CN111374936A - Method for preparing saussurea involucrate cell extract by using multi-component liquid system and application thereof - Google Patents
Method for preparing saussurea involucrate cell extract by using multi-component liquid system and application thereof Download PDFInfo
- Publication number
- CN111374936A CN111374936A CN202010254583.6A CN202010254583A CN111374936A CN 111374936 A CN111374936 A CN 111374936A CN 202010254583 A CN202010254583 A CN 202010254583A CN 111374936 A CN111374936 A CN 111374936A
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- Prior art keywords
- saussurea involucrate
- butanediol
- cell extract
- extraction
- water
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Abstract
The invention discloses a method for preparing a snow lotus cell extract by a multi-component liquid system, which mainly adopts a water, butanediol and propylene glycol multi-component liquid system to extract effective components in snow lotus cells, wherein the butanediol refers to 1, 3-butanediol, and the propylene glycol refers to 1, 3-propylene glycol; in the multi-component liquid system, the concentration range of butanediol is 0-70%, the content of propanediol is 0-70%, and the content of water is 20-90%. The method avoids adverse factors caused by complete water extraction and ethanol extraction, increases product stability, improves product quality, and makes saussurea involucrate cell have outstanding skin care effect in cosmetics due to skin care effects of butanediol and propylene glycol.
Description
Technical Field
The invention belongs to the field of plant extraction biotechnology, and relates to a method for preparing a snow lotus cell extract by using a multi-component liquid system and application thereof.
Background
The Tianshan herba Saussureae Involueratae is distributed in the southern and northern slopes of Tianshan, Altai mountain and Kunlun mountain in Xinjiang, with altitude of 4000-5000 m. Is an excellent Chinese medicinal material, and has therapeutic effects on menoxenia, toothache, rheumatic arthritis, sexual impotence, leukorrhagia, and metrorrhagia. The seeds of saussurea involucrate germinate at zero deg.C and grow at 3-5 deg.C, and the seedlings can be subjected to severe cold at-21 deg.C. Although it can bloom after growing for 5 years, the main growth period is only 8 months in practice. In recent years, the number of saussurea involucrate is greatly reduced due to excessive mining and digging, and the saussurea involucrate is a secondary protection plant endangered by the country.
The wild saussurea involucrate is expensive and the use of the wild saussurea involucrate is limited. The saussurea involucrate cultured by the plant cell culture technology has the characteristics of low use cost and high efficacy content. The invention is based on the saussurea involucrate cell to extract the functional component of the saussurea involucrate cell and the application.
The existing special use mostly adopts a water extraction or ethanol extraction mode to extract functional active ingredients from saussurea involucrate cells, but the water extraction process easily causes the color of an extracting solution to become dark, and meanwhile, a large amount of sugar is contained in an aqueous extract, so that the aqueous extract is easily influenced by polyalcohol to generate precipitates in the process of using the aqueous extract to prepare cosmetics; the ethanol extract is limited in use in the cosmetic preparation process due to the irritation of the ethanol, and the ethanol reagent usually contains methanol, so that the methanol content test is carried out before the ethanol extract is used, and the use cost of the product is increased.
Disclosure of Invention
The invention adopts a multi-component liquid system of water, butanediol and 1, 3-propylene glycol to extract the effective components in the saussurea involucrate cells, avoids the adverse factors generated by complete water extraction and ethanol extraction, increases the stability of the product, improves the quality of the product, and simultaneously has more prominent skin care effect of the saussurea involucrate cells in the cosmetics due to the skin care effect of the butanediol and the propylene glycol.
