CN109350643B

CN109350643B - Medical application of aglaia odorata extract in resisting ischemic cerebral apoplexy

Info

Publication number: CN109350643B
Application number: CN201811441732.9A
Authority: CN
Inventors: 曾克武; 姜勇; 屠鹏飞; 王竞康
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2018-11-29
Filing date: 2018-11-29
Publication date: 2021-08-17
Anticipated expiration: 2038-11-29
Also published as: CN109350643A

Abstract

The invention relates to a new medical application of a aglaia odorata leaf extract, in particular to an application of the aglaia odorata leaf extract in preparing a medicament for treating ischemic cerebral apoplexy. The invention also provides application of the aglaia odorata leaf extract in preparing a medicament for preventing and/or treating acute and chronic complications caused by cerebral ischemia.

Description

Medical application of aglaia odorata extract in resisting ischemic cerebral apoplexy

Technical Field

The invention relates to medical application of a aglaia odorata extract, in particular to application of the aglaia odorata extract in preparing a neuroprotective medicament, and in particular relates to application of the aglaia odorata extract in preparing a medicament for resisting ischemic cerebral apoplexy and a medicament containing the aglaia odorata extract.

Background

Ischemic cerebral apoplexy is also called apoplexy, is acute neuron necrosis induced by the decrease of cerebral blood flow perfusion, has complex pathogenesis and can cause irreversible severe damage to brain function. Therefore, ischemic cerebral stroke has become the neurological disease with the highest disability rate at present. How to improve the cerebral nerve injury caused by ischemic cerebral apoplexy is always an aim pursued by the medical field, and various currently clinically used neuroprotective agents comprise edaravone, ganglioside, cerebrosid carnosine, butylphthalide and the like, but the curative effect and safety of the agents are further verified, and the development of a novel neuroprotective agent for improving cerebral nerve necrosis caused by stroke is still urgently needed.

When cerebral ischemia occurs, energy exhaustion is the most critical factor for inducing nerve damage, and no specific therapeutic drug aiming at the pathological process exists at present. In recent years, innovative medicines for treating ischemic cerebral apoplexy based on traditional Chinese medicine are gradually concerned by people, and have wide development prospects in the future.

The Aglaia odorata Lour is evergreen shrub or small arbor of Aglaia of Meliaceae (Meliaceae), namely chloranthus, dendrin, cymbidium and the like, is mainly distributed in India, Malaysia, Australia and other places, and is also commonly cultivated in Guangxi, Guangdong, Yunnan, Guizhou, Fujian, Sichuan and other provinces of China.

The traditional Chinese medicine considers that branches and leaves of the aglaia odorata can be used as medicines, have the effects of promoting blood circulation, removing blood stasis, relieving swelling and pain, can be used for treating rheumatic arthralgia, traumatic injury, carbuncle, cellulitis, pyogenic infections and the like, and has no record of using the branches and leaves of the aglaia odorata for nerve protection. In the research of modern medicine, the current reports about the pharmacological activity of the Mielan plants mainly focus on the activities of anti-inflammation, immune regulation, virus resistance, tumor resistance and the like, and no report for neuroprotection is available.

Disclosure of Invention

The inventor finds that the extract of the leaves of the aglaia odorata has very obvious activity on improving acute neuronal necrosis induced by the reduction of cerebral blood perfusion, is suitable for being used as a neuroprotective agent for improving the neural necrosis caused by stroke, and provides a new effective component for treating ischemic cerebral stroke and acute and chronic complications caused by cerebral ischemia.

The invention specifically provides the following technical scheme:

the aglaia odorata extract can be applied to neuroprotective drugs, shows activity of improving neuronal necrosis caused by ischemia in animal experiments, cell experiments and molecular biological experiments, and can be used as a unique active ingredient for neuroprotective drugs.

The neuroprotective drug is preferably a drug for treating ischemic cerebral apoplexy, but can also be used as a neuroprotective agent for other purposes, such as treating and repairing cerebral nerve injury caused by Parkinson's disease, Alzheimer's disease and trauma, and treating other nerve injury such as vertebra.

