CN1879697A - Pharmaceutical composition for treating senile dementia - Google Patents
Pharmaceutical composition for treating senile dementia Download PDFInfo
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- CN1879697A CN1879697A CNA2006100354815A CN200610035481A CN1879697A CN 1879697 A CN1879697 A CN 1879697A CN A2006100354815 A CNA2006100354815 A CN A2006100354815A CN 200610035481 A CN200610035481 A CN 200610035481A CN 1879697 A CN1879697 A CN 1879697A
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- tanshinone
- danshensu
- protocatechualdehyde
- salvianolic acid
- arasaponin
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Abstract
Disclosed is a medicinal composition for treating senile dementia, wherein 1g of the dried active sites comprises tanshinone IIA 1.79-9.01mg, salvianolic acid B 35.87-225.23mg, danshensu 4.93-45.05mg, protocatechuic aldehyde 0.10-4.50mg, and notoginseng saponin R 13.14-22.50mg.
Description
Technical field
The present invention relates to the plant is the pharmaceutical products of raw material, is specifically related to be used to prepare the medicine for the treatment of senile dementia.
Background technology
Senile dementia is a kind of performance of senilism phase and geratic period human brain function imbalance, it is a kind of disease that is changed to feature with intellectual deterioration and behavior personality, comprise Alzheimer (be called for short AD), vascular dementia (being called for short VD), mixed type is dull-witted and other is as wound, parkinsonism dementia, wherein Alzheimer and vascular dementia are topmost two major types in the senile dementia, and prevalence accounts for more than 90% of all dementias.
The pathological change of AD is two big pathology signs with senile plaque (SP) and neurofibrillary tangles (NFT) mainly, and the former is main and amyloid-beta (A β) gathering, fibrosis and deposition relation are close especially.The nucleus of senile plaque is beta amyloid peptide (β-AP), it forms amino acid residue through different cut modes by amyloid protein precursor (APP) in the processing modification is formed, and the neurotoxicity that the gathering of beta amyloid peptide and fibrous deposition produce is considered to the one of the main reasons of AD morbidity.Therefore disturb amyloid-beta formation and sedimentary research may become one of most promising method of treatment AD.Effect of free radicals damage has appreciable impact to the pathogenic process of AD, and is considered to participate in the death process of patient's AD brain cell, and anti-radical action can alleviate the development of dull-witted pathological changes, so antioxidant has the effect of prevention and treatment AD.
The Western medicine of existing treatment senile dementia is mainly and improves the medicine that cholinergic nerve transmits, first-class as tacrine (tacrine), Donepezil, galantamine, huperzine, since have liver toxicity and to symptom but not to the unsettled curative effect of treatment mode producing of the cause of disease, make it use extremely dispute.The equal treatments that are used for AD and VD such as short in addition metabolic drug, hormonotherapy, anti-inflammatory treatment, antioxidant, anti-amyloid, calcium channel blocker, anti-cerebral ischemia, curative effect remains certainly exploratoryly.
Chinese patent CN1546112A discloses FUFANG DANSHEN PIAN and can be used for treating senile dementia, and the situation master of present clinical use is based on the treatment angina pectoris, its technology and quality standard are at the treatment of cardiovascular system diseases and formulate, since its process conditions determined the active component of its medicine form in the content of tanshinone, salvianolic acid B, danshensu and protocatechualdehyde all lower, cause the curative effect of treatment senile dementia undesirable; The CN1348815A patent has also been mentioned a kind of FUFANG DANSHEN DIWAN and can be applicable to treat senile dementia, but adopt liposoluble constituent such as no tanshinone in the active component that preparation technology extracted of the said FUFANG DANSHEN DIWAN of this patent, the content of protocatechualdehyde and salvianolic acid B seldom, mainly be to contain danshensu, so, and only treatment coronary heart disease, angina pectoris are had curative effect preferably to treatment senile dementia poor effect.
We are with the red rooted salvia (place of production: Shandong, 041105,041106,041202,050101,050104) and the pseudo-ginseng (place of production: Yunnan gets five different lot numbers:, lot number 041204) by the preparation down of 2005 editions pharmacopeia FUFANG DANSHEN PIAN items, Radix Salviae Miltiorrhizae added alcohol heating reflux 1.5 hours, extracting solution filters, filtrate recycling ethanol and simmer down to thick paste, standby; Medicinal residues add 50% alcohol heating reflux 1.5 hours, and extracting solution filters, and filtrate recycling ethanol and simmer down to thick paste are standby; Medicinal residues decoct with water 2 hours, and decocting liquid filters, filtrate simmer down to thick paste.Radix Notoginseng is pulverized and is fine powder, with above-mentioned concentrated extract mixing, the dry dry product that gets.
With the red rooted salvia (with five lot numbers of system FUFANG DANSHEN PIAN, the place of production: Shandong, lot number 041105,041106,041202,050101,050104) and the pseudo-ginseng (place of production: Yunnan, lot number 041204) by under 2005 editions pharmacopeia FUFANG DANSHEN DIWAN item, Radix Salviae Miltiorrhizae, Radix Notoginseng decoct with water, and decocting liquid filters, and filtrate concentrates, add ethanol, leave standstill and make precipitation, get supernatant, reclaim ethanol, be condensed into thick paste, the dry dry product that gets.
The medical active position 1g dry product that above-mentioned two kinds of methods make, the content results of measuring tanshinone, salvianolic acid B, danshensu, protocatechualdehyde and arasaponin sees Table 1.
Table 1 FUFANG DANSHEN PIAN, FUFANG DANSHEN DIWAN medical active position 1g dry product composition measurement result (unit: mg/g)
| Sample | Tanshinone | Salvianolic acid B | Danshensu | Protocatechualdehyde | Arasaponin R1 |
| Fufang Danshen Pian 05001 Fufang Danshen Pian 05002 Fufang Danshen Pian 05003 Fufang Danshen Pian 05004 Fufang Danshen Pian 05005 compound danshen dripping pills 05001 compound danshen dripping pills 05002 compound danshen dripping pills 05003 compound danshen dripping pills 05004 compound danshen dripping pills 05005 | 1.0503 0.9924 1.0460 0.9596 1.1211 - - - - - | 27.6667 26.5460 24.7981 25.2564 28.0789 16.2112 17.7998 18.4700 17.4004 18.4700 | 4.2519 4.0147 3.8986 3.5671 3.8986 4.8631 4.2235 4.2698 4.4650 3.7902 | 0.0954 0.0913 0.1189 0.1008 0.1189 0.0125 0.0141 0.0090 0.0091 0.0104 | 3.7514 3.7670 3.8062 3.9633 3.7857 3.7559 3.7935 3.8427 3.7274 3.8742 |
This shows tanshinone (C in the dry product of FUFANG DANSHEN PIAN medical active position
19H
18O
3) content at 0.96～1.12mg/g, salvianolic acid B (C
36H
30O
16) content is at 24.8～28.08mg/g, the FUFANG DANSHEN PIAN of preparation can reach all that every of regulation contains tanshinone (C under 2005 editions FUFANG DANSHEN PIAN items of the Pharmacopoeia of the People's Republic of China
19H
18O
3), must not be less than 0.20mg, the amount that contains salvianolic acid B must not be less than the requirement of 5.0mg, promptly is equivalent to contain tanshinone in the dry product of 1g medicinal active ingredient and must not be less than 0.9mg, and salvianolic acid B must not be less than 22.5mg.Then its content is on the low side but as medicinal treatment senile dementia; FUFANG DANSHEN DIWAN does not have the tanshinone composition, and content of danshinolic acid B is on the low side.
Document " Chinese J Pharmacol Toxicol " 1994,8 (1): 19 has reported that tanshinone can suppress the brain cell damage that cerebral ischemia causes, and has antioxidation, and cranial nerve cell is had the better protect effect.Document " Phytomedicine " 2003,10 (4): 286 finds that TANSHINONES can be dwindled the cerebral tissue ischemic areas in the focal cerebral ischemia research of rat, alleviate the symptom that cerebral ischemia causes, cerebral protection is arranged.At document " Free Radic Biol Med " 1996,20 (6): think in 801 that TANSHINONES is to rat brain microsome Na
+-K
+-atpase activity has facilitation.
