CN101596232A - The purposes of the Herba Leonuri extract of purification in the nerve protection medicine of preparation brain injury - Google Patents
The purposes of the Herba Leonuri extract of purification in the nerve protection medicine of preparation brain injury Download PDFInfo
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Abstract
The invention belongs to pharmaceutical field; relate to the new purposes of Chinese medicine Herba Leonuri extract in pharmacy; be specifically related to the purposes of Herba Leonuri extract in the brain-protection drugs of preparation treatment middle cerebral artery occlusion of purposes, the especially purification of Herba Leonuri extract in the nerve protection medicine of preparation brain injury of purification.The Herba Leonuri extract of described purification is through zoopery, the result shows, after using the Herba Leonuri extract of purification, the cerebral infarction volume significantly descends, and compares with matched group, and the delayed ischemic neurological deficits scoring reduces, the total antioxidation agent concentration increases in the blood plasma, the DNA oxidative damage reduces, and reduces apoptosis and pro apoptotic protein expression, increases anti-apoptotic proteins content simultaneously.The Herba Leonuri extract of described purification can prepare the nerve protection medicine of brain injury, can effectively treat apoplexy.
Description
Technical field
The invention belongs to pharmaceutical field; relate to the new purposes of Chinese medicine Herba Leonuri extract in pharmacy; be specifically related to the purposes of Herba Leonuri extract in the brain-protection drugs of preparation treatment middle cerebral artery occlusion of purposes, the especially purification of Herba Leonuri extract HL (pHL) in the nerve protection medicine of preparation brain injury of purification.
Background technology
Prior art cerebral infarction is disclosed because the minimizing of blood flow or fully blocking-up cause local blood glucose and oxygen supply is not enough causes.The most effective Therapeutic Method is a thrombolytic therapy at present, yet has only the part patients with cerebral apoplexy can accept this therapy.Therefore, exploitation treatment window width, can prevent multiple neuro chemistry reaction cause the irreversibility brain injury nerve protection medicine become the task of top priority.
Under the normal physiological state, endogenous antioxidant regulation and control free radical level.Oxidative stress will appear when antioxidant protection ability and free radical level are unbalance.Studies have shown that in a large number the pathomechanism of neurodegenerative diseases relates to oxidative stress.During cerebral ischemia, oxyradical can destroy the 26S Proteasome Structure and Function integrity of cell by number of ways, causes serious cell injury even causes cell death or apoptosis.As everyone knows, cerebral tissue oxygen consumption height, antioxidant level is low, and regeneration power is poor, and is especially responsive to oxidative damage.
In recent years, natural product has been received widely the therapeutical effect of neurodegenerative diseases and has been paid close attention to.Especially relevant have the endogenous of an enhancing antioxidant activity, reduces free radical and generate, by in the non-enzyme means and the natural product of free radical effect.There is report to disclose Folium Ginkgo and has regulation and control ischemic region apoptosis cascade reaction, the effect of treatment persistence middle cerebral artery occlusion.
According to traditional Chinese medical science theory, Herba Leonuri all has good effect for conditioning menstruation, gynecological's childbirth and blood circulation promoting and blood stasis dispelling.There is research report Herba Leonuri to have myocardium protecting action, is used for the treatment of high blood viscosity and myocardial infarction.The most important thing is that report HL such as Liu have antioxidant activity.In the test,, find that HL has powerful superoxides and removes ability in vivo with electron spin resonance (ESR) spin trapping technology for detection.
By scientific methods purification HL crude drug, mainly contain stachydrine (C
7H
13NO
2), Quercetin (C
15H
10O
7), nimbecetin (C
15H
10O
6), leonurine (C
14H
21N
3O
5) and 5,7,4 '-three hydroxyl (base) flavone (C
15H
10O
5).The Herba Leonuri extract HL (pHL) that prior art has been reported purification has antioxidant activity, and by antioxidation protection ischemic myocardium.Now, pHL goes on the market (Kardigen, heart treasure lives), is acknowledged as to have the protection heart, reduces the supplement of risk of cardiovascular diseases.But up to now, as yet the HL (pHL) of not relevant purification can prevent multiple neuro chemistry reaction cause the irreversibility brain injury the report of nerve protection medicine.
