CN105348375A - Tea seed glucoprotein, preparation method therefor and application of tea seed glucoprotein - Google Patents
Tea seed glucoprotein, preparation method therefor and application of tea seed glucoprotein Download PDFInfo
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- CN105348375A CN105348375A CN201510837017.7A CN201510837017A CN105348375A CN 105348375 A CN105348375 A CN 105348375A CN 201510837017 A CN201510837017 A CN 201510837017A CN 105348375 A CN105348375 A CN 105348375A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
The invention belongs to the field of medicines and discloses tea seed glucoprotein, a preparation method therefor and application of the tea seed glucoprotein. The preparation method comprises the following steps: carrying out reflux extraction on defatted tea seed cakes by an ethanol aqueous solution, carrying out filtrating, distilling off ethanol from filtrated residue, then, adding a phosphoric acid buffer into the filtrated residue, carrying out extracting, and carrying out filtrating; and carrying out ultrafiltration on extracted and filtrated filtrate through an ultrafiltration membrane, and carrying out freeze drying on an intercepted part, thereby obtaining the tea seed glucoprotein. The tea seed glucoprotein prepared by the method is high in extraction ratio and activity and is liable to industrial production. Besides, the glucoprotein can be used for improving kinetism as an antifatigue drug.
Description
Technical field
The invention belongs to field of medicaments, be specifically related to a kind of tea seed glycoprotein and preparation method thereof and the application of this glycoprotein as anti-fatigue medicament.
Background technology
Glycoprotein is the associated proteins that a class is connected with covalent linkage by carbohydrate with protein or polypeptide and is formed, and has important physiological action.Non-natural glycoprotein has immunity moderation, antitumor, anti-inflammatory, hypoglycemic, reducing blood-fat, anti-oxidant, health care wait for a long time effect.
Polysaccharide component in tea seed exists mainly with the form of glycoprotein greatly.Tea seed polysaccharide is by multiple mixed polysaccharide formed such as pectinose, semi-lactosi, seminose, glucose, and tea seed protein is then containing 17 seed amino acids, and wherein 8 kinds is essential amino acid.The extracting method of tea seed polysaccharide mainly contains acid degradation method, oxidation degradation method and enzyme liberating method, but these methods all destroy wherein protein active.The extracting method of tea seed albumen mainly contains isoelectric point method, ion exchange method and ultrafiltration process, but these methods to protein extracting ratio less than 50%, and do not obtain polysaccharide fraction.
Fatigue is a kind of sub-health state, and it occurs main owing to energy substance exhaustion, metabolism disorder, internal milieu imbalance etc.Glycoprotein can not only provide motion institute energy requirement, can also scavenging free radicals, reaches the object of antifatigue.
The buffering salt extracting method of tea seed glycoprotein and have no report as the application of anti-fatigue medicament.
Summary of the invention
In order to overcome the shortcoming of prior art with not enough, primary and foremost purpose of the present invention is the preparation method providing a kind of tea seed glycoprotein.
Another object of the present invention is to provide the tea seed obtained by above-mentioned preparation method glycoprotein.
Another object of the present invention is to provide the described application of tea seed glycoprotein in anti-fatigue medicament.
Object of the present invention is achieved through the following technical solutions: a kind of preparation method of tea seed glycoprotein, comprises the steps:
(1) by degreasing leached tea oil slag aqueous ethanolic solution refluxing extraction, filter, obtain filter residue and ethanol-water extraction liquid; Then filter residue is steamed ethanol to without alcohol taste, for subsequent use;
(2) by the filter residue phosphoric acid buffer heat-insulation soaking of step (1), filter, obtain filtrate; The pH of described phosphoric acid buffer is 8 ~ 10; The concentration of described phosphoric acid buffer is 0.05 ~ 0.1M;
(3) by the ultra-filtration membrane ultrafiltration of filtrate 2000Da, then ultra-filtration membrane will retain partial freeze drying, obtain tea seed glycoprotein.
Degreasing leached tea oil slag described in step (1) is degreasing camellia seed meal or the degreasing tea-seed dregs of rice;
The liquid-solid ratio of aqueous ethanolic solution and degreasing leached tea oil slag described in step (1) is (10 ~ 20) mL:1g; Described extraction conditions is extract 0.5 ~ 3 hour at 60 ~ 80 DEG C.
