CN108815153A - Composition and its application in the HK-2 cell that protection is damaged by calcium oxalate monohydrate - Google Patents
Composition and its application in the HK-2 cell that protection is damaged by calcium oxalate monohydrate Download PDFInfo
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Abstract
The present invention relates to drug fields, in particular to a kind of composition and its application in the HK-2 cell that protection is damaged by calcium oxalate monohydrate.Composition mainly includes following components by mass percentage：5-11% Wei Caining -1,8-15% Wei Caining -2,8-15% Wei Caining -3,15-60% Schaftoside, 23-45% Isoschaftoside.Composition provided in an embodiment of the present invention protects the HK-2 cell damaged by calcium oxalate monohydrate by the collaboration cooperation of five compounds, is further able to be used to prepare the drug for the treatment of kidney stone.Composition can be the effective component for playing drug effect in conjunction with the specific site on HK-2 cell.
Description
Technical field
The present invention relates to drug fields, are damaged in particular to a kind of composition and its in protection by calcium oxalate monohydrate
HK-2 cell in application.
Background technique
Desmodium styracifolium is pulse family beggar-ticks plant Desmodium styracifolium Desmodium styracifolium (Osb.) Merr.
Dry aerial parts, Pharmaceutical name is derived from《South of the Five Ridges herbal》.Cold nature, it is sweet in flavor, light, return bladder, liver, gallbladder, kidney channel, has
The effect of inducing diuresis for treating strangurtia, removing damp-heat, row's stone.Clinic is mainly used for urinary infection, stone in urinary system, cholelithiasis, acute Huang
Subcutaneous ulcer type hepatitis etc., has a good effect.Currently, the drug action for being only limitted to general flavone basis to the research of Desmodium styracifolium is ground
Study carefully, the research of effective component is had not been reported yet.
Summary of the invention
The purpose of the present invention is to provide a kind of composition and its applications in protection HK-2 cell, are intended to provide
A kind of composition protects the active constituent of HK-2 cell.
The present invention provides a kind of technical solution：
A kind of composition, composition mainly include following components by mass percentage：
5-11% Wei Caining -1,8-15% Wei Caining -2,8-15% Wei Caining -3,15-60% Schaftoside, 23-45%
Isoschaftoside.
In other embodiments of the invention, above-mentioned composition is selected from Desmodium styracifolium general flavone through ODS column chromatographic elution
Obtained 25% elution position or/and 85% elution position.
In other embodiments of the invention, above-mentioned composition is mainly made by following steps：Desmodium styracifolium alcohol extract
The Desmodium styracifolium general flavone is obtained through macroreticular resin joint polyamide separation and purification；By the Desmodium styracifolium general flavone and ODS
Mixing isolates 25% elution position, 85% elution position using methanol-water solvent gradient elution.
In other embodiments of the invention, above-mentioned Desmodium styracifolium alcohol extract is mainly made by the following method：
After impregnating Desmodium styracifolium 30-40min using the ethyl alcohol of 45-55vol%, refluxing extraction 2-3 times depressurizes extracting solution
It is concentrated into no alcohol taste.
The present invention also provides a kind of technical solutions：
Application of the combinations of the above object in the HK-2 cell that protection is damaged by calcium oxalate monohydrate.
In other embodiments of the invention, above-mentioned composition is applied to reduce the HK-2 cellular oxidative metabolic product
It accumulates, promote anti-oxidant generation, reduce the lipid peroxidation injury of the HK-2 cell.
In other embodiments of the invention, above-mentioned composition is applied to promote the occludin on the HK-2 cell membrane
The expression of albumen, ZO-1 albumen.
In other embodiments of the invention, above-mentioned composition is applied to raise the mitochondrial membrane electricity of the HK-2 cell
Position, reduces the HK-2 cell activity oxygen content.
In other embodiments of the invention, cell culture fluid is added in above-mentioned composition, to what is damaged by calcium oxalate monohydrate
HK-2 cell is cultivated.
Application of the combinations of the above object in preparation treatment or/and prevention kidney stone drug.
Composition provided in an embodiment of the present invention and the beneficial effect of its application in protection HK-2 cell are：
Composition provided in an embodiment of the present invention is protected and is damaged by calcium oxalate monohydrate by the collaboration cooperation of five compounds
HK-2 cell, be further able to be used to prepare the drug for the treatment of kidney stone.Composition can be with the specific site on HK-2 cell
In conjunction with, be play drug effect effective component.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 shows mtt assay evaluation TFDS and different parts to the toxic effect of HK-2；
Protective effect Fig. 2 shows mtt assay evaluation TFDS and different parts to COM damage HK-2；
Fig. 3 shows the influence of TFDS and different parts to COM damage HK-2 cell；
Fig. 4, which shows TFDS and different parts, influences the HK-2 cell ROS that COM is damaged；
Fig. 5, which shows TFDS and different parts, causes HK-2 mitochondrial membrane potential in anoxic to influence COM；
Fig. 6, which shows TFDS and different parts, causes HK-2 Apoptosis to influence COM；
Fig. 7 shows occludin and ZO-1 protein expression on the HK-2 cell membrane that TFDS and different parts stick COM
It influences.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention 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, according to normal conditions or manufacturer builds
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.
Application of the composition of the embodiment of the present invention in protection HK-2 cell is specifically described below.
A kind of composition, composition mainly include component once by mass percentage：
5-11% Wei Caining -1,8-15% Wei Caining -2,8-15% Wei Caining -3,15-60% Schaftoside, 23-45%
Isoschaftoside.
In the present embodiment, above-mentioned composition is selected from 25% that Desmodium styracifolium general flavone is obtained through ODS column chromatographic elution
Elute position or/and 85% elution position.
In detail, above-mentioned elution position is made by following steps：Desmodium styracifolium alcohol extract is combined poly- through macroreticular resin
Amide isolates and purifies to obtain the Desmodium styracifolium general flavone；The Desmodium styracifolium general flavone is mixed with ODS, using methanol-water
Solvent gradient elution isolates 25% elution position, 85% elution position, again isolated composition.