Prior art related to the present invention has:
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Summary of the invention
The purpose of this invention is to provide the new purposes of Chinese medicine Herba Leonuri extract in pharmacy; the purposes of the Herba Leonuri extract HL (pHL) that is specifically related to purification in the nerve protection medicine of preparation brain injury especially treated the purposes in the brain-protection drugs of middle cerebral artery occlusion.
The Herba Leonuri of purification of the present invention (pHL) prepares by following method,
The Herba Leonuri crude drug originates from Chinese Sichuan Province, and the Herba Leonuri of purification is in Singapore's listing (pHL) (trade name ' Kardigen ' " heart treasure lives ", the peculiar limit of Singapore He Bo company).The pHL powder is available from the peculiar limit of Singapore He Bo company.Mainly contain following chemical constitution in the described extract: stachydrine (C
7H
13NO
2), Quercetin (C
15H
10O
7), nimbecetin (C
15H
10O
6), leonurine (C
14H
21N
3O
5) and 5,7,4 '-three hydroxyl (base) flavone (C
15H
10O
5).
Among the present invention, also press preparation technology (the Sun et al of the disclosed pHL of prior art, 2005) preparation, at first carry out the extraction of Herba Leonuri crude drug, decocting in water obtained water extract after 45 minutes, water extract concentrate under 50 ℃ in vacuum (BUCHI revolves and steams instrument R-144, water-bath B-480), 80 ℃ concentrate spend the night after, again liquid is made freeze-dried powder.(API 365LC-MS system, Applied Biosystems USA) analyze extract with liquid chromatograph-ionization-mass spectrometry LC-ESI-MS.
The present invention adopts the Herba Leonuri (pHL) of purification that the apoplexy of testing Wistar rat persistence middle cerebral artery occlusion (MCAO) has been carried out therapeutic test, before rat MCAO operation, and pre-two weeks, postoperative continues administration 7 days.Primary study of the present invention antioxidation and the apoptosis regulation effect of pHL, detected rat cerebral infarction volume and delayed ischemic neurological deficits, total antioxidation agent concentration and DNA oxidative damage; In addition, also carried out the immunohistochemical staining analysis of TUNEL detection and apoptosis of many kinds associated protein.The result shows: the experimental rat post-stroke is after using pHL, and the cerebral infarction volume significantly descends, and is decreased to 15.19 ± 0.02% (p＜0.05) by 20.75 ± 0.03%.Compare with matched group, the delayed ischemic neurological deficits scoring is decreased to 1.82 by 2.43.And after the pHL treatment, the total antioxidation agent concentration increases to 0.42 ± 0.05mM (p＜0.05) from 0.31 ± 0.03mM in the blood plasma, and the DNA oxidative damage is reduced to 1.19 ± 0.03 (p＜0.05) from 1.78 ± 0.03.In addition, pHL reduces apoptosis and pro apoptotic protein expression, increases anti-apoptotic proteins content simultaneously.Experimental result confirms that the Herba Leonuri of purification of the present invention (pHL) can prepare the nerve protection medicine of brain injury, especially the brain-protection drugs of preparation treatment middle cerebral artery occlusion.
Description of drawings
Fig. 1 is an Infarction volume of respectively organizing rat,
Wherein, a) infarcted region after the TTC dyeing; B) image analysis system is analyzed Infarction volume (Scionimage for windows), compares with the apoplexy model group, and pHL treatment back Infarction volume significantly reduces * p＜0.05 apoplexy pHL treatment group vs. apoplexy model group.
Fig. 2 is the delayed ischemic neurological deficits scoring,
Wherein, it is high more to mark, and dyskinesia is serious more, and apoplexy model group rat delayed ischemic neurological deficits score is the highest, and the 7th day after surgery neural dysfunction score of pHL treatment apoplexy rat significantly descends.
Fig. 3 is the influence of pHL to antioxidant activity,
Wherein, a: each organizes total antioxidation agent concentration (mM) in the blood plasma, and the total antioxidation agent concentration of sham-operation and apoplexy group all increases; * p＜0.05 apoplexy model group vs. sham operated rats; ##p＜0.01 apoplexy pHL treatment group vs. apoplexy model group;
B:DNA oxidative damage level, the pHL treatment significantly alleviates post-stroke DNA oxidative damage, but does not change the basic oxidative damage level under the health status; * p＜0.01 apoplexy model group vs. sham operated rats; ##p＜0.01 apoplexy pHL treatment group vs. apoplexy model group.
