CN114344216B - Preparation method and application of lithospermum root extract with stable color - Google Patents
Preparation method and application of lithospermum root extract with stable color Download PDFInfo
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- Cosmetics (AREA)
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Abstract
The invention discloses a preparation method of a lithospermum root extract with stable color, which comprises the following steps: the feed liquid ratio is 1g: mixing radix Arnebiae with 50% ethanol solution by 30mL, treating under high pressure, and filtering to obtain radix Arnebiae crude extract; petroleum ether-ethyl acetate-methanol-water is taken according to the volume ratio of 5-7:3-5:4-6:4-6, uniformly mixing, standing and layering to prepare a stationary phase and a mobile phase; pumping the stationary phase, the mobile phase and the crude radix Arnebiae extract into a high-speed countercurrent chromatograph respectively to obtain high-purity radix Arnebiae extract; preparing high-purity radix Arnebiae extract into radix Arnebiae extract lyophilized powder; mixing radix Arnebiae extract lyophilized powder with octyl dodecanol myristate to obtain radix Arnebiae extract for cosmetic. The preparation method has the characteristics of simplifying steps and saving time, and the obtained extract has the characteristics of lasting and stable color, higher purity of active substances and better anti-aging effect.
Description
Technical Field
The invention relates to a radix arnebiae extract, in particular to a preparation method and application of a radix arnebiae extract with stable color.
Background
Radix Arnebiae (Lithospermum erythrorhizon Sieb. Et Zucc.) is a perennial herb of the genus Lithospermum of the family Lithospermaceae, and is used as a medicine with its dry root. The plant name of lithospermum is recorded in mountain sea Jingxi mountain Jing, the name is perylene, and the use is dyeing; radix Arnebiae is used as a traditional Chinese medicine and is a middle-grade product among three products; the description of Ben Cao gang mu is: lithospermum erythrorhizon is used for treating plague and poxvirus, promoting blood circulation, cooling blood and benefiting large intestine. The Chinese medicinal composition has the effects of cooling blood, promoting blood circulation, clearing heat and detoxicating, and relaxing bowel, and is mainly used for treating blood heat toxin exuberance, epidemic prevention, plague, purple black, water-fire damage, epidemic disease and ulcer. The research at present shows that the traditional Chinese medicine lithospermum has various biological activities, such as anti-inflammatory, antibacterial, wound healing promotion, antiviral, anticancer, antitumor and the like, and has wide clinical application.
The main chemical components of lithospermum are two main types, namely naphthoquinone pigment, which mainly comprises shikonin and derivatives thereof, and structurally takes 5, 8-dihydroxynaphthoquinone as a parent nucleus and has isohexide chains, wherein the difference is the position of hydroxyl in the side chains, hydroxy esterified acid and optical activity; the other is polysaccharide component, mainly radix Arnebiae polysaccharide. Wherein, the naphthoquinone component represented by shikonin and its derivatives is used as its main active component, has good activities of antioxidation, anti-inflammatory, antibacterial and anti-tumor, and promoting wound healing, and is used as good natural pigment in food and cosmetics.
Most of the conventional extraction methods of naphthoquinone pigments comprise a traditional reflux method, a microwave method, a percolation method, a cold leaching method and the like, and the common extraction methods have the defects of complicated operation steps, long extraction time, high temperature, low extraction rate and the like, and the reflux and microwave extraction can cause the destruction of the total naphthoquinone components of lithospermum at high temperature, and the percolation and cold leaching methods have the problems of low extraction rate, long time consumption, large solvent consumption and low solvent recovery rate of the naphthoquinone components.
Disclosure of Invention
The invention aims to provide a preparation method and application of a lithospermum root extract with stable color. The preparation method has the characteristics of simplifying steps and saving time, and the obtained extract has the characteristics of lasting and stable color, higher purity of active substances and better anti-aging effect.
