CN103083210A - Medicine composition with skin whitening effect as well as preparation method and application thereof - Google Patents
Medicine composition with skin whitening effect as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a medicine composition with a skin whitening effect as well as a preparation method and application thereof. The medicine composition with the skin whitening effect is prepared from the following raw materials in parts by weight: 1-60 parts of pyracantha fortuneana fruit, 1-60 parts of liquorice, 1-60 parts of mulberry,1-60 parts of angelica and 1-60 parts of saxifrage. The medicine composition has the obvious skin whitening effect and no irritation and sensitization to skin.
Description
Technical field
The present invention relates to a kind of pharmaceutical composition and preparation method thereof, particularly a kind of used for cosmetic pharmaceutical composition with skin whitening effect and its preparation method and application, be suitable for the cosmetics that make an addition to cream class, white class, newborn class, liquid class (except perfume).
Background technology
As the saying goes " one white hide three ugly ", great beauty, face are white as a jade, are yearned for by people from ancient times.Nails is always advocated pale skin, and skin whitening is that each girl pursues, though the occidentals does not pursue whitening, hankers after the removal to various mottles.The market of skin-lightening cosmetic is increasingly active, and its production marketing grows with each passing day, and has become one of main flow kind of skin protection cosmetics.The whitening chemicals refer to certain stage by the formation that acts on dermal melanin, metabolic pathway, and control, check melanin generate, thereby reduce skin colourity or alleviate pigmentation, to reach skin-whitening as the chemicals of the natural or synthetic of purpose.The whitening chemicals that are used at present cosmetics both at home and abroad are mainly hydroquinone, kojic acid, fruit acid, arbutin and vitamin C derivatives etc., hydroquinone and mercury salt can reach rapid whitening effect, but its toxic and side effects is large, and life-time service can cause the permanent decolouring of skin and contact dermatitis.The life-time service kojic acid has cytotoxicity, and can increase the incidence rate of the gonad cell chromosome interchange and chromosomal aberration, but the kojic acid derivative whitening function is stronger, and toxic and side effects is less, and present skin-lightening cosmetic is many as effective ingredient; The arbutin action temperature and, but using dosage is excessive the toxic and side effects similar with kojic acid arranged.Vitamin C derivatives has poor stability and the shortcoming such as expensive.(Nerya?O，Musa?R，Khatib?s，et?a1.Chalcones∞potent?tyrosinasc?inhibitors：the?effect?of?hydroxyl?positions?and?numbers[J].Phytochemistry，2004，65：1389-1395)
Plant whitening is according to its pure natural property, the popularity in source, and nontoxic, the advantage that has no side effect wide in variety more and more receives consumers in general's concern.(Smit N, Vicanova J, Pavel S.The hunt for natural skin whitening agents[J] .Iht J Mol Sci, 2009,10 (12): 5326-5349; Kamakshi R.Fairness via formulations:a review ofcosmetic skin-lightening ingredients[J] .J Cosmet Sci, 2012,63 (1): 43-54) existing 2,000 years history of Chinese medicine beauty treatment, accumulated rich experience, at present, the cosmetics that contain natural Chinese medicinal herb in Japan have accounted for more than 50% of whole Cosmetic Market.Therefore, seek exploitation and efficiently and to the natural Chinese medicinal herb skin-whitening and speckle eliminating preparation that human body has no side effect or side effect is little become the focus that present cosmetic field is researched and developed.(Shu Shumiao, Xiao Feng, Pan Qin. the progress of whitening Chinese medicament and plant amedica. external medical plant amedica fascicle, 2007,22 (6): 252-255) as patent 200810024411.9, this patent discloses a kind of Chinese medicine composition and application thereof with whitening function, this Chinese medicine composition by Radix Scutellariae, Rhizoma Polygoni Cuspidati, Cortex Moutan extract is composite forms.As patent 200910192274.4, this patent discloses a kind of whitening and spot eliminating cream and preparation method thereof, active component is Chinese medicine extraction liquid, and Chinese medicine is comprised of the component of following Chinese medicine percent: Rhizoma Gymnadeniae 5～55%, Flos Ginseng 15～35%, Flos Albiziae 5～15%, Flos Lonicerae 5～15%, Flos Rosae Rugosae 15～20%, Flos Chrysanthemi 5～10%.As patent 201010261392.9, this patent discloses a kind of compositions for skin whitening, plant extraction lightening compositions and has had the compositions of whitening function, said composition comprises the spermidine derivant of effective dose, and beauty treatment or the upper acceptable vehicle of pharmacology.
Summary of the invention
The objective of the invention is in order to overcome weak point of the prior art, a kind of pharmaceutical composition and preparation method and purposes with skin whitening effect is provided.
