CN108815251A - Russia color fruit and its extract are preparing the purposes in blood lipid-lowering medicine - Google Patents
Russia color fruit and its extract are preparing the purposes in blood lipid-lowering medicine Download PDFInfo
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- 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 present invention provides Russia color fruits and its extract to prepare the purposes in blood lipid-lowering medicine.Pharmacodynamic test proves that Russia's color fruit the different extracted parts of the present invention can reduce TG and TC in various degree, wherein more obvious with the Lipid-lowering activities at methylene chloride position and petroleum ether part.
Description
Technical field
The present invention relates to the new applications of Russia color fruit, specifically preparing the purposes in blood lipid-lowering medicine.
Background technique
The present invention relates to Russia color fruit be rosaceous plant Malus toringoides Hughes Malus toringoides (Rehd.) Hughes. and
The dry mature fruit of tibetan crabapple Malus transitoria (Batal.) Schneid..In Ganzi Tibetan area, Russia color fruit, which is practised, to be claimed
More than watt, more than red watt, more than white watt etc., it furthermore is also known as great Bai stone jujube, little Bai stone jujube, puckery Chinese date etc. in Gansu, another there are also floral leaf birch-leaf pears
The alias such as (northern Shensi), horse birch-leaf pear, thin and delicate Malus spectabilis.Russia color fruit exists《Newly repair Jingzhubencao》,《China's hiding book on Chinese herbal medicine》With《The big diction of Zang Han
Allusion quotation》In it is on the books, have " hepatopathy, hypertension, diarrhea, eye disease, irregular menstruation are controlled in stomach invigorating, blood pressure lowering " the effects of.Russia color fruit
Medicinal history in Tibetan area is long, but its modern pharmacology Effect study is very few, document report Russia color fruit mouse caused by alcohol
Acute liver damage shows certain protective effect.(see Zeng Jun, etc. Tibetan medicine " Russia color fruit " induces HL-7702 liver cell to CCl4
The protective effect of damage, the national Chinese medicine commodity science conference collection of thesis of the 5th, Chinese commodity association, 2017-05-12).
It there is no Russia's color fruit active component for treating the relevant report of reducing blood lipid at present.
Summary of the invention
The present invention relates to the present invention relates to the new applications of Russia color fruit, specifically preparing the purposes in blood lipid-lowering medicine.
The present invention provides Russia color fruits and its extract to prepare the purposes in blood lipid-lowering medicine.
Wherein, the Russia color fruit be rosaceous plant Malus toringoides Hughes Malus toringoides (Rehd.) Hughes and
The dry mature fruit of tibetan crabapple Malus transitoria (Batal.) Schneid..
Wherein, Russia's color berry extract is total extract position, water extract part, n-butanol extract part, acetic acid
Ethyl ester position, methylene chloride extract part, petroleum ether extract part.
Wherein, Russia's color berry extract is methylene chloride position or petroleum ether part.
Wherein, the preparation method of Russia's color berry extract is:
A, it takes Russia color fruit to be sieved, 95% ethyl alcohol is added and extracts, filtering, residue adds water decoction, filters;Alcohol extract depressurizes back
It receives ethyl alcohol to no alcohol taste and obtains alcohol-extracted extract;Disperse alcohol-extracted extract with Aqueous extracts concentrate and be concentrated to give medicinal extract, extract dry is total
Extract part;
B, alcohol-extracted extract is dispersed into alcohol-extracted extract with Aqueous extracts concentrate, makes its full and uniform dissolution, sequentially adds petroleum
Ether, methylene chloride, ethyl acetate, water saturated extracting n-butyl alcohol, extraction remainder are water section, and it is molten to recycle each position extraction
Agent on Rotary Evaporators in being evaporated, then is dried under vacuum to dry, then finally obtains water, water saturated positive fourth according to polarity size order
Alcohol, ethyl acetate, methylene chloride, petroleum ether part.
Pharmacodynamic test proves that Russia's color fruit the different extracted parts of the present invention can reduce TG and TC (P < 0.05, P in various degree
< 0.01), wherein more obvious with the Lipid-lowering activities at methylene chloride position and petroleum ether part.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention.