The aglaia odorata extract can of course also be used in the preparation of a medicament for the prevention and/or treatment of complications resulting from cerebral ischemia. Acute and chronic complications caused by nerve injury caused by cerebral ischemia generally include one or more of dementia, blindness, aphasia, hand and foot weakness, hemiplegia, cerebral infarction and cerebral hemorrhage, and the medicaments for treating the diseases are mainly used for treating the nerve injury, so the aglaia odorata extract can play a good role in treatment.

The extract of aglaia odorata of the present invention may be an extract of various parts of an aglaia odorata plant, and among them, an extract of an aglaia odorata leaf is preferred. The specific extraction method of the extract is not particularly limited, and may be an aqueous extract, an ethanol extract, or an organic solvent extract. From the viewpoint of both solvent residue and extraction efficiency, ethanol extract is preferred.

The aglaia odorata extract of the present invention may include one or more pharmaceutically acceptable excipients, regardless of the preparation of the pharmaceutical product. The pharmaceutically acceptable excipients are not particularly limited, and generally include solvents (such as water, ethanol, propylene glycol, oil for injection, etc.), diluents (such as starch, sugar powder, dextrin, lactose, pregelatinized starch, microcrystalline fiber, inorganic calcium salts (such as calcium sulfate, calcium hydrogen phosphate, calcium carbonate for pharmaceutical use, etc.), mannitol, etc., vegetable oils, polyethylene glycol, etc.), binders (such as water, ethanol, starch slurry, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose and ethyl cellulose, hydroxypropyl methylcellulose, etc.), disintegrants (such as dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, etc.), lubricants (such as magnesium stearate, aerosil, talc, hydrogenated vegetable oils, polyethylene glycols, magnesium lauryl sulfate, etc.), and the like which are conventional in the pharmaceutical field, Absorption enhancer (such as surfactant, Azone, EDTA, salicylic acid, amino acid ethylamine derivatives, acetoacetates, beta-dicarboxylate, aromatic acid compounds, and fatty acids), antiseptic (such as benzoic acid, hydroxypropyl butyl ester, hydroxypropyl methyl ester, phenol, and m-cresol), and correctant (such as sucrose and steviosin).

When used as a pharmaceutical, the aglaia odorata extract may be combined with an active ingredient conventionally used for the treatment of ischemic cerebral apoplexy, and such an active ingredient is not particularly limited, and one or more kinds selected from the group consisting of ginkgo biloba extract, aspirin, warfarin, clopidogrel, ticlopidine and dipyridamole may be used in combination.

The extract of aglaia odorata of the present invention can be used as an oral preparation or a non-oral preparation when applied to the preparation of a pharmaceutical product, and is not particularly limited. For example, the composition can be prepared into tablets, capsules, dripping pills, granules, powder, oral films or oral liquid which are used as oral preparations, and also can be prepared into one or more of injections, ointments, creams and suppositories which are used as non-oral preparations. In view of bioavailability and speed of action, injection is preferable. From the viewpoint of convenience in use and good patient compliance, various oral preparations are preferable.

When the medicament is used for treating ischemic cerebral apoplexy or preventing and/or treating acute and chronic complications caused by cerebral ischemia, a human or a mammal is taken as an administration object of the medicament. For this purpose, the intake mass or administration mass of the drug of the present invention is usually 120 to 700mg per day per person, more preferably 300 to 400mg per person, calculated as 60 to 70kg of adult body weight based on the extract from the leaves of aglaia odorata.

Pharmacological tests show that the extract of the leaves of the aglaia odorata can obviously reduce the cerebral ischemic area of rats caused by ischemia-reperfusion and reduce neurobehavioral scores. The aglaia odorata extract shows the caspase apoptosis signal pathway activation inhibition activity in pharmacological experiments, which provides a mechanism explanation of molecular biology level for the neuroprotection mechanism of the aglaia odorata extract.

Drawings

FIG. 1 is a photograph of brain slices of each group of test animals in example 2, wherein the "sham" group is a sham group, the "model" group is a model group, and the "Milan" group is a leaf extract administration group of Milan (300 mg/kg).

FIG. 2 shows the relative cerebral ischemic area percentage values of the groups of test animals in example 2, wherein the "sham" group was a sham group, the "model" group was a model group, and the "Milan" group was a leaf extract administration group of Milan (300 mg/kg).

