CN105285314A - Extraction method of 13KDa rice alcohol soluble protein - Google Patents
Extraction method of 13KDa rice alcohol soluble protein Download PDFInfo
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Abstract
The present invention relates to an extraction method of 13KDa rice alcohol soluble protein to solve the problems that the existing extraction method of alcohol soluble protein is low in extraction rate, the purity of the extracted alcohol soluble protein is relatively low, and the existing extraction method cannot be applied to the mass industrial production. The extraction method includes: 1, grinding rice into flour with a grinder; 2, de-fatting the rice flour with n-hexane to obtain defatted rice flour; 3, leaching the defatted rice flour with elution liquid, and then washing the precipitates with pure water; 4, leaching the washed precipitates obtained in step 3 with n-propyl alcohol solution with a concentration of 60%, and collecting supernatant; 5, treating the supernatant with trichloroacetic acid solution to precipitate protein; and 6, performing vacuum freeze drying on the precipitates obtained in step 5, thereby obtaining the 13KDa rice alcohol soluble protein. The purity of the extracted rice alcohol soluble protein can reach 90%-94%, and the extraction method is suitable for large-scale industrialized extraction of the rice alcohol soluble protein.
Description
Technical field
The present invention relates to a kind of extracting method of rice prolafnin.
Background technology
Rice protein has hypoallergenic feature, is easy to digested.And rice protein has reducing blood lipid, norcholesterol, anti-oxidant, preventing obesity function, particularly current research shows that rice protein has effect of prevention diabetes, thus rice protein is considered to a kind of plant protein resource with high Development volue, receives the concern of whole world researchers.
Alcohol soluble protein is the major storage protein of rice protein, molecular weight is mainly 10,13,16KDa, wherein, 13KDa albumen is the major protein body of rice prolafnin, has important wholesome physiological regulating control function.Research shows, rice prolafnin has the effects such as stronger absorption of bile acid, effectively can prevent the diseases such as high fat of blood, and, due to the low digestibility feature of rice prolafnin, be the good plant albumen of diabetic population first-selection.But up to now, because the separation of 13KDa rice prolafnin is more difficult, the more difficult control of purity, therefore, still belongs to shortage to the extractive technique of high-purity 13KDa rice prolafnin.
Existing extractive technique shows, the method extracting alcohol soluble protein often adopts ethanol, glycerine, butanediol equal solvent, has recovery rate lower, assorted band is more, the drawbacks such as purity is lower, and waste loss is more, are difficult to the large-scale industrial production of propulsion functions rice prolafnin.
Summary of the invention
The present invention is that will to solve the extracting method recovery rate of existing alcohol soluble protein lower, and purity is lower, cannot the problem of large-scale industrial production, provides a kind of extracting method of 13KDa rice prolafnin.
The extracting method of 13KDa rice prolafnin of the present invention, carry out according to the following steps:
One, rice pulverizer is ground to form ground rice;
Two, by ground rice n-hexane degreasing 3 ~ 5 times, degreasing ground rice is obtained;
Three, by the eluent lixiviate 3 ~ 5 times of degreasing ground rice, then by precipitation pure water cleaning 3 ~ 5 times;
Four, the precipitation concentration after step 3 being cleaned is the normal propyl alcohol solution lixiviate of 60% (v/v), gets supernatant;
Five, be that the solution of trichloroacetic acid of 10% ~ 12% (w/v) is by albumen precipitation by supernatant concentration;
Six, by the precipitation vacuum freeze drying of step 5,13KDa rice prolafnin is namely obtained.
Beneficial effect of the present invention:
The present invention adopts that normal propyl alcohol extracts, trichloroacetic acid precipitation, and its advantage is can by the main body of rice prolafnin---13KDa rice prolafnin fully extracts, and higher, pollution-free without assorted band, purity, without harm, and do not need to adjust pH.
Method of the present invention is simple, quick, efficient, can extract high-purity 13KDa rice prolafnin, and its rice prolafnin purity extracted can reach 90% ~ 94%, rice prolafnin extraction rate reached 95% ~ 97%.Be suitable for heavy industrialization and extract rice prolafnin.
Accompanying drawing explanation
Fig. 1 is the protein electrophoresis figure in embodiment 1.
