CN117883347A - Extraction and conversion method for preparing rare ginsenoside and application thereof - Google Patents
Extraction and conversion method for preparing rare ginsenoside and application thereof Download PDFInfo
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- CN117883347A CN117883347A CN202410284475.1A CN202410284475A CN117883347A CN 117883347 A CN117883347 A CN 117883347A CN 202410284475 A CN202410284475 A CN 202410284475A CN 117883347 A CN117883347 A CN 117883347A
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- ginseng root
- ginseng
- extraction
- enzymolysis
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Links
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- 239000000344 soap Substances 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 229920001864 tannin Polymers 0.000 description 1
- 235000018553 tannin Nutrition 0.000 description 1
- 239000001648 tannin Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- UOJAEODBOCLNBU-UHFFFAOYSA-N vinaginsenoside R4 Natural products C1CC(C2(CC(O)C3C(C)(C)C(OC4C(C(O)C(O)C(CO)O4)OC4C(C(O)C(O)C(CO)O4)O)CCC3(C)C2CC2O)C)(C)C2C1C(C)(CCC=C(C)C)OC1OC(CO)C(O)C(O)C1O UOJAEODBOCLNBU-UHFFFAOYSA-N 0.000 description 1
- UZSYIEXAKKPDPN-LMILTKGHSA-N zingibroside r-1 Chemical compound O([C@@H]1C[C@H](O)[C@H](O[C@H]1O[C@H]1CC[C@]2(C)[C@H]3CC=C4[C@@]([C@@]3(CC[C@H]2C1(C)C)C)(C)CC[C@]1(CCC(C[C@H]14)(C)C)C(O)=O)C(O)=O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O UZSYIEXAKKPDPN-LMILTKGHSA-N 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/63—Steroids; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/20—Chemical, physico-chemical or functional or structural properties of the composition as a whole
- A61K2800/26—Optical properties
- A61K2800/262—Transparent; Translucent
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/81—Preparation or application process involves irradiation
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- 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
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Abstract
The application discloses an extraction and conversion method for preparing rare ginsenoside and application thereof, which comprises the steps of putting dried ginseng root whiskers into a pulverizer to pulverize to obtain ginseng root coarse powder, pouring the ginseng root coarse powder into a screen for shaking to obtain ginseng root fine powder; microwave heating and extracting to obtain radix Ginseng extractive solution; and (3) adding a flocculating agent after centrifuging the rough filtrate, and centrifuging the flocculated liquid for the second time. Adding the enzyme preparation into the ginseng root mother liquor, heating, preserving heat and performing enzymolysis; centrifuging the inactivated radix Ginseng enzymolysis solution, filtering, sterilizing, removing impurities, and packaging the filtered radix Ginseng extract into a sterilizing barrel for storage. The ginseng root extract obtained after enzymolysis contains various rare saponins, has good anti-wrinkle tightening effect, and the solution is super-transparent; meanwhile, the production process adopts a pure water scheme, and the low-temperature process can retain the most active ingredients, so that the conversion speed is high and the efficiency is high; and the method adopts microfiltration purification, and is green and efficient.
Description
Technical Field
The invention relates to the field of cosmetics, in particular to an extraction and conversion method for preparing rare ginsenoside and application thereof.
Background
The existing extraction method generally uses organic solvent to extract ginseng, and the ginseng root extract produced in general is prepared by extracting ginseng root with alcohol, drying to obtain powder, and dissolving to obtain ginseng root extract.
In the conventional enzymolysis process for extracting the effective components of the ginseng by water, the ginseng is generally ground into powder, water is added for extraction, and an enzyme preparation is added for enzymolysis in the step, so that the operation is complex, the enzymolysis is not bottoming, and finally the produced ginseng extract is unstable and is easy to separate out solids. Meanwhile, the existing water extraction method for the effective components of the ginseng has the defects that the types of saponins in the ginseng extract are relatively less, rare saponins are hardly contained, and the obtained ginseng extract has weak skin care effect. Therefore, there is a need in the market for a water extraction method of ginseng that is low cost, low pollution, and has a high efficacy.
Disclosure of Invention
Aiming at a safe and efficient extraction and conversion method for preparing rare ginsenoside, which adopts enzymolysis and rapid conversion, contains various rare saponins and has ultra-transparent mother liquor, and application thereof.
