CN113425635A - Elsholtzia extract, its preparation and use - Google Patents
Elsholtzia extract, its preparation and use Download PDFInfo
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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
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- A—HUMAN NECESSITIES
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- A61K8/00—Cosmetics or similar toiletry preparations
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- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
- A61K8/4973—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
- A61K8/498—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom having 6-membered rings or their condensed derivatives, e.g. coumarin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
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- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/74—Biological properties of particular ingredients
- A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
- A61K2800/782—Enzyme inhibitors; Enzyme antagonists
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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
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Abstract
The invention provides an elsholtzia extract, a preparation method and application thereof, belonging to the technical field of plant extracts and comprising the following components: 0.62-3.78mg/g of apigenin, 2.19-4.44mg/g of baicalein-7-methyl ether, 30-120 mu g/g of quercetin, 0.79-3.29mg/g of kaempferol, 34-120 mu g/g of luteolin and 4.22-6.67 mu g/g of chrysoeriol, and is prepared by a percolation auxiliary enzymolysis process. The elsholtzia extract can achieve the whitening effect by inhibiting the activity of tyrosinase.
Description
Technical Field
The invention belongs to the technical field of plant extracts, and particularly relates to a whitening plant extract and a preparation method and application thereof.
Background
With the improvement of living standard and aesthetic standard of people, smooth and white skin is more and more advocated by women, and the proportion of whitening products in the daily chemical product market is also more and more increased. Because natural functional substances have safe and healthy performance, daily chemical products containing natural plant whitening functional components gradually become the product guide of consumers and industries which are worried by people, and the commonly commercialized natural whitening extracts comprise glycyrrhiza glabra root extract, peony root extract, tea extract and the like.
The tyrosinase inhibitor from natural sources is safe and effective, can effectively slow down and prevent melanin generation, and is more and more widely concerned by people, but the types of the natural tyrosinase inhibitors which can be used for commercial use at present are few, and the relatively accepted whitening agents arbutin and VC still have certain limitations, so that the development of a pure natural whitening agent which is strong in stability, green, safe and efficient and does not have adverse reaction on a human body is urgently needed in the daily chemical industry.
Elsholtzia splendens is an annual herb and belongs to the Labiatae family. The Chinese pharmacopoeia specifies that the medicinal plants include 2 varieties of Mosla chinensis Maxim and Mosla chinensis Jiiangxiangru, the medicinal parts are dry aerial parts of the plants, the former is called as 'Qingxiang Mosla', and the latter is called as 'Jianxiang Bing'. This product is pungent in flavor and slightly warm; enters lung and stomach meridians, has the efficacies of inducing sweat, relieving exterior syndrome, eliminating dampness and regulating the middle warmer, and is a plant medicine used as both medicine and food. Although the research on elsholtzia has entered the field of finished product medicine, the chemical components and biological activity of plants need to be further researched because the chemical components are various and have significant biological activity function.
The chrysoeriol is a natural flavonoid compound, and is present in many plants, such as the chrysoeriol can be separated from fresh leaves and roots of rehmannia glutinosa, artemisia sphaerocephala, scutellaria viscida, honeysuckle, alfalfa leaves, vines, wintercherry, asteraceae echinacea, palm leaves, roots of carrots, peony petals, areca nuts, chrysanthemum nankingense, epimedium, rabdosia rubescens and the like. Chrysoeriol has antiallergic and antiinflammatory activities, and has been reported in the cosmetic field to be effective in protecting against damage caused by ultraviolet rays, and has strong ultraviolet absorption, antiinflammatory, antiallergic, etc., but has not been reported to have whitening activity.
Disclosure of Invention
The invention aims to provide an elsholtzia extract, an extraction and separation method and application thereof.
The technical purpose of the invention is realized by the following technical scheme:
an elsholtzia extract comprises the following components: 0.62-3.78mg/g of apigenin, 2.19-4.44mg/g of baicalein-7-methyl ether, 30-120 mu g/g of quercetin, 0.79-3.29mg/g of kaempferol, 34-120 mu g/g of luteolin and 4.22-6.67 mu g/g of chrysoeriol.
The second purpose of the invention is to provide the method for extracting and separating the elsholtzia extract, which comprises the following steps: (1) placing the elsholtzia powder in an ethanol solution, extracting by a percolation method to obtain an ethanol extracting solution, and recovering an ethanol solvent to obtain a first extract; (2) adding a small amount of water into the first extract to form a suspension, and adjusting the pH value of the suspension to be 4.5-6.0; (3) adding a proper amount of cellulase into the suspension, and carrying out water bath reaction at 40-50 ℃ for 60 min; (4) placing the transfer reaction solution in Soxhlet extractor, adding 95% ethanol, extracting for 90min, cooling, filtering, separating, and concentrating the filtrate to obtain the desired extract. The percolation assisted enzymolysis process has high flavonoid component transfer rate and high extract purity.
