CN110317774A - Aqueous extract of Chinese herbal medicine and combinations thereof and purposes - Google Patents

Abstract

The present invention discloses aqueous extracts of Chinese herbal medicines and compositions and uses thereof, and specifically relates to aqueous extracts of Hedysarum polybotrys Hand.-Mazz., Alisma plantago-aquatica (Sam.) Juzep, Momordica charantia L. or Eisenia foetida, and combinations thereof, and new uses in cell culture. The aqueous extracts of the above-mentioned Chinese herbal medicines can replace some functions of animal serum in cell culture medium, thereby reducing the amount of animal serum used in cell culture medium, thereby reducing the production cost of cells, and reducing the health risks caused by the use of animal serum. The present invention also relates to anti-wrinkle and anti-skin aging cosmetic formulations containing the aqueous extracts of Chinese herbal medicines.

Description

Aqueous extract of Chinese herbal medicine and its composition and use

technical field

The present invention relates to new uses of aqueous extracts of Chinese herbal medicines, especially aqueous extracts of a single Chinese herbal medicine, or a combination of aqueous extracts of several Chinese herbal medicines, new compositions and new uses in cell culture, and in New compositions and uses in the fight against skin wrinkles and skin aging.

Background technique

Fibroblasts exist in many tissues in animals, and are used to synthesize extracellular matrix and collagen, maintain the basic structure of connective tissue, and play an important role in wound healing. Fibroblasts also respond to migration and proliferation due to the cytokines secreted by themselves or surrounding cells.

The cultured fibroblasts have the effect of promoting tissue repair and can be used in a wide range of applications, including acute and chronic wounds, as well as cosmetic and reconstructive surgery, all of which have good clinical results. For wound repair, fibroblasts can be cultured on scaffolds made of biodegradable materials to make skin substitute products. There are many skin substitutes on the market, but their safety and quality need to be strictly planned and managed. For example, whether the cell source is human body, whether the product is sterile, whether it is contaminated, whether it is still active after freezing, and if it is a non-autologous source, whether there is a possibility of rejection or virus infection, etc., are all parts that need to be strictly managed. In addition, how to minimize the cost and maximize the applicability of the product is also a subject that people in the relevant technical fields are eager to solve.

At present, in the treatment of medical aesthetics, autologous fibroblasts are also used as tissue fillers by injection, replacing the traditional injection of bovine collagen, which may have the risk of rejection or infection. Injection of hyaluronic acid may also produce subcutaneous excessive immune response, resulting in redness and swelling. Therefore, injection of autologous fibroblasts has become a treatment method worthy of development in the field of medical cosmetology. Injection of autologous fibroblasts can improve facial wrinkles and scars, and there is no need to consider the risk of adverse reactions such as rejection, and injection of living cells can prolong the therapeutic effect and reduce the number of treatments (Weiss, R.A. et al., Dermatol. Surg. 33(3):263-268).

In response to the application of fibroblasts in autologous injection, it is necessary to reduce the dosage of fetal bovine serum (FBS) under the premise of effectively cultivating human fibroblasts. Currently, fibroblasts are mainly cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS, in which fetal bovine serum is used to provide nutrients and growth factors required for the growth of fibroblasts. However, when fibroblast preparations are used, either by injection or by mesotherapy, they need to be introduced into the patient, and components of fetal bovine serum may enter the cells during culture Inside, the safety is difficult to estimate, and there may be risks of rejection, allergies, and even disease infections. Moreover, the unit price of fetal bovine serum is very high. Therefore, reducing the amount of fetal bovine serum used in the culture process of fibroblasts is a very important issue at present. At present, some studies use the serum isolated from autologous blood as the nutrient source for culturing fibroblast culture medium. Although this method is relatively safe, large amounts of culture serum are required for culturing fibroblasts. If the patient you want to treat is anemic, you can't get enough blood to separate enough serum. What's more, in clinical practice, fibroblasts may need to be subcultured multiple times, and the demand for serum is higher at this time. In the above cases, the patient may not be able to proceed with the treatment smoothly. Therefore, there is an ardent demand in the industry for a substitute for fetal bovine serum that can promote the growth of fibroblasts, or a cell culture supplement that can reduce the amount of fetal bovine serum.

Cell culture supplements from non-animal sources have the advantages of low risk of disease transmission, low cost, and abundant sources. Farizhandi et al. proposed that plant protein hydrolyzates of soybean and rice could be used as culture supplements of human skin fibroblasts (Farizhandi, A.A.K.et al., Adv.Environ.Biol.8(3 Spec.Issue), 838-843). WO2004/083400A2 discloses a medium supplement for culturing animal cells, which is extracted from groundnut, hazelnut, almond and other plant seeds, and can be used as a substitute or supplement for fetal bovine serum. Japanese Patent Application No. 2-49579 proposes to use gluten hydrolyzate and yeast extract in combination with basal medium for culturing animal cells.

Contents of the invention

Dermal fibroblasts are specialized in producing extracellular matrix such as collagen and elastin to maintain the elasticity and density of the skin. The dermis has a sufficient number of fibroblasts to secrete enough collagen to smooth out skin wrinkles and maintain skin elasticity. Therefore, the number and vitality of dermal fibroblasts are closely related to the health and firmness of the skin. The inventors of the present application established an in vitro cell model through primary human dermal fibroblast cell culture to screen out the cells that can improve the survival rate of fibroblasts and/or promote their secretion of collagen , and/or promote its secretion of growth factors, and/or enhance its anti-oxidation ability of medicinal ingredients, used in skin care products, in order to achieve skin anti-wrinkle and anti-aging effects.