In order to achieve the purpose, the invention discloses the following technical contents:
a method for preparing saussurea involucrate cell extract by a multi-component liquid system is characterized by comprising the following steps:
(1) accurately weighing 10g of fresh saussurea involucrate cell, respectively adding multiple solvents with different concentrations for extraction at 70-100 deg.C for 1-3h or standing at room temperature for 24h, centrifuging at 6500rpm for 10-15min, and collecting supernatant to obtain saussurea involucrate cell extract;
(2) performing component analysis and detection on the extracts extracted by the multi-component solvents with different concentration ratios;
the extraction method comprises high-temperature extraction, leaching and ultrasonic extraction;
the multi-component solvents with different concentrations refer to: in the multi-component system, the concentration ratio of butanediol is 0-70%, the concentration ratio of propanediol is 0-70%, and the concentration ratio of water is 20-90%.
The butanediol refers to 1, 3-butanediol, and the propylene glycol refers to 1, 3-propylene glycol.
The method of the invention is characterized in that the saussurea involucrate cell used for extraction is dried saussurea involucrate cell or saussurea involucrate fresh cell containing water.
The invention further discloses application of the snow lotus cell extract prepared by the method in improving the antioxidation, anti-inflammation and moisturizing effects of the snow lotus cells, and experimental results show that the snow lotus cell extract has obvious antioxidation, anti-inflammation and moisturizing effects.
And (3) comparison test:
and (4) conclusion: the saussurea involucrate cell extract prepared by the method has the advantages of anti-corrosion effect and strong stability, the sugar content is greatly reduced compared with that of water, and irritation caused by ethanol in the ethanol extract and detection of the content of methanol are avoided.
The invention mainly solves the problem that the current market mainly uses water or ethanol as a main extraction solvent to prepare the snow lotus extract, and mainly considers different concentration ratios of butanediol, propylene glycol and water in a multi-component solvent in the preparation process of the snow lotus cell extract, and the main difficulty lies in the ratio research of the three solvents in the multi-component solvent.
The invention is described in more detail below:
1. accurately weighing 10g of fresh saussurea involucrate cell, respectively adding 50mL of water, ethanol, butanediol, propylene glycol and a multi-element solvent (water: butanediol: propylene glycol =5:2:3 (m: m: m)) into different solvents for extraction, wherein the extraction temperature is 100 ℃ (the ethanol is boiling 76 ℃), the extraction time is 1h, then carrying out 6500rpm centrifugation for 10min, retaining the supernatant fluid, namely the saussurea involucrate cell extract, carrying out component analysis and detection on the extracts extracted by different solvents, carrying out parallel detection for 3 times, and carrying out the detection method and the detection result as follows:
the detection method of the total flavonoids comprises the following steps:
1) drawing a standard curve: accurately weighing 10mg of rutin reference substance, dissolving with methanol, diluting to 100mL, and preparing 0.2mg/mL of rutin reference substance solution. Precisely absorbing 1mL, 2mL, 3mL, 4mL, 5mL and 6 mL of rutin reference substance solution, respectively placing in 25mL measuring bottles, respectively adding water to 6 mL, adding 1mL of 5% sodium nitrite solution, shaking up, placing for 6 minutes, adding 1mL of 10% aluminum nitrate solution, shaking up, placing for 6 minutes, adding 10mL of 4% sodium hydroxide test solution, adding pure water to a constant volume of 25mL, shaking up, placing for 15 minutes, taking corresponding reagents as blanks, and measuring absorbance at a wavelength of 500 nm by a spectrophotometry method.
2) And (3) determining the total flavone content of the sample: detecting the content of total flavone of the cell extract of saussurea involucrate with different solvents by the above method, and calculating the content of the total flavone.
The total phenolic acid detection method comprises the following steps:
1) drawing a standard curve: accurately weighing 2.7mg of chlorogenic acid, dissolving with absolute ethyl alcohol, fixing the volume to 10mL, precisely sucking 200, 300, 400, 500, 600, 700 and 800uL of reference solution, respectively placing in 25mL measuring bottles, and adding absolute ethyl alcohol to 5 mL; adding 2mL of 0.3% sodium dodecyl sulfate solution, shaking up, adding 1mL of a mixed solution of 0.9% potassium ferricyanide and 0.6% ferric trichloride solution, shaking up, standing in the dark for 5 minutes, adding 0.1mol/L hydrochloric acid solution to the scale mark, shaking up, and standing in the dark for 20 minutes. The absorbance was measured at a wavelength of 760nm with a blank of 5mL volume of solution to which absolute ethanol was added, and a linear regression equation was calculated with the absorbance and its corresponding mass, Y = kx + b.
