A traditional Chinese medicine composition with whitening and freckle-removing effects, preparation method and application thereof

Abstract

The present invention discloses a Chinese medicine composition with whitening and freckle removal effects, a preparation method and application. The Chinese medicine composition is composed of a peony bark extract, a gallnut extract and a breviscapus breviscapus extract in a mass ratio of 2 to 4:1 to 2:1. Combined with an optimized extraction process, efficient extraction and purification of active ingredients are achieved to obtain a Chinese medicine composition that is in the form of a yellow-white powder, has high clarity after dissolution, has no obvious odor, has a low ion concentration, and is safe and non-irritating. The Chinese medicine composition not only significantly reduces the impurity content and solves the problem of "coarse and black" Chinese medicine extracts, but also achieves synergistic enhancement between the ingredients through the scientific ratio of the three extracts, significantly enhancing the whitening and freckle removal skin care effects. The Chinese medicine composition of the present invention has excellent whitening and freckle removal effects, good adaptability and natural non-irritating properties, can be widely used in a variety of cosmetic dosage forms, and provides a new raw material selection for the cosmetics industry.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and in particular, relates to a traditional Chinese medicine composition with whitening and freckle-removing effects, a preparation method and application thereof. Background Art

[0002] Whitening and anti-freckle products have become an important part of the cosmetics industry. When choosing products, consumers not only expect them to have significant whitening and anti-freckle effects, but also pay more attention to the safety, mildness, non-irritation of the products, and whether they are derived from natural ingredients. Chinese herbal extracts have attracted much attention in the cosmetics industry because of their natural, mild and multi-effect characteristics. However, the preparation processes of many Chinese herbal extracts on the market are relatively simple, such as direct grinding into powder, simple water boiling and filtration or alcohol extraction and filtration, etc. These methods result in low content of active ingredients in the extracts and contain a large amount of impurities, such as pigments, cellulose, starch, etc. These impurities not only affect the appearance and texture of the product, but also may weaken its application efficacy, and even irritate the skin due to excessive impurity content, causing allergic reactions. In addition, the composition of Chinese medicine is complex. How to effectively enrich its effective ingredients, remove ineffective ingredients to reduce the dosage, and ensure the stability and safety of Chinese herbal extracts has become the key to current research and development. At the same time, the color, smell, clarity, ion content and skin feel of Chinese herbal extracts will directly affect their adaptability in cosmetics.

[0003] For example, invention patent CN 102293733 B discloses an invention called "whitening and freckle removal skin care product". The Chinese medicinal raw materials of this product mainly include ethanol extract concentrates of Angelica dahurica, Panax notoginseng, Erigeron breviscapus and white Poria cocos. Its preparation process is relatively simple, and the main steps include ethanol extraction, activated carbon decolorization and concentration to the same weight as the raw material. However, due to the relatively extensive preparation process, the amount of the obtained concentrate is large, and the content of ineffective ingredients is high. In order to ensure the use effect of the product, the amount added to the cosmetics needs to reach 10-20%, which is a relatively high proportion. Despite the decolorization step, the color of the concentrate is still dark due to the lack of further refining process. Therefore, the concentrate has the problem of "coarse and black" (i.e. large amount, dark color, and not delicate appearance), which is not conducive to the production of cosmetics and has poor adaptability.

[0004] For another example, invention patent CN 102028752 B discloses a Chinese herbal compound freckle-removing and whitening composition and its external preparation. The composition is composed of Chinese herbal medicines such as Poria cocos, Salvia miltiorrhiza, Chinese Angelica sinensis and Leonurus japonicus. Among them, Poria cocos and Leonurus japonicus are extracted by water, precipitated with alcohol, filtered, concentrated and dried; while Salvia miltiorrhiza and Chinese Angelica sinensis are extracted by alcohol, filtered, concentrated and dried. This invention still has the problem of relatively simple preparation process, resulting in the disadvantages of darker color and stronger smell of the composition, as well as poor compatibility with other cosmetic ingredients.

[0005] In summary, at present, there are some common problems in the application of Chinese herbal extracts in cosmetics, such as too many ineffective ingredients, dark color, strong odor, high ion concentration, and high irritation. These problems not only weaken the efficacy of Chinese herbal extracts in cosmetics, but also pose challenges to their safety and adaptability, thereby limiting their wide application in the field of cosmetics. In order to cope with the deficiencies of the prior art and meet the needs of consumers, it is urgent to provide a Chinese herbal extract with high clarity after dissolution, no obvious odor, low ion content, and safety and non-irritation, which can be widely used in the field of cosmetics.

[0006] In whitening and freckle removal products, peony bark, gallnut and Erigeron breviscapus have been used, such as the invention contents involved in invention patents CN105342933B, CN102283793B and CN101125155B, but these patents mainly focus on the application of single medicinal materials or compositions composed of other medicinal materials. CN105342933B discloses a Chinese medicine compound cleansing effervescent tablet containing components such as mung beans, tribute chrysanthemum, Poria cocos, licorice, and peony bark and a preparation method thereof, and the compound has the effects of cleaning and moisturizing the skin, removing oily shine, inhibiting bacteria, whitening and removing freckles, etc. CN102283793B discloses a Chinese medicine compound with whitening function made of raw materials such as gallnut charcoal, Phellodendron chinense, and Lithospermum officinale, and its preparation method and application. Experimental results show that the compound can effectively inhibit tyrosinase activity, inhibit melanogenesis, inhibit cell proliferation and promote cell apoptosis. CN101125155B discloses the application of methanol extract of Erigeron breviscapus in skin whitening and freckle prevention and treatment. Experiments have shown that the extract can effectively inhibit tyrosinase activity and melanin production, and has no side effects on the skin. It can whiten tanned skin and prevent, improve and treat various skin pigmentation symptoms caused by sun exposure.

[0007] Although these patents respectively demonstrate the effects of Paeonia suffruticosa, Galla chinensis and Erigeron breviscapus in whitening and removing freckles, to date, there has been no report on the compounding of Paeonia suffruticosa extract, Galla chinensis extract and Erigeron breviscapus extract to form a synergistic enhanced composition and its successful application in cosmetics with whitening and freckle-removing effects. Summary of the invention

[0008] The purpose of the present invention is to overcome the above defects and deficiencies in the prior art and provide a Chinese medicine composition with whitening and freckle removal effects.

[0009] The second object of the present invention is to provide a method for preparing the traditional Chinese medicine composition.

[0010] The third object of the present invention is to provide an application of the Chinese medicine composition.

[0011] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0012] The present invention first provides a traditional Chinese medicine composition, which is composed of a peony bark extract, a gallnut extract and a breviscapus breviscapus extract in a mass ratio of 2-4:1-2:1; the peony bark extract is obtained by mixing extracts obtained by sequentially extracting volatile oil compounds and moutan bark total glycosides from the peony bark; the gallnut extract and the breviscapus breviscapus extract are obtained by respectively extracting with an alcohol solvent and then decolorizing and purifying them.

[0013] The present invention is based on the theory of traditional Chinese medicine and the effects and properties of the chemical components of traditional Chinese medicine. Three medicinal materials, namely, peony bark, gallnut and breviscapus, are selected from traditional Chinese medicinal materials to form a formula. A traditional Chinese medicine composition with whitening and freckle-removing effects is prepared through the steps of extraction, filtration, purification, refinement, inclusion, drying and mixing.

[0014] The peony bark described in the present invention is the dried root bark of Paeonia suffruticosa Andr., a plant of the Ranunculaceae family. The gallnut is the galls on the leaves of Rhus chinensis Mill., Rhus potaninii Maxim. or Rhus punjabensis Stew.var.sinica (Diels) Rehd.et Wils., a plant of the Anacardiaceae family. The breviscapus is the dried whole herb of Erigeron breviscapus (Vant.) Hand.-Mazz., a plant of the Asteraceae family.

