CN105193787B - The application of byak-angelicin - Google Patents
The application of byak-angelicin Download PDFInfo
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- CN105193787B CN105193787B CN201510772274.7A CN201510772274A CN105193787B CN 105193787 B CN105193787 B CN 105193787B CN 201510772274 A CN201510772274 A CN 201510772274A CN 105193787 B CN105193787 B CN 105193787B
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Abstract
The present invention relates to technical field of traditional Chinese medicines, the present invention discloses application of the byak-angelicin in injury of kidney cell is protected, so that clinically to prevent injury of kidney from providing a kind of more safely and effectively medications selections.Byak-angelicin can effectively protect injury of kidney cell in safe dose, can be further developed as injury of kidney protection medicine, be with a wide range of applications.
Description
Technical field
The present invention relates to pharmaceutical technology field, and in particular to the application of byak-angelicin.
Background technology
Kidney is the vitals of human body, is that metabolism is normally carried out for ensureing the stabilization of organismic internal environment.
One of common acute disease of Urology Surgery, appropriate therapeutic scheme is made to injury of kidney during injury of kidney, the condition of the injury to mitigating patient, is changed
Kind prognosis situation is most important.Therefore, injury of kidney protects medicine in continuous research and development in recent years.
Research finds that Chinese medicine has significant protective effect to injury of kidney, and for Western medicine, Chinese medicine more heats
And safety, side effect are smaller.Notopterygium root, Umbelliferae, notopterygium root platymiscium are warm-natured, dissipate exterior cold；Wind-damp dispelling；Sharp joint；Analgesic.It is main outer
Feel chill；Have a headache lossless；Geomantic omen edema；Sore swollen toxin.For impotence and seminal emission, enuresis frequent micturition, waist and knee crymodynia, deficiency of the kidney and dyspnea, five
More diarrhea；External application curing vitiligo, alopecia areata.Relieving exterior syndrome and dispelling cold, expelling wind and eliminating dampness, analgesic.Contain various active material, such as Khellinum in notopterygium root
Lactone, RADIX PEUCEDANI aglycon, isopimpinellin, anisic acid para hydroxybenzene ethyl ester, byak-angelicin (byak-angelicin), bergaptol
Deng these active materials have a variety of pharmacologic actions.Wherein, byak-angelicin, which is reported, can treat pancreatitis and cholecystitis,
The application in treating pancreatitis medicine is being prepared as patent CN104188960A discloses a kind of byak-angelicin.In addition, it is combined
Thing is used to prevent and/or treat a variety of pain by caused by energy stagnation and blood stasis, such as stomachache, hypochondriac pain, headache and dysmenorrhoea, especially suitable
In tension headache, it may also be used for prevention and/or treatment depression, as patent 201110332447.5 disclose a kind of Chinese medicine into
It is grouped compound and application thereof.But there is not yet document report byak-angelicin has the effect of preventing injury of kidney.
The content of the invention
In view of this, it is an object of the invention to provide the new opplication of byak-angelicin, byak-angelicin to have anti-
Only the effect of injury of kidney, protection injury of kidney cell, it can be applied in the preparation of injury of kidney protection medicine.
Byak-angelicin, alias byak-angelicin, chemical structural formula are as follows：
Wherein, preferably, the injury of kidney is renal damage caused by cis-platinum.
Byak-angelicin acquiring way can both extract from the natural materials containing the composition such as the root of Dahurain angelica, notopterygium root,
It can be subject to using chemical synthesis process artificial synthesized.Preferably, byak-angelicin of the present invention extracts from notopterygium root
Obtain, it is further preferred that taking following methods to obtain：
Step 1, notopterygium root rhizome being crushed, add ethanol solution extraction, after extract solution filtering, concentration plus salt acid for adjusting pH is 2,
It is heated to reflux and concentrates to be evaporated acquisition crude extract；
Step 2, crude extract add ethanol solution and redissolved, and are adsorbed by macroporous resin column, successively 4 column volumes of water elution,
40% column volume of ethanol elution 3,60% column volume of ethanol elution 6,60% ethanol eluate is collected, remove solvent, concentration
To medicinal extract, ethyl acetate dissolving, filtering are then added, filtrate removes solvent, concentration, dried, and obtains notopterygium root extract；
After step 3, notopterygium root extract add methanol to dissolve, add in the thick silica gel of equivalent and stir, be poured into after drying
In 200-300 mesh silicagel columns containing 4~5 times of amounts；After petroleum ether balance D-101 pillars, petroleum ether-ethyl acetate 50 is utilized:
1、20:1、10:1 respectively elutes 4 column volumes, collects 10:1 elution position concentration is evaporated, and after adding methanol to dissolve, centrifugation, takes supernatant
Liquid, preparative separation is carried out using high pressure preparative liquid chromatography, obtains byak-angelicin.
