CN110314122B - Alisma extract and preparation method and application thereof - Google Patents
Alisma extract and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an alisma extract and a preparation method and application thereof. Specifically, the invention discloses an alisma extract, which contains active ingredients: alisol B and 23-acetyl alisol B, and impurities; wherein the impurities comprise alisol A and 24-acetyl alisol A; wherein the total content of alisol B and 23-acetyl alisol B is more than or equal to 5%. The invention also discloses a preparation method of the extract. The extract of the invention has good whitening effect and no toxicity to skin.
Description
Technical Field
The invention relates to the field of cosmetics and medical treatment, in particular to an alisma orientale extract and a preparation method and application thereof.
Background
Rhizoma alismatis (academic name: alisma plantago-aquatica Linn.), perennial aquatic or marsh herb. The whole plant is toxic, and the underground tuber is more toxic.
The literature search shows that the traditional method adopts a boiling method, and the active ingredients cannot be fully extracted. Although some researchers respectively perform ethanol reflux method, methanol ultrasonic method, microwave extraction method, supercritical carbon dioxide extraction and the like, the extraction rate of total triterpenes (alisol A, alisol B, 23-acetyl alisol B, 24-acetyl alisol A and the like) in alisma orientale by the ethanol reflux method, the methanol ultrasonic method and the microwave extraction method is not more than 2%; although the extraction rate of the supercritical carbon dioxide extraction method is slightly higher (not more than 4 percent), the method is expensive and the extraction rate is still relatively low.
The invention still needs to develop a high-efficiency extraction method of the active ingredients of the alisma orientale.
Disclosure of Invention
The invention aims to provide an alisma extract with excellent performance.
The invention also aims to provide an extraction method of the extract.
The invention aims to provide application of the extract.
The invention provides a rhizoma alismatis extract, which contains active ingredients: alisol B and 23-acetyl alisol B, and impurities; wherein the impurities comprise alisol A and 24-acetyl alisol A; and the mass percentage of the active ingredients is more than or equal to 5 percent.
In another preferred embodiment, the mass percentage of the active ingredients is 5-50%; preferably, 10% to 50%; more preferably, it is 9% to 20%.
In another preferred embodiment, the mass percentage of the alisol A and the 24-acetyl alisol A is less than or equal to 5%; preferably, less than or equal to 2%; more preferably less than or equal to 1%.
In another preferred embodiment, the mass ratio of the active ingredient to alisol a and 24-acetyl alisol a in the impurities is 20:1 to 400:1; preferably, 50:1 to 300:1; preferably, 150:1 to 250:1; more preferably, it is 180:1 to 220:1.
in another preferred example, the impurities further comprise one or more of plant pigment, terpenes, phytosterol glycoside, fatty acid amino acid, and inorganic salt ion.
In another preferred embodiment, the content ratio of alisol B and 23-acetyl alisol B is 1.
In another preferred embodiment, the content ratio of alisol B to 23-acetyl alisol B is 1; more preferably, 1.
In a second aspect, the present invention provides a composition comprising component (a): alisol B and 23-acetyl alisol B, and impurities (B): alisol A and 24-acetyl alisol A; wherein the mass ratio of the component (a) to the impurity (b) is 20:1 to 400:1.
in another preferred example, the mass ratio of the component (a) to the impurity (b) is 50:1 to 300:1; preferably, 150:1 to 250:1; more preferably, it is 180:1 to 220:1.
in another preferred embodiment, in the component (a), the content ratio of alisol B to 23-acetyl alisol B is 1; preferably, 1; more preferably, 1.
In a third aspect, the present invention provides a pharmaceutical or cosmetic composition comprising an alisma orientale extract according to the first aspect of the present invention or a composition according to the second aspect of the present invention.
In another preferred embodiment, the composition is one or more of an emulsion, a liquid, an ointment, a cream, a paste, a cake, and a powder.
In another preferred example, the cosmetic composition is one or more of BB cream, sunscreen cream, primer, makeup cream, foundation cream, concealer.