The concentration of volume percent of step (1) described aqueous ethanolic solution is 80 ~ 90%.
The liquid-solid ratio of the degreasing leached tea oil slag of step (2) described phosphoric acid buffer and step (1) is (15 ~ 30) mL:1g.
The condition of step (2) described heat-insulation soaking is in 50 ~ 60 DEG C of insulation 1 ~ 3h.
The described cryodesiccated condition of step (3) is-30 ~-50 DEG C of lyophilizes 4 ~ 8 hours.
A kind of tea seed glycoprotein is prepared by aforesaid method, and its sugared content is 40 ~ 70%, protein content 30 ~ 60%.
Described tea seed glycoprotein is applied to the pharmaceutical preparation of preparation antifatigue.
Described pharmaceutical preparation is any one in the formulation such as oral preparations (pulvis or liquid) or injection formulations;
Described pharmaceutical preparation is folk prescription containing tea seed glycoprotein or compound preparation.
The present invention has following advantage and effect relative to prior art:
(1) the present invention adopts same raw material first to extract through 80 ~ 90% aqueous ethanolic solutions and eliminates saponin and other fat-soluble components in degreasing raw material, then extracts glycoprotein in filter residue through phosphate buffered saline buffer, can significantly improve the purity of glycoprotein;
(2) the present invention adopts alkaline phosphatase buffer extraction glycoprotein, has both kept pH to stablize, thus has remained the activity of glycoprotein, is dissolved by glycoprotein again, makes it fully to be extracted; Remove freshen by ultra-filtration membrane again, thus product purity and activity are significantly improved;
(3) energy supplement can be carried out the long period because tea seed glycoprotein utilizes speed comparatively slow, maintain glucose level, and osmotic pressure is lower than monose, be more suitable for as sports supplement, therefore tea seed glycoprotein of the present invention can make anti-fatigue medicament or functional additive;
(4) preparation technology of the present invention is simple, and reaction conditions is gentle, and constant product quality, is convenient to suitability for industrialized production.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1
Get the commercially available degreasing tea-seed dregs of rice of 1kg, add the aqueous ethanolic solution of the volume fraction 85% of 12L, in 65 DEG C of refluxing extraction 1 hour, filter, then filter residue is steamed ethanol to without alcohol taste in 80 DEG C; By filter residue 20LpH be the 0.1M phosphoric acid buffer immersion of 8 again and in 55 DEG C of insulation 3h, filter, obtaining soaking the filtrate after filtering; The ultra-filtration membrane ultrafiltration of filtrate 2000Da after filtering will be soaked, ultra-filtration membrane will retain partial freeze drying (-50 DEG C lyophilize 4 hours), obtain tea seed glycoprotein 180g.
Embodiment 2
Get the commercially available degreasing camellia seed meal of 1kg, add the aqueous ethanolic solution of the volume fraction 80% of 10L, in 70 DEG C of refluxing extraction 3 hours, filter, then filter residue is steamed ethanol to without alcohol taste in 80 DEG C; By filter residue 30LpH be the 0.05M phosphoric acid buffer immersion of 10 again and in 60 DEG C of insulation 3h, filter, obtaining soaking the filtrate after filtering; The ultra-filtration membrane ultrafiltration of filtrate 2000Da after filtering will be soaked, ultra-filtration membrane will retain partial freeze drying (-30 DEG C lyophilize 8 hours), obtain tea seed glycoprotein 190g.
Embodiment 3
Get the commercially available degreasing tea-seed dregs of rice of 1kg, add the aqueous ethanolic solution of the volume fraction 82% of 20L, 65 DEG C of refluxing extraction 2 hours, filter, then filter residue is steamed ethanol to without alcohol taste in 80 DEG C; Be the 0.1M phosphoric acid buffer immersion of 9 again by filter residue 15LpH, 60 DEG C of insulation 2h, filter, and obtain soaking the filtrate after filtering; The ultra-filtration membrane ultrafiltration of filtrate 2000Da after filtering will be soaked, ultra-filtration membrane will retain partial freeze drying (-40 DEG C lyophilize 6 hours), obtain tea seed glycoprotein 165g.