Wherein, the volume mass of notopterygium root rhizome and ethanol solution described in step 1 is than preferably 1g:6-10mL.
High pressure preparative liquid chromatography condition is preferably as follows：
The type preparative chromatograph of Agilent 1260, chromatographic column are Fuji C18 (250 × 50mm)；Mobile phase is acetonitrile-water；Inspection
Survey wavelength is 310nm；Elution program is 0~60min, acetonitrile 30%, water 70%；Flow velocity is 30mLmin-1；Column temperature is 30
℃。
It is preferably that crude extract adds 1 times of amount of notopterygium root rhizome gross weight that crude extract described in step 2, which adds ethanol solution and redissolved,
40% ethanol solution dissolves.
It is preferably D-101 type macroporous resin columns to state macroporous resin column.
The macroporous resin column weight is preferably 2 times of notopterygium root rhizome gross weight.
The concentration expressed in percentage by volume of ethanol solution described in step 1 is preferably 70%.
Cis byak-angelicin and trans byak-angelicin the HK-2 cells renal injury model caused by cis-platinum
The protective effect to the security of normal HK-2 cells and to injury of kidney cell is shown in experiment.Therefore, the present invention carries
For application of the byak-angelicin in injury of kidney protection medicine is prepared.
From above technical scheme, the invention provides application of the byak-angelicin in injury of kidney cell is protected,
So that clinically to prevent injury of kidney from providing a kind of more safely and effectively medications selections.Byak-angelicin energy in safe dose
It is enough effectively to protect injury of kidney cell, injury of kidney protection medicine can be further developed as, is with a wide range of applications.
Brief description of the drawings
Fig. 1 show notopterygium root extract high pressure preparative liquid chromatography figure.
Embodiment
The invention discloses the application of byak-angelicin, those skilled in the art can use for reference present disclosure, suitably change
Enter technological parameter realization.In particular, all similar replacements and change are aobvious for a person skilled in the art
And be clear to, they are considered as being included in the present invention.Product of the present invention and preparation method and application by compared with
Good embodiment is described, related personnel substantially can not depart from present invention, in spirit and scope to as described herein
Methods and applications are modified or suitably changed with combining, to realize and using the technology of the present invention.
The application with regard to byak-angelicin provided by the present invention is described further below.
Embodiment 1：The extraction separation of byak-angelicin
Notopterygium root rhizome is ground into coarse powder, 6~10 times of 70% ethanol solution is added by volume/mass and is extracted twice, often
Secondary 1.5 hours, merge filtered fluid, be concentrated into 1 times that medicinal material weight is added water to after no alcohol taste；Concentrate adds concentrated hydrochloric acid regulation pH
To 2,0.5 hour is heated to reflux, concentration is evaporated, and is added 40% ethanol solution of 1 times of amount of medicinal material weight and is completely dissolved it.Water
Solve D-101 macroporous resin column (blade diameter length ratio 1 of the liquid by 2 times of medicinal material weight:8) after adsorbing, 4 column volumes of water elution, 40%
3 column volumes of ethanol elution, 60% column volume of ethanol elution 6.60% ethanol eluate merges, and recycling design, is concentrated into leaching
Cream.Medicinal extract adds ethyl acetate solution, makes its dissolving complete, filtration, recycling design, is concentrated into medicinal extract, 60 DEG C of vacuum drying, powder
It is broken to produce notopterygium root extract.
After notopterygium root extract adds proper amount of methanol to dissolve, add in the thick silica gel (80-100 mesh) of equivalent and stir, dry
It is poured into afterwards in silica gel (200-300 mesh) post containing 4~5 times of amounts.After petroleum ether balance D-101 pillars, petroleum ether-second is utilized
Acetoacetic ester 50:1、20:1、10:1 respectively elutes 4 column volumes, collects 10:The concentration of 1 position is evaporated.Petroleum ether-ethyl acetate 10:1
After elution position adds methanol to dissolve, centrifugation, supernatant is taken, preparing liquid phase using high pressure is prepared, and collects byak-angelicin
Chromatographic peak, byak-angelicin is obtained after concentrate drying.
High pressure preparative liquid chromatography condition：The type preparative chromatograph of Agilent 1260, chromatographic column be Fuji C18 (250 ×
50mm)；Mobile phase is acetonitrile (A)-water (B)；Detection wavelength is 310nm；Elution program is 0~60min, A30%, B70%；Stream
Speed is 30mLmin- 1；Column temperature is 30 DEG C.