[0015] As the main drug of this prescription, peony bark has the effects of clearing heat and cooling blood, promoting blood circulation and removing blood stasis. It can clear the heat of the blood and prevent the formation of spots caused by blood heat. At the same time, its effect of promoting blood circulation and removing blood stasis helps to improve skin microcirculation, promote pigment metabolism, and reduce the deposition of spots. Peony bark mainly contains chemical components such as paeonol and paeoniflorin. Paeonol can inhibit the production and transportation of melanin and reduce the formation of spots. Peaeoniflorin has the effects of improving microcirculation and promoting blood flow, which helps to accelerate pigment metabolism. Gallnut has the effects of astringent hemostasis, antibacterial and antibacterial, and antioxidant. The ingredients such as tannic acid and gallic acid in gallnut have a strong astringent effect, which can tighten the skin and reduce enlarged pores. At the same time, its antibacterial and antibacterial effects help reduce skin inflammation and promote skin health. The antioxidant effect can remove free radicals and reduce skin aging. As an adjuvant, gallnut can enhance the antibacterial and antibacterial and antioxidant effects of the prescription, thereby further improving the skin condition and reducing the formation of spots. Breviscapus has the effects of promoting blood circulation and removing blood stasis, and improving microcirculation. Flavonoids such as breviscapine in Breviscapus can improve microcirculation, promote blood flow, thereby accelerating pigment metabolism and reducing the formation of spots. As an adjuvant, Breviscapus can enhance the effects of promoting blood circulation and removing blood stasis and improving microcirculation of this prescription, thereby accelerating pigment metabolism and improving whitening and freckle removal effects. In short, the Chinese medicine composition provided by the present invention combines the advantages and effects of three medicinal materials: Paeonia suffruticosa, Galla chinensis and Breviscapus. According to the theory of traditional Chinese medicine, this prescription has the effects of clearing heat and cooling blood, promoting blood circulation and removing blood stasis, and astringing and stopping bleeding, and can effectively improve skin microcirculation and promote pigment metabolism. From the perspective of chemical composition, the active ingredients in this prescription can inhibit the formation and transport of melanin, scavenge free radicals, exert antibacterial and antibacterial effects, and thus maintain skin health. In summary, this prescription can achieve a significant effect of whitening and freckle removal as a whole.

[0016] The Chinese medicine composition of the present invention is composed of Paeonia suffruticosa extract, Galla chinensis extract and Erigeron breviscapus extract in a scientific mass ratio (mass ratio of 2 to 4: 1 to 2: 1). Specifically, the present invention realizes the efficient extraction, enrichment and purification of the active ingredients in these three Chinese medicinal materials through an optimized extraction process, and obtains a Chinese medicine composition that is in the form of a yellow-white powder, has high clarity after dissolution, has no obvious odor, has a low ion content, and is safe and non-irritating. Compared with the prior art, the preparation process of the present invention is not only green and environmentally friendly, but also significantly reduces the impurity content through fine processing, successfully solving the problems of dark color and many impurities in most Chinese medicine extracts. The obtained Chinese medicine composition is in the form of a yellow-white powder, has high clarity after dissolution, has no obvious odor, has a low ion content, and is safe and non-irritating. Furthermore, by conducting tyrosinase inhibition and human whitening and freckle removal efficacy evaluation tests on a single Chinese herbal extract, a composition of two-by-two Chinese herbal extracts, and a composition of the other three Chinese herbal extracts in different proportions, the results showed that the composition of the three Chinese herbal extracts in precise proportions achieved synergistic enhancement among the ingredients, significantly enhancing multiple skin care effects such as whitening and freckle removal. In summary. The Chinese herbal composition provided by the present invention has excellent whitening and freckle removal efficacy, good adaptability, and natural non-irritating properties, and is very suitable for wide application in the field of cosmetics. The Chinese herbal composition can be flexibly applied to a variety of cosmetic dosage forms such as water-based agents, emulsions, creams, and essences, providing a new choice for the cosmetics industry.

[0017] Furthermore, the extraction of the volatile oil compounds is performed by crushing the peony bark, moistening it, and steam distilling it to obtain the volatile oil, and then dehydrating and refining it, and encapsulating it with β-cyclodextrin to obtain the volatile oil compound extract; the peony bark total glycosides substance is obtained by taking the peony bark medicinal material after steam distillation, and subjecting it to hydrothermal extraction and purification to obtain the peony bark total glycosides substance extract.

[0018] Preferably, the parameters of the steam distillation are to control the steam flow rate at 1 to 3 liters per minute, the distillation temperature at about 100° C., and the distillation time at 3 to 5 hours.

[0019] Preferably, the dehydration refining is carried out using anhydrous sodium sulfate.

[0020] Preferably, the β-cyclodextrin inclusion is carried out using β-cyclodextrin equivalent to 8 to 12 times the mass of volatile oil.

[0021] Preferably, the hydrothermal extraction is performed by hydrothermal reflux extraction at normal pressure twice, followed by filtration and combining the filtrates to obtain a preliminary extract of total glycosides of Paeonia lactiflora.

[0022] Preferably, the purification is performed using a macroporous resin. Further preferably, the macroporous resin is D101 macroporous resin.

[0023] Preferably, the purification is to pour the extract after hydrothermal extraction into a column filled with pre-treated macroporous resin, retain the drug solution in the column for half an hour, and then wash away the unabsorbed components and impurities such as sugars with 3-5BV of distilled water and 3-5BV of 15-25% ethanol in sequence, with an elution flow rate of 0.5BV / h. Then desorb with 55-75% ethanol solution, with an elution flow rate of 0.5BV / h, collect 55-75% ethanol desorption solution, concentrate the collected 55-75% ethanol desorption solution, remove ethanol and water, and obtain a concentrated solution; dry the concentrated solution to constant weight.

[0024] Furthermore, the gallnut extract is prepared by extracting the gallnut medicinal material with ethanol and then adsorbing and purifying it using a combination of granular white clay and carrier activated carbon.

[0025] Furthermore, the preparation of the Breviscapus breviscapus extract is to obtain a Breviscapus breviscapus extract by reflux extraction with ethanol, and then to obtain the extract by decolorization with activated carbon and purification by macroporous resin adsorption.

[0026] Preferably, the Erigeron breviscapus extract is obtained by extracting twice with ethanol under normal pressure and heating under reflux, filtering, and combining the filtrates.

[0027] Preferably, the amount of activated carbon used for decolorization is 1-3% of the mass of the Erigeron breviscapus extract.

[0028] Preferably, the macroporous resin is D101 macroporous resin.

[0029] Preferably, the macroporous resin adsorption purification is to pour the decolorized Breviscapus extract into a column filled with pretreated macroporous resin at a flow rate of 1.0 BV / h, retain the liquid in the column for half an hour, then elute with 3-5 BV of distilled water at a flow rate of 0.5 BV / h, discard the water eluate, and then elute with 3-5 BV of 45-65% ethanol at a flow rate of 2 BV·h -1 , collect 45-65% ethanol eluate, recover ethanol, and concentrate until the concentrate: medicinal material (mass ratio) is 1:2-5 to obtain a concentrate. Centrifuge the concentrate twice to obtain a precipitate, and obtain the Breviscapus breviscapus extract.

[0030] The present invention provides use of any of the above-mentioned traditional Chinese medicine compositions in the preparation of whitening and anti-freckle cosmetics.

[0031] Furthermore, the cosmetic is an aqueous solution, emulsion, cream or essence.

[0032] The present invention provides a cosmetic comprising any one of the above-mentioned traditional Chinese medicine compositions.

[0033] Furthermore, the mass proportion of the traditional Chinese medicine composition in the cosmetic is 1 to 10%.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. The present invention first provides a traditional Chinese medicine composition, which is composed of a peony bark extract, a gallnut extract and a breviscapus extract in a mass ratio of 2 to 4:1 to 2:1. In the traditional Chinese medicine composition, the peony bark has the effects of clearing heat and cooling blood, promoting blood circulation and removing blood stasis, the gallnut has the effects of astringent hemostasis, antibacterial and antibacterial, and antioxidant, and the breviscapus can improve microcirculation and promote blood flow. The present invention achieves synergistic enhancement among the components by accurately proportioning the extracts of these three traditional Chinese medicines. The combination of these three traditional Chinese medicine extracts in a precise proportion has a whitening and freckle removal effect that is significantly better than using only one or two of them.

[0036] 2. The preparation process of the Chinese medicine extract of the present invention can efficiently extract and purify the active ingredients in the three medicinal materials, significantly reduce the impurity content, and the resulting composition has strong adaptability. The optimized extraction process realizes the efficient extraction and purification of the active ingredients of Chinese medicine. The resulting Chinese medicine composition not only significantly reduces the impurity content, such as pigments, cellulose, starch, etc., but also successfully solves the problems of deep color, many impurities, and strong odor that are common in existing Chinese medicine extracts. The composition is in the form of a yellow-white powder, has high clarity after dissolution, no obvious odor, low ion concentration, mild and non-irritating, thereby greatly improving its adaptability and application effect in cosmetics.