Detailed description of the invention
Technical solution of the present invention is not limited to following cited detailed description of the invention, also comprises any combination between each detailed description of the invention.
Detailed description of the invention one: the extracting method of present embodiment 13KDa rice prolafnin, carry out according to the following steps:
One, rice pulverizer is ground to form ground rice;
Two, by ground rice n-hexane degreasing 3 ~ 5 times, degreasing ground rice is obtained;
Three, by the eluent lixiviate 3 ~ 5 times of degreasing ground rice, then by precipitation pure water cleaning 3 ~ 5 times;
Four, the precipitation concentration after step 3 being cleaned is the normal propyl alcohol solution lixiviate of 60% (v/v), gets supernatant;
Five, be that the solution of trichloroacetic acid of 10% ~ 12% (w/v) is by albumen precipitation by supernatant concentration;
Six, by the precipitation vacuum freeze drying of step 5,13KDa rice prolafnin is namely obtained.
Detailed description of the invention two: present embodiment and detailed description of the invention one unlike: the particle diameter of ground rice described in step one is 70 ~ 90 orders.Other is identical with detailed description of the invention one.
Detailed description of the invention three: present embodiment and detailed description of the invention one unlike: in step 2 by the concrete grammar of ground rice n-hexane degreasing be:
A, ground rice to be mixed with the ratio of n-hexane according to 1g:4 ~ 6mL, stirring at room temperature 1 hour, leave standstill 1 hour, then collecting precipitation after the centrifugal 20min of 4000rpm;
B, repeat steps A 2 ~ 4 times, be deposited in 30 DEG C dry what collect, obtain degreasing ground rice.Other is identical with detailed description of the invention one.
Detailed description of the invention four: present embodiment and detailed description of the invention one unlike: in step 3 by the concrete grammar of degreasing ground rice eluent lixiviate be:
A, mixed with the ratio of eluent according to 1g:10 ~ 12mL by degreasing ground rice, 25 DEG C of 130rpm vibrate 30min, remove supernatant and leave precipitation after centrifugal;
B, repetition steps A 2 ~ 4 times, obtain precipitation.Other is identical with detailed description of the invention one.
Detailed description of the invention five: present embodiment and detailed description of the invention one unlike: described eluent is the KH of 50mM and pH6.5
2pO
4-NaOH buffer solution, wherein containing 0.5MNaCl and 1mMEDTA-2Na.Other is identical with detailed description of the invention one.
Detailed description of the invention six: to be the concrete grammar of the normal propyl alcohol solution lixiviate of 60% unlike: the precipitation volume fraction after step 3 being cleaned in step 4 be for present embodiment and detailed description of the invention one:
1. the precipitation after step 3 being cleaned is that 60% normal propyl alcohol solution mixes according to the ratio of 1g:10 ~ 12mL with volume fraction, 25 DEG C of 130rpm vibration 8h, after 4 DEG C of centrifugal 20min of 10000g, and cleer and peaceful precipitation in collection;
2. by step 1. in precipitation be that 60% normal propyl alcohol solution mixes according to the ratio of 1g:10 ~ 12mL with volume fraction, 25 DEG C of 130rpm vibrate 8h, after 4 DEG C of centrifugal 20min of 10000g, cleer and peaceful precipitation in collection;
3. by step 2. in precipitation be that 60% normal propyl alcohol solution mixes according to the ratio of 1g:10 ~ 12mL with volume fraction, 25 DEG C of 130rpm vibrate 4h, after 4 DEG C of centrifugal 20min of 10000g, collect supernatant.Other is identical with detailed description of the invention one.
Detailed description of the invention seven: present embodiment and detailed description of the invention one unlike: the concrete grammar of albumen precipitation is by the solution of trichloroacetic acid being 10% by supernatant mass concentration in step 5:
The supernatant that step 4 is collected is added the solution of trichloroacetic acid that concentration is 10% (w/v), the volume ratio 3:10 of supernatant and solution of trichloroacetic acid, after concussion mixing, 15 ~ 20min is left standstill in 0 ~ 4 DEG C of ice bath reaction under lucifuge condition, then centrifugal 20min under 4 DEG C of 10000g conditions, abandons supernatant and leaves precipitation.Other is identical with detailed description of the invention one.
