CN106138293A - A kind of red raspberry leaf has the extraction process of the total flavones of anti-AD activity - Google Patents
A kind of red raspberry leaf has the extraction process of the total flavones of anti-AD activity Download PDFInfo
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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Abstract
The invention discloses the extraction process of the total flavones in a kind of red raspberry leaf with anti-AD activity, by red raspberry leaf raw material with 50% 100% alcohol solution dippings, again to its heating and refluxing extraction, water suspendible is used after being concentrated under reduced pressure to give crude extract, can tentatively obtain thick total flavones through macroporous resin with water alcohol system elutions, then be obtained the total flavones of higher purity by polyamide and CHP20 resin column chromatography.The raw material that the present invention uses is red raspberry by-product Fructus Rubi leaf, with low cost；The total flavones that the present invention extracts has significantly suppression aβ protein aggregation activity, and shows good anti-AD activity in the water maze laboratory of Senile Dementia Model Rats；The inventive method is reasonable in design, has the advantages that technique is simple, low cost, energy consumption are low and production process pollution is low.
Description
Technical field
The present invention relates to plant pharmaceutical technology field, particularly relate to the total flavones in a kind of red raspberry leaf with anti-AD activity
Extraction process.
Background technology
Senile dementia is a kind of nonspecific diseases complex, and patient's mainly cognitive function sustains damage thus table
Now being decreased obviously for abilities such as memory, language, Alzheimer (AD) is then the most topmost a kind of symptom.The disease of AD
Becoming the most not clearly, β sample amyloid proteins (A β) is considered as the crucial inducible factor that AD occurs in evolution, can cause
The pathologic of impaired memory function and brain tissue changes, and the Therapeutic Method now concerning AD is also many, wherein choline
Esterase inhibition medicine is still the Therapeutic Method of first-selection.But these medicines can only improve the symptoms such as the cognition of patient, memory dysfunction,
And action target spot is single, can not fundamentally block the development process of AD, so finding new effectively treatment AD medicine still
It it is the vast medicine important goal in research of scholar.Chinese medicine Therapeutic Method has the applicating history of thousand of years in China, due to
Its effective ingredient is more, has the action character of Mutiple Targets, too many levels, thus naturally becomes most potential in terms for the treatment of AD
One of mode.China's plant resources enriches, and finds anti-AD active component and be always phytochemistry and natural product in plant
Learn the main flow of research.
Red raspberry (R.idaeus L.) is also known as Fructus Rubi corchorifolii Immaturus, rasp berry etc., for the perennial deciduous fruit tree of Rosaceae rubus,
In its fruit in addition to the nutritional labeling abundant containing these, possibly together with the functional component that some are special, such as volatile oil, polyphenol
Class, terpenoid, flavonoid etc., and higher than the SOD of other fruit, salicylic acid and Fructus Rubi ketone etc..These active component, make Fructus Rubi have
There is the highest medical health care function.China application Fructus Rubi is as the history of the existing more than one thousand years of Chinese medicine (Fructus Rubi), at " book on Chinese herbal medicine guiding principle
Mesh ", Shennong's Herbal, medical value to Fructus Rubi is documented in the ancient book such as " Mingyi Bielu ".Remember according to Compendium of Material Medica
Carrying: its (Fructus Rubi) stem, root, leaf, seed all can be used as medicine, sweet in the mouth is put down, nontoxic；Reinforce the kidney essence, nourishing liver and improving eyesight, reducing urination.Among the people for controlling
Treat diarrhoea, conjunctival congestion, alcoholism etc..In ancient Greek, China, India and America (American Indian) area, people use Fructus Rubi
Leaf treatment wound, is used as uterine relaxant and analeptic when suffering from diarrhoea and give a birth；Among the people in Europe, Fructus Rubi aerial parts and leaf
It is also used for treating hypertension.In China, Fructus Rubi among the people root is used for treating hypertension, as Xinjiang Mongolian People commonly use its treatment hypertension,
Hepatitis etc..The fruit of Fructus Rubi can be utilized the most, but the value of its leaf not yet causes the enough attention of people.
At present the researches and exploitation of red raspberry is concentrated mainly on red raspberry fruits, because it is rich in nutritional labeling and work
Property material and deeply favored by researcher, and Fructus Rubi leaf is rejected most of the time, is not exploited, and has not yet to see Rhizophora apiculata Blume
Total flavones process of enriching and the relevant report of anti-senile dementia activity in certain kind of berries leaf.