Further, above-mentioned Desmodium styracifolium alcohol extract is mainly made by the following method：
After impregnating Desmodium styracifolium 30-40min using the ethyl alcohol of 45-55vol%, refluxing extraction 2-3 times depressurizes extracting solution
It is concentrated into no alcohol taste.
Composition provided in an embodiment of the present invention can be protected by the collaboration of five compounds and be damaged by calcium oxalate monohydrate
HK-2 cell, be further able to be used to prepare the drug for the treatment of kidney stone.Composition can be with the specific site on HK-2 cell
In conjunction with, be play drug effect effective component.
The present invention also provides a kind of technical solutions：
Application of the combinations of the above object in the HK-2 cell that protection is damaged by calcium oxalate monohydrate.
In other embodiments of the invention, above-mentioned composition is applied to reduce the HK-2 cellular oxidative metabolic product
It accumulates, promote anti-oxidant generation, reduce the lipid peroxidation injury of the HK-2 cell.
In other embodiments of the invention, above-mentioned composition is applied to promote the occludin on the HK-2 cell membrane
The expression of albumen, ZO-1 albumen.
Composition is applied to the HK-2 cell that protection is damaged by calcium oxalate monohydrate.The composition is to calcium oxalate monohydrate
The HK-2 cell membrane that (Calcium oxalate. abbreviation COM) sticks has protective effect.
Close connection (TJ) refers to the closed protein in the plasma membrane of epithelial cell top side with sealing albumen in iuntercellular structure
At sealed connection, be made of transmembrane protein and film peripheral proteins, transmembrane protein include occludin, claudins, connection adherency
Molecule (junctional adhesion molecules, JAM)；Film peripheral proteins includes Zonula occludens (ZO-1, ZO-
2, ZO-3).
In other embodiments of the invention, above-mentioned composition is applied to raise the mitochondrial membrane electricity of the HK-2 cell
Position, reduces the HK-2 cell activity oxygen content.The mitochondrial membrane potential damage that COM stimulation HK-2 cell generates is effectively reduced,
Have the function of protecting mitochondria, reduces damage and the apoptosis of cell.
In other embodiments of the invention, cell culture fluid is added in above-mentioned composition, to what is damaged by calcium oxalate monohydrate
HK-2 cell is cultivated.
Application of the combinations of the above object in preparation treatment or/and prevention kidney stone drug.
Composition has protective effect to the HK-2 cell damaged by calcium oxalate monohydrate, therefore has to the treatment of kidney stone
Positive effect can be used for preparing treatment or/and prevention kidney stone drug.With reference to embodiments to feature and property of the invention
It can be described in further detail.
Test example
Desmodium styracifolium general flavone respectively elutes the preparation at position：
It weighs that Desmodium styracifolium medicinal material is appropriate, presses liquid-to-solid ratio (30 with 50% ethyl alcohol:1) after impregnating 30min, refluxing extraction 2
Secondary, each 1.5h, extracting solution is concentrated under reduced pressure into no alcohol taste and Desmodium styracifolium general flavone (TFDS) is made.
TFDS is purified using the method for macroreticular resin joint polyamide.The Desmodium styracifolium concentrate of extraction is adjusted in right amount
Concentration is purified using DM-130 macroreticular resin, is recycled the polyamide of 60-100 mesh to be further purified, is obtained TFDS.It is purple
Outer visible spectrophotometry carries out content detection, obtains TFDS content up to 75%.
The TFDS being prepared is eluted with ODS.30g ODS powder is taken, after pure methanol soaked overnight, wet process dress
Column, with the 5% methanol water balance ODS column 20min of initial concentration of elution.By the initial solvent dissolution of load weighted sample elution
Afterwards, ODS powder is added to carry out mixing sample, when medicinal powder and ODS are mixed to drift sand sample, dry method loading.With the methanol-water of different gradients
Solvent system is eluted, and gropes its elution requirement, and injection high performance liquid chromatograph is analyzed, isolate 5% elution position,
25% elution position, 85% elution position, 100% elution position, low temperature drying is at powder.
Verifying of the different elution positions TFDS 25%, 85% to the protective effect of COM damaging cells
Establish COM damage HK-2 cell model
10mM calcium chloride and 10mM sodium oxalate are prepared, is uniformly mixed, is placed on 4 DEG C of refrigerators standings after three days, 3000 turns of centrifugations
10min, three times, 60 DEG C of dryings in vacuum desiccator are put under ultraviolet lamp overnight sterile water washing, i.e. acquisition COM crystal powder
End.Precision weighs the COM powder 3mg prepared, after placing the 30min that sterilizes under ultraviolet lamp, prepares 100 μ with blank cultures
The COM suspension of g/mL concentration, addition cell in adherent 24 hours 6 orifice plates, are incubated for for 24 hours, and COM has cell apparent glutinous
Attached effect, damaging cells membrane structure, and the activity and form of cell are affected, cell becomes spindle shape from shape of paving the way.
Mtt assay measures the protective effect of TFDS and different parts to COM damaging cells
Cell dissociation is collected, is resuspended after centrifugation, with 8 × 103The density in a/hole is inoculated in 96 orifice plates, is cultivated to thin
Born of the same parents are merged up to 80%.Complete medium carefully is sucked, blank cultures are added in normal group, and 100 μ of COM suspension is added in model group
G/mL, administration group are separately added into the TFDS and different parts medicine of various concentration gradient in addition to 100 μ g/mL of COM suspension is added
Object.It is incubated for and carries out MTT viability examination afterwards for 24 hours.
The measurement of MDA, GSH, SOD
It is arranged blank group (C), 100 μ g/mL, TFDS treatment group of model group (M)：50μg/mL(TFDS-L),100μg/mL
(TFDS-M), 200 μ g/mL (TFDS-H), 25% elution site treatment group：50μg/mL(P1-L),100μg/mL(P1-M),200
μ g/mL (P1-H), 85% elution site treatment group：50 μ g/mL (P2-L), 100 μ g/mL (P2-M), 200 μ g/mL (P2-H)) and
Solvent control group.The HK-2 cell of logarithmic growth phase, with cell number 3 × 106The number in a/hole is inoculated in 6 orifice plates, culture
For 24 hours, after by cultivating for 24 hours in grouping administration postposition incubator, cell supernatant is discarded, collects cell, pre-cooling PBS cleaning with pancreatin
2 times, after group of cells is collected by centrifugation in 1000r/min, cell, ultrasonication lytic cell is resuspended with isometric PBS.According to
MDA (malonaldehyde), GSH (glutathione), SOD (superoxide dismutase) specification require to measure and calculate GSH in cell,
SOD, MDA content.
