CN101991090A - Chrysanthemum stem and leaf extract and preparation method and application thereof - Google Patents
Chrysanthemum stem and leaf extract and preparation method and application thereof Download PDFInfo
- Publication number
- CN101991090A CN101991090A CN2009101847160A CN200910184716A CN101991090A CN 101991090 A CN101991090 A CN 101991090A CN 2009101847160 A CN2009101847160 A CN 2009101847160A CN 200910184716 A CN200910184716 A CN 200910184716A CN 101991090 A CN101991090 A CN 101991090A
- Authority
- CN
- China
- Prior art keywords
- chrysanthemum
- extract
- leaf
- stem
- extraction
- 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.)
- Pending
Links
Landscapes
- Cosmetics (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention relates to a chrysanthemum stem and leaf extract and a preparation method and application thereof, belonging to the technical field of extraction of natural antioxidants. The invention relates to the application of the chrysanthemum stem and leaf extract as a natural antioxidant and the preparation method thereof. The preparation method of the chrysanthemum stem and leaf extract comprises the following steps: pulverizing chrysanthemum stems and leaves; adding a water-based solvent for extraction; filtering or centrifuging to obtain the solution; drying the extracting solution to obtain a primary extract of the chrysanthemum stems and leaves; carrying out absorbent resin chromatography on the extracting solution or the primary extract of the chrysanthemum stems and leaves; eluting by an ethanol water solution; and drying the eluent to obtain the total flavone extract of the chrysanthemum stems and leaves. The method has the characteristics of simplicity, low cost and the like, and is suitable for industrial production. The chrysanthemum stem and leaf extract has notable antioxidation activity, and can be applied as the natural antioxidant to food, medicine and cosmetics.
Description
Technical field the invention belongs to natural extractive technique field, is specifically related to a kind of method and purposes that obtains extract from Chinese medicine chrysanthemum stem, leaf.
Background technology chrysanthemum (Chrysanthemum morifolium Ramat) is the dry capitulum of feverfew chrysanthemum, be China's tradition conventional Chinese medicine material, main effect has diffusing wind heat-clearing, and flat liver makes eye bright, detoxifcation, step-downs etc. cure mainly anemopyretic cold, and it is dizzy to have a headache, red eye, swell pain, it is dim-sighted to make eye bright, and it is sweet, bitter, cool in nature to distinguish the flavor of.The Pharmacopoeia of the People's Republic of China one one of version in 2005 is loaded with tribute chrysanthemum (Gongju), Chu chrysanthemum (Chuju), Bo chrysanthemum (Boju) and 4 kinds of main medicinal chrysanthemums of Hang Ju (Hangju), and is called four big medicinal chrysanthemums of China; Nowadays, Hang Ju, Bo chrysanthemum, tribute chrysanthemum, Chu chrysanthemum, Qi Ju, bosom chrysanthemum, Ji chrysanthemum, Huang Ju are eight big main flow commodity of the medicinal chrysanthemum of China.But the application study of its cauline leaf is less, and document record chrysanthemum leaf has treatment sore, ulcer, a wind, dizzy effect.Research to the comprehensive utilization of chrysanthemum cauline leaf is less, and Lin Xiaohua, Qiu Yuqi adopt supercritical technology to extract chrysanthemum cauline leaf active ingredient (extracting process of effective components in chrysanthemum root, stem and leaf, application number 01132299.3); Wei Qiang, Wang Qingjian adopt ethanol water to extract Chu chrysanthemum stem leaf total flavones class material, but it rests on laboratory level, and ethanol water is higher as the extractant cost, do not study simultaneously the purposes (Study on extraction of general flavone in Chu chrysanthemum stem, the leaf, Chinese veterinarian's medical magazine .2009.1) of Chu chrysanthemum stem leaf total flavones class material.The report that preparation of industrialization chrysanthemum stem-leaf extract is not arranged at present, as yet.