Pharmaceutical composition with skin whitening effect of the present invention, the preparation take firethorn fruit, Radix Glycyrrhizae, Fructus Mori, Radix Angelicae Sinensis and Herba Saxifragae as raw material, and be with the preparation of the raw material of following parts by weight:
1～60 part of firethorn fruit
1～60 part, Radix Glycyrrhizae
1～60 part, Fructus Mori
1～60 part of Radix Angelicae Sinensis
1～60 part of Herba Saxifragae
Preferably, described pharmaceutical composition with skin whitening effect is with the preparation of the raw material of following parts by weight:
1～50 part of firethorn fruit
1～50 part, Radix Glycyrrhizae
1～50 part, Fructus Mori
1～50 part of Radix Angelicae Sinensis
1～50 part of Herba Saxifragae
Preferably, described pharmaceutical composition with skin whitening effect is with the preparation of the raw material of following parts by weight:
1～40 part of firethorn fruit
1～40 part, Radix Glycyrrhizae
1～40 part, Fructus Mori
1～40 part of Radix Angelicae Sinensis
1～40 part of Herba Saxifragae
Preferably, described pharmaceutical composition with skin whitening effect is with the preparation of the raw material of following parts by weight:
2～35 parts of firethorn fruits
2～35 parts, Radix Glycyrrhizae
2～35 parts, Fructus Mori
2～35 parts of Radix Angelicae Sinensis
2～35 parts of Herba Saxifragaes
Preferably, described pharmaceutical composition with skin whitening effect is with the preparation of the raw material of following parts by weight:
8～30 parts of firethorn fruits
8～30 parts, Radix Glycyrrhizae
8～30 parts, Fructus Mori
10～30 parts of Radix Angelicae Sinensis
10～30 parts of Herba Saxifragaes
Most preferred, be with the preparation of the raw material of following parts by weight:
10 parts of firethorn fruits
10 parts, Radix Glycyrrhizae
10 parts, Fructus Mori
10 parts of Radix Angelicae Sinensis
10 parts of Herba Saxifragaes
Most preferred, be with the preparation of the raw material of following parts by weight:
8 parts of firethorn fruits
8 parts, Radix Glycyrrhizae
12 parts, Fructus Mori
12 parts of Radix Angelicae Sinensis
12 parts of Herba Saxifragaes
Most preferred, be with the preparation of the raw material of following parts by weight:
8 parts of firethorn fruits
8 parts, Radix Glycyrrhizae
15 parts, Fructus Mori
15 parts of Radix Angelicae Sinensis
15 parts of Herba Saxifragaes
Most preferred, be with the preparation of the raw material of following parts by weight:
10 parts of firethorn fruits
10 parts, Radix Glycyrrhizae
12 parts, Fructus Mori
12 parts of Radix Angelicae Sinensis
12 parts of Herba Saxifragaes
Most preferred, be with the preparation of the raw material of following parts by weight:
8 parts of firethorn fruits
8 parts, Radix Glycyrrhizae
10 parts, Fructus Mori
10 parts of Radix Angelicae Sinensis
15 parts of Herba Saxifragaes
Preparation method with pharmaceutical composition of skin whitening effect of the present invention comprises the following steps:
(1) each above-mentioned raw material being placed in volumetric concentration is that 20%～80% ethanol soaked 2～48 hours, and the ethanol consumption that soaks use be 10～200 times of each raw material gross weight, and filtration gets soak;
Preferably, filtration residue is 20%～80% soak with ethanol 2～48 hours with volumetric concentration again, merges the solution after soaking, and gets soak.
(2) soak is concentrated into 2～5% of former weight, and concentrated solution macroporous resin adsorption, then the ethanol of using volumetric concentration 5～95% eluting are repeatedly collected eluent, are described pharmaceutical composition with skin whitening effect;
Preferably, eluting is 40%～95% with the volumetric concentration of ethanol;
Described macroporous resin adopts the HP20 type macroporous resin of Mitsubishi chemical company take polystyrene one divinylbenzene as matrix; The concentrated method of soak is conventional concentrating under reduced pressure.
Evidence, the pharmaceutical composition with skin whitening effect of the present invention has multiple action, can be used in the cosmetics of preparation skin whitening maintenance and the curative drug of skin.
The invention still further relates to a kind of skin care cosmetics, comprise described the have pharmaceutical composition of skin whitening effect and the adjuvant that cosmetic field uses.
The invention still further relates to a kind of curative drug of skin, comprise that claim is described and have the pharmaceutical composition of skin whitening effect and a medically acceptable carrier.
Especially can for the preparation of skin care cosmetics, comprise cream, emulsion, astringent, gel, facial film, liniment or lotion;
During the preparation skin care cosmetics, can be with the extracting solution compositions that makes, through being evaporated to 10%～50% of former weight, obtain composition concentrated solution, mix mutually with the substrate of known cosmetics and medicine or adjuvant, carrier, additive, method according to routine is prepared, and wherein, described composition concentrated solution accounts for 5%～10% of cosmetics gross weight.
The present invention is according to tcm theory, from a large amount of natural drugs, having selected firethorn fruit, Radix Glycyrrhizae, Fructus Mori, Radix Angelicae Sinensis and Herba Saxifragae is raw material, prepared described compositions with skin whitening effect, wherein, described firethorn fruit is selected the fruit of evergreen wild undershrub plant Pyracantha fortuneana (Maxim.) Li of Rosaceae Maloideae Pyracantha; Radix Glycyrrhizae is selected dry root and the rhizome of glycyrrhizic legume Glycyrrhiza uralensis Fisch.; Fructus Mori is selected the dry fruit ear of moraceae plants Mulberry Morus albaL.; Herba Saxifragae is selected the herb of saxifragaceae plant Herba Saxifragae Saxifiagga stolonifera Curt.; Radix Angelicae Sinensis is selected the dry root of the umbrella plant Radix Angelicae Sinensis Angelica sinensis of section (Oliv.) Diels.
According to " Chinese pharmacopoeia and document record:
Firethorn fruit is the fruit of evergreen wild undershrub plant Pyracantha fortuneana (Maxim.) Li of Rosaceae Maloideae Pyracantha, firethorn fruit has another name called Fructus Pyracanthae (Fructus Pyracanthae Fortuneanae), torch fruit etc., mainly is distributed in mountain region and the hilly country in the zones such as China southeast, southwest and northwest.The firethorn fruit edible, the sweet acid of nature and flavor has the effects such as spleen invigorating removing food stagnancy, promoting the production of body fluid to quench thirst.The firethorn fruit extract is rich in the flavone compounds such as rutin and hyperin, also contains in addition aminoacid, plurality of inorganic salt and trace element etc.Once reported abroad that firethorn fruit had significant whitening function, and went out skin-lightening cosmetic as the raw material develop.
