CN106265793B - Application of the ocean mangrove acanthus ilicifolius extract in preparation treatment senile dementia - Google Patents
Application of the ocean mangrove acanthus ilicifolius extract in preparation treatment senile dementia Download PDFInfo
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Abstract
The present invention provides application of the ocean mangrove acanthus ilicifolius extract in preparation treatment and/or prevention senile dementia, wherein the old mouse le extract is prepared by following methods: 1) being set old mouse le leaf in extractor, the w/v of the Lao Shu le leaf of the alcohol reflux for being 30%~95% with concentration extraction and solvent is 1:10~30, and extraction time 1~3 hour, extraction time 1~3 time, extracting solution merges, it is concentrated under reduced pressure into no alcohol taste, is centrifuged, supernatant is collected；The supernatant that step 1) is obtained passes through polyamide chromatography column, wherein the polyamide chromatography column is filled with 5% acetic acid aqueous solution suspension polyamide, polyamide filler dosage is 1~2 times of Lao Shu le leaf weight, successively with water, 10% column volume of ethanol elution 3, then with 70% ethanol elution, 5 column volumes, collect 70% ethyl alcohol section, it is concentrated under reduced pressure, obtains Lao Shu le extract.
Description
Technical field
The present invention relates to natural drug preparation fields, more particularly to the new application of plant extracts.
Background technique
Senile dementia (also known as alzheimer's disease, Alzheimer ' s Disease, abbreviation AD) is a kind of progressive hair
The lethal neurodegenerative disease of exhibition, clinic can behave as cognition and memory function constantly deteriorates, activity of daily living into
Row decline, and can have various neuropsychic symptoms and behavior disorder.It is shown according to related data, early in 2006, the whole world was about
There are 26,000,000 people with Alzheimer's disease, every 85 people in the whole world just has 1 people to suffer from the disease to the year two thousand fifty estimation.According to incomplete system
Meter, there are about 5,000,000 Alzheimer Disease patients at present in China, account for a quarter in the world.With the extension of human longevity, the disease
Disease incidence increasingly rise, it has also become one of main fatal disease of the elderly is to be only second to heart disease, cancer, apoplexy to be led
Cause the fourth-largest killer of the elderly's death.
There are mainly two types of the drugs for treating senile dementia on the market at present: cholinesterase inhibitor (AChEI) and NMDA
The antagonist of receptor.The former mainly passes through the activity of acetylcholine esterase inhibition, the degradation of acetylcholine is reduced, to improve
Intracerebral cholinergic tone improves the cognitive power of patient, represents drug as donepezil.As the antagonist of nmda receptor, U.S. dollar
Just (memantine, Namenda) was approved by the FDA in the United States listing in 2003.It is big although these drugs are widely used
Symptom can only mostly be mitigated, improve the cognitive power of patient, can not be used to treat or prevent the deterioration process of disease.Therefore urgent
The drug for needing to act on new mechanism occurs.
Lao Shu le (Acanthus illicifolius L) is a kind of mangrove plant for being grown in seashore tideland band,
Belong to Acanthaceae brankursine, be distributed mainly on the Southeast Asian countries such as India, Philippine, Malaysia, in China Guangdong, extensively
The provinces such as west, Fujian and Hainan are also widely distributed.The plant is one of the medical mangrove plant that " national Chinese herbal medicine compilation " is included.Its
Nature and flavor are micro- salty, cool；With clearing heat and detoxicating, dispersing swelling and dissipating binds, relieving cough and asthma and other effects.Cure mainly lymph enlargement, acute, chronic hepatitis, liver and spleen
Enlargement, stomachache, cough, asthma etc..Acute and chronic liver is widely used in the Southeast Asian countries such as India, Philippine and China Hainan are civil
Scorching treatment.
Chinese patent application 200810071371.3 is related to " extracting methods of medical mangrove acanthus total flavonoids ", makes
Vacuum distillation, which is extracted, with 50~70% EtOH Sonicates obtains Lao Shu le general flavone.Chinese patent application 201310267606.7 is related to
A kind of preparation method of mangrove acanthus ilicifolius extract is extracted using 70~80% ethanol refluxing processes, obtains Lao Shu le crude extract I
And residue obtains Lao Shu le crude extract II then by resulting residue by ultrasonic extraction, then will twice Lao Shu le crude extract into
Row decoloration is concentrated to get Lao Shu le extract is arrived.
The present inventor's Lao Shu le of discovery in finding novel anti-senile dementia disease drug has certain anti-senile dementia disease
Effect, and by Lao Shu le leaf method for extraction and purification and the active correlation research of anti-senile dementia disease, it finds by this hair
The Lao Shu le extract obtained under bright extraction conditions has the effect of excellent anti-senile dementia disease, and by experiment, obtains
For the experimental animal effective dose of anti-senile dementia disease control efficiency.