2) And (3) measuring the total phenolic acid content of the sample: detecting the content of total phenolic acid in the cell extract of saussurea involucrate with different solvents by the method, and calculating the content of the total phenolic acid.
The total sugar detection method comprises the following steps:
1) drawing a standard curve: accurately weighing 100mg of anhydrous glucose, adding water to dissolve, and fixing the volume to 100 mL. Precisely measuring 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.6 mL and 0.8mL of glucose standard solution, placing the glucose standard solution in a 10mL test tube with a plug, adding distilled water to 1.0mL, adding 4.0mL of anthrone sulfuric acid solution, placing the test tube with the plug in a boiling water bath for reaction for 10min, immediately cooling, placing the test tube at room temperature for 10min, detecting the absorbance of a sample after the reaction, and detecting the wavelength of 620 nm. And drawing a standard curve by taking the mass of the glucose as an abscissa and the absorbance value as an ordinate, and calculating a linear regression equation by taking the absorbance and the corresponding mass, wherein Y = kx + b.
2) And (3) determining the total sugar content of the sample: the total sugar content of the saussurea involucrate cell extracts with different solvents is detected by the method, and the total sugar content is calculated.
TABLE 1 influence of different solvent extractions on the components of the saussurea involucrate cell extract (UV detection)
TABLE 2 influence of different solvent extractions on the components of the saussurea involucrate cell extract (UPLC assay)
2. Accurately weighing 10g of fresh saussurea involucrate cell cells, respectively adding multiple solvents with different concentration ratios for extraction, wherein the ratio of the water to the butanediol to the propylene glycol is ①, the ratio of the butanediol to the propylene glycol is =1:1:0, the ratio of the ② water to the butanediol to the propylene glycol is =1:0:1, the ratio of the ③ water to the butanediol to the propylene glycol is =0:1:1, after the mixture is leached and placed for 24 hours at normal temperature, the mixture is centrifuged at 6500rpm for 10 minutes, supernatant fluid is reserved, namely the saussurea involucrate cell extract, and the extracts extracted by the multiple solvents with different concentration ratios are subjected to component analysis and detection for 3 times, and the detection results are as follows:
TABLE 3 influence of different solvent extractions on the components of the saussurea involucrate cell extract (UV detection)
TABLE 4 influence of different solvent extractions on the components of the saussurea involucrate cell extract (UPLC assay)
3. Accurately weighing 10g of saussurea involucrate cell fresh cells, respectively adding multiple solvents with different concentration ratios for extraction, wherein the ratio of the water to the butanediol to the propanediol is ①, the ratio of the butanediol to the propanediol is =1:1:1, the ratio of the ② water to the butanediol to the propanediol is =2:1:1, the ratio of the ③ water to the butanediol to the propanediol is =5:2:3, the extraction temperature is 100 ℃, the extraction time is 1h, then the centrifugation is carried out at 6500rpm for 10min, the supernatant is reserved, namely the saussurea involucrate cell extract, the extracts extracted by the multiple solvents with different concentration ratios are subjected to component analysis and detection, the parallel detection is carried out for 3 times, and the detection results are as follows:
TABLE 5 Effect of different solvent extractions on the ingredients in the saussurea involucrate cell extract (UV detection)
TABLE 6 influence of different solvent extractions on the components of the saussurea involucrate cell extract (UPLC assay)
1. Experiment on antioxidant Effect
Sample preparation: accurately weighing 1g of saussurea involucrate cell stem cells, adding 30 times of volume of a multi-element solvent for extraction, wherein water comprises butanediol, propylene glycol =5:2:3 (m: m: m), the extraction temperature is 100 ℃, the extraction time is 1h, then performing centrifugation at 6500rpm for 10min, and reserving supernatant, namely saussurea involucrate cell extract, and performing an oxygen radical scavenging experiment on the saussurea involucrate cell extract.