Radix Glycyrrhizae is dry root and the rhizome of glycyrrhizic legume Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat. Glycyrrhiza inflate Bat. or Glycyrrhiza glabra L. Glycyrrhiza glabra L., Radix Glycyrrhizae is clinical the most frequently used Chinese medicine, be commonly called as " Herba Achnatheri Inebriantis of state ", the good reputation that " ten side's nine grass " are arranged, Radix Glycyrrhizae property is flat, sweet in the mouth is returned 12 warps.Detoxifcation arranged, eliminate the phlegm, pain relieving, spasmolytic be so that the pharmacological action such as anticancer.On the traditional Chinese medical science, Radix Glycyrrhizae invigorating the spleen and replenishing QI, relieving cough and moistening lung, emergency detoxifcation, mediation hundred medicines.Clinical practice divides " give birth to and use " and " processed with honey " not.Give birth to curing mainly laryngopharynx swelling and pain, cellulitis skin infection, gastrointestinal ulceration and solution poison of drug, alimentary toxicosis etc.; Processed with honey cures mainly spleen and stomach function and goes down, and large loose stool is thin, weak heating and cough, cardiopalmus etc.
Fructus Mori is the dry fruit ear of moraceae plants Mulberry Morus alba L., and the traditional Chinese medical science thinks that the Fructus Mori sweet in the mouth is sour, and cold nature enters the heart, liver, kidney channel, is strengthening by means of tonics, the good fruit of the heart nourishing and intellengence improving.Have the YIN nourishing of enriching blood, promoting the production of body fluid to quench thirst, the effect such as intestine moistening is dry cures mainly deficiency of YIN-blood and having a dizzy spell of causing, tinnitus cardiopalmus, irritated insomnia, soreness of the waist and knees, early whitening of beard and hair, the xerostomia of quenching one's thirst, the diseases such as constipation with dry stool.
When the dry root that is classified as the umbrella plant Radix Angelicae Sinensis Angelicasinensis of section (Oliv.) Diels.Radix Angelicae Sinensis sweet in the mouth, suffering, warm in nature.Can enrich blood, invigorate blood circulation, regulating menstruation, pain relieving, intestine moistening.Be used for blood deficiency, shallow complexion, dizzy cardiopalmus; Blood deficiency, or have the stagnant menoxenia of the stasis of blood, amenorrhea, dysmenorrhea concurrently; Deficiency and coldness stomachache, angina pectoris, rheumatic arthralgia, traumatic injury etc. are demonstrate,proved bitterly; The dryness of the intestine constipation; Chronic cough asthma.In recent years, the physician has carried out scientific validation to " married woman's face medicine " of anti-ageing speckle removing, skin health-care in the supplement to Thousand Golden Prescriptions of Tang Dynasty in China Sun Simiao work, therefrom filter out the highest medicine of frequency of utilization, result shows, the function of the aqueous solution restraint of tyrosinase activity of Radix Angelicae Sinensis is very strong, thereby the formation of energy check melanin, and treatment chloasma, freckle isochrome disposition dermatosis are produced effects well, have defying age and beautification function, help to make people's youth resident.Radix Angelicae Sinensis is added in cosmetic cream, QUBAN SHUANG, and in capsule snow phenanthrene in the hope of skin nutrition, prevent pachylosis, prevent and treat the effects such as acne, chloasma, freckle, without any side effects through observing.Radix Angelicae Sinensis can also promote natural on-off cycles of hair growth, with hair conditioner, the hair cream that Radix Angelicae Sinensis is made, can make hair soft shinny, is easy to combing.
Herba Saxifragae is the herb of saxifragaceae plant Herba Saxifragae Saxifiagga stolonifera Curt..Little hardship, suffering, cold, slightly poisonous are dispeled the wind, heat clearing away, removing pathogenic heat from blood and toxic substance from the body.Control rubella, eczema, otitis media, erysipelas, cough is spitted blood, lung abscess, metrorrhagia, haemorrhoids.In recent years, it is found that and be rich in arbutin in Herba Saxifragae, effectively the active degree of check melanin blast cell, just as giving the melanin hypnosis, have whitening, the effect of light speckle.
At present, the compositions that there is not yet both at home and abroad firethorn fruit, Radix Glycyrrhizae, Fructus Mori, Radix Angelicae Sinensis and Herba Saxifragae extract is used for having the relevant report of skin whitening effect.
In sum, the inventor has read lot of documents, according to the ultimate principle of skin-whitening agents, utilizes cell model to carry out high flux screening, finally finds to have the pharmaceutical composition of remarkable skin whitening function.Pharmaceutical composition of the present invention confirms that through clinical trial effect is remarkable, and safety is good.Related composition has that effect is clear and definite, safety good, without any side effects and in cosmetic formulations stable characteristics; Comparing with existing like product, have the effect outstanding feature, is a kind of novel compositions with effective natural drug extract of skin whitening effect.
The specific embodiment
Embodiment 1
At room temperature, get fire sour jujube fruit, Radix Glycyrrhizae, Fructus Mori, Radix Angelicae Sinensis and Herba Saxifragae each 10 kilograms (totally 50 kilograms), 600 kilograms of immersions of ethanol with volumetric concentration 70% are extracted, soak time is 24 hours, residue extracted with 600 kilograms of immersions of ethanol of volumetric concentration 70% in 24 hours again, then the solution after soaking merges, after 200 purpose strainer filterings, 20 times of concentrating under reduced pressure, again with macroporous resin on concentrated solution, with the ethanol elution of volumetric concentration 50%, contain needed effective ingredient, i.e. pharmaceutical composition in prescription of the present invention in the eluent of collection.
