CN102229967B - Method for preparing cartharein and isocartharein from naringenin - Google Patents
Method for preparing cartharein and isocartharein from naringenin Download PDFInfo
- Publication number
- CN102229967B CN102229967B CN2011100885016A CN201110088501A CN102229967B CN 102229967 B CN102229967 B CN 102229967B CN 2011100885016 A CN2011100885016 A CN 2011100885016A CN 201110088501 A CN201110088501 A CN 201110088501A CN 102229967 B CN102229967 B CN 102229967B
- Authority
- CN
- China
- Prior art keywords
- naringenin
- isocarthamidin
- carthamin
- potato
- aspergillus niger
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/582—Recycling of unreacted starting or intermediate materials
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for preparing cartharein and isocartharein from naringenin. The method adopts the microbial transformation process comprising the following steps: culture of a microorganism, transformation of a substrate and collection and purification of the final product. The microorganism is a Aspergillus niger strain 3.4628 (CGMCC No.3.4628), and the substrate is naringenin. The transforming strain Aspergillus niger 3.4628 used in the invention is easily available and can be used repeatedly with low cost. The naringenin is rapidly transformed to produce cartharein and isocartharein within a short time period; and the transformation process causes less pollution and has a simple process, and the naringenin as the substrate can be recycled. The method makes preparation of the two substances (cartharein and isocartharein) in a large scale with low cost possible, and provides an important contribution to theoretical studies and wide applications of medicinal value of cartharein and isocartharein.
Description
Technical field
The present invention relates to a kind of method of being obtained carthamin and isocarthamidin by naringenin, specifically, relate to a kind ofly by microbial transformation, obtained the method for carthamin (carthamidin) and isocarthamidin (isocarthamidin) by naringenin, belong to biological pharmacy technical field.
Background technology
Take ectogenic natural or synthetic organic compound as substrate, be added in the living things system or enzyme system that is in growth conditions, under suitable condition, cultivate, so that the enzyme in substrate and the living things system interacts, thereby the generation structural modification, this process is called bio-transformation.Bio-transformation transforms chemical composition of Chinese materia medica with the enzyme system of multiple different catalysiss, can produce new natural compounds, or the raising Effective Component of Chinese Medicine is water-soluble, reduce the toxicity of compound, by combining with the pharmacological screening means, can therefrom find new high reactivity or hypotoxic natural radioactivity lead compound again.
All contain contiguous two hydroxyls in carthamin (carthamidin) and isocarthamidin (isocarthamidin) structure, so have very strong anti-oxidant activity, isocarthamidin particularly, its anti-oxidant activity is almost suitable with α-Tocopherol.The chemical structural formula of Carthamidin and isocarthamidin is as follows:
At present, carthamidin and isocarthamidin mainly extract from the natural phant such as safflower, Herba Scutellariae Barbatae, the root of large-flowered skullcap and obtain.Extract this two compounds if rely on merely the commerial growing of natural phant, not only taken the arable land, also increased extraction cost.Therefore, how using the Chinese traditional medicine biology technology, improve the yield of carthamidin and isocarthamidin, is the effective way of alleviating herb resource pressure.On the other hand, also significant to the production cost, the raising quality of the pharmaceutical preparations that reduce carthamidin and isocarthamidin like this.
Naringenin is a kind of flavonoid compound that extensively is present in plant and Chinese medicine, utilizes naringenin as transforming raw material, and aspergillus niger is as transforming bacterial strain, and the method for preparing carthamidin and isocarthamidin it is not yet seen relevant report.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of by microbial transformation, is obtained the method for carthamin (carthamidin) and isocarthamidin (isocarthamidin) by naringenin.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of method of being obtained carthamin and isocarthamidin by naringenin is characterized in that: be microbe transformation method, comprise the cultivation of microorganism, the conversion of substrate and the collection purification step of product; Described microorganism is aspergillus niger 3.4628 (Aspergillus niger, CGMCC 3.4628); Described substrate is naringenin.
The described method of being obtained carthamin and isocarthamidin by naringenin comprises following concrete steps:
A) cultivate Aspergillus niger strain
1. after Aspergillus niger strain being used the sterilized water mixing, be tiled on the potato solid slant culture base, in 20～40 ℃ of (preferred 25～37 ℃) activation culture 1～7 day (preferred 1～3 day);
2. from potato solid slant culture base, the bacterial strain access is equipped with in the triangular flask of potato liquid nutrient medium, and (1～7 day (preferred 1～3 day) is cultivated in preferred 90～150r/min), 20～40 ℃ (preferred 25～37 ℃) lower concussion at 75～200r/min;
B) add the substrate naringenin in above-mentioned nutrient solution, (preferred 90～150r/min), 20～40 ℃ (preferred 25～37 ℃) lower concussion is cultivated 1～7 day (preferred 1～3 day) and is transformed at 75～200r/min in continuation;
C) cultivate end, collect conversion fluid, use isopyknic ethyl acetate extraction, combined ethyl acetate extraction liquid, concentrating under reduced pressure dry ethyl acetate;
D) will concentrate the residue dissolve with methanol that obtains, separation and purification namely gets the mix products of carthamin and isocarthamidin.