Embodiment 4
Get the commercially available degreasing camellia seed meal of 1kg, add the aqueous ethanolic solution of the volume fraction 90% of 15L, 60 DEG C of refluxing extraction 2 hours, filter, then filter residue is steamed ethanol to without alcohol taste in 80 DEG C; Be the 0.08M phosphoric acid buffer immersion of 8.5 again by filter residue 15LpH, 50 DEG C of insulation 2h, filter, and obtain soaking the filtrate after filtering; The ultra-filtration membrane ultrafiltration of filtrate 2000Da after filtering will be soaked, ultra-filtration membrane will retain partial freeze drying (-35 DEG C lyophilize 7 hours), obtain tea seed glycoprotein 165g.
Embodiment 5
Get the commercially available degreasing tea-seed dregs of rice of 1kg, add the aqueous ethanolic solution of the volume fraction 87% of 10L, 70 DEG C of refluxing extraction 2.5 hours, filter, then filter residue is steamed ethanol to without alcohol taste in 80 DEG C; By filter residue 25LpH be the 0.1M phosphoric acid buffer immersion of 9.5 again and in 55 DEG C of insulation 2h, filter, obtaining soaking the filtrate after filtering; The ultra-filtration membrane ultrafiltration of filtrate 2000Da after filtering will be soaked, ultra-filtration membrane will retain partial freeze drying (-45 DEG C lyophilize 5 hours), obtain tea seed glycoprotein 187g.
Embodiment 6
Get the commercially available degreasing camellia seed meal of 1kg, add the aqueous ethanolic solution of the volume fraction 90% of 15L, 65 DEG C of refluxing extraction 1.5 hours, filter, then filter residue is steamed ethanol to without alcohol taste in 80 DEG C; Be the 0.05M phosphoric acid buffer immersion of 8.5 again by filter residue 20LpH, 60 DEG C of insulation 3h, filter, and obtain soaking the filtrate after filtering; The ultra-filtration membrane ultrafiltration of filtrate 2000Da after filtering will be soaked, ultra-filtration membrane will retain partial freeze drying (-50 DEG C lyophilize 6 hours), obtain tea seed glycoprotein 183g.
Embodiment 7 component content measures
Sugar and protein content determination in embodiment 1 ~ 6 product
Experimental technique: (1) adopts sulfuric acid-phynol method to measure sugar degree.Accurate absorption 0.1mg/mL Glucose standards solution 0,0.05,0.1,0.2,0.3,0.4mL, adds water and mends to 0.5mL, add 0.3mL6% phenol respectively and mix, add vitriol oil 1.5mL fast, after eddy current mixing, put in boiling water and heat 20min, in ice bath, after cooling, measure absorbancy with microcolorimetric ware in 490nm.With standard glucose content (Y, μ g) to absorbancy (X, ABS) drawing standard curve.Accurate absorption concentration known sample liquid 0.5mL, by the operation of standard curve determination method, measures absorbancy, and calculates total sugar content.
(2) BCA (bicinchoninicacid, bicinchoninic acid) method is adopted to measure protein content.Draw bovine serum albumin standard substance 0,10,20,40,60,80 μ L respectively, 100 μ L are supplied with deionized water, add 2mLBCA working standard liquid respectively, in 60 DEG C of water-baths, temperature incubates 15min, letting cool to room temperature, take distilled water as blank, measures 562nm absorbancy, with the amount of protein (μ g) for ordinate zou y, absorbancy is X-coordinate x drawing standard curve.Accurate absorption concentration known sample liquid 100 μ L, by the operation of standard curve determination method, measures absorbancy, and calculates protein content.
Experimental result: Standard for Sugars curve is Y=46.857X-0.263 (r=0.9997), protein standard curve is y=101.32X-3.376 (r=0.9998).In sample, total sugar content and protein content are in table 1, and result shows, the main component of embodiment 1 ~ 6 product is made up of sugar and protein, and total sugar content is 40 ~ 70%, and protein content is 30 ~ 60%.
