CN108210532A - Bupleurum extract is preparing the application in adjusting the drug of metabolism and fat reducing health products - Google Patents
Bupleurum extract is preparing the application in adjusting the drug of metabolism and fat reducing health products 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
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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Abstract
The present invention relates to pharmaceutical field, more particularly to a kind of bupleurum extract is preparing the application in adjusting the drug of metabolism and fat reducing health products.The application of bupleurum extract is excavated, application of the bupleurum extract in adjusting metabolism has been opened up, has especially been their ability to the expression of up-regulation fibroblast growth factor FGF21, promotes liver intake HDL, then the adjusting of realization lipid-metabolism.
Description
Technical field
The present invention relates to pharmaceutical field, more particularly to a kind of bupleurum extract is preparing the drug for adjusting metabolism and fat reducing
Application in health products.
Background technology
Long-term high fat diet custom can cause the excessive accumulation of fat to lead to obesity, same to be supported often along with insulin
The diseases such as anti-, fatty liver, atherosclerosis, nowadays the drug of most tune fat is lipid-lowering statins in the market, CETP inhibits
The chemicals of the list targeted therapy such as agent, have the drawbacks such as side effect is big.Fibroblast growth factor 21
(Fibroblastgrowthfactor21, FGF21) is novel regulation and control of the specific effect in tissues such as liver, pancreas, fat
The factor, it is related to obesity, diabetes, insulin resistance, fatty liver diseases, significantly reduce fasting blood-glucose, insulin, sweet
The effects that oily three esters, weight levels and benign change lipoprotein, exogenous FGF21 can be done directly in vivo, cause a system
Row physiological effect.But the exogenous FGF21 prices of recombination are costly, patient buys difficult, and does not use recombination classes Western medicine also not
Conducive to physical function.Exploitation studies its extraction and prepares crucial skill with the drug for adjusting the HypercholesterolemicRats intervention functions factor
Art, activity rating and the application in terms of metabolic syndrome is prevented and treated have Important Economic and social value.
Invention content
The purpose of the present invention is to provide application of the bupleurum extract in the drug for adjusting metabolism is prepared, can be further
Improve application range of the bupleurum extract in medicine.
The present invention relates to application of the bupleurum extract in the drug for adjusting metabolism is prepared.
It is related to application of the bupleurum extract in fat reducing health products are prepared.
Beneficial effects of the present invention are：
The present invention excavates the application of bupleurum extract, has opened up bupleurum extract in the application for adjusting metabolism, spy
It is not their ability to the expression of up-regulation fibroblast growth factor FGF21, promotes liver intake HDL, then realizes lipid-metabolism
It adjusts.
Description of the drawings
It in order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to institute in embodiment
Attached drawing to be used is needed to be briefly described.
Fig. 1 is radix bupleuri water extract chromatogram；
Fig. 2 is radix bupleuri alcohol extracting saponin(e chromatogram；
Fig. 3 is IncuCyteZOOM cell observation timing shooting results；
Fig. 4 is influence (40 ×) of the radix bupleuri water extract to HepG2 cellular uptakes DiI-HDL；
Fig. 5 is influence (40 ×) of the radix bupleuri alcohol extracting saponin(e to HepG2 cellular uptakes DiI-HDL；
Fig. 6 is that flow cytometry evaluates the influence figure that radix bupleuri water extract absorbs HepG2 DiI-HDL；
Fig. 7 is that flow cytometry evaluates the block diagram that radix bupleuri water extract absorbs HepG2 the influence of DiI-HDL；
Fig. 8 is that flow cytometry evaluates the influence figure that radix bupleuri alcohol extracting saponin(e absorbs HepG2 DiI-HDL；
Fig. 9 is that flow cytometry evaluates the block diagram that radix bupleuri alcohol extracting saponin(e absorbs HepG2 the influence of DiI-HDL；
Figure 10 is radix bupleuri water extract cell membrane solid phase chromatography stacking chart (λ=204nm)；
Figure 11 is radix bupleuri water extract cell membrane solid phase chromatography stacking chart (λ=254nm)；
Figure 12 is the secondary ion flow graph (saikoside b2) for dissociating ingredient P3；
Figure 13 is the secondary ion flow graph (saikoside b1) for dissociating ingredient P4；
Figure 14 is radix bupleuri alcohol extracting saponin(e cell membrane solid phase chromatography stacking chart (λ=204nm)；
Figure 15 carries out micrograph (HE × 400) for mouse left lobe of liver pathological tissue；
Figure 16 summarizes for the differential gene of radix bupleuri administration group and model group；
Figure 17 is BCA kit standard curves；
Figure 18 is the expression of induction FGF21 after the administration of radix bupleuri water extract.
Specific embodiment
Purpose, technical scheme and advantage to make the embodiment of the present invention are clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, builds according to normal condition or manufacturer
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Some researches show that reverse cholesterol transport (Reversecholesteroltransport, RCT) can prevent cholesterol
Injury of blood vessel caused by accumulation.Reverse cholesterol transport refers to that cholesterol extra in vivo flows out to apolipoprotein from peripheral cells,
Liver is transported to by high-density lipoprotein (High-densitylipoprotein, HDL), lipid therein is absorbed by liver cell
And the process removed.Internal RCT includes liver intake HDL, Cholesterol Efflux, cholesterol esterification and cholesterol and removes four mistakes
Journey, HDL play obvious action during this.To atherosclerosis (As), (lipidosis is Atherosclerosis to HDL
Change lesion basis) prevention be beneficial, RCT can be promoted by being primarily due to HDL.Relative to low-density lipoprotein (Low-
Densitylipoprotein, LDL), HDL is considered to have anti-As effects, after fully LDL levels are reduced, HDL levels and the heart
Angiogenesis rate is negatively correlated.
