CN1709498A - Ginseng-astragalus blood-sugar lowering soft capsule, and its preparing and detecting method - Google Patents
Ginseng-astragalus blood-sugar lowering soft capsule, and its preparing and detecting method Download PDFInfo
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Abstract
The present invention discloses a Chinese medicine Shenqi Jiangtang soft capsule preparation with obvious effect for curing diabetes. Said soft capsule preparation is made up by using 11 Chinese medicinal materials of ginseng (stem and leaf) saponin, astragalus root, rehmannia root, ophiopogon tuber, trichosanthes root, lyceum berry, Chinese yam and others.
Description
Technical field
The present invention relates to a kind of Chinese medicinal soft capsule for the treatment of diabetes, specifically a kind of ginseng-astragalus blood-sugar lowering soft capsule relates to the preparation method of this medicine simultaneously.
Background technology
Diabetes are a kind of complex diseases because of syndrome, be since in the body hormone of insulin deficit or antagonism insulin increase, or insulin can not be brought into normal play physiological action and a kind of syndrome of the glucose, protein and the lipid metabolic disorder that cause in target cell.It is characterized by the unusual rising of concentration of glucose and glucose in urine in the blood circulation, occur typical " three-many-one-little " symptom when blood glucose is too high, promptly repeatedly, polyuria, polyphagia and lose weight, and with fatigue and weak.Ketoacidosis, hypertonicity diabetic coma can take place in severe patient, and easily merge multiple infection.Along with the prolongation of the course of disease, its metabolism disorder can cause the chronic pathological changes of histoorgans such as eye, kidney, nerve, blood vessel and heart.If can not get timely, appropriate treatment, heart change, cerebrovascular disease, renal failure then take place, lose the sight of both eyes, situation such as lower limbs necrosis, become disable, lethal main cause.And onset diabetes rate height, the national sampling survey of 1995-1996, natural crowd's prevalence is up to 3.21% more than 20 years old, the whole nation 300,000 census of the population results in 1980, prevalence 4.21% more than 60 years old, single Jiangsu elderly population diabetes, impaired glucose tolerance prevalence then are respectively 14.49% and 10.21%, and no matter are developed country or developing country, and the sickness rate of diabetes is all rising year by year.
Mostly the medicine of clinical treatment 2 types is chemicals, widely used sulphanylureas, biguanides, other antidiabetic drugs and the adjuvant drug of being broadly divided into except that insulin at present.The sulfonylureas drugs for diabetes thing is topmost Remedies for diabetes.Can make that hepatic glycogen is synthetic to be increased,, surrounding tissue be strengthened to the sensitivity of insulin, the picked-up of glucose is increased, thereby reach the effect of blood sugar lowering again by the effect behind pair cell receptor or the receptor.But more easily cause untoward reaction such as hypoglycemia, granulocytopenia and cardiovascular disease.Biguanides antidiabetic drug heavy dose can cause digestive tract reaction, has the patient of pathological changes easily to cause lactic acidosis at lung, liver, kidney.Bring hidden danger for the extensive patients drug safety, for a change this situation for a long time, attempts treating with protocol in the plant clinically always.The traditional Chinese medical science is consistent to the cause of disease view of diabetes, thinks it mainly is that surfeit delicious food, overaction of the five emotions, chamber do not save, the dry and not enough Several Factors of natural endowment of calentura fire.
The treatment that current SHENQI JIANGTANG preparation clinically is widely used in type ii diabetes obtains curative effect preferably, and has no side effect, and promotional value is widely arranged; The SHENQI JIANGTANG oral formulations of present clinical use is still based on oral liquid, granule, capsule, tablet, because dosage form itself, it exists following problem:
It is few that oral liquid has dosage, easily takes, absorb fast, advantages such as good effect.But in real work, we find that the SHENQI JIANGTANG oral liquid is prone to muddiness, flocculent deposit equistability problem in storage process.Astragaloside, ginsenoside belong to saponins simultaneously, facile hydrolysis, and active ingredient is damaged, downgrade.And carry, take inconvenience, production cost height.
Granule is inconvenient to carry and take, and tablet, capsule exist that bioavailability is low, onset waits problem slowly.Soft capsule content is liquid or semisolid, and oral being dispersed or dissolved in very soon in the gastro-intestinal Fluid helps absorbing.
At the problem of above existence, for make the SHENQI JIANGTANG preparation clinically better, clearer and more definite application, we need a kind of determined curative effect, the little SHENQI JIANGTANG preparation easy to carry of consumption to satisfy the clinical application needs.
Summary of the invention
The objective of the invention is to overcome above the deficiencies in the prior art, a kind of efficient, quick-acting SHENQI JIANGTANG preparation is provided, another object of the present invention provides the preparation detection method of this Chinese medicine preparation.
For achieving the above object, we have adopted following technical scheme:
Chinese medicinal soft capsule of the present invention is to be made by the raw material of following weight ratio:
Ginsenoside 4-8 weight portion Radix Astragali 80-150 weight portion Radix Rehmanniae 150-250 weight portion
Radix Ophiopogonis 45-80 weight portion Radix Trichosanthis 40-80 weight portion Fructus Lycii 80-160 weight portion
Fructus Schisandrae Chinensis 40-80 weight portion Rhizoma Dioscoreae 40-80 weight portion Fructus Rubi 20-50 weight portion
Poria 40-80 weight portion Rhizoma Alismatis 40-80 weight portion.
The weight ratio of the raw material of Chinese medicine composition of the present invention is preferably:
Ginsenoside's 6 weight portion Radixs Astragali 124 weight portion Radix Rehmanniae 186 weight portions
Radix Ophiopogonis 62 weight portion Radix Trichosanthis, 62 weight portion Fructus Lyciis, 124 weight portions
Fructus Schisandrae Chinensis 62 weight portion Rhizoma Dioscoreaes 62 weight portion Fructus Rubies 31 weight portions
Poria 62 weight portion Rhizoma Alismatis 62 weight portions.
In order to make this product make our required soft capsule, in the preparation process, need to add various required adjuvants, we mainly select Polyethylene Glycol or refined plant oil in the adjuvant, wherein raw material extract obtained with the adjuvant weight ratio be 1: 0.4-1: 5; Both are preferably ratio: 1: 0.5-1: 1; Wherein with the Macrogol 4000 better effects if, the optimum weight ratio of extract and Macrogol 4000 is 1: 0.7.
Mainly containing effective component content in the soft capsule of the present invention is:
Every soft capsule contains ginsenoside Re 0.05-20mg.
Every soft capsule contains astragaloside 0.02-1mg.
Preparation of the present invention (ginseng-astragalus blood-sugar lowering soft capsule)
(1) ginsenoside Re's assay method is:
The preparation of reference substance solution: it is an amount of that precision takes by weighing ginsenoside Re's reference substance, adds methanol and make the solution that contains the about 0.2-0.8mg of ginsenoside Re among every 1ml, in contrast product solution;
The preparation of need testing solution: get this product 2-20 grain, cut off, scrape and get content, mixing, rubber absolute ethanol washing, content add water 10-50ml, filter, the water saturated n-butyl alcohol 10-50ml of accurate adding extracts 10-40min, and precision is measured supernatant 10-50ml, adds the ammonia solution triplication, shake up, place, divide and get upper solution, evaporate to dryness, residue add methanol makes dissolving, quantitatively moves in the 2-10ml measuring bottle, add methanol and be diluted to scale, shake up, promptly;
Chromatographic condition: with octadecylsilane chemically bonded silica is filler; Volume ratio is that 99: 400 acetonitrile-0.05% phosphoric acid solution is a mobile phase; Detect wavelength 203nm;
Algoscopy: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject hplc determination;
(2) assay method of astragaloside is:
Chromatographic condition: octadecylsilane chemically bonded silica is a filler; Evaporative light scattering detector;
Mobile phase: ratio is acetonitrile-water-oxolane of 33: 67: 4;
The preparation of reference substance solution: get the astragaloside reference substance and add methanol and make the solution that every 1ml contains about 0.1-2mg, shake up;
The preparation of need testing solution: get this product 2-20 grain, cut off, scrape and get content, mixing, rubber absolute ethanol washing, content add water 10-50ml, filter, filtrate is used defat with petroleum ether 1-3 time, and (each 10-50ml) extracted in water saturated n-butyl alcohol jolting 2-6 time, merges n-butanol extracting liquid, extract 1-2 time with ammonia solution, each 10-40ml discards ammonia solution, the water 10-40ml saturated with n-butyl alcohol washes, and discards water liquid, n-butyl alcohol liquid evaporate to dryness, residue adds water 1-25mL makes dissolving, puts coldly, passes through D
101Type macroporous adsorptive resins (internal diameter 1.5cm, long 18cm) is with water elution, discard water liquid, reuse 10-40% ethanol 10-100ml eluting discards ethanol elution, continue with 50-90% ethanol 50-150ml eluting, collect eluent, evaporate to dryness is with dissolve with methanol and be transferred in the 2-10ml measuring bottle, add methanol to scale, shake up, filter, promptly.