In order to achieve the technical purpose, the scheme of the invention is as follows: an extraction and transformation method for preparing rare ginsenoside comprises the following specific steps:
S1, crushing and sieving, namely putting the dried ginseng rootlets into a crusher to crush to obtain ginseng rootlet coarse powder, and pouring the ginseng rootlet coarse powder into a screen to vibrate to obtain ginseng rootlet fine powder;
s2, extracting by microwaves, accurately weighing ginseng root fine powder, pouring the ginseng root fine powder into a microwave extractor, adding pure water, stirring uniformly, and leaching at normal temperature; after leaching, opening a microwave extractor, and performing microwave heating extraction to obtain ginseng root extract;
Step S3, coarse filtration, namely, introducing the ginseng root extract into a plate-and-frame filter press for coarse filtration to obtain coarse filtrate;
step S4, centrifuging, namely centrifuging the coarse filtrate in an inlet pipe type centrifuge, feeding pure water for ejection after the centrifugal feeding is finished, and combining the centrifugate;
Step S5, flocculating, namely adding a flocculating agent into the centrifugate after heating, uniformly stirring, and keeping the temperature and standing to obtain a flocculated liquid;
S6, performing secondary centrifugation, namely performing secondary centrifugation on the flocculation liquid by using a flocculation liquid leading-in type centrifuge, and then feeding pure water for ejection after centrifugal feeding is finished to obtain ginseng root mother liquor;
Step S7, performing enzymolysis, namely adding an enzyme preparation into the ginseng root mother liquor, stirring uniformly, heating, preserving heat, and performing enzymolysis overnight to obtain ginseng root enzymolysis liquor; the enzyme preparation is prepared by compounding cellulase, pectase, medium-temperature amylase, viscosity-reducing enzyme, protease, lysozyme, glucanase and tannase;
the addition amount of the enzyme preparation corresponding to each gram of ginseng root refined powder is as follows: 0.3-0.5ul of cellulase, 0.1-0.4mg of pectase, 1-3mg of medium-temperature amylase, 0.10-0.35mg of viscosity reduction enzyme, 10-30mg of protease, 0.5-3mg of lysozyme, 1-10mg of glucanase and 0.05-0.1mg of tannase;
step S8, inactivating, namely heating and inactivating enzyme activity of the ginseng root enzymatic hydrolysate, standing and cooling to room temperature;
step S9, centrifuging for three times, namely tubular centrifuging the inactivated ginseng root enzymolysis liquid in a tubular centrifuge, and combining centrifugates to obtain ginseng root enzymolysis supernatant;
Step S10, regulating the pH value, namely adding a specified amount of sodium citrate into the ginseng root enzymolysis supernatant, uniformly stirring, and regulating the pH value to 6.48-6.52 to obtain a ginseng root extract mother solution;
Step S11, ultra-nanofiltration, namely introducing the prepared ginseng root extract mother liquor into a ultra-sodium filter, and filtering, sterilizing and removing impurities to obtain a ginseng root extract;
And S12, canning and detecting, namely filling the filtered ginseng root extract into a sterilizing barrel, sealing and preserving at room temperature, and taking a canned sample for detection.
Preferably, in step S4, the pH of the combined solutions is 5.5±0.5;
In step S6, the pH value of the ginseng root mother liquor is 4.76+/-0.1.
Preferably, the pH of the solution after the enzyme preparation is added in step S7 is 4.5-5.5.
Preferably, in the step S2, the flocculant is prepared by weighing acetic acid, adding pure water for constant volume, and preparing an acetic acid solution with the mass percent of 1%; and weighing chitosan, adding 1% acetic acid solution, and stirring and dissolving in water bath at 80 ℃ to obtain the flocculant, wherein the mass part ratio of the chitosan to the acetic acid solution is 1:100.
Preferably, in the step S2 of microwave extraction, the mass part ratio of the ginseng root fine powder to the water is 1:10-40; in the flocculation in the step S5, the mass part ratio of the ginseng root fine powder to the chitosan is 40-50:1.
Preferably, in the first centrifugation in the step S4, the rotation speed of the centrifuge is 12000-16000rpm; s6, during the second centrifugation, the rotating speed of the centrifugal machine is 12000-16000rpm; in the third centrifugation in the step S9, the rotation speed of the centrifugal machine is 12000-16000rpm.
Preferably, in the step S2, the microwave heating is carried out to 80+/-5 ℃ for 30min;
In the step S5, heating the centrifugate to 55+/-5 ℃ and then adding a flocculating agent; in the step S7, heating to 45-50 ℃, and preserving heat and performing enzymolysis for 16-24 hours; and in the step S8, heating to 85-95 ℃, and preserving heat for 30min to inactivate enzyme.
A radix Ginseng extract is prepared by extracting and converting method for preparing rare ginsenoside.
The application of Ginseng radix extract in anti-wrinkle and skin care product is provided.
The ginseng root extract contains various rare saponins, has good anti-wrinkle and tightening effects, and the solution is super-transparent; the process adopts a pure water scheme, does not contain organic solvents, adopts microwaves to extract ginseng roots, adopts a low-temperature process, has high conversion speed, can retain most active components, adopts microfiltration purification, and is green and efficient; the enzymolysis technology is adopted, and various rare ginsenosides are added before and after the enzymolysis technology compared with the existing commercial products, and meanwhile, the product has good anti-wrinkle tightening effect; the ginseng root extract saponin produced by the method of the application has rich varieties, and the product is clear, transparent, stable and not easy to precipitate.
Drawings
FIG. 1 is a flow chart of the present invention;
FIG. 2 is a HPLC chart of the non-enzymatically hydrolyzed ginseng root stock solution of the present invention;
FIG. 3 is a HPLC chart of the ginseng root extract obtained after the enzymolysis of the present invention;
FIG. 4 is a graph showing the total ion flow comparison of ginsenoside in five samples according to the present invention;
FIG. 5 is a relative expression scale of genes of the present invention.
Detailed Description
The application will be described in further detail with reference to fig. 1-5 and the specific examples.