Although the liver of traditional Chinese medicine is not equal to anatomical liver, the two relevance is very strong, and traditional Chinese medical science Viscera-State Doctrine thinks liver
Main catharsis, main store blood, happiness item reach and dislike depression.Liver controlling conveyance and dispersion, referring to irritability has the comprehensive lifes such as dredging, item reaches, liter is sent out, freely lets out
Function is managed, the metabolism of liver inner lipid, harmonizing emotions can be stablized, liver dysfunction can then lead to the symptoms such as disorders of lipid metabolism, depressed emotion.
So for treating the Chinese medicine of abnormalities of sugar/lipid metabolism, most doctors, which accept, adjusts fat to adjust liver in the ban, for close with lipid metaboli
Cutting relevant disease has doctor to propose that the viewpoint of treat from liver is also based on the theory of traditional Chinese medical science of liver controlling conveyance and dispersion.《Dong-eui-bo-gam》Meaning
" residual air of liver is let out in courage, is gathered into essence ", illustrating the generation of bile acid in reverse cholesterol transport link depends on irritability catharsis,
Once liver dysfunction certainly will influence reverse cholesterol transport.
Radix bupleuri (BupleuriRadix) is Umbelliferae (Umbelliferae) plant radix bupleuri (Bupleurumchinense)
Or the dry root of radix bupleuri scorzoneraefolii (BupleurumscorzonerifoliumWilld.), acrid flavour, hardship are slightly cold, channel tropism is in liver, lung
And gallbladder channel.Clinical data shows, is the bupleurum powder for relieving liver-qi of monarch drug in a prescription, Xiao Chaihu Tang and radix bupleuri Wendan Tang in non-alcoholic fat using radix bupleuri
There is significant curative effect in the treatment of fat liver, therefore, inventor guesses that HypercholesterolemicRats process is adjusted in radix bupleuri.But existing skill
Radix bupleuri is to carry out the effect of compatibility realizes dispersing stagnated hepatoqi with other drugs substantially in art, and for radix bupleuri itself or its extraction
The dispersing stagnated hepatoqi of object promotes the research of lipid-metabolism also undisclosed.
To sum up, it finds that bupleurum extract can effectively facilitate reverse cholesterol transport by the research of the present inventor, improves liver
The dirty intake to HDL, then regulating lipid metabolism, finally realizes the adjusting of human metabolism function.And preferably, which extracts
Object is radix bupleuri water extract.
Specifically, the crude drug of radix bupleuri mainly contains saikosaponin a (Saikosaponina, SSa) and saikoside d
(Saikosaponind, SSd), at 2015 editions《Chinese Pharmacopoeia》In to the quality standard of Radix Bupleuri using saikosaponin a, d to refer to
Mark property ingredient, illustrates in the prior art substantially using primary saponin(e a, d of radix bupleuri as index, and is the ingredient of chief active.But
It is to find that radix bupleuri easy recurring structure in decoction process changes by research, saikosaponin a and saikoside d can be transformed into bavin
Hu saponin(e b2 (Saikosaponinb2, SSb2) and saikoside b1 (Saikosaponinb1, SSb1), carries in particular with water
Saponin constituent is mainly saikoside b2 and saikoside b1 in the radix bupleuri water extract being prepared, and radix bupleuri is carried out using alcohol
Extract in obtained alcohol extracting thing is mainly saikosaponin a and saikoside b.Inventor studies the water extract for finding only radix bupleuri
Can be with regulating lipid metabolism, and be the expression for promoting related endogenous metabolic factors FGF21, and increase intake of the liver to HDL,
Then regulating lipid metabolism.
Specifically, the present invention provides application of the bupleurum extract in the drug for adjusting metabolism is prepared.Further, the tune
The drug of section metabolism is the drug of regulating lipid metabolism.
The drug of regulating lipid metabolism is by promoting reverse cholesterol transport regulating lipid metabolism.
The drug of regulating lipid metabolism is by adjusting FGF21 regulation of secretion lipid-metabolisms.
And the bupleurum extract of the present invention is saikoside class compound, the preferably secondary saponin(e of radix bupleuri, more preferably bavin
Hu saponin(e b1 and saikoside b2.
It by radix bupleuri and water according to mass ratio is 1 that the extract of radix bupleuri of the present invention, which is,:It is heated to reflux after the ratio mixing of 8-12
It is prepared after water-bath concentration after 1-2 hours.And the temperature being heated to reflux is 75-90 DEG C, and is soaked before being heated to reflux
Bubble causes the abundant water swelling of radix bupleuri in 30-45 minutes, is dissolved out convenient for active constituent.The concentrate that water-bath is concentrated to give is carries originally
Take the 1/3-1/4 of liquid.
The present invention also provides a kind of application of bupleurum extract in fat reducing health products are prepared.