Described potato solid slant culture base composed as follows: potato filtrate 1.0g/L+ glucose 20.0g/L+ agarose 15.0g/L.
Described potato liquid nutrient medium composed as follows: potato filtrate 1.0g/L+ glucose 20.0g/L.
Described potato filtrate is by with the 200g peeled potatoes, adds 1.0L water, filters after boiling 30min, filtrate is supplied the 1.0L preparation again and gets.
Step b) naringenin in is recommended to add with naringenin-DMSO solution form, and strength of solution is recommended as 0.1～20g/L (preferred 5～15g/L); The add-on of naringenin is recommended as and makes its final concentration is 1～100mg/L (10～60mg/L).
Steps d) separation and purification in can be adopted filtering with microporous membrane, centrifugation or column chromatography for separation.
The aperture of described millipore filtration is recommended as 0.45 μ m.
Compared with prior art, the present invention has following beneficial effect:
1) the conversion bacterial strain aspergillus niger 3.4628 of selecting (Aspergillus niger CGMCC 3.4628) is easy to buy, and can reuse, and cost is lower.
2) the naringenin conversion rate is fast, can obtain carthamidin and isocarthamidin in the short period.
3) conversion process is polluted little; technique is simple; the all right recycle of substrate-naringenin makes mass-producing and low-cost preparation carthamin and these two kinds of materials of isocarthamidin become possibility, and the theoretical investigation of carthamin and isocarthamidin and the extensive utilization of pharmaceutical use thereof are had significant contribution.
Description of drawings
Fig. 1 is the HPLC collection of illustrative plates of laboratory sample, and among the figure: A is the HPLC collection of illustrative plates of blank sample, adds naringenin in the transformation system that is:, but does not access the sample of bacterial strain; B is the HPLC collection of illustrative plates of the sample of the inventive method acquisition; The HPLC collection of illustrative plates of the negative control sample of C that is: accesses bacterial strain in the transformation system, but does not add the sample of naringenin; Applied sample amount is 20 μ l; 1 is the isocarthamidin peak in the collection of illustrative plates, and 2 is the carthamidin peak.
Fig. 2 is carhamidin in the sample that obtains of the inventive method and the LC-MS total ion current figure of isocarthamidin.
Fig. 3 is mass spectrum corresponding to tR=13.39min.
Fig. 4 is mass spectrum corresponding to tR=14.69min.
Fig. 5 is the picture that the DPPH thin-layer method is identified the anti-oxidant activity of naringenin (naringenin), isocarthamidin, carthamidin.
Embodiment
Below in conjunction with drawings and Examples the present invention is done in further detail explanation, but therefore do not limit content of the present invention.
Embodiment 1
One, the cultivation of Aspergillus niger strain
The fungi transformation plant aspergillus niger 3.4628 that the present invention selects (Aspergillus niger CGMCC 3.4628), the ball particulate state is white in color in the potato liquid nutrient medium, in solid medium, be yellow tabular, can continually and steadily growth in the liquid solid substratum.
Cultural method is as follows:
1, with Aspergillus niger strain with behind the sterilized water mixing, be tiled on the potato solid slant culture base, in 28 ℃ of activation culture 3 days;
2, from potato solid slant culture base, the bacterial strain access is equipped with in the triangular flask of 100mL potato liquid nutrient medium, cultivated 2 days in 120r/min, 28 ℃ of lower concussions.
Described potato solid slant culture base composed as follows: potato filtrate 1.0g/L+ glucose 20.0g/L+ agarose 15.0g/L.
Described potato liquid nutrient medium composed as follows: potato filtrate 1.0g/L+ glucose 20.0g/L.
Described potato filtrate is by with the 200g peeled potatoes, is cut into small pieces, and adds 1.0L water, filters after boiling 30min, filtrate is supplied the 1.0L preparation again and gets.
Two, the conversion of naringenin
Add 100 μ L naringenin-DMSO solution (concentration of this solution is 10g/L) in the liquid medium of above-mentioned Aspergillus niger strain, making its final concentration in culture system is 10mg/L, continues to cultivate 1 day in 120r/min, 28 ℃ of lower concussions.