As seen, tanshinone is the effective ingredient of control AD and VD.
Document " Acta Pharmaceutica Sinica " 1992,27 (2): 96 and " Acta Pharmacol Sin " 2000, reported salvianolic acid B have very strong removing free radical antioxidative effect at 21 (8): 463, can pass through antioxidation neuroprotective cell, and beta-amyloid aggregation is had the obvious suppression effect; " Acta Pharmaceutica Sinica " 2000,35 (12): 881-885 salvianolic acid class mixture has the better protect effect to cerebral ischemia, can suppress synaptosome Giu and discharge, and removing oxygen-derived free radicals (ROS) be arranged and improve effect such as learning memory disorder; " medical Leader " 2004; think salvianolic acid to brain injury that animal brain ischemias such as mice rat and ischemia-reperfusion cause have protective effect at 23 (6): 355; can dwindle ischemic areas; reduce MDA content in the cerebral tissue; alleviate the behavioristics's obstacle that causes owing to cerebral ischemia, and the memory dysfunction that causes is thus improved significantly.Chemical substance damage memory acquisition disturbance there is certain improvement effect.
As seen salvianolic acid B is that the salvianolic acid constituents of representative is the effective ingredient of control AD and VD.
At document " Shanghai Medical Univ's journal " 1987,14 (1): 25; " Shanghai Second Emdical University's journal " 1990,10 (3): 208; " combination of Chinese and Western medicine magazine " 1983,3 (5): 297; " Acta Pharmaceutica Sinica " 1982,17 (3): 226; " combination of Chinese and Western medicine magazine " 1984,4 (9): 565; " Chinese Journal of Immunology " 1995,11 (6): 370; " Pharmacology and Clinics of Chinese Materia Medica " 1990,6 (4): but 31 reporting danshensu microcirculation improvement obstacle respectively and reducing blood plasma lactic acid content; Improve body anticoagulant and fibrinolytic; , tangible blood coagulation resisting function is arranged; Remarkable diastole animal coronary artery; Suppress platelet synthetic with discharge prostaglandins angiogenic substance such as TXA2; By regulating action to the mononuclear cell secrete inflammatory cytokines; Suppress the interior miscarriage of calcium and give birth to the effect of antiinflammatory and enhancing human body immunity adjusting.
As seen, danshensu also is the effective ingredient of control AD and VD.
Numerous document: Chinese Medical Journal, 1973,53 (4): 204-205. Suzhou Medical College journal, 1982,2:1-2. day disclosure special permission communique (A), clear 58-83619,1983-05-19. syndrome of blood stasis and blood circulation promoting and blood stasis dispelling research [M]. Beijing: Xueyuan Press, 1990,198-200.Acta Univ.Palackianae Olomucensis, 1958,2 (14): 135-143. has reported that protocatechualdehyde has coronary artery dilator, reduces myocardial oxygen consumption, anticoagulant, removing free radical and effects such as the oxidation of lipotropism matter, antiinflammatory.We have found also that by research protocatechualdehyde has effect of significant DPPH free radical scavenging and ultra-oxygen anion free radical inhibitory action, and clearance rate and suppression ratio all are higher than salvianolic acid B.
So protocatechualdehyde also is control AD and the indispensable a kind of effective ingredient of VD.
Document " Chinese clinical rehabilitation " 2004, reported Radix Notoginseng total arasaponins have antioxidation at 8 (34): 7876, and can alleviate cell to the neurotoxicity reaction of A beta-amyloyd peptide and the effect of promotion cell process growth, show that Radix Notoginseng total arasaponins can have antagonism to the pathology development of alzheimer disease; In addition, at document " Acta Pharmaceutica Sinica " 2005,40 (5): 385; " Chinese Medical Sciences University's journal " 2000,29 (3): 170; " contemporary Chinese application pharmacy " 2002,19 (2): 101; " Zunyi Medical College's journal " 2003,26 (4): 325; University Of Nanhua's journal (medicine), 2001,29 (2): 113 respectively report think that the ginsenoside Rg1 can raise Ach level and M-cholinoceptor number in the brain, has nootropic effect; The lipid peroxidation that free radical causes can cause crosslinked between cell component, can make whole neuron loss of function.The ginsenoside has the free radical resisting Oxidation, and hippocampal neuron is had tangible antioxidation, alleviates the Ultrastructural infringement in positions such as Hippocampus is reduced cell mortality; In the cerebral ischemia mouse experiment, the ginsenoside can suppress the generation of free radical, reduces apoplexy index and mortality rate, prolongs the time-to-live, and ischemic brain injury is had the certain protection effect; Radix Ginseng total saponins can weaken by the rat layer neuronal damage due to the free radical, and along with its protective effect of increasing of dosage also strengthens thereupon; The ginsenoside can reduce after the cerebral ischemia content of malonaldehyde (MDA) in the cerebral tissue, increases the vigor of superoxide dismutase (SOD), illustrate that the ginsenoside has antioxidant radical and damages the effect of anti peroxidation of lipid in cerebral ischemia.
Illustrated that more than arasaponin R1 is that the saponin component of representative is the effective ingredient of control AD and VD.
Therefore, in the red sage formulation for the treatment senile dementia, provide a kind of product that can quantitatively control effective ingredient amounts such as tanshinone, salvianolic acid B, danshensu, protocatechualdehyde, arasaponin R1 necessary.
Summary of the invention
The objective of the invention is with Radix Salviae Miltiorrhizae, Radix Notoginseng is primary raw material, by certain technology and method of quality control, provides a kind of new drug that senile dementia has significant curative effect that is used to prevent and treat.
To achieve the object of the present invention, we at first use the alcohol extract of the fat-soluble position of Radix Salviae Miltiorrhizae and water soluble part, Radix Notoginseng, by the preparation of uniform Design arrangement sample, carry out external effect experiment with the senile dementia correlation model, be identified for the composition of the medicinal active ingredient of senile dementia prevention and cure.
Adopt external high-throughput screening method to determine effective ingredient and content and the ratio relevant with curative effect.
The preparation of 1 laboratory sample
1.1 the preparation of Radix Salviae Miltiorrhizae liposoluble extract: the dry root and rhizome of Labiatae salvia Radix Salviae Miltiorrhizae Salvia miltiorrhizaBge, broken is segment less than 1.5cm, adds soak with ethanol 1h, 70 ℃ of hot reflux 0.5h filter, and reclaim ethanol, lyophilization, promptly.After measured, liposoluble extract only contains fat soluble ingredient of red sage root such as tanshinone, cryptotanshinone, dihydrotanshinone, Tanshinone I, no salvianolic acid B, danshensu, protocatechualdehyde composition.
1.2 the preparation of water-soluble extract of red sage root I: the dry root and rhizome of Labiatae salvia Radix Salviae Miltiorrhizae Salviamiltiorrhiza Bge, broken is fine powder, soaks 12h, stirs, filter, lyophilization, promptly.After measured, water solubility extract I only contains salvianolic acid B and danshensu, protocatechualdehyde, fat soluble ingredient of red sage root such as no tanshinone, cryptotanshinone, dihydrotanshinone, Tanshinone I.
1.3 the preparation of water-soluble extract of red sage root II: the dry root and rhizome of Labiatae salvia Radix Salviae Miltiorrhizae Salviamiltiorrhiza Bge, broken is segment less than 1.5cm, soaks 1h, 100 ℃ of hot reflux 20h filter, lyophilization, promptly.After measured, water solubility extract II only contains danshensu, protocatechualdehyde and minute quantity salvianolic acid B composition (can ignore), fat soluble ingredient of red sage root such as no tanshinone, cryptotanshinone, dihydrotanshinone, Tanshinone I.