Detailed description of the invention
Morphological contrast map (wherein, A Normal group (10 × 20) before and after the normal L-02 hepatocytes induction modeling of Fig. 1;B doctor
With Fat Emulsion induction group (model group) (10 × 20))
Fig. 2 Russia color fruit and E Seye total extract part the intracellular TG content of rouge denaturation L-02 is influenced (N=3)
Fig. 3 Russia color fruit and E Seye total extract part the intracellular TC content of rouge denaturation L-02 is influenced (N=3)
Each solvent extraction position of Fig. 4 Russia color fruit to rouge denaturation the intracellular TG content of L-02 variation (N=3)
Each solvent extraction position of Fig. 5 Russia color fruit to rouge denaturation the intracellular TC content of L-02 variation (N=3)
Each solvent extraction position Fig. 6 E Seye and monomer to the variation of the rouge denaturation intracellular TG content of L-02 (N=
3)
Each solvent extraction position Fig. 7 E Seye and monomer to the variation of the rouge denaturation intracellular TC content of L-02 (N=
3)
Specific embodiment
The preparation of the Russia's color fruit active component of the present invention of test example 1
It takes Russia color fruit, E Seye to cross No. 2 sieve each 100g of powder, 95% second of 2000mL is added by the solid-liquid ratio of 1 ﹕ 20 respectively
Alcohol reflux extracts 2 times, and each 2h is filtered while hot, and residue adds 10 times of amount water to decoct 2 times again, and each 2h is filtered while hot;Merge 2 times
Ethyl alcohol is recovered under reduced pressure to no alcohol taste in alcohol extracting filtrate;Merge 2 water to mention filtrate and be concentrated to give medicinal extract, is total after medicinal extract vacuum drying
Extract part (the mixing medicinal extract that alcohol extracting and water mention).Medicinal extract is dispersed into alcohol-extracted extract with Aqueous extracts concentrate, keeps its full and uniform
Dissolution, sequentially adds petroleum ether, methylene chloride, ethyl acetate, water saturated extracting n-butyl alcohol, each 300mL, continuous extraction 3
Secondary, extraction remainder is water section, recycles each position extractant in being evaporated on Rotary Evaporators, then be dried under vacuum to it is dry,
Then water, water saturated n-butanol, ethyl acetate, methylene chloride, petroleum ether etc. 5 extractions finally are obtained according to polarity size order
Position sample powder.
Testing below by way of specific pharmacodynamic test proves beneficial effects of the present invention.
Instrument and material
1.1 experimental material
Russia color fruit, E Seye, phloridzin, the source of phloretin are commercially available.Propranolol hydrochloride tablets (lot number:1704159) it purchases
From Jiangsu Yabang Aipusen Pharmaceutical Co., Ltd..
1.2 cell strain
Normal liver cell's L-02 cell strain derives from U.S. ATCC company.
1.3 reagent reagents
Medical fat cream (lot number F17020110) is purchased from Kelun Pharm Ind Co., Ltd., Sichuan, dimethyl sulfoxide
(DMSO) it is purchased from BioFROXX company of Germany, total cholesterol (TC) kit is purchased from Beijing Puli lema gene Technology Co., Ltd.,
Triglycerides (TG) kit, total protein quantitative determination reagent kit (microplate method) are purchased from Nanjing and build up Bioengineering Research Institute,
RPMI-1640 culture medium, phosphate buffer (PBS) and trypsase are purchased from HESlone company, and fetal calf serum (FBS) is purchased from
Huhehaote City grassland green field biological engineering material Co., Ltd.Remaining reagent is that analysis is pure.
1.4 instrument and equipment
Inverted microscope (German leica company), cell incubation case (Thermo company of the U.S.), multi-function microplate reader (beauty
Thermo company of state), pipettor (German Eppendorf company), AllegraX-12series centrifuge (U.S. BECK MAN
Coulter company), superclean bench (Beijing hundred million reaches Kechuang Science and Technology Ltd.), excellent general serial Superpure water machine (the ultrapure section in Chengdu
Skill Co., Ltd).
The preparation of 1.5 solution
The preparation of complete culture solution:Fetal calf serum 10mL and RMPI-1640 culture medium 90mL are mixed up to (containing
100U.mL-1Penicillin, 100 μ g.mL-1Streptomysin).