[0037] 3. The Chinese medicine composition of the present invention can be applied to cosmetics of different dosage forms such as water-based, emulsions, creams, essences, etc., and has a wide range of uses. The Chinese medicine composition of the present invention is safe and non-irritating, and is suitable for use on various skin types. Its natural and non-irritating properties make the composition have a wide range of application prospects in the field of cosmetics. Whether it is a variety of cosmetic dosage forms such as water-based, emulsions, creams or essences, the Chinese medicine composition can be flexibly added to enhance the whitening and freckle-removing effect and overall quality of the product. In summary, the technical solution of the present invention not only solves the deficiencies of the prior art, but also provides a new raw material selection for the cosmetics industry, and has extremely high application value and market potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a powder diagram of the Chinese medicine composition prepared in Examples 1 to 5. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0040] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0041] Example 1

[0042] 1. Preparation of Paeonia suffruticosa Extract

[0043] The main chemical components of peony bark include volatile oils, glycosides, alkaloids and terpenes, among which volatile oils and total glycosides of paeonol (represented by paeoniflorin) are particularly rich. The main component of volatile oil is paeonol, accounting for up to 88%. Studies have shown that paeonol can effectively absorb UVB ultraviolet rays, so it has an excellent sunscreen effect. At the same time, paeonol also has strong antioxidant properties, can remove skin free radicals, and inhibit skin aging caused by free radicals. In addition, paeonol also has significant antibacterial and anti-inflammatory effects, can effectively inhibit a variety of bacteria and fungi such as Escherichia coli and Staphylococcus aureus, and can reduce the level of skin inflammation, inhibit the expression of inflammatory factors, and has a good preventive and therapeutic effect on skin diseases such as eczema and dermatitis. On the other hand, total glycosides of paeonol can inhibit the activity of tyrosinase and reduce the production of melanin in the skin, thereby achieving a whitening effect. At the same time, total glycosides of paeonol can also significantly relieve the itching and swelling of organisms in the acute stage, and have the effect of antipruritic and anti-inflammatory. It can be used as a soothing ingredient to effectively relieve inflammatory symptoms such as skin redness, pain and itching.

[0044] Therefore, the preparation process takes into account both the volatile oil and the total glycosides of Paeonia lactiflora. Considering that the volatile oil is insoluble in water and is not conducive to mixing with the total glycosides of Paeonia lactiflora, Galla chinensis extract, and Erigeron breviscapus extract, the volatile oil is subjected to β-cyclodextrin inclusion treatment. Specifically, the process includes the following steps:

[0045] 1.1 Preparation, purification and inclusion of volatile oil compounds mainly based on paeonol

[0046] (1) Medicinal material processing

[0047] Accurately weigh 1 part of the medicinal material of Paeonia suffruticosa, and crush it into coarse powder so that water vapor can better penetrate and the volatile oil can be released more easily. Add an appropriate amount of water (about 1 to 2 times the weight of the medicinal material) to the crushed medicinal material, and moisten it for 1.5 hours to allow the medicinal material to fully absorb water and swell, which is convenient for the extraction of volatile oil during steam distillation.

[0048] (2) Steam distillation

[0049] The moistened medicinal materials are placed in a distillation device, heated to allow water vapor to pass through the medicinal material layer, and the volatile oil components in the medicinal materials are distilled out. During the distillation process, the water vapor flow rate is controlled at 1 liter / minute, the distillation temperature is controlled at about 100°C, and the distillation time is 3 hours, until the amount of distilled volatile oil no longer increases significantly. The distilled steam is condensed into liquid through a condenser and collected in a separator. In the separator, the volatile oil and water layers are separated, with the upper layer being volatile oil and the lower layer being water. The upper layer of volatile oil is collected for the next step of refining.

[0050] (3) Volatile oil refining

[0051] Add 10% of the mass of the volatile oil in anhydrous sodium sulfate for dehydration and purification, and obtain refined volatile oil after filtration. The refined volatile oil is clear and transparent without peculiar smell.

[0052] (4) β-cyclodextrin inclusion

[0053] The refined peony bark volatile oil was mixed evenly with an appropriate amount of 95% ethanol to prepare a volatile oil ethanol solution. β-cyclodextrin equivalent to 8 times the mass of the volatile oil was weighed and dissolved in an appropriate amount of distilled water to prepare a saturated solution. The volatile oil ethanol solution was slowly added dropwise to the β-cyclodextrin solution, and stirring was maintained during the addition process to ensure sufficient mixing. After that, the entire mixed solution was transferred to an ultrasonic cleaner and ultrasonically treated for 30 minutes at an ultrasonic power of 150 watts and a temperature of 30°C to promote further mixing and dispersion of the solution. After the treatment, the product was placed in a 4°C refrigerator for 24 hours. Afterwards, suction filtration was performed, and the filter cake was washed with an appropriate amount of 95% ethanol to remove the unencapsulated volatile oil and impurities. Finally, the obtained precipitate was placed in a vacuum drying oven and dried at 60°C for 2 hours to obtain a β-cyclodextrin-encapsulated peony bark volatile oil product.

[0054] 1.2 Preparation and purification of extracts based on total glycosides of Paeonia lactiflora

[0055] (1) Distilled water reflux extraction

[0056] The above-mentioned Paeonia suffruticosa root medicinal material from which the volatile oil has been extracted is transferred to a reflux extraction device, and distilled water 10 times the weight of the medicinal material is added, and reflux extraction is performed twice under normal pressure and 100°C, and each extraction time is 1 hour. The extracts from the two extractions are combined, filtered to remove the medicinal material residue, and finely filtered through a 0.45 μm filter membrane to obtain a preliminary Paeonia suffruticosa root total glycoside extract.

[0057] (2) Purification by D101 macroporous resin

[0058] Take 10 times the amount of D101 macroporous resin of the quality of the peony bark medicinal material, clean it with distilled water, then soak it with 95% ethanol for 24 hours, fully soak and swell, wet-load the column, and then rinse it with distilled water until there is no ethanol taste, and set it aside. The preliminary total glycosides of Paeonia suffruticosa peony extract is slowly poured into the column equipped with the pre-treated macroporous resin D101 at a flow rate of 1.0BV / h, and the liquid is retained in the column for half an hour to ensure that the liquid can be evenly distributed on the resin surface. Then, 3BV of distilled water and 3BV of 15% concentration of ethanol are used to wash away impurities such as unabsorbed components and sugars, and the elution flow rate is 0.5BV / h. Then desorb with 55% ethanol solution, and the elution flow rate is 0.5BV / h, collect 55% ethanol desorption liquid, and concentrate the collected 55% ethanol desorption liquid to remove ethanol and water to obtain a concentrated solution. The concentrated solution is then placed in a vacuum drying oven and dried at 60°C to a constant weight, thereby obtaining a refined extract mainly containing total glycosides of Paeonia lactiflora.

[0059] 1.3 Mixing

[0060] The above-mentioned "β-cyclodextrin-encapsulated peony bark volatile oil product" and the "peony bark extract mainly containing peony bark total glycosides" are mixed evenly to obtain the peony bark extract.

[0061] 2. Preparation of Galla Chinensis Extract

[0062] The main components of gallnut are natural polyphenol compounds, including tannins and gallic acid. These natural polyphenol compounds have unique chemical and physiological activities and play multiple roles in skin care products. They can astringe the skin, resist oxidation, resist aging, resist UV rays, whiten and moisturize. In addition, these compounds also show unique effects on skin aging problems caused by various factors, such as wrinkles and pigmentation.

[0063] The content of natural polyphenol compounds in gallnut is very high, accounting for about 60% to 70%, sometimes even up to 80%. Due to such a rich content, there is no need for excessive enrichment and purification during the process. Based on this feature, this preparation process mainly uses physical adsorption for decolorization. Specifically, it includes the following steps:

[0064] (1) Preparation of Gallnut Extract

[0065] Accurately weigh 1 part of Gallic acid medicinal material, grind it into coarse powder, add 5 times the amount of 95% edible alcohol, stir and soak at 60℃ for 2 hours, filter to remove the Gallic acid residue, and obtain the Gallic acid extract.

[0066] (2) Activation of granular clay and functionalization of carrier activated carbon

[0067] Accurately weigh 0.5 parts of granular clay, heat it in an oven at 160°C with forced air for 2 hours to activate it, and then place it in a desiccator to cool.

[0068] Accurately weigh 0.5 parts of carrier activated carbon, treat it with chelating agent EDTA-2Na by impregnation method to prepare loaded activated carbon.