The problem that summary of the invention the present invention need solve is: 1, a kind of simple extracting method that can be used for suitability for industrialized production of preparation extract from the chrysanthemum cauline leaf in a large number is provided.2, provide and utilize said method to obtain the chrysanthemum stem-leaf extract.3, provide this chrysanthemum stem-leaf extract to be used for food, medicine and cosmetic use as natural.Described chrysanthemum cauline leaf is a remaining cauline leaf after chrysanthemums such as Hang Ju, Bo chrysanthemum, tribute chrysanthemum, Chu chrysanthemum, Qi Ju, bosom chrysanthemum, Ji chrysanthemum, Huang Ju are gathered, and described chrysanthemum stem-leaf extract is chrysanthemum cauline leaf primary extract or chrysanthemum stem leaf total flavones extract.
Technical scheme of the present invention:
(1) with chrysanthemum cauline leaf drying, pulverize, add the aqueous solvent of 8-100 times of chrysanthemum cauline leaf weight, extract;
(2) filtration or centrifugal removal insoluble matter obtain extract, and drying obtains chrysanthemum cauline leaf primary extract;
(3) chrysanthemum cauline leaf primary extract or extract are passed through the polymeric adsorbent chromatography, with the ethanol water wash-out of percent by volume 10%-95%, the eluent drying obtains chrysanthemum stem leaf total flavones extract.
The described aqueous solvent of step (1) is that pure water or mass percent are the sodium metabisulfite solution of 0.01%-0.4%.
The described extraction of step (1) is a kind of of extracting modes such as cold soaking extraction, seepage extraction, Soxhlet extraction, hot reflux extraction, microwave assisted extraction or ultrasonic wave assisted extraction.
The extraction temperature of the described extraction of step (1) is 20 ℃-100 ℃, divides and carries out for 1 time-6 times, extracts time spent 10min-5h at every turn, merges extract then.
The polymeric adsorbent that step (3) is adopted is the macroporous absorbent resin of polyamide or various models.
Step (2), step (3) and the described drying of step (4) are drying under reduced pressure or spray-drying.
The chrysanthemum stem-leaf extract has very strong oxidation resistance, add 0.1% chrysanthemum cauline leaf primary extract, it suppresses the rotten lipid oxidation rate of refrigeration cold cuts is 93.5%, adds 0.02% chrysanthemum stem leaf total flavones extract, and it suppresses the rotten lipid oxidation rate of refrigeration cold cuts is 92.3%; Mass percentage concentration is that the efficient of 0.1% chrysanthemum cauline leaf primary extract solution removal DPPH free radical is 67.2%, and mass percentage concentration is that the efficient that 0.02% chrysanthemum stem leaf total flavones extract is removed the DPPH free radical is 89.26%; The chrysanthemum cauline leaf primary extract of 10mg/mL, the TAC of chrysanthemum stem leaf total flavones extract are equivalent to 8.65 and 11.36mmol/LFeSO
4The chrysanthemum stem-leaf extract can be used as natural and is used for food, medicine and cosmetics.
The present invention has the following advantages: the present invention adopts aqueous solvent to extract, need not solvent recovery, and cost is low; Extracting mode is simple, and easy operating is compared with the supercritical extract mode, and equipment is cheap; The chrysanthemum stem-leaf extract has very strong non-oxidizability, can be used as natural and uses; The chrysanthemum cauline leaf is extracted, realize comprehensive utilization, help increasing farmers' income, accelerate the chrysanthemum industrialization process.
The specific embodiment
Embodiment 1
(1) the dry chrysanthemum cauline leaf of 2Kg is pulverized, with the water microwave assisted extraction of 12 times of chrysanthemum cauline leaf weight 2 times, power 500W, each 10min extracts 60 ℃ of temperature, the merging extract;
(2) filter the removal insoluble matter, drying under reduced pressure obtains chrysanthemum cauline leaf primary extract 330g;
(3) get chrysanthemum cauline leaf primary extract 300g and carry out the open chromatography of macroreticular resin, with the ethanol water wash-out of percent by volume 10%, the eluent drying obtains chrysanthemum stem leaf total flavones extract 190g.
