CN1880955A - Crocin finger spectrum analysis method - Google Patents
Crocin finger spectrum analysis method Download PDFInfo
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- CN1880955A CN1880955A CNA2005100770055A CN200510077005A CN1880955A CN 1880955 A CN1880955 A CN 1880955A CN A2005100770055 A CNA2005100770055 A CN A2005100770055A CN 200510077005 A CN200510077005 A CN 200510077005A CN 1880955 A CN1880955 A CN 1880955A
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- crocin
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Abstract
The invention discloses a fingerprint atlas analyzing method; which is characterized by the following: adding buffer in the inversed-phase chromatogram system for west carthamin; possessing stable chemical property for west carthamin-1, west carthamin-2, west carthamin-4 in the flow phase; displaying precise quantitative property and stable reserving time for west carthamin-3; satisfying the request of fingerprint atlas analysis.
Description
Technical field
The present invention relates to the analytical approach of a kind of Chinese medicine and extract thereof, particularly adopt high performance liquid chromatography crocin to be carried out the method for fingerprint map analyzing.
Background technology
Crocin is the principal ingredient of Chinese medicine west safflower medicinal material, in saffron extract and injection crocin, its main compound comprises crocin-1, crocin-2, crocin-3, crocin-4, and chemical structural formula and molecular formula that they are concrete are as follows:
Crocin-1 molecular formula C
44H
64O
24
Crocin-2 molecular formula C
38H
54O
19
Crocin-3 molecular formula C
30H
42O
14
Crocin-4 molecular formula C
26H
34O
8
The record crocin has effect promoting blood circulation and removing blood stasis, as to scatter knot in the prior art; Be used for apoplexy syndrome of blood stasis (brain vessle-Bi symptom-complex plug) etc.
The finger-print experimental technique of prior art is to test according to the high performance liquid chromatography of an appendix VID of Chinese Pharmacopoeia version in 2000 defined to carry out, but the high performance liquid chromatography of an appendix VID of Chinese Pharmacopoeia version in 2000 defined is a general rule, not to that concrete kind descriptive analysis method.
General provision is:
1, chromatographic condition and system suitability test
Make filling agent with reverse-phase chromatographic column; Organic phase-buffer solution is moving phase, the moving phase processing of should filtering, outgas, and the detection wavelength is 440nm.It is 2.8-3.3 minute that the adjustment flow velocity makes the retention time at crocin-1 peak, and number of theoretical plate calculates with crocin-1 peak should be not less than 1500.
2, the peak at the total peak of technical parameter number and relative retention time: 1 (0.67-0.72): 2 (0.80-0.85); 3 (S is with reference to peak-crocins-11.0), 4 (1.1-1.2), 5 (crocin-21.4-1.6), 6 (crocin-42.4-2.6), 7 (crocin-36.4-7.0), 8 (7.4-8.1).Peak area ratio: 5: S=(0.40-0.75).
3, the about 5mg of crocin-1 reference substance is got in the preparation of object of reference solution, accurate claims surely, puts in the measuring bottle of 25ml, with dissolve with methanol and be diluted to scale, shakes up.Precision is measured above-mentioned crocin-1 reference substance solution 2.0ml and is put in the 25ml measuring bottle, adds moving phase and is diluted to scale, shakes up, and promptly gets object of reference solution.
4, the preparation of need testing solution
The west safflower medicinal material is got the about 20mg of the dried fine powder of this product, puts in the apparatus,Soxhlet's, adds methyl alcohol 50ml, and reflux is to extracting liquid colourless, put coldly, use the methanol wash extraction apparatus, merge extract and cleansing solution and put in the 100ml measuring bottle, add moving phase and be diluted to scale, shake up, promptly.Crocin is got the about 20mg of this product, puts in the measuring bottle of 25ml, with dissolve with methanol and be diluted to scale, shakes up.Get this liquid 1.0ml and put in the 25ml measuring bottle, add moving phase and be diluted to scale, shake up, promptly.
The injection crocin is got 2 bottles of this product, with moving phase dissolving and be diluted in the measuring bottle of putting 50ml.Get this liquid 1.0ml and put in the 50ml measuring bottle, add moving phase and be diluted to scale, shake up, promptly.