Byak-angelicin qualification result is as follows：
1H NMR(400MHz,DMSO)δ:6.34 (1H, d, J=9.8Hz, H-3), 8.19 (1H, d, J=9.8Hz, H-4),
4.17(3H,s,5-OCH3), 8.10 (1H, d, J=2.3Hz, H-2 '), 7.39 (1H, d, J=2.3Hz, H-3 '), 5.00 (1H,
D, J=5.7Hz, H-2 "), 4.45 (1H, dd, J=10.0,2.2Hz, H-2 "), 3.64 (1H, ddd, J=8.0,5.7,2.3Hz,
H-3″),4.38(1H,s,3″-OH),4.20(1H,s,4″-OH)。13C NMR(100MHz,DMSO)δ:160.1(C-2),112.9
(C-3),140.2(C-4),144.5(C-5),114.9(C-6),150.0(C-7),127.3(C-8),143.6(C-9),107.3
(C-10),146.8(C-2′),106.1(C-3′),61.3(5-OCH3),76.3(C-2″),77.1(C-3″),71.2(C-4″),
24.9(C-5″),27.7(C-6″)。
Embodiment 2：Byak-angelicin causes the protective effect of HK-2 cell renal injury models to cis-platinum
1. materials and methods
1.1 cell people renal cells HK-2, purchased from China typical culture collection center.
1.2 main agents and instrument
Cis-platinum is purchased from sigma companies；Required basic culture solution (Dulbecco ' the s minimum essential of Dulbecco
Medium, DMEM)/F12 nutrient solutions, hyclone (fetal bovine serum, FBS), trypsase is purchased from U.S. Gibco
Company；MTT is purchased from green skies company.
The culture of 1.3HK-2 cells
HK-2 cells carry out cellar culture, 5%CO in the DMEM/F12 nutrient solutions containing 10%FBS2, 37 DEG C of cultures are to melting
The cell of conjunction phase is digested with 0.25% pancreatin, and passage, the cell for growth of taking the logarithm is tested.
Influence of 1.4 byak-angelicins to HK-2 cell growths：
Take the logarithm growth period HK-2 cell, with 1 × 104Cells/100 μ l/ holes are inoculated in 96 well culture plates, treat cell
More than 80% is grown to, being changed to serum free medium culture 12h makes to be in synchronous regime, is separately added into 6.25,12.5,25,50,
The various concentration byak-angelicins of 100,150 μ g/ml are cultivated under normal condition, control group：Cultivated in normal incubation medium,
It is placed in after 24h under inverted microscope and observes cellular morphology change, the μ l of MTT solution 10 is added per hole, are continued in cell culture incubator
4h is incubated, supernatant is abandoned in suction, adds 150 μ l DMSO dissolving first a ceremonial jade-ladle, used in libations, and using ELIASA using 570nm as Detection wavelength, 630nm is ginseng
Than wavelength, absorbance is read, every group sets 6 multiple holes, and each group is averaged, and experiment is repeated 3 times, and experimental result is shown in Table 1.
Influence OD value x ± s of the byak-angelicin of table 1 to normal HK-2 cell growths
| Drug concentration (μ g/mL) | Byak-angelicin (n=6) |
| control | 0.65±0.01 |
| 6.25 | 0.58±0.04 |
| 12.5 | 0.62±0.02 |
| 25 | 0.64±0.01 |
| 50 | 0.66±0.02 |
| 100 | 0.64±0.03 |
| 150 | 0.59±0.01 |
The * compared with normal group：P ＜ 0.05
As seen from the results in Table 1, growth of the byak-angelicin to normal HK-2 cells is no obvious compared with control group
Influence, it is safe.
1.5 protective effect of drug
Take the logarithm growth period HK-2 cell, with 1 × 104Cells/100 μ l/ holes are inoculated in 96 well culture plates, treat cell
More than 80% is grown to, being changed to serum free medium culture 12h makes to be in synchronous regime, adds 80 μm of olL-1DDP of people respectively,
The various concentration byak-angelicin normal condition cultures of 0,6.25,12.5,25,50,100,150 μ g/ml are separately added into simultaneously, it is right
According to group：Cultivated in normal incubation medium, be placed in after 24h under inverted microscope and observe cellular morphology change, MTT solution is added per hole
10 μ l, continue to be incubated 4h in cell culture incubator, supernatant is abandoned in suction, adds 150 μ l DMSO dissolving first a ceremonial jade-ladle, used in libations, using ELIASA with
570nm is Detection wavelength, and 630nm is reference wavelength, reads absorbance, and every group sets 6 multiple holes, and each group is averaged, experiment
It is repeated 3 times, experimental result is shown in Table 2.