Embodiment 2
at room temperature, get fire 8 kilograms of sour jujube fruits, 8 kilograms, Radix Glycyrrhizae, 12 kilograms, Fructus Mori, 12 kilograms of Radix Angelicae Sinensis and 12 kilograms of Herba Saxifragaes (totally 52 kilograms), 1200 kilograms of immersions of ethanol with volumetric concentration 50% are extracted, soak time is 24 hours, residue extracted with 1200 kilograms of immersions of ethanol of volumetric concentration 50% in 24 hours again, then the solution after soaking merges, after 200 purpose strainer filterings, 40 times of concentrating under reduced pressure, again with macroporous resin on concentrated solution, ethanol elution with volumetric concentration 40%, contain needed effective ingredient in prescription of the present invention in the eluent of collecting, it is pharmaceutical composition.
Embodiment 3
at room temperature, get fire 8 kilograms of sour jujube fruits, 8 kilograms, Radix Glycyrrhizae, 15 kilograms, Fructus Mori, 15 kilograms of Radix Angelicae Sinensis and 15 kilograms of Herba Saxifragaes (totally 61 kilograms), 1000 kilograms of immersions of ethanol with volumetric concentration 80% are extracted, soak time is 24 hours, residue extracted with 1000 kilograms of immersions of ethanol of volumetric concentration 80% in 24 hours again, then the solution after soaking merges, through concentrating 25 times after 200 purpose strainer filterings, again with macroporous resin on concentrated solution, ethanol elution with volumetric concentration 60%, contain needed effective ingredient in prescription of the present invention in the eluent of collecting, it is pharmaceutical composition.
Embodiment 4
at room temperature, get fire 10 kilograms of sour jujube fruits, 10 kilograms, Radix Glycyrrhizae, 12 kilograms, Fructus Mori, 12 kilograms of Radix Angelicae Sinensis and 12 kilograms of Herba Saxifragaes (totally 56 kilograms), 2000 kilograms of immersions of ethanol with volumetric concentration 30% are extracted, soak time is 24 hours, residue extracted with 2000 kilograms of immersions of ethanol of volumetric concentration 30% in 24 hours again, then the solution after soaking merges, through concentrating 50 times after 200 purpose strainer filterings, again with macroporous resin on concentrated solution, ethanol elution with volumetric concentration 50%, contain needed effective ingredient in prescription of the present invention in the eluent of collecting, it is pharmaceutical composition.
Embodiment 5
at room temperature, get fire 8 kilograms of sour jujube fruits, 8 kilograms, Radix Glycyrrhizae, 10 kilograms, Fructus Mori, 10 kilograms of Radix Angelicae Sinensis and 15 kilograms of Herba Saxifragaes (totally 51 kilograms), 1200 kilograms of immersions of ethanol with volumetric concentration 60% are extracted, soak time is 24 hours, residue extracted with 1200 kilograms of immersions of ethanol of volumetric concentration 60% in 24 hours again, then the solution after soaking merges, through concentrating 30 times after 200 purpose strainer filterings, again with macroporous resin on concentrated solution, ethanol elution with volumetric concentration 80%, contain needed effective ingredient in prescription of the present invention in the eluent of collecting, it is pharmaceutical composition.
Embodiment 6
The liquid that embodiment 1 is obtained adopts concentrating under reduced pressure, is concentrated into 10%～50% of former weight, gets the pharmaceutical composition concentrated solution, and for the preparation of the essence with skin whitening effect, the percentage by weight of component is as follows:
Production technology: under stirring, A is heated to respectively 70 ℃ mutually with B, to whole dissolving mix homogeneously, then under 70 ℃ of conditions, B is added to A mutually in, to forming W/Q type Emulsion.After stirring, be cooled to room temperature and get final product.
Embodiment 7
Pharmaceutical composition has the cell experiment research report of skin whitening effect: in following experiment, the compositions of drug extract used is the pharmaceutical composition of embodiment 1.
1 materials and methods
1.1 reagent and instrument RPMI1640, Gibco company product; Hyclone (FBS), Hangzhou Ilex purpurea Hassk.[I.chinensis Sims company product; The B-16 mouse black-in tumor cell, Chinese Academy of Sciences's cell provides, and vitamin C is provided by Beijing Berry Lai Si Biochemics Inc., meets cosmetic material medicine standard; L-GLUTAMINE, levodopa (L-Dopa), dimethyl sulfoxide (DMSO), trypsin, TritonX-100 is the Sigma product, Thiazolyl blue (MTT), Serva product; CO
2Incubator, U.S. Bamstead company; The TDL-40B centrifuge, Anting Scientific Instrument Factory, Shanghai; Tissue Culture Plate, U.S. Greiner product; The DG5031 enzyme-linked immunosorbent assay instrument, East China Electronics Co., Ltd pipe factory product.
1.2 method
1.2.1 the strain of cell culture B-16 melanoma cells is purchased from cell institute of the Chinese Academy of Sciences, treats that Growth of Cells to the fusion state, goes down to posterity through 0.25% trypsinization, is inoculated in 25cm
2In culture bottle, be placed in 37 ℃ of CO with the RPMI1640 culture fluid
2Incubator, 5%CO
2The saturated humidity environment is cultivated.Same passage cell is taken from experiment each time, and the initial inoculation cell concentration is 10
5The left and right.After cell inoculation 6h, add tested whitening agent, respectively establish 5 dosage groups, changed identical pastille culture fluid in the 2nd day, harvesting after continuous culture 3d is expected blue dyeing counting total cellular score with tongue, then cell is used for following experiment.