Chinese herbal medicine is a treasure of the long history of Chinese culture, containing thousands of years of accumulated knowledge of medicinal plants and animals. Using the above-mentioned in vitro cell model to test single or compound combinations of various Chinese herbal medicine extracts, the inventors of the present application found that Hedysarum polybotrys Hand.-Mazz., Alisma plantago-aquatica (Sam. .) Juzep), bitter melon (Momordica charantia L.), and earthworm (Eisenia foetida), aqueous extracts of traditional Chinese herbs, either alone or in combination, can be grown under in vitro culture conditions containing only low amounts of fetal bovine serum Promote the growth of human or animal cells, especially the growth of fibroblasts, for example, the growth of human dermal fibroblasts. Accordingly, the aqueous extract of the above-mentioned Chinese herbal medicine can be used to reduce the amount of fetal bovine serum used in the cell culture medium, thereby reducing the production cost of animal cells. In particular, when the fibroblasts produced according to the present application are introduced into the human body, the potential health risks caused by the introduction of fetal bovine serum into the human body can be significantly reduced.

Therefore, according to a first aspect of the present invention, there is provided a cell culture medium supplement, characterized in that the supplement comprises an aqueous extract from one or more biological species selected from the group consisting of Astragalus (Hedysarum polybotrys Hand.-Mazz.), Alisma plantago-aquatica (Sam.) Juzep, Momordica charantia L. and Eisenia foetida.

In a preferred specific embodiment, the aqueous extract is selected from the combination of Astragalus polymorpha and Earthworm aqueous extracts, the combination of Astragalus polymorpha and Alisma and bitter melon aqueous extracts, Alisma Combination with aqueous extracts of bitter melon and earthworm, combination of Astragalus polyme and aqueous extracts of bitter melon and earthworm, and combination of aqueous extracts of Astragalus polyme and Alisma and aqueous extracts of bitter melon and earthworm. In a more preferred embodiment, the aqueous extract is selected from a combination of aqueous extracts of Astragalus polymorpha and Earthworm.

According to the second aspect of the present invention, it provides a kind of cell culture medium, it is characterized in that: said culture medium comprises aforementioned cell culture medium supplement; And a basal medium, it is supplemented with The basis is substantially less than 10% by volume of animal serum, preferably substantially less than 5% by volume of animal serum, more preferably substantially less than 1% by volume of animal serum, for example about 0.5% by volume of animal serum.

According to a third aspect of the present invention, it provides a method for culturing cells, characterized in that:

Said method comprises the following steps:

(a) contacting said cells with said medium; and

(b) culturing said cells under conditions suitable to support culturing said cells in vitro.

In a preferred embodiment, said cells are human or animal cells. In a more preferred embodiment, said cells are fibroblasts. In a more preferred embodiment, said cells are human dermal fibroblasts.

The inventors of the present application further found that the combination of Astragalus root and Earthworm water-based extracts can not only promote the proliferation of fibroblasts, but also promote the secretion of collagen by fibroblasts, and can also increase the number of cells treated with H ₂ O ₂ survival rate. This combination of Chinese herbal medicines has great potential to maintain the number and vitality of dermal fibroblasts to prevent wrinkles.

Therefore, according to a fourth aspect of the present invention, it provides an anti-wrinkle cosmetic formulation, characterized in that the cosmetic formulation comprises: an aqueous extract of Hedysarum polybotrys Hand.-Mazz., Aqueous extract of earthworm (Eisenia foetida), and a cosmetically acceptable carrier.

According to the fifth aspect of the present invention, it also provides a method for preventing or improving skin wrinkles, characterized in that the method comprises: applying the aforementioned anti-wrinkle cosmetic formulation on the skin surface.

The inventors of the present application also found that the aqueous extract of Earthworm can not only increase the activity of superoxide dismutase (SOD), but also increase the cell survival rate in the cell aging model induced by H ₂ O ₂ . In addition, the combination of Astragalus polymorpha and Earthworm aqueous extracts, the combination of bitter melon and Earthworm aqueous extracts, and the combination of bitter melon and Alisma alisma can also increase after H ₂ O ₂ treatment cell viability. These herbal combinations have great potential for maintaining the survival of dermal fibroblasts and preventing their aging.

Therefore, according to a sixth aspect of the present invention, there is provided a cosmetic formulation for combating skin aging, characterized in that said cosmetic formulation comprises: an aqueous extract from one or more biological species selected from Aqueous extracts of astragalus, combination of aqueous extracts of Astragalus polymorpha and aqueous extracts of Earthworm, combinations of aqueous extracts of bitter melon and aqueous extracts of Earthworm, and aqueous extracts of bitter melon and aqueous Alisma a combination of extracts; and a cosmetically acceptable carrier.

According to the seventh aspect of the present invention, it further provides a method for preventing or improving skin aging, which is characterized in that the method comprises: applying the aforementioned anti-aging cosmetic formulation on the skin surface.

The purpose, features and advantages of the present invention will be described in detail through embodiments in conjunction with the accompanying drawings.

Description of drawings

Figure 1 is a bar graph showing the cell survival rate obtained by unilaterally culturing fibroblasts with various Chinese herbal extracts for 3 days;

Figure 2 is a bar graph showing the cell survival rate obtained by unilaterally culturing fibroblasts with various Chinese herbal medicine extracts for 6 days;

Figure 3 is a bar graph showing the amount of collagen secretion obtained by unilaterally culturing fibroblasts with extracts of various Chinese herbal medicines for 3 days;

Figure 4 is a bar graph showing the amount of collagen secretion obtained by unilaterally culturing fibroblasts with extracts of various Chinese herbal medicines for 6 days;

Figure 5 is a bar graph showing the secretion of vascular endothelial growth factor (vascular endothelial growth factor; VEGF) obtained by culturing fibroblasts unilaterally for 3 days with extracts of various Chinese herbal medicines;

Figure 6 is a bar graph showing the secretion of vascular endothelial growth factor obtained by culturing fibroblasts with various Chinese herbal medicine extracts for 6 days;