The sugar of table 1 embodiment 1 ~ 6 product and protein content
| Sample source | Sugar content (%) | Protein content (%) |
| Embodiment 1 | 68.4 | 30.5 |
| Embodiment 2 | 57.3 | 41.2 |
| Embodiment 3 | 61.2 | 35.8 |
| Embodiment 4 | 41.5 | 57.3 |
| Embodiment 5 | 54.8 | 39.4 |
| Embodiment 6 | 45.2 | 52.7 |
Embodiment 8 effect experimental
The tea seed glycoprotein that embodiment 1 ~ 6 obtains is used for the experiment of animal antifatigue.
Method: Kunming healthy adult mouse (18 ~ 22g) is divided into 8 groups at random, often organizes 10, is respectively the tea seed glycoprotein group that normal group, control group, embodiment 1 ~ 6 are obtained, carries out exhaustion swimming time and Biochemical Indexes respectively.Normal group and control group mice gavage tap water, administration group mouse 100mg/kg every day gavage tea seed glycoprotein, continuous gavage is after three days, and except normal group, other groups carry out exercise testing.Before motion, each treated animal gastric infusion of 30min once, often organizes mouse and to bear a heavy burden 5% body weight iron hoop, measure exhaustion swimming time (water temperature 30 DEG C).Blood urea nitrogen (BUN) and liver starch (HG) content is measured after each group of 90min.
Result: the results are shown in Table 2.The mouse comparatively control group of the tea seed glycoprotein that gavage embodiment 1 ~ 6 is obtained, exhaustion swimming time significant prolongation, hepatic glycogen content increases, blood urea nitrogen content obviously reduces.Show that the tea seed glycoprotein that embodiment 1 ~ 6 obtains has obvious antifatigue effect.
Table 2 tea seed glycoprotein on the impact of mouse movement ability (
n=10)
* p<0.05, compares with control group.
Embodiment 9-12 pharmaceutical preparation
Embodiment 9
Take the tea seed glycoprotein 50g that embodiment 1 ~ 6 is obtained, add the mixture 450g of medical starch, Icing Sugar and dextrin 7:2:1 composition in mass ratio, mix, water use regulation, granulation, obtains the granule containing tea seed glycoprotein 10%, can be used as antifatigue granule.
Embodiment 10
The tea seed glycoprotein 2g that Example 1 ~ 6 is obtained, mixes with 8g mixture and Magnesium Stearate 0.1g, and mixture is the 8:1:1 configuration in mass ratio of starch, lactose, crystalline cellulose, and mixing is obtained; 3 kinds of tablets are made through tabletting machine.Each tablet (0.5g), containing tea seed glycoprotein 100mg, can be used as antifatigue tablet.
Embodiment 11
Take the tea seed glycoprotein 10g that embodiment 1 ~ 6 is obtained, add Yelkin TTS 5g, glycerine 10g, injection soybean oil 975g, high-speed stirring, make 1% emulsion.This emulsion can injection for intravenous, as antifatigue emulsion.
Embodiment 12
The tea seed glycoprotein 2g that Example 1 ~ 6 is obtained, add medical starch 8g, mix, wet granulation, regulate with ethanol, obtained loose particles crosses 20 mesh sieves, dries.Filled capsules after dry, obtains 3 kinds of capsules.Each capsule (0.5g), containing tea seed glycoprotein 100mg, can be used as antifatigue capsule agent.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (9)
1. a preparation method for tea seed glycoprotein, is characterized in that comprising the steps:
(1) by degreasing leached tea oil slag aqueous ethanolic solution refluxing extraction, filter, obtain filter residue and ethanol-water extraction liquid; Then filter residue is steamed ethanol to without alcohol taste, for subsequent use;
(2) by the filter residue phosphoric acid buffer heat-insulation soaking in step (1), filter, obtain filtrate; The pH of described phosphoric acid buffer is 8 ~ 10, and the concentration of described phosphoric acid buffer is 0.05 ~ 0.1M;
(3) by the ultra-filtration membrane ultrafiltration of filtrate 2000Da, then partial freeze drying will be retained, obtain tea seed glycoprotein.