Detailed description of the invention eight: present embodiment and detailed description of the invention one unlike: the cryodesiccated condition of step 6 hollow is: vacuum≤15Pa, and condenser temperature is-60 ~-50 DEG C, and sample temperature-30 ~-20 DEG C, freeze-drying time is 20 ~ 24h.Other is identical with detailed description of the invention one.
For verifying effect of the present invention, carry out following test:
Embodiment 1:
The extracting method of the present embodiment 13KDa rice prolafnin, carry out according to the following steps:
One, rice pulverizer is ground to form ground rice, the particle diameter of ground rice is 80 orders.
Two, by ground rice n-hexane degreasing 3 times, obtain degreasing ground rice, concrete grammar is:
A, ground rice to be mixed with the ratio of n-hexane according to 1g:5mL, stirring at room temperature 1 hour, leave standstill 1 hour, then collecting precipitation after the centrifugal 20min of 4000rpm;
B, the precipitation that steps A is collected to be mixed with the ratio of n-hexane according to 1g:5mL, stirring at room temperature 1 hour, standing 1 hour, then collecting precipitation after the centrifugal 20min of 4000rpm;
C, the precipitation that step B collects to be mixed with the ratio of n-hexane according to 1g:5mL, stirring at room temperature 1 hour, standing 1 hour, then collecting precipitation after the centrifugal 20min of 4000rpm; Be deposited in 30 DEG C dry what collect, obtain degreasing ground rice.
By the eluent lixiviate 5 times of degreasing ground rice, concrete grammar is:
A, mixed with the ratio of eluent according to 1g:11mL by degreasing ground rice, 25 DEG C of 130rpm vibrate 30min, remove supernatant and leave precipitation after centrifugal;
B, the precipitation of above-mentioned steps A mixed with the ratio of eluent according to 1g:11mL, 25 DEG C of 130rpm vibrate 30min, remove supernatant and leave precipitation after centrifugal;
C, the precipitation of above-mentioned steps B mixed with the ratio of eluent according to 1g:11mL, 25 DEG C of 130rpm vibrate 30min, remove supernatant and leave precipitation after centrifugal;
D, the precipitation of above-mentioned steps C mixed with the ratio of eluent according to 1g:11mL, 25 DEG C of 130rpm vibrate 30min, remove supernatant and leave precipitation after centrifugal;
E, the precipitation of above-mentioned steps D mixed with the ratio of eluent according to 1g:11mL, 25 DEG C of 130rpm vibrate 30min, remove supernatant and leave precipitation after centrifugal; Again precipitation pure water is cleaned 3 times.
Described eluent is the KH of 50mM and pH6.5
2pO
4-NaOH buffer solution, wherein containing 0.5MNaCl and 1mMEDTA-2Na.
Four, the precipitation volume fraction after step 3 being cleaned is the normal propyl alcohol solution lixiviate of 60%, and concrete grammar is:
1. the precipitation after step 3 being cleaned is that 60% normal propyl alcohol solution mixes according to the ratio of 1g:10mL with volume fraction, 25 DEG C of 130rpm vibration 8h, after 4 DEG C of centrifugal 20min of 10000g, and cleer and peaceful precipitation in collection;
2. by step 1. in precipitation be that 60% normal propyl alcohol solution mixes according to the ratio of 1g:10mL with volume fraction, 25 DEG C of 130rpm vibrate 8h, after 4 DEG C of centrifugal 20min of 10000g, cleer and peaceful precipitation in collection;
3. by step 2. in precipitation be that 60% normal propyl alcohol solution mixes according to the ratio of 1g:10mL with volume fraction, 25 DEG C of 130rpm vibrate 4h, after 4 DEG C of centrifugal 20min of 10000g, collect supernatant.
Five, by supernatant mass concentration be the solution of trichloroacetic acid of 10% by albumen precipitation, concrete grammar is:
Whole supernatants that step 4 is collected are merged, add the solution of trichloroacetic acid that concentration is 10% (w/v), the volume ratio 3:10 of supernatant and solution of trichloroacetic acid, after concussion mixing, 20min is left standstill in a DEG C ice bath reaction under lucifuge condition, then centrifugal 20min under 4 DEG C of 10000g conditions, abandons supernatant and leaves precipitation.