Embodiment 2
(1) the dry chrysanthemum cauline leaf of 2Kg is pulverized, with the water ultrasonic wave assisted extraction of 9 times of chrysanthemum cauline leaf weight 2 times, power 1000W, each 25min extracts 35 ℃ of temperature, the merging extract;
(2) filter the removal insoluble matter, drying under reduced pressure obtains chrysanthemum cauline leaf primary extract 370g;
(3) get chrysanthemum cauline leaf primary extract 300g and carry out the open chromatography of polyamide, with the ethanol water wash-out of percent by volume 30%, the eluent drying obtains chrysanthemum stem leaf total flavones extract 210g.
Embodiment 3
(1) the dry chrysanthemum cauline leaf of 2Kg is pulverized, extracted 2 times with 0.1% sodium metabisulfite solution seepage of 9 times of chrysanthemum cauline leaf weight, each 4h extracts 50 ℃ of temperature, merges extract;
(2) filter the removal insoluble matter, drying under reduced pressure obtains chrysanthemum cauline leaf primary extract 300g;
(3) get chrysanthemum cauline leaf primary extract 280g and carry out the open chromatography of macroreticular resin, with the ethanol water wash-out of percent by volume 50%, the eluent drying obtains chrysanthemum stem leaf total flavones extract 220g.
Embodiment 4
(1) the dry chrysanthemum cauline leaf of 2Kg is pulverized, extracted 3 times with the cold soaking of 9 times of chrysanthemum cauline leaf weight, each 5h extracts 25 ℃ of temperature, the merging extract;
(2) filter the removal insoluble matter, drying under reduced pressure obtains chrysanthemum cauline leaf primary extract 290g;
(3) get chrysanthemum cauline leaf primary extract 240g and carry out the open chromatography of macroreticular resin, with the ethanol water wash-out of percent by volume 80%, the eluent drying obtains chrysanthemum stem leaf total flavones extract 190g.
Embodiment 5
(1) the dry chrysanthemum cauline leaf of 2Kg is pulverized, extracted 2 times with the hot reflux of 9 times of chrysanthemum cauline leaf weight, each 3h extracts 60 ℃ of temperature, merges extract;
(2) filter the removal insoluble matter, drying under reduced pressure obtains chrysanthemum cauline leaf primary extract 320g;
(3) get chrysanthemum cauline leaf primary extract 300g and carry out the open chromatography of polyamide, with the ethanol water wash-out of percent by volume 90%, the eluent drying obtains chrysanthemum stem leaf total flavones extract 260g.
As above described the preparation method of chrysanthemum stem-leaf extract disclosed in this invention, wherein comprise variation and change that those skilled in the art understand, these variations and changing in the scope of the present invention that also comprises claim and limited but need to understand.
Embodiment 6
One, the rotten lipid oxidation experiment of the inhibition of chrysanthemum stem-leaf extract of the present invention refrigeration cold cuts
Get fresh thin pork, be cut into the square about 2cm, add 2%NaCl, a certain amount of example 1 chrysanthemum stem-leaf extract in the meat, put into universal mixer and stir 5min and become meat gruel, 50g/ part is packed in the beaker, and beaker seals with polyethylene film.90 ℃ of water-bath to beaker central temperatures reach 80 ℃, keep 10min.Taking-up is cooled to room temperature with running water, puts into and takes out after 4 ℃ of refrigerators are preserved 72h, puts into universal mixer and pulverizes 3 times, each 10s.Get the meat sample 0.5g after the pulverizing, add 0.02% Vc solution 10ml, put into ultrasonic 5min of supersound washing groove prime time, the centrifugal 10min of 5000rpm gets supernatant, and fixedly Δ λ is 70nm, excites slit 10nm, emission slit 10nm, mensuration synchronous fluorescent spectrum.Each sample determination three times is got average.Be calculated as follows the lipid oxidation inhibiting rate:
Inhibiting rate=(Int
0-Int
s)/(Int0-Int
f) * 100%
In the formula: Int
0, when not adding antioxidant behind the rotten 4 ℃ of storage 72h of cold cuts in the synchronous fluorescence intensity at 326nm place;
Int
f, fresh meat is not in the synchronous fluorescence intensity at 326nm place when adding antioxidant;
Int
s, when adding antioxidant the rotten 4 ℃ of storages of cold cuts after 3 days in the synchronous fluorescence intensity at 326nm place.