1.4 the preparation of Radix Notoginseng extract: the dry root of araliaceae ginseng plant Radix Notoginseng Panax notoginseng (Burk.) F.H.Chen, broken is fine powder, 70% soak with ethanol 1h, 70 ℃ of hot reflux 1h filter, and reclaim ethanol, lyophilization, promptly.After measured, Radix Notoginseng extract contains compositions such as arasaponin R1, ginsenoside Rg1, ginsenoside Rb1.
1.5 commercially available FUFANG DANSHEN PIAN extract: result after measured: every contains tanshinone 0.2854mg, cryptotanshinone 0.2930mg, dihydrotanshinone 0.1235mg, Tanshinone I 0.2744mg, salvianolic acid B 6.1117mg, danshensu 1.9302mg, protocatechualdehyde 0.0038mg, arasaponin R1 0.5544mg, ginsenoside Rg1 3.6967mg, ginsenoside Rb1 2.7958mg
1.6 tanshinone, cryptotanshinone, dihydrotanshinone, Tanshinone I, salvianolic acid B, danshensu, protocatechualdehyde, arasaponin R1, ginsenoside Rg1, ginsenoside Rb1's reference substance are all available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
The experiment of 2 in-vitro screenings
2.1beta-the screening of secretase (BACE) inhibitor and effective ingredient proportioning
2.1.1 experiment material and instrument:
Sodium-acetate buffer (PH4.0).Recombinant human B ACE-1, fluorogenic substrate IV is (all available from R﹠amp; D company).Instrument: Beckman2000 robot, POLARSTAR GALAXY microplate optical detecting instrument, fluoremetry 384 orifice plates (BMG product).
2.1.2 experimental technique:
2.1.2.1 sample preparation
Press U
12(12
5) uniform designs table ratio preparation screening sample.
Table 1U
12(12
4) the dried cream of uniform designs table unit: mg/2.33g
| Numbering | 1 | 2 | 3 | 4 |
| Tanshinone | Salvianolic acid B | Danshensu * | Arasaponin | |
| 1 2 3 4 5 6 7 8 9 10 11 12 | 6.5 3.5 4.5 5 4 1.5 2.5 1 6 5.5 2 3 | 50 10 90 20 80 120 40 70 60 110 30 100 | 25 15 10 30 17.5 12.5 7.5 27.5 5 22.5 20 32.5 | 10 8 12 4 1 5 2 6 7 3 11 9 |
* be in the extract of index, to contain the protocatechualdehyde that is equivalent to danshensu amount 2% with the danshensu.
2.1.2.2 experimental procedure
Add sample solution at 384 orifice plates successively, sodium-acetate buffer, BACE, 25 ℃ of reaction temperatures add fluorogenic substrate IV again, substrate final concentration 10 μ M, enzyme final concentration 4 μ g/L measure wavelength Ex=320nm, Em=405nm, the record fluorescent value changes slope as enzymatic activity, relatively calculates suppression ratio to what enzyme with standard control.8 holes in contrast in 384 orifice plates.
2.1.3 experimental result:
The suppression ratio (per diem taking dose screening) of table 2beta-secretase (BACE) inhibitor screening sample
| Numbering | Sample | Suppression ratio % |
| 1 2 | Even 1 even 2 | 39.08 13.05 |
| 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 | The commercially available Fufang Danshen Pian extract of even 3 even 4 even 5 even 6 even 7 even 8 even 9 even 10 even 11 even 12 tanshinone IIA tanshin polyphenolic acid B danshensu notoginsenoside R Cryptotanshinone dihydro tanshinone and tanshinone I protocatechualdehyde ginsenoside Rg1 ginsenoside Rb1s * * | 37.26 47.19 53.07 71.42 41.30 43.13 35.08 48.54 40.10 63.47 41.44 59.07 29.53 14.44 10.89 18.16 9.68 26.49 33.39 12.68 18.79 |
* is the meansigma methods that 2 parallel sample are measured
The uniform Design sample is selected into threshold value 1 with the SPSS10.0 software processes, rejects threshold value 1, gets multiple linear regression equations: Y=24.4356+0.2857X
2+ 0.4752X
3-4.5488X
4, F
0.05(3,8)=5.6241＞F
0.05(3,8)=4.07, equation is remarkable, multiple correlation coefficient r=0.83 (X wherein
2Be salvianolic acid B, X
3Be danshensu, X
4Be arasaponin R1, Y is a suppression ratio).
Found that: salvianolic acid B and content of Danshensu height, arasaponin R1 content are low, and is favourable to improving suppression ratio; Tanshinone is like irrelevant with beta-secretase inhibitory action.The suppression ratio of comprehensive actual experiment sample is found, former FUFANG DANSHEN PIAN suppression ratio 18.79%, and the suppression ratio of laboratory sample surpasses 50%, reaches as high as 71.42%.The theoretical suppression ratio of best proportion sample is 69.61%, and is approaching with the sample that actual suppression ratio is the highest.
2.2 butyrylcholinesterase inhibitor screening model
2.2.1 experiment material and instrument:
Butyrylcholine esterase testing cassete (bio-engineering research institute is built up in Nanjing), the two nitrobenzoic acids (Sigma company) of two sulfur.Instrument: 721 spectrophotometers.
2.2.2 experimental technique:
2.2.2.1 sample preparation
In table 1 ratio preparation screening sample.
2.2.2.2 experimental procedure
With the phosphate buffer of 0.05M dilution in 1: the 80 human plasma enzyme liquid that is applied.After at first candidate compound 5 μ l and enzyme liquid 10 μ l temperature being incubated 30min, add the two nitrobenzoic acids (final concentration 0.25mM) of substrate chlorination butyryl sulfydryl choline test solution (final concentration 3mM) and developer two sulfur successively, mixing is incubated in 30 ℃ of temperature, reads colourimetric number OD412.
2.2.3 experimental result:
The suppression ratio of table 3 inhibitor screening sample (per diem taking dose screening)
| Numbering | Sample | Suppression ratio % |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 | Even 1 even 2 even 3 even 4 even 5 even 6 even 7 even 8 even 9 even 10 even 11 even 12 tanshinone IIA tanshin polyphenolic acid B danshensu notoginsenoside R Cryptotanshinone dihydro tanshinone and tanshinone I protocatechualdehyde ginsenoside Rg1 ginsenoside Rb1s | 12.19 15.27 18.57 14.06 67.92 14.50 16.37 7.07 30.56 5.97 13.62 12.68 6.19 17.78 10.00 20.55 18.73 24.46 5.53 6.91 16.12 15.42 |
| 23 | Commercially available FUFANG DANSHEN PIAN extract ** | 16.92 |
* is the meansigma methods that 2 parallel sample are measured
According to interpretation: former FUFANG DANSHEN PIAN suppression ratio 16.92%, the suppression ratio of laboratory sample surpasses 20%, reaches as high as 67.92%.Wherein higher 5, No. 9 sample suppression ratio of tanshinone content estimate that than higher effective site mainly is that fat soluble ingredient of red sage root is main.With monomer suppression ratio under the concentration be from high to low: dihydrotanshinone＞arasaponin R1＞cryptotanshinone＞ginsenoside Rg1.
2.3 antioxidation model-DPPH (hexichol trinitrophenyl-hydrazine) free radical scavenging
2.3.1 instrument: Zenyth 200rt microplate reader.
2.3.2 experimental technique:
2.3.2.1 sample preparation
In table 1 ratio preparation screening sample.
2.3.2.2 experimental technique
With certain density DPPH 190ul, add 10 μ l samples then.The blank group adds 10ul ethanol, and cumulative volume 200ul shakes up, and after lucifuge under the room temperature was placed 30min, microplate reader was measured the DPPH mixed solution at 517nm place light absorption value (A).Calculate the clearance rate of DPPH.
