CN105287474A - Application of p-hydroxyphenethylanisate - Google Patents
Application of p-hydroxyphenethylanisate Download PDFInfo
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- CN105287474A CN105287474A CN201510770791.0A CN201510770791A CN105287474A CN 105287474 A CN105287474 A CN 105287474A CN 201510770791 A CN201510770791 A CN 201510770791A CN 105287474 A CN105287474 A CN 105287474A
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Abstract
The invention relates to the technical field of traditional Chinese medicine and discloses application of p-hydroxyphenethylanisate in protection of renal injury cells, thereby providing a safe and effective medication option for clinical prevention of renal injury. P-hydroxyphenethylanisate can effectively protect renal injury cells with a safe dosage, can be further developed into a protection drug for renal injury and has wide application prospects.
Description
Technical field
The present invention relates to medical art, be specifically related to the application of anisic acid para hydroxybenzene ethyl ester.
Background technology
Kidney is the vitals of human body, for ensureing the stable of organismic internal environment, is that metabolism is normally carried out.One of emergency case that during injury of kidney, Urology Surgery is common, makes suitable therapeutic scheme to injury of kidney, to the traumatic condition alleviating patient, improves prognosis situation most important.Therefore, the medicine of injury of kidney protection is in recent years in constantly researching and developing.
Research finds that Chinese medicine has significant protective effect to injury of kidney, and relative to Western medicine, the gentleer safety of Chinese medicine, side effect is less.Rhizoma Et Radix Notopterygii, Umbelliferae, Notopterygium plant, warm in nature, loose exterior cold; Wind-damp dispelling; Profit joint; Pain relieving.Main affection of exogenous wind-cold; Have a headache lossless; Edema due to wind pathogen edema; Sore swollen toxin.For impotence and seminal emission, enuresis frequent micturition, chills and pain of the waist and kness, suffers from a deficiency of the kidney and breathes heavily, Diarrhoea; External curing vitiligo, alopecia areata.Induce sweat cold expelling, expelling wind and dampness, pain relieving.Containing various active material in Rhizoma Et Radix Notopterygii, as khellactone, RADIX PEUCEDANI aglycon, isopimpinellin, anisic acid para hydroxybenzene ethyl ester, byak-angelicin, bergaptol etc., these active substances have multiple pharmacologic action.Wherein, anisic acid para hydroxybenzene ethyl ester not yet reports what concrete active function it has.Meanwhile, also have no bibliographical information anisic acid para hydroxybenzene ethyl ester and there is the effect preventing injury of kidney.
Summary of the invention
In view of this, the object of the present invention is to provide the new opplication of anisic acid para hydroxybenzene ethyl ester, anisic acid para hydroxybenzene ethyl ester has the effect preventing injury of kidney, protection injury of kidney cell, can be applied in the preparation of injury of kidney protection medicine.
Wherein, as preferably, described injury of kidney is renal damage caused by cis-platinum.
Anisic acid para hydroxybenzene ethyl ester acquiring way both can from containing the natural materials of this composition as extracted Rhizoma Et Radix Notopterygii, also can utilize chemical synthesis process in addition synthetic.As preferably, anisic acid para hydroxybenzene ethyl ester of the present invention extracts and obtains from Rhizoma Et Radix Notopterygii, still more preferably, takes following methods to obtain:
Step 1, pulverizing Rhizoma Et Radix Notopterygii rhizome, add alcoholic solution and extract, and extracting liquid filtering, concentrated rear salt adding acid for adjusting pH are 2, and reflux also concentrates evaporate to dryness and obtains crude extract;
Step 2, crude extract add alcoholic solution and redissolve, adsorbed by macroporous resin column, successively water elution 4 column volumes, 40% ethanol elution 3 column volume, 60% ethanol elution 6 column volume, collect 60% ethanol elution, except desolventizing, be concentrated into extractum, then acetic acid ethyl dissolution, filtration is added, filtrate is except desolventizing, concentrated, dry, obtains Rhizoma Seu Radix Notopterygii extract;
After step 3, Rhizoma Seu Radix Notopterygii extract add dissolve with methanol, add in the thick silica gel of equivalent and stir, be poured in the 200-300 order silicagel column containing 4 ~ 5 times amount after oven dry; After petroleum ether balance D-101 pillar, utilize each eluting of petroleum ether-ethyl acetate 50:1,20:1,10:1 4 column volumes, collect 10:1 eluting position and concentrate evaporate to dryness, after adding dissolve with methanol, centrifugal, get supernatant, utilize high pressure preparative liquid chromatography to be prepared separation, obtain anisic acid para hydroxybenzene ethyl ester.
Wherein, Rhizoma Et Radix Notopterygii rhizome described in step 1 and the volume mass of alcoholic solution are than being preferably 1g:6-10mL.