Fig. 4 is MCAO cerebral cortex apoptosis dyeing (20x object lens) after 7 days,
Wherein, section is observed under wavelength 520 ± 20nm to detect green fluorescence; Under wavelength 460nm, observe and detect DAPI dyeing; Image integration is to detect the configuration of cell; A) sham operated rats; B) sham-operation pHL treatment group; C) apoplexy model group; D) apoplexy pHL treatment group.
Fig. 5 is MCAO cerebral cortex immunohistochemical staining (40x object lens) after 7 days,
Wherein, short apoptosis protein (b) BAX and (c) FAS, anti-apoptotic proteins (d) BCL-2 and (e) BCL-XL, (a) negative control;
Coloration result shows, post-stroke, and the immunoreation of short apoptosis protein strengthens, and the immunoreation of anti-apoptotic proteins weakens; After the HL treatment, the immunoreation of anti-apoptotic proteins is strengthened, and the immunoreation of short apoptosis protein weakens; From left side i) sham operated rats; Ii) sham-operation pHL treatment group; Iii) apoplexy model group; Iv) apoplexy pHL treatment group.
Specific embodiments
Testing program of the present invention authenticates by ethics association, meets international ethical standard.Laboratory animal (available from NUS's Experimental Animal Center).
The Wistar rat of 84 heavy 220g～250g is being raised in cages under the light conditions in the daytime, gives competent food and water.Rat is divided into four groups at random: sham operated rats [Sham], sham-operation pHL treatment group (400mg/kg/day) [Sham+pHL], apoplexy model group [Vehicle], apoplexy pHL treatment group (400mg/kg/day) [Sroke+pHL].Every day gastric infusion.The pre-administration of operation the last fortnight, inaccessible intraluminal middle cerebral artery occlusion in rats (MCAO) causes apoplexy.At first use ketamine/xylazine mixture anesthetized rat (0.1ml/100gi.p.), isolate middle cerebral artery, electricity irons blocking-up and locates this section middle cerebral artery branch with inferior cerebral vein infall to close lentiform nucleus striatum branch source, guarantees complete permanent occlusion.Sew up wound is kept rat temperature at 37 ± 0.5 ℃ with electric blanket in the whole surgery process.The sham operated rats rat is given same operation, but does not cause MCAO.Postoperative continues one week of administration, puts to death and gathers sample.
The cerebral infarction cubing
TTC (2,3,5-triphenyltetrazolium chloride) dyeing is adopted in the measurement of cerebral infarction volume.After cerebral tissue is removed, remove cerebellum and brain mesentery.Cerebral tissue is cut into 8 thin slices that 2mm is thick, is dipped in 4% formalin solution.The cerebral infarction volume is converted into the true ischemic injuries infarcted region in the full brain hemisphere with image analysis system analysis (Scion image forwindows) after edema and atrophy rectification.
The result shows, each Infarction volume of organizing rat as shown in Figure 1.The same as expection, sham operated rats and sham-operation pHL treatment group do not have brain injury, thus do not have infarcted region (Figure 2ai, ii).And the middle cerebral artery occlusion operation causes the rat ischemia damage, and infarcted region (Figure 2aiii) all appears in left cerebral cortex and striatum.After the apoplexy pHL treatment, Infarction volume significantly is decreased to 15.19 ± 0.02% (Fig. 1 b) by 20.75 ± 0.03%.
The evaluation of delayed ischemic neurological deficits
Adopt delayed ischemic neurological deficits scoring system scoring (Lu et al, 2005), the neurologic impairment after the damage of assessment apoplexy, the scoring scope is from 0-5, is used for estimating behavior and the motion change of animal behind the MCAO.Fixedly rat tails makes its suspension, if rat behavior is normal, all stretches to ground as forelimb, then is chosen as 0 fen.If the forelimb of rat MCAO offside remains flexing in the suspension process, there are not other Deviant Behavioies in addition, then be chosen as 1 fen.Next, rat is placed on the ground, allows them act on one's own.Rat moves from being sent to all directions, but when gently drawing the Mus tail, a side is unidirectional turn-takes along paralysing, and then is chosen as 2 fens.Continue the spontaneous rat that turn-takes along the MCAO offside and be cited as 3 fens.And those physical weakness, only the rat that just moves under stimulating is cited as 4 fens.Those rats in scoring death on the same day are cited as 5 fens.The rat that score is high shows all symptoms of the low rat of score.Therefore, score is high more, and the rat delayed ischemic neurological deficits is serious more.