The measurement of LDH (lactic dehydrogenase)
By HK-2 cell inoculation in 24 orifice plates, density is 4 × 104A/hole, volume is 500 μ L, in 37 DEG C, 5%CO2Training
It is cultivated under the conditions of feeding case for 24 hours, after 2.5.2 grouping administration, after cultivating for 24 hours in incubator, collects group of cells supernatant, be added
0.1%TritionX-100 lysate cracks attached cell, collects cell pyrolysis liquid, measures LDH slip.
The assay of intracellular ROS (active oxygen)
Cell is with 3 × 106The number in a/hole is inoculated in 6 orifice plates, and culture for 24 hours, is grouped by 2.5.2 and is administered, set incubator
In for 24 hours after, clean the free COM of non-adherent cell with PBS, clean 2-3 times.Final concentration of 10 will prepared with blank cultures
μM molecular probe 2', 7'- dichlorofluorescein diacetate (H2DCFDA) solution be added cell in, 37 DEG C, 5%CO2Incubator
Middle incubation 20min, carefully sucks probe solution, with blank cultures rinse cell, removes not with the probe of cell combination, reduces
Background interference.Then 500 μ L blank cultures, the observation of InCucyte ZOOM long-time cell imaging instrument and function is added in every hole
Analysis system (Channel green) captured in real-time, and calculate cell ROS fluorescent value.
The detection of intracellular MMP
After cell culture to logarithmic phase, cell is collected, by cell with 6 × 103A/hole is inoculated in 96 orifice plates, it is adherent for 24 hours
Afterwards, it is grouped and is administered by 2.5.2；After discarding culture medium, cell is washed with PBS, removes not with the COM of cell combination, 100 μ L is added
Cell culture fluid adds 100 μ L JC-1 dyeing working fluids, mixes well to be placed in cell incubator and is incubated for 20min；It abandons
Working solution is removed, is washed twice with the JC-1 dye solution of pre-cooling, dye solution is discarded, after 100 μ L cell culture fluids are added,
The observation of InCucyte ZOOM long-time cell imaging instrument and Functional assay system (Channel green/red) captured in real-time are glimmering
Light detects JC-1 polymer, and calculates cell fluorescence density.
The bis- dye Apoptosis by Flow Cytometries of AnnexinV-FITC/PI
Logarithmic growth phase HK-2, with cell number 3 × 105A/hole is inoculated in 6 orifice plates, culture for 24 hours after, respectively plus
COM (100 μ g/mL) the suspension modeling for entering same concentrations, adds the administration of various concentration as treatment group, while sky is arranged
White group and model group；37 DEG C, 5%CO2After culture is incubated for for 24 hours under the conditions of relative humidity 95%, cell supernatant is collected, pancreatin disappears
Change, 800rpm is centrifuged 5min and collects cell, crosses 300 mesh nylon wires and removes the COM to dissociate after digestion, centrifugation, pre-cooling PBS cleaning 3
After secondary, cell is resuspended with 1 × Binding Buffer buffer, adjustment cell concentration is 1 × 106It is thin to draw 400 μ L by a/mL
5 μ L Annexin V are added in streaming pipe in born of the same parents' suspension.After being incubated at room temperature 10-15min, cell is washed with buffer, is laid equal stress on
Outstanding cell；5 μ L PI are added into cell re-suspension liquid, gently blows and beats and is protected from light incubation 15min in 2-8 DEG C of environment of uniform postposition, then plus
Enter to mix gently after 10 μ L PI dyeing liquors 2-8 DEG C be protected from light under the conditions of be incubated for 5min, immediately through flow cytometer in excitation wavelength
The apoptosis of cell is detected at 488nm, launch wavelength 530nm.
ZO-1 and occludin on the HK-2 cell membrane that Western Blot method detection TFDS and different parts stick COM
Protein expression
The preparation of total protein of cell
Logarithmic growth phase HK-2, with cell number 3 × 105A/hole is inoculated in 6 orifice plates, after culture for 24 hours, is not added
COM (100 μ g/mL) suspension modeling of same concentrations, adds the administration of various concentration as treatment group, while blank is arranged
Group and model group；After culture for 24 hours, liquid is discarded supernatant, with PBS rinse cell 1-2 after, 100 μ L protein lysates is added and (contain
PMSF), after cracking 30min on ice, cell pyrolysis liquid is collected, 4 DEG C, 12000r/min is centrifuged 5min, and Aspirate supernatant surveys its egg
White concentration is put in ultra low temperature freezer after packing and saves.
The measurement of protein concentration
Using BCA kit measurement determination of protein concentration, BSA standard items are diluted using PBS by kit specification requirement,
Protein standard liquid is configured to 0,0.0626,0.125,0.25,0.5,1,2 μ g/ μ L concentration gradients and establishes BSA protein standard song
Line will extract after resulting each group total protein dilutes 10 times and detect, the protein concentration of each group sample is calculated using standard curve.
SDS-PAGE electrophoresis
Protein sample is diluted to same concentrations using PBS, to guarantee that albumen applied sample amount is consistent.Albumen sample after taking dilution
100 μ L of product, after mixing with 5 × Loading Buffer buffer, the 100 μ L of 4 times of volumes, 100 DEG C are boiled 10min, and cold water is pre-
Cold, 10000r is centrifuged 5min, takes supernatant, dispenses up to albumen loading sample.Prepare 10% separation gel and 5% concentration glue, system
It after glue, is put into electrophoresis tank, electrophoretic buffer, sample-adding is added, each sample albumen applied sample amount is 20 μ g, injection volume one
It causes.SDS-PAGE electrophoresis, concentrate glue voltage 60V, when bromophenol blue electrophoresis to separation gel and concentration glue are handed over are carried out after sample-adding
When at boundary, constant pressure 110V electrophoresis is replaced, stops electrophoresis when dyestuff reaches gel bottom margin.