Preparation method of the present invention is prepared by following steps:
A uses warm water lixiviate 1-3 time Radix Ophiopogonis, and each 0.5-3 hour, merge lixiviating solution, filter, filtrate is concentrated into the thick paste that relative density is 1.03-1.40;
B Fructus Schisandrae Chinensis 40-70% ethanol, percolation, percolate reclaim ethanol, are concentrated into thick paste;
C Fructus Lycii, the Radix Astragali, Radix Rehmanniae, Rhizoma Alismatis, Rhizoma Dioscoreae, Radix Trichosanthis, Fructus Rubi, Poria water decoct 1-4 time, each 0.5-3 hour, merging decoction liquor, filtration, filtrate, to be concentrated into relative density be 1.20 thick paste, adding ethanol makes and contains the alcohol amount and be 40%-80%, leave standstill, the leaching supernatant, reclaim ethanol, concentrate
D merges drying, pulverizing and ginsenoside with above-mentioned extractum and merges, and mixing is got adjuvant Macrogol 4000 or refined plant oil and added the extract powder mixing, moves in the encapsulating machine storage tank, is pressed into ball, selects ball, promptly.
The technology of steps d can be replaced with following technology: extractum is merged drying, pulverizing and ginsenoside merge, mixing, get adjuvant Macrogol 4000 or refined plant oil and add the extract powder mixing, move in the encapsulating machine medicinal liquid storage tank, put gelatin solution in the gelatin storage tank and be incubated 60 ℃, make ball 40-50 ℃ following of water dropper temperature, select ball, drying, packing, promptly.
Drug treatment of diabetic of the present invention has better curative effect.For showing medicine of the present invention to the treatment of diabetes effect, we have done a large amount of experimentatioies, below experimental example be used to further specify the present invention.
One, the present invention is to causing the influence of kidney of rats upper parathyrine hyperglycemia model
Type ii diabetes is common complaint among the elderly, frequently-occurring disease.Through clinical efficacy and experiment confirm, the present invention has certain hypoglycemic activity.This experiment is to quicken glycogen and steatolysis in the rat body with the adrenal gland, makes that concentration of glucose increases in the rat serum.Epinephrine can also suppress the intravital insulin of rat and discharge.Irritate the doses glucose to rat more simultaneously and quicken to cause hyperglycemia model.From experimental result, administration group and matched group relatively, the present invention has hypoglycemic activity clearly, we and QIZHI JIANGTANG JIAONANG are as positive controls simultaneously.
Material
Animal: the Wistar rat, male and female half and half, body weight are 230 ± 30g
Medicine: 1, the present invention, drug effect dosage is respectively 50 times, 37.5 times, 25 times of clinical application amount).
2, QIZHI JIANGTANG JIAONANG, middleization Siping City pharmaceutical factory produces.
Method and result:
Get 50 of rats, be divided into 5 groups at random, 10 every group.First group of negative matched group gives distilled water.Second group of positive matched group, QIZHI JIANGTANG JIAONANG.Third and fourth, five groups be respectively high, medium and low three dosage groups of the present invention, dosage is respectively 50 times, 37.5 times, 25 times of clinical application amount, the animal gastric infusion, medicine journey one month, after the last administration 1 hour, dosage injection epinephrine by 500 μ g/kg, cause adrenal gland's hyperglycemia model, irritate stomach with the dosage of glucose 1g/kg simultaneously, the foundation of acceleration model, adopt glucose oxidase method to measure empty stomach, the change of blood sugar of half an hour, 1 hour, 2 hours, 3 hours respectively, it the results are shown in Table 1.
Table 1 the present invention is to the influence of epinephrine moulding rat blood sugar content
Blood sugar content (mg%) X ± SD
1 hour 2 hours 3 hours half an hour on an empty stomach
82.8±18.3 233.5±65.3 247.6±57.1 144.0±67.6 95.9±10.3
Matched group
(n＝12) (n＝10) (n＝10) (n＝10) (n＝11)
94.1±31.8 195.6±39.1 220.0±39.3 150.4±57.7 90.7±21.0
Positive controls
(n＝10) (n＝11) (n＝10) (n＝11) (n＝10)
The high agent 97.9 of the present invention ± 23.4 244.6 ± 44.3 154.6 ± 35.4 101.9 ± 33.3 100.1 ± 14.3
Amount group (n=10) is (n=13) (n=12)
* *(n=13) (n=12)
Agent 95.9 among the present invention ± 22.9 236.8 ± 42.7 185.0 ± 42.1 109.2 ± 32.6 99.1 ± 10.1
Amount group (n=10) is (n=10) (n=10)
* *(n=9) (n=9)
The present invention hangs down agent 95.4 ± 22.5 224.1 ± 41.7 156.5 ± 48.0 113.8 ± 47.5 98.4 ± 10.4
Amount group (n=10) is (n=8) (n=10)
* *(n=9) (n=10)
***P＜0.001
Result and discussion:
The animal hyperglycemia model is a kind of method that increases with simulation irritability blood glucose due to the epinephrine, is equivalent to type ii diabetes patient's the post-stimulatory morbidity state of spiritual irritability of being grown up, so has certain clinical meaning.
QIZHI JIANGTANG JIAONANG can obviously reduce adrenal gland's disposition hyperglycemia and confirm for institute in the past.Therefore be selected to the positive control of this experiment as Chinese medicine, experimental result shows that the blood glucose response curve of QIZHI JIANGTANG JIAONANG group is low than negative control group, illustrates that this experimental result is believable.The blood glucose response curve that three dosage groups of the present invention are presented is low than negative control group also, especially with 1 hour difference highly significant (P＜0.01).
This experimental result shows that the present invention can suppress to increase reaction by the rat blood sugar due to the epinephrine preferably.
Two, the present invention is to the influence of mice alloxan diabetes model
Materials and methods:
1, medicine: alloxan moulding dosage 100mg/kg (being produced by Japan and light pharmaceuticals industry Co., Ltd.), all the other reagent are national market sale product.
2, medicine: powder of the present invention is a light brown, sweet in the mouth, little puckery.Drug dose is equivalent to 50 times, 37.5 times, 25 times of clinical adult's consumption respectively.
QIZHI JIANGTANG JIAONANG, middleization Siping City pharmaceutical factory produces.
3, animal: 50 of outbreeding system Kunming mouses, body weight 20 ± 2g.
4, grouping: pairing is divided equally five groups after measuring fasting glucose.Be grown up 50 times of consumption of first group of the present invention, be grown up 37.5 times of consumption of second group of the present invention, be grown up 25 times of consumption of the 3rd group of the present invention, the 4th group of QIZHI JIANGTANG JIAONANG, the 5th group of matched group gives distilled water (calculating by heavy dose of dosage).
5, pathology moulding and medication: animal ig administration, 1 time/day, continuous 7 days, ihr is by the tail vein injection alloxan after the administration in seven days, dosage 100mg/kg causes diabetes model, and in the 9th, 10,14,18,22,24 day difference rathole vena orbitalis posterior blood sampling from childhood mensuration blood sugar content, simultaneously, record animal dead number.
6, observation index: by aforementioned blood sampling day mensuration blood sugar content, after putting to death animal on the 24th day, examine pancreas at once, carry out A, B, cell tissue chemical staining, counting after fixing with BovwShi.
The result:
1, each treated animal survival condition
The injection alloxan after second day, each is organized blood glucose and obviously raises, death condition sees Table 1.Injection alloxan the 4th day (be the full course of treatment the 11st day), negative control group and QIZHI JIANGTANG JIAONANG group begin to occur dead, the negative control group general mortality rate was 70% when finished the course of treatment, QIZHI JIANGTANG JIAONANG group general mortality rate is 50%, high dose group of the present invention (adult consumption 50 times) and in dosage group (adult's consumption 37.5 times) general mortality rate be 20%, low dose group (adult consumption 25 times), it is dead not have 1 example.
2, each treated animal blood glucose situation
Each group blood glucose after moulding all has obvious rising, but total trend the present invention and QIZHI JIANGTANG JIAONANG group all are lower than matched group, and owing to matched group is fallen ill serious mice in viewing duration death, so mean blood glucose does not significantly raise in matched group.Even so; after moulding the 11st day; low dose group of the present invention, middle dosage group and QIZHI JIANGTANG JIAONANG group blood glucose and matched group compare, and still there were significant differences (P＜0.01), illustrate that the present invention has certain protection effect and hypoglycemic activity to alloxan moulding mice sugar disease.
3, islets of langerhans A, B cell pathology check result
Matched group, the islets of langerhans decreased number, form is irregular, a small amount of non-viable non-apoptotic cell fragment in the still visible islets of langerhans in the HE sheet, lymphocyte and histiocytic infiltrate, the A cytosis, the B cell reduces, and the B cell reduces in most of islets of langerhans.
Low dose group of the present invention, most of specimen islets of langerhans number is normal substantially, and (3 example) A cell and B cell proportion are normal in the part islets of langerhans, and other sections belong to moderate lesion, and the B cell has minimizing slightly, the A cytosis.
The dosage group belongs to the minor injury among the present invention, occurs the B cell in the part islets of langerhans and reduces the A cytosis.
High dose group of the present invention belongs to the minor injury, compares with positive controls, and lesion and damage obviously alleviates.
QIZHI JIANGTANG JIAONANG: 1 example is slight pathological changes, and all the other are the moderate pathological changes.