In another preferred embodiment, the composition further comprises other whitening or freckle-removing components.
In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient; and/or the cosmetic composition further comprises a cosmetically acceptable carrier or excipient.
In another preferred embodiment, the cosmetically acceptable carrier or excipient is selected from the group consisting of: a humectant, an antioxidant, an anti-ultraviolet agent, a preservative, a film forming agent, an oil-soluble gelling agent, an organically modified clay mineral, a resin, an antibacterial agent, an essence, a salt, an antioxidant, a pH adjusting agent, a chelating agent, a cooling agent, an anti-inflammatory agent, a skin beautifying ingredient, a vitamin, an amino acid, a nucleic acid, a hormone, an inclusion compound, or a combination thereof.
In a fourth aspect, the present invention provides a use of the alisma orientale extract of the first aspect, or the composition of the second aspect, or the composition of the third aspect, for:
(1) Preparing a medicament or cosmetic for whitening, spot-removing or skin care;
(2) Inhibiting melanin;
(3) Inhibiting tyrosine.
In a fifth aspect, the present invention provides a method for preparing the alisma orientale extract according to the first aspect of the present invention, the method comprising the steps of:
(1) Extraction:
extracting Alismatis rhizoma powder with organic solvent; collecting extractive solution, and concentrating to obtain first extract containing Alismatis rhizoma extract;
(2) And (3) extraction:
suspending the first extract containing the alisma extract obtained in the step (1) with water, and then extracting with an organic solvent; collecting organic phase, and concentrating to obtain second extract containing Alismatis rhizoma extract;
(3) And (3) purification:
purifying the second extract containing the Alisma extract by gel column chromatography to obtain the Alisma extract.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the comparison of the crude Alismatis rhizoma extract obtained in step (1) of example 1, the refined Alismatis rhizoma extract obtained in step (3) of example 1 and a standard. In the figure, the upper diagram is mixed standard (alisol B and 23-acetyl alisol B from left to right), the middle diagram is crude extract, and the lower diagram is refined extract.
FIG. 2 shows that the refined extract of Alisma orientale has no significant toxicity to the survival rate of melan-a melanocytes.
FIG. 3 shows that the common crude extract of Alisma orientale has obvious toxicity on the cell survival rate of melan-a melanocytes.
FIG. 4 shows the melanin synthesis inhibitory effect of the refined extract of Alisma orientale on melan-a melanocytes.
FIG. 5 shows that alisol A or 24-acetyl alisol A removed by the preparation process has no inhibitory effect on melanin synthesis of melanocyte.
Figure 6 shows that the refined extract of alisma has no significant toxicity to the artificial skin model.
Figure 7 shows the whitening effect of the refined extract of alisma on an artificial skin model.
FIG. 8 shows that the purified extract of Alisma orientale can significantly inhibit the intracellular tyrosinase activity of Melan-a.
Detailed Description
The inventor of the invention unexpectedly discovers a safe and nontoxic alisma orientale extract with good whitening and freckle removing effects for the first time through extensive and intensive research. The present invention has been completed based on this finding.
Alismatis rhizoma extract
The alisma extract of the present invention can be prepared by a method comprising the steps of:
(1) Extraction: extracting Alismatis rhizoma powder with organic solvent; collecting the extract, and concentrating to obtain a first extract containing Alismatis rhizoma extract;
in another preferred example, in step (1), the rhizoma alismatis powder is fine powder collected after the rhizoma alismatis sample is sieved (for example, a 60-mesh sieve).
In another preferred example, in the step (1), the mass volume ratio of the alisma orientale powder to the organic solvent is 1:1 to 1:10; preferably, 1:2 to 1:8.
in another preferred example, in the step (1), the extraction is performed for 1 to 5 times; preferably, it is 1 to 3 times.
In another preferred example, in the step (1), the extraction is performed for 0.1 to 10 hours each time; preferably, each extraction is carried out for 0.5 to 5 hours or 0.5 to 1.5 hours.
In another preferred example, in step (1), the organic solvent is acetone.