Embodiment 1
It is close to weigh Radix Bupleuri 20.0g, it puts in 500ml Backflow bottles, adds water 200ml, impregnate 30min, be heated to reflux 1h,
Filtration, water-bath are concentrated to give bupleurum extract, and the multiple of concentrate is the 1/3 of original extracting solution multiple, the temperature being heated to reflux
It is 75 DEG C.
Embodiment 2
It is close to weigh Radix Bupleuri 20.0g, it puts in 500ml Backflow bottles, adds water 160ml, impregnate 45min, be heated to reflux
1.5h, filtration, water-bath are concentrated to give bupleurum extract, and the multiple of concentrate is the 1/4 of original extracting solution multiple, is heated to reflux
Temperature is 90 DEG C.
Embodiment 3
It is close to weigh Radix Bupleuri 20.0g, it puts in 500ml Backflow bottles, adds water 240ml, impregnate 40min, be heated to reflux 2h,
Filtration, water-bath are concentrated to give bupleurum extract, and the multiple of concentrate is the 1/4 of original extracting solution multiple, the temperature being heated to reflux
It is 85 DEG C.
Comparative example：Radix bupleuri is lifted using alcohol, precision weighs Radix Bupleuri 20.0g, puts in 250ml conical flask with cover, adds and contain
The methanol solution 100ml of 8% strong ammonia solution, close plug, ultrasonic 30min, filtration, the dregs of a decoction add the methanol solution of 8% strong ammonia solution again
100ml, close plug, ultrasonic 30min, filtration merge filtrate twice.
Experimental example
By the bupleurum extract of embodiment 1,50ml measuring bottles are transferred to, are diluted with water to scale, are shaken up, labeled as radix bupleuri water
Extract.
By the filtrate water bath method of comparative example twice, residue is dissolved in water and is transferred to 50ml measuring bottles, is diluted with water to quarter
Degree, shakes up, labeled as radix bupleuri alcohol extracting thing.
Experimental example 1：Characterization
The bupleurum extract of embodiment 1 and comparative example is characterized, referring to Fig. 1 and Fig. 2, Fig. 1 is radix bupleuri water extract
Collection of illustrative plates when chromatogram, wherein A are 204nm, collection of illustrative plates when B is 254nm；Fig. 2 is radix bupleuri alcohol extracting saponin(e chromatogram, and wherein A is
Collection of illustrative plates during 204nm, collection of illustrative plates when B is 254nm.
As can be seen from FIG. 1, radix bupleuri water extract characteristic spectrum visible 8 shared peaks (Fig. 2-A) under wavelength 204nm, through pair
Confirm according to product, it may be determined that peak S6 is saikosaponin a.In addition peak S6 (saikosaponin a) separating degree is good, and peak type is preferable, therefore selects
It calculates the relative retention time of each characteristic peak as the reference peak under wavelength 204nm.The opposite reservation of each characteristic peak
Time fixes tentatively：1.000 (peak S6), 0.370 (peak S1), 0.503 (peak S2), 0.690 (peak S3), 0.811 (peak S4), 0.875
(peak S5), 1.034 (peak S7), 1.317 (peak S8).
By the visible 6 shared peaks (Fig. 2-B) of the chromatogram of wavelength 254nm, confirm through reference substance, it is radix bupleuri soap to learn peak P3
Glycosides b2, peak P4 are saikoside b1, and in addition peak P3 (saikoside b2) peak area is larger, and separating degree and peak type are preferable, therefore are selected
It is selected as the reference peak under wavelength 254nm, and calculates the relative retention time of each characteristic peak.When each characteristic peak retains relatively
Between fix tentatively：1.000 (peak P3), 0.358 (peak P1), 0.877 (peak P2), 1.095 (peak P4), 1.197 (peak P5), 1.274 (peaks
P6)。
As can be seen from FIG. 2, radix bupleuri alcohol extracting saponin(e characteristic spectrum visible 7 shared peaks (Fig. 2-A) under wavelength 204nm, warp
Reference substance confirms, it may be determined that peak M4 is saikosaponin a, and peak M6 is saikoside d.In addition peak M4 (saikosaponin a) separating degree is good
Good, peak type is preferable, therefore the reference peak being selected as under wavelength 204nm, and calculates the relative retention time of each characteristic peak.Respectively
The relative retention time of a characteristic peak is fixed tentatively：1.000(M4)、0.704(M1)、 0.819(M2)、0.889(M3)、1.184
(M5), 1.257 (M6) and 1.312 (M7).
By the visible 2 shared peaks (Fig. 2-B) of the chromatogram of wavelength 204nm, confirm through reference substance, it may be determined that retention time
The chromatographic peak of 53.9min is saikoside b2.Compared to wavelength 204nm and 254nm, radix bupleuri alcohol extracting saponin(e 204nm characteristic peaks compared with
It is more, prompt radix bupleuri alcohol extracting saponin(e that wavelength 204nm is preferably selected to detect.
In conclusion the ingredient in radix bupleuri water extract based on saikoside b1 and saikoside b2, exists simultaneously less
The saikosaponin a of amount, and in alcohol extracting saponin(e based on saikosaponin a and saikoside d.
Experimental example 2：Liver cell absorbs the detection and analysis of DiI-HDL
Precision pipettes radix bupleuri water extract and radix bupleuri alcohol extracting thing respectively, and storing solution is placed in 5ml centrifuge tubes, nitrogen in right amount
Residue adds appropriate DMSO hydrotropies after drying, adds in blank culture solution, and concussion 0.22 μm of the mistake after residue fully dissolves that is vortexed is sterile
Filter membrane to obtain the final product.