Summary of the invention
This team carries out activity by suppression aβ protein aggregation activity and guides tracking, finds the 70% of red raspberry leaf extract
D101 macroporous resin elution fraction has preferably suppression aβ protein aggregation activity, then through polyamide and CHP20 resin etc. to having
Effect position is enriched with further, makes general flavone content reach more than 80%, and activity is greatly improved simultaneously.Determine that this effective site is (total
Flavone) it is the effective site assembled of the suppression aβ protein of red raspberry leaf, meanwhile, we have carried out old age to the total flavones after refined
The water maze laboratory of dementia rats, result display red raspberry leaf flavonoids has significant anti-AD activity.
A kind of red raspberry leaf has the extraction process of the total flavones of anti-AD activity, comprises the following steps:
A, take red raspberry leaf raw material and dry in the shade, extract 2-3 time with the ethanol water of 5-10 times of volume, the body of ethanol solution
Volume concentrations is 50%-100%, extracts and uses heating and refluxing extraction；
B, by extracting solution merge after concentrating under reduced pressure, remove ethanol, concentrating under reduced pressure is drying to obtain extractum；
C, extractum add water after suspendible, upper D101 macroporous resin, use volumetric concentration 50%-again after 5-8 volume of water elution
It is colourless that 70% ethanol solution is eluted to effluent, collects ethanol elution, and decompression recycling ethanol, concentrate drying i.e. obtains crude flavonoid powder
Extractum (general flavone content more than 35%)；
After D, upper step gained extractum add alcohol dissolving, mixing with the polyamide filler of 2 times of weight, room temperature is mixed sample and is dried, and takes 5 times
The polyamide filler of weight, as column packing, after 10%-20% 5-8 volume of ethanol solution eluting, then uses concentration 70%-90%
Ethanol solution to be eluted to effluent colourless, collect ethanol elution, decompression recycling ethanol, it is higher that concentrate drying obtains purity
Total flavonoids extract (general flavone content more than 60%)；
E, upper step gained extractum add water after suspendible, and upper CHP20 resin uses volumetric concentration after 5-8 volume of water elution again
It is colourless that 40%-60% ethanol solution is eluted to effluent, collects ethanol elution, and decompression recycling ethanol, concentrate drying i.e. obtains
High-purity Refined flavonoid powder extractum (general flavone content more than 80%).
The extract of gained utilizes FeCl3Color reaction is positive, and HCl-Mg reaction is positive, and determines extract obtained
For total flavones.Utilize visible spectrophotometry (one annex V of Chinese PharmacopoeiaA), with rutin (self-control, purity 98%) for comparison
Product, and use sodium nitrite-aluminum nitrate-sodium hydroxide colorimetry, react the red network of generation according to flavone compound and aluminium salt
Compound, detects wavelength 510nm, for detecting the content of each obtained total flavones of chromatography eluant post.
The present invention is by red raspberry leaf raw material 50%-100% alcohol solution dipping, then to its heating and refluxing extraction, reduces pressure
Use water suspendible after being concentrated to give crude extract, can tentatively obtain thick total flavones through macroporous resin with water alcohol system elutions, then by poly-
Amide and CHP20 resin column chromatography obtain the total flavones of higher purity.
This programme having the beneficial effect that compared to traditional scheme
(1) raw material that the present invention uses is red raspberry by-product Fructus Rubi leaf, and with low cost, its value not yet causes people's
Enough attention, its manufactured goods the most not yet exist.
(2) utilize the resourceful feature of China Fructus Rubi, study the new technique of red raspberry leaf processing and utilization, new technology, with
The novel Fructus Rubi leaf processed goods of output of seeking survival is the approach breaking through Fructus Rubi current development limitation, from the point of view of long-range development,
Greatly develop our own Fructus Rubi leaf secondary industry to have a high potential, have a extensive future.
(3) carried out sufficiently carrying to the effective site in crude drug under the guidance that suppression aβ protein inhibitory activity is followed the trail of
Pure, concentrate and be enriched with, obtain its total flavones (its effective site), utilize spectrophotometry general flavone content, its content reaches
To 80%, it is much better than its crude extract and is directly used as medicine.
(4) total flavones obtained by this method, has significantly suppression aβ protein aggregation activity, and at senile dementia mould
Showing good anti-AD activity in the water maze laboratory of type rat, meanwhile, Fructus Rubi is as a kind of medicinal and edible plant, and its poison is secondary
Effect is inevitable less, certainly will have broad prospects in the research and development of anti-senile dementia disease drug.
(5) the inventive method is reasonable in design, has the spy that technique is simple, low cost, energy consumption are low and production process pollution is low
Point.