Figure 7 is a bar graph showing the superoxide dismutase (SOD) activity obtained by unilaterally culturing fibroblasts with various Chinese herbal extracts for 3 days;

Fig. 8 is a bar graph showing the cell survival rate obtained by culturing fibroblasts for 3 days with various Chinese herbal medicine extract compounds;

Fig. 9 is a bar graph showing the cell viability obtained by culturing fibroblasts for 6 days with compound extracts of various Chinese herbal medicines;

Fig. 10 is a bar graph showing the amount of collagen secretion obtained by culturing fibroblasts for 3 days with compound extracts of various Chinese herbal medicines;

Fig. 11 is a bar graph showing the amount of collagen secretion obtained by culturing fibroblasts for 6 days with compound extracts of various Chinese herbal medicines;

Figure 12 is a bar graph showing the amount of vascular endothelial growth factor secreted by culturing fibroblasts for 3 days with various Chinese herbal medicine extract compounds;

Figure 13 is a bar graph showing the secretion of vascular endothelial growth factor obtained by culturing fibroblasts for 6 days with various Chinese herbal medicine extract compounds;

Figure 14 is a bar graph showing the superoxide dismutase (SOD) activity obtained by culturing fibroblasts for 3 days using various Chinese herbal medicine extract compounds;

Figure 15 is a bar graph showing the effects of each Chinese herbal extract single and compound on cells treated with H ₂ O ₂ ;

Fig. 16 is a fluorescence microscope image, showing the image of cells expressing FSP-1 after unilaterally treating fibroblasts with extracts of various Chinese herbal medicines; and

Fig. 17 is a bar graph showing the percentage of cells expressing FSP-1 after treatment of fibroblasts with each Chinese herbal extract single and compound.

Detailed ways

Unless otherwise stated, the following terms used in the specification and claims of this application have the definitions given below. Please note that the singular term "a" used in the specification and claims of this application is intended to cover one and more than one items, such as at least one, at least two or at least three, and does not mean only having A single recorded matter. In addition, open conjunctions such as "comprising" and "having" used in the claims indicate that in the combination of components or components described in the claims, other components or components not specified in the claims are not excluded. It should also be noted that the term "or" generally includes "and/or" unless the content clearly indicates otherwise. The terms "about" or "substantially" used in the description and claims of this application are used to modify any error that can be slightly changed, but such a slight change will not change its essence.

This application relates to the use of traditional Chinese herbal medicines such as Astragalus polysemen, Alisma, bitter gourd and earthworm in cell culture and skin care.

In this specification, the terms "Astragalus polybotrys" and "Astragalus" are used interchangeably to cover freshly harvested, unprocessed and processed species of the plant Hedysarum polybotrys Hand.-Mazz. (abbreviated as H. polybotrys) Whole plants or plant parts, especially the underground parts of said plants, such as the dried roots of said plants sold in traditional Chinese herbal medicine stores. It should be noted that Astragalus membranaceus (Fisch.) Bge.var.mongholicus (Bge.) Hsiao) commonly used on the market includes Astragalusmembranaceus (Fisch.) Bge. The "Astragalus polysensus" used belonged to different species. The "Astragalus polycysticum" used in this application has been identified by the Industrial Technology Research Institute of Taiwan, China, and confirmed to belong to the species.

The term "Alisma plantago-aquatica (Sam.) Juzep; or simply A. plantago-aquatica)" as used in this specification covers freshly harvested, unprocessed and processed whole plants of this plant species Or a plant part, especially the underground part of the plant, such as the tuber part of the plant sold in traditional Chinese herbal medicine shops. Usually, the tuber part of the plant is excavated when the stems and leaves start to wither in winter, washed and dried with low heat to remove fibrous roots and rough skin, sliced with water, and then dried in the sun. Alisma is usually sold in traditional Chinese herbal medicine stores as the dried tuber part after being processed by bran frying or salt water frying.

The term "Momordica charantia L.; or simply M. charantia" as used in this specification covers freshly harvested, unprocessed and processed whole plants or plant parts of this plant species, especially those The fruit part of the plant. Bitter gourd is a common fruit vegetable in the fruit and vegetable market. The balsam pear used in this application has been identified by the Industrial Technology Research Institute of Taiwan, and confirmed to belong to the species.

The term "Eisenia foetida; or simply E. foetida", also known as Eisenia foetida, as used in this specification covers the freshly harvested, raw and processed whole worms of this animal species Or insect parts, such as unprocessed whole insects sold as bait in fishing supplies stores, or processed whole insects or insect fragments sold in traditional Chinese herbal medicine stores. In the traditional processing process, earthworms are usually caught in summer and autumn. After the abdomen is cut open, the viscera and sediment are washed away, and then exposed to the sun or dried at low temperature. Dried worms can be directly used for medicinal purposes, or further add 12.5 kg of rice wine to every 100 kg of earthworm, stir fry until the surface turns brown, and let it cool for later use.

The term "aqueous extract" in this application refers to a composition prepared by contacting the aforementioned one or more medicinal materials with an aqueous solvent and following standard extraction procedures. The term "derived from" used in this specification is used to indicate the source of the medicinal material from which the aqueous extract is prepared. Examples of the aqueous solvent include but are not limited to water, C _1-6 alkanols (C _{1-6 alkanols} ), C _1-4 alkylene glycols (C _1-4 alkylene glycols), aqueous carbohydrate solutions, which salt solutions, and their mixtures. In a preferred embodiment, the aqueous solvent is selected from water and its salt solutions. In order to improve the extraction efficiency, the medicinal materials can be ground first, and the temperature and/or pressure can be increased during extraction. In a preferred embodiment, the aqueous extract is obtained by extracting with deionized water at high temperature, for example, extracting at about 100° C. under normal pressure. The duration of the extraction depends on the yield of the extraction, and usually lasts from 30 minutes to 1 day, preferably from 1 to 12 hours, such as 1 to 3 hours. The term "aqueous extract" covers crude extracts obtained by a single aqueous extraction procedure as well as crude extracts further subjected to one or more separation and/or purification procedures, including by subjecting the crude extract to one or more Active components or fractions derived from an additional extraction, concentration, filtration, distillation or other purification process. When the aqueous extract comes from more than two medicinal materials, individual medicinal materials can be extracted separately, and then the individual extracts can be mixed, or these medicinal materials can be mixed first, and then jointly extracted. The aqueous extracts may be in liquid form, such as a solution, concentrate or distillate, or in semi-liquid form, such as a gel. If desired, the aqueous extract can be freeze-dried and stored in sterile containers. Before use, add an aqueous solvent such as deionized water for rehydration.