[0069] (3) Combined adsorption and purification of granular clay and carrier activated carbon

[0070] The activated granular clay and the functionalized carrier activated carbon are respectively loaded into the chromatography column to form an adsorption column system, and a constant temperature heating system and a conveying system are installed to form a closed loop with the adsorption column system. The gallnut extract is preheated to 60°C and conveyed by a peristaltic pump to flow through the granular clay and carrier activated carbon adsorption columns in turn, and the cycle continues for 2 hours. After the cycle is stopped, the adsorbed gallnut extract is concentrated and dried to obtain the gallnut extract. (This process is slightly modified with reference to the following literature: Xu Hao, Zhang Zonghe, Zhong Chongmao, et al. Research on purification of gallnut tannin by physical adsorption [J]. Chemistry and Industry of Forest Products, 2008, 28(06): 79-82.)

[0071] 3. Preparation of Erigeron Breviscapus Extract

[0072] Brevifolia mainly contains flavonoids, the most important of which is scutellarin, including scutellarin A and scutellarin B (scutellarin). Studies have shown that scutellarin is the most critical active ingredient in Brevifolia, and its content accounts for more than 90% of the total flavonoids in Brevifolia extract. Studies have also found that flavonoids in Brevifolia exhibit significant free radical scavenging and antioxidant activities. In cosmetic applications, they can effectively reduce the damage to the skin caused by sunlight, prevent and delay the process of skin sagging and aging. In addition, scutellarin B also has multiple activities such as improving microcirculation, inhibiting platelet aggregation, promoting fibrinolytic activity, anti-thrombosis, and promoting blood flow. These properties help accelerate pigment metabolism, thereby reducing the formation of spots.

[0073] In view of these important effects of flavonoids from Erigeron breviscapus, this preparation process mainly focuses on extracting total flavonoids from Erigeron breviscapus and performing decolorization and purification. The specific steps include:

[0074] (1) Extraction of medicinal materials

[0075] Grind the medicinal material of Breviscapus breviscapus into coarse powder, accurately weigh 1 portion, add 10 times the mass of 65% ethanol, and perform reflux extraction twice under normal pressure and 100°C, with each extraction time being 2 hours. Filter the two extracts, combine the filtrates, recover the ethanol and concentrate until there is no alcohol taste, thus obtaining the Breviscapus breviscapus extract for later use.

[0076] (2) Activated carbon decolorization

[0077] Take the above Breviscapus breviscapus extract, add 1% activated carbon by weight of the extract, mix well, boil for 30 minutes, and filter to obtain Breviscapus breviscapus decolorized filtrate.

[0078] (3) Macroporous resin purification

[0079] The D101 macroporous resin with 10 times the quality of the drug material of the scutellaria breviscapus was cleaned with distilled water, and then soaked in 95% ethanol for 24 hours. After being fully soaked and swollen, the column was wet-loaded, and then rinsed with distilled water until there was no ethanol smell, and it was set aside. The above-mentioned scutellaria breviscapus decolorization filtrate was slowly poured into the column containing the pre-treated macroporous resin D101 at a flow rate of 1.0BV / h. The drug solution was retained in the column for half an hour to ensure that the drug solution could be evenly distributed on the resin surface. Then 3BV of distilled water was used for elution, and the elution flow rate was 0.5BV / h. The water eluent was discarded, and then 3BV of 45% ethanol was used for elution, and the elution flow rate was 2BV·h-1. The 45% ethanol eluent was collected, the ethanol was recovered, and concentrated to a concentrated solution: drug material (mass ratio) of 1:2 to obtain a concentrated solution. The above concentrate was placed in a 4°C refrigerator for 12 hours, and then centrifuged at 4000 r / min for 15 min to obtain a precipitate. Distilled water 3 times the mass of the precipitate was added and stirred evenly. The concentrate was centrifuged at 4000 r / min for 15 min, and the supernatant was discarded. The operation was repeated until the supernatant was clear, and the precipitate was collected to obtain the Erigeron breviscapus extract.

[0080] 4. Mixed preparation of three Chinese herbal medicine extracts

[0081] The above-mentioned Paeonia suffruticosa root extract, Galla chinensis extract and Erigeron breviscapus flower extract are uniformly mixed in a weight ratio of 4:1:1 to obtain a whitening and freckle-removing traditional Chinese medicine composition.

[0082] Example 2

[0083] 1. The preparation methods of Paeonia suffruticosa Andrews extract, Galla chinensis extract and Erigeron breviscapus extract are exactly the same as those in Example 1.

[0084] 2. Mixed preparation of three Chinese herbal medicine extracts

[0085] The above-mentioned Paeonia suffruticosa root extract, Galla chinensis extract and Erigeron breviscapus flower extract are uniformly mixed in a weight ratio of 4:2:1 to obtain a whitening and freckle-removing Chinese medicinal composition.

[0086] Example 3

[0087] 1. The preparation method of the peony bark extract is basically the same as that of the peony bark extract in Example 1, except that:

[0088] In the preparation, purification and inclusion of volatile oil compounds mainly composed of paeonol, the parameters of step (2) steam distillation are to control the steam flow rate at 2 liters / minute, the distillation temperature at about 100°C, and the distillation time for 4 hours; in step (4) β-cyclodextrin inclusion, β-cyclodextrin equivalent to 10 times the mass of the volatile oil is weighed for inclusion.

[0089] In the preparation and purification of the extract mainly composed of total glycosides of Paeonia lactiflora, the parameters for elution, recovery and concentration of the drug solution in step (2) D101 macroporous resin refining and purification are:

[0090] 4 BV of distilled water and 4 BV of 20% ethanol were used to wash away the unabsorbed components and impurities such as sugars, with an elution flow rate of 0.5 BV / h. Then, 65% ethanol solution was used for desorption, with an elution flow rate of 0.5 BV / h, and the 65% ethanol desorption solution was collected, and the collected 65% ethanol desorption solution was concentrated to remove ethanol and water to obtain a concentrated solution.

[0091] 2. The preparation method of the gallnut extract is exactly the same as that of the gallnut extract in Example 1.

[0092] 3. The preparation method of the Breviscapus breviscapus extract is basically the same as the preparation method of the Breviscapus breviscapus extract in Example 1, except that:

[0093] In step (1), 10 times the mass of 75% ethanol is added during the extraction of medicinal materials; and in step (2), 2% the mass of activated carbon is added during the decolorization of activated carbon.

[0094] Step (3) The parameters for eluting, recovering and concentrating the Chinese medicine solution by macroporous resin refining and purification are:

[0095] Elute with 4 BV of distilled water at a flow rate of 0.5 BV / h, discard the water eluent, and then elute with 4 BV of 55% ethanol at a flow rate of 2 BV·h -1 , collect 55% ethanol eluate, recover ethanol, and concentrate until the concentrate: medicinal material (mass ratio) is 1:3 to obtain a concentrate.

[0096] 4. Mixed preparation of three Chinese herbal medicine extracts

[0097] The above-mentioned Paeonia suffruticosa root extract, Galla chinensis extract and Erigeron breviscapus flower extract are mixed evenly in a weight ratio of 3:2:1 to obtain a whitening and freckle-removing Chinese medicinal composition.

[0098] Example 4

[0099] 1. The preparation method of the peony bark extract is basically the same as that of the peony bark extract in Example 1, except that:

[0100] In the preparation, purification and inclusion of volatile oil compounds mainly composed of paeonol, the parameters of the steam distillation in step (2) are to control the steam flow rate at 3 liters / minute, the distillation temperature at about 100°C, and the distillation time for 5 hours; in the β-cyclodextrin inclusion in step (4), β-cyclodextrin equivalent to 12 times the mass of the volatile oil is weighed.

[0101] In the preparation and purification of the extract mainly composed of total glycosides of Paeonia lactiflora, the elution, recovery and concentration parameters of the purified Chinese medicine solution by D101 macroporous resin in step (2) are:

[0102] 5BV of distilled water and 5BV of 25% ethanol were used to wash away the unabsorbed components and impurities such as sugars, with an elution flow rate of 0.5BV / h. Then, 75% ethanol solution was used for desorption, with an elution flow rate of 0.5BV / h, and the 75% ethanol desorption solution was collected and concentrated to remove ethanol and water to obtain a concentrated solution.

[0103] 2. The preparation method of the gallnut extract is exactly the same as that of the gallnut extract in Example 1.