The technical scheme of the invention is as follows: a method for preparing color-stable radix Arnebiae extract comprises the following steps:
s1, drying, crushing and sieving radix arnebiae, adding an ethanol solution with the volume fraction of 50%, wherein the feed liquid ratio of radix arnebiae to the ethanol solution is 1:30 (g/mL) to obtain product A;
s2, carrying out 300MPa high-pressure treatment on the product A for 10min, and filtering residues after pressure relief to obtain a crude radix arnebiae extract;
s3, petroleum ether-ethyl acetate-methanol-water is taken according to the volume ratio of 5-7:3-5:4-6:4-6, uniformly mixing, standing for layering, taking an upper phase and a lower phase, respectively carrying out ultrasonic degassing, and preparing a stationary phase and a mobile phase of the HSCCC two-phase solvent system;
s4, respectively pumping the stationary phase and the mobile phase into a high-speed countercurrent chromatograph, injecting the crude radix arnebiae extract into the high-speed countercurrent chromatograph after balancing, detecting the wavelength to be 254nm, and collecting fractions according to a chromatogram to obtain a high-purity radix arnebiae extract;
s5, recovering the solvent from the high-purity radix arnebiae extract under reduced pressure, and freeze-drying to obtain radix arnebiae extract freeze-dried powder;
s6, freeze-dried powder of the lithospermum extract is subjected to octyl dodecanol myristate or cocoyl-caprylate/caprate with a feed liquid ratio of 1:99 (m/m) mixing, stirring, and making into radix Arnebiae extract for cosmetic.
In the aforementioned preparation method of the color-stable radix Arnebiae extract, in the step S1, radix Arnebiae is dried in a constant temperature drying oven at 50deg.C, pulverized, and sieved with 80 mesh sieve.
In the aforementioned preparation method of the color-stable radix Arnebiae extract, in step S4, the fraction is collected according to a chromatogram, and detected by HPLC, and shikonin, β -hydroxyisovaleryl shikonin, acetyl shikonin, and β -dimethylacryl shikonin are collected.
In the preparation method of the lithospermum extract with stable color, the detection condition of HPLC detection is that the chromatographic column is Venusil-ASB-C18, the detection wavelength is 254nm, the mobile phase is acetonitrile/water=40% to 60% isocratic elution, the flow rate is 1.0mL/min, and the sample injection amount is 20 mu L.
In the preparation method of the color-stable radix Arnebiae extract, in the step S4, the pumping flow rate of the stationary phase is 20mL/min, and the pumping flow rate of the mobile phase is 2.0-4.0mL/min.
In the preparation method of the color-stable radix Arnebiae extract, in the step S4, the rotation speed of the high-speed countercurrent chromatograph is 850rpm.
The application of radix Arnebiae extract in preparing cosmetics is provided, wherein the radix Arnebiae extract is prepared by the above preparation method.
Compared with the prior art, the method adopts a mode of combining ultrahigh pressure extraction and liquid-liquid partition chromatography technology-high-speed countercurrent chromatography technology purification to extract and purify the radix arnebiae, and better extracts shikonin and derivatives thereof in the radix arnebiae; the method has the advantages of simplifying operation steps, reducing extraction difficulty, reducing solvent consumption, effectively avoiding thermal degradation or isomerization of shikonin, obviously shortening extraction time and effectively improving extraction rate and purity; and no solid carrier exists during the preparation, so that the sample loss caused by adsorption can be avoided, and the stability of naphthoquinone components can be protected.
The test shows that the prepared radix Arnebiae extract has the advantages of lasting and stable color, higher purity of active substances, more outstanding functionality and easier absorption by skin, effectively reduces the ROS content in skin after UV irradiation, thereby inhibiting the synthesis of MMPs, effectively controlling the MMP-1 expression level caused by external UVA stress, preventing skin collagen from being damaged and wrinkles from being generated, resisting non-enzymatic glycosylation and related oxidative stress, having the effects of protecting light, preventing photodamage, absorbing ultraviolet rays and delaying skin aging, and being used as an effective component in sun-screening and after-sun repair cosmetics.
Drawings
FIG. 1 is a graph of UV versus MMP-1 expression in different groups of mouse cells.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to be limiting.