HepG2 cells add in the sugared culture solutions of a certain amount of DMEM high (containing 10% fetal calf serum), are placed in culture bottle, 37
DEG C, it is cultivated in 95% incubator of 5%CO2 relative humidity.The cell strain category human liver cell system, the adherent growth in culture solution, one
As 2-4d or so HepG2 cells are adherent reaches more than 80%, visible polygon, refractivity are good under inverted phase contrast microscope
It is primary to replace culture solution per 2d for bright cell.
(1) timed shooting HepG2 cellular uptakes DiI-HDL processes：It is thin that human liver cell HepG2 is inoculated in Corning6 holes
Born of the same parents' culture plate, 10 × 104/hole of cell density are incubated for 24 hours in cell incubator, and after cell is adherent, 10 μ are added in per hole
GDiI-HDL puts IncuCyteZOOM long-times cell observation and Functional assay system (Channelgreen) captured in real-time, point
It does not pinpoint and shoots in 0,1,2,3,4 and 5h, amplification factor 20 ×.Specific testing result is referring to Fig. 3, Fig. 3 IncuCyteZOOM
Cell observation timing shooting result.
From the figure 3, it may be seen that after DiI-HDL fluorescence lipoprotein is incubated 5h jointly with HepG2 cells, absorbed through liver cell from extracellular
(Fig. 3-A) is transferred to intracellular (Fig. 3-F), and (A-F is followed successively by 0h, 1h, 2h, 3h, 4h, 5h) and fluorescence intensity has no quenching phenomenon,
Later experiments can this cell behavior and fluorescent characteristic be used to evaluate influence of the saikoside extract to liver cell intake HDL.
(2) fluorescence microscope each group HepG2 cellular uptakes DiI-HDL is utilized：Human liver cell HepG2 is inoculated in
MilliporeMillicellEZSlide8 holes laser co-focusing culture dish, inoculum density treat that cell pastes per 2.5 × 104, hole
After wall plus relative medicine is handled for 24 hours, adds in 10 μ gDiI-HDL per hole thereafter.After 37 DEG C are incubated 5h, culture solution, sterile PBS are discarded
After washing 2 times, glass cover-slip is covered, puts and just puts fluorescence microscopy Microscopic observation HepG2 cellular uptake DiI-HDL situations, detected
As a result referring to Fig. 4-Fig. 5, Fig. 4 is influence (40 ×) of the radix bupleuri water extract to HepG2 cellular uptakes DiI-HDL, wherein, A is
Control group；B is low dose group (5mg/mL)；C is middle dose group (10mg/mL)；D is high dose group (15mg/mL).Fig. 5 is
Influence (40 ×) of the radix bupleuri alcohol extracting saponin(e to HepG2 cellular uptakes DiI-HDL, wherein, A is control group；B is low dose group
(5mg/mL)；C is middle dose group (10mg/mL)；D is high dose group (15mg/mL).
Fluorescence microscopy Fig. 4 shows that HepG2 cells are after the effect of radix bupleuri water extract, compared with the control group, administration group intracellular
The trend for first enhancing and weakening afterwards is presented with the raising of dosage in fluorescence intensity, tentatively judges radix bupleuri water extract to a certain degree
The ability of upper adjustable HepG2 cellular uptake lipids.
Fluorescence microscopy Fig. 5 shows, compared with the control group, radix bupleuri alcohol extracting saponin(e administration group fluorescence intensity and quantity it is apparent under
Drop, prompts to show inhibiting effect, and certain dose-dependant is presented to HepG2 cellular uptake HDL links, with dosage
It is more apparent to improve inhibiting effect, tentatively judges the ability that radix bupleuri alcohol extracting saponin(e inhibits HepG2 cellular uptake lipids to a certain extent.
(3) flow cytomery HepG2 cellular uptakes DiI-HDL：Human liver cell HepG2 is inoculated in Corning24 holes
Tissue culture plate, 5 × 104/hole, 37 DEG C, after being cultivated for 24 hours under the conditions of 5%CO2, PBS rinsings cell 2 times adds in corresponding dense
The serum-free DMEM high glucose mediums of drug containing are spent, while the DMSO high glucose mediums containing corresponding concentration are added in control wells.37
DEG C, after being cultivated for 24 hours under the conditions of 5%CO2 relative humidity 95%, 2 μ gDiI-HDL, 37 DEG C of incubation 5h are added in per hole.It is floated with cold PBS
It washes cell 1 time, PBSs of the 1mL containing 0.5%BSA and 2mMEDTA is separately added into per hole, 1h is placed in 4 DEG C.It gently blows and beats and collects
Cell is to streaming pipe, and for cell suspension in 4 DEG C, 800rpm centrifuges 10min.Cell is resuspended in 0.5mLPBS, crosses 300 mesh nylon
Fluorescent value through flow cytomery cell after net.Each sample average detects 10000 cells, every group of cell it is relatively glimmering
Luminous intensity represents that specific testing result evaluates radix bupleuri water extract referring to Fig. 6-9, Fig. 6 for flow cytometry with logarithmic integral figure
Influence to HepG2 intakes DiI-HDL, wherein, A is control group；B is low dose group (5mg/mL)；C is middle dose group
(10mg/mL)；D is high dose group (15mg/mL).Fig. 7 evaluates radix bupleuri water extract for flow cytometry and HepG2 is absorbed
The block diagram of the influence of DiI-HDL.Fig. 8 is that flow cytometry evaluates the shadow that radix bupleuri alcohol extracting saponin(e absorbs HepG2 DiI-HDL
It rings, wherein, A is control group；B is low dose group (5mg/mL)；C is middle dose group (10mg/mL)；D is high dose group (15mg/
mL).Fig. 9 is that flow cytometry evaluates the block diagram that radix bupleuri alcohol extracting saponin(e absorbs HepG2 the influence of DiI-HDL.