[0104] 3. The preparation method of the Breviscapus breviscapus extract is basically the same as the preparation method of the Breviscapus breviscapus extract in Example 1, except that:

[0105] In step (1), 10 times the weight of 85% ethanol is added to the extraction of medicinal materials; in step 2 (2), 3% weight of activated carbon is added to the activated carbon decolorization;

[0106] Step (3) The parameters for eluting, recovering and concentrating the Chinese medicine solution by macroporous resin refining and purification are:

[0107] Use 5 BV of distilled water for elution at a flow rate of 0.5 BV / h, discard the water eluent, and then use 5 BV of 65% ethanol for elution at a flow rate of 2 BV·h -1 , collect 65% ethanol eluate, recover ethanol, and concentrate until the ratio of concentrated solution to medicinal material (mass ratio) is 1:5 to obtain a concentrated solution.

[0108] 4. Mixed preparation of three Chinese herbal medicine extracts

[0109] The above-mentioned peony bark extract, gallnut extract and Erigeron breviscapus extract are mixed evenly in a weight ratio of 2:1:1 to obtain a whitening and freckle-removing traditional Chinese medicine composition.

[0110] Example 5

[0111] 1. The preparation method of the Paeonia suffruticosa Andrews extract is exactly the same as that of the Paeonia suffruticosa Andrews extract in Example 4.

[0112] 2. The preparation method of the gallnut extract is exactly the same as that of the gallnut extract in Example 1.

[0113] 3. The preparation method of the Erigeron breviscapus extract is exactly the same as the preparation method of the Erigeron breviscapus extract in Example 4.

[0114] 4. Mixed preparation of three Chinese herbal medicine extracts

[0115] The above-mentioned peony bark extract, gallnut extract and Erigeron breviscapus extract are mixed evenly in a weight ratio of 2:2:1 to obtain a whitening and freckle-removing traditional Chinese medicine composition.

[0116] Comparative Example 1

[0117] In this comparative example, the method steps and parameters of "preparation of gallnut extract" and "preparation of Erigeron breviscapus extract" are exactly the same as those in Example 3, and "Peony bark extract" is not prepared. In the "mixed preparation of three Chinese medicine extracts" part, the weight ratio is different from that in Example 3. Specifically, the Peony bark extract, gallnut extract and Erigeron breviscapus extract of this comparative example are mixed uniformly according to a weight ratio of 0:4:2 (no Peony bark extract is added, and the total amount of the composition is the same as that in Example 3), and the whitening and freckle-removing Chinese medicine composition of this comparative example is obtained.

[0118] Comparative Example 2

[0119] In this comparative example, the method steps and parameters of "preparation of peony bark extract" and "preparation of Erigeron breviscapus extract" are exactly the same as those in Example 3, and "gallothinus extract" is not prepared. In the "mixed preparation of three Chinese medicine extracts" part, the weight ratio is different from that in Example 3. Specifically, the peony bark extract, gallnut extract and Erigeron breviscapus extract of this comparative example are mixed uniformly according to a weight ratio of 4.5:0:1.5 (gallothinus extract is not added, and the total amount of the composition is the same as that in Example 3), and the whitening and freckle-removing Chinese medicine composition of this comparative example is obtained.

[0120] Comparative Example 3

[0121] In this comparative example, the method steps and parameters of "preparation of peony bark extract" and "preparation of gallnut extract" are exactly the same as those in Example 3, and "Erigeron breviscapus extract" is not prepared. In the "mixed preparation of three Chinese medicine extracts" part, the weight ratio is different from that in Example 3. Specifically, the peony bark extract, gallnut extract and Erigeron breviscapus extract of this comparative example are mixed uniformly according to a weight ratio of 3.6:2.4:0 (without adding Erigeron breviscapus extract, and the total amount of the composition is the same as that in Example 3), and the whitening and freckle-removing Chinese medicine composition of this comparative example is obtained.

[0122] Comparative Example 4

[0123] In this comparative example, the method steps and parameters of "preparation of Erigeron breviscapus extract" are exactly the same as those in Example 3, and "Paeonia suffruticosa extract" and "Gallac extract" are not prepared. In the "mixed preparation of three Chinese medicine extracts" part, the weight ratio is different from that in Example 3. Specifically, the Paeonia suffruticosa extract, Galllac extract and Erigeron breviscapus extract of this comparative example are mixed uniformly according to a weight ratio of 0:0:6 (only Erigeron breviscapus extract is used, and the total amount is the same as that in Example 3), and the whitening and freckle-removing Chinese medicine composition of this comparative example is obtained.

[0124] Comparative Example 5

[0125] In this comparative example, the method steps and parameters of "preparation of gallnut extract" are exactly the same as those in Example 3, and "peony bark extract" and "Erigeron breviscapus extract" are not prepared. In the "mixed preparation of three Chinese medicine extracts" part, the weight ratio is different from that in Example 3. Specifically, the peony bark extract, gallnut extract and Erigeron breviscapus extract of this comparative example are mixed uniformly according to a weight ratio of 0:6:0 (only gallnut extract is used, and the total amount is the same as that in Example 3), and the whitening and freckle-removing Chinese medicine composition of this comparative example is obtained.

[0126] Comparative Example 6

[0127] In this comparative example, the method steps and parameters of "preparation of peony bark extract" are exactly the same as those in Example 3, and "gallothinus chinensis extract" and "Erigeron breviscapus extract" are not prepared. In the "mixed preparation of three Chinese medicine extracts" part, the weight ratio is different from that in Example 3. Specifically, the peony bark extract, gallnut extract and Erigeron breviscapus extract of this comparative example are mixed uniformly according to a weight ratio of 6:0:0 (only peony bark extract is used, and the total amount is the same as that in Example 3), and the whitening and freckle-removing Chinese medicine composition of this comparative example is obtained.

[0128] Comparative Example 7

[0129] In this comparative example, the method steps and parameters of "preparation of peony bark extract", "preparation of gallnut extract" and "preparation of breviscapus extract" are exactly the same as those in Example 3. In the "mixed preparation of three Chinese medicine extracts" part, the weight ratio is different from that in Example 3. Specifically, the peony bark extract, gallnut extract and breviscapus extract of this comparative example are mixed uniformly according to a weight ratio of 5:1:1 (and the total amount of the composition is the same as that in Example 3), so as to obtain the whitening and freckle-removing Chinese medicine composition of this comparative example.

[0130] Comparative Example 8

[0131] In this comparative example, the method steps and parameters of "preparation of peony bark extract", "preparation of gallnut extract" and "preparation of breviscapus extract" are exactly the same as those in Example 3. In the "mixed preparation of three Chinese medicine extracts" part, the weight ratio is different from that in Example 3. Specifically, the peony bark extract, gallnut extract and breviscapus extract of this comparative example are mixed uniformly according to a weight ratio of 1:4:1 (and the total amount of the composition is the same as that in Example 3), so as to obtain the whitening and freckle-removing Chinese medicine composition of this comparative example.

[0132] Comparative Example 9

[0133] In this comparative example, the method steps and parameters of "preparation of peony bark extract", "preparation of gallnut extract" and "preparation of breviscapus extract" are exactly the same as those in Example 3. In the "mixed preparation of three Chinese medicine extracts" part, the weight ratio is different from that in Example 3. Specifically, the peony bark extract, gallnut extract and breviscapus extract of this comparative example are mixed uniformly according to a weight ratio of 1:1:4 (and the total amount of the composition is the same as that in Example 3), so as to obtain the whitening and freckle-removing Chinese medicine composition of this comparative example.

[0134] Application Example 1

[0135] An aqueous solution containing a whitening and freckle-removing Chinese medicine composition and a preparation method thereof:

[0136] The raw material ratio and preparation method are shown in Table 1.

[0137] Table 1 A kind of aqueous preparation raw material ratio and preparation method containing whitening and anti-freckle Chinese medicine composition

[0138]

[0139]

[0140] Application Example 2

[0141] An emulsion containing a whitening and freckle-removing Chinese medicine composition and a preparation method thereof:

[0142] The raw material ratio and preparation method are shown in Table 2.

[0143] Table 2 A kind of emulsion raw material ratio and preparation method containing whitening and anti-freckle Chinese medicine composition

[0144]

[0145]

[0146] Application Example 3

[0147] A cream containing a whitening and freckle-removing Chinese medicine composition and a preparation method thereof:

[0148] The raw material ratio and preparation method are shown in Table 3.

[0149] Table 3 Raw material ratio and preparation method of a cream containing a whitening and anti-freckle Chinese medicine composition

[0150]

[0151]

[0152] Application Example 4

[0153] An essence containing a whitening and freckle-removing Chinese medicine composition and a preparation method thereof:

[0154] The raw material ratio and preparation method are shown in Table 4.