An extraction and transformation method for preparing rare ginsenoside comprises the following specific steps:
s101, checking and accepting raw materials, namely pale yellow appearance of ginseng root whiskers, no foreign matters visible to naked eyes, strong ginseng taste, no other abnormal odors, sampling and checking, wherein the moisture of the ginseng root is lower than 12%;
S102, crushing, namely putting the dried ginseng rootlets into a crusher, wherein the working period is 10S, the working time is 3S, and repeating for 3 times;
S103, sieving, pouring the coarse powder of the ginseng roots which are coarsely crushed into a 80-mesh screen, and vibrating to obtain refined ginseng root powder;
S104, leaching, namely accurately weighing 2300g of ginseng root fine powder, pouring the 2300g of ginseng root fine powder into a microwave extractor, adding pure water according to the mass ratio of 1:40, stirring uniformly, and leaching for 30min at normal temperature;
s105, microwave extraction, namely, after leaching, opening a microwave extractor, and heating the microwave extractor to 80 ℃ for 30min to obtain ginseng root extract;
S106, coarse filtering, namely, introducing the ginseng root extract into a plate-and-frame filter press, and filtering through a filter cloth with 100-300 meshes to obtain coarse filtrate;
s107, centrifuging, namely introducing the coarse filtrate into a centrifugal machine, centrifuging at 12000-16000rpm, feeding 5L of pure water for ejection after the centrifugal feeding is finished, and combining the centrifugate, wherein the pH value of the centrifugate is 5.5+/-0.5;
S108, preparing a flocculant, (1) preparing a 1% acetic acid solution: 46g of acetic acid was weighed out, and 4600g of pure water was added to fix the volume. (2) preparing chitosan: 46g of chitosan (medicinal material: chitosan mass ratio=50:1) was weighed, 4600g of 1% acetic acid solution was added, and the mixture was dissolved by stirring in a water bath at 80 ℃.
S109, flocculating, heating the centrifugate to 55 ℃, adding 4600g of flocculant, stirring for 3min, and keeping the temperature and standing for 30min; then, obtaining flocculation liquid; the application adopts water extraction and flocculation by using flocculant, thereby effectively avoiding ethanol precipitation. The traditional preparation method of the ginseng root extract often involves ethanol precipitation, so that an ethanol explosion-proof workshop is required to be built in industrial production, and the production cost and the production risk are greatly increased. The method of the application avoids the use of ethanol in the production process, and greatly reduces the production risk and the production cost by optimizing the flocculant process.
S110, performing secondary centrifugation, namely performing secondary centrifugation on the flocculated liquid at 12000-16000rpm in a tube-type centrifuge, adding 5L of pure water for ejection after centrifugal feeding is finished, and combining the secondary centrifugate to obtain ginseng root mother liquor, wherein the pH value of the ginseng root mother liquor is 4.76+/-0.1.
S111, preferably eight enzyme preparations are compounded in scientific proportion. The enzyme preparation is prepared according to the table, the enzyme addition amount is calculated, the enzyme is precisely called, and the enzyme preparation is obtained after mixing, and the enzyme preparation is shown in the table 1.
Table 1, enzyme preparation formulation table
| Enzyme variety | Enzyme activity (U/g) | The addition amount of the enzyme preparation is corresponding to each g of ginseng root refined powder |
| Cellulase enzymes | 10000 | 0.5ul |
| Pectase enzyme | 60000 | 0.4mg |
| Middle temperature amylase | 4000 | 1mg |
| Viscosity reducing enzyme | 65000 | 0.35mg |
| Protease enzyme | 600000 | 0.01g |
| Lysozyme | 2000000 | 3mg |
| Glucanase | 300 | 0.01g |
| Tannase | 300 | 0.10mg |
Wherein the protease is compounded by neutral protease and acid protease according to the mass ratio of 1:1.
The enzyme engineering process is used to replace the traditional fermentation process to produce rare ginsenoside. One pain point of the fermentation process is that the growth state of bacteria is difficult to control, so that fermentation sales amount floats greatly among different batches, and the yield of rare saponins is not constant. Meanwhile, carbon sources and nitrogen sources are required to be added in the bacterial fermentation technology, so that the impurity content in the process of extracting ginsenoside is increased, and the product is unstable. The common ginsenoside is effectively converted into rare ginsenoside by the complex enzyme technology. Parameters such as the activity and the concentration of the enzyme can be precisely controlled, so that the properties of the ginseng root extract among batches are highly repeated, and the production process is stabilized.
The adoption of the multiple complex enzyme processes can greatly increase the stability of the aqueous solution of the ginseng root extract. At present, common ginseng extraction water agents in the market all have the problem of long-time standing and precipitation, and the problem is mainly caused by the fact that the extract is mixed with high-content sugar and protein. These biomacromolecule substances can be continuously aggregated within a period of several weeks, and soluble and insoluble precipitates are generated, so that not only is turbidity caused by the products, but also the ginsenoside content can be reduced by the precipitates. Protein, tannin and polysaccharide can be degraded in a full-path and multi-channel way through the 9 enzymes. The types and the dosage of the compound enzyme are repeatedly optimized according to a large number of experimental results, so that enzyme residues are avoided, and the ginseng root solution is clarified and does not precipitate after long-term storage.