Transferring film
Filter paper, foam-rubber cushion are placed in transferring film liquid and impregnate 30min, while clip PVDF identical with separation gel size in advance
Film.Pvdf membrane methanol is impregnated into 30s, is then placed into transfer liquid and impregnates.The glue that electrophoresis is completed is removed, concentration glue is cut
Part is transferred to and has been covered on foam-rubber cushion and filter paper, then places pvdf membrane, while draining air, is sequentially followed successively by foam-rubber cushion-filter
Paper-glue-film-filter paper-foam-rubber cushion, and black and white board clamping is put in vertical transfer groove, notice that positive and negative anodes are aligned.Use Bio-Rad
Wet type membrane-transferring device transferring film 90min, transferring film electric current are 150mA.After transferring film, blotting membrane 3 times are washed with TBST, each 5min.
Closing
It uses TBST to prepare 5% skim milk as confining liquid, film is put into confining liquid, is slowly shaken on shaking table, room
Temperature closing 120min.It is used TBST rinse 3 times after closing, each 5min.According to the position clip target protein of pre-dyed albumen and interior
Join the corresponding pvdf membrane of albumen, carries out the incubation of corresponding antibodies respectively.
Antibody closing
TBST detergent bar band 3 times after each 5min, matches antibody processed with milk confining liquid.The dilution ratio of antibody：ZO-1 mono-
Anti- dilution ratio：1/3000, molecular weight：182kDa；The dilution ratio of occludin primary antibody：1/5000, molecular weight：80kDa；
β-actin primary antibody dilution ratio：1/1000, molecular weight：42kDa；The dilution ratio of secondary antibody：1/3000.4 DEG C of overnight incubations, are returned
It is washed after receiving an antibody with TBST, 5min is slowly shaken on shaking table, washed 3 times altogether.It is added after washing and is matched with milk
The secondary antibody set, shaking table, which slowly shakes, is incubated for 2h.Secondary antibody is recycled, 0.1%TBST washs 10min, washs 3 times altogether.
Colour developing
Preparing developer liquid A liquid mixes in equal volume with B liquid.The band washed is taken out with tweezers, was sucked with blotting paper
It after more liquid, is put into magazine, configured good developer solution is added, so that developer solution is covered on item and takes, after placing 1-2min,
Film is taken, film is placed into chemical imaging instrument, is taken pictures.
Gray scale measurement
Image-Pro Plus 6.0 carries out gray analysis to protein band.By ZO-1 and occludin band and β-
The relative expression levels of the gray level ratio of actin band albumen as a purpose, calculate and the gray scale of comparison model group and administration group
Value.
Data processing
Cell experiment repeats 2-3 times, and each group of data is indicated with mean ± standard deviation, soft using Graphpadprism5.0
Part drawing, SPSS20.0 statistical software is for statistical analysis, and group difference compares using one-way analysis of variance (ANNOVA)；When
Think that difference has statistical significance when P < 0.05
Mtt assay, which measures TFDS and different elution positions, influences cell activity
Effect of the experiment using mtt assay measurement TFDS, P1, P2 to cell.Fig. 1 shows mtt assay evaluation TFDS and difference
Toxic effect (n=6) of the position to HK-2, wherein A figure represents TFDS；B figure represents P1；Figure C represents P2.
Such as Fig. 1-A it is found that when TFDS concentration is in 0-500 μ g/mL, the vigor of cell is 107.30%, with normal cell
Compared to there was no significant difference.Therefore the selection of TFDS concentration should be in 0-500 μ g/mL；It is found that P1 is in 0-500 μ g/ from Fig. 1-B
When mL, the vigor of cell is 99.78%, is illustrated under this concentration range, drug is to normal cell without obvious inhibiting effect；And P2
In 0-250 μ g/mL, the vigor of cell is 97.98%, is illustrated under low strength range, and drug makees normal cell unrestraint
With.
Therefore, when TFDS, P1 are in 0-500 μ g/mL concentration range, P2 does not have cell in 0-250 μ g/mL concentration range
It influences.But with the increase of concentration, inhibiting effect gradually is presented to cell.The half-inhibitory concentration of TFDS, P1, P2 is respectively：
18.90±0.63mg/mL,22.67±0.58mg/mL,6.059±0.29mg/mL.Not influence later stage evaluation TFDS, P1, P2
Effect to COM damaging cells should be administered using low concentration.
The protective effect of TFDS and different parts to COM damage HK-2 cell
COM sticks HK-2 cell, and cell viability is caused to reduce, and for research TFDS and different parts, to stick HK-2 to COM thin
The protective effect of born of the same parents, experiment influence cell viability using mtt assay detection TFDS and different parts.
Protective effect (n=6) Fig. 2 shows mtt assay evaluation TFDS and different parts to COM damage HK-2, wherein A schemes
Represent TFDS；B figure represents P1；Figure C represents P2.In figure, P###Indicate normal group P compared with model group<0.001；P*It is represented to
Medicine group is compared with model group, P*<0.05, P**<0.01, P***<0.001。
As can be seen from Figure 2, in COM damage HK-2 cell model, the vigor of cell is reduced, and reaches 70% or so, when
TFDS, P1, P2 administration concentration are in 25 μ g/mL, without apparent difference compared with model cell；When administration concentration is from 50 μ g/mL
When, the vigor of cell starts to enhance, and has significant difference, when administration concentration reaches 200 μ g/mL, the vigor of cell tends to
Normally.It is thus determined that the action concentration of drug is 50 μ g/mL, 50~200 μ g/mL of administration range.
TFDS and different parts influence COM damage HK-2 cell MDA, GSH, SOD
Further to study the influence that TFDS and different parts damage COM, experiment utilizes tri- lipids of MDA, GSH, SOD
Effect of the peroxidating metrics evaluation drug to the cell of damage.