2.3.3 experimental result:
Table 4 antioxidation model-DPPH free radical scavenging activity (per diem taking dose screening)
| Numbering | Sample | Clearance rate % |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 | Even 1 even 2 even 3 even 4 even 5 even 6 even 7 even 8 even 9 even 10 even 11 even 12 tanshinone IIA tanshin polyphenolic acid Bs | 20.82 10.94 6.84 11.70 18.09 17.78 20.21 15.65 11.70 19.45 12.16 12.16 -1.82 52.74 |
| 15 16 17 18 19 20 21 22 23 | The commercially available Fufang Danshen Pian extract of danshensu notoginsenoside R Cryptotanshinone dihydro tanshinone and tanshinone I protocatechualdehyde ginsenoside Rg1 ginsenoside Rb1** | 49.24 1.98 13.98 1.82 8.21 60.03 0.91 1.37 7.75 |
* is the meansigma methods that 2 parallel sample are measured
The uniform Design sample is selected into threshold value 1 with the SPSS software processes, rejects threshold value 1, gets multiple linear regression equations:
Y=38.5833-6.5146X
1-0.0471X
2-12.8129X
4+ 0.5367X
1 2-0.0196X
3 2-0.0123X
1X
2+ 0.0492X
1X
3+ 0.3874X
1X
4+ 0.0115X
2X
4+ 0.4045X
3X
4, F
0.05(10,1)=3486.7＞F
0.05(10,1)=241, equation is remarkable, multiple correlation coefficient r=0.9999 (X wherein
1Be tanshinone, X
2Be salvianolic acid B, X
3Be danshensu, X
4Be arasaponin R1, Y is a clearance rate).
Optimization is calculated as: X
1=6.50, X
2=119.98, X
3=32.49, X
4=12.00, Y=44.11.
Found that: former FUFANG DANSHEN PIAN antioxidation DPPH free radical scavenging activity 7.75%, the clearance rate of laboratory sample can reach 20%, and the theoretical clearance rate of best proportion sample is 44.11%.Reach as high as 60.03% with monomer clearance rate under the concentration.With monomer clearance rate under the concentration be from high to low: protocatechualdehyde＞salvianolic acid B＞danshensu.Multiple main component content height, favourable to improving the effect of removing free radical.
2.4 antioxidation model-ultra-oxygen anion free radical suppresses experiment
2.4.1 experimental apparatus: the microwell plate luminous calculating instrument Topcount that glimmers.
2.4.2 experimental technique:
2.4.2.1 sample preparation
In table 1 ratio preparation screening sample.
2.4.2.2 experimental technique
Adopt xanthine one xanthine oxidase one luminol system to detect.Add 10 μ l screening samples in 96 orifice plates, blank group adds the phosphate buffer of equivalent, adds xanthine-luminol liquid 188ul, adds xanthine oxidase 2 μ l, starts luminously, and continuous measurement 6 times lasting 6 seconds at every turn, is got 6 times meansigma methods.
2.4.3 experimental result:
Table 5 antioxidation model-ultra-oxygen anion free radical suppression ratio (per diem taking dose screening)
| Numbering | Sample | Suppression ratio/% |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 | The commercially available Fufang Danshen Pian extract of even 1 even 2 even 3 even 4 even 5 even 6 even 7 even 8 even 9 even 10 even 11 even 12 tanshinone IIA tanshin polyphenolic acid B danshensu notoginsenoside R Cryptotanshinone dihydro tanshinone and tanshinone I protocatechualdehyde ginsenoside Rg1 ginsenoside Rb1s** | 80 80 80 80 95 80 80 80 80 80 80 80 80 95 95 - - - - 99 - - 80 |
* is the meansigma methods that 2 parallel sample are measured
According to interpretation: the composition of antioxidation model-ultra-oxygen anion free radical effect is mainly red sage root water soluble ingredient and tanshinone, and promptly salvianolic acid B, danshensu, protocatechualdehyde and tanshinone content height are favourable to improving suppression ratio.The monomer suppression ratio is from high to low: protocatechualdehyde＞danshensu＞salvianolic acid B＞tanshinone.
2.5 antioxidation model-lipoid peroxidization resistant experiment
2.5.1 experiment material and instrument: Radix Salviae Miltiorrhizae liposoluble extract, water-soluble extract of red sage root I, water-soluble extract of red sage root II, Radix Notoginseng extract.Tanshinone, cryptotanshinone, dihydrotanshinone, Tanshinone I, salvianolic acid B, danshensu, protocatechualdehyde, arasaponin R1, ginsenoside Rg1, ginsenoside Rb1's reference substance are all available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute.Instrument: 721 spectrophotometers.
2.5.2 experimental technique:
2.5.2.1 sample preparation
In table 1 ratio preparation screening sample.
2.5.2.2 experimental technique
The microsome preparation: the rat overnight fasting, sacrificed by decapitation is earlier with cold TMS buffer (Tris-HCl 0.05mmol.L
-1, sucrose 0.2mmol.L
-1, MgCl23mmol.L
-1, pH 7.4) and wash liver to khaki through portal vein, get liver and make homogenate, the centrifugal 20min of 3000g, again with the centrifugal 60min of supernatant 1.05 * 105g, precipitation is microsome.Hepatomicrosome is resuspended in the TMS buffer, and being diluted to protein content is 15gL
-1(surveying protein concentration with the Coomassie brilliant blue method), it is standby to be stored in-60 ℃ of refrigerators.More than operate under 4 ℃ of conditions and finish.
Contain microsome 0.1ml in the reaction system, add the medicine 0.01ml to be measured (positive control does not add medicine to be measured) of solvent or variable concentrations, cysteine (0.01mol.L
-1) 0.02ml, ferrous sulfate (1mmol.L
-1) 0.05ml (blank does not add ferrous sulfate), use 0.1mol.L
-1It is 1ml that PBS (pH 7.4) is supplemented to cumulative volume.Behind 37 ℃ of common incubation 30min, add trichloroacetic acid and thiobarbituricacid developer, measure the lipid peroxide MDA content.
Suppression ratio (IR%) expression that the work of medicine to be measured generates in order to MDA.
IR%=[(OD positive control-OD blank)-(OD sample-OD blank)]/(OD positive control-OD blank) * 100%
2.5.3 experimental result:
The suppression ratio (per diem taking dose screening) that table 5 lipoid peroxidization resistant MDA generates
| Numbering | Sample | Suppression ratio % |
| 1 2 3 4 5 6 7 8 9 10 | Even 1 even 2 even 3 even 4 even 5 even 6 even 7 even 8 even 9 even 10 | 0.39 2.75 22.09 19.93 5.34 25.23 9.42 16.01 10.16 2.32 |
| 11 12 13 14 15 16 17 18 19 20 21 22 23 | The commercially available Fufang Danshen Pian extract of even 11 even 12 tanshinone IIA tanshin polyphenolic acid B danshensu notoginsenoside R Cryptotanshinone dihydro tanshinone and tanshinone I protocatechualdehyde ginsenoside Rg1 ginsenoside Rb1s** | 0.20 20.64 17.30 11.73 5.57 19.42 16.87 26.64 16.32 25.70 18.09 8.87 8.77 |
* is the meansigma methods that 2 parallel sample are measured
The uniform Design sample is selected into threshold value 3 with the SPSS software processes, rejects threshold value 3, gets multiple linear regression equations:
Y=2.1792+2.4477X
1+ 0.8159X
3-0.4182X
1 2-6.1492X
4 2-0.013X
2X
3+ 0.2916X
2X
4, F
0.01(6,5)=64.23158＞F
0.01(6,5)=10.67, equation highly significant, multiple correlation coefficient r=0.9936 (X wherein
1Be tanshinone, X
2Be salvianolic acid B, X
3Be danshensu, X
4Be arasaponin R1, Y is a clearance rate).
Optimize and be calculated as X
1=2.87, X
2=119.36, X
3=5.28, X
4=2.89, Y=50.59.
Interpretation of result: former FUFANG DANSHEN PIAN suppression ratio 18.79%, the suppression ratio of laboratory sample can reach 50%, reaches as high as 71.42%, and the theoretical clearance rate of optimization ratio sample is 50.59%, actual suppression ratio may be higher than theoretical value, and lipoid peroxidization resistant is remarkable.With monomer suppression ratio under the concentration be from high to low: dihydrotanshinone＞protocatechualdehyde＞ginsenoside Rg1＞arasaponin R1.