According to the present application, aqueous extracts of Astragalus polymorpha, Alisma, Momordica charantia, and Earthworm, as well as compound combinations of these aqueous extracts, can promote human Or the growth of animal cells, especially can promote the growth of fibroblasts, for example, promote the growth of human dermal fibroblasts. This indicates that the aqueous extracts of the above-mentioned Chinese herbal medicines can be used as supplements for animal cell culture media to reduce the amount of animal serum used in the cell culture media. Generally, animal cell culture medium is supplemented with 10% to 20% animal serum to provide proteins, inorganic salts, growth factors, hormones and cell attachment factors required for cell growth. As used herein, "animal serum" means a liquid product derived from the blood of a non-human animal from which hemocytes, fibrinogen and fibrin have been removed for addition to the culture medium to provide the necessary nutrients for cell growth. Nutrients are required, which include but are not limited to fetal bovine serum (FBS), calf serum, adult bovine serum, or serum from other animals, such as serum products from horses and camels.

This application uses human primary dermal fibroblasts as a screening platform for aqueous extracts of Chinese herbal medicines. As shown in Examples 1 and 7 below, from the perspective of promoting cell proliferation, compared with the control group using the basal medium supplemented with 0.5% FBS, in addition to 0.5% FBS, the addition of P. polymorpha, The basal medium of the water-based extracts of Alisma, bitter melon or earthworm alone or their compound combination can increase the number of fibroblasts by 1.5 times when culturing fibroblasts for 3 days, and it can even increase the number of fibroblasts after 3 days. 1.4 to 2.4 times higher, showing that the aqueous extracts of the aforementioned Chinese herbal medicines all have the effect of promoting the growth of fibroblasts. Example 7 further shows that, compared with 5% and 10% FBS, the degree of cell proliferation of the aqueous extract of Chinese herbal medicines alone or in combination with 0.5% FBS can also reach about 50% to 66.7% of the conventional dosage (10%) of FBS . While not wishing to be bound by any theory, we believe that the aqueous extracts of the aforementioned Chinese herbal medicines not only contain nutrients needed for animal cell growth, but may also contain ingredients that can stimulate the release of growth factors from fibroblasts, thereby promoting cell growth. Rapid proliferation. Example 12 also confirms that the fibroblasts treated with the aqueous extract of the aforementioned Chinese herbal medicine will not change their original cell characteristics and differentiate into mature cells while rapidly proliferating.

It is worth noting that, as shown in Example 7, when using the combination of Astragalus polymorpha and Earthworm aqueous extracts, the combination of Astragalus polymorpha and Alisma and bitter melon aqueous extracts, Combination of aqueous extracts of bitter melon and earthworm, combination of aqueous extracts of astragalus and bitter gourd and earthworm, and combination of aqueous extracts of astragalus and alisma and bitter melon and earthworm to cultivate Fibroblasts can not only make the cell proliferation times more than 2 times, but also the cell proliferation effect obtained by the combination of these compounds is better than that obtained by individual single drugs in the combination. While not wishing to be bound by any theory, we believe that the ingredients in this five-combination combination work synergistically in promoting cell proliferation. Therefore, in a preferred embodiment, the aqueous extract is from one of the aforementioned five compound combinations. In a more preferred embodiment, the aqueous extract is a combination of aqueous extracts from Astragalus polymorpha and Earthworm.

Accordingly, the present application provides a cell culture medium supplement comprising: an aqueous extract from one or more biological species selected from the group consisting of Astragalus polymorpha, Alisma, Momordica, and Earthworm . A "cell culture medium supplement" as used herein refers to a composition added to a cell culture medium to help maintain the viability and/or promote the proliferation of cells seeded in said medium. The cell culture medium supplement is suitable for supporting the in vitro culture of human or animal cells, preferably human or animal fibroblasts, for example human dermal fibroblasts in vitro culture. The amount of the aqueous extract of the above-mentioned Chinese herbal medicine in the culture medium can be determined by those of ordinary skill in the technical field involved in the present application through conventional means in the field. Since the aqueous extract of the above-mentioned Chinese herbal medicine can replace part or all of the functions of animal serum in the culture medium, the amount of animal serum in the cell culture medium can be based on the total volume of the cell culture medium being 10% of the conventional The dosage is substantially reduced to substantially less than 10% by volume of animal serum based on the total volume of the cell culture medium, preferably substantially less than 5% by volume of animal serum, more preferably substantially less than 1% by volume. % animal serum by volume, for example about 0.5% animal serum by volume.

Therefore, the present application also provides a cell culture medium, which comprises: the aforementioned cell culture medium supplement; and a basal medium, which is supplemented with substantially less than 10 volume % of Animal serum, preferably substantially less than 5% by volume of animal serum, more preferably substantially less than 1% by volume of animal serum, such as about 0.5% by volume of animal serum. The culture medium is generally in a liquid or semi-liquid form.