1.2.2 reagent preparation is got whitening extract 0.5g of the present invention first with the 1mlDMSO dissolving with the experiment grouping, adds freshly prepared RPMI-1640 and is diluted to that final concentration is 5,0.5,0.05,0.005,0.0005g/L, the final concentration of DMSO is lower than 1%.Vitamin C directly adds culture fluid to be diluted to final concentration, and diluted concentration is respectively 10
-1, 10
-2, 10
-3, 10
-4, 10
-5Mol/L.The blank group only adds dilution medicine corresponding solvent used.
1.2.3 the tyrosinase activity suppression ratio detects take L-Dopa as substrate, measures the cell tyrosinase activity with reference to the method for Maeda etc.Adjust melanocyte density, with 10
4Individual/hole is inoculated in 96 orifice plates, changes liquid after 6h, and every hole adds the drug solution of 200 μ l, and each concentration arranges 4 multiple holes, and matched group adds fresh medium to replace drug solution, 5%CO
237 ℃ hatch 3d after (liquid is changed once in the centre), abandoning supernatant, PBS washes 3 times, every hole adds 45 μ k1%TritonX-100 solution, 10min is with dissolved cell in concussion, and every hole adds the L-Dopa of 0.01mol/L, hatches 60min for 37 ℃, in 450nm wavelength place colorimetric, with the blank well zeroing, survey each hole absorbance at enzyme-linked immunosorbent assay instrument, each experiment repeats 3 times.Tyrosinase activity suppression ratio=(each concentration mean light absorbency value of 1-÷ matched group mean light absorbency value) * 100% uses SPSS13.0 carry out curve fitting and calculate IC
50Value.
Adopt the method for Victoria to measure 1.2.4 the synthetic suppression ratio of melanocyte detects melanin content, adjust melanocyte density, with 5 * 10
5Individual/hole is inoculated in 6 orifice plates, changes liquid after 6h, and every hole adds the drug solution of 1ml, and each concentration arranges 3 multiple holes, and matched group adds fresh medium to replace drug solution, 5%CO
237 ℃ hatch 3d after (liquid is changed once in the centre), abandoning supernatant, every hole adds 0.25% pancreatin 1ml to digest 2min under room temperature, adds the 4ml culture fluid to end digestion, blows and beats into single cell suspension, get 20 μ l and make cell counting, the centrifugal 5min of all the other cell suspension 1500r/min abandons supernatant, adds 1ml1N NaOH solution, concussion 5min, after 80 ℃ of heating in water bath 1h, be transferred to 96 orifice plates, every hole adds 100 μ l, select the 405nm wavelength, with the blank well zeroing, survey absorbance on enzyme-linked immunosorbent assay instrument, each experiment repeats 3 times.Melanocyte synthesizes suppression ratio (%)=[1-(absorbance ÷ medicine hole, medicine hole L cell density) ÷ (control wells absorbance ÷ control wells cell density)] * 100%, uses SPSS13.0 carry out curve fitting and calculate IC
50Value.
1.2.5MTT colorimetric determination cell increment suppression ratio fetching is counted the B-16 cell of trophophase, disperses the preparation single cell suspension through digestion, adjusts cell concentration, and cell is seeded to 96 porocyte culture plates, the initial cell inoculum density is 10
5Individual/hole, 6h is hatched after cell attachment in amount of liquid 200 μ l/ holes, sucks culture fluid, adds and contains variable concentrations medicine culture fluid, and each concentration arranges 6 multiple holes, continues to cultivate 3d (liquid is changed once in the centre).When measuring cell proliferation rate, add 0.5%MTT storage liquid 20 μ l to every hole, put CO
2Incubator continues to cultivate 4h.This moment, MTT was converted into first for (formazan) through the mitochondrial membrane succinate dehydrogenase, a large amount of aubergine acicular crystals are arranged inside and outside the microscopically visible cell, carefully draw culture fluid, every hole adds 200 μ l DMSO, culture plate jolting 10min makes first replace crystallization to dissolve fully.Put immediately microplate reader in 570nm photometry absorption value, each experiment repeats 3 times, calculates with following formula and respectively organizes cell proliferation inhibition rate.Cell proliferation inhibition rate (%)=(1-experimental group 570/ matched group 570) * 100% uses SPSS13.0 carry out curve fitting and calculate IC
50Value.
1.2.6 statistical analysis is to the IC in each group
50Value adopts SPSS to carry out variance analysis, carries out the comparison between sample average.
Result
The in-vitro evaluation of table 1 pharmaceutical composition to whitening function
Annotate: compare * P＜0.05, * * P＜0.01 with the vitamin C group.
Result of study shows that (seeing table 1 for details) pharmaceutical composition of the present invention and vitamin C all have inhibitory action to intracellular tyrosine enzymatic activity and melanogenesis, and has dose-dependence, namely the increase suppression ratio with dosage improves, and the effect of pharmaceutical composition of the present invention will significantly be better than vitamin C (P＜0.05 or P＜0.01).And pharmaceutical composition of the present invention suppresses the value-added IC of cell
50Close with vitamin C, the safety of this explanation pharmaceutical composition cell of the present invention is good.
Adopt the pharmaceutical composition with skin whitening effect of other embodiment to test, result also proves to have identical effect.
Embodiment 8
Pharmaceutical composition has the clinical experimental study report of skin whitening effect: in following clinical trial, the compositions of drug extract used is the essence with skin whitening effect of the cosmetic formulations preparation of embodiment 6.