The result shows, it is 93.5% that the chrysanthemum cauline leaf primary extract that the embodiment 1 of interpolation 0.1% obtains suppresses the rotten lipid oxidation rate of refrigeration cold cuts, it is 92.3% that the chrysanthemum stem leaf total flavones extract that the embodiment 1 of interpolation 0.02% obtains suppresses the rotten lipid oxidation rate of refrigeration cold cuts, therefore, the chrysanthemum stem-leaf extract has the rotten lipid oxidation ability of good restraining refrigeration cold cuts, can be used as antioxidant and is used for meat products.
Two, chrysanthemum stem-leaf extract of the present invention is removed the experiment of DPPH free radical
Preparation 6.5 * 10
-5The DPPH methanol solution of mol/L keeps in Dark Place.After getting 1mL example 3 chrysanthemum stem-leaf extract solution adding 5mLDPPH solution reaction 25min, measure extinction A at the 517nm place
iDistilled water with equal volume replaces chrysanthemum stem-leaf extract solution as blank.Each sample is made 3 parallel samples, gets its mean value.Remove the computing formula of DPPH free radical activity:
P＝(A
0-A
i+A
j)/A
0×100％
In the formula, A
iBe the absorbance of sample, A
jBe the absorbance of sample liquid under 517nm, A
0Blank absorbance.
The result shows, mass percentage concentration is that the efficient of the chrysanthemum cauline leaf primary extract solution removal DPPH free radical that obtains of 0.1% embodiment 3 is 67.2%, and mass percentage concentration is that the efficient that chrysanthemum stem leaf total flavones extract that 0.02% embodiment 3 obtains is removed the DPPH free radical is 89.26%; This explanation chrysanthemum stem-leaf extract has the effect of very strong removing DPPH free radical, can be used as antioxidant and uses.
Three, the TAC of chrysanthemum stem-leaf extract of the present invention
With reference to FRAP method (Benzie IFF, Strain JJ.The ferric reducing ability of plasma as a measure of " antioxidantpower ": the FRAP assay[J] .Anal Biochem, 1996,239:70～76.), and improve to some extent.This method principle is than under the low ph environment, Fe
3+-three pyridines three azines (TPTZ) can be reduced to the ferrous iron form by reducing substances in the polyphenoils, present tangible blueness, and have maximum light absorption in the 593nm place, calculate the power of sample antioxidation activity according to the size of absorbance.0.1ml example 5 chrysanthemum stem-leaf extract solution+0.3ml water+3ml are preheated to 37 ℃ FRAP working solution (10mmol/LTPTZ, 20mmol/LFeCl
3, the 0.3mmol/L sodium-acetate buffer mixes with 1: 1: 10 ratio), shake up back 37 ℃ of water-bath 10min, ice bath 1 minute is immediately surveyed its light absorption value in 593nm then; In addition with 0.5mmol/L～2.0mmol/L FeSO
4Standard liquid replace chrysanthemum stem-leaf extract solution to make calibration curve, obtain regression equation y=0.6869x+0.0715, coefficient R
2=0.9996.The TAC of sample is with mmol/L FeSO
4Expression.Every part of sample replication 3 times.
The result shows, the TAC of the chrysanthemum cauline leaf primary extract that the BHT of 10mg/mL, embodiment 5 obtain, the chrysanthemum stem leaf total flavones extract that embodiment 5 obtains is equivalent to 12.77,8.65,11.36mmol/LFeSO
4, the height of chrysanthemum stem leaf total flavones extract oxidation resistance than chrysanthemum cauline leaf primary extract is described, this illustrates that also they have very strong confession proton ability, can be used as antioxidant and use.
Claims (9)
1. a chrysanthemum stem-leaf extract is characterized in that: be chrysanthemum cauline leaf primary extract or chrysanthemum stem leaf total flavones extract.