The term "basal medium" refers to any liquid medium that does not contain animal serum but contains basic nutrients such as inorganic salts, amino acids and vitamins for supporting the in vitro culture of human or animal cells. In a preferred embodiment, said basal medium does not contain growth factors. Examples of basal media suitable for use in the present invention include, but are not limited to, Basal Medium Eagles (Basal Medium Eagles; BME), Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (Dulbecco's Modified Eagle's Medium; DMEM), Nutrient Mixed Medium F-10 (HAM's F-10) and Nutrient Mixed Medium F-12 (HAM's F-12). In a more preferred embodiment, the basal medium is DMEM.

The present application also provides a method for culturing cells using the aforementioned culture medium, which comprises the following steps: (a) contacting the cells with the aforementioned culture medium; and (b) culturing the cells in vitro The cells were cultured under conditions. The term "contacting" as used herein refers to placing cells into a culture vessel filled with medium. The means of bringing the cells into contact with the medium are routine and routine in the technical field to which this application pertains and include, but are not limited to, mixing the cells with the medium, pipetting the medium into the culture vessel containing the cells , So that the cells are immersed in the culture medium. As used herein, the term "culturing" refers to maintaining cells under in vitro conditions favorable for their growth, survival and/or differentiation. In a preferred embodiment, said cells are human or animal cells. In a more preferred embodiment, said cells are fibroblasts. In a more preferred embodiment, said cells are human dermal fibroblasts. The means of culturing cells in vitro belong to conventional technical means in the technical field to which this application relates. In general, the optimum temperature for animal cells is about 35 to 37°C. Deviate from this temperature range, the normal metabolism and growth of cells will be affected, and even lead to death. When the temperature is above 43°C, most of the cells will die. Low temperature has little effect on cells, cells can still survive and grow at 25 to 35°C, but at a slow rate. Generally, cells are cultured in a constant temperature and aerobic environment, and a certain concentration of carbon dioxide is given to maintain the pH value of the medium. In a preferred embodiment, the cells are cultured in a carbon dioxide incubator. The carbon dioxide incubator is usually set at constant temperature (eg 37°C), stable _CO level (eg 5%), constant pH (eg pH 7.2-7.4) and high relative saturation humidity (eg 95%) , to simulate the growth environment of cells in vivo.

In this application, the first-generation human dermal fibroblasts are used as a screening platform to further screen out the aqueous extract of Chinese herbal medicines with anti-skin wrinkle effects. It is generally believed that the formation of wrinkles is caused by the free radicals and active oxygen in the environment (such as sunlight ultraviolet rays, cigarette smoke and air pollution) destroying the intermolecular connection between collagen and elastin in the dermis of the skin, and causing cell membrane Lipid peroxidation. Therefore, the aqueous extract of Chinese herbal medicine, which can promote the proliferation of fibroblasts and promote their secretion of collagen, has the potential of anti-wrinkle. As shown in Example 8 below, the combination of the aqueous extracts of Astragalus polymorpha and Earthworm can significantly promote the secretion of collagen by culturing fibroblasts for 6 days, and the effect can even reach that obtained by using 10% FBS 2 to 3 times the amount of secretion, thus helping to reduce the loss of collagen in the skin. It is worth noting that the combination of the aqueous extracts of Astragalus polymorpha and Earthworm has the effect of promoting the secretion of collagen in cells, which is much higher than the effect obtained by using the extracts of Astragalus polymorpha and Earthworm alone. The results of this experiment pointed out that the aqueous extracts of Astragalus polymorpha and Earthworm produced a synergistic effect on promoting the secretion of collagen from fibroblasts. Example 7 also shows that this compound combination has the effect of promoting the growth of fibroblasts. Example 11 further shows that this compound combination can increase the survival rate of cells treated with H ₂ O ₂ .

Accordingly, the present application also provides an anti-wrinkle cosmetic formulation, which comprises: an aqueous extract of Astragalus polymorpha, an aqueous extract of Earthworm, and a cosmetically acceptable carrier. "Anti-wrinkle" as used herein means preventing wrinkles from occurring on the skin and/or improving wrinkles that have already occurred on the skin. It should be noted that "prevention" and "improvement" do not mean that the skin to which the anti-wrinkle cosmetic formulation is applied is never wrinkled, but that it may have fewer and/or appearance wrinkles than skin to which the cosmetic formulation is not applied. Less noticeable wrinkles.

On the other hand, the antioxidant molecules and enzymes naturally produced in human skin will gradually decrease with age, making the skin's antioxidant capacity unable to completely remove environmental free radicals and reactive oxygen species. In addition, superoxide dismutase (SOD), an enzyme capable of catalyzing the conversion of superoxide into oxygen and hydrogen peroxide through a dismutation reaction, is a major antioxidant in fibroblasts. Excessive external stimuli (such as sunlight ultraviolet rays) may not only lead to excessive free radicals, but may also cause SOD to lose its activity. These factors all contribute to skin aging. This application also uses the first generation of human dermal fibroblasts as a screening platform to screen out the water-based extract of Chinese herbal medicines with anti-aging effects on the skin. First of all, the present application refers to the literature data of Shatuo et al. and Qiu et al., using hydrogen peroxide to induce oxidative stress on cells, thereby establishing an in vitro model of cell aging (Satoh, A. et al., J.Pharm. Pharmacol. 57(10):1335-1343; and Choi, MJ et al., Biol. Pharm. Bull. 33(3):421-426). As shown in Example 11 below, the single prescription of the extract of Earthworm, the combination of Astragalus polymorpha and the aqueous extract of Earthworm, the combination of the aqueous extract of Bitter Melon and Earthworm, and the aqueous extract of Bitter Melon and Alisma The combination of extracts can increase the survival rate of cells in H ₂ O ₂ -induced cellular aging model. Example 5 further shows that the groundworm aqueous extract alone can also increase the activity of superoxide dismutase. These herbal combinations have great potential for maintaining the survival of dermal fibroblasts and preventing their aging.