Referring to Fig. 6-7 it is found that stream data is shown, compared with the control group, HepG2 cells are a concentration of in radix bupleuri water extract
5th, 10 and 15mg/mL be respectively to the opposite uptake ratio of DiI-HDL (168.8 ± 1.415) %, (207.6 ± 4.286) % and
(173.3 ± 4.678) %.It can be seen that HepG2 cells can selectively absorb HDL after the effect of radix bupleuri water extract for 24 hours, with compareing
Group is remarkably reinforced compared to intake.
By Fig. 8 and Fig. 9 it is found that in radix bupleuri alcohol extracting saponin(e group, compared with the control group, HepG2 cells drug concentration for 5,
10 and 15mg/mL be respectively to the opposite uptake ratio of DiI-HDL (90.98 ± 3.673) %, (79.60 ± 1.158) % and
(20.47 ± 2.341) %.Compared with control group, radix bupleuri alcohol extracting saponin(e effect HepG2 cells for 24 hours after, to the intake of HDL not
There is apparent facilitation or even inhibiting effect occur.
In conclusion integrated fluorescence micrograph (qualitative) and Flow cytometry experiments (quantitative) result, it can be seen that extraction side
The difference of formula, drug action is also different therewith, and there is the active constituent for adjusting liver cell intake HDL in radix bupleuri.Radix bupleuri passes through
Water decoct gained extract in the administration range of reasonable benefit/risk can by promote reverse cholesterol transport link (liver absorb
HDL) realize that lipid-metabolism is adjusted, so as to inhibit the formation of atherosclerosis.At the same time, even if in the safe range of administration
Interior, radix bupleuri alcohol extracting saponin(e remains inhibiting effect to HepG2 cellular uptakes HDL, and dose-dependant phenomenon is presented.
Experimental example 3：Promote the Active Components of liver intake HDL
Operating procedure：DMEM high sugar culture solution dilution saikoside extracts, the final concentration of 60mg/ of radix bupleuri water extract
ML, the final concentration of 30mg/mL of alcohol extracting saponin(e.It adds in HepG2 cells, is placed in 75cm2 culture bottles, 37 DEG C, 5%CO2 is opposite
2h is cultivated in 95% incubator of humidity.
After 2h, culture medium is discarded, adds in 4mLPBS buffer solutions, cell is washed, discards cleaning solution, clean repeatedly, until washing
Wash liquid peak without reserve.
Citric acid-disodium hydrogen phosphate buffer solution of addition pH=4 after cell elution, 37 DEG C, 5%CO2 relative humidity 95%
1h is cultivated in incubator.Occur to dissociate and discharge into dissociation solution with the active constituent of cell combination under acid condition.After dissociation
Cell is blown and beaten, collects cell and dissociation solution, 10000r/min centrifuges 10min, takes supernatant spare.
Dissociation solution is collected, nitrogen blows recycling design, and 100 μ L chromatographies methanol redissolve, and 13000r/min high speed centrifugations take supernatant,
HPLC-MS analyses are carried out by following chromatography and Mass Spectrometry Conditions：
LUBEXEcosilC18 chromatographic columns (4.6mm × 250mm, 5 μm), acetonitrile-water carry out gradient elution, second for mobile phase
Nitrile (B)-water (A), 0-15min, 15%-25%B；15-55min, 25%-40%B；55-80min, 40%-55%B；80-
85min, 55%-15%B；85-95min, 15%-15%B；Flow velocity is 1.0mL/min, 25 DEG C of column temperature, 20 μ L of sample size；
Data acquisition scheme：5600+LC30+CDS；Scan type：TOFMS；Scanning range：100-1000；Detection pattern：
Negative；Atomization gas：55psi；Assist gas：55psi；Gas curtain gas：35psi；Atomization temperature：550℃；Ion source：
DuoSpray electric spray ion sources；Ionizing voltage：4500V；Remove cluster voltage：100V；Impact energy：10.
Testing result referring to Figure 10-Figure 14, Figure 10 for radix bupleuri water extract cell membrane solid phase chromatography stacking chart (λ=
204nm), wherein A is saikoside d, and B is saikosaponin a, and C is λ=204nm radix bupleuri water extract characteristic spectrums, D PBS
1st eluent, E are the 9th eluent of PBS, and F is dissociation solution.Figure 11 is superimposed for radix bupleuri water extract cell membrane solid phase chromatography
Scheme (λ=254nm), wherein A is saikoside b2, and B is saikoside b1；C is λ=254nm radix bupleuri water extract characteristic patterns
Spectrum；D is the 1st eluent of PBS；E is the 2nd eluent of PBS；F is the 9th eluent of PBS；G is dissociation solution.Figure 12 is solution
Secondary ion flow graph (saikoside b2) from ingredient P3.Figure 13 is the secondary ion flow graph (saikoside for dissociating ingredient P4
b1).Figure 14 is radix bupleuri alcohol extracting saponin(e cell membrane solid phase chromatography stacking chart (λ=204nm)；A is saikoside d；B is saikoside
a；C is λ=204nm radix bupleuri water extract characteristic spectrums；D is the 1st eluent of PBS；E is the 9th eluent of PBS；F is solution
Chaotropic.