1. materials and methods
1.1 the selection of tested object
Altogether include 20 female volunteers of 24～42 years old in, inclusive criteria: the skin type belongs to the IV type, and unglazed allergies are without the cosmetic allergic contact dermatitis history.Exclusion standard: known to being subjected to the composition allergy sufferers in trial product; The psychotic; Be in period of pregnancy, age of sucking or childbirth interior person later six months; Serious hepatorenal disease, autoimmune disease (as systemic lupus erythematosus (sle), rheumatoid arthritis), HIV the infected are arranged: be diagnosed as cutaneous tumor and curer in nearly 12 months; Using and/or in front 14 days of test oral or external the medicine (glucocorticoid, immunosuppressant and antiallergic agent) of test forbidding; Using or testing and used other like products person in front 1 month.
1.3 test instrunment adopts Lab colorimeter system spectrophotometer (Minolta spectrophotometer CM-2002) to measure the variation of skin color.
1.4 test index L=116 (Y/Y
n)
1/3-16; A=500[(X/X
n)
1/3-(Y/Yn)
1/3]; B=200[(Y/Y
n)
1/3-(Z/Z
n)
1/3]; C
ab=(a
2+ b
2)
1/2Hab=arctg (b/a), in formula, L, a, b are the coordinate figure of three-dimensional cartesian coordinate system system; X, Y, Z are the tristimulus values of XYZ colour system; Xn, Yn, Zn are the tristimulus values of persect reflecting diffuser.L: be brightness, be worth greatlyr, color is deflection white more, otherwise, deflection black.A: be red, green product ,+a is red direction, and-a is green direction.B: be yellow, blue product ,+b is yellow direction, and-b is blue direction.C: be saturation, in order to represent the deep or light of color of object surface.H: be hue angle, the colored characteristic of dividing mutually each other of expression.In the Lab colorimeter system, represent two kinds of aberration Δ E between stimulation with difference Δ L, Δ a, the Δ b of coordinate L, a, b
ab, i.e. distance between the colour space two color dot.ΔE
ab＝[(ΔL)
2+(Δa)
2+(Δb)
2]
1/2。In formula: due to Δ E
abBe the aggregative index of L, a, b, represented that the solid of colourity changes, so be to estimate the more suitable index that the colour of skin changes.
1.5 the smearing method tested object uses continuously, smears the cosmetics of appointment at face after washing one's face before sleeping in every morning, evening, applying amount gets final product slightly to see smear layer.Cervical region is not coated with cosmetics, is used as own control.
Testing time make apply some make up before 1d, use after the 30th, 60,90d uses spectrophotometer to measure the variation of skin color.Follow-on test 3 times is averaged.
Test position forehead (place between the eyebrows top 2cm); Left and right buccal (cheekbone back lower place 1cm); Cervical region (Adam's apple below 2cm)
1.6. safety evaluatio carries out the untoward reaction evaluation to the experimenter when further consultation, whether the former symptom of inquiry experimenter's target site increases the weight of or new skin lesion occurs, reaches whether hinder daily life as symptoms such as pruritus, drying, desquamation, twinge, redness, desquamations.Severe: untoward reaction hinders daily routines, and experimenter's subjective symptoms is remarkable, is impatient at, and needs to stop using; Moderate: untoward reaction hinders daily life, and experimenter's subjective symptoms is obvious, but can stand, and need not stop using is subjected to trial product; Slightly: but the Intolerance reaction, and the experimenter occasionally can experience.To the experimenter of untoward reaction occurring, the doctor carries out patch test to it. to determine the cause effect relation of untoward reaction and test products.Responsible doctor thinks that the experimenter is not suitable for continuing test, or the experimenter requires to stop, and namely stops this experimenter's test, and records stop reason and time.
1.7. statistical procedures adopts SSPS11.5 software to carry out self front and back paired t-test.
2. result
2.1 different parts shows the color difference table 2 of pharmaceutical composition different time, the buccal aberration raises after using pharmaceutical composition of the present invention, after using 90d, significance variation (P＜0.05) appears in the buccal aberration, and the variation at all the other positions is without significance (P＞0.05).
Table 2 different parts make pharmaceutical composition different time aberration Δ E comparison (
N=20)
Annotate: relatively front with use, * P＜0.05.
2.2 use the data show of the change list 3 of different time skin of cheek color value, use that L-value and the b value of buccal all raises after pharmaceutical composition 30 of the present invention, 60,90d, relatively front with use, difference has significance (P＜0.05), and a value all has the attenuating (P＜0.01) of utmost point significance after use 60,90d.The c value significance occurs and raises (P＜0.05) after using 60d, there are no significant in the whole process of the test variation (P＞0.05) of h value.This proves absolutely, the volunteer is after using pharmaceutical composition of the present invention, and a value of its cervical region descends, and L-value continues to rise, and the rising of b value shows that the colour of skin changes to the HUANGBAI(sic) direction; The c value raises and makes colour of skin HUANGBAI(sic) saturation increase after this brand skin-lightening cosmetic is used in explanation continuously; The h value changes little, illustrates that the variation of the colour of skin is more consistent.
Table 3 make pharmaceutical composition different time skin of cheek color value comparison (
N=20)
Annotate: relatively front with use, * P＜0.05, * * P＜0.01.
Before and after Product Safety assessment treatment, untoward reaction not occuring all, comes off without 1 routine patient.The Adverse Event relevant to this test products do not occur in whole research process.
The pharmaceutical composition that adopts other embodiment to have the skin whitening effect carries out clinical trial, and result also proves to have identical effect.