2. chrysanthemum cauline leaf as claimed in claim 1 is characterized in that: be chrysanthemums such as Hang Ju, Bo chrysanthemum, tribute chrysanthemum, Chu chrysanthemum, Qi Ju, bosom chrysanthemum, Ji chrysanthemum, the Huang Ju remaining cauline leaf in back of gathering.
3. chrysanthemum stem-leaf extract as claimed in claim 1 is characterized in that being obtained by following preparation method:
(1) with chrysanthemum cauline leaf drying, pulverize, add the aqueous solvent of 8-100 times of chrysanthemum cauline leaf weight, extract;
(2) filtration or centrifugal removal insoluble matter obtain extract, and drying obtains chrysanthemum cauline leaf primary extract;
(3) chrysanthemum cauline leaf primary extract or extract are passed through the polymeric adsorbent chromatography, with the ethanol water wash-out of percent by volume 10%-95%, the eluent drying obtains chrysanthemum stem leaf total flavones extract.
4. the preparation method of chrysanthemum stem-leaf extract according to claim 3 is characterized in that: the described aqueous solvent of step (1) is that pure water or mass percent are the sodium metabisulfite solution of 0.01%-0.4%.
5. the preparation method of chrysanthemum stem-leaf extract according to claim 3 is characterized in that: the described extraction of step (1) is a kind of of extracting modes such as cold soaking extraction, seepage extraction, Soxhlet extraction, hot reflux extraction, microwave assisted extraction or ultrasonic wave assisted extraction.
6. the preparation method of chrysanthemum stem-leaf extract according to claim 3 is characterized in that: the extraction temperature of the described extraction of step (1) is 20 ℃-100 ℃, divides and carries out for 1-6 time, extracts time spent 10min-5h at every turn, merges extract then.
7. the preparation method of chrysanthemum stem-leaf extract according to claim 3 is characterized in that: the polymeric adsorbent that step (3) is adopted is the macroporous absorbent resin of polyamide or various models.
8. the preparation method of chrysanthemum stem-leaf extract according to claim 3 is characterized in that: step (2), step (3) and the described drying of step (4) are drying under reduced pressure or spray-drying.
9. the application of the described chrysanthemum stem-leaf extract of claim 1 is characterized in that being applied in food, cosmetics and the medicine as natural.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101847160A CN101991090A (en) | 2009-08-20 | 2009-08-20 | Chrysanthemum stem and leaf extract and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101847160A CN101991090A (en) | 2009-08-20 | 2009-08-20 | Chrysanthemum stem and leaf extract and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101991090A true CN101991090A (en) | 2011-03-30 |
Family
ID=43782237
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009101847160A Pending CN101991090A (en) | 2009-08-20 | 2009-08-20 | Chrysanthemum stem and leaf extract and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101991090A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103054026A (en) * | 2013-01-08 | 2013-04-24 | 滁州学院 | Low temperature extraction technology of active components of Chuzhou chrysanthemum |
| CN105136967A (en) * | 2015-07-23 | 2015-12-09 | 北京市药品检验所 | Reversed-phase thin-layer chromatographic method for identifying florists chrysanthemum and bo chrysanthemum |
| CN105175376A (en) * | 2015-10-26 | 2015-12-23 | 湖北来凤腾升香料化工有限公司 | Method for extracting luteolin from Laifeng crossostephium leaves |
| CN106974862A (en) * | 2017-04-14 | 2017-07-25 | 张仕英 | A kind of preparation method of microwave radiation exaraction whitewood melon extract solution |
| CN109010169A (en) * | 2018-08-28 | 2018-12-18 | 安徽科技学院 | A kind of preparation method of chu chrysanthemum eye cream |
| CN109221269A (en) * | 2018-11-08 | 2019-01-18 | 浙江中医药大学 | A kind of FLOS CHRYSANTHEMI ALBA from Haizhou of China leaf extract and preparation method thereof and the application in restraining and sterilizing bacteria |
-
2009