DPPH oxygen radical scavenging assay
Preparing a DPPH ethanol solution: 3.94mg of DPPH is accurately weighed, added with absolute ethyl alcohol to be dissolved and fixed to a volume of 100mL volumetric flask to prepare 0.1mM DPPH solution, and the solution is stored at low temperature (0-4 ℃) in dark. Measuring saussurea involucrate cell extracts with different volumes, adding the saussurea involucrate cell extracts into 3mL of DPPH solution, uniformly mixing, adding water to complement to 4mL, measuring the absorbance at 517nm, and calculating the clearance rate, wherein the calculation formula is as follows:
wherein the control A is the solution without sample, and only the absorbance of 1.0mL of purified water is added; sample a is the absorbance of different volumes of sample solution.
The experimental result is shown in figure 1, and the saussurea involucrate cell extract has stronger oxygen radical scavenging capacity on DPPH free radicals.
ABTS free radical scavenging efficacy experiment
Required solution formulation 7.4 mM ABTS solution: accurately weighing 0.0384 g of ABTS, placing the ABTS in a 10mL brown volumetric flask, and fixing the volume of purified water; 2.6 mM potassium persulfate solution: accurately weighing 0.0066 g of potassium persulfate, placing the potassium persulfate in a 10mL volumetric flask, and fixing the volume of purified water; ABTS working solution: the 7.4 mM ABTS solution and the 2.6 mM potassium persulfate solution are mixed according to the volume ratio of 1:1, kept in the dark at room temperature for 12 hours for standby, and diluted by 80% ethanol solution until the absorbance is 0.7 +/-0.02 before use.
In the ABTS oxygen free radical removal experiment of the sample, different volumes of the saussurea involucrate cell extracts are accurately measured and added into 3.4mL of ABTS working solution, then water is added to the ABTS working solution to be constant volume to 4.0mL, the mixture is uniformly mixed, the mixture is kept stand at room temperature for 30min, then spectrophotometric detection is carried out at 734nm, the removal rate is calculated through absorbance change, and the calculation formula is as follows:
wherein the control A is the solution without sample, and only the absorbance of 1.0mL of purified water is added; sample a is the absorbance of different volumes of sample solution.
The experimental result is shown in fig. 2, and the result shows that the saussurea involucrate cell extract has good clearance rate for the oxygen free radical of ABTS, and simultaneously shows that the saussurea involucrate cell extract has obvious free radical clearing capacity.
2. Anti-inflammatory efficacy experiments
Sample preparation: accurately weighing 1g of saussurea involucrate cell stem cells, adding 30 times of volume of a multi-element solvent for extraction, wherein water comprises butanediol and propylene glycol =5:2:3 (m: m: m), the extraction temperature is 100 ℃, the extraction time is 1h, then performing centrifugation at 6500rpm for 10min, and reserving supernatant, namely the saussurea involucrate cell extract, and performing an anti-inflammatory effect experiment on the saussurea involucrate cell extract.
Cell culture: RAW 264.7 mouse macrophage cells were cultured in high-glucose DMEM medium, which was supplemented with 10% fetal bovine serum and 1% diabody, at 37 deg.C and 5% CO 2. And (5) carrying out passage or liquid change according to the growth state of the cells.
NO and IL-6 assay logarithmic growth phase cells were collected and adjusted to a concentration of 4 × 105Single cell suspension of individual cells/mL, seeded in 96-well plates at 100. mu.l per well, and cultured overnight. The culture medium was discarded and divided into 3 groups: blank group, LPS model group and saussurea involucrate cell extract experimental group. LPS model group and EWH extract experimental group were cultured for 1h with 180. mu.l of serum-containing DMEM medium containing 1. mu.g/mLLPS. Then adding 20 μ l of herba Saussureae Involueratae cell extract with different concentrations into experimental group of herba Saussureae Involueratae cell extract, and adding the blank group and LPS experimental group into experimental group without containingSerum in DMEM medium 20. mu.l. Each group was prepared into 5 parallel wells, and after 24 hours of stimulation, 50. mu.l of the culture supernatant in a 96-well plate was aspirated, and the content of NO and IL-6 in the supernatant was measured by the ELISA kit method.