Accompanying drawing explanation
Fig. 1: red raspberry folic acid flavone impact figure (mean ± SD, n=10) preclinical on rat in water maze laboratory,
Note: compared with sham operated rats, * * p < 0.01；Compared with model group, * * p < 0.01.
Fig. 2: red raspberry essence flavone on rat in water maze laboratory target quadrant the treated time affect figure (mean ±
SD, n=10), note: compared with sham operated rats, * * p < 0.01；Compared with model group, * * p < 0.01.
Fig. 3: red raspberry essence flavone on rat in water maze laboratory through position of platform number of times affect figure (mean ± SD, n
=10), note: compared with sham operated rats, * * p < 0.01；Compared with model group, * * p < 0.01.
Detailed description of the invention
The enrichment of embodiment 1 red raspberry leaf flavonoids
Taking red raspberry leaf 1000g to pulverize, add 8000mL 60% ethanol solution, reflux, extract, 2 hours, filtered filtration residue is again
Add 3000mL 60% ethanol solution reflux, extract, 1 hour, merge twice filtrate, decompression recycling ethanol, obtain extractum.This is soaked
Cream with D101 macroporous resin upper after 1000mL water suspendible, is that 60% ethanol is molten by volume fraction after 5 volumes of water elution the most respectively
It is the most colourless that liquid is eluted to effluent, collects eluent, decompression recycling ethanol, is spray-dried and obtains dried powder, obtain 95.2g total
Flavone.
Utilize FeCl3 color reaction to be positive, and HCl-Mg reaction is positive, and determines extract obtained for total flavones.Profit
With visible spectrophotometry (one annex V of Chinese PharmacopoeiaA), it is reference substance with rutin (self-control, purity 98%), and uses Asia
Sodium nitrate-aluminum nitrate-sodium hydroxide colorimetry, reacts generation red complex according to flavone compound and aluminium salt, detects ripple
Long 510nm, in testing result display gained fraction, its general flavone content is 38%.Inhibited aβ protein focusing experiment, specifically real
Proved recipe method is as follows:
1, the pretreatment of A β
The A β pre-saved in-80 DEG C of refrigerators is taken out, is dissolved in HFIP with the concentration of 1mg/mL.Left at room temperature
30min is after its secondary structure is removed completely, and HFIP is removed in vacuum lyophilization, is placed in-20 DEG C of refrigerators preserving.
2, prepared by phosphate buffer (PBS, pH=7.4,0.1M)
The preparation (PBS, buffer) of buffer: pH=7.4, concentration is 0.1mol/mL.With reference to People's Republic of China's medicine
Allusion quotation second annex of version in 2010, takes potassium dihydrogen phosphate 1.36g, adds 0.l mol/L sodium hydroxide solution 79mL, be diluted with water to
200mL, is corrected with Accurate pH acidometer, to obtain final product.
3, the preparation of A beta response test solution
Take pretreated A β lyophilized powder, be configured to the A β examination of 50 μm ol/L with the 0.1mol/mL PBS of pH=7.4
Liquid ,-20 DEG C frozen standby.
4, developer configuration
Precision weighs Th-T powder, and with the 0.1mol/mL PBS of pH=7.4, preparation becomes the Th-T test solution of 10 μm ol/L,
Matching while using.
5, the preparation of compound solution
With 0.1mol/mL PBS and DMSO of pH=7.4, it is 10 that VE and testing sample are respectively configured concentration-3, 10-4,
10-5, 10-6With 10-7The final volume mark of mol/L, DMSO is not more than 0.001.
6, assay method
Group is arranged:
(1) blank group: PBS 60 μ L, A β 10 μ L, PBS+DMSO 10 μ L
(2) sample suppression group: PBS 60 μ L, A β 10 μ L, medicine 10 μ L
(3) blank background group: PBS 60 μ L, PBS 10 μ L, PBS+DMSO 10 μ L
(4) group at the bottom of sample copy: PBS 60 μ L, PBS 10 μ L, medicine 10 μ L
It is separately added into above-mentioned each group of solution in 96 orifice plates, is placed in constant-temperature incubation case 37 DEG C and hatches 24h, then in each group
Add 80 μ L Th-T solution, be placed in constant-temperature incubation case 37 DEG C and hatch 5min, by microplate reader in (λ ex=450nm；λ em=
485nm) place measures each group of fluorescent value.Often group is repeated 3 times.