Derive in conjunction with the direct analysis of screening sample according to above-mentioned five experimental results: in the screening of beta-Secretase inhibitors, arasaponin R1 content is low, to improve suppression ratio favourable outside, each model all has the effective component content height to improving the favourable phenomenon of index (activity).In addition, be in the extract of index with the danshensu, contain the protocatechualdehyde that is no less than danshensu amount 2%.Therefore, according to the situation of danshensu, the effective ingredient protocatechualdehyde also has the content height to improving active favourable situation in each model.In beta-Secretase inhibitors screening experiment, No. 5 samples of uniform Design are at tanshinone: salvianolic acid B: danshensu: protocatechualdehyde: arasaponin R1 is 4: 80: 17.5: (be in the medical active position dry product of 1g contain tanshinone 1.79mg at 0.35: 1, salvianolic acid B 35.87mg, danshensu 4.93mg, protocatechualdehyde 0.10mg,) time existing 53% suppression ratio, though arasaponin R1 does not have beta-secretase inhibitory action, have significant free radical resisting activity.Consider the beta-Secretase inhibitors be applied in the AD control to be important approach, to think that in this ratio and arasaponin R1 median dose be the lower limit of effective ingredient.Draw thus in the medical active position dry product of 1g and contain tanshinone 1.79～9.01mg, salvianolic acid B 35.87～225.23mg, danshensu 4.93～45.05mg, protocatechualdehyde 0.10～4.50mg, arasaponin R1 3.14～22.50mg, tanshinone: salvianolic acid B: danshensu: protocatechualdehyde: the weight ratio of arasaponin R1 is 4～20: 80～500: 11～100: 0.2～10: 7～50.Contain tanshinone 2.83～9.01mg in the medical active position dry product of optimum curative effect, salvianolic acid B 53.81～225.23mg, danshensu 7.85～45.05mg, protocatechualdehyde 0.16～4.5mg, arasaponin R1 3.14～22.50mg, tanshinone: salvianolic acid B: danshensu: protocatechualdehyde: the weight ratio of arasaponin R1 is 6.3～20: 120～500: 17.5～100: 0.3～10: 7～50.
Technical scheme of the present invention is: the pharmaceutical composition that is used to prevent and treat senile dementia, the oral formulations of making by medical active position and conventional pharmaceutic adjuvant process of Chinese medicine preparation routinely, its medical active position is mainly to be prepared from by Radix Salviae Miltiorrhizae, pseudo-ginseng, contain tanshinone 1.79～9.01mg in the medical active position dry product of 1g, salvianolic acid B 35.87～225.23mg, danshensu 4.93～45.05mg, protocatechualdehyde 0.10～4.50mg, arasaponin R1 3.14～22.50mg; Tanshinone: salvianolic acid B: danshensu: protocatechualdehyde: the weight ratio of arasaponin R1 is 4～20: 80～500: 11～100: 0.2～10: 7～50.For the curative effect that makes medicine better, preferably contain tanshinone 2.83～9.01mg in the dry product of medical active position, salvianolic acid B 53.81～225.23mg, danshensu 7.85～45.05mg, protocatechualdehyde 0.16～4.5mg, arasaponin R1 3.14～22.50mg; Tanshinone: salvianolic acid B: danshensu: protocatechualdehyde: the weight ratio of arasaponin R1 is 6.3～20: 120～500: 17.5～100: 0.3～10: 7～50.
Contain cryptotanshinone 1.52～9.00mg, dihydrotanshinone 0.63～4.50mg, Tanshinone I 1.35～9.00mg, ginsenoside Rg1 18.83～135.14mg, ginsenoside Rb1 15.70～112.61mg with recording in the dry product of the said 1g medical active of high effective liquid chromatography for measuring the present invention position; Better when also containing cryptotanshinone 2.42～9.00mg, dihydrotanshinone 0.99～4.50mg, Tanshinone I 2.11～9.00mg, ginsenoside Rg1 18.83～135.14mg, ginsenoside Rb1 15.70～112.61mg in the dry product of 1g medical active position.
Product of the present invention, said medical active position, it can be the method that adopts as above-mentioned in vitro tests, use the Radix Salviae Miltiorrhizae liposoluble extract, water-soluble extract of red sage root I, water-soluble extract of red sage root II, composite forming such as Radix Notoginseng extract and tanshinone, cryptotanshinone, dihydrotanshinone, Tanshinone I, salvianolic acid B, danshensu, protocatechualdehyde, arasaponin R1, ginsenoside Rg1, ginsenoside Rb1.In order to obtain simple preparation method, guarantee the quality of medicine again, we study preparation process condition, can preferably obtain following preparation method: said medical active position is to get red rooted salvia, and broken is segment, uses soak with ethanol 0.5～2 hour, hot reflux 0.5～1 hour, filter, collect ethanol filtrate, reclaim ethanol and concentrate extractum I; Medicinal residues filter with 80% alcohol heat reflux 0.5～1 hour, collect 80% ethanol filtrate, reclaim ethanol and concentrate extractum II; Medicinal residues refluxed 1～2 hour with hydro-thermal again, filtrate concentrate extractum III; Radix Notoginseng powder is broken into fine powder more than 80 orders, mixes drying thoroughly with Radix Salviae Miltiorrhizae extractum I, II and III; Dry 36～83 hours is good under 70～90 ℃, the medical active position dry product that gets, moisture≤5%.
Red rooted salvia need be pulverized and be segment, is more fully to extract for the salvianolic acid constituents that will mainly be distributed in xylem in extraction time; Use soak with ethanol 0.5～2 hour earlier, can reduce the time that Radix Salviae Miltiorrhizae extracts with alcohol heat reflux, reduce the easily loss of the tanshinone of degraded of being heated, improved the content of tanshinone component; Salvianolic acid is heated at leaching process can be decomposed into danshensu, the salvianolic acid of 1 molecule decomposes the danshensu that can obtain 3 molecules, so the time of heating and the time of extract dry are extracted in essential control when extracting, at different baking temperatures, dry heat makes the salvianolic acid class be converted into danshensu and protocatechualdehyde regularly, find that through experiment danshensu and protocatechualdehyde are one of main components of drug activity, certain content regulation need be arranged in product, for guaranteeing salvianolic acid B, danshensu and protocatechualdehyde three content all reach requirement, thus baking temperature and drying time scope need control.
Pharmaceutical composition provided by the invention is to get it filled to use the active site dry product, add conventional pharmaceutic adjuvant, Chinese patent medicine preparation technology is routinely made oral formulations, said oral formulations is preferably made tablet, capsule, granule, pill, and its pill can be the watered pill, small honey pill, big honeyed pills, concentrated pill, drop pill etc.
The content assaying method that is used for tanshinone of the present invention, salvianolic acid B, danshensu, protocatechualdehyde, arasaponin R1, ginsenoside Rg1, Rb1, cryptotanshinone, dihydrotanshinone, Tanshinone I all adopts high performance liquid chromatography (HPLC).
Assay method is as follows:
1, tanshinone (can measure cryptotanshinone, dihydrotanshinone, Tanshinone I simultaneously) content assaying method
Chromatographic condition and system suitability condition: C18 post; Mobile phase: methanol-water (80: 20); Detect wavelength: 254nm.Number of theoretical plate calculates by the tanshinone peak should be not less than 2000.
It is an amount of that tanshinone, dihydrotanshinone, cryptotanshinone, Tanshinone I are got in the preparation of reference substance solution, the accurate title, decide, put in the brown measuring bottle, add methanol and make every 1ml and contain tanshinone 2.4 μ g, dihydrotanshinone 2.7 μ g, cryptotanshinone 2.1 μ g, Tanshinone I 2.1 μ g mixed solutions, promptly.