High pressure preparative liquid chromatography condition is preferably as follows:
Agilent 1260 type preparative chromatograph, chromatographic column is FujiC18 (250 × 50mm); Mobile phase is acetonitrile-water; Determined wavelength is 310nm; Elution program is 0 ~ 60min, acetonitrile 30%, water 70%; Flow velocity is 30mLmin
-1; Column temperature is 30 DEG C.
Crude extract described in step 2 adds alcoholic solution and redissolves and be preferably crude extract and add 40% dissolve with ethanol solution of Rhizoma Et Radix Notopterygii rhizome gross weight 1 times amount.
State macroporous resin column and be preferably D-101 type macroporous resin column.
Described macroporous resin column weight is preferably 2 times of Rhizoma Et Radix Notopterygii rhizome gross weight.
Described in step 1, the concentration expressed in percentage by volume of alcoholic solution is preferably 70%.
Cis anisic acid para hydroxybenzene ethyl ester and trans anisic acid para hydroxybenzene ethyl ester all show the safety of normal HK-2 cell and the protective effect to injury of kidney cell in the test of HK-2 cell renal injury model caused by cisplatin.Therefore, the invention provides the application of anisic acid para hydroxybenzene ethyl ester in preparation injury of kidney protection medicine.
From above technical scheme, the invention provides the application of anisic acid para hydroxybenzene ethyl ester in protection injury of kidney cell, thus for preventing injury of kidney from providing the medication safely and effectively of many one to select clinically.Anisic acid para hydroxybenzene ethyl ester can effectively protect injury of kidney cell in safe dose, can be developed as injury of kidney protection medicine further, be with a wide range of applications.
Accompanying drawing explanation
Figure 1 shows that Rhizoma Seu Radix Notopterygii extract high pressure preparative liquid chromatography figure.
Detailed description of the invention
The invention discloses the application of anisic acid para hydroxybenzene ethyl ester, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Product of the present invention and preparation method and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
Just the application of anisic acid para hydroxybenzene ethyl ester provided by the present invention is described further below.
Embodiment 1: the extraction and isolation of anisic acid para hydroxybenzene ethyl ester
Rhizoma Et Radix Notopterygii rhizome is ground into coarse powder, and by volume/quality adds the 70% alcoholic solution extraction twice of 6 ~ 10 times, each 1.5 hours, merging filtrate, being concentrated into 1 times without adding water to medical material weight after alcohol taste; Concentrated solution adds concentrated hydrochloric acid and regulates pH to 2, reflux 0.5 hour, and concentrated evaporate to dryness, 40% alcoholic solution adding medical material weight 1 times amount makes it dissolve completely.After hydrolyzed solution is adsorbed by the D-101 macroporous resin column (blade diameter length ratio is 1:8) of 2 times of medical material weight, water elution 4 column volumes, 40% ethanol elution 3 column volume, 60% ethanol elution 6 column volume.60% ethanol elution merges, and recycling design, is concentrated into extractum.Extractum adds ethyl acetate solution, makes it dissolve completely, and filter, recycling design, is concentrated into extractum, and 60 DEG C of vacuum dryings, pulverize and obtain Rhizoma Seu Radix Notopterygii extract.
Rhizoma Seu Radix Notopterygii extract adds in the thick silica gel (80-100 order) of equivalent and stirs, be poured in silica gel (200-300 order) post containing 4 ~ 5 times amount after oven dry after adding proper amount of methanol dissolving.After petroleum ether balance D-101 pillar, utilize each eluting of petroleum ether-ethyl acetate 50:1,20:1,10:1 4 column volumes, collect 10:1 position and concentrate evaporate to dryness.After petroleum ether-ethyl acetate 10:1 eluting position adds dissolve with methanol, centrifugal, get supernatant, utilize high pressure to prepare liquid phase and be prepared, collect anisic acid para hydroxybenzene ethyl ester chromatographic peak, after concentrate drying, obtain anisic acid para hydroxybenzene ethyl ester.
High pressure preparative liquid chromatography condition: Agilent 1260 type preparative chromatograph, chromatographic column is FujiC18 (250 × 50mm); Mobile phase is acetonitrile (A)-water (B); Determined wavelength is 310nm; Elution program is 0 ~ 60min, A30%, B70%; Flow velocity is 30mLmin-
1; Column temperature is 30 DEG C.