Fig. 3 shows the influence of TFDS and different parts to COM damage HK-2 cell, and in figure, A figure represents MDA；B schemes generation
Table GSH；C figure represents SOD；D figure represents LDH.Table 1 shows the influence of TFDS and different parts to COM damage HK-2 cell.
By Fig. 3-A it is found that effect shown in TFDS and different parts is different.Malonaldehyde (MDA) is as measurement cell
Lipid peroxide index can indirectly reflect the degree of injury of cell by measuring intracellular level of lipid peroxidation.
In MDA index, the effect that the low middle high dose of TFDS is showed is preferable, compared with model group, tool extremely significant difference
(P<0.001)；It can be found from table 1, the TFDS under low dosage has been able to significantly reduce the generation of MDA.And P1 and model group
It compares, there is notable difference (P in middle high dose<0.01).In tri- dosage of P2, the generation of intracellular MDA can be reduced,
Especially in high dose effect, compared with model group group, great significant difference (P<0.001).
By observing GSH index in Fig. 3-B and table 1, it is found that the TFDS of high dose is strong to the protective effect of COM damaging cells
(P<0.001), illustrate that certain ingredients in TFDS have facilitation to the generation of GSH, enhance body resists lipidization energy
Power.And two positions P1, P2 also can promote the generation of GSH.It takes temperature from containing for GSH is generated, the effect of P2 is better than P1.
The height of SOD vigor indirectly reflects the ability of body scavenging activated oxygen, also reflects the physiology shape of cell
State.Cell SOD index determining result in Fig. 3-C and table 1 obtains：After P2 regional administration, the vigor of SOD is significantly increased, especially
In middle high dose (P<0.001), effect is best.
TFDS and different parts influence LDH leakage rate in COM damage HK-2 cell
As shown in Fig. 3 and table 1, model group is compared to the blank group compared with the cell LDH slip of model group obviously rises (P<
0.001), leakage rate reaches 40% or more.Compared with model group, the LDH slip of three administration groups is substantially reduced, and is had aobvious
Write sex differernce (P<0.001), wherein P2 is better than TFDS, P1 of same concentrations to the reduction effect of LDH slip under low dosage
Group.
The influence of 1 TFDS of table and different parts to COM damage HK-2 cell
Note：# indicate M group compared with C group,##P<0.01,###P<0.001；* indicate administration group compared with M group,*P<
0.05,**P<0.01,***P<0.001
TFDS and different parts influence the intracellular ROS of COM damage HK-2
The effect of HK-2 cell is damaged further to inquire into TFDS and different parts ingredient to COM, experiment uses active oxygen
Measurement further probe into active oxygen upon administration COM damage HK-2 cell model in variation.Experiment uses fluorescence probe
DCFH-DA is measured intracellular ROS active o content.Fig. 4 shows the HK-2 that TFDS and different parts damage COM
Cell ROS influences.
Fig. 4 is as the result is shown：Compared with normal cell, the active o content in model cell is much higher than blank group, reaches
3.40 times or so, there is significant statistical significance (P<0.001) after, illustrating COM modeling, lead to cell oxidative damage, cause
The a large amount of generations and accumulation of reactive oxygen species.When giving three groups of drugs and being intervened, TFDS and different parts ingredient energy
It is horizontal effectively to lower cell ROS.Wherein, P2-L (50 μ g/mL) group is compared with model group, and indifference is anisotropic, but P1-M (100 μ g/
ML), the active o content of P1-H (200 μ g/mL) reaches：2.6 times, 1.9 times are lowered respectively compared with model group：0.8
Again, 1.5 times；P2-L (50 μ g/mL), P2-M (100 μ g/mL), P2-H (200 μ g/mL) active o content reach：3.3 times,
It is horizontal to have lowered intracellular ROS respectively compared with model group for 2.5 times, 1.6 times:0.1 times, 0.9 times, 1.8 times；TFDS-L
(50 μ g/mL), TFDS-M (100 μ g/mL), TFDS-H (200 μ g/mL) active o content be：2.7 times, 1.6 times, 1.1 times；
Respectively compared with model group, it is horizontal that intracellular ROS has been lowered respectively：0.7 times, 1.8 times, 2.3 times.
As a result illustrate that TFDS can significantly reduce active oxygen generation, protection COM sticks cellular damage caused by HK-2 and drawn
The oxidative damage risen.Compared with model group, TFDS just has significant difference (P since low concentration<0.05), middle high dose
Under, great significant difference (P<0.001)；The effect of position P1, P2 at low concentration (50 μ g/mL) to active oxygen, with model
Group compares no significant difference, when reaching high dose, has significant difference (P<0.001), great significant difference.Therefore,
In the reduction effect for the HK-2 reactive oxygen species that TFDS and different parts stick COM, the effect of TFDS is best.And P1 with
P2 compares, and under middle high dose, P2 is better than P1 to the adjustment effect of active oxygen.
TFDS and different parts, which stick HK-2 cell MMP to COM, to be influenced
Experiment uses cell membrane inner mitochondria film current potential (MMP), and the change of cell membrane potential is detected by JC-1 fluorescence probe
Change, detecting TFDS and different parts as Testing index influences COM damage HK-2 mitochondrial membrane potential in anoxic.
Experimental result is as shown in figure 5, compared with normal cell group, after giving COM pharmaceutical intervention, in model group cell
Red green fluorescence ratio significantly reduce to 0.68 (P<0.001), illustrate that COM can significantly reduce mitochondrial membrane potential in anoxic.When
When TFDS and different parts intervene the HK-2 cell model that COM sticks, the mitochondrial membrane potential of cell is had occurred significantly
Variation.Three concentration in TFDS can significantly raise the red green fluorescence ratio of mitochondrial membrane potential：1.06(P<0.05),1.59
(P<0.01),2.52(P<0.001)；But the red green fluorescence ratio of the mitochondrial membrane potential of administration group P2-H is up to 2.65 (P<
0.001), the high dose of the slightly excellent general flavone of effect, can conspicuousness (P<0.001) the red green fluorescence ratio of regulation mitochondrial membrane potential
It is horizontal.