Summary of the invention
It is an object of the present invention to provide treat and/or prevent senile dementia in preparation with ocean mangrove acanthus ilicifolius extract
The purposes of disease drug.
According to an aspect of the present invention, ocean mangrove acanthus ilicifolius extract can be by extracting Lao Shu le leaf through ethyl alcohol
Afterwards, it is obtained through polyamide column chromatography.
Specifically, the preparation of ocean mangrove acanthus ilicifolius extract includes the following steps:
(1) Lao Shu le leaf medicinal material is set in extractor, is extracted with the alcohol reflux that concentration is 30%~95%, Lao Shu le leaf
The w/v of medicinal material and solvent is 1:10~30, and extraction time 1~3 hour, extraction time 1~3 time, extracting solution merged,
It is concentrated under reduced pressure into no alcohol taste, is centrifuged, obtains supernatant.
(2) by supernatant in step (1) by polyamide chromatography column, wherein the polyamide chromatography column is with 5% acetic acid
The filling of aqueous solution suspension polyamide, polyamide filler dosage are 1~2 times of Lao Shu le leaf medicinal material, and successively with water, 10% ethyl alcohol is washed
3 column volumes are taken off, then with 70% ethanol elution, 5 column volumes, collect 70% ethyl alcohol section, are concentrated under reduced pressure, as in the present invention
Lao Shu le extract.
Preferably, the preparation of ocean mangrove acanthus ilicifolius extract can advanced optimize are as follows:
I) Lao Shu le leaf medicinal material is set in extractor, with concentration be 70% alcohol reflux extract, Lao Shu le leaf medicinal material with
The w/v of solvent is 1:20, and extraction time 2 hours, extraction time 2 times, extracting solution merged, and is concentrated under reduced pressure into no alcohol taste,
Centrifugation, obtains supernatant.
Ii) by supernatant in step (1) by polyamide chromatography column, polyamide filler dosage is Lao Shu le leaf medicinal material
1.5 times, successively with water, 10% column volume of ethanol elution 3, then with 70% ethanol elution, 5 column volumes, collect 70% ethyl alcohol
Section is concentrated under reduced pressure, as Lao Shu le extract in the present invention.
The Lao Shu le extract prepared in aforementioned manners has the effect of excellent anti-senile dementia disease, therefore Lao Shu le mentions
Take object that there is applied to clinical treatment and/or prevent the potential using value of senile dementia.
Specific embodiment
Essence for a better understanding of the invention is described, in detail below by better embodiment of the present invention
Illustrate the present invention but does not limit the present invention.
Material and reagent used in the following embodiment remove specified otherwise, are common commercially available.
The preparation of the Lao Shu le extract 1 of 1: of embodiment
Lao Shu le leaf medicinal material (purchased from Guangzhou mangrove seedling base) 5kg is taken to set in extractor, adding 100L concentration is 70%
Alcohol reflux extract, extract 2 times, 2 hours each extraction times, extracting solution merge, be concentrated under reduced pressure into no alcohol taste, be centrifuged, obtain
Supernatant.By supernatant by being equipped with 7.5L with 5% acetic acid aqueous solution suspension polyamide filler (model: PA66, specification: 30
~60 mesh) filling chromatographic column, successively with water, 10% column volume of ethanol elution 3, then with 70% ethanol elution, 5 column volumes,
70% ethyl alcohol section is collected, is concentrated under reduced pressure, obtains Lao Shu le extract 370g.
The preparation of the Lao Shu le extract 2 of 2: of embodiment
It takes Lao Shu le leaf medicinal material 5kg to set in extractor, the alcohol reflux that 80L concentration is 80% is added to extract, extraction 3 times, often
3 hours secondary extraction times, extracting solution merge, and are concentrated under reduced pressure into no alcohol taste, are centrifuged, obtain supernatant.By supernatant by being equipped with 10L
With 5% acetic acid aqueous solution suspension polyamide filler (model: PA66, specification: 30~60 mesh) fill chromatographic column, successively with
Water, 10% column volume of ethanol elution 3, then with 70% ethanol elution, 5 column volumes, 70% ethyl alcohol section is collected, it is concentrated under reduced pressure, obtains
To Lao Shu le extract 390g.
The preparation of the Lao Shu le extract 3 of 3: of embodiment
It takes Lao Shu le leaf medicinal material 5kg to set in extractor, the alcohol reflux that 80L concentration is 80% is added to extract, extraction 3 times, often
3 hours secondary extraction times, extracting solution merge, and are concentrated under reduced pressure into no alcohol taste, are centrifuged, obtain supernatant.By supernatant by being equipped with 10L
The chromatographic column for taking water as a solvent suspension polyamide filler (model: PA66, specification: 30~60 mesh) filling, successively with water, 10% second
Alcohol elutes 3 column volumes, then with 70% ethanol elution, 5 column volumes, collects 70% ethyl alcohol section, is concentrated under reduced pressure, obtains Lao Shu le
Extract 338g.