Examples:
a method for preparing radix Arnebiae extract with stable color,
the method comprises the following steps:
s1, taking radix arnebiae, drying in a constant temperature drying oven at 50 ℃, crushing, sieving with a 80-mesh sieve, putting into a transparent polyethylene bag, adding an ethanol solution with the volume fraction of 50%, wherein the feed liquid ratio of radix arnebiae to the ethanol solution is 1:30 Sealing and mixing uniformly to obtain a product A;
experimental study shows that when 50% ethanol solution is adopted as the optimal extraction solvent and the ratio of feed to liquid is 1:30 (g/mL), the concentration around the lithospermum particles is reduced, the concentration gradient of the inner side and the outer side of the cell wall is increased, the diffusion rate of naphthoquinone compounds is increased, the dissolution of active ingredients is facilitated, the solubility of naphthoquinone substances is optimal, the extraction rate of naphthoquinone pigments is 50-75%, and the dissolution of pigments is inhibited due to the excessively high and excessively low volume fractions and the excessively low ethanol solution, so that the extraction rate is reduced.
S2, placing the product A into a pressurizing cavity of an ultrahigh pressure container, performing 300MPa high-pressure treatment, maintaining the pressure for 10min, and filtering residues through vacuum filtration after pressure relief to obtain a radix arnebiae crude extract;
the cell walls and the cell membranes of the cells are fully destroyed by adopting 300MPa high-pressure treatment and 10min dwell time, so that the active ingredients are quickly diffused into the solvent to reach the balance inside and outside the cells, the release of excessive impurities and the destruction of other active ingredients are avoided, the extraction of naphthoquinone substances is facilitated, and the energy consumption is reduced.
S3, petroleum ether-ethyl acetate-methanol-water is taken according to the volume ratio of 6:4:5:5, uniformly mixing, standing for layering, taking an upper phase and a lower phase, respectively carrying out ultrasonic degassing for 30min to remove bubbles, and preparing an upper stationary phase and a mobile phase of the HSCCC two-phase solvent system;
s4, pumping the stationary phase into a spiral tube of a high-speed countercurrent chromatograph at a pumping flow rate of 20mL/min, starting the chromatograph after the upper phase is filled with the spiral tube, adjusting the host to rotate clockwise, pumping the mobile phase into the high-speed countercurrent chromatograph at a pumping flow rate of 3.0mL/min after the rotating speed is stabilized at 850rpm, and injecting the crude radix arnebiae extract into the high-speed countercurrent chromatograph from a sample injection ring after the lower phase flows out from an outlet, namely the upper phase and the lower phase in the spiral tube are balanced, starting a detector and a recorder, detecting the wavelength of 254nm, collecting fractions according to the chromatogram, and detecting by HPLC to obtain the high-purity radix arnebiae extract;
the HPLC detection condition is that the chromatographic column is Venusil-ASB-C18, the detection wavelength is 254nm, the mobile phase is acetonitrile/water=40:60% isocratic elution, the flow rate is 1.0mL/min, and the sample injection amount is 20 mu L.
The high-purity radix arnebiae extract contains the following components by detection: shikonin, beta-hydroxyisovaleryl shikonin, acetyl shikonin, and beta-beta dimethylacryl shikonin are shown in table 1.
TABLE 1 high purity Lithospermum erythrorhizon extract ingredients
| Name of compound | Retention time(min) | Rate of area(％) |
| Shikonin | 6.5 | 4.6 |
| Beta-hydroxy isovaleryl shikonin | 8.0 | 18.6 |
| Acetyl shikonin | 11.5 | 32.8 |
| Beta-dimethyl-acryloylshikonin | 24.7 | 3.9 |
S5, recovering the solvent from the high-purity radix arnebiae extract under reduced pressure, and freeze-drying to obtain radix arnebiae extract freeze-dried powder;
s6, freeze-dried powder of the lithospermum root extract and octyl dodecanol myristate are mixed according to a feed liquid ratio of 1:99 (m/m) mixing, stirring, and making into radix Arnebiae extract for cosmetic.
Measurement of the performance of the radix Arnebiae extract:
the performance of the radix Arnebiae extract obtained in the example was measured.
1. Thermal stability test
6 parts of 100mL radix Arnebiae extract were taken, and placed in ovens set at different temperatures, and the color of the extract was observed, and the results are shown in Table 2.
TABLE 2 Heat stability of Lithospermum erythrorhizon extracts
| Temperature/. Degree.C | 30 | 40 | 50 | 60 |
| Color of | Magenta red | Magenta red | Magenta red | Magenta red |
| Color after 1 month | Magenta red | Magenta red | Magenta red | Magenta red |
| Color after 2 months | Magenta red | Magenta red | Magenta red | Magenta red |
| Color after 3 months | Magenta red | Magenta red | Magenta red | Magenta red |
The results show that after 3 months, the color of the radix arnebiae extract is still not changed, indicating that the radix arnebiae extract has good stability to heat.