Anisic acid para hydroxybenzene ethyl ester qualification result is as follows:
1HNMR(400MHz,CH3OD)δ:7.92(2H,d,J=8.8Hz,H-2,6),6.96(2H,d,J=8.8Hz,H-3,5),7.09(2H,d,J=8.4Hz,H-2′,6′),6.72(2H,d,J=8.4Hz,H-3′,5′),4.40(2H,t,J=7.0Hz,H-2″),2.94(2H,t,J=7.0Hz,H-3″),3.84(3H,s,-OMe);
13CNMR(100MHz,CD3OD)δ:123.7(C-1),132.6(C-2,C-6),114.8(C-3,C-5),165.2(C-4),130.1(C-1′),131.0(C-2′,6′),116.3(C-3′,C-5′),157.1(C-4′),167.9(C-1″),66.9(C-2″),35.4(C-3″),56.0(-OMe)。
Embodiment 2: anisic acid para hydroxybenzene ethyl ester causes the protective effect of HK-2 cell renal injury model to cisplatin
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
Cisplatin is purchased from sigma company; The required basic culture solution of Da Erbaike (Dulbecco ' sminimumessentialmedium, DMEM)/F12 culture fluid, hyclone (fetalbovineserum, FBS), trypsin purchased from American Gibco company; MTT is purchased from green skies company.
The cultivation of 1.3HK-2 cell
HK-2 cell carries out cellar culture, 5%CO in containing the DMEM/F12 culture fluid of 10%FBS
2, 37 DEG C of cell 0.25% trypsinizations being cultured to incorporating period, go down to posterity, and the cell of growth of taking the logarithm is tested.
1.4 anisic acid para hydroxybenzene ethyl esters are on the impact of HK-2 Growth of Cells:
To take the logarithm trophophase HK-2 cell, with 1 × 10
4cells/100 μ l/ hole is inoculated in 96 well culture plates, treat more than Growth of Cells to 80%, be changed to serum-free medium cultivation 12h to make to be in synchronous regime, add 6.25 respectively, 12.5, 25, 50, 100, 150 μ g/ml various concentration anisic acid para hydroxybenzene ethyl ester is cultivated under normal condition, matched group: cultivate in normal incubation medium, 24h is placed on observation of cell metamorphosis under inverted microscope, every hole adds MTT solution 10 μ l, continue to hatch 4h in cell culture incubator, supernatant is abandoned in suction, add 150 μ lDMSO and dissolve first a ceremonial jade-ladle, used in libation, microplate reader is used to take 570nm as determined wavelength, 630nm is reference wavelength, read absorbance, often group establishes 6 multiple holes, average for each group, experiment repetition 3 times, experimental result is in table 1.
Table 1 anisic acid para hydroxybenzene ethyl ester affects OD value x ± s to normal HK-2 Growth of Cells
Drug level (μ g/mL) | Anisic acid is to hydroxy methacrylate (n=6) |
control | 0.63±0.03 |
6.25 | 0.62±0.06 |
12.5 | 0.63±0.02 |
25 | 0.64±0.01 |
50 | 0.65±0.02 |
100 | 0.66±0.03 |
150 | 0.61±0.01 |
*: P < 0.05 is compared with normal group
As seen from the results in Table 1, the growth of anisic acid para hydroxybenzene ethyl ester on normal HK-2 cell does not significantly affect compared with matched group, and safety is high.
1.5 protective effect of drug
To take the logarithm trophophase HK-2 cell, with 1 × 10
4cells/100 μ l/ hole is inoculated in 96 well culture plates, treat more than Growth of Cells to 80%, be changed to serum-free medium cultivation 12h to make to be in synchronous regime, add people 80 μm of olL-1DDP respectively, add 0 respectively simultaneously, 6.25, 12.5, 25, 50, 100, 150 μ g/ml various concentration anisic acid para hydroxybenzene ethyl ester normal condition is cultivated, matched group: cultivate in normal incubation medium, 24h is placed on observation of cell metamorphosis under inverted microscope, every hole adds MTT solution 10 μ l, continue to hatch 4h in cell culture incubator, supernatant is abandoned in suction, add 150 μ lDMSO and dissolve first a ceremonial jade-ladle, used in libation, microplate reader is used to take 570nm as determined wavelength, 630nm is reference wavelength, read absorbance, often group establishes 6 multiple holes, average for each group, experiment repetition 3 times, experimental result is in table 2.
Table 2 anisic acid para hydroxybenzene ethyl ester is to the protective effect of injury of kidney cell
Drug level (μ g/mL) | Anisic acid is to hydroxy methacrylate (n=6) |
control | 0.66±0.05 |
Model | 0.49±0.04 ## |
6.25 | 0.50±0.03 |
12.5 | 0.51±0.02 |
25 | 0.51±0.03 |
50 | 0.55±0.02* |
100 | 0.56±0.03* |
150 | 0.50±0.01 |
##:P < 0.01 is compared with normal group; *: p<0.05 is compared with model group; *: P < 0.01
As seen from the results in Table 2, model group is compared matched group and is had significant difference, shows that cisplatin causes HK-2 cell renal injury model and successfully constructs.And when anisic acid para hydroxybenzene ethyl ester drug level is 50,100 μ g/ml, it has significant protective effect to injury of kidney cell, show that anisic acid para hydroxybenzene ethyl ester can be applied in the preparation of injury of kidney protection medicine.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. the application of anisic acid para hydroxybenzene ethyl ester in preparation injury of kidney protection medicine.
