 0.05-0.25MPa, and ultrafiltrate is for subsequent use;
C, get the ultrafiltrate that step b obtains, add appropriate hydrochloric acid adjust pH to 4.0～7.0, leave standstill more than 8～24 hours, adopting the molecular retention amount is that 1000～50,000 daltonian ultrafilter membranes carry out ultrafiltration, pressure 0.05-0.25MPa, and ultrafiltrate is for subsequent use;
Further preferably, the water that adds in step a is 8～12 times of medical material; The microporous filter membrane aperture of using is 0.45 μ m; Ultrafilter membrane among described step b and the c is selected from respectively ceramic membrane, cellulose acetate membrane, poly (ether sulfone) film, polyamide membrane or poly-fluorine film.
5. each described Leonurus Heterophyllus injection according to claim 1～4, it is characterized in that the chemical compound of described pharmaceutically acceptable adjusting osmotic pressure is selected from one or more in glucose, sodium chloride, magnesium chloride, calcium chloride, sodium lactate, xylitol, sorbitol and/or the dextran;
Preferably, described chemical compound is selected from one or more in glucose, sodium chloride, calcium chloride, dextran and/or the sodium lactate.
6. each described Leonurus Heterophyllus injection is characterized in that according to claim 1～5, and the osmotic pressure of described injection and people's plasma osmotic pressure equate.
7. the preparation method of a Leonurus Heterophyllus injection is characterized in that, described preparation method may further comprise the steps:
Get Herba Leonuri, decoct with water, merging filtrate, centrifugal treating or process through microporous filter membrane is carried out ultrafiltration, and ultrafiltrate adds the chemical compound accent of pharmaceutically acceptable adjusting osmotic pressure etc. and oozes, and adds water for injection or concentrated, sterilization, fill;
Preferably, said method comprising the steps of:
A, get Herba Leonuri, decoct with water 2～5 times, collecting decoction, 1500～5000 rev/mins of centrifugal treating 5～30 minutes or process through microporous filter membrane, the supernatant of getting after the processing is for subsequent use;
B, get the supernatant that step a obtains, adopting the molecular retention amount is that 10,000～100,000 daltonian ultrafilter membranes carry out ultrafiltration, pressure 0.05-0.25MPa, and ultrafiltrate is for subsequent use;
C, get the ultrafiltrate that step b obtains, add appropriate hydrochloric acid adjust pH to 4.0～7.0, leave standstill more than 8～24 hours, adopting the molecular retention amount is that 1000～50,000 daltonian ultrafilter membranes carry out ultrafiltration, pressure 0.05-0.25MPa, and ultrafiltrate is for subsequent use;
D, get the chemical compound accent etc. that ultrafiltrate that step c obtains adds pharmaceutically acceptable adjusting osmotic pressure and ooze, add water for injection or concentrated, fill nitrogen, sterilization, fill.
8. according to claim 6 or 7 described preparation methoies, it is characterized in that the water that adds among the described step a is 8～12 times of medical material;
The microporous filter membrane aperture of preferably, using among the step a is 0.45 μ m;
Preferably, the ultrafilter membrane among described step b and the c is selected from respectively ceramic membrane, cellulose acetate membrane, poly (ether sulfone) film, polyamide membrane or poly-fluorine film.
9. each described preparation method according to claim 6～8, it is characterized in that the chemical compound of described pharmaceutically acceptable adjusting osmotic pressure is selected from one or more in glucose, sodium chloride, magnesium chloride, calcium chloride, sodium lactate, xylitol, sorbitol and/or the dextran;
Preferably, described chemical compound is selected from one or more in glucose, sodium chloride, calcium chloride, dextran and/or the sodium lactate.
10. the detection method of total alkaloids in the Leonurus Heterophyllus injection is characterized in that, described detection method comprises the content that detects leonurine and stachydrine hydrochloride in the described Leonurus Heterophyllus injection;
Preferably, described detection method is according to high-performance liquid chromatogram determination, and described chromatographic condition comprises:
Take octadecylsilane chemically bonded silica as filler; Take 0.1% phosphoric acid solution (regulating pH4.0 with triethylamine)-acetonitrile-methanol (40: 30: 30) as mobile phase, column temperature is 30 ℃; The detection wavelength is 276nm; Flow velocity: 0.8mL/min; Sample size 20 μ l; Number of theoretical plate calculates by the stachydrine hydrochloride peak and is not less than 3000.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104055840A (en) * | 2014-06-18 | 2014-09-24 | 广州白云山星群(药业)股份有限公司 | Motherwort extracting solution refining method |
| CN105380945A (en) * | 2015-10-27 | 2016-03-09 | 江苏大学 | Application of leonurus stachydrine to preparation of medicine for preventing and treating atherosclerosis |
| CN109655565A (en) * | 2019-01-07 | 2019-04-19 | 南京海昌中药集团有限公司 | The fingerprint atlas detection method of yimucao paste |
| CN113030359A (en) * | 2021-01-28 | 2021-06-25 | 成都第一制药有限公司 | Detection method for various index components in motherwort injection and quality control method of motherwort injection |
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| CN1557347A (en) * | 2004-01-13 | 2004-12-29 | 吴良信 | Medicine for treating cardiac and cerebral vascular diseases and its preparation |
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| CN1973854A (en) * | 2006-10-16 | 2007-06-06 | 成都市时代第一药物研究所有限公司 | Process of preparing motherwort injection |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104055840A (en) * | 2014-06-18 | 2014-09-24 | 广州白云山星群(药业)股份有限公司 | Motherwort extracting solution refining method |
| CN105380945A (en) * | 2015-10-27 | 2016-03-09 | 江苏大学 | Application of leonurus stachydrine to preparation of medicine for preventing and treating atherosclerosis |
| CN109655565A (en) * | 2019-01-07 | 2019-04-19 | 南京海昌中药集团有限公司 | The fingerprint atlas detection method of yimucao paste |
| CN109655565B (en) * | 2019-01-07 | 2020-10-30 | 南京海昌中药集团有限公司 | Fingerprint spectrum detection method of motherwort herb paste |
| CN113030359A (en) * | 2021-01-28 | 2021-06-25 | 成都第一制药有限公司 | Detection method for various index components in motherwort injection and quality control method of motherwort injection |
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| Publication number | Publication date |
|---|---|
| CN102846704B (en) | 2014-09-17 |
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