CN107929280A - Application of the andrographolide in the medicine for preparing treatment depression - Google Patents

Abstract

The present invention relates to pharmaceutical technology field, is specifically application of the andrographolide in the medicine for preparing treatment depression.The present invention is studied using a variety of animal models of depression with test method, it was demonstrated that the intraperitoneal injection of andrographolide, which is given, can substantially reverse many behavior depressions caused by depression model, and efficacy strength is not weaker than classical clinical antidepressants Prozac；Therefore find a kind of brand-new pharmacological effect of andrographolide：Effective remissive treatment depression.

Description

Application of the andrographolide in the medicine for preparing treatment depression

Technical field

It is andrographolide in the medicine for preparing treatment depression specifically the present invention relates to pharmaceutical technology field Application.

Background technology

Depression is common disturbance of emotion type mental illness, its incidence can be in any more always；The mental illness is not only Personal work and study and quality of life are influenced, also because of its higher introgression, drastically influence the stabilization of family and society； It is mainly shown as (the bibliography such as depressed, interest attenuating, pessimistic, decrease of memory, shortage initiative：[1].Bowden, C.L.,2005.Treatment options for bipolar depression.J.Clin.Psychiatry 66,3-6. [2].Krishnan,V.,Nestler,E.J.,2008.The molecular neurobiology of depression.Nature 455,894-902.[3].Shelton,R.C.,2007.The molecular neurobiology of depression.Psychiatr.Clin.North Am.30,1-11.).Current clinical antidepressants Medicine is mostly chemical synthetic drug, mainly including tricyclic antidepressant, tetracyclic antidepressants, the resistance of selective serotonin reuptake Stagnant dose and monoamine oxidase inhibitors；Although species is many, effective percentage is deposited often less than 70%, and side effect is many In the possibility [4,5] of recurrence.Therefore, preferable antidepressant is found and developed from natural drug and Chinese medicinal formulae, more and more Cause the concern of researcher.

For the pathogenesis of depression, last century be thought that be maincenter monoamine energy mediator (such as serotonin) system work( The generation of energy defect mediation depression, but clinically drug effect is all there are for a series of antidepressants of this system development The shortcomings of cycle is long, cure rate is not high, recurrent exerbation (bibliography：[4].Berton,O.,Nestler,E.J.,2006.New approaches to antidepressant drug discovery:beyond monoamines.Nat.Rev.Neurosci.7,137-151.[5].Hirschfeld,R.M.,2012.The epidemiology of depression and the evolution of treatment.J.Clin.Psychiatry73,5-9.).Academia proposes new viewpoint in 21 century, i.e. neurotrophy is false Say：Think that depression and maincenter brain-derived neurotrophic factor (BDNF) are closely related.BDNF has widely distributed, regulation and control in intracerebral The internal important physiology course such as nerve growth development, synaptic plasticity.Known BDNF can be with its receptor tyrosine kinase B (TrkB) With reference to passing through downstream mitogen-activated protein kinase (MAPK)/extracellular regulated kinases (ERK), phosphatidylinositol-3-kinase (PI-3K)/protein kinase B (AKT) and Ca2+/cam kinase (CaM) several signal paths activate intranuclear cycle phosphoric acid Adenosine response element binding protein (CREB) (bibliography：[6].Qi X,Lin W,Wang D,et al.A role for the extracellular signal-regulated kinase signal pathway in depressive-like behavior.Behav Brain Res.2009,199:203-209.).More research report confirmations, depressive patient and depression Bdnf protein expression quantity is significantly lower than normal individual in the serum of animal model and in hippocampal neuron；Clinical antidepressants The means such as long-term use of and electro-shock therapy (ECT) can reverse these to change (bibliography：[7].Duman RS, Monteggia LM.A neurotrophic model for stress-related mood disorders.Biol Psychiatry.2006,59,1116-1127.[8].Molteni R,Calabrese F,Bedogni F,et al.Chronic treatment with fluoxetine up-regulates cellular BDNF mRNA expression in rat dopaminergic regions.Int J Neuropsychopharmacol.2006,9,307- 317.)。