The bis- dye Apoptosis by Flow Cytometries of AnnexinV-FITC/PI influence
Fig. 6, which shows TFDS and different parts, causes HK-2 Apoptosis to influence COM.
It can be found from Fig. 6 cell apoptosis assay, the Apoptosis of model group reaches 14.0%, and normal cell group apoptosis reaches
1.8%, model group Apoptosis conspicuousness increases (P<0.001).TFDS and each elution regional administration can effectively inhibit cell
The low middle high dose of apoptosis, TFDS reduces the apoptosis of cell to 8.4% (P<0.05), 3.7% (P<0.01), 3.7% (P<
0.01)；And the low middle high dose of P1 inhibits the apoptosis rate of cell to respectively reach：7.0% (P<0.01), 5.9% (P<0.01),
2.8% (P<0.001)；The P2 of low middle high dose makes apoptosis rate reduce by 3.5%, 3.1%, 2.4%, has compared with model group
There is significant difference (P<0.001).By Fig. 5 it is evident that TFDS and different parts administration inhibit Apoptosis, and have
Concentration dependent.Wherein, the apoptosis rate of P2 inhibits apoptotic effect better than TFDS under same dose, is secondly TFDS.
On Western Blot method detection TFDS and the HK-2 cell membrane that sticks to COM of opposed polarity position occludin with
ZO-1 protein expression influences
Fig. 7 shows occludin and ZO-1 protein expression on the HK-2 cell membrane that TFDS and different parts stick COM
It influences.A represents occludin protein expression；B represents ZO-1 protein expression.
As shown in fig. 7, occludin and ZO-1 albumen table compared with normal cell, in calcium oxalate COM model group cell
It is lowered up to significant, occludin albumen is lowered to 0.46 (P<0.001), ZO-1 albumen is lowered to 0.45 (P<0.001).?
In occludin Protein Assav, administration group TFDS concentration is respectively 50,200 μ g/mL, compared with model group, respectively on be transferred to：
0.65(P<0.05), 1.21 (P<0.01)；P1 concentration is respectively 50,200 μ g/mL, compared with model group, respectively on be transferred to：
0.79(P<0.001)0.90(P<0.05)；And in P2, respectively on be transferred to：0.79(P<0.05), 1.23 (P<0.01).In ZO-
In 1 Protein Assav, administration group TFDS concentration be 50,200 μ g/mL, compared with model group, respectively on be transferred to：0.97(P<
0.05), 1.45 (P<0.001)；P1 concentration be 50,200 μ g/mL, compared with model group, respectively on be transferred to：1.02(P<
0.001), 1.11 (P<0.001)；And in P2, respectively on be transferred to：1.29(P<0.001), 1.67 (P<0.001).
Therefore, in the experiment of kidney stone cell model, COM causes occludin and ZO-1 albumen on cell membrane to lower,
By the processing at TFDS and different elution positions, discovery occludin and ZO-1 albumen have up-regulation trend, as a result illustrate TFDS
And different elution positions have the function of promoting occludin and ZO-1 protein upregulation, TFDS treatment kidney stone may be by adjusting
Section occludin and ZO-1 reach curative effect.And in TFDS and different elution positions, compared with other two groups.It was found that P2 is in height
Dosage (200 μ g/mL) is most strong to the adjustment effect of occludin albumen, and in ZO-1 adjustment effect, P2 pairs of low high dose
The up-regulation effect of occludin and ZO-1 albumen is strong.
By above-mentioned it can be proved that oxidative damage, thorn occur for cell when HK-2 cell membrane surface is sticked by COM
The generation of activity oxygen leads to a series of generation of Petoxidation products, the content decline of antioxidant.It can be sent out from experiment
It is existing.85% elution position (P2) of TFDS can reduce COM and stick active o content caused by HK-2 cell, lower ability
By force, and TFDS also shows stronger downward effect, damage of the active oxygen to cell can be reduced, protect cell membrane.And TFDS
25% elution position (P1) can also lower the content of active oxygen to a certain extent, but lower ability and be weaker than the above two.Wide money
Careless general flavone ODS column 25% elutes position or/and Desmodium styracifolium general flavone ODS column 85% elutes position and has protection HK-2 cell
Effect.The mitochondrial membrane potential damage that COM stimulation HK-2 cell generates can be effectively reduced, there is the function of protection mitochondria
Energy；Occludin is raised, the protein content expression of ZO-1 regulates and controls barrier function and the osmotic pressure effect of TJ, subtracts and hurt COM to cell membrane
Damage.Reduce damage and the apoptosis of cell.
Desmodium styracifolium general flavone 25%ODS elutes the detection at position and 85%ODS elution position active constituent
Cell administration
After cell dissociation in exponential phase of growth is prepared into single cell suspension, with 5 × 105The density of a/mL is inoculated with
In 6 orifice plates, culture to exponential phase of growth.The TFDS powder that suitable purity is 75% is weighed, it is molten with DMEM-F12 culture medium
37 DEG C of cell, 5%CO is added at stock solution, 30mg/mL concentration liquid in solution2, cultivate 2h in 95% incubator of relative humidity after, show
The growth conditions of micro mirror observation cell.
TFDS active constituent and HK-2 cell combination
After cell dissociation in exponential phase of growth is prepared into single cell suspension, it is inoculated with into 75cm2In culture bottle, by it
It is divided into blank control group and TFDS administration group.When cell confluency 90%, with the fraction of dead cell of PBS rinse cell surface
Afterwards, after the TFDS that addition has been diluted with DMEM-F12 culture solution in TFDS administration group, final concentration of 30mg/mL, blank group is only
Isometric DMEM-F12 culture solution is added, is placed in cell incubator and cultivates 4h, condition is 37 DEG C, 5%CO2, it is relatively wet
Degree 95%.
TFDS is not associated with the elution of ingredient
After 2h, blanc cell blank culture solution, administration cell culture solution containing TFDS are discarded, 5mL PBS buffer solution is added,
Cell is washed, cleaning solution is discarded, cleans repeatedly, until cleaning solution peak without reserve, collects last eluent.