The aqueous extracts of the aforementioned Chinese herbal medicines can resist the oxidative stress caused by hydrogen peroxide, and can even increase the activity of superoxide dismutase, that is, they have the potential to resist skin cell aging. Accordingly, the present application further provides a cosmetic formulation for anti-aging skin, which comprises: an aqueous extract from one or more biological species, which is selected from the aqueous extract of Dilonga, the aqueous Combinations of extracts and aqueous extracts of Earthworm, combinations of aqueous extracts of Bitter Melon and aqueous extracts of Earthworm, and combinations of aqueous extracts of Bitter Melon and aqueous extracts of Alisma; and Cosmetic Acceptability carrier.

The aforementioned anti-wrinkle cosmetic formulations and anti-skin aging cosmetic formulations may be in any convenient form without specific limitations. In a preferred embodiment, the cosmetic formulation is presented as a maintenance solution, lotion, cream, gel, ointment, foam, soap, eye shadow, lip balm, body wash, shaving cream, etc. for topical application. In the form of a product or cosmetic, it can be applied via application to the surface of the skin. In a preferred embodiment, the cosmetic formulation is in the form of an infusion solution for subcutaneous or intravenous injection, such as suspension, intravenous infusion, dry powder injection, suspension injection, dry powder suspension injection, etc. .

The term "cosmetically acceptable carrier" in this application means an inert substance used as a carrier of the aqueous extract of Chinese herbal medicine, which has no toxicity, irritation, or pyrogenicity to the site of application (such as the skin) , antigenicity and hemolyticity, and has no substantial pharmacological activity, and will not hinder the beneficial effect of the Chinese herbal medicine aqueous extract. Generally, the amount of cosmetically acceptable carrier is from about 1% to about 99.9%, preferably from about 50% to about 99%, based on the total weight of the cosmetic formulation. The type of cosmetically acceptable carrier that is suitable depends on the form of the cosmetic formulation.

The following examples are provided only to illustrate the present invention, but not to limit the scope of the present invention. The experimental results shown in each embodiment are compared with the control group by Student's t test, #: 0.05<p<0.1; *: p<0.05.

Preparation Example 1: Primary culture of human dermal fibroblasts

Skin samples were obtained with the subject's consent. Skin samples were cut into skin fragments 0.5 to 1 mm cubic in size. Subsequently, the skin fragments were placed in DMEM containing FBS, collagenase and dispase, and incubated at 37° C. for 16 to 18 hours. Centrifugation was performed at 1,500 rpm for 5 minutes. Then, the supernatant was removed to replace the broken cells and skin pieces supplemented with FBS, and the broken skin pieces and cells were evenly distributed in Erlenmeyer flasks, and cultured at 37°C. Cells were subcultured when the cell growth density reached 80%. The cells of the third to sixth passages were used for the experiments described below.

Preparation Example 2: Preparation of Chinese herbal medicine extract

Astragalus and Alisma were purchased from traditional Chinese medicine shops. Earth dragons were purchased from area fishing supply stores. Bitter melon was purchased from the regional fruit and vegetable market.

100 grams of Astragalus polyphylla was added to 500 grams of secondary deionized water, followed by boiling at normal pressure for 2 hours. Weigh to get 210 grams of Astragalus polysequence suspension.

100 grams of Alisma was added to 500 grams of secondary deionized water, followed by boiling under normal pressure for 2 hours. Weigh 242 grams of Alisma suspension.

100 grams of balsam pear fruit was added to 500 grams of secondary deionized water, followed by boiling at normal pressure for 2 hours. Weigh 150 grams of bitter gourd suspension.

100 grams of unprocessed whole worms of Earthworm were added to 500 grams of secondary deionized water, followed by boiling under normal pressure for 2 hours. Weigh 170 grams of earthworm suspension.

Each suspension mentioned above was passed through gauze to filter out impurities. The filtrate was collected and centrifuged at 6000 rpm for 30 minutes. The supernatant was taken, passed through a 0.2 micron pore size filter, and weighed to record. The filtrate was collected and used as the test stock solution of each Chinese herbal medicine extract, sealed with a sterile serum bottle and stored in a -20°C refrigerator. In order to prevent the Chinese herbal medicine extracts from containing a large amount of heavy metals from affecting the experimental results, the heavy metal content of each Chinese herbal medicine extract has been tested. The test results found that the lead, mercury, and cadmium contents of the four Chinese herbal medicine extracts were all lower than the detection limit of 2ppm (mg/kg).

In the following examples, the stock solution of each Chinese herbal medicine extract will be diluted several times with the medium of the control group, and then the diluted solution will be used to treat the cells. For example, Astragalus 100X refers to the dilution obtained by diluting the stock solution of Astragalus polymorpha extract 100 times with the medium of the control group.

Example 1: Unilateral treatment of cells with extracts

Take quantitative fibroblasts obtained in Preparation Example 1, plant them in a 96-well flat culture dish, and in the presence of the individual Chinese herbal medicine extracts prepared in Preparation Example 2, culture them in a medium containing 0.5% heat-deactivated calf serum (FBS; Gibco, NY, USA) in Duchenne's Modified Eagle's Medium (DMEM; Sigma Chemical Co., St. Louis, MO, USA) for 3 to 6 days. The control group was the fibroblasts of Preparation Example 1 cultured in DMEM containing 0.5% or 1.0% FBS. The supernatant and cells were harvested and analyzed separately.