Andrographolide (molecular formula C20H30O5, Andrographolide) is the main effective of natural plants Herba Andrographitis Composition, at home traditional Chinese medicine in the market be widely used, such as andrographolide tablet, dripping pills of andrographolide, andrographolide glue Capsule etc..The medical instrument has clearing heat and detoxicating, cool blood detumescence, antipyretic and anti-inflammatory, antibacterial and other effects, available for gastroenteritis, catches a cold, tracheitis, Pneumonia, pertussis, pulmonary tuberculosis, lung abscess, cholecystitis, hypertension, oropharynx swelling and pain, venomous snake bite etc..Recently studies have reported that claiming Andrographolide can promote the expression of intracellular BDNF to synthesize, and prompt it to possess antidepressant curative effect (bibliography： [9].Xu F,Wu H,Zhang K,et al.Pro-neurogenic effects of andrographolide on RSC96Schwan cells in vitro.Mol Med Repl.2016,14,3573-3580.)。

But the application on andrographolide in the medicine for preparing treatment depression yet there are no report.

The content of the invention

It is an object of the invention to provide the new medical usage of andrographolide.

The first aspect of the present invention, there is provided application of the andrographolide in the medicine for preparing treatment depression.

Preferably, the medicine of the treatment depression is either included using andrographolide as sole active composition The pharmaceutical composition of andrographolide.

Preferably, any formulation can be made with pharmaceutically common auxiliary material in the medicine or pharmaceutical composition.

The second aspect of the present invention, there is provided a kind of medicine for treating depression, can by andrographolide and pharmaceutically connect The auxiliary material composition received.

The beneficial effects of the present invention are：

The present invention is studied using a variety of animal models of depression with test method, it was demonstrated that the abdominal cavity note of andrographolide Penetrate and give (30,60mg/kg) and can substantially reverse many behavior depressions caused by depression model, and efficacy strength is not weaker than Classical clinical antidepressants Prozac；A kind of present invention discover that brand-new pharmacological effect of andrographolide：Effective remissive treatment Depression.

Brief description of the drawings

The structure diagram of Fig. 1 societies engaged test case.

Fig. 2 andrographolides are in forced swimming test (FST) with showing that notable antidepression is imitated in outstanding tail test (TST) Should.To normal C57BL/6J mouse, first solvent (physiological saline), Prozac (positive control, 20mg/ are given in intraperitoneal injection respectively Kg) or andrographolide (30,60mg/kg), the test of FST/TST/ autonomic activitieses is carried out after 30 minutes.(A) andrographolide Injection give the dead time for significantly reducing normal C57BL/6J mouse in FST tests, and effect is not weaker than Prozac. (B) andrographolide is significantly reduced dead time of the normal C57BL/6J mouse in TST tests, and effect is not weaker than fluorine west Spit of fland.(C) andrographolide does not influence the autonomic activities of mouse.Experimental data represents (every group 10 with average value ± standard error Only)；**P<0.01vs. solvent control groups.

Fig. 3 andrographolides unpredictable stress show potent antidepressant effect chronic in (CUMS) depression model.Make CUMS models are manufactured with C57BL/6J mouse, 8 weeks by a definite date, and carried out daily give in last 2 weeks and solvent (physiology salt is injected intraperitoneally Water), Prozac (positive control, 20mg/kg) or andrographolide (30,60mg/kg), carry out Behavior test afterwards.(A) wear The dead time for significantly reducing CUMS depression models mouse in FST tests is given in the injection of heart lotus lactone, and effect is not weaker than fluorine Xi Ting.(B) andrographolide substantially reduces dead time of the CUMS depression models mouse in TST tests, and effect is not weaker than fluorine Xi Ting.(C) the syrup preference rate of andrographolide while significantly raised CUMS depression models mouse, reverses its anhedonia behavior table It is existing.Experimental data is represented (every group 10) with average value ± standard error；*P<0.05, * * P<0.01vs. solvent control groups；^#P< 0.05,^##P<0.01vs.CUMS models+solvent group.