7, interpretation of result:
After individually hatching using A β with Th-T measure 485nm place fluorescent value as comparison: in order to avoid compound itself tool
The interference to result of some fluorescence, to deduct the fluorescent value of independent test-compound Th-T mensuration as background.Suppression ratio
Computing formula is as follows:
Result shows: it reaches 49.5% to the suppression ratio of this albumen, and wherein positive control drug (curcumin) suppression ratio is
60.1%, result shows that the total flavones that the present embodiment obtains suppresses the activity of aβ protein to be slightly weaker than positive drug.
The enrichment of embodiment 2 red raspberry leaf flavonoids
Crude flavonoid 95.2g prepared by Example 1, after adding alcohol dissolving, mixes with the polyamide filler of 2 times of weight,
Room temperature is mixed sample and is dried, and takes the polyamide filler of 5 times of weight as column packing, after 15% ethanol solution eluting 5 volume, then with dense
It is colourless that the ethanol solution of degree 80% is eluted to effluent, collects ethanol elution, and decompression recycling ethanol, concentrate drying obtains purity
Higher total flavonoids extract 62.8g.
Utilize visible spectrophotometry (one annex V of Chinese PharmacopoeiaA), with rutin (self-control, purity 98%) for comparison
Product, and use sodium nitrite-aluminum nitrate-sodium hydroxide colorimetry, react the red network of generation according to flavone compound and aluminium salt
Compound, detects wavelength 510nm, and testing result display gained fraction general flavone content is 62%.Inhibited aβ protein focusing experiment,
Specific experiment method is with reference to embodiment 1:
Result shows: it reaches 59.2% to the suppression ratio of this albumen, and wherein positive control drug (curcumin) suppression ratio is
60.1%, result shows that the activity of the total flavones suppression aβ protein that the present embodiment obtains is suitable with positive drug.
The enrichment of embodiment 3 red raspberry leaf flavonoids
Flavone 62.8g prepared by Example 2, after the suspendible that adds water, upper CHP20 resin, use again after 5 volumes of water elution
Volumetric concentration 55% ethanol elution is colourless to effluent, collects ethanol elution, and decompression recycling ethanol, concentrate drying had both obtained height
Purity Refined flavonoid powder extractum 33.8g.
Utilize visible spectrophotometry (one annex V of Chinese PharmacopoeiaA), with rutin (self-control, purity 98%) for comparison
Product, and use sodium nitrite-aluminum nitrate-sodium hydroxide colorimetry, react the red network of generation according to flavone compound and aluminium salt
Compound, detects wavelength 510nm, and testing result display gained fraction general flavone content is 82%.Inhibited aβ protein focusing experiment,
Specific experiment method is with reference to embodiment 1.
Result shows: it reaches 87.4% to the suppression ratio of this albumen, and wherein positive control drug (curcumin) suppression ratio is
60.1%, result shows that the total flavones that the present embodiment obtains suppresses the activity of aβ protein to be better than positive drug.
Take the refined rear general flavone content sample more than 80% of this embodiment, carry out the Senlie dementia model of embodiment 4
The water maze laboratory of rat.
The water maze laboratory of embodiment 4 Senile Dementia Model Rats
Take SD rat, male, body weight 250-300g, set up rat Senlie dementia model, animal is randomly divided into 6 groups:
Sham operated rats (Sham): animal not modeling, gavage is to normal saline, once a day, successive administration 30 days；
Model group (Model): after animal model, gavage is to normal saline, once a day, successive administration 30 days；
Positive controls (Donepezil): after animal model, gavage is to donepezil 1.5mg/kg, once a day, continuously
It is administered 30 days；
Three kinds of chromatographic column refined total flavonoids (RLTF) low dose group of red raspberry leaf: after animal model, gavage is to red raspberry leaf
Total flavones 10mg/kg, once a day, successive administration 30 days；
Dosage group in three kinds of chromatographic column refined total flavonoids (RLTF) of red raspberry leaf: after animal model, gavage is to red raspberry leaf
Total flavones 50mg/kg, once a day, successive administration 30 days；
Three kinds of chromatographic column refined total flavonoids (RLTF) high dose group of red raspberry leaf: after animal model, gavage is to red raspberry leaf
Total flavones 100mg/kg, once a day, successive administration 30 days.