Above result shows that the present invention and QIZHI JIANGTANG JIAONANG group have certain protective role to alloxan moulding mouse islets B cell, and can reduce injury of pancreas degree and mouse death rate, and wherein medication group low dosage is the most obvious.
Three, the present invention is to the influence of lipid peroxide, monoamine oxidase, MAO, superoxide dismutase
Lipid peroxide (LPO) is in the chemical compound or the free unsaturated fatty acid peroxide that generated by the free radical effect, and maturity-onset diabetes is because dysbolismus (sugar and fat matter) causes the lipid peroxide increase, and can promote the development of diabetic complications.Therefore, measuring lipid peroxidation confrontation observation curative effect of the present invention is an important index.
Monoamine oxidase, MAO (Monoamine oxdase MAO) is that a class extensively is present in the monoamine oxidase, MAO class in the organism different tissues, and it has the oxidation deoxidation of the dissimilar monoamines of catalysis.The activity that someone finds monoamine oxidase, MAO in human brain blood and the platelet (WAO, EC, 1,4,1,3) with advancing age and showed increased, particularly since the seventies, substantial connection has been arranged owing to find the activity of aging course and some Senile disease and monoamine oxidase-B (MAO-B).People seek to use suitable oxidase inhibitor (MAOI) always and selectively suppress MAO-B to reach the purpose for the treatment of some Senile disease.
Superoxide dismutase (SOD) is to have one of main enzyme of eliminating the free radical effect, thereby reduces the level of lipid peroxide.Under the situation of some pathology, for example: radiation damage and aging can cause increasing of lipid peroxide when free radical increases.Therefore measure the metabolism situation that LPO in serum or the tissue and SOD can understand free radical.The blood vessel injury complication of diabetics is relevant with LPO and SOD, and the purpose of this experiment promptly is to understand the present invention's effect in this regard.
Materials and methods:
Use the LCR mouse inbred lines, body weight 25-30g, normal control group and positive controls (QIZHI JIANGTANG JIAONANG) are established in the pairing grouping, and the present invention establishes high, medium and low three dosage groups.High dose (adult consumption 50 times), middle dosage (adult's consumption 37.5 times), low dosage (adult's consumption 25 times).Route of administration is irritated stomach, once a day, and sacrificed by exsanguination after two weeks of administration.Gather respectively that blood, brain, liver are MAO-B, LPO, SOD measures.
One, the mensuration of LPO, Hepar Mus or brain are made 10% tissue homogenate, get 0.1ml → add 8.1%SDS 0.2ml; PH3.5,20%HACbufferl? 1.5ml; 0.8%TBA 1.5ml; 1 hour → water of the back 95 ℃ of water-baths of distilled water 0.5ml → stirring cooled off → adds n-butyl alcohol pyridine (15: 1) 3ml vibration back centrifugal (3000 rev/mins) 15 minutes → measure optical density value (O, D) under the 532nm wavelength towards test tube
Two, the mensuration of MAO-B, get → organize the cold PH7.4 of giving of 10 times of volumes that weighs → add, 0.2MP.B buffer grind to form homogenate → 1000g4 ℃ centrifugal 10 minutes → remove precipitation to get 4 ℃ of following 17000g of supernatant centrifugal 30 minutes → get precipitation to suspend with the 0.3m10.2MP.B buffer and promptly get thick enzyme, add the 0.3ml80mM benzylamine in having the test tube of ground lid: 2.4ml0.2MPH7.4P.B → 37 ℃ water-bath vibration 3 hours → add the 60%PCAO0.3ml cessation reaction added 3 minutes → 3000 rev/mins of cyclohexane extraction 3ml vibrations centrifugal 10 minutes → get supernatant in 242nm wavelength photometry density (O.D value) up and down again.
Three, the mensuration of SOD:
Refer to that with reference to the auspicious trace in Dinke the blood superoxide dismutase rapid assay methods carries out.
Result and discussion:
The present invention is to the influence of mouse brain LPO
Group (n=8) X ± SD (0.D value) L P
Normal control 0.55 ± 0.046
Positive control (QIZHI JIANGTANG JIAONANG) 0.220 ± 0.064 11.84＜0.01
Low dose group 0.424 ± 0.034 6.23＜0.01
Middle dosage group 0.315 ± 0.073 7.70＜0.01
High dose group 0.250 ± 0.061 11.10＜0.01
The present invention is to the influence of Mouse Liver LPB
Group (n=8) X ± SD (0.D value) L P
Normal control 0.54 ± 0.035
Positive control (QIZHI JIANGTANG JIAONANG) 0.370 ± 0.064 6.59＜0.01
Low dose group 0.460 ± 0.083 2.51＜0.05
Middle dosage group 0.430 ± 0.075 3.76＜0.01
High dose group 0.355 ± 0.085 5.69＜0.01
The present invention is to the influence of mouse brain MAOB
Group (n=8) X ± SD (0.D value) L P
Normal control 0.466 ± 0.07
Positive control (QIZHI JIANGTANG JIAONANG) 0.213 ± 0.078 6.28＜0.01
Low dose group 0.332 ± 0.023 4.38＜0.01
Middle dosage group 0.315 ± 0.046 4.42＜0.01
High dose group 0.217 ± 0.055 7.23＜0.01
The present invention is to the influence of SOD in Mice
Group (n=8) X ± SD (0.D value) L P
Normal control 0.147 ± 0.013
Positive control (QIZHI JIANGTANG JIAONANG) 0.171 ± 0.011 3.99＜0.01
Low dose group 0.165 ± 0.014 2.66＜0.05
Middle dosage group 0.169 ± 0.010 3.79＜0.01
High dose group 0.170 ± 0.015 3.28＜0.01
Above-mentioned experimental result shows, the present invention has the effect of obvious reduction mouse brain, liver lipid peroxide, for the development that stops diabetic complications provides experimental basis, simultaneously the mouse brain monoamine oxidase-B there is tangible reduction effect, played the effect of oxidase inhibitor, superoxide dismutase has been had tangible potentiation.These effects of the present invention may provide experimental basis for clinical prevention diabetes and complication thereof.
Four, the present invention is to the influence of bone marrow cells in mice Insulin receptor INSR and adrenal cortical hormone receptor
Materials and methods
Animal
(1) gets pure lines I CL mice, 6 monthly ages, body weight (18 ± 2) g, male 10, divide equally 4 groups, I organizes negative contrast, and the II group is known drug positive control (QIZHI JIANGTANG JIAONANG), III group is low dose group of the present invention (adult's consumption 12.5 times), and the IV group is high dose group of the present invention (adult's consumption 25 times).
(2) get pure lines I CL mice, 18 monthly ages, body weight (35 ± 2) g, male 40, divide equally 4 groups, every group of medicining condition be with (1), more than each group be and measure Insulin receptor INSR and use.
(3) the Weistar rat is 20, and is male, and body weight (200 ± 10) g divides equally 4 groups, and each organizes the administration situation with (1), is used to measure adrenal cortical hormone receptor.
1, reagent:
(1) the single iodine atom of I-14-A-insulin is provided by Sichuan Huaxi Medical Univ Isotope Lab.Activity 223 μ ci/mg, the non-marked insulin is the Sigma product.
(2) 6,7-[A] the Atdosterone activity is that 72ci/nnik (New England Nucleor Comp) non-marked Atdosterone (Slgma CO) produces.
3, assay method:
(1) Insulin receptor INSR is measured: get bone marrow cells in mice and make cell suspension according to a conventional method and make into 5-7 * 10
7The total liquid measure of/ml reaction tube is 1ml, and wherein the I insulin is 0.1ml, and the CPM value is amount adjustment on demand, and add 1000 times non-marked thing, hatched 2 hours 0 ℃ of ice-water bath stopped reaction for 22 ℃, put 49 fiber type filter membrane sucking filtration, clean rearmounted Fj2003 type immunity enumerator, measure CPM value and calculating.
CV 2.44 also draws Scatchard Plot, and to get 7 points between [I] insulin 20nM to 20nM, the result has obvious saturated phenomenon, and Scatchard Plot result of calculation sees Table 1.Measure respectively 6 monthly ages and 18 the monthly age mice medullary cell Insulin receptor INSR number (getting one point method 10nM) the results are shown in Table II.
(2) adrenal cortical hormone receptor is measured: behind the rat anesthesia with cold buffer liquid A (20nM Na-Phosphate PH7.41mM disodium EDTA 2mM 2-hydroxyethymercaptane and 10% giycerol (Wt/Vol) heart perfusion, dehematize.Operation in 0 ℃ of cold house, get brain, weigh, add the 2ml buffer A, use glass homogenizer homogenate, centrifugal 85000 * 9 * 45min gets supernatant homogenate and measures protein content with the Lowryis method, and Dexamethasone gets 100 times of non-marked thing competitions of 6 points (0.5nM-10nM), with the hydroxyapatite is separating medium, centrifugal 10000 * g 10min gets supernatant and adds mensuration CPM in the scintillation solution, and makes Schathard Plot, the results are shown in Table III.
Result and discussion:
1, the present invention can make 18 monthly ages bone marrow cells in mice Insulin receptor INSR numbers rise, but 6 monthly ages were not had effect, and aged Mus Insulin receptor INSR number has with the gesture that increases age and descend, so the present invention plays restitution.