Fig. 4 andrographolides are defeated in chronic society to show potent antidepressant effect in (CSDS) depression model.Make CSDS models are manufactured with C57BL/6J mouse, 10 days by a definite date, then give intraperitoneal injection solvent (physiological saline), Prozac daily (positive control, 20mg/kg) or andrographolide (30,60mg/kg), continues 14, after carry out Behavior test.(A) The dead time for significantly reducing CSDS depression models mouse in FST tests is given in the injection of andrographolide, and effect is not weaker than Prozac.(B) andrographolide substantially reduces dead time of the CSDS depression models mouse in TST tests, and effect is not weaker than Prozac.(C) the social time of contact of the significantly raised CSDS depression models mouse of andrographolide, reverses its social phobia behavior table It is existing.(D) the syrup preference rate of andrographolide while significantly raised CSDS depression models mouse, reverses its anhedonia behavior table It is existing.Experimental data is represented (every group 10) with average value ± standard error；*P<0.05, * * P<0.01vs. solvent control groups；^#P< 0.05,^##P<0.01vs.CSDS models+solvent group.

The continuous injection of Fig. 5 andrographolides, which is given, significantly to reverse caused by CUMS depression models in hippocampal neuron BDNF signal paths level (BDNF, pTrkB, pCREB) reduces.Experimental data represents (every group 5 with average value ± standard error Only)；**P<0.01vs. solvent control groups；^##P<0.01vs.CUMS models+solvent group.

The continuous injection of Fig. 6 andrographolides, which is given, significantly to reverse caused by CSDS depression models in hippocampal neuron BDNF signal paths level (BDNF, pTrkB, pCREB) reduces.Experimental data represents (every group 5 with average value ± standard error Only)；**P<0.01vs. solvent control groups；^##P<0.01vs.CSDS models+solvent group.

The antidepressant effects that Fig. 7 andrographolides are showed in FST (A) and TST (B) tests are blocked by BDNF paths Agent K252a is substantially blocked.Experimental data is represented (every group 10) with average value ± standard error；**P<0.01vs. solvent controls Group.

The antidepressant effects that Fig. 8 andrographolides are showed in CUMS depression models are by BDNF path blocking agents K252a It is obvious to block.(A) forced swimming behavioral test data result.(B) tail behavioral test data result is hanged.(C) syrup consumes Behavioral test data result.Experimental data is represented (every group 10) with average value ± standard error；**P<0.01vs. solvents pair According to group；^##P<0.01vs.CUMS models+solvent group.

The antidepressant effects that Fig. 9 andrographolides are showed in CSDS depression models are by BDNF path blocking agents K252a It is obvious to block.(A) forced swimming behavioral test data result.(B) tail behavioral test data result is hanged.(C) society's contact Behavioral test data result.(D) syrup consumption behavioral test data result.Experimental data is with average value ± standard error table Show (every group 10)；**P<0.01vs. solvent control groups；^##P<0.01vs.CSDS models+solvent group.

Embodiment

Elaborate with reference to embodiment to embodiment provided by the invention.

Embodiment 1

First, experimental method：

1. chronic unpredictable Stress model (CUMS)

With male C57BL6 mouse (8 week old) for experimental subjects, random packet, every group 10, except normal group is per 5, cage Outside, the equal single cage raising of remaining group.Model process continues 8 weeks, and a kind of stimulation of mouse, including fasting 24h, taboo water is given once daily 24h, folder tail 1 minute, wet environment, overturn round the clock, outstanding tail 5 minutes, frozen water swimming (4 DEG C, 4 minutes).Average every kind of stimulation is every Week is each once, and each all stimulation sequences are different, prevent mouse from which kind of stimulation predicted.Opened to while stimulation within last two weeks Begin to be administered by group and weight daily, in a manner of being injected intraperitoneally, by 10ml/kg dosage, administration process continues 14 days.Terminate laggard The test of row forced swimming, outstanding tail test and syrup consumption test.

2. chronic society's property defeated Stress model (CSDS)

First choice selects suitable ICR/CD1 mouse as aggressive instrument mouse, in before experiment.Raising in one mouse cage One CD1 male mouse (50 week old) and a CD1 Female Rats, allow its pairing to cohabit, so that male CD1 mouse produce manor meaning Know.After a week, it is aggressive that it is tested male CD1 mouse：Pairing CD1 Female Rats in mouse cage are taken out, are subsequently placed into one Strange C57 male mouses are as manor invader；If CD1 mouse begin to, to the latter's offensive attack, be considered as within 1min Aggressive CD1 mouse.