(2) And (3) extraction: suspending the first extract containing the alisma extract obtained in the step (1) with water (such as ultrasonic suspension), and then extracting with an organic solvent; collecting organic phase, and concentrating to obtain second extract containing Alismatis rhizoma extract;
in another preferred example, in the step (2), the addition amount of the water is 1/10-1/1 of the rhizoma alismatis powder; preferably 1/5 to 1/2.
In another preferred example, in the step (2), the addition amount of the organic solvent is 1/10 to 1/1 of the alisma orientale powder; preferably 1/5 to 1/2.
In another preferred example, in the step (2), the extraction is performed for 1 to 5 times; preferably, it is 1 to 3 times.
In another preferred example, in the step (2), the organic solvent is a mixed solvent of petroleum ether and dichloromethane.
In another preferred example, in the step (2), the volume ratio of the petroleum ether to the dichloromethane in the organic solvent is 2:1.
(3) And (3) purification: purifying the second extract containing the Alisma extract by gel column chromatography to obtain the Alisma extract.
In another preferred example, in the step (3), the chromatographic purification is an elution purification with an organic solvent.
In another preferred example, in the step (3), the organic solvent is a mixed solvent of petroleum ether, dichloromethane and methanol.
In another preferred example, in the step (3), the volume ratio of petroleum ether, dichloromethane and methanol in the organic solvent is 1:2:1.
in another preferred embodiment, in step (3), the flow rate of the eluent during purification is 2-3ml/min.
In another preferred example, in step (3), after elution and purification, the eluate is collected and then concentrated.
In another preferred example, in step (3), the concentration is carried out at 30 to 50 ℃.
In another preferred embodiment, in the alisma extract obtained in step (3), the total extraction rate of alisol B and 23-acetyl alisol B is 30% -70%.
Applications of
The alisma extract has the performance of inhibiting melanin synthesis and tyrosine activity, and can be used for preparing a pharmaceutical composition or a cosmetic composition.
It is possible to prepare the Alisma extract of the present invention into a pharmaceutical composition such as a tablet, a capsule, a powder, a fine granule, a solution, a lozenge, a jelly, a cream preparation, a spirit, a suspension, a tincture, a cataplasm, a liniment, a lotion, and an aerosol. The drug can be prepared by a generally known preparation technique, and a suitable pharmaceutical additive can be added to the drug.
Examples of the pharmaceutical additives include excipients, binders, disintegrating agents, lubricants, flow aids, suspending agents, emulsifiers, stabilizers, warming (wetting) agents, preservatives, solvents, solubilizers, preservatives, flavoring agents, sweeteners, dyes, flavors, propellants and the like, and these pharmaceutical additives may be selected and added in an appropriate amount within a range not affecting the effect of the present invention.
It is possible to prepare the alisma extract of the present invention into cosmetic compositions such as emulsion, liquid, ointment, cream, paste, cake, powder and the like.
Other ingredients used in general cosmetics, such as film-forming agents, oil-soluble gelling agents, organically modified clay minerals, resins, moisturizers, preservatives, antibacterial agents, perfumes, salts, antioxidants, pH adjusters, chelating agents, cooling agents, anti-inflammatory agents, skin beautifying ingredients (whitening agents, cell activators, skin roughness improvers, blood circulation improvers, skin astringents, anti-lipid leakage agents, and the like), vitamins, amino acids, nucleic acids, hormones, inclusion compounds, and the like may be added to the cosmetic of the present invention within a range that does not interfere with the effects of the present invention.
The oil-soluble gelling agent is selected from metal soaps such as aluminum stearate, magnesium stearate, and zinc myristate; amino acid derivatives such as N-lauroyl-L-glutamic acid, alpha, gamma-di-N-butylamine, and the like; cyclodextrin fatty acid esters such as cyclodextrin palmitate, cyclodextrin stearate, and cyclodextrin 2-ethylhexanoate palmitate; sucrose fatty acid esters such as sucrose palmitate and sucrose stearate; benzylidene derivatives of sorbitol such as monobenzylidene sorbitol and dibenzylidene sorbitol; one or two or more gelling agents such as an organically modified clay mineral such as dimethylbenzyldodecylammonium montmorillonite clay or dimethyloctacosylammonium montmorillonite clay may be used as necessary.