Red rooted salvia 0.2g is got in the preparation of need testing solution, adds 75% methanol 25ml, and reflux 1h filters, and filtrate is concentrated into 1ml, promptly.Get DANSHEN KELI 1g, accurate claim surely, put in the tool plug brown bottle, the accurate methanol 25ml that adds, close plug claims decide weight, and supersound process is put coldly, and weight decided in title, supplies the weight that subtracts mistake with methanol, shakes up, and gets subsequent filtrate, puts in the brown bottle, promptly.Get red sage formulation, grind and be fine powder, the accurate title, decide 1g, puts in the tool plug brown bottle, the accurate methanol 25ml that adds, and close plug claims to decide weight, and supersound process is put coldly, and weight decided in title, supplies the weight that subtracts mistake with methanol, shakes up, and gets subsequent filtrate, puts in the brown bottle, promptly.
2 danshensus (calculate with danshensu sodium, can measure protocatechualdehyde simultaneously) content assaying method
Chromatographic condition and system suitability condition: C
18Post; Mobile phase: 0.5% acetic acid water-methanol (80: 20); Detect wavelength: 280nm.Number of theoretical plate calculates by the danshensu peak should be not less than 2000.
Danshensu sodium is got in the preparation of reference substance solution, protocatechualdehyde is an amount of, accurate claims surely, adds methanol and makes every 1ml and contain danshensu 100 μ g, protocatechualdehyde 100 μ g solution, promptly.
Red rooted salvia 0.2g is got in the preparation of need testing solution, adds methanol 15ml, and supersound process is filtered, and puts coldly, claims to decide weight, supplies the weight that subtracts mistake with methanol, promptly.Get DANSHEN KELI 1g, accurate claim fixed, the accurate methanol 15ml that adds, close plug claims decide weight, supersound process 15min is put coldly, weight decided in title, supplies the weight that subtracts mistake with methanol, promptly.Get red sage formulation, grind and be fine powder, the accurate title, decide 1g, puts in the tool plug brown bottle, the accurate methanol 15ml that adds, and close plug claims to decide weight, and supersound process is put coldly, and weight decided in title, supplies the weight that subtracts mistake with methanol, promptly.
3 content of danshinolic acid B assay methods
Chromatographic condition and system suitability condition: C18 post; Mobile phase: methanol-acetonitrile-formic acid-water (30: 10: 1: 59); Detect wavelength: 286nm.Number of theoretical plate calculates by the salvianolic acid B peak should be not less than 2000.
It is an amount of that salvianolic acid B is got in the preparation of reference substance solution, accurate claims surely, puts in the brown measuring bottle, adds water and make every 1ml and contain salvianolic acid B 60 μ g solution, promptly.
The preparation of need testing solution is got red rooted salvia powder 0.2g and is put in the tool plug conical flask, and the accurate 75% methanol 50ml that adds claims decide weight, and reflux 1h takes out, and puts coldly, claims to decide weight again, supplies the weight that subtracts mistake with 75% methanol, and filtration is got subsequent filtrate, promptly.Get DANSHEN KELI 0.15g, accurate claim surely, put in the tool plug conical flask, add water 50ml, close plug claims decide weight, and supersound process is put coldly, and weight decided in title, and water is supplied the weight that subtracts mistake, shakes up, and gets subsequent filtrate, promptly.Get red sage formulation, grind and be fine powder, accurately claim surely, put in the tool plug conical flask, add water 50ml, close plug claims decide weight, and supersound process is put coldly, and weight decided in title, and water is supplied the weight that subtracts mistake, shakes up, and gets subsequent filtrate, promptly.
4 arasaponin R1s (can measure ginsenoside Rg1, Rb1 simultaneously) content assaying method
Chromatographic condition and system suitability condition: C18 post; Mobile phase: acetonitrile is a mobile phase A, and water is Mobile phase B, carries out 0～12min, mobile phase A-Mobile phase B (19: 81), 12～60min, mobile phase A-Mobile phase B (19～36: 81～64) by gradient elution; Detect wavelength: 203nm.Number of theoretical plate calculates by the arasaponin R1 peak should be not less than 4000.
It is an amount of that arasaponin R1, ginsenoside Rg1, ginsenoside Rb1 are got in the preparation of reference substance solution, accurately claims surely, adds methanol and make every 1ml and contain arasaponin R1 0.1mg, ginsenoside Rg1 0.4mg, ginsenoside Rb1 0.1mg mixed solution, promptly.
Red rooted salvia powder 0.6g is got in the preparation of need testing solution, and accurate the title decides, and the accurate methanol 50ml that adds claims to decide weight, and placement is spent the night, put in 80 ℃ of water-baths and to keep little 2h of boiling, put coldly, claim again to decide weight, supply the weight that subtracts mistake with methanol, filter, shake up, get subsequent filtrate, promptly.Get DANSHEN KELI powder 1g, accurate claim surely, the accurate methanol 50ml that adds claims decide weight, and placement is spent the night, and puts to keep little 2h of boiling in 80 ℃ of water-baths, puts coldly, claims to decide weight again, supplies the weight that subtracts mistake with methanol, and filtration shakes up, and gets subsequent filtrate, promptly.Get red sage formulation, grind and be fine powder, accurately claim decide 1g, the accurate methanol 50ml that adds claims decide weight, and placement is spent the night, and puts to keep little 2h of boiling in 80 ℃ of water-baths, puts coldly, and weight decided in title again, supplies the weight that subtracts mistake with methanol, and filtration shakes up, and gets subsequent filtrate, promptly.
Beneficial effect of the present invention: 1, compositions provided by the present invention, the senile dementia of control AD and VD is treated definite, more remarkable than existing FUFANG DANSHEN PIAN, FUFANG DANSHEN DIWAN effect; 2, effective ingredient composition, content and the ratio of product are clear and definite, and controllable product quality and operability are good; 3, can be mass-produced.
Further set forth technical scheme of the present invention and effect below by embodiment
The specific embodiment
Embodiment 1:
Get the red rooted salvia (place of production: Shandong, lot number 0411053) 100Kg, the pseudo-ginseng (place of production: Yunnan, lot number 0412041) 30Kg, red rooted salvia is broken to be segment, refluxed 0.5 hour with 1 hour post-heating of 8 times of amount soak with ethanol, filter, collect ethanol filtrate, reclaiming ethanol and being concentrated into relative density is 1.35 extractum I; Medicinal residues filter with 80% alcohol heat reflux 1 hour, collect 80% ethanol filtrate, and reclaiming ethanol and being concentrated into relative density is 1.36 extractum II; Refluxed 1.5 hours with hydro-thermal at last, it is 1.29 extractum III that filtrate is concentrated into relative density; Radix Notoginseng powder is broken into fine powder more than 80 orders, with Radix Salviae Miltiorrhizae extractum I, II and III mixing, dry 36h about 90 ℃, medical active position dry product (dried cream) 55Kg that makes, moisture≤5%.Measure with above-mentioned high performance liquid chromatography method, contain tanshinone 2.66mg in the dried cream of every 1g, salvianolic acid B 48.81mg, danshensu 7.15mg, protocatechualdehyde 0.12mg, arasaponin R1 5.34mg, cryptotanshinone 2.72mg, dihydrotanshinone 1.12mg, Tanshinone I 2.00mg, ginsenoside Rg1 22.66mg, ginsenoside Rb1 19.87mg); Tanshinone: salvianolic acid B: danshensu: protocatechualdehyde: the weight ratio of arasaponin R1 is 6: 109: 16: 0.27: 12.Add adjuvant (13% dextrin, 24% Icing Sugar, 22% simple syrup, 4% starch, 0.5% magnesium stearate) and make granule 65Kg, prepare tabletting with common process, tablet.Every heavy 0.3g of this tablet, every contains tanshinone 0.675mg, salvianolic acid B 12.34mg, danshensu 1.81mg, arasaponin R1 1.38mg, cryptotanshinone 0.69mg, dihydrotanshinone 0.28mg, Tanshinone I 0.51mg, protocatechualdehyde 0.03mg, ginsenoside Rg1 5.76mg, ginsenoside Rb1 5.05mg.Oral dose: each 3, every day 3 times.