2, the present invention can disturb adrenal cortical hormone receptor, makes it adhesion and descends, and Bamx reduces, and this also is favourable for preventing and treating diabetes.
Above result shows that old Mus that the present invention is used for Insulin receptor INSR hypofunction state can make it receptor binding capacity and raise, and therefore, the present invention can be used as insulin receptor regulator and takes an entrance examination in clinical.
Table II the present invention is to the effect of bone marrow cells in mice insulin not of the same age
(one point method 10nM) P divides into groups to be counted out
Negative control 693 ± 392
Positive control 839 ± 198＞0.05
6 monthly ages
The present invention's (low) 910 ± 466＞0.05
The present invention's (height) 899 ± 446＞0.05
Negative control 383 ± 230
Positive control 1027 ± 441＜0.01
18 monthly ages
The present invention's (low) 1130 ± 430＜0.01
The present invention's (height) 1240 ± 340＜0.01
Table III the present invention is to the effect of adrenal gland's sebum hormone receptor
r kd(m) Bmax fmol mg prot
Group
I II I II I II
1.1×
Negative control 0.89 0.95 12 * 10 289 110
10
Positive control 0.87 0.85 1.1 3.1 130 102
The present invention's (low) 0.88 0.87 1.0 2.9 120 100
The present invention's (height) 0.87 0.86 0.9 2.1 110 96
I glucocorticoid receptor (GR) II mineralcorticoid receptor
Five, the present invention is to the influence of human body embryo lung diploid fibroblastic growth and PAS reaction
Materials and methods:
One, cell culture human embryonic lung fibroblast (2BS cell) is provided by Beijing Biological Product Inst., and the life-span of going down to posterity was 55 ± 10 generations.40 ages in generation of recovery human embryonic lung fibroblast from liquid nitrogen are with the EagleMEM culture fluid that contains 10% calf serum, 10%NaHCO
3, accent PH is 7.2-7.4, adds the 2ml-glutamine, 37 ℃ of incubations, treat that cell growth merges into monolayer after, with 0.25% trypsinization 2 minutes, abandon pancreatin and add culture fluid piping and druming and make cell suspension, going down to posterity to cultivate with 1: 2 ratio is equipped with experiment usefulness.
Two, the preparation of medicine: the present invention gets 10g and is dissolved in the 200ml redistilled water, through magnetic agitation 24 hours, filtering is dissolved particles not, filtered solution adds the MEM culture medium dry powder, the G6 filter sucking filtration sterilization of dissolving back, furnishing contains 10% calf serum, PH=7.2, filtered solution is a water-soluble portion of the present invention, and original liquid concentration is 50mg/ml.
Three, method and grouping
(1) acute toxicity testing get growth conditions good 40 age in generation 2BS cell number bottle, through 0.25% trypsinization, abandon pancreatin and add 10ml culture fluid piping and druming, be prepared into cell suspension, through cell counting, being diluted to cell concentration is 200,000/ml suspension, and getting 30 bottles of floor spaces is 13.8cm
2Inoculation bottle, every bottle graft kind 1ml cell suspension, 3ml contains the medicine culture medium, 3 bottles of matched groups, 3 bottles totally 33 bottles of each concentration of dosing group, the dosing group is initial concentration with 37.5mg/ml, below each concentration all by the dilution of 1: 0: 7 ratio, through 37 ℃ of incubations 4 hours, cell counting, gained result machine as calculated handles, and obtains the median effective dose (EDx) of medicine pair cell card wall rate influence.
(2) the present invention is to all same acute toxicity testing of the preparation of 2BS cell growth inhibition test, cell culture, cell suspension, this experiment is the present invention's first dosage to the 2BS cell growth inhibition test with the concentration that does not influence cell card wall rate in the toxicity test, concentration is 1050 μ g/ml, following concentration was by ratio dilution in 1: 0.5, and getting 24 bottles of floor spaces is 13.8cm
2Little square vase, 3 bottles is one group, one group of contrast, each group of experiment all adds the 1ml cell, and PAS reacts and descends in the cell, has illustrated that synthetic polysaccharide ability reduces or the catabolism enhancing in the cell, from this result of experiment, the present invention can significantly increase late content for polysaccharide in the cell, and this is that this useful experiment also shows regulating aspect the carbohydrate metabolism body, and the present invention can directly make a difference to carbohydrate metabolism at cellular level.This can confirm the effect of Insulin receptor INSR in integral level mutually in conjunction with preamble the present invention.
Following embodiment is further openly the present invention, need to prove that these embodiment only for optimal way of the present invention, do not limit the scope of protection of present invention.
Embodiment 1
Ginsenoside 6g Radix Astragali 124g Radix Rehmanniae 186g
Radix Ophiopogonis 62g Radix Trichosanthis 62g Fructus Lycii 124g
Fructus Schisandrae Chinensis 62g Rhizoma Dioscoreae 62g Fructus Rubi 31g
Poria 62g Rhizoma Alismatis 62g.
Method for making: a Radix Ophiopogonis is with warm water lixiviate 2 times, and each 2 hours, merge lixiviating solution, filter, it is 1.20 thick paste that filtrate is concentrated into relative density;
B Fructus Schisandrae Chinensis 50% ethanol, percolation, percolate reclaim ethanol, are concentrated into thick paste;
C Fructus Lycii, the Radix Astragali, Radix Rehmanniae, Rhizoma Alismatis, Rhizoma Dioscoreae, Radix Trichosanthis, Fructus Rubi, Poria water decoct 2 times, each 1.5 hours, merging decoction liquor, filtration, filtrate, to be concentrated into relative density be 1.20 thick paste, add ethanol and make that to contain the alcohol amount be 60%, leave standstill, the leaching supernatant, reclaim ethanol, concentrate
D merges dry, pulverizing with above-mentioned extractum and merges with the ginsenoside, and mixing gets extract powder 100g, adds the extract powder mixing after getting adjuvant Polyethylene Glycol PEG4000 70g heating and melting, in the immigration encapsulating machine storage tank, is pressed into ball, selects ball, promptly.Assay method is:
(1) ginsenoside Re's assay method is:
The preparation of reference substance solution: it is an amount of that precision takes by weighing ginsenoside Re's reference substance, adds methanol and make the solution that contains the about 0.2mg of ginsenoside Re among every 1ml, in contrast product solution;
The preparation of need testing solution: get 2 of this product, cut off, scrape and get content, mixing, rubber absolute ethanol washing, content add water 10ml, filter, the water saturated n-butyl alcohol 10ml of accurate adding extracts 10min, and precision is measured supernatant 10ml, adds the ammonia solution triplication, shake up, place, divide and get upper solution, evaporate to dryness, residue add methanol makes dissolving, quantitatively moves in the 2ml measuring bottle, add methanol and be diluted to scale, shake up, promptly;
Chromatographic condition: with octadecylsilane chemically bonded silica is filler; Volume ratio is that 99: 400 acetonitrile-0.05% phosphoric acid solution is a mobile phase; Detect wavelength 203nm;
Algoscopy: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject hplc determination;
(2) assay method of astragaloside is:
Chromatographic condition: octadecylsilane chemically bonded silica is a filler; Evaporative light scattering detector;
Mobile phase: ratio is acetonitrile-water-oxolane of 33: 67: 4;
The preparation of reference substance solution: get the astragaloside reference substance and add methanol and make the solution that every 1ml contains about 0.1mg, shake up;
The preparation of need testing solution: get 2 of this product, cut off, scrape and get content, mixing, rubber absolute ethanol washing, content add water 10ml, filter, filtrate is used defat with petroleum ether 1 time, and (each 10ml) extracted in water saturated n-butyl alcohol jolting 2 times, merges n-butanol extracting liquid, extract 1 time with ammonia solution, each 10ml discards ammonia solution, the water 10ml saturated with n-butyl alcohol washes, and discards water liquid, n-butyl alcohol liquid evaporate to dryness, residue adds water 5mL makes dissolving, puts coldly, passes through D
101Type macroporous adsorptive resins (internal diameter 1.5cm, long 18cm) is with water elution, discard water liquid, reuse 10% ethanol 30ml eluting discards ethanol elution, continue with 50% ethanol 50ml eluting, collect eluent, evaporate to dryness is with dissolve with methanol and be transferred in the 2ml measuring bottle, add methanol to scale, shake up, filter, promptly.
The result: every contains ginsenoside Re 0.2mg, contains astragaloside 0.06mg.