As can be seen from FIG. 10, since being washed the 1st time, the chromatographic peak of unbonded ingredient gradually weakens, until the 9th elution
Substantially there is no chromatographic peak.Dissociation solution (λ=204nm) chromatogram shows that there is no the chromatographic peaks of binding constituents.Figure 11 (λ=
It 254nm) shows, since being washed the 1st time, the chromatography peak-to-peak signal of unbonded ingredient gradually weakens, until the 9th elution is basic
There is no chromatographic peak.Dissociation solution chromatogram shows that HepG2 cells are after the processing of radix bupleuri water extract, and there are 2 colors in binding component
Spectral peak is peak P3 and peak P4 respectively.It is further compared through the characteristic spectrum with water extract, peak P3 and peak P4 are existed simultaneously in bavin
In the characteristic spectrum of Hu water extract, peak P3 and peak P4 in radix bupleuri water extract is prompted to belong to the active constituent at the position, it can
Specific binding is generated, and generate physiological effect with HepG2 cell membranes.
Cell pyrolysis liquid is analyzed by LC-Q-TOF/MS/MS, it has been found that there are 2 chromatographies in HepG2 cell membrane extractions
Peak, and the chromatographic peak is not detected in last 1 eluent, it can thus be assumed that both chemical compositions are from dissection and physiology shape
What HepG2 cellular portions receptor or channel under state disintegrated down, it is washed when using acidity Na2HPO4 buffer solutions (pH4.0)
When, HepG2 cell receptors or channel will inactivate, and binding constituents therefrom release.Cell dissociation buffer is analyzed using LC-TOF/MS
The comparison of (Figure 12 and Figure 13) and reference substance retention time (Figure 10 and Figure 11), ingredient P3 and P4 can be identified as radix bupleuri soap
Glycosides b2 and saikoside b1.
As shown in Figure 14, it is compared by the radix bupleuri alcohol extracting saponin(e characteristic spectrum that early period establishes, HepG2 cell pyrolysis liquids
Do not detect with characteristic spectrum tie element, and have no the main component saikosaponin a and d of alcohol extracting saponin(e, prompt saikosaponin a and
D fails to generate specific adsorption with liver cell.
In conclusion characteristic spectrum and last 1 eluent by comparing radix bupleuri water extract, find the extraction of radix bupleuri water
There are 2 chromatographic peaks (peak P3 and peak P4) in dissociation solution after object processing, through reference substance comparison and Mass Spectrometer Method, peak P3 and peak P4
Differentiate respectively as saikoside b2 and saikoside b1, both for the secondary saponin(e of radix bupleuri, and the primary saponin(e saikoside of radix bupleuri
A and saikoside d do not detect in eluent.Liver intake HDL effect experiment show, the bavin based on the secondary saponin(e of radix bupleuri
Hu water extract has facilitation, and the alcohol extracting thing based on the primary saponin(e of radix bupleuri does not have facilitation, thus can speculate
It is the active constituent for promoting reverse cholesterol transport in radix bupleuri to go out the secondary saponin(e of radix bupleuri.
Experimental example 4：Influence of the radix bupleuri water extract to high fat diet mouse liver gene expression
SPF grades of healthy adult C57BL/6 mouse 24, from Traditional Chinese Medicine University Of Guangzhou's Experimental Animal Center.Experimental animal
Certificate Of Conformance：NO.440058000；Zoopery credit number：SYXK (Guangdong) 2013-0085.
Animal model：Grow up C57BL/6 mouse (8-10 week old), is randomly divided into 3 groups, control group, model group and administration group,
Every group 8.Each group mouse adaptability is raised 1 week, ad lib, drinking-water.Model group is given high lipid food nursing with administration group and is held
Continuous modeling 20 weeks, control group mice feeding normal diet.Administration group is given radix bupleuri water by crude drug amount 0.325g/10g and is carried after modeling
Take object 4 weeks, gavage volume 0.2mL/10g, control group and model group give the distilled water of equivalent, record the weight of mouse weekly.
Mouse liver hematoxylin eosin staining：After each group mouse takes blood, heart perfusion is carried out using physiological saline, treats liver
Dirty perfusion is put in 10% formalin after liver (left lobe of liver) and fixes to after khaki, taking, routine paraffin wax embedding, 5 μm of companies of row
Continuous slice, hematoxylin eosin staining, the pathological change of optical microphotograph Microscopic observation liver.Coloration result is：Cytoplasm is in purplish red
Color, nucleus are in taupe.
Transcript profile sequencing evaluation murine liver tissue gene expression：After mouse dissection, liver other parts are (in right lobe of liver, liver
Leaf and liver caudal lobe) it preserves in juxtaposition ultra low temperature freezer after packing, wherein right lobe of liver part next day is sent to Hua Da cara gene
RNA-sequence detections are carried out, in addition the detection of liver middle period and liver caudal lobe part later stage as protein level.