In conclusion, the invention has the following beneficial effects:
1. the elsholtzia extract is prepared by a percolation-assisted enzymolysis process, the extraction rate of the method is higher than that of other solvent extraction methods, and the extracted extract has higher tyrosinase inhibitory activity, so that the extract has better whitening activity.
2. The elsholtzia extract disclosed by the invention comprises the following components: 0.62-3.78mg/g of apigenin, 2.19-4.44mg/g of baicalein-7-methyl ether, 30-120 mu g/g of quercetin, 0.79-3.29mg/g of kaempferol, 34-120 mu g/g of luteolin and 4.22-6.67 mu g/g of chrysoeriol. The tyrosinase inhibitory activity and the melanocyte inhibitory activity of the extract are higher than those of any component in the extract, and the compound in the extract has a synergistic effect.
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FIG. 1 is UPLC-DAD-QTOF MS chromatogram of herba Moslae extract (graph A: 330nm graph B: 254nm graph)
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the terms used herein in the specification of the present invention are for the purpose of describing particular embodiments only and are not intended to limit the present invention.
Example 1 preparation of Elsholtzia extract
(1) Placing the elsholtzia powder in a 60% ethanol solution, extracting by a percolation method to obtain an ethanol extracting solution, and recovering an ethanol solvent to obtain a first extract; (2) adding a small amount of water into the first extract to form a suspension, and adjusting the pH value of the suspension to be 4.5-6.0; (3) adding a proper amount of cellulase into the suspension, and carrying out water bath reaction at 45 ℃ for 60 min; (4) the transfer reaction solution was placed in a Soxhlet extractor, extracted with 95% ethanol for 90min, cooled, filtered and separated, and the filtrate was concentrated to obtain the objective extract (hereinafter referred to as sample 1).
Example 2 comparison of Elsholtzia extract in different preparation methods
Extracting herba Moslae powder with 60% ethanol solution by percolation to obtain ethanol extractive solution, recovering ethanol solvent to obtain ethanol extractive sample (hereinafter referred to as sample 2)
Extracting herba Moslae powder with 60% ethanol solution by percolation method to obtain ethanol extractive solution, recovering ethanol solvent to obtain first extract, extracting the first extract with ethyl acetate, mixing extractive solutions, and recovering to obtain ethyl acetate sample (hereinafter referred to as sample 3)
Extracting herba Moslae powder with 60% ethanol solution by percolation to obtain ethanol extractive solution, recovering ethanol solvent to obtain first extract, extracting the first extract with n-butanol, mixing extractive solutions, and recovering n-butanol sample (hereinafter referred to as sample 4)
Extracting herba Moslae powder with methanol solution by percolation to obtain methanol extractive solution, recovering methanol solvent to obtain methanol extractive sample (hereinafter referred to as sample 5)
Respectively calculating the extraction rate and the purity of the flavonoid compounds in the sample according to the following formulas
Extraction yield (%). The amount of the flavonoid compound in the crude extract/the amount of the flavonoid compound in the raw material X100
Flavone purity (%) ═ amount of flavonoid compound in crude extract/crude extract amount × 100
TABLE 1 comparative study of extraction efficiency for different preparation methods
Total Flavonoids transfer Rate (%) | Extraction purity (%) | |
Sample 1 | 91 | 63 |
Sample 2 | 91 | 32 |
Sample 3 | 86 | 50 |
Sample No. 4 | 73 | 53 |
Sample No. 5 | 85 | 46 |
As can be seen from the results of comparing samples 1-5, sample 1 was prepared by a percolation assisted enzymatic hydrolysis process, which resulted in high transfer rate of flavonoid components and high purity of extract.
Example 3 comparison of tyrosinase Activity by different preparation methods
It is known to those skilled in the art that the compounds contained in the fractions extracted by different extraction methods have different activities. The activity of the samples prepared in examples 1 and 2 was compared by tyrosine activity, provided that the chemical composition was not completely defined.
Determination of Tyrosinase (TYR) activity: 3mL of 50mmol/L PBS (pH 6.8) was measured, 2.9mL of 1 mmol/L levodopa solution was added, the mixture was placed in a water bath at 32 ℃ and incubated for 20min, 0.1mL of TYR solution (0.01g/L) was added and mixed, and the absorbance increase curve of the mixture with time was measured at a constant temperature. The absorbance value of the reaction solution at 475nm per minute was defined as an increase of 0.001 of TYR activity unit (U/min), and kojic acid was used as a positive control.