2. Light stability test
Respectively collecting 50ml of radix Arnebiae extract, packaging in 4 color comparison tubes, and storing at room temperature under direct sunlight, fluorescent lamp irradiation, indoor astigmatism and darkness for 3 months, and observing color change.
TABLE 3 stability of radix Arnebiae extract to light
| Illumination conditions | Direct sunlight | Fluorescent lamp irradiation | Indoor astigmatism | Dark conditions |
| Color of | Magenta red | Magenta red | Magenta red | Magenta red |
| Color after 1 month | Magenta red | Magenta red | Magenta red | Magenta red |
| Color after 2 months | Magenta red | Magenta red | Magenta red | Magenta red |
| Color after 3 months | Magenta red | Magenta red | Magenta red | Magenta red |
The result shows that after 3 months, the color of the radix arnebiae extract is still not changed, which indicates that the radix arnebiae extract has good stability to light.
The radix Arnebiae extract prepared by the above preparation method can be used in preparation of anti-aging cosmetics, and the cosmetic containing radix Arnebiae extract is tested as follows:
3. animal experiment
A large amount of antioxidants such as peroxidase (CAT) and superoxide dismutase (SOD) exist in a human body, so that excessive free radicals in the human body can be removed, and the generation and removal of the free radicals are in dynamic balance. When the skin is irradiated by ultraviolet rays, free radicals are excessively generated. If the antioxidants are insufficient, too many free radicals cannot be scavenged, skin aging may result. MDA (malondialdehyde) in the skin is a metabolite of oxygen radicals after lipid peroxidation with unsaturated fatty acids in biological membranes, and MDA content decreases when radical scavengers are present. SOD can remove superoxide anion free radical, and CAT plays a role in removing peroxide free radical, and when skin is damaged by free radical or SOD and CAT are damaged, the content of SOD and CAT is reduced. These indicators can be used to measure the extent to which skin is damaged by oxidation.
Preparation of a mouse skin photodamage model:
in a constant temperature and humidity environment (temperature 25 ℃ and humidity 50%), 4 male mice were adaptively fed for two days and randomly divided into a blank group, a model group, an experimental group and a positive control group. The left and right backs of the mice were dehaired to an area of 2cm. Times.2 cm. The left side of the mouse No. 1 and the left side of the mouse No. 2 are blank groups, ultraviolet rays are not irradiated, any skin care product is not smeared, and other groups are irradiated with the ultraviolet rays; the right side was used as a model group to which distilled water alone (0.5 mL/day) was applied. Left side of mice No. 3 and No. 4 as positive control group was smeared with octyl dodecanol myristate (0.5 m L/day); the right side was used as the experimental group to smear radix Arnebiae extract (0.5 mL/day). The other groups were exposed to ultraviolet light for 2 hours daily, with 13 weeks of continuous exposure, except for the blank group. After the observation is finished, the mice are killed by neck breaking, back skin is sheared off, the skin is sheared after cleaning, and the mice are ground into powder by liquid nitrogen and used for measuring indexes such as MDA, SOD and the like. The specification of the kit is referred to measure the indexes such as MDA, SOD and the like in the skin.
Experimental results:
TABLE 4 skin conditions
| Group of | Skin condition |
| Blank group | The skin surface is smooth, has no wrinkles, clear lines and elastic pressing |
| Model group | Rough skin surface, reddening and blacking, unclear texture and poor pressing elasticity |
| Control group | The skin surface is smoother, reddish, disordered in texture and general in pressing elasticity |
| Experimental group | The skin surface is smooth, the lines are clear, and the pressing elasticity is good |
The result shows that the radix arnebiae extract has a certain anti-photoaging effect and can delay the aging change of the skin.
TABLE 5 MDA content and antioxidant enzyme Activity changes in skin
p <0.05 has statistical significance compared to the blank: #p <0.05, #p <0.01; compared with the model group: * p <0.05, p <0.01.
The result shows that the application of the radix arnebiae extract inhibits lipid peroxidation in skin, improves the activity of SOD and CAT, and delays the aging effect of skin.