The preparation of 0.2% serum RPMI-1640 culture solution:By fetal calf serum 0.2mL and RMPI-1640 culture medium
99.8mL mixing up to (containing 100U.mL-1Penicillin, 100 μ g.mL-1Streptomysin).
The preparation of medical fat cream induction liquid:Fetal calf serum 10mL and 20% medical fat cream 5mL is mixed, serum-free is used
RMPI-1640 be diluted to 100mL to get.
2 methods
The preparation of 2.1 samples
2.1.1 each solvent extraction position of " Russia's color " Different plant parts prepares (preparation of embodiment 1)
2.1.2 each solvent extraction position of " Russia's color " Different plant parts is prepared
After Russia color fruit and the total extract part of E Seye and 5 extraction position sample powders are dissolved with DMSO respectively, Yu Shi
It is configured to low dose group (25 μ g.mL respectively with the RPMI-1640 culture solution of 0.2% serum before testing-1), middle dose group (50 μ
g.mL-1), high dose group (100 μ g.mL-1) 3 concentration test sample, 0.22 μm of miillpore filter filtration, packing to sterile vials
It is sealed spare to 4 DEG C of refrigerators.
2.1.3 the preparation of phloridzin, phloretin and Atorvastatin test sample
Precision weighs appropriate phloridzin and phloretin, is configured to low dose respectively with the RPMI-1640 culture solution of 0.2% serum
Amount group (12.5 μ g.mL-1), middle dose group (25 μ g.mL-1), high dose group (50 μ g.mL-1) 3 concentration test sample.Precision claims
Appropriate Atorvastatin is taken, being configured to concentration respectively with the RPMI-1640 culture solution of 0.2% serum is 10 μ g.mL-1For examination
Product.
2.2L-02 cell culture is passed on, is frozen, recovering
2.2.1 the culture of cell
Using 25cm2Tissue Culture Flask L-02 cell is cultivated, be placed in 37 DEG C, saturated humidity and 5%CO2Cell
In incubator, to carry out routine culture containing 10% FBS RPMI-1640 culture medium.Cellular morphology and upgrowth situation are seen
It examines, according to its growth conditions, change fresh medium is primary within every 1~2 day, and experimental implementation is carried out in aseptic operating platform.
2.2.2 the passage of cell
The growing state that cell is observed under inverted microscope covers with bottom of bottle when observing that L-02 cell is covered with or approached
When, it in aseptic superclean bench, discards old culture solution, after PBS rinse 1~2 time, 0.25% pancreatin 1mL is added, 37 DEG C to thin
Born of the same parents digest, and after about 1~2min, gently shake culture bottle, microscopically observation is rounded to cellular morphology, and intercellular spaces become larger, a
When other cell detachment, illustrate that cell dissociation is more abundant, the complete medium that pre-temperature is added immediately terminates digestion.Use liquid relief
Rifle blows and beats the bottom of culture bottle, so that cell is shattered into cell suspension, is transferred in 10mL centrifuge tube, 1000rpm.min-1Centrifugation
3min abandons supernatant, and fresh culture 1mL is added, and piping and druming, which mixes, is dispersed in cell individually, passes 3 or 1 ratio for passing 5, transfer in 1
Part suspension sucks 3mL~4mL fresh complete medium, is put into secondary culture in cell incubator, about into new culture bottle
Passage in 1~2 day is primary.
2.2.3 cell freezes
The L-02 cell (proxima luce (prox. luc) changes old culture solution) of logarithmic growth phase, discards cell culture medium, by this chapter
Digestion method under " 2.2.2 " item is digested, 1000rpm.min-1It is centrifuged 3min, supernatant is inhaled and is abandoned, frozen stock solution is added
(DMSO:Complete medium=1:9), gently piping and druming is allowed to even suspension, and cell suspension is dispensed by the amount of every pipe 1mL to cell
It is saved in cryopreservation tube, after gradient cooling (4 DEG C of 30min, -20 DEG C of 2h, -80 DEG C overnight), is put into liquid nitrogen container and freezes.