Then begin to formally test, with male C57BL6 mouse (8 week old) for experimental subjects, random packet.Except control group Outside per cage 2 mouse, remaining equal single cage raising, first normal diet drinks water one week to adapt to environment.

Stress process it is as follows：A model group C57 mouse is taken first, puts it into the mouse cage of a strange male CD1 mouse In, and the CD1 Female Rats with the pairing of male CD1 mouse are removed at the same time, make continuation of the C57 mouse by male CD1 mouse Attack and last about 10min；Then the two is separated with wire netting, each other still can phase though making two mouse not contact directly Mutually smelt depending on information, which continues 2h；Then this C57 mouse is put back into former cage, and puts back to CD1 Female Rats, this should for first day Swash.The C57 mouse are put into the cage of another strange CD1 male mouses again within second day, giving in the same way stress；Put again within 3rd day In the cage for entering the 3rd strange CD1 male mouse, giving again in the same way stress.By such Coping style continuously stress 10d, And ensure that C57 mouse are subjected to the attack of different CD1 male mouses daily.Normal group C57 mouse then maintain normal diet to drink water, and keep 2 cages, mutually replace object of living together daily.

After continuous 10d stress stimulations, start to be administered by group and weight daily, in a manner of being injected intraperitoneally, by 10ml/ Kg dosage, administration process continue 14 days, then carry out forced swimming test, outstanding tail test, syrup consumption test and society and connect Touch test.

3. forced swimming tests (FST)

Test mouse is put into high 20cm, diameter 15cm, depth of water 10cm, water temperature is to be forced it in 25 ± 1 DEG C of water drum Swimming 6 minutes, with the dead time of mouse in 4 minutes after stopwatch bulk registration, standard：Mouse systemic only has head to expose water Face, four limbs it is motionless in floating or be motionless state for the done mild action that makes oneself not being drowned.Every mouse assay is complete After dry, put back in cage.Keep quite during experiment in room, light is soft.Experiment is measured using blind, to ensure to test The objectivity of data.

4. outstanding tail test (TST)

Will at test mouse tail end tip 1cm with immobilization with adhesive tape on iron, body is in projecting state, head distance Face about 50cm, is around isolated with plastic plate, it is disturbed with excluding surrounding enviroment.Observing time continues 6 minutes, is added up with stopwatch After record in 4 minutes mouse dead time, standard：Mouse four limbs trunk is completely motionless.Keep quite during experiment in room, light Line is soft.Experiment is measured using blind, to ensure the objectivity of experimental data.

5. syrup consumption test

The C57 mouse every for making to want acceptance test first before test match somebody with somebody one bottle 1% per cage all in single cage dwelling state Sucrose water and one bottle of pure water simultaneously keep free diet to drink water two days, during which exchange the position of syrup bottle and pure water bottle every 12h, Prevent C57 mouse from having Place Preference, this is syrup preference training.Two bottles of water and feed are all withdrawn since the 3rd day, are allowed C57 mouse receive 24h fasting for solids and liquids.Official testing in 4th day, first all puts back to two bottles of water of every cage (in syrup bottle and pure water bottle Liquor capacity equivalent, and measure in advance), allow C57 mouse to remove two bottles of water again after freely drinking 1h；It is remaining molten that its is measured respectively Liquid accumulates, and calculates syrup consumption volume A and pure water consumption volume B, show that A/ (A+B) is exactly the syrup of every C57 mouse with this Hobby degree.The numerical value is directly proportional to animal pleasant sensation：Indicate that animal is more normal more greatly；It is on the contrary then represent anhedonia it is more obvious, Animal is more depressed.

6. social touching act test

Experimental principle：Since muroid belongs to koinotropic type animal, every normal mice can be frequently in contact with it after running into strange mouse, The contact ac time of the two can be long in unit interval；And the C57 mouse after chronic social stress can produce orphan keep away, Escape strike and the depressed sample symptom such as social phobia, can strongly be avoided after running into strange mouse to avoid can suffer from it is similar before Phase undergoes (being attacked by CD1 mouse), and contact exchange time can be shorter in unit period.By testing C57 in computational rules period The time length that mouse contacts with strange mouse evaluates whether the C57 mouse produce depressed sample symptom.