The result is as shown in Figure 2: sham operated rats and sham-operation pHL treatment group rat do not have delayed ischemic neurological deficits, and scoring is 0 minute; Postoperative apoplexy model group rat score is the highest, and the Infarction volume that shows maximum with them is consistent; Apoplexy pHL treatment group, first day neural dysfunction gets proportion by subtraction model group low (2.22vs.2.43) behind the MCAO, and along with further treatment, the delayed ischemic neurological deficits score further descends, and postoperative the 7th day is reduced to 1.81 (Fig. 2) by 2.22.
The total antioxidation agent content is analyzed
Gather plasma sample and be used to analyze the influence of pHL the endogenous antioxidant system.PHL to the influence of endogenous antioxidant system with total antioxidation agent detection kit analyze (615700, Calbiochem, Germany).Operate according to the test kit description.The principle of test kit is that antioxidant suppresses MetMb Ferrimyoglobins. oxidation ABTS
TMSubstrate is ABTS
TM+Product.Therefore the content and the detected ABTS of antioxidant in the sample
TM+Content is inversely proportional to.ABTS
TM+Content can detect by the absorbance that detects wavelength 600nm.
The result shows, the sham operated rats antioxidant concentration be 0.38 ± 0.08mM (Fig. 3 is a). the antioxidant concentration of sham-operation pHL treatment group increases to 0.5 ± 0.2mM, and (Fig. 3 a).Apoplexy model group, antioxidant concentration significantly are reduced to 0.31 ± 0.03mM, and (Fig. 3 a).Apoplexy pHL treatment group antioxidant concentration returns to 0.42 ± 0.05mM, even (Fig. 3 a) also to omit height than sham operated rats.
GC/MS analyzing DNA oxidative damage
Adopt benzene phenol-chloroform method extracting DNA from the blood sample of each treated animal, DNA oxidation adduct can be considered as the sign of oxidative stress.Adopt GC/MS (Agilent 6890 gas chromatograies and Agilent MSD5973) methods analyst dna sample.The meansigma methods of dna adduct is calculated in the summation of DNA oxidation adduct amount in each sample.
The result is shown in Fig. 3 b, and sham operated rats is almost consistent with the DNA oxidative damage level of sham-operation pHL treatment group, is respectively 0.98 ± 0.05 and 1.08 ± 0.06, and visible pHL treatment can not change basic DNA oxidative damage level.The DNA oxidative damage the most serious (1.78 ± 0.03) of apoplexy model group is shown in Fig. 3 b.After the pHL treatment, the DNA oxidative damage level that apoplexy causes significantly drops to 1.19 ± 0.03 (Fig. 3 b) and sham operated rats basically identicals.
The TUNEL detection method
Treatment is final, with fixing animal tissue, gets cerebral tissue with 2% formalin perfusion, continues to fix two hours with 2% formalin.15% sucrose phosphate buffer soaked overnight, 30% sucrose phosphate buffer soaks dehydration.With
(Leica microscopic system, IL USA) make cerebral tissue the thin slice of thick 20 μ m to the CM1510 freezing microtome.Use DeadEnd
TMFluorescence TUNEL system (G3250, Promega, USA) the nuclear dna break that causes of kit detection cell apoptosis, and under fluorescence microscope, observe.Section is used
Fluorescence microscope also takes pictures that (Leica microscopic system, IL USA), observe the green fluorescence of wavelength 520 ± 20nm and the blue-fluorescence of wavelength 460nm.
TUNEL dyeing can be indicated apoptotic cell.After the TUNEL dyeing, apoptotic nucleus sends very strong green fluorescence.The result shows, only detects apoptosis in left cerebral cortex infarcted region, and (Fig. 4 a) does not all detect apoptotic cell with sham-operation pHL treatment group rat (Fig. 4 b) non-infarcted region, sham operated rats rat.Apoplexy model group apoptosis labelling is (Fig. 4 c) at most.With the Infarction volume maximum of this group, the most serious unanimity of nerve injury.Apoplexy pHL treatment group infarcted region apoptosis labelling significantly reduces (Fig. 4 d).