Three, the collection purifying of converted product
Cultivate to finish, collect conversion fluid (vacuum filtration or centrifugation), with isopyknic ethyl acetate extraction 3 times, combined ethyl acetate extraction liquid, concentrating under reduced pressure dry ethyl acetate; With the concentrated residue 1mL dissolve with methanol that obtains, with 0.45 μ m filtering with microporous membrane, namely get the mix products of carthamin and isocarthamidin.
Four, the affirmation analysis of sample structure
A) HPLC analyzes
Chromatographic column: Plastisil ODS column (5 μ m, 250mm * 4.6mm)
Agilent 1100 high performance liquid chromatographs, the DAD detector;
Detect wavelength: 280nm; Column temperature: 30 ℃; Flow velocity: 1ml/min; Sample size: 20 μ l;
Moving phase: A-acetonitrile; B-formic acid (0.1wt%);
Gradient condition:
| Time | The ratio of A | The ratio of B |
| 0min | 25％ | 75％ |
| 10min | 38％ | 62％ |
| 30min | 38％ | 62％ |
Fig. 1 is the HPLC collection of illustrative plates of laboratory sample, and among the figure: A is the HPLC collection of illustrative plates of blank sample, adds naringenin in the transformation system that is:, but does not access the sample of bacterial strain; B is the HPLC collection of illustrative plates of the sample of the inventive method acquisition; The HPLC collection of illustrative plates of the negative control sample of C that is: accesses bacterial strain in the transformation system, but does not add the sample of naringenin; Applied sample amount is 20 μ l; 1 is the isocarthamidin peak in the collection of illustrative plates, and 2 is the carthamidin peak.As seen from Figure 1: 23.0min is naringenin substrate peak, and 13.29min and 14.54min have two converted product peaks, and 1 is isocarthamidin, and 2 is carthamidin.
B) LC-MS analyzes
Chromatographic column: Plastisil ODS column (5 μ m, 250mm * 4.6mm)
Agilent 1100 high performance liquid chromatographs, the DAD detector;
Detect wavelength: 280nm; Column temperature: 30 ℃; Flow velocity: 1ml/min; Sample size: 20 μ l;
Moving phase: A-acetonitrile; B-formic acid (0.1wt%);
Gradient condition:
| Time | The ratio of A | The ratio of B |
| 0min | 25％ | 75％ |
| 10min | 38％ | 62％ |
| 30min | 38％ | 62％ |
The MS analytical applications is with the ion trap instrument of Xcalibur workstation, MS and multistage MS analyze and select negative ion mode, carry out under the following conditions: capillary voltage 19V, spray voltage 4.5V, 300 ℃ of capillary temperatures, sheath gas velocity 20Arb, assisted gas flow velocity 5Arb, full scan mass spectrum scope parent ion is m/z100～1000, and daughter ion is m/z50～500.The LC-MS data analysis adopts instrument Finnigan Xcalibur 1.3 softwares going along with to carry out.
Fig. 2 is carhamidin in the sample that obtains of the inventive method and the LC-MS total ion current figure of isocarthamidin, and Fig. 3 is mass spectrum corresponding to tR=13.39min, and Fig. 4 is mass spectrum corresponding to tR=14.69min.By Fig. 2～4 as can be known: the molecular weight of two converted products all is 288, and the molecular weight of naringenin is 272, so can infer that two converted products has added respectively a hydroxyl in certain position.
Embodiment 2
One, investigates the conversion fate to the impact of converted product yield
Carry out transformation experiment by embodiment 1 described method, it is 10mg/L that the add-on of naringenin makes its final concentration in culture system, samples altogether six days.Experimental result is shown in Table 1.
Table 1 transforms fate to the impact of converted product yield
By as seen from Table 1: after adding the substrate naringenin, transform and carried out two days, efficiency of pcr product just reaches the highest.
Two, investigate the add-on of naringenin to the impact of converted product yield
Carry out transformation experiment by embodiment 1 described method, the add-on of naringenin makes its final concentration in culture system be respectively 10mg/L, 20mg/L, 30mg/L, 40mg/L, 50mg/L, 60mg/L.Experimental result is shown in Table 2.
The add-on of table 2 naringenin is on the impact of converted product yield
By as seen from Table 2: when the naringenin amount that adds in the culture system made its final concentration be 40mg/L, the converted product yield just reached the highest.