FIG. 3 shows the neurobehavioral scores of the groups of test animals in example 3, wherein the "sham" group was the sham group, the "model" group was the model group, and the "Milan" group was the leaf extract administration group of Milan (300 mg/kg).

FIG. 4 shows the molecular mechanism of the neuroprotective effect of the leaf extract of Milan in example 4, wherein 4(A) shows that the leaf extract of Milan inhibits apoptosis due to hypoxia/glucose-deficient reperfusion, 4(B) shows that the leaf extract of Milan inhibits mitochondrial damage due to hypoxia/glucose-deficient reperfusion, and 4(C) shows that the leaf extract of Milan inhibits activation of caspase apoptosis signaling pathway due to hypoxia/glucose-deficient reperfusion.

Detailed Description

The invention is described below with reference to specific figures and examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagent materials used in the following examples are all commercially available products unless otherwise specified. Wherein, the purchase conditions of part of raw materials and reagents are as follows:

the Michelia is purchased from Kunming plant Biotechnology Limited;

ethanol, petroleum ether, ethyl acetate, and n-butanol were all analytically pure and purchased from Beijing chemical plant.

Example 1Preparation of extract of leaves of aglaia odorata

Taking 200g of the leaves of the aglaia odorata, and grinding by a grinder. Ultrasonic extracting with 8 times of 95% ethanol for 3 times, each for 1 hr. Recovering solvent under reduced pressure to obtain 95% ethanol extract, and volatilizing solvent until no alcohol smell exists to obtain 5g extract of Saylaria Mira leaf.

Example 2Extract of Mira young leaves for ratsProtective effect of cerebral ischemia reperfusion injury

2.1 test animals: SD rat, 8 weeks, male, beijing vindolicha laboratory animal technology ltd.

2.2 tested drugs:

the extract of the leaves of the aglaia odorata was dissolved in a 0.5% CMCNa solution to prepare a 5mg/mL drug solution.

2.3 animal grouping and handling:

SD rats 24, after 1 week of acclimatization, 8 per group, were randomized into 3 groups: sham group (sham group, only epidermis was incised without artery ligation), model group (physiological saline 300ml/kg), and Mira's extract group (300 mg/kg). The model group and the aglaia odorata leaf extract group were subjected to the following operations:

SD rats isolated the right Common Carotid Artery (CCA), Internal Carotid Artery (ICA) and External Carotid Artery (ECA), tied off ECA and cut off, CCA was temporarily blocked by a knot tied with silk thread, ICA was closed with an artery clamp, then incision was cut off at ECA and nylon thread was inserted to cause the middle cerebral artery to block ischemia. After 1.5h, the nylon thread is drawn out, and the CCA thread is released to realize reperfusion. At the same time, each group of rats was gavaged with the corresponding test drug. After 24 hours, the animals are anesthetized, the brains are taken out after euthanasia, cut into 8 brain slices on average, and incubated in TTC staining solution at 37 ℃ in the dark for about 15 mm. The ischemic area was white and the non-ischemic area rose red. And (4) scanning the ischemic areas of the brains of all groups, calculating the relative ischemic area value of the brains, and performing statistical analysis.

At the time of sacrifice of the above groups of rats, sham groups of rats were simultaneously euthanized, and brain sections were obtained according to the same procedure, followed by scanning and data processing.

2.4 test results

A black and white photograph of each group of brain slices is shown in figure 1. The photograph of fig. 1 shows that the brain sections of the rats of the normal control group are all dark, and have no ischemic region; large areas of white color appeared in the brain sections of the model group, indicating successful molding. The cerebral ischemic area of the Sasa Veitchii leaf extract group (300mg/kg) was significantly reduced compared to the model group.

Relative ischemic area (%) of the brain of each group of rats is shown in table 1 and fig. 2.

TABLE 1 relative ischemic area of rat brain for each group (mean. + -. standard deviation)

Note: # indicates P < 0.01 compared to sham; p < 0.01, as compared to model groups.

The data of table 1 is shown in connection with fig. 2: the area of cerebral ischemia was significantly reduced in the case of the milan leaf extract group (300mg/kg) compared to the model group (. about.p < 0.01).

The results of this example illustrate that:

the extract of the leaves of the aglaia odorata can effectively reduce the area of cerebral ischemia after cerebral ischemia reperfusion. The Mianea zealand leaf extract is suggested to be capable of well improving the brain injury caused by cerebral ischemia reperfusion.