Chinese patent application 200810071371.3 discloses " extracting methods of medical mangrove acanthus total flavonoids ",
And Chinese patent application 201310267606.7 discloses " preparation method of mangrove acanthus ilicifolius extract ", uses respectively
50~80% and 70~80% ethyl alcohol extract.It is extracted in industry in botanical medicine, usually selects 70~80% or so ethyl alcohol conduct
Extraction solvent, because the ethyl alcohol extraction efficiency of the concentration is higher, and the ethyl alcohol of various concentration extracts for conventional medicinal material
The composition difference of the extract effective component arrived is little, essentially consists in content difference.For this purpose, comparative study of the present invention and patent
200810071371.3 the content with general flavone in the obtained extract of patent 201310267606.7.
It extracts to obtain extract 4 fully according to 200810071371.3 method of patent respectively, fully according to patent
201310267606.7 methods are extracted to obtain extract 5, using common ultraviolet spectrophotometry, distinguish by reference substance of rutin
The general flavone content in the above extract 1-4 is measured, the result is as follows:
General flavone content in Lao Shu le extract that table Different Extraction Method obtains
| Number | Extract 1 | Extract 2 | Extract 3 | Extract 4 | Extract 5 |
| Content (%) | 56.3 | 55.5 | 40.6 | 23.1 | 19.8 |
By the above comparative illustration, Lao Shu le medicinal material of the present invention is after ethyl alcohol extracts, then passes through polyamide column chromatography
Afterwards, general flavone content is greatly improved.However, the polyamide column chromatography method for taking water as a solvent filling routinely used
(such as embodiment 3) although the general flavone content in old mouse le extract can be greatly improved, highest content be can only achieve
40% or so, by further investigation revealed that Lao Shu le can be mentioned with 5% acetic acid aqueous solution filling polyamide chromatography column
It takes the general flavone content in object to be increased to 55% or more, fullys meet Chinese medicine, 5 effective kind part new drug of natural drug is not less than
50% requirement.
The Lao Shu le extract of 4: of embodiment is to Α β25-35The PC-12 cellular damage model repair of induction
Lao Shu le extract is prepared according to the method for embodiment 1.
Cell fishplate bar culture: taking well-grown PC-12 cell, cleaned with PBS balance solution, digests through 0.25% pancreatin
Cell suspension is made, it is 1 × 10 that cell concentration is adjusted after counting5A/mL.By this cell suspension inoculation in 96 porocyte culture plates
In, every hole is inoculated with 100 μ L, and 100 μ L D-hank ' s balanced salt solutions are respectively added in each hole.Culture plate after inoculating cell is placed in
5%CO2, cultivate 24 hours in 37 DEG C of constant incubators, it is spare.
By 96 porocyte culture plates after inoculation press column split, successively set blank group, model group, administration group 1, administration group 2,
Administration group 3 etc..20 μ L of water-containing cell nutrient solution is added in the every hole of blank group, adds 30 μ L sterile distilled waters；The every hole of model group adds
Enter 20 μ L of water-containing cell nutrient solution, adds 30 μ L Α β25-35(75 μm of ol/L) solution (amyloid beta 25-35 segment commodity
Name: Α β25-35, it is purchased from Sigma company)；The 20 μ L of drug containing cell nutrient solution of various concentration is added in the every hole of administration group, adds 30 μ
LΑβ25-35(75 μm of ol/L) solution；The above each group is put into 5%CO2, cultivate 48 hours in 37 DEG C of constant incubators.
In group of cells modeling and after administration culture 48 hours, every hole is separately added into each 20 μ L of 0.5%MTT solution, continues
It is put into 5%CO2, cultivate 4 hours in 37 DEG C of constant incubators, inhale and abandon liquid in hole, be separately added into 150 μ L DMSO solutions, immediately
It shakes 10min, sets measurement OD value at microplate reader 570nm (experimental result is shown in Table 1).
The 1 anti-Α β of Lao Shu le extract of table25-35The PC-12 cellular damage result of induction
(* * indicates p < 0.05 compared with model group)
Experimental result: Lao Shu le extract under various concentration all has the significant anti-beta induced PC12 cellular damage of A and makees
With.