Embodiment 2: get red rooted salvia (place of production: Shandong, lot number 0501001) 150Kg, pseudo-ginseng (place of production: Yunnan, lot number 0412041) 45Kg, red rooted salvia is broken to be segment, uses soak with ethanol 2 hours, hot reflux 0.5 hour, filter, collect ethanol filtrate, reclaim ethanol and concentrate extractum I; Medicinal residues filter with 80% alcohol heat reflux 0.5 hour, collect 80% ethanol filtrate, reclaim ethanol and concentrate extractum II; Refluxed 1.5 hours with hydro-thermal at last, filtrate concentrate extractum III.Radix Notoginseng powder is broken into fine powder more than 80 orders, with Radix Salviae Miltiorrhizae extractum I, II and III mixing, dry 50h about 80 ℃.The 72Kg that gets dry extract altogether, moisture≤5%.
Measure with above-mentioned high performance liquid chromatography method, contain tanshinone 2.98mg in the dried cream of every 1g, salvianolic acid B 45.79mg, danshensu 7.44mg, arasaponin R1 5.27mg, cryptotanshinone 3.02mg, dihydrotanshinone 1.30mg, Tanshinone I 2.29mg, protocatechualdehyde 0.13mg, ginsenoside Rg1 23.81mg, ginsenoside Rb1 20.78mg; Tanshinone: salvianolic acid B: danshensu: protocatechualdehyde: the weight ratio of arasaponin R1 is 6.6: 102: 17: 0.29: 12.
Embodiment 3: get red rooted salvia 100Kg (place of production: Shandong, lot number 0501021), pseudo-ginseng (place of production: Yunnan, lot number 0501001) 30Kg, red rooted salvia is broken to be segment, soak with ethanol 1 hour, hot reflux 1 hour, filter, collect ethanol filtrate, reclaim ethanol and concentrate extractum I; Medicinal residues filter with 80% alcohol heat reflux 0.5 hour, collect 80% ethanol filtrate, reclaim ethanol and concentrate extractum II; Refluxed 2 hours with hydro-thermal at last, filtrate concentrate extractum III.Radix Notoginseng powder is broken into fine powder more than 80 orders, with Radix Salviae Miltiorrhizae extractum I, II and III mixing, dry 70h about 75 ℃, the 50.66Kg that gets dry extract altogether, moisture≤5%.
Measure with above-mentioned high performance liquid chromatography method, contain tanshinone 2.56mg in the dried cream of every 1g, salvianolic acid B 50.03mg, danshensu 7.34mg, arasaponin R1 6.15mg, cryptotanshinone 2.60mg, dihydrotanshinone 1.04mg, Tanshinone I 1.90mg, protocatechualdehyde 0.16mg, ginsenoside Rg1 24.25mg, ginsenoside Rb1 19.99mg; Tanshinone: salvianolic acid B: danshensu: protocatechualdehyde: the weight ratio of arasaponin R1 is 5.7: 112: 16: 0.36: 14.
Embodiment 4: get red rooted salvia 20Kg (place of production: Shandong, lot number 0502002), pseudo-ginseng (place of production: Yunnan, lot number 0502001) 6Kg, red rooted salvia is broken to be segment, soak with ethanol 1.5 hours, hot reflux 0.5 hour, filter, collect ethanol filtrate, reclaim ethanol and concentrate extractum I; Medicinal residues filter with 80% alcohol heat reflux 0.5 hour, collect 80% ethanol filtrate, reclaim ethanol and concentrate extractum II; Refluxed 1.5 hours with hydro-thermal at last, filtrate concentrate extractum III.Radix Notoginseng powder is broken into fine powder more than 80 orders, with Radix Salviae Miltiorrhizae extractum I, II and III mixing, dry 83h about 70 ℃, the 9.70Kg that gets dry extract altogether, moisture≤5%.
Measure with above-mentioned high performance liquid chromatography method, contain tanshinone 2.75mg in the dried cream of every 1g, salvianolic acid B 50.46mg, danshensu 7.00mg, protocatechualdehyde 0.18mg, arasaponin R1 5.55mg; Tanshinone: salvianolic acid B: danshensu: protocatechualdehyde: the weight ratio of arasaponin R1 is 6: 113: 16: 0.4: 12.
The preparation of embodiment 5, preparation
Granule: get the dried cream 7.2Kg of embodiment 2, add adjuvant (Icing Sugar) and make granule 20.2Kg.This granule is measured every g with the HPLC method and is contained tanshinone 0.83mg, salvianolic acid B 19.72mg, danshensu 3.09mg, protocatechualdehyde 0.05mg, arasaponin R1 1.82mg.Oral dose: each 2g, every day 3 times.
Capsule: get the dried cream 7.2Kg of embodiment 2, add adjuvant (starch) and make granule 9.4Kg, encapsulated.This capsule particle is measured every g with the HPLC method and is contained tanshinone 1.78mg, salvianolic acid B 42.37mg, danshensu 6.64mg, arasaponin R1 3.91mg.Oral dose: each 1～2 capsule, every day 3 times.
Piller: get the dried cream 10Kg of embodiment 3, add adjuvant (starch) and make piller 12.7Kg (the heavy 0.3g of per 10 balls).Piller is measured 10 balls with the HPLC method and is contained tanshinone 0.70mg, salvianolic acid B 13.79mg, danshensu 1.93mg, protocatechualdehyde 0.03mg, arasaponin R1 1.25mg.Oral dose: each 30 balls, every day 3 times.
The watered pill: get the dried cream 10Kg of embodiment 3, add adjuvant (starch) and make watered pill 12.0Kg.Per 10 balls of this watered pill contain tanshinone 0.68mg, salvianolic acid B 13.77mg, danshensu 1.97mg, protocatechualdehyde 0.03mg, arasaponin R1 1.32mg.Oral dose: each 30 balls, every day 3 times.
Small honey pill: get the dried cream 10Kg of embodiment 3, add adjuvant (Mel, starch) and make small honey pill 13Kg.Per 10 balls of this small honey pill contain tanshinone 0.71mg, salvianolic acid B 14.09mg, danshensu 1.93mg, protocatechualdehyde 0.03mg, arasaponin R1 1.18mg.Oral dose: each 30 balls, every day 3 times.
Big honeyed pills: get the dried cream 10Kg of embodiment 3, add adjuvant (Mel) and make big honeyed pills 12.2Kg.This every ball contains tanshinone 2.09mg, salvianolic acid B 42.52mg, danshensu 5.83mg, protocatechualdehyde 0.10mg, arasaponin R1 3.75mg.Oral dose: each 1 ball, every day 3 times.
Below with the effect of in vivo test checking product of the present invention to the treatment senile dementia:
1 experiment material
Laboratory animal: the SD rat, body weight 300-380g, male and female are regardless of, the SPF level.
Medicine and reagent: get content lower limit group and upper limit group that the product of embodiment 2, commercially available FUFANG DANSHEN PIAN, effective site are pressed the preparation of composition content.Pulverize before the above drug study, cross 100 mesh sieves, be dissolved in the water recently distilled, be made into concentration and be 6% aqueous solution and be stored in 4 ℃ of refrigerators standby.D-galactose (packing of AMRSCO company), Ibotenic acid (IBO) (Sigma company).
Experimental apparatus: rat founds position finder, the dentistry drill carriage.
2 experiment contents
2.1 influence (experiment of Morris water maze behavioristics) to senile dementia animal spatial cognition obstacle
2.1.1 experimental technique
Adopt Ibotenic acid (IBO) to cause rat senile dementia model, build up model after, surviving animals is divided into 10 groups at random.Each organizes continuous irrigation stomach 2 months, carries out the experiment of Morris behavioristics, continuous 6 days.The experiment phase I: 1～5 day is the training stage, the experiment second stage: carried out behavioristics and test in the 6th day.