2.2.4 the recovery of cell
L-02 cell cryopreservation tube is taken out from liquid nitrogen container rapidly, makes its thawing in 37 DEG C of water-baths, is melted completely to it,
It is quickly transferred in aseptic superclean bench and is operated, cell suspension is transferred in centrifuge tube, be put into 10 times of culture mediums, gently
Mixing is played in featheriness, the 1000rpm.min in centrifuge-1It is centrifuged 3min, discards supernatant liquid, 4~5mL of complete medium is added, gently
It after mixing is played in featheriness, is transferred in culture bottle, is put into culture in cell incubator.
The foundation of the external hyperlipidemia model of 2.3 L-02 hepatocytes
0.25% trypsin digestion of the L-02 hepatocytes of logarithmic growth phase, is matched after cell count with normal culture solution
At individual cells suspension, with every hole 3 × 105The density of a cell is inoculated in 6 well culture plates, and every hole nutrient solution volume 2mL is moved
Enter in cell incubator, in 37 DEG C, saturated humidity and 5%CO2Under the conditions of, culture 48h makes its abundant adherent growth, long to cell
To when 80% fusion, changing the only RPMI-1640 culture solution culture containing 0.2% serum into for 24 hours, makes cell cycle synchronization.Just
Normal control group continues to give the RPMI-1640 culture solution culture 48h containing 10%FBS, and model group gives the medical rouge containing 10%FBS
After fat cream RPMI-1640 induction broth culture 48h, the external hyperlipidemia model of L-02 hepatocytes can be established.
2.4 groupings and administration
It by L-02 hepatocytes according to " 2.3 " the method, is inoculated in 6 well culture plates, if blank group, model group, Russia's color
Each solvent extraction position fruit, E Seye, phloridzin, phloretin and positive control Atorvastatin group.Blank group is every with model group
Continue to give the normal culture solution of 2mL in hole;The culture solution 2mL of drug containing respective concentration progress is given in administration group and the every hole of positive controls
Culture.3 multiple holes of each concentration, incubation time 48h.
The measurement of TC, TG content in 2.5L-02 cell
After cultivating 48h, culture medium is discarded, is washed cell 1~2 time, extra PBS is blotted, every hole adds with the PBS of pre-cooling
Enter 200 μ L cell pyrolysis liquids, mix, stands 10min.The lysate of collection presses total cholesterol (TC) kit, triglycerides
(TG) kit measurement TG and TC content.
2.6 statistical method
Experimental result is with " mean+SD "It indicates.It is mapped, is used using GraphPad Prism 6
SPSS18.0 software is analyzed, and one-way analysis of variance is used between group, is compared two-by-two using LSD method.With P<0.05 table
Show that difference has statistical significance.
3 results
3.1 establish L-02 cellular fat fibrosis models
After medical fat cream induces L-02 cell 48h, observed under inverted microscope, control group:Iuntercellular combines tight
Close, edge clear is seamless, and intracellular fat drips are less (Figure 1A).Model group:Compared with normally group cell, model group cell is bright
Aobvious enlargement, cell outline is fuzzy, and iuntercellular, which combines, owes close, and intracellular fat drips are more and circlewise surround cell, have fat drips to melt
It closes phenomenon (Figure 1B).Lipid within endothelial cells assay is the results show that intracellular TG, TC content of medical fat cream induction group significantly rises
Height (P<0.01) it, is shown in Table 1, table 2 and table 3, shows after inducing 48h by medical fat cream, has been successfully established L-02 cellular fat
Fibrosis models.
3.2 each solvent extraction positions of " Russia's color " Different plant parts influence 3.2.1 Russia color fruit, Russia to TG, TC in liver cell
The total extract part of color Ye influences TG, TC in liver cell
Compared with blank control group, model group TG, TC significantly rises (P<0.01), illustrate L-02 hepatocytes mould high in fat in vitro
Type modeling success.Compared with model group, it is thin that Russia's color fruit middle dose group and high dose group can significantly reduce steatosis liver L-02
Content (the P of TG intracellular<0.05, P<0.01), Russia's color fruit low dose group and E Seye low, middle and high dose groups cannot reduce carefully
The content of TG intracellular.E Seye low, middle and high dose groups and Russia's color fruit low dose group and high dose group can reduce steatosis liver
Content (the P of the intracellular TC of L-02<0.01), Russia's color fruit middle dose group not can significantly reduce intracellular TC content (P > 0.05).Knot
Fruit is shown in Table 1, Fig. 2 and Fig. 3.