Embodiment 2
Ginsenoside 4g Radix Astragali 150g Radix Rehmanniae 250g
Radix Ophiopogonis 80g Radix Trichosanthis 80g Fructus Lycii 160g
Fructus Schisandrae Chinensis 80g Rhizoma Dioscoreae 80g Fructus Rubi 50g
Poria 80g Rhizoma Alismatis 80g
Method for making: a Radix Ophiopogonis is with warm water lixiviate 3 times, and each 3 hours, merge lixiviating solution, filter, it is 1.40 thick paste that filtrate is concentrated into relative density;
B Fructus Schisandrae Chinensis 70% ethanol, percolation, percolate reclaim ethanol, are concentrated into thick paste;
C Fructus Lycii, the Radix Astragali, Radix Rehmanniae, Rhizoma Alismatis, Rhizoma Dioscoreae, Radix Trichosanthis, Fructus Rubi, Poria water decoct 4 times, and each 3 hours, merging decoction liquor, filtration, filtrate, to be concentrated into relative density be 1.20 thick paste, add ethanol and make that to contain the alcohol amount be 80%, leave standstill, the leaching supernatant, reclaim ethanol, concentrate
D merges drying, pulverizing and ginsenoside with above-mentioned extractum and merges, mixing gets extract powder 175g, get adjuvant Macrogol 4000 175g, add the extract powder mixing behind the heating and melting, move in the encapsulating machine medicinal liquid storage tank, put gelatin solution in the gelatin storage tank and be incubated 60 ℃, make ball 50 ℃ following of water dropper temperature, select ball, drying, packing, promptly.
Assay method is:
(1) ginsenoside Re's assay method is:
The preparation of reference substance solution: it is an amount of that precision takes by weighing ginsenoside Re's reference substance, adds methanol and make the solution that contains ginsenoside Re 0.8mg among every 1ml, in contrast product solution;
The preparation of need testing solution: get 20 of this product, cut off, scrape and get content, mixing, rubber absolute ethanol washing, content add water 50ml, filter, the water saturated n-butyl alcohol 50ml of accurate adding extracts 40min, and precision is measured supernatant 50ml, adds the ammonia solution triplication, shake up, place, divide and get upper solution, evaporate to dryness, residue add methanol makes dissolving, quantitatively moves in the 10ml measuring bottle, add methanol and be diluted to scale, shake up, promptly;
Chromatographic condition: with octadecylsilane chemically bonded silica is filler; Volume ratio is that 99: 400 acetonitrile-0.05% phosphoric acid solution is a mobile phase; Detect wavelength 203nm;
Algoscopy: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject hplc determination;
(2) assay method of astragaloside is:
Chromatographic condition: octadecylsilane chemically bonded silica is a filler; Evaporative light scattering detector;
Mobile phase: ratio is acetonitrile-water-oxolane of 33: 67: 4;
The preparation of reference substance solution: get the astragaloside reference substance and add methanol and make the solution that every 1ml contains 2mg, shake up;
The preparation of need testing solution: get 20 of this product, cut off, scrape and get content, mixing, rubber absolute ethanol washing, content add water 50ml, filter, filtrate is used defat with petroleum ether 3 times, and (each 50ml) extracted in water saturated n-butyl alcohol jolting 6 times, merges n-butanol extracting liquid, extract 2 times with ammonia solution, each 40ml discards ammonia solution, the water 40ml saturated with n-butyl alcohol washes, and discards water liquid, n-butyl alcohol liquid evaporate to dryness, residue adds water 25mL makes dissolving, puts coldly, passes through D
101Type macroporous adsorptive resins (internal diameter 1.5cm, long 18cm) is with water elution, discard water liquid, reuse 40% ethanol 100ml eluting discards ethanol elution, continue with 90% ethanol 150ml eluting, collect eluent, evaporate to dryness is with dissolve with methanol and be transferred in the 10ml measuring bottle, add methanol to scale, shake up, filter, promptly.
The result: every contains ginsenoside Re 2mg, contains astragaloside 1mg.
Embodiment 3
Ginsenoside 8g Radix Astragali 80g Radix Rehmanniae 250g
Radix Ophiopogonis 80g Radix Trichosanthis 80g Fructus Lycii 160g
Fructus Schisandrae Chinensis 80g Rhizoma Dioscoreae 80g Fructus Rubi 50g
Poria 80g Rhizoma Alismatis 80g
Method for making: a Radix Ophiopogonis is with warm water lixiviate 1 time, and each 3 hours, merge lixiviating solution, filter, it is 1.30 thick paste that filtrate is concentrated into relative density;
B Fructus Schisandrae Chinensis 50% ethanol, percolation, percolate reclaim ethanol, are concentrated into thick paste;
C Fructus Lycii, the Radix Astragali, Radix Rehmanniae, Rhizoma Alismatis, Rhizoma Dioscoreae, Radix Trichosanthis, Fructus Rubi, Poria water decoct 3 times, each 1.5 hours, merging decoction liquor, filtration, filtrate, to be concentrated into relative density be 1.20 thick paste, add ethanol and make that to contain the alcohol amount be 50%, leave standstill, the leaching supernatant, reclaim ethanol, concentrate
D merges drying, pulverizing and ginsenoside with above-mentioned extractum and merges, and mixing gets extract powder 210g, gets adjuvant Macrogol 4000 105g and adds the extract powder mixing, moves in the encapsulating machine storage tank, is pressed into ball, selects ball, promptly.
Assay:
(1) ginsenoside Re's assay method is:
The system of reference substance solution each: it is an amount of that precision takes by weighing ginsenoside Re's reference substance, adds methanol and make the solution that contains the about 0.5mg of ginsenoside Re among every 1ml, in contrast product solution;
The preparation of need testing solution: get 10 of this product, cut off, scrape and get content, mixing, rubber absolute ethanol washing, content add water 30ml, filter, the water saturated n-butyl alcohol 30ml of accurate adding extracts 30min, and precision is measured supernatant 30ml, adds the ammonia solution triplication, shake up, place, divide and get upper solution, evaporate to dryness, residue add methanol makes dissolving, quantitatively moves in the 5ml measuring bottle, add methanol and be diluted to scale, shake up, promptly;
Chromatographic condition: with octadecylsilane chemically bonded silica is filler; Volume ratio is that 99: 400 acetonitrile-0.05% phosphoric acid solution is a mobile phase; Detect wavelength 203nm;
Algoscopy: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject hplc determination;
(3) assay method of astragaloside is:
Chromatographic condition: octadecylsilane chemically bonded silica is a filler; Evaporative light scattering detector;
Mobile phase: ratio is acetonitrile-water-oxolane of 33: 67: 4;
The preparation of reference substance solution: get the astragaloside reference substance and add methanol and make the solution that every 1ml contains about 1mg, shake up;
The preparation of need testing solution: get 10 of this product, cut off, scrape and get content, mixing, rubber absolute ethanol washing, content add water 30ml, filter, filtrate is used defat with petroleum ether 2 times, and (each 30ml) extracted in water saturated n-butyl alcohol jolting 4 times, merges n-butanol extracting liquid, extract 2 times with ammonia solution, each 25ml discards ammonia solution, the water 25ml saturated with n-butyl alcohol washes, and discards water liquid, n-butyl alcohol liquid evaporate to dryness, residue adds water 15mL makes dissolving, puts coldly, passes through D
101Type macroporous adsorptive resins (internal diameter 1.5cm, long 18cm) is with water elution, discard water liquid, reuse 30% ethanol 50ml eluting discards ethanol elution, continue with 70% ethanol 100ml eluting, collect eluent, evaporate to dryness is with dissolve with methanol and be transferred in the 5ml measuring bottle, add methanol to scale, shake up, filter, promptly.
The result: every contains ginsenoside Re 20mg, contains astragaloside 1mg.
Embodiment 4
Ginsenoside 8g Radix Astragali 150g Radix Rehmanniae 150g
Radix Ophiopogonis 80g Radix Trichosanthis 80g Fructus Lycii 160g
Fructus Schisandrae Chinensis 80g Rhizoma Dioscoreae 80g Fructus Rubi 50g
Poria 80g Rhizoma Alismatis 80g
Method for making: a Radix Ophiopogonis is with warm water lixiviate 3 times, and each 2.5 hours, merge lixiviating solution, filter, it is 1.20 thick paste that filtrate is concentrated into relative density;
B Fructus Schisandrae Chinensis 55% ethanol, percolation, percolate reclaim ethanol, are concentrated into thick paste;
C Fructus Lycii, the Radix Astragali, Radix Rehmanniae, Rhizoma Alismatis, Rhizoma Dioscoreae, Radix Trichosanthis, Fructus Rubi, Poria water decoct 2 times, and each 1 hour, merging decoction liquor, filtration, filtrate, to be concentrated into relative density be 1.20 thick paste, add ethanol and make that to contain the alcohol amount be 70%, leave standstill, the leaching supernatant, reclaim ethanol, concentrate
D merges drying, pulverizing and ginsenoside with above-mentioned extractum and merges, mixing gets extract powder 160g, get adjuvant Macrogol 4000 800g and add the extract powder mixing, move in the encapsulating machine medicinal liquid storage tank, put gelatin solution in the gelatin storage tank and be incubated 60 ℃, make ball 45 ℃ following of water dropper temperature, select ball, drying, packing, promptly.