The humectant comprises: glycerin, sorbitol, propylene glycol, dipropylene glycol, 1,3-butanediol, glucose, xylitol, maltitol, polyethylene glycol, hyaluronic acid, chondroitin sulfate, pyrrolidone carboxylate, polyoxyethylene methyl glucoside, polyoxypropylene methyl glucoside, and the like.
The antibacterial preservative comprises: alkyl parabens, benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, phenoxyethanol, and the like, and antibacterial agents such as: benzoic acid, salicylic acid, carbolic acid, sorbic acid, alkyl parabens, parachloro-metacresol, hexachlorophene, benzalkonium chloride, chlorhexidine chloride, trichloro-carbanilide, triclosan, a photosensitizer, phenoxyethanol, and the like.
The antioxidant is as follows: tocopherol, butyl hydroxy anisole, dibutyl hydroxy toluene, phytic acid and the like, and pH regulators comprise: lactic acid, citric acid, glycolic acid, succinic acid, tartaric acid, dl-malic acid, potassium carbonate, sodium bicarbonate, ammonium bicarbonate, and the like, as chelating agents, alanine, sodium ethylenediaminetetraacetate, sodium polyphosphate, sodium metaphosphate, phosphoric acid, and the like, as cooling agents: l-menthol, camphor, etc., and the anti-inflammatory agents include: allantoin, glycyrrhetinic acid, glycyrrhizic acid, tranexamic acid, and Azulene (Azulene).
The skin beautifying components are as follows: whitening agent such as placenta extract, arbutin, glutathione, and herba Saxifragae extract; cell activator such as Lac Regis Apis, photosensitizer, cholesterol derivative, calf blood extractive solution, etc.; an agent for improving rough skin; blood circulation promoters such as valeriana nonanoate, benzyl nicotinate, beta-butoxyethyl nicotinate, capsaicin, zingerone, tincture of cantharides, ichthammol, caffeine, tannic acid, alpha-borneol, tocopherol nicotinate, inositol hexanicotinate, cyclamate, cinnarizine, tolazoline, acetylcholine, verapamil, cepharanthine, and gamma-oryzanol; skin astringents such as zinc oxide and tannic acid; sulfur, and the like antilipemic agents, and the like, the vitamins include: vitamin A oil, rosin oil, acetic acid rosin oil, palmitic acid rosin oil, and the like; vitamin B2 compounds such as riboflavin, riboflavin butyrate, and flavin adenine nucleotide; vitamin B6 such as pyridoxine hydrochloride, pyridoxine dioctanoate, and pyridoxine tripalmitate, vitamin B12 and its derivatives, and vitamin B15 and its derivatives; vitamin C compounds such as L-ascorbic acid, L-ascorbyl dipalmitate, L-ascorbic acid-2-sodium sulfate, and L-ascorbic acid phosphoric acid diester dipotassium; vitamin D compounds such as ergocalciferol and cholecalciferol; vitamin E compounds such as alpha-tocopherol, beta-tocopherol, gamma-tocopherol, dl-alpha-tocopherol acetate, dl-alpha-tocopherol nicotinate, dl-alpha-tocopherol succinate, and the like; vitamin H; a vitamin P; nicotinic acids such as nicotinic acid, benzyl nicotinate and nicotinamide; pantothenic acids such as calcium pantothenate, D-panthenol, panthenyl ethyl ether, and acetyl panthenyl ethyl ether; biotin, and the like.
The amino acids are: glycine, valine, leucine, isoleucine, serine, threonine, phenylalanine, arginine, lysine, aspartic acid, glutamic acid, cystine, cysteine, methionine, tryptophan, etc., nucleic acids include deoxyribonucleic acid, etc., and hormones include estradiol, vinylestradiol, etc.