Example 2: The extract unilaterally promotes cell growth

Use a commercially available XTT analysis kit (Sigma Chemical Co., St. Louis, Missouri, U.S.A.) and refer to the kit guide to process the cells obtained in Example 1, and then obtain the readings of each well in the flat culture dish at 450nm to obtain the cell viability . Figure 1 and Figure 2 show that when fibroblasts were cultured for 3 days and 6 days using the extracts of Astragalus, Alisma and Earthworm respectively, a significant increase in the number of cells could be observed compared to the 0.5% FBS control group, in which When cultured for 3 days, the proliferation rate was only about 1.2 times that of the 0.5% FBS control group, and when cultured for 6 days, it was even higher than 1.5 times that of the control group. In the case of using bitter melon extract, the proliferation of fibroblasts was significantly promoted after 6 days of culture. This example shows that the four Chinese herbal extracts used in this application have the ability to promote the proliferation of human dermal fibroblasts.

Example 3: The effect of extract unilaterally on the secretion of collagen by fibroblasts

Use the commercially available human recombinant type I collagen test kit (Gibco, New York, USA; kit number PRO-479A) and refer to the kit guide to process the supernatant obtained in Example 1 to obtain the fibroblasts in each experimental group of collagen secretion.

Figure 3 shows the amount of collagen secreted by culturing fibroblasts with extracts of individual Chinese herbal medicines for 3 days. Compared with the 0.5% FBS control group, the astragalus extract can significantly stimulate the collagen secretion of fibroblasts at three test concentrations, and the 200X dilution has the best effect. Alisma extract can significantly promote the secretion of collagen when diluted 4800X. Earthworm extract can also significantly stimulate fibroblasts to secrete collagen when diluted at 4000X and 2000X. Bitter melon extract did not have the ability to stimulate fibroblasts to secrete collagen at the tested concentrations. Figure 4 shows the amount of collagen secretion obtained by culturing fibroblasts with individual Chinese herbal medicines for 6 days. Among them, only the groups of Astragalus 200X and Dilong 8000X can significantly promote the secretion of collagen. This example shows that the extracts of Astragalus and Earthworm combined with 0.5% FBS can promote the secretion of collagen by fibroblasts, and the promoting effect even exceeds the effect achieved by 1.0% FBS.

Example 4: The effect of extract unilaterally on the secretion of growth factors by fibroblasts

Using a commercially available VEGF test kit (R&D Systems, Minnesota, USA; VEGF ELISA Kit, No. DY293B) and the supernatant obtained in Example 1 were processed according to the kit instructions to obtain the VEGF secretion amount of fibroblasts in each experimental group.

Figure 5 and Figure 6 show the VEGF secretion obtained by culturing fibroblasts with individual Chinese herbal extracts for 3 days and 6 days, respectively. Compared with the 0.5% FBS control group, the effect of Chinese herbal medicine extract on the secretion of VEGF in fibroblasts, no matter whether it was cultured for 3 days or 6 days, there was no significant difference.

In the unilateral test experiments of Examples 1 to 4, the extract of Astragalus extract works best when diluted 400X, the extract of Earthworm extract works best when diluted 2000X, and the extract of Alisma extract works best when diluted 4800X , and bitter melon extract works best when diluted 2500X. Therefore, in the subsequent examples, Astragalus 400X, Dilong 2000X, Alisma 4800X and Bitter Melon 2500X were used for testing.

Embodiment 5: Antioxidant activity of extract unilaterally

Using a commercially available reagent kit (Cayman Chemical, Ann Arbor, Michigan, U.S., kit number #706002) and referring to the kit guide to process the supernatant obtained in Example 1, to obtain the results of each experimental group Superoxide dismutase (SOD) activity. As shown in Figure 7, Earthworm 2000X has the ability to promote SOD activity significantly compared to the control group, which indicates that the earthworm extract has the effect of enhancing the antioxidant capacity of cells.

Example 6: Treatment of Cells Using Extract Compounds

Repeat the process described in Example 1, but the four extract dilutions of Astragalus 400X, Earthworm 2000X, Alisma 4800X and Bitter Melon 2500X are used alone or in combination for culturing fibroblasts for 3 to 6 sky. The control group was the fibroblasts of Preparation Example 1 cultured in DMEM containing 0.5% or 1.0% FBS. The supernatant and cells were harvested and analyzed separately.

Example 7: Extract compound promotes cell growth

The cells obtained in Example 6 were treated with a commercially available XTT assay kit (Sigma Chemical Co., St. Louis, Missouri, USA), and then readings were taken at 450 nm for each well in the flat culture dish to obtain the cell survival rate. Figure 8 and Figure 9 show that using the above four extract dilutions to culture fibroblasts for 3 days and 6 days, no matter single or compound, can promote the proliferation of fibroblasts. When cultured for 3 days, each group can promote the proliferation of fibroblasts by more than 1.5 times, and even up to 2 times when cultured for 6 days. This example also shows that these four extract dilutions do not interfere with each other to inhibit cell production when culturing fibroblasts. It is worth noting that, as shown in Figure 9, when using Astragalus + Dilong, Astragalus + Alisma + Bitter Gourd, Alisma + Bitter Gourd + Dilong, Astragalus + Bitter Gourd + Dilong, and Astragalus + Alisma + When the combination of bitter gourd + earthworm is used to culture fibroblasts for 6 days, the proliferation of the cells can be more than doubled, and the effect of cell proliferation obtained by the compound combination is better than that obtained by individual ingredients in the combination. Compared with the results obtained with 5% and 10% FBS, various Chinese herbal medicinal aqueous extracts alone or in combination with 0.5% FBS can also achieve a cell proliferation effect of about 50% to 66.7%.

Example 8: The effect of the extract compound on the secretion of collagen by fibroblasts

Using a commercially available collagen test kit (Gibco, New York, U.S.; kit number PRO-479A) and referring to the kit guide to process the supernatant obtained in Example 6, the collagen secretion of fibroblasts in each experimental group was obtained .