Experiment carries out (such as Fig. 1) in a small-sized spacious case (44 × 44cm), is put into one toward the center of side in spacious case first Wire mesh cage (10 × 6cm, for placing strange mouse), and divide around the cage rectangular area as contact area (14 × 26cm), in test time of contact is included in once testing C57 mouse and being contacted into this region with strange mouse.

The test is divided into two benches：

The 1.target absent stages:Strange mouse is not first put into, and wire mesh cage is empty, and an experiment C57 mouse is put into case In；Using 5min as prescribed period of time, calculating C57 mouse enter the time length in contact area in this 5min, are set to A；

The 2.target present stages：A strange CD1 heros mouse is put into wire mesh cage, and (this mouse is not previous any one Aggressive CD1 mouse, completely strange), then same experiment C57 mouse is put into case, using 5min as the stipulated time, it is calculated again The time length in contact area is entered in this 5min, is set to B；

It is more obvious by the use of the ratio of B and A as behavioral indexes, the depressed sample symptom of the smaller explanation animal of the ratio.

7. Open field test (open field test)

Test mouse is put into spacious case center, the horizontal lattice number passed through carries out observation statistics at the appointed time to it, should Index is widely used for judging the autonomic activities degree of animal, and every three feet of experiment regulation mouse enter a lattice and calculate one point, measure Time continues 3 minutes.Carried out using blind, the peace and quiet of environment are kept in experimentation, are carried out in darkroom, open distance above case The light bulb of a 50W is hung at 40cm.Spacious case thoroughly clean to eliminate smell to next after every zoometry The interference of sample.

8.Western blotting western blot tests

Histone is extracted, is then quantitatively inactivated, carries out western blot test.

(1) glue.

(2) electrophoresis liquid (1000ml distilled waters 3.03g containing Tris-base, glycine 18.8g, SDS1g) is prepared, will be made Glue be fixed in electrophoresis tank, add electrophoresis liquid, liquid level do not had filamentary silver, pulled out plastic comb, added albumen sample and Marker； Electrophoresis 90min under 100V voltage conditions.

(3) transferring film liquid (1000ml distilled waters methanol containing 200ml, Tris-base 3.03g, glycine 14.4g), electricity are prepared After swimming, glue is cut by the selection of molecular weight of albumen size, and prepare filter paper and nitrocellulose membrane；By filter paper, tunica fibrosa, sponge and Glue is overlayed on clamping plate in order, and subsequent strap is in transferring film groove, transferring film (260mA, 90min) on ice.

(4) after transferring film, nitrocellulose membrane is taken out, is immersed in 5% skim milk TBST, closing 2h is shaked under room temperature.

(5) after closing, TBST washes 3 × 10min of film, and primary antibody is incubated, and closing is shaked at 4 DEG C overnight；Then take out nitre Sour tunica fibrosa simultaneously recycles primary antibody, and TBST washes 3 × 10min of film, and secondary antibody is incubated 2h, and TBST washes 3 × 10min of film again.

(7) developing room develops.

2nd, experimental result：

1. andrographolide has antidepressant effect in FST and TST

The antidepressant effect of andrographolide is tested in (FST) in forced swimming studied first, and FST is the ten of application Divide a kind of extensive laboratory facilities for being used for detecting the potential antidepression ability of medicine.We using Prozac as positive control medicine, Solvent control group, Prozac group (20mg/kg), andrographolide low dose group (30mg/kg) and the high agent of andrographolide are set 4 groups of amount group (60mg/kg).Experimental result finds that andrographolide shows obvious antidepression sample effect in FST (Fig. 2A), wherein 60mg/kg andrographolides cause the reduction of mouse dead time about 40%, similar with Prozac.

Then we test (TST) using outstanding tail, and TST is similar with FST, are another quick, reliable antidepressants Screening technique.TST results are similar to FST results, and andrographolide gives the dead time (Fig. 2 B) for significantly reducing mouse, And the effect of 60mg/kg andrographolides is similar to Prozac.

It is the spontaneous activity by influencing mouse to exclude antidepressant effect of the andrographolide in FST and TST And produce, we have carried out open field test to exclude the possibility of this respect again.The results show that each group mouse is in testing either Through region quantity all being not significantly different property (Fig. 2 C) at spacious case center or edge.