In reduction steatosis L-02 hepatocytes in terms of TG content, Russia color fruit is substantially better than E Seye;Reducing fat
It is denaturalized in L-02 hepatocytes in terms of TC content, E Seye is better than Russia's color fruit.Russia color fruit can reduce TG, TC content simultaneously, and Russia's color
Leaf is only capable of reducing TC content, therefore Russia color fruit is better than E Seye for the curative effect of steatosis L-02 hepatocytes, can preferably mitigate
The steatosis of L-02 hepatocytes.
1 Russia color fruit of table and E Seye total extract part intracellular TG, TC content of rouge denaturation L-02 is influenced (N=3)
Note:Compared with blank group, * * P<0.01, * P<0.05;Compared with model group,##P<0.01,#P<0.05, similarly hereinafter
3.2.2 each solvent extraction position Russia's color fruit, E Seye influences TG, TC in liver cell
Compared with model group, Russia's color fruit water extract part (high dose group) and ethyl acetate extract part (low dose group) are removed
The content (P > 0.05) of TG in steatosis L-02 hepatocytes is not can significantly reduce outside, remaining each dosage group can significantly reduce
Content (the P of intracellular TG<0.05 or P<0.01).Compared with model group, Russia's color fruit water extract part (low dose group), n-butanol
Extract part (low, high dose group), ethyl acetate extract part (middle dose group), methylene chloride extract part (high dose group),
Petroleum ether extract part (middle and high dosage group) can significantly reduce the content (P of TC in steatosis L-02 hepatocytes<0.05 or P
<0.01), remaining each group administration front and back, TC content there are no significant difference (P > 0.05).It the results are shown in Table 2 and table 3, Fig. 4, figure
5, Fig. 6, Fig. 7.
In terms of reducing TG content, each solvent extraction position of Russia color fruit can significant low intracellular TG content, wherein dichloro
Methane extract part and petroleum ether extract part are more excellent, and ethyl acetate extract part takes second place, and water extract part and n-butanol extract
It is relatively weak that position reduces the effect of TG content;In terms of reducing TC content, each solvent extraction position of Russia color fruit can be in various degree
The intracellular TC content of reduction, wherein petroleum ether extract part and methylene chloride extract part are more excellent, water extract part, n-butanol
Extract part, ethyl acetate extract part take second place.Totally it was found that, Russia's color fruit methylene chloride extract part and petroleum ether extract
Position lipid-lowering effect is preferable.Each solvent extraction position result of Russia color fruit can significantly reduce TC, TG with the Russia total extract part of color fruit and contain
Amount, and reduce TG effect relatively to make good use of result almost the same by reduction TC.
2 each solvent extraction position of Russia color fruit of table to rouge denaturation intracellular TG, TC content of L-02 variation (N=3)
Each solvent extraction position 3 E Seye of table and monomer to the variation of rouge denaturation intracellular TG, TC content of L-02 (n
=3)
4 discuss
Hyperlipidemia refers to blood cholesterol (TC) and/or triglycerides (TG) is excessively high or high-density lipoprotein cholesterol
(HDL-C) too low, modern medicine is referred to as dyslipidemia.TC and TG is the most lipid components of people's in-vivo content, content
Exception is the important pathologic basis of hyperlipidemia morbidity, therefore this experiment emphasis has detected influence of each drug to TC, TG content.
The L-02 hepatocytes rouge varying model TG concentration established using medical fat cream molding method is high, and steatosis rate is high, carefully
Born of the same parents' form is good, and medical fat breast valency lattice are low, low to cytotoxicity, draws materials and easy to use, is that a kind of good liver L-02 is thin
Born of the same parents' steatosis inducer.After this experiment is incubated for L-02 cell 48h using the complete medium of the cream Han 1% medical fat, induction
It forms external cell model high in fat, and stability of characteristics.Lipid within endothelial cells level is able to reflect the height of lipids contents, therefore will
In-vitro screening and verifying of the cell model high in fat for blood lipid-lowering medicine, can substantially shorten the period of fat-reducing effect evaluation.