Six, by the precipitation vacuum freeze drying of step 5, vacuum is 10Pa, and condenser temperature is-55 DEG C, sample temperature-25 DEG C, and freeze-drying time is 24h, namely obtains 13KDa rice prolafnin.
Rice in step one is commercially available rice.
Vacuum freeze drying described in step 6 adopts vacuum freeze drier to carry out, and vacuum freeze drier is the LGJ-12 freeze drier of Beijing development in science and technology Co., Ltd of Song Yuan Huaxing.
The KH of the preparation of eluent: 50mmol
2pO
4be dissolved in 950mL deionized water, adjust pH to 6.5, then add 0.5molNaCl, 1mmolEDTA-2Na, be stirred to abundant dissolving, be settled to 1000mL.
Detect:
13KDa rice prolafnin prepared by the present embodiment carries out protein component analysis by SDS-PAGE electrophoresis.The rice prolafnin prepared by the present embodiment is dissolved in sample-loading buffer, and carry out SDS-PAGE electrophoresis after then keeping 3min in the water-bath of 100 DEG C, gel strength chooses 15%.Wherein in sample-loading buffer, the concentration of Tris-HCl is 0.125mol/L, SDS mass percent concentration is 4%, the concentration of volume percent of 2 mercapto ethanol is 5%, the molar concentration of urea is the pH=6.8 of 8mol/L, buffer solution.
As shown in Figure 1, in Fig. 1, swimming lane 1 is the non-pre-dyed Marker of albumen to protein electrophoresis figure, and swimming lane 2 is the rice holoprotein of alkalinity extraction, and swimming lane 3 extracts the alcohol soluble protein obtained for the present embodiment.The albumen that the present embodiment extracts as seen from Figure 1 is mixed and is with, and illustrates that the alcohol soluble protein carried is purer.Band is relatively thicker, illustrates that alcohol soluble protein extracted amount is many.Measure by analysis, the rice prolafnin purity that the present embodiment extracts is 92.16%.Alcohol soluble protein extraction rate reached 96.34%.
Claims (8)
1. an extracting method for 13KDa rice prolafnin, is characterized in that carrying out according to the following steps:
One, rice pulverizer is ground to form ground rice;
Two, by ground rice n-hexane degreasing 3 ~ 5 times, degreasing ground rice is obtained;
Three, by the eluent lixiviate 3 ~ 5 times of degreasing ground rice, then by precipitation pure water cleaning 3 ~ 5 times;
Four, the precipitation concentration after step 3 being cleaned is the normal propyl alcohol solution lixiviate of 60% (v/v), gets supernatant;
Five, be that the solution of trichloroacetic acid of 10% ~ 12% (w/v) is by albumen precipitation by supernatant concentration;
Six, by the precipitation vacuum freeze drying of step 5,13KDa rice prolafnin is namely obtained.
2. the extracting method of a kind of 13KDa rice prolafnin according to claim 1, is characterized in that the particle diameter of ground rice described in step one is 70 ~ 90 orders.
3. the extracting method of a kind of 13KDa rice prolafnin according to claim 1, it is characterized in that in step 2 by the concrete grammar of ground rice n-hexane degreasing be:
A, ground rice to be mixed with the ratio of n-hexane according to 1g:4 ~ 6mL, stirring at room temperature 1 hour, leave standstill 1 hour, then collecting precipitation after the centrifugal 20min of 4000rpm;
B, repeat steps A 2 ~ 4 times, be deposited in 30 DEG C dry what collect, obtain degreasing ground rice.
4. the extracting method of a kind of 13KDa rice prolafnin according to claim 1, it is characterized in that in step 3 by the concrete grammar of degreasing ground rice eluent lixiviate be:
A, mixed with the ratio of eluent according to 1g:10 ~ 12mL by degreasing ground rice, 25 DEG C of 130rpm vibrate 30min, remove supernatant and leave precipitation after centrifugal;
B, repetition steps A 2 ~ 4 times, obtain precipitation.
5. the extracting method of a kind of 13KDa rice prolafnin according to claim 1, is characterized in that described eluent is the KH of 50mM and pH6.5
2pO
4-NaOH buffer solution, wherein containing 0.5MNaCl and 1mMEDTA-2Na.