Protective effect of the byak-angelicin of table 2 to injury of kidney cell
| Drug concentration (μ g/mL) | Byak-angelicin (n=6) |
| control | 0.68±0.04 |
| Model | 0.50±0.02## |
| 6.25 | 0.50±0.01 |
| 12.5 | 0.50±0.03 |
| 25 | 0.51±0.02 |
| 50 | 0.56±0.01* |
| 100 | 0.55±0.06* |
| 150 | 0.49±0.03 |
The ## compared with normal group：P ＜ 0.01；The * compared with model group：p<0.05；**：P ＜ 0.01
As seen from the results in Table 2, model group has significant difference compared to control group, shows that cis-platinum causes HK-2 cell injury of kidney moulds
Type successfully constructs.And when byak-angelicin drug concentration is 50,100 μ g/ml, it has significant protect to injury of kidney cell
Shield acts on, and shows that byak-angelicin can be applied in the preparation of injury of kidney protection medicine.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (9)
- The application of 1.50 μ g/ml or 100 μ g/ml byak-angelicins in renal damage caused by cis-platinum protection medicine is prepared.
- 2. apply according to claim 1, it is characterised in that the byak-angelicin is extracted from notopterygium root and obtained.
- 3. apply according to claim 2, it is characterised in that the byak-angelicin is obtained by following methods extraction separation ：Step 1, notopterygium root rhizome is crushed, add ethanol solution extraction, after extract solution filtering, concentration plus salt acid for adjusting pH is 2, heating Flow back and concentrate and be evaporated acquisition crude extract；Step 2, crude extract add ethanol solution and redissolved, and are adsorbed by macroporous resin column, successively 4 column volumes of water elution, 40% 3 column volumes of ethanol elution, 60% column volume of ethanol elution 6,60% ethanol eluate is collected, remove solvent, be concentrated into leaching Cream, ethyl acetate dissolving, filtering are then added, filtrate removes solvent, concentration, dried, and obtains notopterygium root extract；After step 3, notopterygium root extract add methanol to dissolve, add in the thick silica gel of equivalent and stir, be poured into after drying containing 4~ In the 200-300 mesh silicagel columns of 5 times of amounts；After petroleum ether balance D-101 pillars, petroleum ether-ethyl acetate 50 is utilized:1、20: 1、10:1 respectively elutes 4 column volumes, collects 10:1 elution position concentration is evaporated, and after adding methanol to dissolve, centrifugation, takes supernatant, profit Preparative separation is carried out with high pressure preparative liquid chromatography, obtains byak-angelicin.
- 4. apply according to claim 3, it is characterised in that the volume mass of notopterygium root rhizome and ethanol solution described in step 1 Than for 1g:6-10mL.
- 5. apply according to claim 3, it is characterised in that the concentration expressed in percentage by volume of ethanol solution described in step 1 is 70%.
- 6. apply according to claim 3, it is characterised in that the high pressure preparative liquid chromatography condition is as follows：The type preparative chromatograph of Agilent 1260, chromatographic column are Fuji C18 (250 × 50mm)；Mobile phase is acetonitrile-water；Detect ripple A length of 310nm；Elution program is 0~60min, acetonitrile 30%, water 70%；Flow velocity is 30mLmin-1；Column temperature is 30 DEG C.
- 7. apply according to claim 3, it is characterised in that crude extract described in step 2 adds ethanol solution and redissolved slightly to carry Thing adds the 40% ethanol solution dissolving of 1 times of amount of notopterygium root rhizome gross weight.
- 8. apply according to claim 3, it is characterised in that the macroporous resin column is D-101 type macroporous resin columns.
- 9. apply according to claim 3, it is characterised in that the macroporous resin column weight is 2 times of notopterygium root rhizome gross weight.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1422159A (en) * | 2000-04-10 | 2003-06-04 | 宝生物工程株式会社 | Remedies |
| CN101693716A (en) * | 2009-10-20 | 2010-04-14 | 南京荣世医药科技有限公司 | Process for preparing byak-angelicol |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1422159A (en) * | 2000-04-10 | 2003-06-04 | 宝生物工程株式会社 | Remedies |
| CN101693716A (en) * | 2009-10-20 | 2010-04-14 | 南京荣世医药科技有限公司 | Process for preparing byak-angelicol |
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