[0155] Table 4: Raw material ratio and preparation method of an essence containing a whitening and anti-freckle Chinese medicine composition

[0156]

[0157]

[0158] Test Example 1 Skin irritation test

[0159] The Chinese medicine compositions involved in Examples 1 to 5 were tested for skin irritation, and the method and results are as follows:

[0160] 1. Test method basis: In accordance with the "Skin Irritation Test Method" in the "Technical Specifications for Safety of Cosmetics (2015 Edition)".

[0161] 2. Experimental principle: Apply the test substance once to the skin of the test animal, observe the degree of local irritation to the animal skin at a specified time interval and score it. Use self-control to evaluate the irritation of the test substance to the skin.

[0162] 3. Test samples and animals: The test substance is a 10% ethanol solution of the Chinese medicine composition involved in Examples 1 to 5. The test animals are ordinary New Zealand white rabbits purchased from Guangdong Medical Experimental Animal Center.

[0163] 4. Experimental steps:

[0164] 4.1 25 white New Zealand rabbits were selected and randomly divided into 5 groups, 5 rabbits in each group, which were respectively Groups 1 to 5 of Examples.

[0165] 4.2 About 24 hours before the test, trim the hair on both sides of the spine of the New Zealand rabbit's back without damaging the epidermis. The hair removal area is about 3cm×3cm on the left and right sides.

[0166] 4.3 Take about 0.5 mL of the test substance and apply it directly to the skin, then cover it with two layers of gauze (2.5 cm × 2.5 cm) and a layer of cellophane, and then fix it with non-irritating adhesive tape and bandage. Use the other side of the skin as a control. Use a closed test with a duration of 4 hours. After the test, use warm water to remove the residual test substance.

[0167] 4.4 Observe the skin reaction at the application site 1, 24, 48 and 72 hours after the removal of the test substance, and score the skin reaction according to Table 5. The average score of the test animals is used for comprehensive evaluation, and the skin irritation intensity is determined according to Table 6 based on the average of the highest scores at each observation time point of 24, 48 and 72 hours.

[0168] Table 5 Skin irritation reaction scores

[0169]

[0170] Table 6 Skin irritation intensity classification

[0171]

[0172] 5. Results and Analysis

[0173] Within 1 to 72 hours after administration of the Chinese medicine compositions of the embodiments of the present invention, no erythema or edema was observed on the skin of the New Zealand rabbits. According to the skin irritation intensity classification in Table 2, it is shown that the Chinese medicine compositions of the embodiments of the present invention are non-irritating to the skin.

[0174] Test Example 2: Compatibility Evaluation

[0175] The compatibility of Chinese herbal medicine extracts as cosmetic raw materials is restricted by many factors, mainly including color, solubility, odor and ionicity.

[0176] In terms of color. If the color of Chinese herbal extracts is too dark, it may affect the appearance and texture of cosmetics, making them appear less clear, which in turn affects the consumer's experience. Dark-colored extracts may contain high pigment components, which may have a potential impact on skin absorption and metabolism, reducing compatibility.

[0177] Solubility. The solubility of Chinese herbal extracts determines their dispersibility and stability in cosmetics. If the solubility is poor, the extracts may precipitate or precipitate, affecting the texture and stability of the cosmetics. In addition, solubility is closely related to the cosmetic formula design and production process. Mismatches may lead to formula instability or stratification.

[0178] Smell: Chinese herbal extracts usually have a unique smell. If the smell is too strong or irritating, it may make consumers uncomfortable and affect sensory evaluation.

[0179] Ionicity. Generally, the total concentration of water-soluble ingredients in cosmetics, including salts, minerals, metal ions, etc., should be controlled within a certain range to prevent irritation to the skin or adverse reactions. When the concentration of these ingredients is higher than this range, especially when the salt or metal ion content is too high, it may cause dry skin, dehydration, and even allergic reactions. Traditional Chinese medicine extracts may contain a certain amount of salts or metal ions. If the content is too high, it may also cause problems such as dry skin, allergies or inflammation. In a cosmetic formula, if the ion concentration is too high, it may also interfere with the stability of other ingredients, causing precipitation, stratification or discoloration of the cosmetics, ultimately affecting the texture, appearance of the product and the user's skin feel.

[0180] In summary, in order to ensure the quality and safety of cosmetics, the above factors must be considered comprehensively and sufficient testing and evaluation must be carried out when selecting and using Chinese herbal extracts.

[0181] This test example evaluates the compatibility of the Chinese medicine compositions involved in Examples 1 to 5 from four aspects: color, solubility, odor, and ion content. The method and results are as follows:

[0182] Color evaluation:

[0183] Method: The color of the Chinese medicine compositions and solutions of Examples 1 to 5 were observed by visual evaluation to evaluate the color depth.

[0184] Results: The powder colors of the Chinese medicine compositions of Examples 1 to 5 are slightly different, but the colors are all lighter, yellow-white, see Figure 1 Take 1 part of the extract powder and dissolve it in 10 times the amount of 30% ethanol. The resulting solution is clear and slightly yellowish white, which meets the requirements of cosmetics for the color of raw materials and is easy to adapt.

[0185] Solubility evaluation:

[0186] Method: The Chinese medicine compositions of Examples 1 to 5 were added into the cosmetic matrix in a certain proportion, and their dispersibility and stability were observed.

[0187] Results: The Chinese medicine compositions of Examples 1 to 5 were completely dissolved in 30 times the amount of cold water, 10 times the amount of hot water, and 10 times the amount of 30% ethanol, were evenly dispersed, and had good stability, meeting the requirements of cosmetics for the solubility of raw materials and facilitating adaptation.

[0188] Odor evaluation:

[0189] Method: 10 sensory evaluators were organized to evaluate the odor of the Chinese herbal extracts of Examples 1 to 5 and to evaluate their odor intensity.

[0190] Results: The Chinese medicine compositions of Examples 1 to 5 have a light smell, only a slight herbal smell, which is completely within the acceptable range. They meet the requirements of cosmetics for the smell of raw materials and are easy to adapt.

[0191] Ionicity evaluation:

[0192] Method: The Chinese medicine compositions of Examples 1 to 5 were dissolved in 10 times the amount of 30% ethanol respectively, and the conductivity of these solutions was measured using a conductivity meter.

[0193] In general, the conductivity of cosmetic solutions (i.e., the expression of ion concentration) should be kept within a certain range and should not be too high. Usually, formulas with too high conductivity may show discomfort to the skin, especially when the conductivity reaches above 1000μS / cm, it may cause discomfort and irritation to the skin.

[0194] Results: The conductivity of the solutions of the Chinese medicine compositions of Examples 1 to 5 were all less than 200 μS / cm, which was within the safe range, met the requirements of cosmetics for ionicity, and was conducive to adaptation. See Table 7 for details.

[0195] Table 7 Conductivity of the Chinese medicine composition solution of Examples 1 to 5 ( n=5)

[0196]

[0197] In summary, the Chinese medicine compositions of Examples 1 to 5 show good adaptability in terms of color, solubility, odor and ionicity, and are suitable for use as cosmetic raw materials.

[0198] Test Example 3 In vitro whitening and freckle removal efficacy test

[0199] In vitro whitening and freckle removal efficacy tests were performed on Examples 1 to 5 and Comparative Examples 1 to 9.

[0200] 1. Methodology

[0201] The test is carried out in accordance with "T / GDCA 006-2021 Test Method for Inhibition of Tyrosinase Activity by Cosmetic Raw Materials (In Vitro Method)".

[0202] 2. Test Principle

[0203] The production process of melanin is finely regulated by tyrosinase. Tyrosinase can catalyze the conversion of L-tyrosine into DOPA, and further oxidize DOPA into DOPAquinone. This series of reactions ultimately leads to the synthesis of melanin. Therefore, tyrosinase plays a vital role in the production of melanin. It controls the production process of melanin, and its activity level is directly related to the deposition of pigment in the skin. Inhibiting the activity of tyrosinase is of great significance in delaying the browning process of the skin and reducing the production of melanin, and is one of the effective ways to whiten the skin.

[0204] Those test substances that inhibit tyrosinase activity can effectively slow down the rate at which tyrosinase catalyzes the conversion of L-tyrosine to dopaquinone. By measuring the absorbance value of dopaquinone at a wavelength of 475nm, the inhibitory effect of the test substance on tyrosinase activity can be quantitatively evaluated. The ability of a substance to inhibit tyrosinase activity can, to a certain extent, be used as an indicator to measure its whitening potential. Specifically, the higher the degree of inhibition of tyrosinase activity, the more significant the whitening and freckle removal effect of the substance.