Embodiment 3
The isocarthamidin that obtains with DPPH thin-layer method qualitative identification the inventive method and the anti-oxidant activity of carthamidin sample: methanol solution 1 μ l, the 2 μ l, the 5 μ l that get respectively the naringenin that concentration is 1mg/mL (naringenin), isocarthamidin, carthamidin carry out point sample, with DPPH (1,1-phenylbenzene-2-trinitrophenyl-hydrazine) colour developing, colour developing the results are shown in shown in Figure 5.
Spot among Fig. 5 represents the colour developing result of carthamidin, isocarthamidin, naringenin from top to bottom successively, represents successively from left to right the colour developing result of the different point sample amounts of same sample.As seen from Figure 5: the isocarthamidin that is obtained by the inventive method and the purity of carthamidin are higher, all have preferably anti-oxidant activity.
Claims (8)
1. a method of being obtained carthamin and isocarthamidin by naringenin is characterized in that: be microbe transformation method, comprise the cultivation of microorganism, the conversion of substrate and the collection purification step of product; Described microorganism is aspergillus niger 3.4628(Aspergillus niger, and CGMCC 3.4628); Described substrate is naringenin; Comprise following concrete steps:
A) cultivate Aspergillus niger strain
1. with Aspergillus niger strain with behind the sterilized water mixing, be tiled on the potato solid slant culture base, in 20～40 ℃ of activation culture 1～7 day;
2. from potato solid slant culture base, the bacterial strain access is equipped with in the triangular flask of potato liquid nutrient medium, cultivated 1～7 day in 75～200r/min, 20～40 ℃ of lower concussions;
B) in above-mentioned nutrient solution, add the substrate naringenin, continue to cultivate and transformed in 1～7 day in 75～200r/min, 20～40 ℃ of lower concussions;
C) cultivate end, collect conversion fluid, use isopyknic ethyl acetate extraction, combined ethyl acetate extraction liquid, concentrating under reduced pressure dry ethyl acetate;
D) will concentrate the residue dissolve with methanol that obtains, separation and purification namely gets the mix products of carthamin and isocarthamidin;
Step b) naringenin in adds with naringenin-DMSO solution form.
2. the method for being obtained carthamin and isocarthamidin by naringenin according to claim 1 is characterized in that, comprises following concrete steps:
B) cultivate Aspergillus niger strain
1. with Aspergillus niger strain with behind the sterilized water mixing, be tiled on the potato solid slant culture base, in 25～37 ℃ of activation culture 1～3 day;
2. from potato solid slant culture base, the bacterial strain access is equipped with in the triangular flask of potato liquid nutrient medium, cultivated 1～3 day in 90～150r/min, 25～37 ℃ of lower concussions;
B) in above-mentioned nutrient solution, add the substrate naringenin, continue to cultivate and transformed in 1～3 day in 90～150r/min, 25～37 ℃ of lower concussions;
C) cultivate end, collect conversion fluid, use isopyknic ethyl acetate extraction, combined ethyl acetate extraction liquid, concentrating under reduced pressure dry ethyl acetate;
D) will concentrate the residue dissolve with methanol that obtains, separation and purification namely gets the mix products of carthamin and isocarthamidin.
3. the method for being obtained carthamin and isocarthamidin by naringenin according to claim 1 and 2 is characterized in that, described potato solid slant culture base composed as follows: potato filtrate 1.0g/L+ glucose 20.0g/L+ agarose 15.0g/L.
4. the method for being obtained carthamin and isocarthamidin by naringenin according to claim 1 and 2 is characterized in that, described potato liquid nutrient medium composed as follows: potato filtrate 1.0g/L+ glucose 20.0g/L.
5. according to claim 3 or the 4 described methods of being obtained carthamin and isocarthamidin by naringenin, it is characterized in that: described potato filtrate is by with the 200g peeled potatoes, adds 1.0L water, filters after boiling 30min, filtrate is supplied the 1.0L preparation again and gets.
6. the method for being obtained carthamin and isocarthamidin by naringenin according to claim 1, it is characterized in that: the concentration of described naringenin-DMSO solution is 0.1～20g/L, it is 1～100mg/L that the add-on of naringenin makes its final concentration.
7. the method for being obtained carthamin and isocarthamidin by naringenin according to claim 6, it is characterized in that: the concentration of described naringenin-DMSO solution is 5～15g/L, it is 10～60mg/L that the add-on of naringenin makes its final concentration.