Preferred examples of the cosmetic of the present invention include: skin care cosmetics, makeup cosmetics, and ultraviolet-shielding cosmetics. Such as basic cosmetics such as milky lotion, cream, lotion, sunscreen agent, mask material, face toilet, essence, etc.; makeup cosmetics such as foundation, powdery skin, blush, etc.
The form of the product is not particularly limited, and may be liquid, emulsion, cream, solid, paste, gel, powder, multi-layer, mousse (mousse), spray, or the like.
The invention also provides a whitening, freckle removing or skin care method, which comprises the following steps: administering the alisma orientale extract of the present invention or the composition of the present invention to a subject in need thereof.
In another preferred embodiment, the effective concentration range of the alisma orientale extract is 100 mug/ml to 50mg/ml.
In another preferred embodiment, the method is a method for removing sunburn, chloasma, freckles, discoloration, post-inflammatory hyperpigmentation, and the like.
The main advantages of the invention are:
1. the invention provides a method for effectively enriching active components alisol B and 23-acetyl alisol B in alisma, which mainly comprises three steps of extraction, extraction and purification, can improve the extraction efficiency by nearly 100 times, can effectively enrich alisol B and 23-acetyl alisol B components in alisma, and reduce the content of other components.
2. The alisma orientale extract disclosed by the invention has good whitening and freckle removing effects on human body surface skin, and has no obvious damage to the surface skin.
3. The alisma extract can effectively inhibit the synthesis of melanin, but has no obvious toxicity to melanocytes.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
The test materials and reagents used in the following examples are commercially available without specific reference.
EXAMPLE 1 preparation of Alisma extract
(1) Extraction: pulverizing Alismatis rhizoma, and sieving with 60 mesh sieve. Taking 100g of rhizoma alismatis fine powder, adding 500ml of acetone, and carrying out ultrasonic extraction for 3 times, wherein each time lasts for 1 hour. Mixing the three extractive solutions, and concentrating under reduced pressure below 40 deg.C until no acetone is contained to obtain extract of Alismatis rhizoma crude extract.
(2) Extraction: adding 25-50ml of purified water into the extract of the crude extract of the rhizoma alismatis obtained in the step (1), carrying out ultrasonic suspension, and transferring the suspension into a proper separating funnel. Using petroleum ether: dichloromethane =2:1 mixed solvent 25-50ml extracted 3 times. Mixing the organic phase layer solutions, and concentrating under reduced pressure below 40 deg.C to obtain Alismatis rhizoma extract.
(3) And (3) purification: gel column chromatography preceded by petroleum ether: dichloromethane: methanol = 1. Dissolving the extract of the rhizoma alismatis extract obtained in the step (2) by using a small amount of dichloromethane, and purifying and eluting by using a gel column, wherein an eluent is petroleum ether: dichloromethane: methanol =1, flow rate controlled at 2-3ml/min. Performing HPLC tracking detection, mixing the required parts, and concentrating under reduced pressure below 40 deg.C to obtain refined extract of Alismatis rhizoma 3g.
High performance liquid chromatography analysis as shown in FIG. 1:
in the refined extract, the main effective components are alisol B and 23-acetyl alisol B, and most of alisol A and 24-acetyl alisol A are removed, wherein the proportion of alisol B and 23-acetyl alisol B is about 1:1. the total mass content of alisol B and 23-acetyl alisol B is increased from 0.1-0.2% in the crude extract of alisma to 10-15% in the refined extract of alisma. The ratio of the total content of alisol B and 23-acetyl alisol B to the total content of alisol a and 23-acetyl alisol a is increased from the original about 5:1 (alisma crude extract) to about 200 (alisma refined extract). The other components comprise phytochrome, terpenes, phytosterol glycoside, fatty acid amino acid, inorganic salt ions and the like.