[0205] 3. Materials and Reagents

[0206] The Chinese medicine compositions were prepared according to the methods of Examples 1 to 5 and Comparative Examples 1 to 9, and dried and stored for later use.

[0207] Mushroom tyrosinase, purchased from Macklin, specification 25KU. L-Tyrosine, purchased from Macklin, purity ≥ 99.0%, biochemical reagent BR. Kojic acid, purchased from Macklin, purity ≥ 99.0%, biochemical reagent BR. The remaining reagents were all commercially available analytical grade. Microplate reader (Thermo scientific 1530 model).

[0208] 4. Experimental steps:

[0209] 4.1 Solution preparation

[0210] Phosphate buffer solution (PBS), pH=6.8, 0.1 mol / L: Weigh 1.361 g of potassium dihydrogen phosphate and add distilled water to a total volume of 100 ml to obtain solution A. Weigh 1.78 g of disodium hydrogen phosphate and add distilled water to 100 ml to obtain solution B. Then, mix solution A and solution B in a ratio of 1:1 to obtain a phosphate buffer solution (PBS).

[0211] L-tyrosine solution: Weigh 0.025 g of L-tyrosine, dissolve it in the PBS buffer prepared above, and perform ultrasonic treatment to promote dissolution, and finally adjust the volume to 50 ml. This solution must be prepared and used immediately to ensure its freshness and effectiveness.

[0212] Tyrosinase solution: Use PBS buffer as solvent to prepare mushroom tyrosinase to 500U / mL. After preparation, divide the solution into portions and store it at -20 degrees Celsius. At the same time, be careful to avoid secondary freezing and thawing of the solution to ensure the activity of the enzyme.

[0213] Positive control kojic acid: PBS buffer was used to dilute kojic acid into a series of mass concentration gradient solutions, with specific concentrations of 0.080 g / L, 0.040 g / L, 0.020 g / L, 0.010 g / L, 0.008 g / L, 0.005 g / L, 0.002 g / L, and 0.001 g / L.

[0214] Traditional Chinese medicine composition solution: The traditional Chinese medicine compositions in Examples 1 to 5 and Comparative Examples 1 to 9 were prepared into solutions with a concentration of 1.0 mg / mL using PBS buffer for later use.

[0215] Distilled water was used as blank control.

[0216] 4.2 Experimental Grouping

[0217] Four types of wells are set up on the 96-well ELISA plate: solvent background wells (marked as Ta), solvent reaction wells (marked as Tb), sample background wells (marked as Tc), and sample reaction wells (marked as Td). Among them, the Ta group is the solvent background group, and its operation is not to add the substrate L-tyrosine solution, and no sample solution is added at the same time; the Tb group is the solvent reaction group, and the substrate L-tyrosine solution needs to be added, but the sample solution is not added; the Tc group is set as the sample background group, and its operation is not to add the substrate L-tyrosine solution, but the sample solution is added; and the Td group is the sample reaction group, and the substrate L-tyrosine solution and the sample solution need to be added at the same time. Three replicate wells are set up for each of the above experiments to ensure the accuracy and reliability of the data.

[0218] 4.3 Test steps

[0219] According to the reagent addition amount provided in Table 8, L-tyrosine solution, sample solution / solvent and PBS buffer were added to each well in turn. After the addition is completed, the contents of each well are fully mixed and placed in a constant temperature environment of 37°C for incubation for 10 minutes. Subsequently, 20 μL of tyrosinase solution was added to each well, and the mixing reaction was continued at 37°C to ensure that the reaction time was 5min±5s. After the reaction was completed, the ELISA plate was immediately placed in the ELISA instrument to measure the absorbance values ​​of different samples and positive controls at a wavelength of 475nm. Three plate wells were set for each group for measurement, and the average value was taken. From the addition of the tyrosinase solution to the final measurement of the absorbance, the operation time of all wells remained consistent, all of which were 5min±5s.

[0220] Table 8 Tyrosinase activity inhibition experiment sample addition table

[0221]

[0222] Calculation of tyrosinase activity inhibition rate:

[0223] Suppression rate = [1-(Td-Tc) / (Tb-Ta)] × 100%

[0224] Where:

[0225] Td—absorbance of sample reaction well;

[0226] Tc—absorbance of sample background hole;

[0227] Tb—average absorbance of solvent reaction wells;

[0228] Ta—average absorbance of solvent background holes.

[0229] The results are shown in Table 9.

[0230] Table 9 Tyrosinase activity inhibition rate ( n=3)

[0231]

[0232] 5. Results and Analysis

[0233] As can be seen from the data in Table 9, the Chinese medicine compositions of Examples 1 to 5 exhibited a good inhibitory effect on tyrosinase activity, with an inhibition rate between 71.25% and 73.58%, which was comparable to the inhibition rate of 72.86% of the positive control group (kojic acid), showing a strong whitening effect. In contrast, the inhibition rates of the Chinese medicine compositions of Comparative Examples 1 to 9 were lower, ranging from 31.24% to 56.18%. This comparison shows that there are significant differences in the inhibitory effects between the Examples and the Comparative Examples.

[0234] The inhibition rate of the positive control group (kojic acid) was 72.86%, indicating that the positive control had a strong tyrosinase inhibition effect. The inhibition rate of the blank control group was only 0.25%, which fully demonstrated the effectiveness of the experimental design and further verified the reliability of the experimental results.

[0235] In-depth analysis of the Chinese medicine compositions in Examples 1 to 5 and Comparative Examples 1 to 9 shows that the main difference lies in the different ratios of the three Chinese medicine extracts in the Chinese medicine compositions. In Examples 1 to 5, the weight ratio of Paeonia suffruticosa extract: Galla chinensis extract: Erigeron breviscapus extract is 2 to 4: 1 to 2: 1, while some of the Chinese medicine compositions in Comparative Examples 1 to 9 contain only one (Comparative Examples 4 to 6) or two Chinese medicine extracts (Comparative Examples 1 to 3), and some contain three Chinese medicine extracts (Comparative Examples 7 to 9), but their ratios are quite different from the ratios in the embodiments. This shows that when the ratios of the three Chinese medicine extracts in the Chinese medicine composition are within a certain range (such as the ratios in the embodiments), a synergistic effect will occur between them, making the whitening and freckle removal effect of the composition more significant. However, when one or two of the extracts are missing, or the ratios of the three extracts are inappropriate, this synergistic effect may be weakened, resulting in a reduction in the whitening and freckle removal effect.

[0236] Test Example 4 Human body whitening and freckle removal efficacy evaluation test

[0237] 1. Methodology

[0238] The test is carried out in accordance with the "UV-induced human skin darkening model freckle removal and whitening efficacy test method" in the "Technical Specifications for Safety of Cosmetics (2022 Edition)".

[0239] 2. Test Principle

[0240] The color of the skin is mainly determined by melanin, hemoglobin, carotene and other common factors. Ultraviolet rays can activate tyrosinase, thereby promoting the production of melanin. Therefore, using low-dose ultraviolet rays to irradiate the skin can induce the skin to darken.

[0241] The test method first uses low-dose ultraviolet rays to irradiate human skin to simulate the skin melanization process in the natural environment, activate tyrosinase in the skin, and promote the production of melanin, thereby establishing a skin melanization model. Subsequently, the freckle-removing and whitening cosmetics to be tested are applied to the subject's skin and used continuously for a period of time. During this period, the skin color is measured regularly by instrument detection, and the changes in skin color before and after the use of cosmetics are recorded and analyzed. By comparing the skin color evaluation results before and after use, including calculating parameters such as the individual type angle of skin color (ITA°), it is determined whether the cosmetics significantly improve the skin color. If the skin color after the use of cosmetics is significantly improved compared with that before use, and this improvement is statistically significantly different from the control group that does not use cosmetics, it can be considered that the cosmetics have freckle-removing and whitening effects.

[0242] 3. Materials and Reagents

[0243] The Chinese medicine compositions were prepared according to the methods of Examples 1 to 5 and Comparative Examples 1 to 9, and dried and stored for later use.

[0244] 7% ascorbic acid (vitamin C) was used as a positive control and prepared according to the formula in Appendix A of the "UV-induced human skin darkening model freckle removal and whitening efficacy test method" in the "Technical Specifications for Safety of Cosmetics (2022 Edition)". Solar Light 601 daylight simulator, Skin-colorimeter CL 400 skin colorimeter, Mexameter MX18 skin melanin detector.