Example 3Mianella leaf extract for improving neurobehavioral characteristics caused by cerebral ischemia reperfusion of rats

3.1 test animals: SD rat, 8 weeks, male, beijing vindolicha laboratory animal technology ltd.

3.2 tested drugs:

3.3 animal grouping and handling:

SD rats 24, after 1 week of acclimatization, 8 per group, were randomized into 3 groups: sham group (sham group, only epidermis was incised without artery ligation), model group (physiological saline 300ml/kg), and Mira's extract group (300 mg/kg). The animals of the model group and the animals of the Mira's orchid leaf extract group are subjected to the following operations:

SD rats isolated the right Common Carotid Artery (CCA), Internal Carotid Artery (ICA) and External Carotid Artery (ECA), tied off ECA and cut off, CCA was temporarily blocked by a knot tied with silk thread, ICA was closed with an artery clamp, then incision was cut off at ECA and nylon thread was inserted to cause the middle cerebral artery to block ischemia. After 1.5h, the nylon thread is drawn out, and the CCA thread is released to realize reperfusion. At the same time, each group of rats was gavaged with the corresponding test drug. After 24 hours, neurobehavioral scores of the rats in each group were counted according to the longa5 scale.

The scoring criteria for longa5 were:

no symptoms of nerve damage, score 0;

the left front paw cannot be fully extended for 1 minute;

turning to the non-ischemic side for 2 minutes;

when walking, the patient is inclined to the non-ischemic side for 3 minutes;

spontaneous walking, coma and consciousness, 4 points.

3.4 test results

Neuro-behavioral scores for each group of rats are shown in table 2 and figure 3.

TABLE 2 behavioral scoring of cranial nerves of rats in each group (mean. + -. standard deviation)

The data in table 2 and figure 3 show that the neurobehavioral scores for the aglaia odorata leaf extract group (300mg/kg) were very significantly lower than the model group (. about.p < 0.01).

The results of this example illustrate that:

the extract of the leaves of the aglaia odorata can well improve neurobehavioral injury caused by cerebral ischemia-reperfusion.

Example 4Mechanism for realizing neuroprotective effect of aglaia odorata leaf extract

The inventor conducts various molecular biological experiments, and in the exploration of a plurality of activity mechanisms, the inventor finds that the aglaia odorata extract shows obvious activity in the aspect of the regulation of mitochondrial apoptosis signal pathways. The relevant experimental procedures are as follows:

the PC12 cell line was purchased from cell center of Chinese academy of medicine and was cultured in high-glucose DMEM medium (containing 10% fetal bovine serum, 100 U.L)^-1Penicillin, 100. mu.g.L^-1Streptomycin) is subjected to subculture,passage 1 every 2 days. The aglaia odorata leaf extract was dissolved in DMSO to prepare a stock solution.

PC12 cells were treated with Saylaria Mirabilis leaf extract (5, 10, 50ng/mL) under hypoxic/sugar-deficient conditions for 2h prior to reperfusion injury. And then, continuously culturing the cells for 24 hours under normal conditions, carrying out cell fluorescent staining or collecting the cells, and analyzing protein expression by a western blot method to carry out action mechanism research.

The experimental conditions of Hoechst33258 cell fluorescent staining are as follows: PC12 cells were seeded in 24-well plates, model groups induced hypoxia/glucose-deprivation reperfusion injury, and aglaia odorata leaf extract treated groups treated cells at the concentrations described above. Reoxygenation is carried out for 24 hours after oxygen deficiency. Adding 4% paraformaldehyde, and fixing for 30 min; absorbing paraformaldehyde, adding PBS for cleaning, and cleaning for 3 times; discarding PBS, adding Hoechst33258 dye solution, and dyeing for 30 min; the dye solution was aspirated, washed with PBS for 5min each time for 3 times. The nuclear morphology of the (× 200) cells was observed with a fluorescence microscope (excitation wavelength 352nm, emission wavelength 461 nm). Finally, 4(A) pictures are obtained.