All experimental datas are for statistical analysis using GraphPadPrism6.0 statistical softwares, and major experimental data are come
It tests, is represented with mean ± SD, P from 3 repetitions<0.05 is significant for difference.
Testing result：
Each group mouse is in feeding process weight in growing steadily, and normal group and administration group mouse hair are in the experimentation later stage
Hair darkly light tone has gloss, model group atrichia gloss, and this group of some animals weight reaches 45g.
It takes a picture to liver organization morphological change, is specifically shown in Figure 15, Figure 15 is carried out for mouse left lobe of liver pathological tissue
Micrograph (HE × 400), wherein, A is control group；B is model group；C puies forward saponin(e group for radix bupleuri water, and arrow shows fat vacuole.By
Figure 15 is it is found that according to group mouse liver cell marshalling, and in polygon, endochylema enriches, and nucleus is high-visible, and lobuli hepatis structure is complete
It is whole, pathological change unobvious.Structure gets muddled in model group lobuli hepatis, occurs apparent steatosis, visible in liver cell
Apparent fat vesicle, cell boundaries are unintelligible, and nucleus is placed in the middle, and part of hepatocytes lacks nucleus.Administration group lobuli hepatis knot
Structure is complete, liver harden structure queueing discipline in leaflet, has no apparent steatosis and necrosis, liver cell structural integrity, extremely slightly
Steatosis, polygon is presented, endochylema enriches, and it is central that nucleus is located at cell.It can be seen that radix bupleuri water extract can inhibit liver
Generation fat cavity improves significantly to the fat lesion tool of high fat diet induction.
According to the gene data of administration group and model group compare, find high fat diet C57BL/6 mouse through radix bupleuri water
After extract gavages, liver mainly has 13 expressing gene differences extremely significantly (p<0.0000), including Gm3776,
Cyp2b9, Lgals1, Actg1, Gstm3, Pfkfb3, Col3a1, Derl3, Moxd1, FGF21, BC090627, Gm15441 and
8 genes such as G0s2 etc., wherein Gm3776, Cyp2b9, Lgals1, Actg1, Gstm3, Pfkfb3, Col3a1, Derl3 are in bavin
(p is extremely significantly lowered after being administered recklessly<0.0000), 5 differential genes such as FGF21, Moxd1, BC090627, Gm15441 and G0s2
Extremely significantly up-regulation (p<0.0000).As shown in figure 16, Figure 16 summarizes (p for the differential gene of radix bupleuri administration group and model group<
0.0000)。
In conclusion pathological section is the results show that compared with the control group, apparent fat occurs for the liver of high in fat group of mouse
Fat is denaturalized, and visible apparent fat vesicle in liver cell, in addition radix bupleuri water extract administration group mouse liver fat lesion is unknown
It is aobvious, show that radix bupleuri water extract has improvement result to the fat lesion of liver.HDL is absorbed with reference to liver cell and cell membrane is consolidated
Phase chromatographic results, the active ingredient of radix bupleuri water extract can be strengthened by generating physiological effect with targeted integration on liver plasma membrane
Liver is to the capture functions of HDL, so as to reach the improvement to fat lesion.
In transcript profile sequencing data, we administration group and model group relatively in detect that 201 genes exist and express
Difference, wherein Gm3776, Cyp2b9, Lgals1, Actg1, Gstm3, Pfkfb3, Col3a1, Derl3, Moxd1, FGF21,
13 gene expression differences such as BC090627, Gm15441 and G0s2 extremely significantly (p<0.0000), wherein Gm3776, Cyp2b9,
8 genes such as Lgals1, Actg1, Gstm3, Pfkfb3, Col3a1, Derl3 significantly lower (p after radix bupleuri administration<
0.0000), 5 differential genes such as Moxd1, FGF21, BC090627, Gm15441 and G0s2 significantly raise (p<0.0000).It surveys
Sequence the result shows that, radix bupleuri promote liver intake HDL may with this 13 genes of differential expression have larger relevance.
The study found that the C57BL/6 mouse of high fat diet feeding through radix bupleuri water extract processing after, internal FGF21 genes
Transcription significantly increases, and prompts the active constituent of radix bupleuri and can inhibit liver lipids synthesis, by the way that FGF21 is induced to generate so as to reach
Treat the effect of fatty liver.In addition, as the endogenous excretion factor, FGF21 belongs to protein coding gene, while there is also the mankind
(Homosapiens) in vivo, this is radix bupleuri in the meeting point of lipid regulation clinical practice, prompts the active constituent of radix bupleuri in human body
The interior expression that can promote FGF21 gene levels increases.
Experimental example 5：The expression of Westernblot verification radix bupleuri water extract inducing mouse liver Fs GF21
Liver organization protein extraction and concentration mensuration：By PMSF (100mM) according to 1：100 ratios are mixed with RIPA lysates
It closes and prepares Tissue lysates.The mouse liver tissue (liver middle period and caudal lobe) for being stored in ultra low temperature freezer is taken out, uses surgical scissors
Clip about 100mg hepatic tissues add in the lysate that 1000 μ L are prepared in advance, will first utilize glass in the glass homogenizer of sterilizing
Liver organization is homogenized by homogenizer on ice, and until 95% tissue is broken, sample is statically placed in trash ice after homogenate
20min is fully dissolved out albumen.Sample is placed on whirlpool instrument and is fully suspended after standing, 4 DEG C, and 12000g/min centrifugation 5min inhale
Supernatant is taken to be put in ultra low temperature freezer and preserve after packing to get tissue total protein product.