- 2009-08-20 CN CN2009101847160A patent/CN101991090A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103054026A (en) * | 2013-01-08 | 2013-04-24 | 滁州学院 | Low temperature extraction technology of active components of Chuzhou chrysanthemum |
| CN105136967A (en) * | 2015-07-23 | 2015-12-09 | 北京市药品检验所 | Reversed-phase thin-layer chromatographic method for identifying florists chrysanthemum and bo chrysanthemum |
| CN105175376A (en) * | 2015-10-26 | 2015-12-23 | 湖北来凤腾升香料化工有限公司 | Method for extracting luteolin from Laifeng crossostephium leaves |
| CN106974862A (en) * | 2017-04-14 | 2017-07-25 | 张仕英 | A kind of preparation method of microwave radiation exaraction whitewood melon extract solution |
| CN109010169A (en) * | 2018-08-28 | 2018-12-18 | 安徽科技学院 | A kind of preparation method of chu chrysanthemum eye cream |
| CN109221269A (en) * | 2018-11-08 | 2019-01-18 | 浙江中医药大学 | A kind of FLOS CHRYSANTHEMI ALBA from Haizhou of China leaf extract and preparation method thereof and the application in restraining and sterilizing bacteria |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Huang et al. | Antioxidant and anti-inflammatory activities of aqueous extract of Centipeda minima | |
| Boaventura et al. | Enhancement of bioactive compounds content and antioxidant activity of aqueous extract of mate (Ilex paraguariensis A. St. Hil.) through freeze concentration technology | |
| CN101991090A (en) | Chrysanthemum stem and leaf extract and preparation method and application thereof | |
| CN106420521B (en) | A kind of extraction process of roxburgh anoectochilus terminal bud flavones and its application in skin-protection product | |
| CN102178848A (en) | Traditional Chinese medicinal dendrobium officinale preparation and preparation method thereof | |
| CN104473828A (en) | Dragon fruit stem moisturizing lotion and preparation method thereof | |
| CN106580778A (en) | Macadamia nut green peel extract, cosmetics employing same as active ingredient and preparation method of macadamia nut green peel extract | |
| CN101828593A (en) | Preparation method of composite green health food of freeze dried purslane and purslane/seaweed | |
| CN101897656A (en) | Application of Saussurea involucrata culture as new raw material in daily chemicals | |
| CN103919712A (en) | Cordyceps militaris extract, and preparation method and application thereof | |
| CN102302553A (en) | Extraction method of key components of Chinese magnoliavine fruit medicament | |
| CN107440963A (en) | A kind of persimmon leaf flavone with whitening anti-aging activity and its preparation method and application | |
| CN112006959A (en) | Antioxidant whitening cream containing dandelion and tea extracts and preparation method thereof | |
| CN103860620A (en) | Antioxidant active extract of snow chrysanthemum and preparation method thereof | |
| Huang et al. | Simultaneous recovery of glycyrrhizic acid and liquiritin from Chinese licorice root (Glycyrrhiza uralensis Fisch) by aqueous two-phase system and evaluation biological activities of extracts | |
| Kim et al. | Anti-aging and anti-diabetes effects of Aconitum pesudo-laeve var. erectum extracts | |
| CN103749812B (en) | A kind of processing method of radix tetrastigme tea | |
| CN103385837B (en) | Myrica rubra leaf proanthocyanidin toning lotion and preparation method of myrica rubra leaf proanthocyanidin toning lotion | |
| CN102578452B (en) | Skin-nourishing nutritional food | |
| CN104523500A (en) | Composition containing double-effect plant active ingredients and preparation method for composition | |
| KR20110048401A (en) | Method for preparing the extract fortified with chalcones from by-product of Angelica keiskei juice | |
| CN102258094B (en) | Tea drink containing natural plants or natural plant extracts, and preparation method and application thereof | |
| CN102165978A (en) | Cordyceps health-preserving tea and making method thereof | |
| CN103751061A (en) | Tomato leaf extract and application thereof in cosmetics | |
| CN113925081A (en) | Natural plant anticorrosion composition, preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20110330 |