S112, performing enzymolysis, namely adding the enzyme preparation into ginseng root mother liquor, stirring and mixing uniformly for 5min, heating to 45-50 ℃, keeping the pH value to 4.5-5.5, and performing heat preservation and enzymolysis overnight (16 hours) to obtain ginseng root enzymolysis liquid;
S113, inactivating the ginseng root enzymolysis liquid, heating to 85-95 ℃, preserving heat for 30min, inactivating enzyme activity, standing, and cooling to room temperature;
S114, centrifuging for three times, introducing the inactivated ginseng root enzymolysis liquid into a tube type centrifuge at 12000-16000rpm, centrifuging in a tube type, and combining the centrifugates to obtain ginseng root enzymolysis supernatant;
s115, regulating the pH, namely adding 1.2kg of sodium citrate into the ginseng root enzymolysis supernatant (volume is about 77L), uniformly stirring, and regulating the pH to 6.48-6.52; to obtain the ginseng root extract mother liquor,
S116, detecting, namely detecting the content of the saponin in the mother solution by a method specified in pharmacopoeia.
S117, performing ultrafiltration, namely introducing the prepared ginseng root extract mother liquor into a sodium ultrafilter, performing membrane filtration on the ginseng root extract with the power of 1 kilodaltons, and performing degerming and impurity removal to obtain the ginseng root extract.
S118, canning, namely filling the filtered ginseng root extract into a sterilized 25Kg barrel, and sealing and preserving at room temperature.
S119, detecting a finished product, namely taking a canned sample to observe color, smell, detection pH, diopter, total number of bacterial colonies, mould and saccharomycetes. And labeling the qualified products for shipment. The produced ginseng root extract is clear and transparent, and the stability test result shows that the ginseng root extract has no precipitation and stable quality.
The method adopts a two-step method for conversion (firstly, obtaining a ginseng root mother liquor containing common ginsenoside through flocculation and centrifugation, and secondly, obtaining a ginseng root extract containing rare ginsenoside through enzymolysis of the ginseng root mother liquor to obtain rare ginsenoside. In the existing production process, the conversion process of common ginsenoside to rare ginsenoside is carried out together with extraction, so that two problems are brought about: firstly, independent common ginsenoside products cannot be obtained, all ginsenosides contain rare ginsenosides, the production cost of the common ginsenosides is lower than that of the rare ginsenosides, and if the common ginsenosides are required to be added in part of products due to cost consideration, additional processes are required to be used for extraction, so that the production flexibility is greatly reduced; the method comprises the steps of obtaining rare ginsenoside in two parts, obtaining common ginsenoside in the first step, and converting the common ginsenoside into the rare ginsenoside by using an enzyme engineering process in the second step, wherein the rare ginsenoside can be adjusted at any time according to the production requirement of the product. Secondly, the content of rare ginsenoside in the existing method can not be regulated, and the method can regulate the content of rare ginsenoside in the final product by regulating enzymolysis conditions, such as prolonging enzymolysis time and the like, comprehensively balancing production time (the cost can be increased when the production time is prolonged) and the required content of rare ginsenoside. The traditional method integrates conversion and extraction, and lacks the capability of flexibly adjusting the content of rare ginsenoside according to the requirement. Meanwhile, the two-step method for converting rare ginsenoside does not need to extract the ginsenoside in a solid form, but is directly carried out in a water phase. The advantages are as follows: firstly, the damage of spray drying to the ginsenoside structure is reduced; secondly, the production time is shortened, and the production period of all products can be controlled to be 24 hours.
The ginseng root extract produced by the method is clear and transparent, the stability test result is free from precipitation, the quality is stable, and the results of the extraction, flocculation, impurity removal, enzymolysis and ph+ adjustment post-filtration tests are shown in table 2.
Table 2 particle size results of different pore size filtration tests
1.1, Ginseng radix extract mother liquor and detection of ginsenoside component in Ginseng radix extract
The application detects ginsenoside components in ginseng extract, and detects non-enzymatic hydrolysis ginseng root mother liquor (light yellow transparent liquid, short for non-enzymatic hydrolysis sample) and ginseng extract obtained after enzymatic hydrolysis (light yellow transparent liquid, short for enzymatic hydrolysis sample).
The testing method comprises the following steps: and detecting the ginseng root extract sample by adopting an ultra-high performance liquid chromatography/high resolution mass spectrometry instrument. The specific operation is as follows:
(1) The model of the instrument is Waters-acquisition-UPLC-Q-TOFSYNAPTG.A liquid chromatography-mass spectrometry (UPLC-Q-TOF/MS) instrument model. (2) The liquid-phase condition chromatographic column is AgilentZORBAX-Extend-C18 (5 um 4.6x250mm) chromatographic conditions, the mobile phase is acetonitrile (A) and 0.1% formic acid water (B), the flow rate is 1mL/min (the split flow into a mass spectrometer is 0.3 mL/min), and the sample injection amount is 10HI. (3) Mass Spectrometry conditions: the electrospray ESI ion source is used for data acquisition under the negative ion mode, the scanning time of the MS mode is 0.2, the scanning range is 50-2000Da, the ion source temperature is 120 ℃, the desolvation gas temperature is 350 ℃, the flow rates of taper hole gas (N 2) and desolvation gas (N 2) are respectively 50L/h and 600L/h, the capillary voltage is 2.2kV, the MS mode has low collision energy of 6V, and the high collision energy of 50-60V. Mass spectrometer calibration was performed using sodium formate and leucine enkephalin was subjected to real-time mass calibration.
Experimental results: (1) The analysis by ultra-high performance liquid chromatography/high resolution mass spectrometry shows that the ginsenoside compound is detected in the non-enzymatic hydrolysis sample which is sent for inspection at this time, as shown in figure 2. Wherein, rare ginseng sapoxib is F1, F3 and Rg3.