Calculation of tyrosinase inhibition:
inhibition ratio (%) - (1- (T-T)0)/(C-C0))*100%
In the formula: t-sample tube light absorption value, namely the light absorption value of the solution after the sample reacts with tyrosinase; t is0-sample background absorbance; average value of light absorption value of C-enzyme reaction tube for 3 times, i.e. no sample is addedThe light absorption value of tyrosinase and dopa reaction in the preparation process; c0-solvent background absorbance.
TABLE 2 comparison of tyrosinase Activity for different preparation methods
As can be seen from the results of comparing samples 1-5, sample 1 was prepared by a percolation assisted enzymatic hydrolysis process, and the tyrosinase inhibitory activity of the sample was higher than that of samples 2-5 and higher than that of the positive control kojic acid, regardless of whether the sample was 1mg/ml or 0.2 mg/ml.
EXAMPLE 4 assay
10 parts of elsholtzia haichowensis from different sources are collected, and samples are prepared according to the method of example 1.
Carrying out UPLC-DAD-QTOF MS chromatographic detection on the sample, wherein the chromatographic conditions are as follows: the chromatographic instrument is as follows: agilent Zorbax SB-C18600 bar; the chromatographic column is C18(ii) a The column temperature was 35 ℃; the flow rate is 0.3 mL/min; the sample injection amount is 5 mu L; the mobile phase was eluted using a gradient of acetonitrile-0.1% aqueous formic acid. The mass spectrum conditions are as follows: mass spectrometer ESI ion source, negative ion mode, atomizer pressure 50psi, dry gas flow rate 10ml/min, dry gas temperature 350 ℃, capillary voltage 4000V, fragment voltage 150V (optimized), MS/MS collision energy 25V (optimized).
The detection result is shown in figure 1, and the detection data under 330nm and 254nm show that the extract contains apigenin 0.62-3.78mg/g, baicalein-7-methyl ether 2.19-4.44mg/g, quercetin 30-120 μ g/g, kaempferol 0.79-3.29mg/g, luteolin 34-120 μ g/g, and chrysoeriol 4.22-6.67 μ g/g.
EXAMPLE 5 comparison of tyrosinase Activity of Components at different ratios
To clarify the activity of the extract, we examined the tyrosinase inhibitory activity of the sample according to the method of example 3.
TABLE 3 comparison of tyrosinase inhibitory Activity
The test results in table 3 show that: the tyrosinase inhibitory activity of the elsholtzia extract (sample 1) prepared by the invention is stronger than that of any single compound in the extract, so that the compounds in the extract have synergistic effect. The chrysoeriol activity is also stronger than that of the positive drug kojic acid, and can be seen as the main active ingredient in the extract.
The extract prepared by the invention has tyrosinase inhibitory activity, tyrosinase has a unique catalytic function, is a key enzyme for controlling melanin generation of organisms, is closely related to aging and beauty treatment, and the tyrosinase inhibitor can be used for common pigmentation skin diseases, and can inhibit melanin synthesis by inhibiting the activity of tyrosinase, so that the whitening effect is realized, and therefore, the elsholtzia extract has the whitening effect.
EXAMPLE 6 sample melanocyte Activity determination Studies
Mouse B16 melanocyte culture: under aseptic conditions, mix 1x105one/mL of B16 cells were inoculated in a flask of RPMI1640 medium containing 10% (volume fraction) calf serum in 5% (volume fraction) CO2The culture was carried out in an incubator and passaged every 3 days by digestion with 0.25% pancreatin. After the cells had grown to log phase, they were washed with PBS, digested with 0.25% pancreatin and counted on a cell counting plate. And observing the morphological change of the cells by an electron microscope. Modulation of cell 2x104one/mL. Inoculating into two 96-well plates (100 μ L per well), and placing at 37 deg.C with 5% CO2After incubation in an incubator for 24h, the supernatant is discarded, and the drugs to be tested are added, wherein the final mass concentration of each group of drugs is 1000mg/L, 500mg/L and 100mg/L respectively. 4 wells were set for each concentration, the control group was not dosed with drug, the same amount of maintenance solution was used instead, and the blank wells were not inoculated with cells. 5% CO at 37 ℃2Incubate in incubator for 72 h.
Determination of tyrosinase activity: inhibiting tyrosinase activity in mouse melanocytesForce test: after 3 days of drug action, the supernatant was discarded and washed twice with PBS pH 6.8, and 90. mu.L of PBS containing 1% (volume fraction) Triton X-100 was added to each well. Ultrasonication in ice bath, adding 10. mu.L of 1X10 to each well-2And (3) incubating at 37 ℃ for 60min by virtue of L-dopa of moL/L, carrying out color comparison at 490nm, adjusting the blank wells to zero, and measuring the absorbance value of each well.