2. andrographolide being capable of reversal therapies CUMS and the behavior depression of CSDS depression models initiation

We further using it is chronic it is unpredictable stress (CUMS) and chronic society defeat stress (CSDS) depression model In come inquire into research andrographolide antidepressant effect；Both depression models of CUMS and CSDS can have in animals Many symptoms of effect simulation human depression, such as anhedonia, social phobia, behavioral despair.We are first manufactured with mouse CUMS/CSDS depression models, then give Prozac or andrographolide is treated, then carry out various actions test.

CUMS model behaviors data result is as shown in figure 3, and CSDS model behavior data results are as shown in Figure 3.From In it will be seen that andrographolide persistently give significantly reverse depression model caused by anhedonia, social phobia with And behavioral despair these behavior depressions, and the effect of 60mg/kg andrographolides is even better than Prozac, in conjunction with Fig. 2 Test result, we demonstrate that andrographolide has antidepressant effects really.

3. andrographolide, which can reverse, recovers the hippocampus BDNF signal paths level that CUMS triggers with CSDS depression models Reduce

To inquire into the antidepressant effect and mechanism of andrographolide, we use Western blotting technical research Its influence to CUMS and the expression of CSDS depression model mouse cerebral hippocampals bdnf protein, albumen internal reference is used as using β-actin.Experiment knot Fruit such as Fig. 5 and Fig. 6 are shown, relative to solvent control mouse, the hippocampus BDNF expressions of CUMS depressions mouse and CSDS depression mouse are all It is remarkably decreased, and continuous give of 30mg/kg and 60mg/kg andrographolides all different degrees of has reversed this change.

Afterwards, we also have detected depression model and andrographolide to hippocampus BDNF downstream signaling pathway key molecules The function influence of (TrkB and pTrkB, CREB and pCREB).As a result as shown in Figure 5 and Figure 6, as BDNF, andrographolide Persistently give and significantly reversed CUMS to be reduced with the hippocampus pTrkB of CSDS stress-induceds and pCREB expressions；Phase Instead, the total TrkB and CREB protein levels between each experimental group are constant.These results synthesis shows that hippocampus BDNF signal paths are very It may participate in the antidepressant effect of andrographolide.

4. the antidepressant effect of andrographolide needs hippocampus BDNF systems

BDNF systems whether are needed to participate in order to further determine the antidepressant effect of andrographolide, we used BDNF acceptor TrkB inhibitor K252a.We first carry out K252a (25 μ g/kg, 1 time a day) to C57BL/6J mouse and continue 3 days Give, then carry out the FST experiments of andrographolide.It turns out that the pretreatment of K252a has substantially blocked andrographolide to exist Antidepressant effect (Fig. 7 A) in FST tests；Similar, K252a pretreatments have also blocked andrographolide in TST tests Antidepressant effect (Fig. 7 B).

In addition, we give CUMS depressions mouse and CSDS depressions mouse the same of 60mg/kg andrographolides and K252a respectively When inject, continue 14 days, then carry out Behavior test afterwards.Fig. 8 data results show that andrographolide is in CUMS depression models Middle showed antidepressant effects are substantially blocked by BDNF path blocking agents K252a；And Fig. 9 data results are shown, in Herba Andrographitis The antidepressant effects that ester is showed in CSDS depression models are substantially blocked by BDNF path blocking agents K252a.So Herba Andrographitis The antidepression curative effect of lactone is by caused by promoting hippocampus BDNF systemic-functions.

The preferred embodiment to the invention is illustrated above, but the invention be not limited to it is described Embodiment, those skilled in the art can also make a variety of equivalent on the premise of without prejudice to the invention spirit Modification or replacement, these equivalent modifications or replacement are all contained in the application claim limited range.

Claims (4)

1. application of the andrographolide in the medicine for preparing treatment depression.

2. application of the andrographolide according to claim 1 in the medicine for preparing treatment depression, it is characterised in that The medicine of the treatment depression is the medicine for using andrographolide as sole active composition or including andrographolide Compositions.

3. application of the andrographolide according to claim 2 in the medicine for preparing treatment depression, it is characterised in that Any formulation can be made with pharmaceutically common auxiliary material in the medicine or pharmaceutical composition.

4. a kind of medicine for treating depression, it is characterised in that be made of andrographolide and pharmaceutically acceptable auxiliary material.