Figure 10 and Figure 11 show the amount of collagen secretion obtained by culturing fibroblasts for 3 days and 6 days using four kinds of Chinese herbal extracts alone or in combination. Compared with the 0.5% FBS control group, the compound combination of Astragalus and Earthworm can increase the secretion of collagen by nearly 3 times, and its promoting effect also greatly exceeds the effect achieved by 1.0% FBS.

Example 9: The effect of compound extracts on growth factors secreted by fibroblasts

Using a commercially available VEGF test kit (R&D Systems, Minnesota, USA; VEGF ELISAKit, number DY293B) and refer to the kit guide to process the supernatant obtained in Example 6 to obtain the VEGF secretion of fibroblasts in each experimental group.

Figure 12 and Figure 13 show the VEGF secretion obtained by culturing fibroblasts for 3 days and 6 days by using four kinds of Chinese herbal medicine extracts alone or in combination. Compared with the 0.5% FBS control group, the effect of the Chinese herbal extract compound on the VEGF secretion of fibroblasts, no matter whether it was cultured for 3 days or 6 days, there was no significant difference.

Embodiment 10: Antioxidant activity of extract unilaterally

Repeat the experimental procedure described in Example 5, but use it to process the supernatant obtained in Example 6 to obtain the SOD activity of each experimental group. Figure 14 shows the SOD activity obtained by culturing fibroblasts for 3 days using four kinds of Chinese herbal extracts alone or in paired combination. Compared with the 0.5% FBS control group, the compound combination of each Chinese herbal medicine extract had no significant effect on the SOD activity.

Embodiment 11: Anti-aging ability test of cells

Referring to the literature data of Sartoh et al. and Qiu et al., a cell model for testing anti-aging ability was established (Satoh, A. et al., J. Pharm. Pharmacol. 57(10): 1335-1343; and Choi, MJ et al ., Biol. Pharm. Bull. 33(3):421-426). First seed the fibroblasts obtained in Preparation Example 1 in a 12-well flat culture dish overnight. After the cells were attached to the culture dish, the dilutions of various Chinese herbal extracts were added. After 72 hours of treatment, cells were stimulated with 300 μM H ₂ O ₂ for 24 hours to induce premature aging of the cells.

In this example, it was observed that under the influence of H ₂ O ₂ , the morphology of fibroblasts will change, the adhesion status will be poor, stress-induced premature senescence will occur, and cell apoptosis will occur (apoptosis). The number of cells calculated by staining with trypan blue is shown in FIG. 15 . Before adding H ₂ O ₂ , the number of cells in the 0.5% FBS control group was significantly higher than that after adding H ₂ O ₂ . Treating cells with extracts of Earthworm, Astragalus + Earthworm, Alisma + Bitter Gourd, or Bitter Gourd + Earthworm for 72 hours can significantly improve the apoptosis caused by H ₂ O ₂ .

Example 12: Effect of extracts on fibroblast properties

In order to confirm that Chinese herbal medicine will not change the properties of fibroblasts, the present embodiment stains the fibroblasts obtained in Example 6 with antibodies to check whether it still expresses fibroblast specific protein 1 (fibroblastspecific protein 1, FSP for short). -1). As shown in FIG. 16 , after being treated with the Chinese herbal medicine extract for 3 days, the fibroblasts in each group expressed the indicator protein FSP-1. Fig. 17 shows the percentage of cells expressing FSP-1 after treatment of fibroblasts with each Chinese herbal extract alone or in combination, that is, the purity of fibroblasts. The results showed that more than 95% of the treated fibroblasts in each group could express FSP-1, which indicated that the single or compound combination of Chinese herbal extracts would not affect the FSP-1 expression of primary dermal fibroblasts. This indicates that the basic properties of fibroblasts will not be changed by the influence of Chinese herbal extracts.

The above embodiments are only for illustrating the present invention, rather than limiting the present invention. Various transformations or changes made by those skilled in the relevant fields without departing from the technical scope of the present invention should also belong to the protection category of the present invention.

Claims (10)

1. A cell culture medium supplement, characterized in that: The supplement comprises aqueous extracts from one or more biological species selected from the group consisting of Hedysarum polybotrys Hand.-Mazz., Alisma plantago-aquatica (Sam. ) Juzep), bitter melon (Momordica charantia L.) and earthworm (Eisenia foetida). 2. The cell culture medium supplement according to claim 1, wherein the aqueous extract is selected from the group consisting of the combination of Astragalus polymorpha and Earthworm aqueous extracts, Atragalus polymorpha and Alisma and Momordica Combination of extracts, combination of Alisma and balsam pear and earthworm aqueous extract, combination of Astragalus polymorpha and balsam pear and earthworm aqueous extract, and astragalus and Alisma and balsam pear and earthworm Combination of dragon water extracts. 3. The cell culture medium supplement according to claim 2, wherein the aqueous extract is selected from the group consisting of A. polymorpha and Earthworm aqueous extracts. 4. A cell culture medium, characterized in that: The medium comprises the cell culture medium supplement according to any one of claims 1-3; and a basal medium supplemented with substantially less than 10% by volume based on the total volume of the cell culture medium animal serum. 5. The cell culture medium according to claim 4, wherein the basal medium is supplemented with substantially less than 5% by volume of animal serum based on the total volume of the cell culture medium. 6. The cell culture medium according to claim 5, wherein the basal medium is supplemented with substantially less than 1% by volume of animal serum based on the total volume of the cell culture medium. 7. The cell culture medium of claim 6, the basal medium supplemented with about 0.5% by volume of animal serum based on the total volume of the cell culture medium. 8. The cell culture medium of claim 4, wherein the cells are fibroblasts. 9. The cell culture medium of claim 8, wherein the cells are human dermal fibroblasts. 10. An anti-wrinkle cosmetic formulation characterized by: The cosmetic formulation comprises an aqueous extract of Astragalus polymorpha, an aqueous extract of Earthworm, and a cosmetically acceptable carrier.