5, accurate respectively above-mentioned object of reference solution and each 10ul of need testing solution of drawing of determination method, inject liquid chromatograph respectively, write down 1 hour chromatogram, calculate the retention time and the peak area of each chromatographic peak, the peak at the total peak of contrast number and relative retention time should be up to specification.
According to the technical requirement of State Food and Drug Administration to the research of traditional Chinese medicine finger-print, when the finger-print of crocin and medicinal material and injection has been carried out analysis, owing to there is the carboxylic acid (seeing following formula) of a part in crocin-3 molecular structure, in organic phase-water, can ionization go out H
+Or and H
+In conjunction with forming carboxylic acid, this mobile equilibrium is unsettled, therefore causes crocin-3 when fingerprint map analyzing, and the retention time of each sample introduction is unsettled, and drift is indefinite, and repeatability is very poor, does not meet the requirement of fingerprint map analyzing.
Summary of the invention
In order to overcome the deficiencies in the prior art part, the object of the present invention is to provide the fingerprint analysis method of a kind of improved Chinese medicine and extract thereof, particularly crocin is carried out the method for fingerprint map analyzing.
In order to finish above-mentioned goal of the invention, the present invention by the following technical solutions:
The analytical approach of the finger-print of a kind of Chinese medicine crocin composition and extract thereof comprises and adopts chromatographic column or reverse-phase chromatographic column high performance liquid chromatography, and organic phase-buffer solution is moving phase, it is characterized in that:
The filler of chromatographic column is selected from C1, C3, C6, C8, C18, CN, NH
2, described organic phase-buffer solution is organic phase-salt or the acids buffer solution of pH value for 1.5-8.5, and wherein the volume ratio of organic phase is 5%-80%, and preferably the volume ratio of organic phase is 10%-60%.
Described organic phase comprises tetrahydrofuran, methyl alcohol, acetonitrile.
The preferred pH value of described organic phase-buffer solution is 2.8-8.0.
Analytical approach of the present invention is to find employing organic phase-buffer solution to make moving phase, for the analytical approach of the finger-print of west safflower glycoside Chinese medicine that contains carboxylic acid structure and extract thereof.The filler of chromatographic column that concrete is is selected from C1, C3, C6, C8, C18, CN, NH
2, moving phase consist of organic phase-buffer solution, pH value wherein is 1.5-8.5, the ratio of organic phase is 5%-80% (v/v), the ratio of buffer solution is 20%-80% (v/v).
What the present invention adopted is that organic phase-buffer solution is moving phase.If not adopting organic phase-buffer solution is moving phase, be moving phase and adopt organic phase-water, then contain the crocin-3 of carboxylic acid, when analyzing is unsettled, and it is unsettled therefore causing each retention time, and drift is indefinite, poor repeatability does not meet the requirement of fingerprint map analyzing.
The present invention's (concentration of salt is that 0.005mol/L-0.2mol/L, pH value of buffer solution scope are 2.0-8.0) when in aqueous solution, adding various salts with corresponding acid or polyprotonic acid formation buffer solution, the retention time of crocin-3 is stable, the retention time of crocin-1, crocin-2, crocin-4 is constant substantially, the stable in properties of compound meets the requirement of fingerprint map analyzing.
Principal ingredient is a crocin-1 in the specimen of the present invention, its stable in properties, and relative content is elected object of reference as more than 50%.
Description of drawings
Fig. 1 is the finger-print of crocin.
Embodiment
Below will describe specific embodiments in detail, following embodiment and non-limiting the solution of the present invention further combined with embodiment.
Measuring 11 samples through following examples obtains respectively (seeing Table 1) and accompanying drawing 1 in the crocin technical data.
Embodiment 1
Instrument Agilent 1100 high pressure liquid chromatographs (comprising G1312A BinPump, G1313AALS, G1314A VWD), the Agilent chromatographic work station.
When adopting method of the present invention that sample 1 is measured, be filling agent (Shimadzu VP-ODS 150mm * 4.6mm, 5 μ m) with octadecylsilane chemically bonded silica; The ammonium acetate solution of methyl alcohol-0.02mol/L (regulating pH4.0 with acetic acid) (55: 45) is a moving phase, and the detection wavelength is 440nm.The adjustment flow velocity makes retention time 2.9-3.3 minute of crocin-1 peak, and number of theoretical plate calculates with crocin-1 peak should be not less than 1500.