The experiments are shown in fig. 3 and 4, and the results show that: the saussurea involucrate cell extract can obviously reduce the content of NO and IL-6 in macrophage induced by LPS, and has the function of inhibiting inflammation
3. Moisture retention efficacy experiment
Sample preparation: accurately weighing 10g of saussurea involucrate cell stem cells, adding 30 times of volume of a multi-element solvent for extraction, wherein water comprises butanediol and propylene glycol =8:1:1 (m: m: m), the extraction temperature is 100 ℃, the extraction time is 1h, then performing centrifugation at 6500rpm for 10min, retaining supernatant fluid, namely saussurea involucrate cell extract, and drying the supernatant fluid to obtain dry powder. The supernatant was subjected to a moisture retention test, and the dried powder was subjected to a moisture absorption test.
Moisture retention efficacy experiment
Accurately weighing 5.0g of water, herba Saussureae Involueratae cell extract, and 20% glycerol, respectively placing into a dryer containing allochroic silica gel and a dryer containing saturated sodium carbonate solution, and weighing at regular intervals.
Moisture retention rate (%) = M2/M 1100, wherein M2: weight after standing, M1: weight before placement.
The results are shown in table 7, and the experimental results show that the saussurea involucrate cell extract can reduce the volatilization amount of water and has certain moisturizing effect compared with water
TABLE 7 moisturizing experiment of saussurea involucrate cell extract
Moisture absorption efficacy experiment: accurately weighing 3 parts of saussurea involucrate cell extract dry powder 1.0g, placing the saussurea involucrate cell extract dry powder in an oven for drying, then placing the dried sample in a dryer filled with saturated sodium carbonate solution, and weighing at intervals.
Moisture absorption rate = (M)1-M0）/m 0100% of where M1For the post-hygroscopic sample mass, M0Is the sample mass before moisture absorption
The experimental results are shown in table 8, and the results show that the saussurea involucrate reaches saturation moisture absorption at 24 hours, the moisture absorption rate is 16.08%, and the saussurea involucrate has a certain moisture absorption effect.
TABLE 8 moisture absorption rate of saussurea involucrate cell extract at different times
Compared with the prior art, the method for preparing the snow lotus cell extract by the multi-element liquid system has the positive effects that:
(1) the invention adopts water solutions containing butanediol and propylene glycol with different concentrations as solvents to extract the saussurea involucrate cell extract, and the saussurea involucrate cell extract prepared in a multi-component liquid system is mainly used in cosmetic products with the functions of removing oxygen free radicals, preserving moisture and resisting inflammation.
(2) The invention avoids the adverse factors of high sugar content, unstable color of the extracting solution, irritation and the like caused by ethanol extraction, and adopts a multi-component liquid system of water, butanediol and propanediol to extract the effective components in the saussurea involucrate cells, thereby avoiding the adverse factors caused by complete water extraction and ethanol extraction, increasing the stability of the product and improving the quality of the product. Meanwhile, the aqueous solution multi-component system containing butanediol and propylene glycol contains a water polar solvent and a low-polarity solvent such as butanediol and propylene glycol, so that the polar functional components can be extracted, the extraction rate of the low-polarity functional components (syringin) can be enhanced, and the component concentration is changed.
Drawings
FIG. 1 DPPH oxygen radical scavenging assay;
FIG. 2 ABTS free radical scavenging assay;
FIG. 3 the effect of saussurea involucrate cell extract on NO content in macrophages;
FIG. 4 the effect of saussurea involucrate cell extract on IL-6 content in macrophages;
FIG. 5 is a schematic diagram of a process for preparing saussurea involucrate cell extract by using a multi-component liquid system.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The raw materials and reagents used in the present invention are commercially available.