4. Fibroblast (HSF) assay
MMP-1 expression plays an important role in photoaging to produce wrinkles, and MMP-1 expression is increased by signaling pathways when UVA increases the activity of JNK/p38 and transcription factor AP-1, resulting in skin collagen deficiency.
Experiment design:
4 bottles of normal-feeding HSF cells were selected as blank, model, control, and experimental groups, respectively.
Blank group: HSF cells in the logarithmic growth phase were digested into single cell suspensions, and the cell concentration was adjusted to (1X 104) cells/ml. Inoculated in another cell culture flask, 5 ml/flask, 10 flasks total. After 24h of culture, the fresh culture solution is replaced, and after 12h of normal feeding, cell culture supernatant and cells are collected and used for detecting related indexes.
Model group: after 36J/cm2UVA irradiation, 5ml of a cell culture medium containing 2% octyldodecanol myristate was added at 37℃under CO 2 After culturing in an incubator for 12 hours, cell culture supernatant and cells were collected for relevant index detection.
Experimental group: adding culture containing 2% radix Arnebiae extract after 36J/cm2UVA irradiationNutrient solution 5ml, CO at 37deg.C 2 After culturing in an incubator for 12 hours, cell culture supernatant and cells were collected for relevant index detection.
The results are shown in figure 1, and the results show that the lithospermum root extract can effectively inhibit MMP-1 expression caused by UVA stress, thereby inhibiting skin wrinkles caused by photoaging.
Claims (6)
1. A preparation method of a color-stable radix arnebiae extract is characterized by comprising the following steps: the method comprises the following steps:
s1, drying, crushing and sieving radix arnebiae, adding an ethanol solution with the volume fraction of 50%, wherein the feed liquid ratio of radix arnebiae to the ethanol solution is 1g:30mL to obtain a product A;
s2, carrying out 300MPa high-pressure treatment on the product A for 10min, and filtering residues after pressure relief to obtain a crude radix arnebiae extract;
s3, petroleum ether-ethyl acetate-methanol-water is taken according to the volume ratio of 5-7:3-5:4-6:4-6, uniformly mixing, standing for layering, taking an upper phase and a lower phase, respectively carrying out ultrasonic degassing, and preparing a stationary phase and a mobile phase of the HSCCC two-phase solvent system;
s4, respectively pumping the stationary phase and the mobile phase into a high-speed countercurrent chromatograph, injecting the crude radix arnebiae extract into the high-speed countercurrent chromatograph after balancing, detecting the wavelength to be 254nm, and collecting fractions according to a chromatogram to obtain a high-purity radix arnebiae extract; detecting by HPLC, and collecting shikonin, beta-hydroxy isovaleryl shikonin, acetyl shikonin and beta-beta dimethyl acryloyl shikonin;
s5, recovering the solvent from the high-purity radix arnebiae extract under reduced pressure, and freeze-drying to obtain radix arnebiae extract freeze-dried powder;
s6, freeze-dried powder of the lithospermum root extract and octyl dodecanol myristate are mixed according to a feed liquid ratio of 1:99, mixing, stirring, and making into radix Arnebiae extract for cosmetic.
2. The method for preparing the color-stable arnebia root extract according to claim 1, wherein: in the step S1, the lithospermum is placed in a constant temperature drying oven at 50 ℃ for drying, crushed and sieved by a 80-mesh sieve.
3. The method for preparing the color-stable arnebia root extract according to claim 1, wherein: the HPLC detection condition is that the chromatographic column is Venusil-ASB-C18, the detection wavelength is 254nm, the mobile phase is acetonitrile/water=40% to 60% isocratic elution, the flow rate is 1.0mL/min, and the sample injection amount is 20 mu L.
4. The method for preparing the color-stable arnebia root extract according to claim 1, wherein: in the step S4, the pumping flow rate of the stationary phase is 20mL/min, and the pumping flow rate of the mobile phase is 2.0-4.0mL/min.
5. The method for preparing the color-stable arnebia root extract according to claim 1, wherein: in the step S4, the rotation speed of the high-speed countercurrent chromatograph is 850rpm.
6. An application of radix arnebiae extract in preparing cosmetics is characterized in that: the radix Arnebiae extract obtained by the preparation method of any one of claims 1-5.
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