Embodiment 2
Instrument Agilent 1100 high pressure liquid chromatographs (comprising G1312A BinPump, G1313AALS, G1315A DAD), the Agilent chromatographic work station.
When adopting method of the present invention that sample 2 is measured, be filling agent (Kromasil-ODS 200mm * 4.6mm, 5 μ m) with octadecylsilane chemically bonded silica; The ammonium acetate solution of methyl alcohol-acetonitrile-0.2mol/L (regulating pH4.5 with acetic acid) (25: 5: 70) is a moving phase, and the detection wavelength is 440nm.The adjustment flow velocity makes retention time 2.8-3.3 minute of crocin-1 peak, and number of theoretical plate calculates with crocin-1 peak should be not less than 1500.
Embodiment 3
Instrument Agilent 1100 high pressure liquid chromatographs (comprising G1312A BinPump, G1313AALS, G1314A VWD), the Agilent chromatographic work station.
When adopting method of the present invention that sample 3 is measured, be filling agent (NOVPAK-ODS 100mm * 3.9mm, 5 μ m) with octadecylsilane chemically bonded silica; The sodium acetate solution of tetrahydrofuran-0.05mol/L (regulating pH2.8 with acetic acid) (20: 80) is a moving phase, and the detection wavelength is 440nm.The adjustment flow velocity makes retention time 2.8-3.3 minute of crocin-1 peak, and number of theoretical plate calculates with crocin-1 peak should be not less than 1500.
Embodiment 4
Instrument Hi-Tech P 4000 Pump systems, Model 525 UV-detector able to programme, Chromtek chromatographic work station.
When adopting method of the present invention that sample 4 is measured, be filling agent (ZORBAX-C8 200mm * 4.6mm, 5 μ m) with octyl silane group silica gel; Methanol-water solution (regulating pH4.0 with phosphoric acid) (60: 40) is moving phase, and detecting wavelength is 440nm.The adjustment flow velocity makes retention time 2.8-3.3 minute of crocin-1 peak, and number of theoretical plate calculates with crocin-1 peak should be not less than 1500.
Embodiment 5
Instrument Agilent 1100 high pressure liquid chromatographs (comprising G1312A BinPump, G1313AALS, G1314A VWD), the Agilent chromatographic work station.
When adopting method of the present invention that sample 5 is measured, be filling agent (Chromasil-C3 200mm * 4.6mm, 5 μ m) with C3 alkyl silane bonded silica gel; The ammonium sulfate of methyl alcohol-0.02mol/L (regulating pH7.0 with sulfuric acid) (55: 45) is a moving phase, and the detection wavelength is 440nm.The adjustment flow velocity makes retention time 2.8-3.3 minute of crocin-1 peak, and number of theoretical plate calculates with crocin-1 peak should be not less than 1500.
Embodiment 6
The instrument Alltech 426 Alltech uvis of HPLC Pump system 200 variable wavelength UV-detector, the Chromtek chromatographic work station.
When adopting method of the present invention that sample 6 is measured, be filling agent (Chromasil-C1 200mm * 4.6mm, 5 μ m) with C1 alkyl silane bonded silica gel; The sodium acetate solution of methyl alcohol-0.1mol/L (regulating pH6.0 with acetic acid) (40: 60) is a moving phase, and the detection wavelength is 440nm.The adjustment flow velocity makes retention time 2.8-3.3 minute of crocin-1 peak, and number of theoretical plate calculates with crocin-1 peak should be not less than 1500.
Embodiment 7
Instrument Agilent 1100 high pressure liquid chromatographs (comprising G1312A BinPump, G1313AALS, G1314A VWD), the Agilent chromatographic work station.
When adopting method of the present invention that sample 7 is measured, be filling agent (HYPER-NH with amino chemically bonded silica
2250mm * 4.6mm, 5 μ m); The liquor sodii citratis of acetonitrile-0.03mol/L (regulating pH5.0 with citric acid) (30: 70) is a moving phase, and the detection wavelength is 440nm.The adjustment flow velocity makes retention time 2.8-3.3 minute of crocin-1 peak, and number of theoretical plate calculates with crocin-1 peak should be not less than 1500.
Embodiment 8
Instrument Agilent 1100 high pressure liquid chromatographs (comprising G1312A BinPump, G1313AALS, G1314A VWD), the Agilent chromatographic work station.