Example 1
The method for preparing the snow lotus cell extract by the multi-component liquid system comprises the following steps:
accurately weighing 10g of fresh saussurea involucrate cell, respectively adding 50mL of water, ethanol, butanediol, propylene glycol, multi-solvent water, butanediol and propylene glycol =5:2:3 (m: m: m) different solvents for extraction at 100 ℃ (wherein the ethanol is boiling at 76 ℃) for 1h, then centrifuging at 6500rpm for 10min, and reserving supernatant fluid, namely the saussurea involucrate cell extract.
Example 2
Accurately weighing 10g of fresh saussurea involucrate cell cells, respectively adding 30 mL of a multi-element solvent for extraction, wherein the mass ratio of the fresh saussurea involucrate cell cells to the multi-element solvent is respectively water to butanediol to propanediol =1:1:1 (m: m: m), extracting at 100 ℃ (wherein ethanol boils for 76 ℃) for 1h, centrifuging at 6500rpm for 10min, and retaining supernatant, namely the saussurea involucrate cell extract
Example 3
Accurately weighing 10g of saussurea involucrate stem cells, respectively adding 60mL of multi-component solvent for extraction, wherein the mass ratio of the multi-component solvent is water to butanediol to propanediol =1:2:3 (m: m: m), leaching at normal temperature for 24h, then centrifuging at 6500rpm for 10min, and reserving supernatant, namely the saussurea involucrate cell extract.
Example 4
Accurately weighing 10g of saussurea involucrate fresh cells, respectively adding 50mL of multi-component solvent for extraction, wherein the mass ratio of the multi-component solvent is water to butanediol to propanediol =1:2:3 (m: m: m), performing ultrasonic extraction for 30min at the ultrasonic temperature of 60 ℃, then performing centrifugation for 10min at 6500rpm, and retaining the supernatant, namely the saussurea involucrate cell extract.
Example 5
Accurately weighing 30g of behenyl alcohol, 20g of polyglycerol-10 pentastearate, 20g of glyceryl monostearate, 50g of beeswax, 50g of cetearyl alcohol, 60g of shea butter, 40g of glycerol, 20g of butanediol, 5g of carbomer, 5g of methylparaben, 2g of allantoin, 30g of snow lotus cell extract and 670g of purified water, and preparing the snow lotus cell extract moisturizing cream. The prepared saussurea involucrate cell extract moisturizing cream is used by female volunteers, so that the effects of the saussurea involucrate cell extract moisturizing cream after use are evaluated, and the female volunteers show that the product has good moisturizing effect and relatively good moisturizing degree, and can lighten canthus wrinkles.
Claims (3)
1. A method for preparing saussurea involucrate cell extract by a multi-component liquid system is characterized by comprising the following steps:
(1) accurately weighing certain mass of saussurea involucrate cells, adding multi-component solvents with different concentrations for extraction, and then centrifuging or filtering to obtain clear transparent liquid, namely the saussurea involucrate cell extract;
(2) performing component analysis and detection on the extracts extracted by the multi-component solvents with different concentrations; the extraction method comprises high-temperature extraction, leaching and ultrasonic extraction; the multi-component solvents with different concentrations refer to: in a multi-component system, the concentration ratio of butanediol is 0-70%, the concentration ratio of propanediol is 0-70%, and the concentration ratio of water is 20-90%; the butanediol refers to 1, 3-butanediol, and the propylene glycol refers to 1, 3-propylene glycol.
2. The method as described in claim 1, wherein the saussurea involucrate cell used for extraction is dried saussurea involucrate cell or saussurea involucrate fresh cell containing water.
3. The saussurea involucrate cell extract prepared by the method of claim 1 is used in cosmetics with antioxidant, anti-inflammatory and moisturizing effects.
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