Prepare citric acid buffer salt solution
Solution A：Precision weighs citric acid 21g, and sterile water dissolution is settled to 1000mL, and 4 refrigerators save.Second liquid：Weigh phosphoric acid
Disodium hydrogen powder 71.63g, sterile water dissolution are settled to 1000mL；It takes solution A 61.45mL, second liquid 38.55mL to be uniformly mixed, uses
Hydrochloric acid adjust its pH value be 4.0 when, cross 0.22 μm of filtering with microporous membrane to obtain the final product.
The dissociation of TFDS and HK-2 cell-binding component
Citric acid-disodium hydrogen phosphate buffer of pH=4,37 DEG C, 5%CO are added after elution2Relative humidity 95% is cultivated
1h is cultivated in case.Occur to dissociate and discharge into dissociation solution with the active constituent of cell combination under acid condition.It is blown and beaten after dissociation
Cell collects cell and dissociation solution.
High performance liquid chromatography detects HK-2 cell-binding component
Chromatographic column Phenomenex Kinetex C18(4.6mm×250mm,5μm)；Mobile phase：0.1% aqueous formic acid
For mobile phase A, using methanol as Mobile phase B, gradient elution：0-30min, 74%A；30-60min, 74-35%A；60-75min,
35-5%A；20-25min, 5%A；Detection wavelength 272nm；Flow velocity：1.0mL·min-1；Column temperature：40℃；10 μ L of sample volume.
UPLC-Q-TOF/MS identifies HK-2 cell-binding component
Liquid-phase condition：Phenomenex Kinetex C18column(100mm×2.1mm,1.7μm)；Mobile phase：01%
- 0.1% formic acid water (B) of formic acid acetonitrile (A), gradient elution；Elution requirement：0-7min, 10%-15%A；7-9min, 15%-
40%A；9-12min, 40%-100%A；12-13min, 100%A；13-14min, 100%-10%A；14-18min, 10%
A.35 DEG C of column temperature, sample volume 1 μ L, flow velocity 0.4mL/min.
Mass Spectrometry Conditions：Data acquisition scheme uses 5600+LC30+CDS；Scan type：TOF MS；Scanning range：100-
1000；Detection pattern：Negtive；Atomization gas：55psi；Assist gas：55psi；Gas curtain gas：35psi；Atomization temperature：550℃；
Ion source：Duo Spray electric spray ion source；Ionizing voltage：4500V；Remove cluster voltage：100V；Impact energy：45V；Collision is lived
Change scanning：15V.
The detection of clasmatosis liquid
By cell and cell dissociation buffer, after the last eluent of cell is placed in 37 DEG C of nitrogen evaporator dryings, with the methanol of 200 μ L
It redissolves, vortex is completely dissolved in ingredient in methanol, and high speed freezing centrifuge is centrifuged 10000r/min, is centrifuged 10min, takes supernatant
Liquid injects HPLC chromatogram instrument and mass spectrograph detection, records chromatographic peak.
The active constituent of HK-2 cell combination after HPLC detection administration
Collected eluent, blanc cell dissociation solution, administration cell dissociation buffer are carried out by high performance liquid chromatography
Preliminary detection, find to illustrate in the last eluent of administration group without specific chromatographic peak last eluent will not with
The TFDS medicine liquid ingredient elution of cell combination is clean.Blanc cell dissociation solution is had detected, the influence of ingredient in blanc cell is excluded.
Illustrate that cell membrane can be specifically bound with the ingredient in TFDS.This is in next step to cell membrane specific binding member
Further identification provide foundation.
Identify the active constituent of TFDS and HK-2 cell combination
For the active constituent of the combination of identification of cell and Desmodium styracifolium general flavone, experiment uses UPLC-Q-TOF/MS method
Analysis comparison is carried out to administration cell pyrolysis liquid, plain lysis liquid, last time eluent and medical fluid, is sent out from total ion current figure
It is existing, there is no the chromatographic peak in the presence of specificity in blanc cell lysate, after administration the total ion current figure of cell pyrolysis liquid it is existing
Specific chromatographic peak.
Using peakView software to blanc cell lysate, TFDS administration group lysate, last eluent and TFDS medicine
Liquid is analyzed, and is retrieved in conjunction with the resulting database of Qualitative Identification of seminar's early period to Desmodium styracifolium chemical component, really
Determine distinctive molecular weight in TFDS administration group, and extract chromatography of ions figure and its second order ms figure, and summarize for table 2, by giving
The fragment ion information and document of specific component in medicine group lysate and the retention time and fragment information of reference substance carry out
It compares.Primarily determine that the compound contained in TFDS administration group dissociation solution is respectively：Wei Caining -2 (p1), Wei Caining -1
(p2), carlinoside (p3), Isoschaftoside (p5), Schaftoside (p6), Saponaretin (p7), astragalin (p9), dye
Expect lignin (p10) and diosmetin (p11).Since lysate also contains molecular weight identical as Schaftoside and cleaved fragment phase
Together, but the different compound of retention time, thus can not its accurate qualitative compound structure, therefore speculate and may contain summer Buddhist
Tower glycosides isomer I (p4), Schaftoside isomer II (p8).This chapter is had by taking the p1-p9 in chromatogram as an example
The structure elucidation of body.
p1:The chromatographic peak molecular ion peak of extraction is m/z 593.15 [M-H]-, its second order spectrum is parsed, is obtained
Fragment ion：295,297,383,473,593,575,591, it compares, is found in retention time and fragment cracking side with reference substance
Formula is consistent, it is thus determined that p1 is compound Wei Caining -2.
p2:The chromatographic peak molecular ion peak of extraction is m/z 563.14 [M-H]-, its second order spectrum is parsed, is obtained
Fragment ion：296,297,325,353,383,395,443,473,563, it is compared with reference substance, discovery is in retention time and broken
Piece cracking mode is consistent, it is thus determined that p1 is compound Wei Caining -1.
p3:The chromatographic peak molecular ion peak of extraction is m/z 579.14 [M-H]-, its second order spectrum is parsed, is obtained
Fragment ion：203, it 298,327,339,369,399,429,471,489,519,531,561,579. compares, sends out with reference substance
Present retention time and fragment cracking mode are consistent, it is thus determined that p3 is compound carlinoside.