2. apply according to claim 1, it is characterized in that, described injury of kidney is renal damage caused by cis-platinum.
3. apply according to claim 1, it is characterized in that, described anisic acid para hydroxybenzene ethyl ester extracts and obtains from Rhizoma Et Radix Notopterygii.
4. apply according to claim 3, it is characterized in that, described anisic acid para hydroxybenzene ethyl ester is obtained by following methods extraction and isolation:
Step 1, pulverizing Rhizoma Et Radix Notopterygii rhizome, add alcoholic solution and extract, and extracting liquid filtering, concentrated rear salt adding acid for adjusting pH are 2, and reflux also concentrates evaporate to dryness and obtains crude extract;
Step 2, crude extract add alcoholic solution and redissolve, adsorbed by macroporous resin column, successively water elution 4 column volumes, 40% ethanol elution 3 column volume, 60% ethanol elution 6 column volume, collect 60% ethanol elution, except desolventizing, be concentrated into extractum, then acetic acid ethyl dissolution, filtration is added, filtrate is except desolventizing, concentrated, dry, obtains Rhizoma Seu Radix Notopterygii extract;
After step 3, Rhizoma Seu Radix Notopterygii extract add dissolve with methanol, add in the thick silica gel of equivalent and stir, be poured in the 200-300 order silicagel column containing 4 ~ 5 times amount after oven dry; After petroleum ether balance D-101 pillar, utilize each eluting of petroleum ether-ethyl acetate 50:1,20:1,10:1 4 column volumes, collect 10:1 eluting position and concentrate evaporate to dryness, after adding dissolve with methanol, centrifugal, get supernatant, utilize high pressure preparative liquid chromatography to be prepared separation, obtain anisic acid para hydroxybenzene ethyl ester.
5. preparation method according to claim 4, is characterized in that, the volume mass of Rhizoma Et Radix Notopterygii rhizome described in step 1 and alcoholic solution is than being 1g:6-10mL.
6. preparation method according to claim 4, it is characterized in that, described in step 1, the concentration expressed in percentage by volume of alcoholic solution is preferably 70%.
7. preparation method according to claim 4, it is characterized in that, described high pressure preparative liquid chromatography condition is as follows:
Agilent 1260 type preparative chromatograph, chromatographic column is FujiC18 (250 × 50mm); Mobile phase is acetonitrile-water; Determined wavelength is 310nm; Elution program is 0 ~ 60min, acetonitrile 30%, water 70%; Flow velocity is 30mLmin
-1; Column temperature is 30 DEG C.
8. preparation method according to claim 4, is characterized in that, crude extract described in step 2 adds alcoholic solution and redissolves for crude extract adds 40% dissolve with ethanol solution of Rhizoma Et Radix Notopterygii rhizome gross weight 1 times amount.
9. preparation method according to claim 4, it is characterized in that, described macroporous resin column is D-101 type macroporous resin column.
10. preparation method according to claim 4, it is characterized in that, described macroporous resin column weight is 2 times of Rhizoma Et Radix Notopterygii rhizome gross weight.
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Cited By (2)
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CN106905385A (en) * | 2017-04-13 | 2017-06-30 | 嘉兴德扬生物科技有限公司 | A kind of low stain prepares the method and its antibacterial applications of anisic acid sugar ester |
CN113244208A (en) * | 2021-05-20 | 2021-08-13 | 云南中医药大学 | Application of HPA in preparation of medicine for treating non-alcoholic fatty liver disease |
Citations (1)
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US20090163545A1 (en) * | 2007-12-21 | 2009-06-25 | University Of Rochester | Method For Altering The Lifespan Of Eukaryotic Organisms |
Non-Patent Citations (4)
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YOU-BO ZHANG等: "Rapid and Sensitive RP-LC Method for the Quantification and Pharmacokinetic Study of p-Hydroxyphenethyl Anisate in Rat Plasma", 《CHROMATOGRAPHIA》 * |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN106905385A (en) * | 2017-04-13 | 2017-06-30 | 嘉兴德扬生物科技有限公司 | A kind of low stain prepares the method and its antibacterial applications of anisic acid sugar ester |
CN113244208A (en) * | 2021-05-20 | 2021-08-13 | 云南中医药大学 | Application of HPA in preparation of medicine for treating non-alcoholic fatty liver disease |
CN113244208B (en) * | 2021-05-20 | 2022-07-12 | 云南中医药大学 | Application of HPA in preparation of medicine for treating non-alcoholic fatty liver disease |
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