High performance liquid chromatography conditions:
waters 2695 liquid chromatograph, 2996 PAD detector
A chromatographic column: c 18 ，250×4.6mm,5μm
Mobile phase: acetonitrile: h 2 O＝73：27
Flow rate: 1.0ml/min
Detection wavelength: 210nm
EXAMPLE 2 Effect of Alisma orientale extract
1.1 mouse Melan-a melanocyte culture and treatment
Melan-a cells were cultured in RPMI1640 (GIBCO, GRAND ISLAND, NY) medium containing 10% fetal bovine serum (GIBCO, GRAND ISLAND, NY) and 1% diabody (penicillin 100U/mL, streptomycin 100 ug/mL), placed at 37 ℃ and 5% CO 2 Culturing under conventional conditions.
The following test substances were dissolved in 100% dmso solution for storage:
refined extract of rhizoma alismatis: example 1 the extract obtained in the step (3);
common crude extract of alisma orientale: example 1 extract obtained in step (1);
glabridin (Glabridin): a positive control;
alisol A:
24-acetyl alisol A:
purified water: blank control.
Diluted to the corresponding concentration with cell culture medium before use. The final DMSO concentration in the medium did not exceed 0.1%.
1.2 CCK-8 method cell viability assay
The CCK-8 method cell viability test is used to determine the concentration of the extract in the safety test.
mu.L Melan-a cells (2X 10) were inoculated 24 hours before the experiment 4 One/well) into a 96-well plate, adding test substances with different concentrations for treatment for 72 hours, washing the cells with PBS to remove the culture medium and the added test substances, adding 10. Mu.L of CCK-8 solution into each well for culture for 2 hours, and reading the OD value of the absorbance value at the wavelength of 450nm by an enzyme linked immunosorbent assay (Bio-Rad).
Cell viability = (experimental OD value-blank OD value)/(control OD value-blank OD value). Each result is represented by the mean ± s.d of three independent experimental results.
The cell viability of Melan-a melanocytes after 72 hours of treatment with the refined extract of alisma orientale was tested by the CCK-8 method. The results are shown in FIGS. 2 and 3. Each value is expressed as mean ± s.d.
The results show that: the refined extract of the alisma orientale has no obvious toxicity on the survival rate of melan-a melanocytes, while the ordinary crude extract of the alisma orientale has obvious toxicity on the cell survival rate of melan-a melanocytes.
1.3 Cell number statistics and cell melanin content test by SRB method
The SRB method was used to count the corresponding cell number before the melanin content test to ensure that the decrease in melanin content was not due to massive cell death.
Inoculation of Melan-a cells (3X 10) 5 One/well) in 12-well plates, after 24 hours of incubation, the media was changed 1 time with α -MSH (1 μ M) with test substance and with fresh α -MSH (1 μ M) without test substance, and incubation was continued for 72 hours.
After the culture was completed, the number of cells in each well was counted by the SRB method: to each well of cell culture medium was added 250. Mu.L of trichloroacetic acid (4 ℃) to fix the cells to 12 Kong Banbi, and after 1 hour of fixation, the cells were washed 5 times with PBS to remove trichloroacetic acid, medium and dead cells. After air-drying the 12-well plate, adding 500. Mu.L of 1%15 acetic acid solution containing 0.4% w/v SRB per well, staining for 30 minutes, and washing 10 times with 1% acetic acid solution to completely remove the SRB crystals not bound to the cells. The 12-well plate was air-dried, 300. Mu.L of purple crystals in 10mM Trisbase-lysed cells were added to each well, shaken for 15 minutes to dissolve sufficiently, transferred to a 96-well plate, and the absorbance value ODS was read at 595nm in an enzyme linked immunosorbent assay (Bio-Rad).
The fixation step of the SRB method allows the total amount of melanin in the cells to be measured after counting the number of cells. Cells were washed 2 times with PBS to remove SRB crystals from the cells, and twelve well plates were air-dried and digested for 1 hour with 300. Mu.L of trypsin. The digestion was stopped by adding 300. Mu.L of medium and the cell suspension was collected in a 1.5mL centrifuge tube, centrifuged at 13500g for 10 min, the supernatant removed, and placed in a lyophilizer for 12 h to completely remove the residual liquid. After completely dissolving melanin in the cells by adding 150. Mu.L of 1N sodium hydroxide solution, the cells were transferred to a 96-well plate, and the absorbance value ODM was read at 595nm using an enzyme linked immunosorbent assay (Bio-Rad).