(2) The analysis experiment of ultra-high performance liquid chromatography/high resolution mass spectrometry shows that the ginsenoside compound is detected in the sample treated by the enzymolysis technology of the current inspection, as shown in figure 3-enzymolysis sample. Wherein, rare ginseng sapoxib is F1, F3, rk3, rh4, 20 (S) -Rg3, 20 (R) -Rg3, rg5, rk1 and Rh2.
(3) In summary, 21 ginseng saponin components are identified in total in the non-enzymatic hydrolysis ginseng root mother liquor sample, and 27 ginseng saponin components are identified in total in the sample treated by the enzymatic hydrolysis process of the current inspection. The products before and after enzymolysis (figures 2 and 3) are compared, and rare ginseng sapoxik 3, rh4, 20 (R) -Rg3, rg5, rk1 and Rh2 are added in the enzymolysis samples after enzymolysis.
Conclusion of experiment: the two products (the ginseng root extract obtained by enzymolysis and the ginseng root mother liquor which is not subjected to enzymolysis) of the application which are sent for inspection all contain part of rare ginseng soap shake. Compared with the non-enzymatic sample, the sample treated by the enzymatic hydrolysis process has higher content of rare ginseng sapoxix and richer variety. As shown in fig. 2 and 3, the saponin change study of the enzymatic sample and the non-enzymatic sample: HPLC (high Performance liquid chromatography) patterns show that the enzymolysis process is at least increased by 6 rare saponins, and the value potential brought by the rare saponins is huge, so that the enzymolysis process is worthy of further development and deep investigation.
1.2 Comparison of the Ginseng radix extract of the present application with the existing products
In order to compare and analyze the condition of the ginseng root extract obtained by enzymolysis of the inventor (the enzymolysis sample for short) and the type of the saponin in the existing product. The applicant sends samples of products A, B, C and D obtained by enzymolysis of other companies to a testing mechanism for liquid chromatography-mass spectrometry analysis, and the ginseng root extract produced by the process has the greatest types of saponins and increases rare saponins.
The method for detecting the ginseng root extract sample by adopting an ultra-high performance liquid chromatography/high resolution mass spectrometer comprises the following steps:
Instrument model: the model of the liquid chromatography-mass spectrometry (UPLC-Q-TOF/MS) instrument is Waters-acquisition-UPLC-Q-TOF-SYNAPTG. (2) liquid phase conditions: the column was Agilent-ZORBAX-Extend-C18 (5 μm, 4.6X1250 mm), chromatographic conditions: the mobile phase is acetonitrile (A) and 0.1% formic acid water (B); the flow rate was 1mL/min (split into mass spectrometer was 0.3 mL/min); the sample loading was 10. Mu.L and the elution gradient was as shown in Table 3.
TABLE 3 gradient of mobile phase elution
| Time (min) | A（%） | B（%） |
| 0-35 | 19 | 81 |
| 35-60 | 19-30 | 81-70 |
| 60-70 | 30 | 70 |
| 70-100 | 30-40 | 70-60 |
| 100-120 | 40-60 | 60-40 |
| 120-125 | 60-19 | 40-81 |
(3) Mass spectrometry conditions: the ESI ion source is subjected to data acquisition in an anion mode, the scanning time of the MSE mode is 0.2s, the scanning range is 50-2000Da, the ion source temperature is 120 ℃, the desolvation gas temperature is 350 ℃, the flow rates of taper hole gas (N 2) and desolvation gas (N 2) are respectively 50L/h and 600L/h, the capillary voltage is 2.2kV, the low collision energy of the MSE mode is 6V, and the high collision energy is 50-60V. Mass spectrometer calibration was performed using sodium formate and real-time mass calibration was performed with leucine enkephalin.
Experimental results: through ultra-high performance liquid chromatography/high resolution mass spectrometry analysis, 20 ginsenosides are detected in the products A, B and C of the current inspection, wherein 11 ginsenosides are common, and 9 rare ginsenosides are rare; 17 ginsenosides are detected in the sample D, wherein 11 ginsenosides are common ginsenosides and 6 rare ginsenosides are rare ginsenosides; 27 ginsenosides are detected in the enzymolysis sample, wherein 18 ginsenosides are common ginsenosides and 9 rare ginsenosides are rare ginsenosides;
the ginsenoside information detected by each manufacturer is shown in table 1. Wherein: rare ginsenoside: f1, F3, rk3, rh4, 20 (S) -Rg3, 20 (R) -Rg3, rg5, rk1 and Rh2.
(3) In sum, 20 ginsenosides are detected in the products A, B and C, 17 ginsenosides are detected in the sample D, 27 ginsenosides are detected in the enzymolysis sample, and compared with other four products, the ginsenosides such as Compound-Ra1/Ra2, malonyl-ginsenoside (Malonyl-ginsenosides) -Re2, malonyl-ginsenoside-Rb1, malonyl-ginsenoside-Rc, malonyl-ginsenoside-Rb2, malonyl-ginsenoside-Rd and isomers thereof are added in the products after enzymolysis. Table 4 shows the ginsenoside content of the samples from different manufacturers.