Embodiment 9
Skin irritation test research report with pharmaceutical composition of skin whitening effect: by animal experiment, observe and of the present inventionly have skin whitening drugs with function compositions to the zest of rabbit skin.In following experiment, the compositions of drug extract used is the pharmaceutical composition of embodiment 4.
1 animal
16 regular grade adult healthy new zealand rabbits, male and female half and half, 2.5kg～3.5kg buys in Guangdong Medical Lab Animal Center, and the laboratory animal production licence number is SCXK (Guangdong) 2008-0002, the animal quality certification number: Guangdong 2012A008.Raise in new southern drug safety assessment centers general area Animal House F4 room (the experimental animal room quality certification number: 0060590.), duration of test, 20 ℃～22 ℃ of room temperatures, humidity 40%～67%, illumination 12h/12h light and shade round the clock replaces.The pharmaceutical composition that uses in this experiment is the pharmaceutical composition of the embodiment of the present invention 1, and concentration is 1.0g crude drug/mL.
2 methods
According to Ministry of Health of the People's Republic of China " cosmetics health standard (version in 2007) ", adopt consubstantiality self left and right sides matching type, the left side gives blank substrate (normal saline), and the right side gives the pharmaceutical composition of 1.0g/mL.Prepare before administration: 24h before administration, adopt mao method of shaving to remove family's rabbit back spinal column both sides by hair, left and right each the about 3cm * 3cm of unhairing scope, application area 2.5cm * 2.5cm, not injured skin.Administration: get tested material approximately 0.5mL spread upon on the skin of right side, the left side is coated with normal saline in contrast, smears every day 1 time, continuously 14d.From second day, will be by hair before smearing at every turn.Medicinal liquid 0.5mL is soaked into the double-deck hospital gauze sheet of 2.5cm * 2.5cm, be covered in plucked skin, cover one deck template, then fix with nonirritant adhesive plaster and fixing band.Fix and approximately remove tested material after 4h, remove residual tested material with warm water.Observed result after one hour.3 results
Under this experiment condition, the pharmaceutical composition of smearing continuously 1.0g/mL reacted (specifically seeing Table 4) to rabbit skin nonirritant in 14 days.
Table 41.0.g/mL pharmaceutical composition is to rabbit irritation test result repeatedly
Embodiment 10
Hypersensitive experimentation report with pharmaceutical composition of skin whitening effect: by animal experiment, observe and of the present inventionly have skin whitening drugs with function compositions to the sensitization of Hartley guinea pig skin.The pharmaceutical composition that uses in this experiment is the pharmaceutical composition of the embodiment of the present invention 5.
1 animal
Select 50 of regular grade Hartley Cavia porcelluss, male and female half and half, weight range 180～250g buys in Guangdong Medical Lab Animal Center, and the laboratory animal production licence number is SCXK (Guangdong) 2008-0002, the animal quality certification number: Guangdong 2012A016.Raise in new southern drug safety assessment centers general area Animal House F4 room (the experimental animal room quality certification number: 0060590.), duration of test, 20 ℃～22 ℃ of room temperatures, humidity 40%～67%, illumination 12h/12h light and shade round the clock replaces.By number Cavia porcellus is carried out skin dyeing, with saturated picric acid solution (yellow) dye marker units, with 2% silver nitrate solution (coffee color) dye marker tens; Separately raise by sex, each cage box is put 5 Cavia porcelluss, and the cage box is outer labelled, indicates special topic numbering, animal classification, group, sex, route of administration, cage number, number of animals and estimates to test the information such as commencement date and deadline.The pharmaceutical composition that uses in this experiment is the pharmaceutical composition of the embodiment of the present invention 1, and concentration is 1.0g crude drug/mL.
2 methods
According to country " medicine registration management way ", " chemicals zest, anaphylaxis and hemolytic investigative technique guideline ".Be divided at random negative control group, positive controls, pharmaceutical composition group, 10 Cavia porcelluss of negative control group wherein, all the other respectively organize 20 Cavia porcelluss.Each organizes Cavia porcellus male and female half and half.Successively induce sensitization to contact experiment with exciting, the eluting phase of 10 days of being separated by between two experiments.The Induction exposure experimental technique is front 24h preserved skin, cut off Cavia porcellus spinal column each 2cm of both sides * 2cm zone by hair.Positive controls: get 2.5%2, the acetone soln 0.2g of 4-dinitro-chloro-benzene, use respectively two-layer gauze (2.5cm * 2.5cm) cover with one deck antistaling film, cover 6h with non-stimulated gauze, remove covering, remove residual given the test agent with warm water, the 7th day and the 14th day, in kind repeat once; Be coated with distilled water 0.4ml in negative control treated animal same area, the pharmaceutical composition group is coated with 0.5ml.Carry out Induction exposure by same step.Exciting contact is after last is to the tested material sensitization contact 14 days (namely testing the 28th day), at experimental group and negative control group guinea pig back preserved skin place's coating 0.5ml, package and fixing, remove tested material after 6h, observe at once, then again observe the skin allergy situation in 24h, 48h, 72h.Positive controls is coated with distilled water 0.4ml with the acetone soln 0.2g of 2.5%2,4-dinitro-chloro-benzene, negative control treated animal same area.24h and 48h observe administration local skin erythema, edema and other abnormal responses at 1h, 24h after sensitization and after exciting.By the skin allergy standards of grading, skin erythema and edema are marked, and calculate the anaphylaxis incidence rate of each group, press skin hypersensitivity evaluation criterion judgement anaphylaxis occurrence degree.Anaphylaxis incidence rate=(occurring the number of animals of skin erythema, edema or systemic anaphylaxis/this treated animal sum in group) * 100%.