And (3) melanin content determination: b16 melanocytes at 1x105one/mL density was seeded in 3 6-well plates. 3mL per well, 5% CO at 37 ℃2After incubation for 24h in an incubator, the supernatant is discarded, and the drugs to be tested are added, wherein the concentration of each drug is 3 holes, and the final drug mass concentration is 1000mg/L, 500mg/L and 100mg/L respectively. After 3 days of drug action, the supernatant was discarded, washed with PBS, and 0.5mL of trypsinized cells were added to each well for 3min, and 2mL of maintenance solution was added to each well to stop digestion. After the mixture was blown and mixed, 0.5mL of each concentration was taken out and counted. The rest cell suspension is centrifuged at 1500r/min for 10min, the supernatant is discarded, 200 mul of distilled water and the volume ratio of 1: 1 (removing some opaque substances), shaking, standing at room temperature for 15min, and centrifuging at 3000r/min for 5 min. The supernatant was discarded, and 1mL of a 1mol/L aqueous NaOH solution containing 10% (by mass) DMSO was added to the precipitate. Heating at 80 deg.C for 30min (with stopper), and measuring absorbance at 490nm with ELISA.
Through tyrosinase activity measurement and melanin content measurement, the sample 1 is stronger than any single compound (apigenin, baicalein-7-methyl ether, quercetin, kaempferol, luteolin and chrysoeriol) in the extract, and the compound in the extract has a synergistic effect. The eriodictyol activity in the extract is stronger than that of the positive drug kojic acid, and the eriodictyol is the main active ingredient in the extract.
Claims (6)
1. The herba elsholtziae extract is characterized by being prepared by a percolation auxiliary enzymolysis process.
2. The elsholtzia extract according to claim 1, wherein said elsholtzia extract is prepared by a process comprising the steps of:
(1) placing the elsholtzia powder in an ethanol solution, extracting by a percolation method to obtain an ethanol extracting solution, and recovering an ethanol solvent to obtain a first extract;
(2) adding a small amount of water into the first extract to form a suspension, and adjusting the pH value of the suspension to be 4.5-6.0;
(3) adding a proper amount of cellulase into the suspension, and carrying out water bath reaction at 40-50 ℃ for 60 min;
(4) placing the transfer reaction solution in Soxhlet extractor, adding 95% ethanol, extracting for 90min, cooling, filtering, separating, and concentrating the filtrate to obtain the desired extract.
3. The elsholtzia extract according to claim 2, wherein the ethanol solution in the preparation step (1) has a concentration of 60-70%.
4. The elsholtzia extract according to claim 2, wherein the amount of cellulase added in the preparation step (3) is 0.25wt% of the mass of the raw material elsholtzia.
5. Elsholtzia ciliate extract according to any one of claims 2 to 4, wherein the extract is subjected to UPLC-DAD-QTOF MS chromatographic detection and comprises the following components: 0.62-3.78mg/g of apigenin, 2.19-4.44mg/g of baicalein-7-methyl ether, 30-120 mu g/g of quercetin, 0.79-3.29mg/g of kaempferol, 34-120 mu g/g of luteolin and 4.22-6.67 mu g/g of chrysoeriol.
6. The use of an extract of Elsholtzia ciliata as claimed in claim 1 in the preparation of whitening products.
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN102977164A (en) * | 2012-11-05 | 2013-03-20 | 河南牧翔动物药业有限公司 | Enzyme-assistant extraction technology of hyperin in leaves of Acanthopanax senticosus Harms |
CN110824084A (en) * | 2019-11-12 | 2020-02-21 | 江西中医药大学 | Establishment of HPLC (high Performance liquid chromatography) fingerprint spectrums of elsholtzia haichowensis in different producing areas and component determination method thereof |
CN111388381A (en) * | 2020-05-21 | 2020-07-10 | 成都蓓乐康生物科技有限公司 | Cosmetic elsholtzia extract and extraction method thereof |
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN102977164A (en) * | 2012-11-05 | 2013-03-20 | 河南牧翔动物药业有限公司 | Enzyme-assistant extraction technology of hyperin in leaves of Acanthopanax senticosus Harms |
CN110824084A (en) * | 2019-11-12 | 2020-02-21 | 江西中医药大学 | Establishment of HPLC (high Performance liquid chromatography) fingerprint spectrums of elsholtzia haichowensis in different producing areas and component determination method thereof |
CN111388381A (en) * | 2020-05-21 | 2020-07-10 | 成都蓓乐康生物科技有限公司 | Cosmetic elsholtzia extract and extraction method thereof |
Non-Patent Citations (2)
Title |
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RONGCHAO ZHANG等: "Whitening activity of constituents isolated from the Trichosanthes Pulp", EVIDENCE-BASED COMPLEMENTARY AND ALTERNATIVE MEDICINE, pages 6 * |
汤洪波等: "香薷中挥发性成分的酶提取及GC-MS分析", 应用化工, vol. 39, no. 10, pages 1 * |
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