TABLE 4 ginsenoside detected by different manufacturers
| Identified | RT/min | Observed m/z | Enzymolysis sample | Product C | Product B | Product A | Product D |
| Ginsenoside Rg1 | 28.33 | 845.4814 | P | P | P | P | P |
| Ginsenoside Re | 31.19 | 945.5471 | P | P | P | P | P |
| Compound Ra1/Ra2 | 45.37 | 885.4775 | P | - | - | - | - |
| Malonyl-ginsenoside Re2 | 50.64 | 1031.5281 | P | - | - | - | - |
| Ginsenoside Rf | 59.84 | 845.4814 | P | P | P | P | P |
| Ginsenoside Ra1 | 64.17 | 1255.6208 | P | P | P | P | P |
| Ginsenoside F3 | 64.93 | 815.4699 | P | P | P | P | - |
| Ginsenoside Rg2 | 64.99 | 783.4787 | P | P | P | P | P |
| Ginsenoside Rb1 | 65.00 | 1107.5828 | P | P | P | P | P |
| Malonyl-ginsenoside Rb1 | 67.53 | 1193.5789 | P | - | - | - | - |
| Ginsenoside Rc | 68.24 | 1123.5667 | P | P | P | P | P |
| Ginsenoside Ro | 70.72 | 955.4746 | P | P | P | P | P |
| Malonyl-ginsenoside Rc | 70.82 | 1193.5487 | P | - | - | - | - |
| Ginsenoside Rb2 | 71.99 | 1123.5665 | P | P | P | P | P |
| Ginsenoside F1 | 73.57 | 683.4396 | P | P | P | P | - |
| Malonyl-ginsenoside Rb2 | 75.54 | 1163.5723 | P | - | - | - | - |
| Ginsenoside Rd | 79.22 | 991.5266 | P | P | P | P | P |
| Malonyl-ginsenoside Rd or iso Malonyl-ginsenoside Rd | 82.08 | 1031.5281 | P | - | - | - | - |
| Malonyl-ginsenoside Rd or iso Malonyl-ginsenoside Rd | 86.58 | 1031.5281 | P | - | - | - | - |
| Ginsenoside Rk3 | 97.05 | 665.4166 | P | P | P | P | P |
| Ginsenoside Rh4 | 100.06 | 665.4166 | P | P | P | P | P |
| Zingibroside R1 | 104.39 | 793.431 | P | P | P | P | P |
| (20S)Ginsenoside Rg3 | 105.52 | 829.4951 | P | P | P | P | P |
| (20R)Ginsenoside Rg3 | 106.60 | 829.4951 | P | P | P | P | P |
| Ginsenoside Rg5 | 115.70 | 811.4812 | P | P | P | P | P |
| Ginsenoside Rk1 | 116.61 | 811.4812 | P | P | P | P | P |
| Ginsenoside Rh2 | 116.70 | 667.4362 | P | P | P | P | - |
Detection result (in table 4: P indicates that the component was detected, -indicates that the component was not detected): 20 ginsenosides are detected in the products A, B and C of the current inspection, 17 ginsenosides are detected in the sample D, 27 ginsenosides are detected in the enzymolysis sample, and compared with other four products (figure 4), 7 ginsenosides are added in the enzymolysis sample after enzymolysis on the basis of retaining rare ginsenosides, and the variety of ginsenosides is more abundant. In FIG. 4, sample A (third left-right bar), sample B (fourth left-right bar), sample C (fifth left-right bar), sample D (first left-right bar, and Time base line almost overlap), and enzymatic hydrolysis sample of the present application (second left-right bar).
1.3 Skin anti-wrinkle, tightening efficacy test
The ginseng root extract of the present application was sent to a detection mechanism for detection as shown in fig. 5 and table 5. Test items: anti-wrinkle and tightening effects, namely promoting test of zebra fish type I collagen gene expression. The test method comprises the steps of exposing 36 zebra fish for 3 days to a 0.1% formula adding concentration ginseng root extract sample solution, setting a blank control group, performing RNA extraction and CDNA synthesis on the zebra fish after 24 hours of exposure, performing real-time PCR amplification, taking B-actin as a housekeeping gene, calculating the relative expression quantity of collala, collalb and colia2 by taking Ct as an amplification result, and performing statistical analysis.
TABLE 5 anti-wrinkle and compactification evaluation Table
Efficacy evaluation table of table 5 above: the samples (ginseng root extract of the present application) had an acceleration rate of 57% (p= 0.000091), 212% (p-0.000099), 225% (p=0.00042) for zebra fish collala, collalb and colla2 gene expression, respectively, at the 0.1% formulation addition concentration.
Conclusion: the sample can obviously promote the expression of the zebra fish type I collagen gene, has the effect of promoting the regeneration of type I collagen, and supports the declaration of anti-wrinkle and tightening effects.
1.4, Keratinocyte scratch repair test
The ginseng root extract of the application is sent to a designated detection mechanism to detect the repairing capability of the extract of the application to cell scratches. Cytotoxicity screening was performed first, and the results of the specific cytotoxicity screening table are shown in table 6.