Assay method is:
(1) ginsenoside Re's assay method is:
The preparation of reference substance solution: it is an amount of that precision takes by weighing ginsenoside Re's reference substance, adds methanol and make the solution that contains the about 0.6mg of ginsenoside Re among every 1ml, in contrast product solution;
The preparation of need testing solution: get 8 of this product, cut off, scrape and get content, mixing, rubber absolute ethanol washing, content add water 25ml, filter, the water saturated n-butyl alcohol 25ml of accurate adding extracts 20min, and precision is measured supernatant 25ml, adds the ammonia solution triplication, shake up, place, divide and get upper solution, evaporate to dryness, residue add methanol makes dissolving, quantitatively moves in the 5ml measuring bottle, add methanol and be diluted to scale, shake up, promptly;
Chromatographic condition: with octadecylsilane chemically bonded silica is filler; Volume ratio is that 99: 400 acetonitrile-0.05% phosphoric acid solution is a mobile phase; Detect wavelength 203nm;
Algoscopy: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject hplc determination;
(2) assay method of astragaloside is:
Chromatographic condition: octadecylsilane chemically bonded silica is a filler; Evaporative light scattering detector;
Mobile phase: ratio is acetonitrile-water-oxolane of 33: 67: 4;
The preparation of reference substance solution: get the astragaloside reference substance and add methanol and make the solution that every 1ml contains about 0.6mg, shake up;
The preparation of need testing solution: get 8 of this product, cut off, scrape and get content, mixing, rubber absolute ethanol washing, content add water 25ml, filter, filtrate is used defat with petroleum ether 2 times, and (each 25ml) extracted in water saturated n-butyl alcohol jolting 3 times, merges n-butanol extracting liquid, extract 2 times with ammonia solution, each 25ml discards ammonia solution, the water 40ml saturated with n-butyl alcohol washes, and discards water liquid, n-butyl alcohol liquid evaporate to dryness, residue adds water 5mL makes dissolving, puts coldly, passes through D
101Type macroporous adsorptive resins (internal diameter 1.5cm, long 18cm) is with water elution, discard water liquid, reuse 40% ethanol 100ml eluting discards ethanol elution, continue with 70% ethanol 100ml eluting, collect eluent, evaporate to dryness is with dissolve with methanol and be transferred in the 5ml measuring bottle, add methanol to scale, shake up, filter, promptly.
The result: every contains ginsenoside Re 20mg, contains astragaloside 0.02mg.
Embodiment 5
Ginsenoside 8g Radix Astragali 150g Radix Rehmanniae 250g
Radix Ophiopogonis 45g Radix Trichosanthis 80g Fructus Lycii 160g
Fructus Schisandrae Chinensis 80g Rhizoma Dioscoreae 80g Fructus Rubi 50g
Poria 80g Rhizoma Alismatis 80g
Method for making: a Radix Ophiopogonis is with warm water lixiviate 2 times, and each 2 hours, merge lixiviating solution, filter, it is 1.40 thick paste that filtrate is concentrated into relative density;
B Fructus Schisandrae Chinensis 50% ethanol, percolation, percolate reclaim ethanol, are concentrated into thick paste;
C Fructus Lycii, the Radix Astragali, Radix Rehmanniae, Rhizoma Alismatis, Rhizoma Dioscoreae, Radix Trichosanthis, Fructus Rubi, Poria water decoct 3 times, and each 2 hours, merging decoction liquor, filtration, filtrate, to be concentrated into relative density be 1.20 thick paste, add ethanol and make that to contain the alcohol amount be 70%, leave standstill, the leaching supernatant, reclaim ethanol, concentrate
D merges drying, pulverizing and ginsenoside with above-mentioned extractum and merges, and mixing gets extract powder 100g, gets adjuvant Macrogol 4000 40g and adds the extract powder mixing, moves in the encapsulating machine storage tank, is pressed into ball, selects ball, promptly.
Content assaying method:
(1) ginsenoside Re's assay method is:
The preparation of reference substance solution: it is an amount of that precision takes by weighing ginsenoside Re's reference substance, adds methanol and make the solution that contains the about 0.5mg of ginsenoside Re among every 1ml, in contrast product solution;
The preparation of need testing solution: get 10 of this product, cut off, scrape and get content, mixing, rubber absolute ethanol washing, content add water 25ml, filter, the water saturated n-butyl alcohol 25ml of accurate adding extracts 30min, and precision is measured supernatant 25ml, adds the ammonia solution triplication, shake up, place, divide and get upper solution, evaporate to dryness, residue add methanol makes dissolving, quantitatively moves in the 5ml measuring bottle, add methanol and be diluted to scale, shake up, promptly;
Chromatographic condition: with octadecylsilane chemically bonded silica is filler; Volume ratio is that 99: 400 acetonitrile-0.05% phosphoric acid solution is a mobile phase; Detect wavelength 203nm;
Algoscopy: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject hplc determination;
(2) assay method of astragaloside is:
Chromatographic condition: octadecylsilane chemically bonded silica is a filler; Evaporative light scattering detector;
Mobile phase: ratio is acetonitrile-water-oxolane of 33: 67: 4;
The preparation of reference substance solution: get the astragaloside reference substance and add methanol and make the solution that every 1ml contains about 0.5mg, shake up;
The preparation of need testing solution: get 10 of this product, cut off, scrape and get content, mixing, rubber absolute ethanol washing, content add water 25ml, filter, filtrate is used defat with petroleum ether 3 times, and (each 25ml) extracted in water saturated n-butyl alcohol jolting 3 times, merges n-butanol extracting liquid, extract 1 time with ammonia solution, each 20ml discards ammonia solution, the water 10ml saturated with n-butyl alcohol washes, and discards water liquid, n-butyl alcohol liquid evaporate to dryness, residue adds water 2mL makes dissolving, puts coldly, passes through D
101Type macroporous adsorptive resins (internal diameter 1.5cm, long 18cm) is with water elution, discard water liquid, reuse 30% ethanol 40ml eluting discards ethanol elution, continue with 80% ethanol 100ml eluting, collect eluent, evaporate to dryness is with dissolve with methanol and be transferred in the 5ml measuring bottle, add methanol to scale, shake up, filter, promptly.
The result: every contains ginsenoside Re 0.05mg, contains astragaloside 1mg.
Embodiment 6
Ginsenoside 8g Radix Astragali 150g Radix Rehmanniae 250g
Radix Ophiopogonis 80g Radix Trichosanthis 40g Fructus Lycii 160g
Fructus Schisandrae Chinensis 80g Rhizoma Dioscoreae 80g Fructus Rubi 50g
Poria 80g Rhizoma Alismatis 80g
Method for making: a Radix Ophiopogonis is with warm water lixiviate 2 times, and each 2 hours, merge lixiviating solution, filter, it is 1.40 thick paste that filtrate is concentrated into relative density;
B Fructus Schisandrae Chinensis 50% ethanol, percolation, percolate reclaim ethanol, are concentrated into thick paste;
C Fructus Lycii, the Radix Astragali, Radix Rehmanniae, Rhizoma Alismatis, Rhizoma Dioscoreae, Radix Trichosanthis, Fructus Rubi, Poria water decoct 3 times, and each 2 hours, merging decoction liquor, filtration, filtrate, to be concentrated into relative density be 1.20 thick paste, add ethanol and make that to contain the alcohol amount be 70%, leave standstill, the leaching supernatant, reclaim ethanol, concentrate
D merges drying, pulverizing and ginsenoside with above-mentioned extractum and merges, mixing gets extract powder 100g, get refined plant oil 70g and add the extract powder mixing, move in the encapsulating machine medicinal liquid storage tank, put gelatin solution in the gelatin storage tank and be incubated 60 ℃, make ball 50 ℃ following of water dropper temperature, select ball, drying, packing, promptly.
Content assaying method:
(1) ginsenoside Re's assay method is:
The preparation of reference substance solution: it is an amount of that precision takes by weighing ginsenoside Re's reference substance, adds methanol and make the solution that contains ginsenoside Re 0.4mg among every 1ml, in contrast product solution;
The preparation of need testing solution: get 15 of this product, cut off, scrape and get content, mixing, rubber absolute ethanol washing, content add water 50ml, filter, the water saturated n-butyl alcohol 50ml of accurate adding extracts 40min, and precision is measured supernatant 50ml, adds the ammonia solution triplication, shake up, place, divide and get upper solution, evaporate to dryness, residue add methanol makes dissolving, quantitatively moves in the 10ml measuring bottle, add methanol and be diluted to scale, shake up, promptly;
Chromatographic condition: with octadecylsilane chemically bonded silica is filler; Volume ratio is that 99: 400 acetonitrile-0.05% phosphoric acid solution is a mobile phase; Detect wavelength 203nm;
Algoscopy: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject hplc determination;
(2) assay method of astragaloside is:
Chromatographic condition: octadecylsilane chemically bonded silica is a filler; Evaporative light scattering detector;
Mobile phase: ratio is acetonitrile-water-oxolane of 33: 67: 4;
The preparation of reference substance solution: get the astragaloside reference substance and add methanol and make the solution that every 1ml contains 0.5mg, shake up;
The preparation of need testing solution: get 15 of this product, cut off, scrape and get content, mixing, rubber absolute ethanol washing, content add water 50ml, filter, filtrate is used defat with petroleum ether 2 times, and (each 50ml) extracted in water saturated n-butyl alcohol jolting 3 times, merges n-butanol extracting liquid, extract 1 time with ammonia solution, each 40ml discards ammonia solution, the water 40ml saturated with n-butyl alcohol washes, and discards water liquid, n-butyl alcohol liquid evaporate to dryness, residue adds water 10mL makes dissolving, puts coldly, passes through D
101Type macroporous adsorptive resins (internal diameter 1.5cm, long 18cm) is with water elution, discard water liquid, reuse 40% ethanol 70ml eluting discards ethanol elution, continue with 70% ethanol 100ml eluting, collect eluent, evaporate to dryness is with dissolve with methanol and be transferred in the 5ml measuring bottle, add methanol to scale, shake up, filter, promptly.