Since the melanin solution and the SRB crystal solution have a linear relationship with the melanin content and the cell number, the result of the inhibition of intracellular melanin by the extract = ODS/ODM. Each result is represented by the mean ± s.d of three independent experimental results. A P value less than 0.05 indicates a significant difference. * P value less than 0.05, indicates P value less than 0.01.
The results of the melanin content per cell in the melan-a cell line after 72 hours of treatment with each test substance are shown in FIGS. 4 and 5. * Represents a significant difference p <0.05 from the control group; * Indicates a significant difference p <0.01 from control; * Indicates a significant difference p <0.001 from the control group.
The results in figure 4 show that the alisma purified extract significantly reduced the melanin content in Melan-a cells after 72 hours of treatment and that the inhibitory effect increased with increasing concentration. The known whitening component glabridin does not so significantly inhibit the content of melanin in Melan-a cells. Therefore, the refined extract of the alisma orientale can generate stronger melanin synthesis inhibition effect than the glabridin.
The results in FIG. 5 show that alisol A (alisol a) and 24-acetyl alisol A (alisol a 24-acetate) removed from the manufacturing process after 72 hours of treatment had no inhibitory effect on melanin synthesis in melanocytes.
1.4 intracellular enzyme Activity assay
Test substance: refined extract of rhizoma alismatis: example 1 extract obtained in step (3).
Inoculation of Melan-a cells (1.5X 10) 5 /well) in 24-well plates, after 24 hours of incubation, the medium was changed 1 time with α -MSH (1 μ M) with the test substance and with fresh α -MSH (1 μ M) without the test substance, and incubation was continued for 72 hours. After washing the cells with PBS, the cells were lysed with 900. Mu.l of PBS lysis solution containing 1% Triton X-100. The cells were frozen at-80 ℃ for 30 minutes, and after thawing the cell lysate, 100. Mu.L of 10mM L-DOPA was added, and incubated at 37 ℃ for 2 hours. The reaction solution was transferred to a 96-well plate, and absorbance was read at 490nm using an enzyme linked immunosorbent assay (Bio-Rad). Each result is represented by the mean ± s.d of the results of three independent experiments. A P value less than 0.05 indicates a significant difference. * Denotes a P value of less than 0.05, denotes a P value of less than 0.01. Inhibition of intracellular tyrosinase activity is the most common way to inhibit melanin content. As shown in fig. 8, the test substance was able to significantly inhibit tyrosinase activity in Melan-a cells.
1.5 cultivation, treatment and result detection of human epidermal model
Mel-300-B is a multi-layered, highly differentiated three-dimensional human epidermal model formed by culturing normal, black-derived epidermal keratinocytes (NHEK) and melanocytes (NHM). Cultures were grown on cell culture inserts at the air-liquid interface to allow simulated local application of the agents to be tested. The model provides an effective in vitro evaluation mode as an alternative to animal experiments.
In this study, the Mel-300-B human epidermal model was cultured in serum-free EPI-100-NMM-113 medium for 14 days. The medium was changed every other day. 25 μ L of Alismatis rhizoma extract (25 μ g/ml) was added directly to the surface of human epidermal model tissue. 25 μ L of purified water was used as a blank control (control). After day 14, the tissue survival rate was examined using the CCK-8 method. Each value is expressed as mean ± s.d.
The results of the experiment are shown in fig. 6 and 7. The results show that the refined extract of Alisma orientale has whitening effect on human epidermis (as shown in figure 7), and the refined extract of Alisma orientale of 25 μ g/ml does not cause significant influence on the survival rate of human epidermis model, which indicates that the concentration is basically nontoxic to human epidermis model (as shown in figure 6)
In summary, the refined extract of alisma orientale obtained by the preparation method of the invention not only shows good melanin inhibition in a melanocyte system, but also shows obvious whitening effect in a three-dimensional human epidermis model, so that the refined extract of alisma orientale can be used as a new whitening component to be added into skin care products and medicines.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.