2. the preparation method of tea seed glycoprotein according to claim 1, it is characterized in that: liquid-solid ratio (15 ~ 30) mL:1g of phosphoric acid buffer and degreasing leached tea oil slag described in step (2), the condition of described heat-insulation soaking is in 50 ~ 60 DEG C of heat-insulation soaking 1 ~ 3h.
3. the preparation method of tea seed glycoprotein according to claim 1, is characterized in that: described in step (1), the concentration of volume percent of aqueous ethanolic solution is 80 ~ 90%; The liquid-solid ratio of aqueous ethanolic solution and degreasing leached tea oil slag described in step (1) is (10 ~ 20) mL:1g.
4. the preparation method of tea seed glycoprotein according to claim 1, is characterized in that: extraction conditions described in step (1) is for extracting 0.5 ~ 3h in 60 ~ 80 DEG C.
5. the preparation method of tea seed glycoprotein according to claim 1, is characterized in that: degreasing leached tea oil slag described in step (1) is degreasing camellia seed meal or the degreasing tea-seed dregs of rice.
6. the tea seed glycoprotein obtained by preparation method described in any one of Claims 1 to 5, is characterized in that: the sugared content of described tea seed glycoprotein is 40 ~ 70%, and protein content is 30 ~ 60%.
7. the application of tea seed glycoprotein according to claim 6, is characterized in that: described tea seed glycoprotein is for the preparation of anti-fatigue medicament preparation.
8. application according to claim 7, is characterized in that: described pharmaceutical preparation is oral preparations and injection formulations.
9. the application according to claim 7 or 8, is characterized in that: described pharmaceutical preparation is folk prescription containing tea seed glycoprotein or compound preparation.
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| CN201510837017.7A CN105348375A (en) | 2015-11-25 | 2015-11-25 | Tea seed glucoprotein, preparation method therefor and application of tea seed glucoprotein |
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| CN201510837017.7A CN105348375A (en) | 2015-11-25 | 2015-11-25 | Tea seed glucoprotein, preparation method therefor and application of tea seed glucoprotein |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106188258A (en) * | 2016-08-08 | 2016-12-07 | 山西中医学院 | A kind of method extracting Radix Polygalae glycoprotein |
| JP2020019756A (en) * | 2018-08-03 | 2020-02-06 | ビーエイチエヌ株式会社 | Nitric oxide production promoter |
| CN115364161A (en) * | 2021-12-29 | 2022-11-22 | 江西环境工程职业学院 | Tea oil glycoprotein saponin nano intestinal probiotic accelerant and preparation method and application thereof |
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| CN1283629A (en) * | 1999-08-05 | 2001-02-14 | 武汉大学 | Antineoplastic gancoderma mycelium-glucoprotein composition and its application and preparing process |
| CN102010460A (en) * | 2010-09-27 | 2011-04-13 | 厦门盛洲植物油有限公司 | Tea seed polypeptide and preparation method thereof |
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2015
- 2015-11-25 CN CN201510837017.7A patent/CN105348375A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1283629A (en) * | 1999-08-05 | 2001-02-14 | 武汉大学 | Antineoplastic gancoderma mycelium-glucoprotein composition and its application and preparing process |
| CN102010460A (en) * | 2010-09-27 | 2011-04-13 | 厦门盛洲植物油有限公司 | Tea seed polypeptide and preparation method thereof |
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| 李婷婷: "油茶籽糖蛋白的分离纯化及其功能活性研究", 《中国博士学位论文全文数据库工程科技I辑》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106188258A (en) * | 2016-08-08 | 2016-12-07 | 山西中医学院 | A kind of method extracting Radix Polygalae glycoprotein |
| JP2020019756A (en) * | 2018-08-03 | 2020-02-06 | ビーエイチエヌ株式会社 | Nitric oxide production promoter |
| JP7296611B2 (en) | 2018-08-03 | 2023-06-23 | ビーエイチエヌ株式会社 | Nitric oxide production accelerator |
| CN115364161A (en) * | 2021-12-29 | 2022-11-22 | 江西环境工程职业学院 | Tea oil glycoprotein saponin nano intestinal probiotic accelerant and preparation method and application thereof |
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Application publication date: 20160224 |