The result: every contains ginsenoside Re 1mg, contains astragaloside 1mg.
Embodiment 7
Ginsenoside 8g Radix Astragali 150g Radix Rehmanniae 250g
Radix Ophiopogonis 80g Radix Trichosanthis 80g Fructus Lycii 80g
Fructus Schisandrae Chinensis 80g Rhizoma Dioscoreae 80g Fructus Rubi 50g
Poria 80g Rhizoma Alismatis 80g
Method for making: a Radix Ophiopogonis is with warm water lixiviate 2 times, and each 2 hours, merge lixiviating solution, filter, it is 1.40 thick paste that filtrate is concentrated into relative density;
B Fructus Schisandrae Chinensis 50% ethanol, percolation, percolate reclaim ethanol, are concentrated into thick paste;
C Fructus Lycii, the Radix Astragali, Radix Rehmanniae, Rhizoma Alismatis, Rhizoma Dioscoreae, Radix Trichosanthis, Fructus Rubi, Poria water decoct 3 times, and each 2 hours, merging decoction liquor, filtration, filtrate, to be concentrated into relative density be 1.20 thick paste, add ethanol and make that to contain the alcohol amount be 70%, leave standstill, the leaching supernatant, reclaim ethanol, concentrate;
D merges drying, pulverizing and ginsenoside with above-mentioned extractum and merges, and mixing gets extract powder 100g, gets refined plant oil 100g and adds the extract powder mixing, moves in the encapsulating machine storage tank, is pressed into ball, selects ball, promptly.
Content assaying method: with embodiment 4
The result: every contains ginsenoside Re 1mg, contains astragaloside 0.05mg.
Embodiment 8
Ginsenoside 8g Radix Astragali 150g Radix Rehmanniae 250g
Radix Ophiopogonis 80g Radix Trichosanthis 80g Fructus Lycii 160g
Fructus Schisandrae Chinensis 40g Rhizoma Dioscoreae 80g Fructus Rubi 50g
Poria 80g Rhizoma Alismatis 80g
Method for making: a Radix Ophiopogonis is with warm water lixiviate 2 times, and each 2 hours, merge lixiviating solution, filter, it is 1.40 thick paste that filtrate is concentrated into relative density;
B Fructus Schisandrae Chinensis 50% ethanol, percolation, percolate reclaim ethanol, are concentrated into thick paste;
C Fructus Lycii, the Radix Astragali, Radix Rehmanniae, Rhizoma Alismatis, Rhizoma Dioscoreae, Radix Trichosanthis, Fructus Rubi, Poria water decoct 3 times, and each 2 hours, merging decoction liquor, filtration, filtrate, to be concentrated into relative density be 1.20 thick paste, add ethanol and make that to contain the alcohol amount be 70%, leave standstill, the leaching supernatant, reclaim ethanol, concentrate
D merges drying, pulverizing and ginsenoside with above-mentioned extractum and merges, mixing gets extract powder 100g, get refined plant oil 50g and add the extract powder mixing, move in the encapsulating machine medicinal liquid storage tank, put gelatin solution in the gelatin storage tank and be incubated 60 ℃, make ball 50 ℃ following of water dropper temperature, select ball, drying, packing, promptly.
Content assaying method: with embodiment 5
The result: every contains ginsenoside Re 20mg, contains astragaloside 0.02mg.
Embodiment 9
Ginsenoside 8g Radix Astragali 150g Radix Rehmanniae 250g
Radix Ophiopogonis 80g Radix Trichosanthis 80g Fructus Lycii 160g
Fructus Schisandrae Chinensis 80g Rhizoma Dioscoreae 40g Fructus Rubi 50g
Poria 80g Rhizoma Alismatis 80g
Method for making: a Radix Ophiopogonis is with warm water lixiviate 2 times, and each 2 hours, merge lixiviating solution, filter, it is 1.30 thick paste that filtrate is concentrated into relative density;
B Fructus Schisandrae Chinensis 50% ethanol, percolation, percolate reclaim ethanol, are concentrated into thick paste;
C Fructus Lycii, the Radix Astragali, Radix Rehmanniae, Rhizoma Alismatis, Rhizoma Dioscoreae, Radix Trichosanthis, Fructus Rubi, Poria water decoct 3 times, and each 2 hours, merging decoction liquor, filtration, filtrate, to be concentrated into relative density be 1.20 thick paste, add ethanol and make that to contain the alcohol amount be 60%, leave standstill, the leaching supernatant, reclaim ethanol, concentrate
D merges drying, pulverizing and ginsenoside with above-mentioned extractum and merges, and mixing gets extract powder 100g, gets refined plant oil 500g and adds the extract powder mixing, moves in the encapsulating machine storage tank, is pressed into ball, selects ball, promptly.
Content assaying method: with embodiment 3
The result: every contains ginsenoside Re 0.5mg, contains astragaloside 0.1mg.
Embodiment 10
Ginsenoside 8g Radix Astragali 150g Radix Rehmanniae 250g
Radix Ophiopogonis 80g Radix Trichosanthis 80g Fructus Lycii 160g
Fructus Schisandrae Chinensis 80g Rhizoma Dioscoreae 80g Fructus Rubi 20g
Poria 80g Rhizoma Alismatis 80g
Method for making: a Radix Ophiopogonis is with warm water lixiviate 2 times, and each 2 hours, merge lixiviating solution, filter, it is 1.30 thick paste that filtrate is concentrated into relative density;
B Fructus Schisandrae Chinensis 50% ethanol, percolation, percolate reclaim ethanol, are concentrated into thick paste;
C Fructus Lycii, the Radix Astragali, Radix Rehmanniae, Rhizoma Alismatis, Rhizoma Dioscoreae, Radix Trichosanthis, Fructus Rubi, Poria water decoct 3 times, and each 2 hours, merging decoction liquor, filtration, filtrate, to be concentrated into relative density be 1.20 thick paste, add ethanol and make that to contain the alcohol amount be 60%, leave standstill, the leaching supernatant, reclaim ethanol, concentrate
D merges drying, pulverizing and ginsenoside with above-mentioned extractum and merges, mixing gets extract powder 100g, get refined plant oil 40g and add the extract powder mixing, move in the encapsulating machine medicinal liquid storage tank, put gelatin solution in the gelatin storage tank and be incubated 60 ℃, make ball 50 ℃ following of water dropper temperature, select ball, drying, packing, promptly.
Content assaying method: with embodiment 4
The result: every contains ginsenoside Re 15mg, contains astragaloside 0.02mg.
Embodiment 11
Ginsenoside 8g Radix Astragali 150g Radix Rehmanniae 250g
Radix Ophiopogonis 80g Radix Trichosanthis 80g Fructus Lycii 160g
Fructus Schisandrae Chinensis 80g Rhizoma Dioscoreae 80g Fructus Rubi 50g
Poria 40g Rhizoma Alismatis 80g
Method for making: a Radix Ophiopogonis is with warm water lixiviate 2 times, and each 2 hours, merge lixiviating solution, filter, it is 1.30 thick paste that filtrate is concentrated into relative density;
B Fructus Schisandrae Chinensis 50% ethanol, percolation, percolate reclaim ethanol, are concentrated into thick paste;
C Fructus Lycii, the Radix Astragali, Radix Rehmanniae, Rhizoma Alismatis, Rhizoma Dioscoreae, Radix Trichosanthis, Fructus Rubi, Poria water decoct 3 times, and each 2 hours, merging decoction liquor, filtration, filtrate, to be concentrated into relative density be 1.20 thick paste, add ethanol and make that to contain the alcohol amount be 60%, leave standstill, the leaching supernatant, reclaim ethanol, concentrate
D merges drying, pulverizing and ginsenoside with above-mentioned extractum and merges, and mixing gets extract powder 100g, gets refined plant 90 oil and adds the extract powder mixings, moves in the encapsulating machine storage tank, is pressed into ball, selects ball, promptly.
Content assaying method: with embodiment 3
The result: every contains ginsenoside Re 10mg, contains astragaloside 0.5mg.
Embodiment 12
Ginsenoside 8g Radix Astragali 150g Radix Rehmanniae 250g
Radix Ophiopogonis 80g Radix Trichosanthis 80g Fructus Lycii 160g
Fructus Schisandrae Chinensis 80g Rhizoma Dioscoreae 80g Fructus Rubi 50g
Poria 80g Rhizoma Alismatis 40g.
Method for making: a Radix Ophiopogonis is with warm water lixiviate 2 times, and each 2 hours, merge lixiviating solution, filter, it is 1.30 thick paste that filtrate is concentrated into relative density;
B Fructus Schisandrae Chinensis 50% ethanol, percolation, percolate reclaim ethanol, are concentrated into thick paste;
C Fructus Lycii, the Radix Astragali, Radix Rehmanniae, Rhizoma Alismatis, Rhizoma Dioscoreae, Radix Trichosanthis, Fructus Rubi, Poria water decoct 3 times, and each 2 hours, merging decoction liquor, filtration, filtrate, to be concentrated into relative density be 1.20 thick paste, add ethanol and make that to contain the alcohol amount be 60%, leave standstill, the leaching supernatant, reclaim ethanol, concentrate
D merges drying, pulverizing and ginsenoside with above-mentioned extractum and merges, and mixing gets extract powder 100g, gets refined plant oil 80g and adds the extract powder mixing, moves in the encapsulating machine storage tank, is pressed into ball, selects ball, promptly.