TABLE 6 cytotoxicity screening table
| Concentration% (v/v) | Cell viability (%) |
| 10 | 20.96±2.79 |
| 9 | 43.75±3.20 |
| 8 | 64.70±4.04 |
| 7 | 51.11±4.04 |
| 6 | 73.54±3.90 |
| 5 | 93.45±4.83 |
| 4 | 99.06±2.82 |
| 3 | 100.99±10.49 |
Cell scratch test, after evenly mixing culture solution containing 3.0-5.0X105/mL suspended cells in a 24-hole plate, adding the mixture into a 24-hole cell culture plate according to 500 mu L/hole, and culturing the mixture for 18-24 hours under the conditions that the temperature is 37 ℃ and the humidity is more than 90% of a carbon dioxide incubator and a CO 2 is adopted. A200. Mu.L pipette tip was used to vertically scribe a flat cell-free area in the center of the well. The cell layer and the scratch edge were washed 3 times with PBS and the detached cells were removed. mu.L of culture medium containing test samples of different concentrations was added to the wells, and the image was collected under a mirror, recorded as 0h, and the collected locations were marked. Collecting pictures at the same marked positions for 0, 24, 30 and 48 hours, changing the liquid of the sample before collecting the pictures for 24 hours, preparing and grouping the same as 2.5, discarding the original culture medium of each group at 48 hours, and adding fresh serum-free culture medium for picture collection. Data analysis: the area of the scratch area was analyzed using IPP map analysis software, and the area Δs was calculated as reduced over 0 days.
Under the experimental conditions, the scratch migration healing of HaCaT cells is obviously improved when the samples are acted for 48 hours at the concentrations of 6%, 3% and 1% (VV) compared with NC groups in the same time point (P < 0.05), and the scratch migration healing of HaCaT cells is obviously improved when the samples are acted for 24 hours, 30 hours and 48 hours at the concentrations of 6% (V) and 1% (VV) compared with NC groups in the same time point (P < 0.05).
TABLE 7 scratch area reduction ΔS results for each group
| Group/concentration (%) (v/v) | 24h | 30h | 48h |
| 6 | 664.15±104.03* | 723.72±103.75* | 957.20±66.56*** |
| 3 | 480.52±60.12 | 526.59±84.21 | 767.11±67.40* |
| 1 | 572.74±56.57* | 632.26±70.96* | 830.09±71.78* |
| NC | 435.68±36.54 | 481.08±25.27 | 616.86±47.44 |
| PC | 833.75±103.53** | 978.63±105.11** | 1,448.92±55.83*** |
The scratch area reduction Δs for each group is shown in table 7, notes: ". Shows statistically significant differences compared to NC group, significant (P < 0.05); ". Times." indicates statistically significant differences compared to NC groups, with very significant significance (P < 0.01); ", indicates statistically significant differences from NC group, with very high significance (P < 0.001).
1.5, Tightening efficacy test
The ginseng root extract of the present application is sent to a designated detecting mechanism to detect the tightening effect of the extract of the present application on the skin.
Experimental principle: elastin is a protein that is present in a variety of connective tissues and has unique physiological elasticity. Although elastin represents only 2% of the total dermal protein, it promotes an important function of skin homeostasis. One of the molecular mechanisms of skin photoaging is that UV radiation can regulate elastase gene expression, resulting in reduced elastin content, such as Matrix Metalloproteinases (MMPs), particularly human macrophage metalloelastase (MMP 12), which is the most active protease in elastin degradation. While elastin production decreases with age or damage caused by solar radiation. In the experiment, the Elastin content change is measured by an ELISA method, and the tightening efficacy of the to-be-measured object is evaluated. Experimental materials: cell line: human skin fibroblasts. Culture solution: DMEM medium containing 10% fetal bovine serum. Test conditions: the temperature of the incubator is 37+/-1 ℃, the humidity is 90+/-5%, and the CO 2 is 5+/-1%. Elastin content determination: normal human skin fibroblasts were inoculated into 96-well plates and cultured for 18-24h. Cell layers were washed 2 times with PBS, and maintenance medium with different concentrations of test substances and maintenance medium without sample were added according to groups, 3 wells were multiplexed, and exposed for 72h. Cell supernatants were collected, stored at-80℃and assayed for Elastin content using ELISA kits. Wherein each cell group has a relative activity as shown in Table 8.
Table 8, relative Activity of groups of cells (mean.+ -. SD)
| Concentration (%) | Viability（%） |
| Control | 100.00±5.26 |
| 20.000 | 4.91±1.42 |
| 6.329 | 80.56±2.81 |
| 2.003 | 94.70±5.18 |
| 0.634 | 107.55±4.41 |
| 0.201 | 107.91±7.77 |
| 0.063 | 111.33±6.94 |
| 0.020 | 109.56±6.59 |
| 0.006 | 105.81±5.91 |
The effect of the test substances on the levels of Elastin in cells is shown in table 9, wherein the differences are statistically significant (p < 0.05) compared to the negative control group (NT). Compared with the negative control group (NT), the positive control group (PC) has obviously increased Elastin relative content (P < 0.05), which indicates that the modeling is successful; the relative levels of Elastin at 5.0% (v/v), 1.0% (v/v), 0.2% (v/v) tested were significantly increased by 56.21%, 52.63%, 47.44% (P < 0.05), respectively, compared to the negative control. In summary, the samples had a tightening effect at 5.0% (v/v), 1.0% (v/v), 0.2% (v/v) of the test concentration.
TABLE 9 influence of the test substances on the levels of cell Elastin (mean.+ -. SD)
The foregoing description is only of the preferred embodiments of the present invention, and is not intended to limit the invention, but any minor modifications, equivalents, and improvements made to the above embodiments according to the technical principles of the present invention should be included in the scope of the technical solutions of the present invention.