8. the method for being obtained carthamin and isocarthamidin by naringenin according to claim 1 and 2 is characterized in that: separation and purification employing filtering with microporous membrane, centrifugation or column chromatography for separation steps d).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100885016A CN102229967B (en) | 2011-04-08 | 2011-04-08 | Method for preparing cartharein and isocartharein from naringenin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100885016A CN102229967B (en) | 2011-04-08 | 2011-04-08 | Method for preparing cartharein and isocartharein from naringenin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102229967A CN102229967A (en) | 2011-11-02 |
| CN102229967B true CN102229967B (en) | 2013-04-10 |
Family
ID=44842572
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011100885016A Expired - Fee Related CN102229967B (en) | 2011-04-08 | 2011-04-08 | Method for preparing cartharein and isocartharein from naringenin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102229967B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559545B (en) * | 2011-12-15 | 2014-05-21 | 北京大北农科技集团股份有限公司 | Composite micro-ecological preparation for aquatic product and premix thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002281995A (en) * | 2001-03-28 | 2002-10-02 | Pokka Corp | Flavonoid compound and method for producing the same |
| CN101089187A (en) * | 2006-06-14 | 2007-12-19 | 浙江工业大学 | Method for hydrolytic preparing biological tangeritin by enzyme |
| CN101092611A (en) * | 2006-09-08 | 2007-12-26 | 颜廷和 | Method for preparing purified Naringoside acid, and application of enzyme preparation |
-
2011
- 2011-04-08 CN CN2011100885016A patent/CN102229967B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002281995A (en) * | 2001-03-28 | 2002-10-02 | Pokka Corp | Flavonoid compound and method for producing the same |
| CN101089187A (en) * | 2006-06-14 | 2007-12-19 | 浙江工业大学 | Method for hydrolytic preparing biological tangeritin by enzyme |
| CN101092611A (en) * | 2006-09-08 | 2007-12-26 | 颜廷和 | Method for preparing purified Naringoside acid, and application of enzyme preparation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102229967A (en) | 2011-11-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9326506B2 (en) | Preparation method, agricultural composition and applications of natural brassinolide analogs | |
| CN108689803B (en) | Comprehensive utilization method of waste tobacco leaves | |
| CN102659912B (en) | Oxygen-rich disesquiterpenes compound, and preparation method and application thereof | |
| CN105567751A (en) | Fermentation method for bacillus subtilis to produce chlorogenic acid | |
| CN104838867A (en) | Chemical regulation method for increasing active ingredient content in polygala tenuifolia | |
| CN104894173B (en) | A kind of preparation method of curcumin derivate | |
| CN102229967B (en) | Method for preparing cartharein and isocartharein from naringenin | |
| CN1283779C (en) | Mucor for producing picropodophyllin and its glucoside and kaempferol flavone kind substance, its method and application | |
| CN103044511B (en) | A kind of isolation technique of ergot sterone in Phellinus bacterium | |
| CN109796394A (en) | A method of extracting auxin from Paenibacillus polymyxa fermentation liquid | |
| CN1534032A (en) | Method of high effect preparing rhoxadunol | |
| CN102757443B (en) | Sulfur-substituted podophyllum derivative and bioconversion, separation and purification method thereof | |
| CN108753626A (en) | One plant of 16 beta-hydroxy -19- of biosynthesis goes bacterial strain and its application of first -4-AD | |
| CN102382866B (en) | Preparation, purification and content detection methods for cerebroside | |
| CN104278070A (en) | Method for improving content of ergosterol in liquid fermentation products of phellinus igniarius | |
| CN102234669A (en) | Biotransformation and purification method of 4-(2,3,5,6-tetramethylpyrazine-1-group)-4'-demethylepipodophyllotoxin | |
| CN102337308A (en) | Method for converting bergenin into special nitrogenous derivative by using penicillium | |
| CN104807925A (en) | Method for extracting and separating allelopathic autotoxicity substances from panax notoginseng rhizosphere soil | |
| CN103923848B (en) | Multiple-shaped nuohan inferior yeast recombinant bacterial strain and the application in Taxol biosynthesis thereof | |
| CN104774773A (en) | Polygonum cuspidatum endogenous bacterial strain aspergillus fumigates J3 bacterial strain for converting polydatin | |
| CN101928291B (en) | Method for separating and purifying ansamitocin from precious orange synnema actinomycetes fermentation liquor | |
| CN105294687A (en) | Ion pair dual water phase method for extracting and separating pyrroloquinoline quinone | |
| CN110527632A (en) | One plant height imitates endogenetic fungal bacterial strain and its application of bioconversion betulic acid | |
| CN113583008B (en) | Method for extracting and separating pterosin | |
| CN105199966B (en) | A kind of conversion ginsenoside Rb1 produces aspergillus and the application of Rd |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130410 Termination date: 20160408 |