Content assaying method: with embodiment 3
The result: every contains ginsenoside Re 10mg, contains astragaloside 0.5mg.
Claims (10)
1, a kind of ginseng-astragalus blood-sugar lowering soft capsule is characterized in that it is prepared from as raw materials of effective components by following:
Ginsenoside 4-8 weight portion Radix Astragali 80-150 weight portion Radix Rehmanniae 150-250 weight portion
Radix Ophiopogonis 45-80 weight portion Radix Trichosanthis 40-80 weight portion Fructus Lycii 80-160 weight portion
Fructus Schisandrae Chinensis 40-80 weight portion Rhizoma Dioscoreae 40-80 weight portion Fructus Rubi 20-50 weight portion
Poria 40-80 weight portion Rhizoma Alismatis 40-80 weight portion.
2, soft capsule as claimed in claim 1 is characterized in that it is prepared from as raw materials of effective components by following:
Ginsenoside's 6 weight portion Radixs Astragali 124 weight portion Radix Rehmanniae 186 weight portions
Radix Ophiopogonis 62 weight portion Radix Trichosanthis, 62 weight portion Fructus Lyciis, 124 weight portions
Fructus Schisandrae Chinensis 62 weight portion Rhizoma Dioscoreaes 62 weight portion Fructus Rubies 31 weight portions
Poria 62 weight portion Rhizoma Alismatis 62 weight portions.
3,, it is characterized in that the extract obtained part by weight with Macrogol 4000 or refined plant oil of raw material is 1: 0.4-1: 5 as soft capsule as described in the claim 1,2.
4,, it is characterized in that the extract obtained ratio with Macrogol 4000 or refined plant oil of raw material is 1: 0.5-1: 1 as soft capsule as described in the claim 1,2.
5,, it is characterized in that this crude drug is 1: 0.7 through extracting extract obtained and ratio Macrogol 4000 as soft capsule as described in the claim 1,2.
6,, it is characterized in that every soft capsule contains ginsenoside Re 0.05-20mg as the described soft capsule of claim 1-5.
7,, it is characterized in that every soft capsule contains astragaloside 0.02-1mg as the described soft capsule of claim 1-5.
8,, it is characterized in that assay method is as claim 6,7 described effective ingredient:
(1) ginsenoside Re's assay method is:
The preparation of reference substance solution: it is an amount of that precision takes by weighing ginsenoside Re's reference substance, adds methanol and make the solution that contains the about 0.2-0.8mg of ginsenoside Re among every 1ml, in contrast product solution;
The preparation of need testing solution: get this product 2-20 grain, cut off, scrape and get content, mixing, rubber absolute ethanol washing, content add water 10-50ml, filter, the water saturated n-butyl alcohol 10-50ml of accurate adding extracts 10-40min, and precision is measured supernatant 10-50ml, adds the ammonia solution triplication, shake up, place, divide and get upper solution, evaporate to dryness, residue add methanol makes dissolving, quantitatively moves in the 2-10ml measuring bottle, add methanol and be diluted to scale, shake up, promptly;
Chromatographic condition: with octadecylsilane chemically bonded silica is filler; Volume ratio is that 99: 400 acetonitrile-0.05% phosphoric acid solution is a mobile phase; Detect wavelength 203nm;
Algoscopy: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject hplc determination;
(2) assay method of astragaloside is:
Chromatographic condition: octadecylsilane chemically bonded silica is a filler; Evaporative light scattering detector;
Mobile phase: ratio is acetonitrile-water-oxolane of 33: 67: 4;
The preparation of reference substance solution: get the astragaloside reference substance and add methanol and make the solution that every 1ml contains about 0.1-2mg, shake up;
The preparation of need testing solution: get this product 2-20 grain, cut off, scrape and get content, mixing, rubber absolute ethanol washing, content add water 10-50ml, filter, filtrate is used defat with petroleum ether 1-3 time, and (each 10-50ml) extracted in water saturated n-butyl alcohol jolting 2-6 time, merges n-butanol extracting liquid, extract 1-2 time with ammonia solution, each 10-40ml discards ammonia solution, the water 10-40ml saturated with n-butyl alcohol washes, and discards water liquid, n-butyl alcohol liquid evaporate to dryness, residue adds water 1-25ml makes dissolving, puts coldly, passes through D
101Type macroporous adsorptive resins (internal diameter 1.5cm, long 18cm) is with water elution, discard water liquid, reuse 10-40% ethanol 10-100ml eluting discards ethanol elution, continue with 50-90% ethanol 50-150ml eluting, collect eluent, evaporate to dryness is with dissolve with methanol and be transferred in the 2-10ml measuring bottle, add methanol to scale, shake up, filter, promptly.
9, a kind of Chinese medicinal soft capsule for the treatment of diabetes is characterized in that being prepared by following steps:
A uses warm water lixiviate 1-3 time Radix Ophiopogonis, and each 0.5-3 hour, merge lixiviating solution, filter, filtrate is concentrated into the thick paste that relative density is 1.03-1.40;
B Fructus Schisandrae Chinensis 40-70% ethanol, percolation, percolate reclaim ethanol, are concentrated into thick paste;
C Fructus Lycii, the Radix Astragali, Radix Rehmanniae, Rhizoma Alismatis, Rhizoma Dioscoreae, Radix Trichosanthis, Fructus Rubi, Poria water decoct 1-4 time, each 0.5-3 hour, merging decoction liquor, filtration, filtrate, to be concentrated into relative density be 1.20 thick paste, adding ethanol makes and contains the alcohol amount and be 40%-80%, leave standstill, the leaching supernatant, reclaim ethanol, concentrate;
D merges drying, pulverizing and ginsenoside with above-mentioned extractum and merges, and mixing is got adjuvant Macrogol 4000 or refined plant oil and added the extract powder mixing, moves in the encapsulating machine storage tank, is pressed into ball, selects ball, promptly.
10, Chinese medicinal soft capsule as claimed in claim 9, the technology that it is characterized in that steps d can be replaced with following technology generations: extractum is merged drying, pulverizing and ginsenoside merge, mixing is got adjuvant Macrogol 4000 or refined plant oil and is added the extract powder mixing, moves in the encapsulating machine medicinal liquid storage tank, put gelatin solution in the gelatin storage tank and be incubated 60 ℃, make ball 40-50 ℃ following of water dropper temperature, select ball, drying, packing, promptly.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103728387A (en) * | 2013-12-27 | 2014-04-16 | 广东万年青制药有限公司 | Shenqi hypoglycemic preparation HPLC standard finger print and construction method thereof |
| CN103844253A (en) * | 2012-12-05 | 2014-06-11 | 娄妍 | Formula for treating diabetes |
| CN106266543A (en) * | 2016-09-10 | 2017-01-04 | 四川金堂海纳生物医药技术研究所 | A kind of Chinese medicine composition treating type Ⅱdiabetes mellitus and preparation method thereof |
| CN106526002A (en) * | 2016-10-12 | 2017-03-22 | 浙江工业大学 | Method for measuring content of Shenqi blood sugar reducing preparation and application thereof in overall quality control |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1245198C (en) * | 2004-09-17 | 2006-03-15 | 鲁南制药集团股份有限公司 | Chinese medicine composition for treating diabetes and its preparing method |
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2005
- 2005-07-06 CN CNB2005100806536A patent/CN100341492C/en active Active
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103844253A (en) * | 2012-12-05 | 2014-06-11 | 娄妍 | Formula for treating diabetes |
| CN103728387A (en) * | 2013-12-27 | 2014-04-16 | 广东万年青制药有限公司 | Shenqi hypoglycemic preparation HPLC standard finger print and construction method thereof |
| CN103728387B (en) * | 2013-12-27 | 2015-08-19 | 广东万年青制药有限公司 | Ginseng-astragalus blood-sugar lowering preparation HPLC standard finger-print and construction method thereof |
| CN106266543A (en) * | 2016-09-10 | 2017-01-04 | 四川金堂海纳生物医药技术研究所 | A kind of Chinese medicine composition treating type Ⅱdiabetes mellitus and preparation method thereof |
| CN106526002A (en) * | 2016-10-12 | 2017-03-22 | 浙江工业大学 | Method for measuring content of Shenqi blood sugar reducing preparation and application thereof in overall quality control |
| CN106526002B (en) * | 2016-10-12 | 2019-04-09 | 浙江工业大学 | Ginseng-astragalus blood-sugar lowering preparation content determining method and its application in global quality control |
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Effective date of registration: 20211210 Address after: 276006 No. 209 Hongqi Road, Shandong, Linyi Patentee after: LUNAN HOPE PHARMACEUTICAL Co.,Ltd. Address before: 276005 No. 209 Hongqi Road, Shandong, Linyi Patentee before: LUNAN PHARMACEUTICAL Group Corp. |