Claims (9)
1. The extraction and transformation method for preparing rare ginsenoside is characterized by comprising the following specific steps:
S1, crushing and sieving, namely putting the dried ginseng rootlets into a crusher to crush to obtain ginseng rootlet coarse powder, and pouring the ginseng rootlet coarse powder into a screen to vibrate to obtain ginseng rootlet fine powder;
s2, extracting by microwaves, accurately weighing ginseng root fine powder, pouring the ginseng root fine powder into a microwave extractor, adding pure water, stirring uniformly, and leaching at normal temperature; after leaching, opening a microwave extractor, and performing microwave heating extraction to obtain ginseng root extract;
Step S3, coarse filtration, namely, introducing the ginseng root extract into a plate-and-frame filter press for coarse filtration to obtain coarse filtrate;
step S4, centrifuging, namely centrifuging the coarse filtrate in an inlet pipe type centrifuge, feeding pure water for ejection after the centrifugal feeding is finished, and combining the centrifugate;
Step S5, flocculating, namely adding a flocculating agent into the centrifugate after heating, uniformly stirring, and keeping the temperature and standing to obtain a flocculated liquid;
S6, performing secondary centrifugation, namely performing secondary centrifugation on the flocculation liquid by using a flocculation liquid leading-in type centrifuge, and then feeding pure water for ejection after centrifugal feeding is finished to obtain ginseng root mother liquor;
Step S7, performing enzymolysis, namely adding an enzyme preparation into the ginseng root mother liquor, stirring uniformly, heating, preserving heat, and performing enzymolysis overnight to obtain ginseng root enzymolysis liquor; the enzyme preparation is prepared by compounding cellulase, pectase, medium-temperature amylase, viscosity-reducing enzyme, protease, lysozyme, glucanase and tannase;
the addition amount of the enzyme preparation corresponding to each gram of ginseng root refined powder is as follows: 0.3-0.5ul of cellulase, 0.1-0.4mg of pectase, 1-3mg of medium-temperature amylase, 0.10-0.35mg of viscosity reduction enzyme, 10-30mg of protease, 0.5-3mg of lysozyme, 1-10mg of glucanase and 0.05-0.1mg of tannase;
step S8, inactivating, namely heating and inactivating enzyme activity of the ginseng root enzymatic hydrolysate, standing and cooling to room temperature;
step S9, centrifuging for three times, namely tubular centrifuging the inactivated ginseng root enzymolysis liquid in a tubular centrifuge, and combining centrifugates to obtain ginseng root enzymolysis supernatant;
Step S10, regulating the pH value, namely adding a specified amount of sodium citrate into the ginseng root enzymolysis supernatant, uniformly stirring, and regulating the pH value to 6.48-6.52 to obtain a ginseng root extract mother solution;
Step S11, ultra-nanofiltration, namely introducing the prepared ginseng root extract mother liquor into a ultra-sodium filter, and filtering, sterilizing and removing impurities to obtain a ginseng root extract;
And S12, canning and detecting, namely filling the filtered ginseng root extract into a sterilizing barrel, sealing and preserving at room temperature, and taking a canned sample for detection.
2. The extraction and transformation method for preparing rare ginsenoside according to claim 1, wherein: in the step S4, the pH value of the centrifugate is 5.5+/-0.5 after the centrifugate is combined;
In step S6, the pH value of the ginseng root mother liquor is 4.76+/-0.1.
3. The extraction and transformation method for preparing rare ginsenoside according to claim 1, wherein: the pH of the solution after the enzyme preparation is added in step S7 is 4.5-5.5.
4. The extraction and transformation method for preparing rare ginsenoside according to claim 1, wherein: in the step S2, flocculating agent, weighing acetic acid, adding pure water to fix volume, and preparing acetic acid solution with the mass percent of 1%;
And weighing chitosan, adding 1% acetic acid solution, and stirring and dissolving in water bath at 80 ℃ to obtain the flocculant, wherein the mass part ratio of the chitosan to the acetic acid solution is 1:100.
5. The extraction and conversion method for preparing rare ginsenoside of claim 4, wherein: in the step S2 of microwave extraction, the mass ratio of the ginseng root fine powder to the water is 1:10-40;
in the flocculation in the step S5, the mass part ratio of the ginseng root fine powder to the chitosan is 40-50:1.
6. The extraction and transformation method for preparing rare ginsenoside according to claim 1, wherein: s4, during the first centrifugation, the rotating speed of the centrifugal machine is 12000-16000rpm;
s6, during the second centrifugation, the rotating speed of the centrifugal machine is 12000-16000rpm;
In the third centrifugation in the step S9, the rotation speed of the centrifugal machine is 12000-16000rpm.
7. The extraction and transformation method for preparing rare ginsenoside according to claim 1, wherein: in the step S2, heating to 80+/-5 ℃ for 30min by microwaves;
in the step S5, heating the centrifugate to 55+/-5 ℃ and then adding a flocculating agent;
in the step S7, heating to 45-50 ℃, and preserving heat for enzymolysis for 16-24 hours;
and in the step S8, heating to 85-95 ℃, and preserving heat for 30min to inactivate enzyme.
8. An extract of ginseng root, characterized in that: the extraction and conversion method for preparing rare ginsenoside as set forth in any one of claims 1 to 7.
9. Use of the ginseng root extract according to claim 8 in anti-wrinkle tightening skin care products.
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