A β is the senile dementia Etiological being currently known, and having numerous studies confirms Α β25-35Almost with endogenous A β
Segment has same toxicity, therefore often passes through Α β25-35Modeling Senlie dementia model.Certain density Α β is added25-35Afterwards,
The growth of PC12 cell is suppressed, and the mean OD value of cell is remarkably decreased, and after Lao Shu le extract of various concentration is added,
Cell OD value is significantly increased compared with model group, illustrates that Lao Shu le extract under various concentration all has the significant anti-beta induced PC- of A
12 cellular damages effect, to prove that the old mouse le of various concentration extracts the activity with anti-senile dementia in cell level.
Influence of the Lao Shu le extract of 5: of embodiment to mouse memory ability
Lao Shu le extract is prepared according to the method for embodiment 1.
Take ICR kind mouse 60 (purchased from Nanjing Jin Lai Biotechnology Co., Ltd), after adaptive feeding 1 week, random point
At 6 groups, every group 10, half male and half female.Respectively Normal group, model control group, positive controls, high dose group, middle dose
Amount group, low dose group.Normal group, model control group distinguish daily stomach-filling 0.5%CMC-Na solution (administered volume,
0.2ml/10g), the daily stomach-filling huperzine solution of positive controls (administered volume, 0.2ml/10g), medicine group distinguish stomach-filling
Without the medical fluid (administered volume, 0.2ml/10g) of concentration.After successive administration 2 weeks, except for the normal group, hyoscine is injected intraperitoneally
Solution 1mg/kg, volume injected 0.1ml/10g, continuous injection 5 days.By Y maze test, escapes and arrive after being shocked by electricity with mouse
The number of safety zone as inspection target, investigate Lao Shu le extract to ability of learning and memory in mice influence (experimental result is shown in
Table 2).
2 mouse Y maze learning memory capability test result of table
(* * indicates p < 0.01 compared with model group；* p < 0.05 compared with model group is indicated)
Test result shows: after mouse is by intraperitoneal injection hyoscine, the ability of learning and memory of model group mouse, and just
Normal group is compared, hence it is evident that decline；And positive control combines administration group mouse, the ability of learning and memory compared to model group mouse obtains
Apparent to improve, wherein huperzine and the correct number of the old mouse le extract group of high middle dosage significantly improve.
Claims (3)
1. the alcohol reflux extract of ocean mangrove acanthus is in preparation treatment and/or prevention senile dementia
Using.
2. application described in claim 1, wherein Lao Shu le extract is prepared using following preparation method:
1) Lao Shu le leaf is set in extractor, is extracted with the alcohol reflux that concentration is 30%~95%, Lao Shu le leaf and solvent
W/v is 1:10~30, and extraction time 1~3 hour, extraction time 1~3 time, extracting solution merged, and is concentrated under reduced pressure into nothing
Supernatant is collected in alcohol taste, centrifugation；
2) supernatant for obtaining step 1) is by polyamide chromatography column, wherein the polyamide chromatography column is with 5% acetic acid water
The filling of solution suspension polyamide, polyamide filler dosage are 1~2 times of Lao Shu le leaf weight, successively with water, 10% ethyl alcohol difference
It elutes 3 column volumes, then with 70% ethanol elution, 5 column volumes, collects 70% ethyl alcohol section, be concentrated under reduced pressure, obtain described old
Shu le extract.
3. application as claimed in claim 2, wherein the preparation method of Lao Shu le extract includes:
I) Lao Shu le leaf is set in extractor, is extracted with the alcohol reflux that concentration is 70%, the weight of Lao Shu le leaf medicinal material and solvent
Amount volume ratio is 1:20, and extraction time 2 hours, extraction time 2 times, extracting solution merged, and is concentrated under reduced pressure into no alcohol taste, is centrifuged, and is received
Collect supernatant；
Ii) by the supernatant obtained in step i) by polyamide chromatography column, wherein the polyamide chromatography column is with 5% acetic acid
The filling of aqueous solution suspension polyamide, polyamide filler dosage are 1.5 times of Lao Shu le leaf medicinal material, successively with water, 10% ethyl alcohol point
Not Xi Tuo 3 column volumes, then with 70% ethanol elution, 5 column volumes, collect 70% ethyl alcohol section, be concentrated under reduced pressure, obtain described
Lao Shu le extract.
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| PL241271B1 (en) | 2019-05-31 | 2022-08-29 | Inst Badawczy Innowacyjno Rozwojowy Biotomed Spolka Z Ograniczona Odpowiedzialnoscia | Application of a compound of the triterpene lupane type for the prevention and treatment of Alzheimer's disease and a pharmaceutical composition |
| CN110812375A (en) * | 2019-12-12 | 2020-02-21 | 中国海洋大学 | Phenylethanoid glycoside extract in Acanthus ilicifolius, preparation method thereof and application of extract as anti-liver injury medicine |
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