Immunohistochemical staining
Above-mentioned frozen tissue is used for immunohistochemical staining (IHC).With the anti-Bax antibody of multi-clone rabbit (sc-493, Santa Cruz, CA, USA), the multi-clone rabbit anti-Fas antibody (sc-715, Santa Cruz, CA, USA) and the anti-Bcl-xS/xL antibody of multi-clone rabbit (CA USA) carries out anti-hatching for sc-634, Santa Cruz.(Lab Vision Corporation, CA USA) carries out antibody staining with Ultravist detection system and HRP/DAB test kit.Use haematoxylin redyeing.Section exists
Observe under the fluorescence microscope and take pictures.
Described immunohistochemical staining is used for the cell death related protein location, and negative control is used to guarantee two anti-specificitys, and (Fig. 5 a).The result shows that pro apoptotic protein BAX (Fig. 5 b) is not found in sham operated rats and the dyeing of sham-operation pHL treatment group; Otherwise, apoplexy model group BAX signal the strongest (Fig. 5 b); Apoplexy pHL treatment group BAX immunoreation is weak (Fig. 5 b), only in infarcted region the BAX positive staining is arranged; FAS coloration result similar to BAX (Fig. 5 c); Behind the MCAO, short apoptosis protein BAX and FAS significantly increase in the apoplexy model group infarcted region, and these short apoptosis proteins only are detected in infarcted region, confirm that ischemia can cause these protein expressions to raise, and are lower than detection level at these expressing quantities of non-infarcted region;
In sham operated rats and sham-operation pHL treatment group the strongest anti-apoptotic proteins BCL-2 and BCL-XL (Fig. 5 d, e) positive signal are arranged; Apoplexy model group BCL-2 positive staining very a little less than, the BCL-XL signal does not then detect (Fig. 5); The pHL treatment makes the positive expression of post-stroke BCL-2 and BCL-XL return to normal level.
Experimental result of the present invention adopts meansigma methods ± S.E.M to represent; Two tail student t-check analysis group differences when p＜0.05, think that group difference is remarkable.
Experimental result has confirmed that pHL has neuroprotective, especially the neuroprotective behind the cerebral infarction.Among the present invention, pHL can reduce Infarction volume, improves function of nervous system behind the MCAO.In addition, it also strengthens the endogenous oxidation resistance, thereby can resist ischemic injuries, reduces DNA oxidative damage and apoptosis.MCAO is the local cerebral ischemia model of extensive use, but the brain injury similar (Miller, 1999) that the infarction that MCAO causes and people's large tracts of land lethal ischemic stroke cause.Left middle cerebral artery is cerebral cortex and striatum blood supply left, and the damage in this zone can cause motion, language, function of deglutition obstacle.Among the present invention, the TTC coloration result has confirmed that left MCAO can cause left cerebral cortex and striatum infarcted region to occur.In addition, the delayed ischemic neurological deficits scoring system evaluation of being adopted shows that left MCAO can cause serious dyskinesia, and after the pHL treatment, Infarction volume significantly reduces, and this result shows with the apoplexy model group and compare that the pHL treatment reduces brain tissue impairment.Therefore, pHL treatment back neurological disorders also alleviates accordingly.
The total antioxidation agent concentration significantly descended after result of the present invention showed MCAO.The result of report such as this result and Antonio (2000) is consistent.After the present invention adopted the pHL treatment, under health and the apoplexy state, plasma antioxidants concentration was significantly increased, and shows whether no matter oxidative stress take place, and pHL has the ability that increases the endogenous antioxidant, thereby formed the protection barrier of antagonism apoplexy.
Result of the present invention shows that the DNA oxidative damage increases behind the MCAO, has proved the enhancing of free radical vigor.Relative, the DNA oxidative damage that apoplexy causes descends the endogenous antioxidant activity.PHL treatment apoplexy rat, not only the endogenous antioxidant concentration improves, and the DNA oxidative damage also obviously alleviates.Therefore confirm that it is closely related that pHL tests the therapeutical effect and its antioxidation of demonstration in vivo, promptly strengthens the endogenous anti-oxidant vigor, alleviates oxidative stress.