This experiment system has carried out lipidemia research to Russia color fruit for the first time both at home and abroad, and research finds the total extract part of Russia color fruit
And each solvent (water, n-butanol, ethyl acetate, methylene chloride, petroleum ether) extract part can reduce medical fat in various degree
TG, TC content in the L-02 hepatocytes of cream induction illustrate Russia color fruit difference extract to triglycerides (TG), cholesterol (TC)
Synthesis all has good regulating and controlling effect, has preferable Lipid-lowering activities.
Experimental result shows that E Seye can significantly reduce TC content in denaturation L-02 hepatocytes, but cannot reduce TG content.
E Seye cannot reduce intracellular TG content under this experimental administration dosage, but increasing dosage may can reduce intracellular TG
Content.Russia color fruit can significantly reduce intracellular TG, TC content simultaneously, although Russia color fruit reduces TC, effect is weaker than E Seye, from
For reducing from the aspect of TG, TC two, Russia's color fruit Lipid-lowering activities are better than E Seye.
5 brief summaries
(1) the total extract part of Russia's color fruit can reduce TG, TC content in rouge denaturation L-02 hepatocytes simultaneously, but drop TC content and make
With being weaker than the total extract part of E Seye.The total extract part of E Seye not can significantly reduce TG content in rouge denaturation L-02 hepatocytes,
But intracellular TC content can be reduced and effect is extremely significant.Russia's color fruit effect for reducing fat is better than E Seye.
(2) each solvent of Russia's color fruit (water, n-butanol, ethyl acetate, methylene chloride, petroleum ether) extract part can different journeys
Degree reduces TG, TC content in rouge denaturation L-02 hepatocytes, has certain Lipid-lowering activities.Wherein in terms of reducing TG content, with
Russia's color fruit methylene chloride extract part, petroleum ether extract part activity are stronger, and ethyl acetate extract takes second place, water position, n-butanol
Position influences intracellular TC content relatively weak;In terms of reducing TC content, petroleum ether part, methylene chloride position activity compared with
By force, water position, n-butanol portion, ethyl acetate extract take second place.Russia's color fruit methylene chloride position and petroleum ether part lipid-lowering effect
It is relatively strong.
Claims (5)
1. Russia color fruit and its extract are preparing the purposes in blood lipid-lowering medicine.
2. purposes according to claim 1, it is characterised in that:The Russia color fruit is rosaceous plant Malus toringoides Hughes
Malus toringoides (Rehd.) Hughes and tibetan crabapple Malus transitoria (Batal.) Schneid.'s is dry
Dry ripening fruits.
3. purposes according to claim 2, it is characterised in that:Russia's color berry extract is total extract position, water
Extract part, n-butanol extract part, ethyl acetate extract, methylene chloride extract part, petroleum ether extract part.
4. purposes according to claim 3, it is characterised in that:Russia's color berry extract is methylene chloride position or stone
Oily ether position.
5. purposes according to claim 3, it is characterised in that:The preparation method of Russia's color berry extract is:
A, it takes Russia color fruit to be sieved, 95% ethyl alcohol is added and extracts, filtering, residue adds water decoction, filters;Second is recovered under reduced pressure in alcohol extract
Alcohol is to no alcohol taste;Water mentions filtrate and is concentrated to give medicinal extract, and extract dry is total extract part;
B, medicinal extract is dispersed into alcohol-extracted extract with Aqueous extracts concentrate, makes its full and uniform dissolution, sequentially adds petroleum ether, dichloromethane
Alkane, ethyl acetate, water saturated extracting n-butyl alcohol, extraction remainder are water section, recycle each position extractant in rotation
It is evaporated on evaporimeter, then is dried under vacuum to dry, then finally obtain water, water saturated n-butanol, acetic acid according to polarity size order
Ethyl ester, methylene chloride, petroleum ether part.
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CN114432370A (en) * | 2020-11-02 | 2022-05-06 | 四川智佳成生物科技有限公司 | Russian fruit extract and pharmaceutical application thereof |
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