3 results
excite contact experiment rear 24, 48h, by the professional who does not know experiment content according to " chemicals zest, anaphylaxis and hemolytic investigative technique guideline " pass judgment on sensitization of skin reaction (table 5) whether occurs, sensitivity response scoring 〉=2 timesharing, judge that this animal skin sensitivity response is positive, calculate sensitization rate (〉=2 minutes number of animals/these treated animal sum * 100%), and by " chemicals zest, anaphylaxis and hemolytic investigative technique guideline " in sensitization of skin reaction test grade scale judge that pharmaceutical composition of the present invention is to the sensitization intensity (table 5) of guinea pig skin.
Table 5 sensitization of skin extent of reaction standards of grading
At first calculate to put each observing time and respectively organize integral mean value, then calculate and observe every animal integral mean value interior every day in time limit, estimate by " chemicals zest, anaphylaxis and hemolytic investigative technique guideline ".Calculate every animal average integral every day by following formula.
Table 6 sensitization of skin extent of reaction grade scale
Excite rear 24h, 48h, the Cavia porcellus of negative control group, pharmaceutical composition group excites position skin erythema, edema situation all not to occur, other abnormal response also do not occur, and the anaphylaxis incidence rate is 0, and the anaphylaxis evaluation is and has no skin allergy.
Excite rear 24h, the positive controls Cavia porcellus excites position skin erythema all to occur, and the anaphylaxis incidence rate is 100%, and anaphylaxis is evaluated as extremely strong sensitization.Excite rear 48h, the positive controls Cavia porcellus excites position skin that erythema is still all arranged, and the anaphylaxis incidence rate is 100%, and the anaphylaxis evaluation is still extremely strong sensitization.
4 conclusions
Under this experiment condition, pharmaceutical composition the Hartley guinea pig skin is carried out 3 sensitization and excite for 1 time after have no skin allergy.
Claims (7)
1. pharmaceutical composition with skin whitening effect is characterized in that: be to be made by the raw material of following weight proportioning:
1～60 part of firethorn fruit
1～60 part, Radix Glycyrrhizae
1～60 part, Fructus Mori
1～60 part of Radix Angelicae Sinensis
1～60 part of Herba Saxifragae
2. the pharmaceutical composition with skin whitening effect according to claim 1 is characterized in that: be to be made by the raw material of following weight proportioning:
1～50 part of firethorn fruit
1～50 part, Radix Glycyrrhizae
1～50 part, Fructus Mori
1～50 part of Radix Angelicae Sinensis
1～50 part of Herba Saxifragae
3. according to claim 1-2 described preparation methoies with pharmaceutical composition of skin whitening effect is characterized in that: comprise the following steps:
The first step: be 20%～80% soak with ethanol with above-mentioned Chinese prescription volumetric concentration, the ethanol consumption that soaks use is 10～200 times of Chinese prescription gross weight, gets soak;
Second step: soak is concentrated into 2～5% of former weight after filtering, and concentrated solution macroporous resin adsorption, then the ethanol of using volumetric concentration 5%～95% eluting are repeatedly collected eluent, and eluent comprises the compositions of Chinese medicine extract.
4. according to claim 3 have U.S. from the preparation method of the pharmaceutical composition of skin effect, and it is characterized in that: in the first step, the described soak with ethanol time is 2～48 hours; Or Chinese prescription first used soak with ethanol 2～48 hours, and residue was used soak with ethanol 2～48 hours again, merged the solution after soaking, and got soak.
5. the preparation method with pharmaceutical composition of skin whitening effect according to claim 3, it is characterized in that: in second step, described eluting is 40%～95% with the volumetric concentration of ethanol.
6. the purposes of the pharmaceutical composition with skin whitening effect that makes of according to claim 3-5 described methods, is characterized in that: for the preparation of skin care cosmetics, comprise cream, emulsion, astringent, gel, facial film, liniment and lotion.
7. the purposes with pharmaceutical composition of skin whitening effect according to claim 6, it is characterized in that: with the pharmaceutical composition that makes through being evaporated to 10%～50% of former weight, obtain composition concentrated solution, for the preparation of skin care cosmetics, wherein, described composition concentrated solution accounts for 5%～10% of cosmetics gross weight.
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| CN105055667A (en) * | 2015-09-06 | 2015-11-18 | 何黎 | Composite whitening composition containing Yunnan mountain tea extract, preparation and preparation methods of composition and preparation |
| CN105169064A (en) * | 2015-10-11 | 2015-12-23 | 淄博齐鼎立专利信息咨询有限公司 | Medicinal preparation for whitening skin |
| CN106236634A (en) * | 2016-08-19 | 2016-12-21 | 蚌埠团结日用化学有限公司 | Draft Chinese medicine face guard frost |
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| CN105055667B (en) * | 2015-09-06 | 2017-12-05 | 何黎 | A kind of composite whitening composition of the leaf extract of camellia containing Yunnan, preparation and preparation method thereof |
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| CN106236634A (en) * | 2016-08-19 | 2016-12-21 | 蚌埠团结日用化学有限公司 | Draft Chinese medicine face guard frost |
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| CN108721179B (en) * | 2017-04-25 | 2021-01-12 | 上海珈凯生物科技有限公司 | Plant extract composition with whitening effect, preparation method and application |
| CN107574044A (en) * | 2017-08-25 | 2018-01-12 | 薛建萍 | A kind of handmade soap of the extract containing Rocket ram jet and preparation method thereof |
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| CN108143683A (en) * | 2018-01-12 | 2018-06-12 | 福建师范大学 | A kind of plant composition with white-skinned face function and preparation method thereof |
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