When adopting method of the present invention that sample 8 is measured, be filling agent (ALLTIMA-CN 200mm * 4.6mm, 5 μ m) with cyano group silane group silica gel; The acetic acid triethylamine solution (regulating pH8.0 with acetic acid) (52: 48) of methyl alcohol-0.01mol/L is a moving phase, and the detection wavelength is 440nm.The adjustment flow velocity makes retention time 2.8-3.3 minute of crocin-1 peak, and number of theoretical plate calculates with crocin-1 peak should be not less than 1500.
Embodiment 9
Employing is measured sample 9 with embodiment 1 identical method, and what obtain the results are shown in Table 1.
Embodiment 10
Adopt embodiment 2 identical methods that sample 10 is measured, what obtain the results are shown in Table 1.
Embodiment 11
Adopt embodiment 4 identical methods that sample 11 is measured, what obtain the results are shown in Table 1.
| Sample | Peak 1 (0.67-0.72) | Peak 2 (0.80-0.85) | Peak S (1.0) | Peak 4 (1.1-1.2) | Peak 5 (1.4-1.6) | Peak 6 (2.4-2.6) | Peak 7 (6.4-7.0) | Peak 8 (7.4-8.1) |
| 1 | 2.21 | 2.62 | 3.20 | 3.64 | 4.82 | 7.99 | 21.21 | 24.90 |
| 2 | 2.20 | 2.61 | 3.19 | 3.63 | 4.80 | 7.96 | 21.12 | 24.82 |
| 3 | 2.20 | 2.61 | 3.18 | 3.61 | 4.79 | 7.94 | 21.04 | 24.73 |
| 4 | 2.20 | 2.61 | 3.18 | 3.60 | 4.78 | 7.92 | 20.98 | 24.64 |
| 5 | 2.20 | 2.61 | 3.17 | 3.61 | 4.79 | 7.93 | 20.95 | 24.58 |
| 6 | 2.20 | 2.61 | 3.17 | 3.60 | 4.78 | 7.91 | 20.89 | 24.51 |
| 7 | 2.20 | 2.61 | 3.18 | 3.60 | 4.78 | 7.90 | 20.82 | 24.52 |
| 8 | 2.20 | 2.61 | 3.17 | 3.61 | 4.77 | 7.88 | 21.02 | 24.62 |
| 9 | 2.20 | 2.61 | 3.18 | 3.60 | 4.78 | 7.90 | 20.82 | 24.52 |
| 10 | 2.20 | 2.61 | 3.16 | 3.559 | 4.75 | 7.86 | 20.97 | 24.34 |
| 11 | 2.20 | 2.60 | 3.15 | 3.58 | 4.73 | 7.81 | 20.99 | 24.20 |
Table 1
Claims (4)
1, the analytical approach of the finger-print of Chinese medicine crocin composition and extract thereof comprises and adopts chromatographic column or reverse-phase chromatographic column high performance liquid chromatography, and organic phase-buffer solution is moving phase, it is characterized in that:
The filler of chromatographic column is selected from C1, C3, C6, C8, C18, CN, NH2, and described organic phase-buffer solution is organic phase-salt or the acids buffer solution of pH value for 1.5-8.5, and wherein the volume ratio of organic phase is 5%-80%.
2, analytical approach according to claim 1 is characterized in that described organic phase comprises the tetrahydrochysene furan food in one's mouth, methyl alcohol, acetonitrile.
3, analytical approach according to claim 1 and 2, the volume ratio that it is characterized in that organic phase is 10%-60%.
4, analytical approach according to claim 1 is characterized in that the pH value of described organic phase-buffer solution is 2.8-8.0.