Claims (12)
1. An alisma orientale extract, characterized in that the extract contains active ingredients: alisol B and 23-acetyl alisol B, and impurities; wherein the impurities comprise alisol A and 24-acetyl alisol A; and the mass percent of the active ingredients is more than or equal to 5 percent; and the mass ratio of the active ingredients to the alisol A and the 24-acetyl alisol A in the impurities is 20:1 to 400:1;
wherein, the alisma orientale extract is prepared by the method comprising the following steps:
(1) Extraction:
extracting Alismatis rhizoma powder with organic solvent acetone; collecting the extract, and concentrating to obtain a first extract containing Alismatis rhizoma extract;
(2) Extraction:
suspending the first extract containing the alisma extract obtained in the step (1) with water, and then extracting with an organic solvent; collecting organic phase, and concentrating to obtain second extract containing Alismatis rhizoma extract;
wherein in the step (2), the addition amount of the water is 1/10-1/1 of the alisma orientale powder; the addition amount of the organic solvent is 1/10-1/1 of the alisma orientale powder, and the organic solvent is a mixed solvent of petroleum ether and dichloromethane;
(3) And (3) purification:
purifying the second extract containing the alisma extract by gel column chromatography to obtain the alisma extract;
wherein the chromatographic purification is an elution purification by using an organic solvent, and the organic solvent is a mixed solvent of petroleum ether, dichloromethane and methanol.
2. The alisma extract of claim 1, wherein the content ratio of alisol B to 23-acetyl alisol B is 1.
3. The alisma extract of claim 1, wherein the mass ratio of the active ingredient to alisol a and 24-acetyl alisol a in the impurities is 150:1 to 250:1.
4. a pharmaceutical or cosmetic composition comprising an alisma orientale extract according to any one of claims 1 to 3.
5. The composition of claim 4, wherein the composition is one or more of a liquid, an ointment, a cream, a paste, a cake, a powder.
6. The composition of claim 4, wherein the composition is an emulsion.
7. The composition of claim 4, further comprising an additional whitening or depigmenting ingredient.
8. The composition of claim 4, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient; and/or the cosmetic composition further comprises a cosmetically acceptable carrier or excipient.
9. The composition of claim 8, wherein the cosmetically acceptable carrier or excipient is selected from the group consisting of: humectant, antioxidant, anti-ultraviolet agent, antiseptic, film forming agent, oil soluble gelling agent, organic modified clay mineral, resin, antibacterial agent, essence, salt, pH regulator, chelating agent, algefacient, antiinflammatory agent, skin beautifying component, vitamin, amino acid, nucleic acid, inclusion compound or their combination.
10. Use of an extract of alisma orientale as claimed in any of claims 1 to 3 or a pharmaceutical or cosmetic composition as claimed in any of claims 4 to 9, characterized by being used for
Preparing into medicine or cosmetic for whitening skin or removing speckle.
11. Use according to claim 10, for whitening, depigmenting, by inhibiting melanin and/or tyrosine.
12. The method for preparing an alisma orientale extract according to claim 1, wherein the method comprises the steps of:
(1) Extraction:
extracting Alismatis rhizoma powder with organic solvent acetone; collecting extractive solution, and concentrating to obtain first extract containing Alismatis rhizoma extract;
(2) And (3) extraction:
suspending the first extract containing the alisma extract obtained in the step (1) with water, and then extracting with an organic solvent; collecting organic phase, and concentrating to obtain second extract containing Alismatis rhizoma extract;
wherein, in the step (2), the addition amount of the water is 1/10-1/1 of the alisma orientale powder; the addition amount of the organic solvent is 1/10-1/1 of the alisma orientale powder, and the organic solvent is a mixed solvent of petroleum ether and dichloromethane;
(3) And (3) purification:
purifying the second extract containing the alisma extract by gel column chromatography to obtain an alisma extract;
wherein the chromatographic purification is an elution purification by using an organic solvent, and the organic solvent is a mixed solvent of petroleum ether, dichloromethane and methanol.
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