2.1.2 experimental result
Result data sees Table 2 with the SPSS10.0 software processes:
The influence that table 2 embodiment 2 red sage formulations improve senile dementia animal pattern spatial cognition obstacle (x ± s)
| Group | n | Dosage g/kg | To all quadrants cross over the number of times of original platform/time | |||
| R | P | L | O | |||
| Operative control group model group huperzine Fufang Danshen Pian compound danshen dripping pills active constituent content lower limit of the present invention group△ | 10 9 8 7 8 8 | - - 2.3×10 5 0.32 0.089 0.30 | 6.70±2.95 4.40±3.37 7.25±4.76 6.67±1.50* 7.18±1.88 7.03±1.25* | 9.25±2.98 5.27±2.00 8.45±2.64 8.11±4.89 6.78±2.45 8.22±3.48 | 9.31±2.75 6.47±2.09 6.83±3.87 8.66±3.40 7.21±3.40 8.88±2.85 | 5.52±2.31 3.55±2.10 4.83±3.37 5.50±2.93 6.99±2.37 6.72±3.17 |
| Active constituent content upper limit group of the present invention △Dosage group embodiment 2 sample high dose group in embodiment 2 sample low dose group embodiment 2 samples | 8 7 9 8 | 0.30 0.16 0.30 0.44 | 8.49±1.83 8.23±0.79** 7.56±1.39* 6.97±3.51 | 7.47±2.69 7.83±2.95 7.90±3.33 8.33±3.55 | 7.90±3.46 9.27±3.12 8.65±3.54 8.23±4.22 | 7.40±2.28 7.77±1.93 7.85±1.09 6.96±1.73 |
Annotate: compare * P＜0.05, * * P＜0.01 with model group;
△For medical active effective site of the present invention is pressed the preparation of composition content.
The result shows that embodiment 2 red sage formulations and FUFANG DANSHEN PIAN are to the spatial cognition obstacle effect of having clear improvement of AD rat.Embodiment 2 low, middle dosage groups, active constituent content lower limit group and the poor opposite sex of model group are remarkable, wherein middle dosage group and the poor different in nature highly significant of model group.High dose group, active constituent content upper limit group and model group be there was no significant difference relatively, illustrates that active constituent content gets final product within the specific limits, and the content that significantly improves them can not increase effectiveness.Radix Salviae Miltiorrhizae drop pill group and model group be there was no significant difference relatively.Embodiment 2 red sage formulations and FUFANG DANSHEN PIAN group are relatively, from embodiment 2 red sage formulations and model group comparative result and FUFANG DANSHEN PIAN and model group comparative result, embodiment 2 red sage formulations are more remarkable than FUFANG DANSHEN PIAN on the difference level, prove better efficacy.
2.2 influence to the improvement of senile dementia animal memory dysfunction
2.2.1 experimental technique
Rat is kept away the camera bellows experiment.Rat is divided into 10 groups at random.After the administration 2 months, place and keep away in the camera bellows camera-lucida, after rat enters camera bellows, shocked by electricity 1 second, take out.1 week back repeated experiments, the record rat enters time of camera bellows and number of times, number by camera-lucida, the results are shown in Table 3.
The influence that the red sage formulation of table 3 embodiment 2 improves senile dementia animal pattern memory dysfunction (x ± s)
| Group | n | Dosage g/kg | Experimental result | ||
| Incubation period/s | Enter number of times in 5 minutes | Enter darkroom Mus number | |||
| Operative control group model group huperzine A FUFANG DANSHEN PIAN FUFANG DANSHEN DIWAN | 10 9 9 9 8 | - - 2.3×10 5 0.32 0.089 | 233.3±125.7** 66.4±102.09 186.8±93.87 243.5±165.5* 177.5±106.3* | 0.70±0.95* 3.40±2.87 0.85±1.76* 0.42±0.79* 1.67±1.42 | 2 8 1 2 1 |
| Active constituent content lower limit group of the present invention △Active constituent content upper limit group of the present invention △Dosage group embodiment 2 sample high dose group in embodiment 2 sample low dose group embodiment 2 samples | 9 7 9 7 8 | 0.30 0.30 0.16 0.30 0.45 | 229.2±136.8* 184.1±100.6* 288.2±113.1** 246.6±104.8** 322.2±59.2** | 0.78±1.40* 1.45±1.66 0.48±0.98* 0.78±0.31** 0.87±0.88* | 3 2 2 3 3 |
Annotate: compare with model group, * P＜0.05, * * P＜0.01:
△For medical active effective site of the present invention is pressed the preparation of composition content.
The result shows that the red sage formulation that embodiment 2 provides is to the memory dysfunction effect of having clear improvement of AD rat.The embodiment 2 basic, normal, high dosage groups and the poor opposite sex of model group be (P＜0.05～0.01) significantly.Positive drug huperzine A, FUFANG DANSHEN PIAN also have certain effect.Embodiment 2 red sage formulations, FUFANG DANSHEN PIAN, active constituent content lower limit group and upper limit group and the poor opposite sex of model group are remarkable, and embodiment 2 basic, normal, high dosage groups on the difference level than FUFANG DANSHEN PIAN more remarkable (being highly significant), prove better efficacy.Active constituent content gets final product within the specific limits, and the content that significantly improves them can not increase effectiveness.
Claims (7)
1, a kind of pharmaceutical composition for the treatment of senile dementia, by the medical active position and and the oral formulations made of conventional pharmaceutic adjuvant process of Chinese medicine preparation routinely, it is characterized in that: contain tanshinone 1.79～9.01mg in the medical active position dry product of 1g, salvianolic acid B 35.87～225.23mg, danshensu 4.93～45.05mg, protocatechualdehyde 0.10～4.50mg, arasaponin R1 3.14～22.50mg; Tanshinone: salvianolic acid B: danshensu: protocatechualdehyde: the weight ratio of arasaponin R1 is 4～20: 80～500: 11～100: 0.2～10: 7～50.
2, pharmaceutical composition according to claim 1, it is characterized in that: contain in the dry product of 1g medical active position in the dry product of said 1g medical active position and contain tanshinone 2.83～9.01mg, salvianolic acid B 53.81～225.23mg, danshensu 7.85～45.05mg, protocatechualdehyde 0.16～4.5mg, arasaponin R1 3.14～22.50mg; Tanshinone: salvianolic acid B: danshensu: protocatechualdehyde: the weight ratio of arasaponin R1 is 6.3～20: 120～500: 17.5～100: 0.3～10: 7～50.
3, pharmaceutical composition according to claim 1 is characterized in that: also contain cryptotanshinone 1.52～9.00mg, dihydrotanshinone 0.63～4.50mg, Tanshinone I 1.35～9.00mg, ginsenoside Rg1 18.83～135.14mg, ginsenoside Rb1 15.70～112.61mg in the dry product of said 1g medical active position.
4, pharmaceutical composition according to claim 3 is characterized in that: also contain cryptotanshinone 2.42～9.00mg, dihydrotanshinone 0.99～4.50mg, Tanshinone I 2.11～9.00mg in the dry product of said 1g medical active position.
5, pharmaceutical composition according to claim 1 is characterized in that: said medical active position is that getting red rooted salvia broken is segment, soak with ethanol 0.5～2 hour, hot reflux 0.5～1 hour, filter, collect ethanol filtrate, reclaim ethanol and concentrate extractum I; Medicinal residues filter with 80% alcohol heat reflux 0.5～1 hour, collect 80% ethanol filtrate, reclaim ethanol and concentrate extractum II; Medicinal residues refluxed 1～2 hour with water again, filtrate concentrate extractum III, Radix Notoginseng powder is broken into fine powder more than 80 orders, with Radix Salviae Miltiorrhizae extractum I, II and III, mix thoroughly, 70～90 ℃ dry 36～83 hours down, the medical active position dry product of gained, moisture≤5%.
6, pharmaceutical composition according to claim 1 is characterized in that: said oral formulations is tablet, capsule, pill, granule.
7, pharmaceutical composition according to claim 6 is characterized in that: said pill is the watered pill, small honey pill, big honeyed pills, concentrated pill, drop pill.
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