Application water maze laboratory observes the cognitive function of rat.Water maze instrument is movably justified by a round pool and one
Shape platform forms, and pool diameter 100cm, depth of water 40cm, water temperature controls at 23 ± 1 DEG C, and platform diameter 15cm, the water surface exceeds platform
2cm.During experiment, platform is positioned over the centre of first quartile, and pond is provided above automatic camera system and connects computer, follows the tracks of
The swimming track of record rat, automatically calculates distance that rat swims across in pond and finds the time required for platform.Just
Before formula experiment, animal is placed on platform and adapts it to 10s, then rat is put in water, observe the motion feelings in 90s
Condition.Record rat finds the time required for platform, if not finding platform in animal 90s, it is positioned over gently on platform also
Continue 15s, then put back in cage.Every day, animal training three times, for three days on end, then started formally to test.Observe rat from putting into water
To the time found required for platform in pond, more than 90s, then the time is calculated as 90s, once a day, and continuous 5 days.After experiment terminates,
Each animal is taken a day off, then carries out memory experiment.Being withdrawn from by platform, rat puts into pond from third quadrant, observes and record
In 120s, each rat is at the time at first quartile place and the number of times through platform position.
Result shows, compares with sham operated rats, during model group rats was tested at 5 days, finds the time of platform substantially to increase
(incubation period), it is decreased obviously through first quartile number of times and time of being treated at first quartile.Compare with model group, red raspberry leaf
Refined total flavonoids 50mg/kg, 100mg/kg dosage group can obviously reduce the incubation period of rat, increases the number of times by first quartile
And extending the time treated at first quartile, difference has significance (P ＜ 0.01).Its effect is organized similar to many piperazines the most together.Specifically
See Fig. 1, Fig. 2 and Fig. 3.
The above, the only present invention preferably detailed description of the invention, but protection scope of the present invention is not limited thereto,
Any those familiar with the art in the technical scope that the invention discloses, according to technical scheme and
Inventive concept equivalent or change in addition, all should contain within protection scope of the present invention.
Claims (4)
1. a red raspberry leaf has the extraction process of the total flavones of anti-AD activity, it is characterised in that comprise the following steps:
A, taking red raspberry leaf raw material and dry in the shade, extract 2-3 time with the ethanol water of 5-10 times of volume, the volume of ethanol solution is dense
Degree is 50%-100%, extracts and uses heating and refluxing extraction；
B, by extracting solution merge after concentrating under reduced pressure, remove ethanol, concentrating under reduced pressure is drying to obtain extractum；
C, extractum add water after suspendible, upper D101 macroporous resin, again by volumetric concentration 50%-70% second after 5-8 volume of water elution
It is colourless that alcoholic solution is eluted to effluent, collects ethanol elution, and decompression recycling ethanol, concentrate drying i.e. obtains crude flavonoid powder extractum；
After D, upper step gained extractum add alcohol dissolving, mixing with the polyamide filler of 2 times of weight, room temperature is mixed sample and is dried, and takes 5 times of weight
Polyamide filler as column packing, after 10%-20% 5-8 volume of ethanol solution eluting, then by the second of concentration 70%-90%
It is colourless that alcoholic solution is eluted to effluent, collects ethanol elution, and decompression recycling ethanol, concentrate drying obtains the higher total Huang of purity
Ketone extractum；
E, upper step gained extractum add water after suspendible, and upper CHP20 resin uses volumetric concentration 40%-after 5-8 volume of water elution again
It is colourless that 60% ethanol solution is eluted to effluent, collects ethanol elution, and decompression recycling ethanol, concentrate drying i.e. obtains high-purity
Refined flavonoid powder extractum.
2. red raspberry leaf as claimed in claim 1 has the extraction process of the total flavones of anti-AD activity, it is characterised in that institute
In step A stated, the volumetric concentration of described ethanol solution is 60%.
3. red raspberry leaf as claimed in claim 1 has the extraction process of the total flavones of anti-AD activity, it is characterised in that institute
In step C stated, the volumetric concentration of described ethanol solution is 60%.
4. red raspberry leaf as claimed in claim 1 has the extraction process of the total flavones of anti-AD activity, it is characterised in that institute
In step D stated, first with after 5-8 volume of ethanol solution eluting of 15%, then it is eluted to stream with the ethanol solution of concentration 80%
Go out liquid colourless.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103623082A (en) * | 2013-11-29 | 2014-03-12 | 江西中医药大学 | Raspberry extract and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103623082A (en) * | 2013-11-29 | 2014-03-12 | 江西中医药大学 | Raspberry extract and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| 徐雅琴等: "红树莓叶片中黄酮类物质提取及抗氧化性的研究", 《中国油脂》 * |
| 杨昱等: "树莓叶片黄酮成分分离纯化研究", 《高校化学工程学报》 * |
| 罗光乾: "《驻颜革命》", 31 January 2009, 京华出版社 * |
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Application publication date: 20161123 |