TUNEL testing result of the present invention shows that green fluorescence only is detected in infarcted region, shows that apoptosis only occurs in affected area.In addition, apoplexy model group rat demonstrates the strongest nuclear green fluorescence, and is consistent with other testing results.PHL treats post-stroke, and the nuclear green fluorescence reduces, and the result who reduces Infarction volume, nervous function damage and DNA oxidative damage with it is consistent, and the TUNEL testing result has further confirmed the therapeutical effect of pHL to middle cerebral artery occlusion wistar rat.
Immunohistochemical staining result of the present invention show pro apoptotic protein BAX and FAS only behind MCAO infarcted region detect, and in healthy individual, detect less than.The apoplexy model group has the highest pro apoptotic protein to express, and is consistent with the strongest nuclear green fluorescence result.Proved that the infarcted region pro apoptotic protein raises, and causes serious cell death and morphology significant change.On the contrary, no matter be sham operated rats or sham-operation pHL treatment group, anti-apoptotic proteins BCL-XL and BCL-2 can detect at whole brain.It is weak that apoplexy model group anti-apoptotic proteins signal is wanted.In apoplexy administration group, the pro apoptotic protein immunoreation significantly reduces and the signal of anti-apoptotic proteins is significantly strengthened.In sum, have the pHL of antioxidant activity, can the anti-apoptotic state of transformant, have the regulating cell effect of apoptosis.
Results suggest of the present invention medication learn to do the effective ways that the damage of shed repair reoxygenation provides apoplexy treatment.
Claims (5)
1, the purposes of the Herba Leonuri extract of purification in the nerve protection medicine of preparation brain injury.
2, the purposes of the Herba Leonuri extract of purification in the brain-protection drugs of preparation treatment middle cerebral artery occlusion.
3, by claim 1 or 2 described purposes, it is characterized in that the Herba Leonuri extract of described purification contains stachydrine C
7H
13NO
2, Quercetin C
15H
10O
7, nimbecetin C
15H
10O
6, leonurine C
14H
21N
3O
5With 5,7,4 '-three hydroxyls (base) flavone C
15H
10O
5
4, a kind of nerve protection medicine of brain injury is characterized in that containing the Herba Leonuri extract of the described purification of claim 3 of effective therapeutic dose.
5, a kind of brain-protection drugs for the treatment of middle cerebral artery occlusion is characterized in that containing the Herba Leonuri extract of the described purification of claim 3 of effective therapeutic dose.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105997975A (en) * | 2016-05-23 | 2016-10-12 | 南开大学 | Application of leonurine to preparation of medicine for treating vascular dementia |
| WO2018072689A1 (en) * | 2016-10-19 | 2018-04-26 | 青岛海洋生物医药研究院股份有限公司 | Stachydrine derivative, preparation method therefor, and applications thereof in preparation of drugs for treating cardiovascular and cerebrovascular diseases |
| CN109350615A (en) * | 2018-08-17 | 2019-02-19 | 上海市浦东新区浦南医院 | Stachydrine hydrochloride is in the new application for preventing and treating ischemic cerebrovascular disease |
-
2009
- 2009-07-03 CN CNA2009100544175A patent/CN101596232A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105997975A (en) * | 2016-05-23 | 2016-10-12 | 南开大学 | Application of leonurine to preparation of medicine for treating vascular dementia |
| WO2018072689A1 (en) * | 2016-10-19 | 2018-04-26 | 青岛海洋生物医药研究院股份有限公司 | Stachydrine derivative, preparation method therefor, and applications thereof in preparation of drugs for treating cardiovascular and cerebrovascular diseases |
| CN107963987A (en) * | 2016-10-19 | 2018-04-27 | 青岛海洋生物医药研究院股份有限公司 | A kind of wood alkali derivant and preparation method thereof and the application in the medicine for preparing treatment cardio-cerebralvascular diseases |
| CN109350615A (en) * | 2018-08-17 | 2019-02-19 | 上海市浦东新区浦南医院 | Stachydrine hydrochloride is in the new application for preventing and treating ischemic cerebrovascular disease |
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Application publication date: 20091209 |