6. the extracting method of a kind of 13KDa rice prolafnin according to claim 1, is characterized in that the concrete grammar being the normal propyl alcohol solution lixiviate of 60% by the precipitation concentration after step 3 cleaning in step 4 is:
1. the precipitation after step 3 being cleaned is that 60% normal propyl alcohol solution mixes according to the ratio of 1g:10 ~ 12mL with volume fraction, 25 DEG C of 130rpm vibration 8h, after 4 DEG C of centrifugal 20min of 10000g, and cleer and peaceful precipitation in collection;
By step 1. in precipitation be that 60% normal propyl alcohol solution mixes according to the ratio of 1g:10 ~ 12mL with volume fraction, 25 DEG C of 130rpm vibrate 8h, after 4 DEG C of centrifugal 20min of 10000g, cleer and peaceful precipitation in collection;
By step 2. in precipitation be that 60% normal propyl alcohol solution mixes according to the ratio of 1g:10 ~ 12mL with volume fraction, 25 DEG C of 130rpm vibrate 4h, after 4 DEG C of centrifugal 20min of 10000g, collect supernatant.
7. the extracting method of a kind of 13KDa rice prolafnin according to claim 1, it is characterized in that being the solution of trichloroacetic acid of 10% ~ 12% (w/v) by supernatant concentration in step 5 by the concrete grammar of albumen precipitation be:
Whole supernatants that step 4 is collected are merged, add the solution of trichloroacetic acid that concentration is 10% ~ 12% (w/v), volume ratio 1:3 ~ 5 of supernatant and solution of trichloroacetic acid, after concussion mixing, 15 ~ 20min is left standstill in 0 ~ 4 DEG C of ice bath reaction under lucifuge condition, then centrifugal 20min under 4 DEG C of 10000g conditions, abandons supernatant and leaves precipitation.
8. the extracting method of a kind of 13KDa rice prolafnin according to claim 1, it is characterized in that the cryodesiccated condition of step 6 hollow is: vacuum≤15Pa, condenser temperature is-60 ~-50 DEG C, and sample temperature-30 ~-20 DEG C, freeze-drying time is 20 ~ 24h.
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| CN1421123A (en) * | 2002-12-25 | 2003-06-04 | 南京农业大学 | Selective breeding method of special rice variety for nephrosis and diabetes patients |
| CN101103114A (en) * | 2004-11-29 | 2008-01-09 | Era生物技术有限公司 | Protein isolation and purification |
| CN101347094A (en) * | 2008-08-22 | 2009-01-21 | 南京农业大学 | Method for breeding rice vairety with polymerized low-glutelin and rice stripe-resistance |
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| CN101861909B (en) * | 2010-05-05 | 2012-09-12 | 长沙理工大学 | Method for modification of rice protein and oryzenin with protein glutaminase |
| CN103242423A (en) * | 2013-05-13 | 2013-08-14 | 江苏大学 | Method for extracting silybum marianum protein |
| CN104086639A (en) * | 2014-07-21 | 2014-10-08 | 天津商业大学 | Purification process of chicken ovomucoid |
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2015
- 2015-09-14 CN CN201510582411.0A patent/CN105285314A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1421123A (en) * | 2002-12-25 | 2003-06-04 | 南京农业大学 | Selective breeding method of special rice variety for nephrosis and diabetes patients |
| CN101103114A (en) * | 2004-11-29 | 2008-01-09 | Era生物技术有限公司 | Protein isolation and purification |
| CN101347094A (en) * | 2008-08-22 | 2009-01-21 | 南京农业大学 | Method for breeding rice vairety with polymerized low-glutelin and rice stripe-resistance |
| CN101861909B (en) * | 2010-05-05 | 2012-09-12 | 长沙理工大学 | Method for modification of rice protein and oryzenin with protein glutaminase |
| CN102335414A (en) * | 2011-09-30 | 2012-02-01 | 哈尔滨工业大学 | Rice prolamin bile acid adsorbent and preparation method thereof |
| CN103242423A (en) * | 2013-05-13 | 2013-08-14 | 江苏大学 | Method for extracting silybum marianum protein |
| CN104086639A (en) * | 2014-07-21 | 2014-10-08 | 天津商业大学 | Purification process of chicken ovomucoid |
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Application publication date: 20160203 |