[0245] 4. Experimental steps:

[0246] 4.1 Subject recruitment and screening

[0247] According to the needs of the test, 30 volunteers were publicly recruited as subjects. Subjects were strictly screened to ensure that they met the following conditions: aged between 18 and 60 years old, regardless of gender; the ITA° value of the skin color of the test site was between 20° and 50°; no allergic diseases, no history of allergies to cosmetics or other topical preparations; no history of photosensitive diseases, and no recent use of drugs that affect photosensitivity; no pigmentation, inflammation, scars, pigmented nevi, hirsutism, etc. on the skin of the test site; able to accept tanning of the skin in the test area using artificial light; able to understand the test process, voluntarily participate in the test and sign a written informed consent.

[0248] Exclusion criteria: pregnant or breastfeeding women, or those who are planning to get pregnant in the near future; those with a history of skin diseases such as psoriasis, eczema, atopic dermatitis, severe acne, etc.; those who have taken anti-inflammatory drugs such as corticosteroids orally or topically in the past month; those who have taken any products or drugs that affect skin color (such as hydroquinone preparations) orally or topically in the past 2 months; those who have participated in similar trials in the past 2 months or participated in similar trials 2 months ago but the melanization marks on the skin at the test site have not completely faded; those who have participated in other clinical trials in the past 2 months; and other patients who are considered unsuitable for participation in the trial by clinical evaluation.

[0249] 4.2 Establishing a human skin darkening model

[0250] Determine the MED (minimum erythemadose) of each subject's test site. Minimal erythemadose (MED) refers to the minimum dose (J / m2) or shortest time (s) of ultraviolet radiation required to cause a clearly visible erythema on the skin, covering most of the irradiation point.

[0251] Each test area was selected at the test site, and the same irradiation point was irradiated once a day with a daylight simulator at a dose of 0.75 times the MED for 4 consecutive days. The 4 days after the irradiation was the skin melanization period, and no treatment was performed.

[0252] 4.3 Application of test article

[0253] On the 5th day after the irradiation, the skin color of each test area was tested by instrument, and the test areas with poor consistency were eliminated.

[0254] The corresponding test substance (whitening and anti-freckle Chinese medicine composition) was smeared on each melanization test area according to the random table, and a negative control (melanization area blank control) and a positive control (7% ascorbic acid product prepared according to the formula) were set up at the same time.

[0255] The coating area should not be less than 6cm 2 , the coating amount is (2.0±0.1)mg / cm 2 or (2.0±0.1)L / cm 2 , the interval between each test area should be no less than 1.0 cm. Apply once in the morning and once in the evening.

[0256] 4.4 Experimental process and evaluation

[0257] The test substance was applied continuously for 4 weeks. The skin color was instrumentally tested every week (1 week, 2 weeks, 3 weeks, 4 weeks ± 1 day...) after application.

[0258] Use a skin colorimeter to measure the L, a, and b* values ​​of each test area and calculate the individual type angle (ITA°); ITA° refers to the parameters of human skin color measured by a skin colorimeter or a reflectance spectrophotometer using the skin L*a*b* color space data to measure the skin. The larger the ITA° value, the lighter the skin color, and vice versa. The calculation formula is as follows:

[0259]

[0260] The melanin index (MI) of each test area was measured using a skin melanin detector. MI refers to a parameter that characterizes the melanin content in the skin by measuring the absorption of a specific wavelength spectrum by the skin surface. The smaller the MI value, the lower the skin melanin content, and vice versa.

[0261] Relevant data are shown in Tables 10 and 11.

[0262] Table 10 ITA ° value of embodiment, comparative example and control group (X ± s, n = 30)

[0263]

[0264]

[0265] Table 11 MI values ​​of the examples, comparative examples and control groups ( n=30)

[0266]

[0267] 5. Results and Analysis

[0268] The ITA° value is positively correlated with the skin color depth, that is, the larger the ITA° value, the lighter the skin color. At the same time, the MI value is negatively correlated with the skin melanin content and skin color depth. The smaller the MI value, the lower the skin melanin content and the lighter the skin color.

[0269] From the data in Tables 10 and 11, it can be seen that after one week of use of the test substance, the ITA° value of each embodiment significantly increased, while the MI value significantly decreased. This indicates that the skin color became significantly lighter within this week, that is, the test substance exhibited a good whitening and freckle removal effect. In the next three weeks, the data remained basically stable with little change, which shows that the embodiment has achieved a good whitening and freckle removal effect after one week of use.

[0270] In contrast, the ITA° values ​​of the comparative examples increased less after one week of use, and in the following three weeks, the data basically showed an increasing trend. At the same time, the MI values ​​of the comparative examples also decreased less after one week of use, and in the following three weeks, the data basically showed a decreasing trend. It was not until four weeks of use that the ITA° and MI values ​​of the comparative examples were close to the levels of the examples. This shows that the improvement effect of the comparative examples on skin color is not as significant and rapid as that of the examples, and the effect of the comparative examples after four weeks of use is close to the effect of the examples after one week of use.

[0271] The t-test further verified that the difference in the change of ITA° value after use between the embodiment and the comparative example was statistically significant (p<0.05), which indicates that the whitening effect of the embodiment is significantly better than that of the comparative example. The reason for this is that the Chinese medicine composition in the embodiment has a reasonable ratio, which produces a synergistic effect, making the whitening and freckle removal effect of the composition more significant. However, when one or two of the extracts are missing, or the ratio of the three extracts is inappropriate (as shown in the comparative example), this synergistic effect may be weakened, resulting in a reduced whitening and freckle removal effect.

[0272] In addition, the reliability of this experimental method can be further verified by comparing the data of the embodiments, comparative examples and control groups. The data of the control group (including the negative control and the positive control) provide verification for the effectiveness and repeatability of the experiment. The negative control did not use any test substance, and the skin color changed slightly; the positive control used a known whitening and freckle-removing ingredient (7% ascorbic acid), and the skin color improvement effect was more obvious. The data of the embodiment is similar to or even better than the positive control, which further proves the reliability of this experimental method and the effectiveness of the test substance.

[0273] In summary, this experiment verified the whitening and freckle-removing effect and its significance of the test substance through reasonable experimental design and data comparison, and also revealed the importance of the rationality of the ratio of the Chinese medicine composition of the present invention.

Claims

1. A Chinese medicine composition, characterized in that: The invention is composed of a peony bark extract, a gallnut extract and a breviscapus breviscapus extract in a mass ratio of 2 to 4:1 to 2:1; the peony bark extract is obtained by mixing extracts obtained by sequentially extracting volatile oil compounds and moutan bark total glycosides from the peony bark; the gallnut extract and the breviscapus breviscapus extract are obtained by respectively extracting with an alcohol solvent and then decolorizing and purifying them.

2. The Chinese medicine composition according to claim 1, characterized in that: The extraction of the volatile oil compounds is carried out by wetting and steam distilling the peony bark after crushing it, obtaining the volatile oil, and then dehydrating and refining it, and encapsulating it with β-cyclodextrin to obtain the volatile oil compound extract; the peony bark total glycosides substance is obtained by hydrothermally extracting and purifying the peony bark total glycosides substance extract after steam distillation of the peony bark medicinal material.

3. The Chinese medicine composition according to claim 2, characterized in that: The β-cyclodextrin inclusion is carried out by using β-cyclodextrin equivalent to 8 to 12 times the mass of volatile oil.

4. The Chinese medicine composition according to claim 2, characterized in that: The purification is performed using a macroporous resin.

5. The Chinese medicine composition according to claim 1, characterized in that: The preparation method of the gallnut extract is as follows: the gallnut medicinal material is extracted with ethanol, and then adsorbed and purified by combining granular white clay and carrier activated carbon.

6. The Chinese medicine composition according to claim 1, characterized in that: The preparation of the Breviscapus breviscapus extract is performed by using ethanol reflux extraction to obtain the Breviscapus breviscapus extract, and then using activated carbon decolorization and macroporous resin adsorption to purify the extract.

7. The Chinese medicine composition according to claim 7, characterized in that: The amount of activated carbon used in the activated carbon decolorization is 1-3% of the mass of the breviscapus breviscapus extract.

8. Use of the Chinese medicine composition according to any one of claims 1 to 7 in the preparation of whitening and anti-freckle cosmetics.

9. A cosmetic, characterized in that: A Chinese medicine composition comprising any one of claims 1 to 7.

10. The cosmetic according to claim 9, characterized in that: The mass proportion of the traditional Chinese medicine composition in the cosmetic is 1 to 10%.

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