JC-1 cell mitochondrion fluorescence staining experimental conditions are as follows: PC12 cells were seeded in 24-well plates, model groups induced hypoxia/glucose-deprivation reperfusion injury, and aglaia odorata leaf extract treated groups treated cells at the concentrations described above. Reoxygenation is carried out for 24 hours after oxygen deficiency, mother liquor is prepared into working solution according to the requirements of the kit, and the working solution is added and dyed for 30min in a dark place; the working solution was aspirated, washed with PBS 3 times, and observed (× 200) for mitochondrial morphology using a fluorescence microscope (excitation wavelength 514nm, emission wavelength 529 nm). Finally, 4(B) pictures are obtained.

Western blot experiment conditions were as follows: the total protein was obtained by collecting the cells from each group and lysing the cells with RIPA lysate (containing protease inhibitors). Then separating the protein by adopting 6-15% SDS-PAGE, transferring the protein to a PVDF membrane by a semidry method, sealing for 20min by 5% skimmed milk powder, adding a primary antibody (1: 1000), incubating for 2h at room temperature, fully washing, adding a secondary antibody (1: 1000), incubating for 1h at room temperature, fully washing, adding an ECL reagent for color development, and taking a picture on a gel imaging system to obtain a 4(C) picture. The absorbance integral analysis of the bands was performed on the 4(C) picture with Gel-Pro software, and a bar graph was drawn. All data are expressed as mean ± standard deviation. T-tests and one-way anova were performed using GraphPad Prism5 statistical software, and P < 0.05 was considered statistically significant.

From the experimental result of Hoechst33258 cell fluorescent staining (as shown in fig. 4 (a)), the cells of the hypoxia/sugar-deficient reperfusion injury group show obvious apoptosis characteristics such as chromatin condensation, cell nucleus condensation and the like, and after the treatment of the aglaia odorata leaf extract, the appearance of the apoptosis characteristics is obviously inhibited, which suggests that the aglaia odorata leaf extract has better effect of resisting hypoxia/sugar-deficient reperfusion injury. In addition, as can be seen from the experimental results of JC-1 cell mitochondrial fluorescence staining (as shown in fig. 4 (B)), the cells of the hypoxia/glucose-deficient reperfusion injury group exhibited significant injury characteristics (green fluorescence) such as mitochondrial membrane depolarization, and the addition of the aglaia odorata leaf extract significantly suppressed the occurrence of the above-mentioned mitochondrial injury characteristics, suggesting that the aglaia odorata leaf extract had a better effect of resisting mitochondrial injury caused by hypoxia/glucose-deficient reperfusion. FIG. 4(C) shows that the extract of leaves of Milan blue can promote the activation of caspase-9, caspase-3 and PARP proteins under the condition of hypoxia/sugar-deficient reperfusion injury, which indicates that the extract of leaves of Milan blue may exert the effect of resisting hypoxia/sugar-deficient reperfusion injury by inhibiting the activation of mitochondria-mediated caspase-9/3/PARP apoptosis signaling pathway. Therefore, it is speculated that the Miraban leaf extract-mediated nerve cell protection effect is probably realized by inhibiting the depolarization of a mitochondrial membrane and further inhibiting a mitochondria-dependent caspase apoptosis signal pathway. The mechanism of action has not been reported before.

The invention provides a new medical application of the extract of the leaves of the aglaia odorata, thereby providing a new choice for clinically treating ischemic cerebral apoplexy and preventing and/or treating acute and chronic complications caused by cerebral ischemia.

Claims

1. Use of ethanol extract of Sasa Veitchii leaf in preparing medicine for treating ischemic cerebral apoplexy is provided.

2. The use of claim 1, wherein the medicament comprises one or more pharmaceutically acceptable excipients.

3. The use of claim 1, wherein the medicament is an oral formulation.

4. The application of the ethanol extract of the leaves of the aglaia odorata in preparing the medicines for preventing and/or treating acute and chronic complications caused by cerebral ischemia.

5. The use according to claim 4, wherein the acute or chronic complications are selected from one or more of dementia, blindness, aphasia, hand and foot weakness, hemiplegia, and cerebral hemorrhage.

6. The use according to claim 4, wherein the medicament for preventing and/or treating acute and chronic complications caused by cerebral ischemia further comprises one or more pharmaceutically acceptable excipients.

7. The use according to claim 4, wherein the medicament for preventing and/or treating acute and chronic complications due to cerebral ischemia is an oral preparation.