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|---|---|---|---|
| CNB2005100770055A CN100495026C (en) | 2005-06-13 | 2005-06-13 | Crocin finger spectrum analysis method |
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|---|---|---|---|
| CNB2005100770055A CN100495026C (en) | 2005-06-13 | 2005-06-13 | Crocin finger spectrum analysis method |
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|---|---|
| CN1880955A true CN1880955A (en) | 2006-12-20 |
| CN100495026C CN100495026C (en) | 2009-06-03 |
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|---|---|---|---|
| CNB2005100770055A Expired - Fee Related CN100495026C (en) | 2005-06-13 | 2005-06-13 | Crocin finger spectrum analysis method |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103512979A (en) * | 2013-10-21 | 2014-01-15 | 山东阿如拉药物研究开发有限公司 | Detection method of pharmaceutical composition Zuozhudaxi |
| CN103529140A (en) * | 2013-10-18 | 2014-01-22 | 山东阿如拉药物研究开发有限公司 | Method for determining content of saffron in medicinal composition |
| CN103601764A (en) * | 2013-11-26 | 2014-02-26 | 威海东宝制药有限公司 | Purification and separation technology of crocin in gardenia extract |
| CN106645529A (en) * | 2016-11-21 | 2017-05-10 | 湖州市中心医院 | Method for evaluating color, flavor and taste multicomponent quantification combination fingerprint quality of saffron |
| CN106706774A (en) * | 2016-11-21 | 2017-05-24 | 湖州市中心医院 | Method for measuring crocin components in stigma croci medicinal materials through area normalization method |
| CN109507343A (en) * | 2018-12-24 | 2019-03-22 | 上海医药集团股份有限公司 | UPLC method quickly measures west safflower active constituent content and identifies illegal dyeing |
| CN110579558A (en) * | 2019-10-09 | 2019-12-17 | 浙江寿仙谷医药股份有限公司 | Detection system and detection method for component content of saffron |
| CN114324701A (en) * | 2021-12-21 | 2022-04-12 | 中南大学 | Method for rapidly and simultaneously determining content of crocin-1, crocin-2, crocin-3 and crocin-4 |
-
2005
- 2005-06-13 CN CNB2005100770055A patent/CN100495026C/en not_active Expired - Fee Related
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103529140A (en) * | 2013-10-18 | 2014-01-22 | 山东阿如拉药物研究开发有限公司 | Method for determining content of saffron in medicinal composition |
| CN103529140B (en) * | 2013-10-18 | 2015-04-15 | 山东金诃药物研究开发有限公司 | Method for determining content of saffron in medicinal composition |
| CN103512979A (en) * | 2013-10-21 | 2014-01-15 | 山东阿如拉药物研究开发有限公司 | Detection method of pharmaceutical composition Zuozhudaxi |
| CN103512979B (en) * | 2013-10-21 | 2014-09-03 | 山东阿如拉药物研究开发有限公司 | Detection method of pharmaceutical composition Zuozhudaxi |
| CN103601764A (en) * | 2013-11-26 | 2014-02-26 | 威海东宝制药有限公司 | Purification and separation technology of crocin in gardenia extract |
| CN106706774A (en) * | 2016-11-21 | 2017-05-24 | 湖州市中心医院 | Method for measuring crocin components in stigma croci medicinal materials through area normalization method |
| CN106645529A (en) * | 2016-11-21 | 2017-05-10 | 湖州市中心医院 | Method for evaluating color, flavor and taste multicomponent quantification combination fingerprint quality of saffron |
| CN106645529B (en) * | 2016-11-21 | 2018-09-11 | 湖州市中心医院 | West safflower color, smell and taste multicomponent quantitatively combines finger-print quality evaluating method |
| CN106706774B (en) * | 2016-11-21 | 2019-05-28 | 湖州市中心医院 | The method that area normalization method measures crocin constituents in west safflower medicinal material |
| CN109507343A (en) * | 2018-12-24 | 2019-03-22 | 上海医药集团股份有限公司 | UPLC method quickly measures west safflower active constituent content and identifies illegal dyeing |
| CN109507343B (en) * | 2018-12-24 | 2021-08-06 | 上海医药集团股份有限公司 | UPLC method for rapidly determining content of effective components of saffron and identifying illegal dyeing |
| CN110579558A (en) * | 2019-10-09 | 2019-12-17 | 浙江寿仙谷医药股份有限公司 | Detection system and detection method for component content of saffron |
| CN110579558B (en) * | 2019-10-09 | 2022-04-05 | 浙江寿仙谷医药股份有限公司 | Detection system and detection method for component content of saffron |
| CN114324701A (en) * | 2021-12-21 | 2022-04-12 | 中南大学 | Method for rapidly and simultaneously determining content of crocin-1, crocin-2, crocin-3 and crocin-4 |
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| CN100495026C (en) | 2009-06-03 |
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