P4, p6 and p8:The chromatographic peak molecular ion peak that three chromatographic peaks extract is 563.14 [M-H]-, from the two of extraction
The cleaved fragment of grade fragment ion INFORMATION DISCOVERY compound is consistent：296,297,325,353,383,395,443,473,563, it says
Its bright cracking mode is identical；Mass spectrometric data is carried out with reference substance Schaftoside to compare, and finds p6 in retention time and fragment ion
It is identical as reference substance Schaftoside, it is reference substance Schaftoside；P4 and p8 retention time tR(min) it is：4.30,7.58, with the summer
The retention time of pagoda glycosides reference substance has differences, thus it is speculated that is Schaftoside isomer, but due to lacking reference substance, fails
Precise Identification structure, therefore tentative p4 and p8 is：Schaftoside isomer I, Schaftoside isomer II.
p5:The chromatographic peak molecular ion peak of extraction is m/z 563.14 [M-H]-, its second order spectrum is parsed, is obtained
Fragment ion：296,297,325,353,383,395,443,473,563, it is isomer with p6, but when reservation with p6
Between different, tR(min) it is 4.79, is compared by reference substance, discovery p5 is compared with reference substance chromatographic data, it is thus determined that p5
For compound Isoschaftoside.
p7:The chromatographic peak molecular ion peak of extraction is m/z 431.10 [M-H]-, its second order spectrum is parsed, is obtained
Fragment ion：117,161,269,281,282,283,311,323,341,431, tR(min) it is 6.75, is compared with reference substance,
It was found that it is consistent in retention time and fragment cracking mode, it is thus determined that p7 is compound Saponaretin.
p9:The chromatographic peak molecular ion peak of extraction is m/z 447.10 [M-H]-, its second order spectrum is parsed, is obtained
Fragment ion：127,183,225,227,255,284,285,329,447, retention time tRIt (min) is 8.67.In conjunction with document report
Road identifies that p9 compound may be astragalin.
Binding constituents fragment ion information in table 2TFDS cell dissociation buffer
Illustrating to be enriched in P1 in TFDS to elute position and the main of P2 elution position includes Wei Caining -1, Wei Caining -2, dimension
Cai Ning -3, Schaftoside, Isoschaftoside composition, can be specifically bound with HK-2 cell membrane, can be used for protecting
HK-2 cell, further for treating kidney stone.
For the ease of control, English breviary vocabulary such as table 3 is enclosed.
The English breviary vocabulary of table 3
Embodiment 1
The present embodiment provides a kind of composition, composition mainly includes following components by mass percentage：
5% Wei Caining -1,8% Wei Caining -2,8% Wei Caining -3,15% Schaftoside, 23% Isoschaftoside and remaining
Under cell culture fluid.
Embodiment 2
The present embodiment provides a kind of composition, composition mainly includes following components by mass percentage：
11% Wei Caining -1,15% Wei Caining -2,15% Wei Caining -3,30% Schaftoside, 25% Isoschaftoside with
And remaining cell culture fluid.
The composition that embodiment 1 and embodiment 2 provide carries out COM damage HK-2 cell using test example providing method
Culture, detection.It was found that the composition that embodiment 1 and embodiment 2 provide can reduce the mitochondria that COM stimulation HK-2 cell generates
Film potential damage, raises occludin, and the protein content expression of ZO-1 reduces damage and the apoptosis of cell.
Illustrate the HK-2 cell that there is the composition that embodiment 1 and embodiment 2 provide protection to be damaged by calcium oxalate monohydrate
Effect.
The foregoing is merely the preferred embodiment of invention, it is not limited to invent, for those skilled in the art
For member, invention can have various modifications and variations.With within principle, made any modification is equal all spirit in invention
Replacement, improvement etc., should be included within the protection scope of invention.
Claims (10)
1. a kind of composition, which is characterized in that the composition mainly includes following components by mass percentage：
5-11% Wei Caining -1,8-15% Wei Caining -2,8-15% Wei Caining -3,15-60% Schaftoside, 23-45% different summer
Pagoda glycosides.
2. composition according to claim 1, which is characterized in that the composition is passed through selected from Desmodium styracifolium general flavone
The 25% elution position or/and 85% elution position that ODS column chromatographic elution obtains.
3. composition according to claim 2, which is characterized in that the composition is mainly made by following steps：Extensively
Herba lysimachiae alcohol extract obtains the Desmodium styracifolium general flavone through macroreticular resin joint polyamide separation and purification；By the Desmodium styracifolium
General flavone is mixed with ODS, using methanol-water solvent gradient elution, isolates 25% elution position, 85% elution position；Again
The isolated composition.
4. composition according to claim 3, which is characterized in that the Desmodium styracifolium alcohol extract is mainly by the following method
It is made：
After impregnating Desmodium styracifolium 30-40min using the ethyl alcohol of 45-55vol%, extracting solution is concentrated under reduced pressure refluxing extraction 2-3 times
To no alcohol taste.
5. composition according to any one of claims 1-4 is in protection by answering in HK-2 cell that calcium oxalate monohydrate damages
With.
6. application according to claim 5, which is characterized in that the composition is applied to reduce the HK-2 cellular oxidation
The accumulation of metabolite promotes anti-oxidant generation, reduces the lipid peroxidation injury of the HK-2 cell.
7. application according to claim 5 or 6, which is characterized in that the composition is applied to promote the HK-2 cell
The expression of occludin albumen, ZO-1 albumen on film.
8. application according to claim 5 or 6, which is characterized in that the composition is applied to raise the HK-2 cell
Mitochondrial membrane potential, reduce the HK-2 cell activity oxygen content.
9. application according to claim 5, which is characterized in that cell culture fluid is added in the composition, to by a water plant
The HK-2 cell of sour calcium damage is cultivated.
10. application of the composition according to any one of claims 1-4 in preparation treatment or/and prevention kidney stone drug.
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