Using BCA kit measurement determination of protein concentration, BSA standards are diluted using PBS by kit specification requirement
Product establish BSA protein standard curves (range of linearity 20-2000 μ g/mL), and each group total protein for extracting gained is diluted 10 times
After detect, the protein concentration of each group sample is calculated using standard curve.
Protein immunoblot (Westernblot) detects：Implement according to following steps：SDS-PAGE electrophoresis-albumen turns
Film-nonspecific binding site closing-antibody incubation and immune detection.
The foundation of protein standard curve：Referring to Figure 17, Figure 17 is BCA kit standards curve (R=0.9968), BCA eggs
The standard curve that white kit is established shows that bovine serum albumin(BSA) (BSA) linear relationship in the range of 20-2000 μ g/mL is good,
Measure available for mouse liver sample total protein content.After calculating each sample total protein concentration using standard curve, use
Each sample is diluted to same concentrations (5 μ g/ μ L) by PBS, for late protein Blot experiment.
For specific testing result referring to Figure 18, Figure 18 is expression (* *, the p ＜ of induction FGF21 after the administration of radix bupleuri water extract
0.01), as can be seen from FIG. 18, the active constituent of radix bupleuri water extract has tune to the rna transcription and protein level of mouse liver
Control acts on, and expression pattern is consistent, can increase FGF21 albumen by raising FGF21 gene transcription levels in mouse liver cell and turn over
It translates, so as to improve FGF21 protein levels in liver, it is seen that radix bupleuri water extract can promote the synthesis of FGF21 albumen to realize liver
The adjusting of lipid disorders.
In conclusion bupleurum extract of the embodiment of the present invention is mainly based on saikoside b1 and saikoside b2, simultaneously
There are a small amount of saikosaponin as.DiI-HDL is absorbed to liver cell by comparing radix bupleuri water extract and radix bupleuri alcohol extracting thing simultaneously
Detection and analysis and promote the Active Components of liver intake HDL, it is known that saikoside b1 and saikoside b2 promote liver to take the photograph
HDL is taken, then realizes the adjusting of lipid-metabolism, meanwhile, pass through transcript profile sequencing and protein blot experiment, it is known that radix bupleuri water extracts
Object can raise the expression of FGF21 in mouse liver, can regulating lipid metabolism after adding FGF21 without external source.
Embodiments described above is part of the embodiment of the present invention, instead of all the embodiments.The reality of the present invention
The detailed description for applying example is not intended to limit the range of claimed invention, but is merely representative of the selected implementation of the present invention
Example.Based on the embodiments of the present invention, those of ordinary skill in the art are obtained without creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.
Claims (10)
1. application of the bupleurum extract in the drug for adjusting metabolism is prepared.
2. application according to claim 1, which is characterized in that described to adjust the medicine that the drug being metabolized is regulating lipid metabolism
Object.
3. application according to claim 2, which is characterized in that the drug of the regulating lipid metabolism is by the way that courage is promoted to consolidate
Alcohol counter transport regulating lipid metabolism.
4. application according to claim 3, which is characterized in that the drug of the regulating lipid metabolism is to pass through adjusting
FGF21 regulation of secretion lipid-metabolisms.
5. apply according to any one of claims 1-4, which is characterized in that bupleurum extract is saikoside class chemical combination
Object.
6. application according to claim 5, which is characterized in that by weight, the saikoside class compound includes
The secondary saponin(e of radix bupleuri.
7. application according to claim 6, which is characterized in that the secondary saponin(e of radix bupleuri includes saikoside b1 and radix bupleuri
Saponin(e b2.
8. application according to claim 7, which is characterized in that the bupleurum extract is according to mass ratio by radix bupleuri and water
It is 1:It is prepared after being heated to reflux 1-2 hours after the ratio mixing of 8-12.
9. application according to claim 8, which is characterized in that be heated to reflux rear water-bath concentration.
10. application of the bupleurum extract in fat reducing health products are prepared.
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| CN114903929A (en) * | 2022-06-02 | 2022-08-16 | 西安交通大学 | Use of bupleuri radix and its main active ingredient in preparation of medicine for treating asthma |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101810654A (en) * | 2009-02-24 | 2010-08-25 | 上海医药工业研究院 | Application of vinegar-prepared radix bupleuri solvent extract in cholesterol-reducing drug preparation |
-
2018
- 2018-02-26 CN CN201810164415.0A patent/CN108210532A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101810654A (en) * | 2009-02-24 | 2010-08-25 | 上海医药工业研究院 | Application of vinegar-prepared radix bupleuri solvent extract in cholesterol-reducing drug preparation |
Non-Patent Citations (2)
| Title |
|---|
| 刘树强: "柴胡水煎过程中皂苷成分的变化规律", 《中药新药与临床药理》 * |
| 李娉婷: "柴胡药对配伍对皂苷成分影响及调节RCT的实验研究", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114903929A (en) * | 2022-06-02 | 2022-08-16 | 西安交通大学 | Use of bupleuri radix and its main active ingredient in preparation of medicine for treating asthma |
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