CN113599320A

CN113599320A - Composition for culturing pluripotent cells, preparation method and application

Info

Publication number: CN113599320A
Application number: CN202110906178.2A
Authority: CN
Inventors: 周守山; 戴稳平; 林晓; 肖炼; 朱单单; 徐燕燕
Original assignee: Zhuhai Jintai Biotechnology Co ltd
Current assignee: Zhuhai Jintai Biotechnology Co ltd
Priority date: 2021-08-09
Filing date: 2021-08-09
Publication date: 2021-11-05

Abstract

The invention belongs to the technical field of cell culture, and discloses a composition for pluripotent cell culture, a preparation method and application thereof. The composition comprises herba Saussureae Involueratae extract, folium Ginkgo extract, and herba Sidae Rhombifoliae extract. The composition contains saussurea involucrate extract, ginkgo leaf extract and calendula officinalis extract, and the three substances have synergistic interaction, so that the composition can remarkably improve the proliferation capacity of skin cells and is further beneficial to repair and update of the skin cells. The composition can be applied to cosmetics or medical products, and can remarkably improve the anti-aging capability of skin.

Description

Composition for culturing pluripotent cells, preparation method and application

Technical Field

The invention belongs to the technical field of cell culture, and particularly relates to a composition for pluripotent cell culture, a preparation method and application thereof.

Background

A stem cell is a pluripotent cell having a self-replicating ability, which can differentiate into cells having various functions under certain conditions. Typical stem cell media include essential amino acids, salts, vitamins, minerals, trace metals, sugars, lipids, and nucleosides. The most conventional culture medium for stem cells is a medium supplemented with fetal bovine serum, however, stem cells cultured with this medium not only grow slowly, but also have greatly reduced potential to differentiate into functional cells after more than 5 passages. The cell culture medium in the prior art has no obvious proliferation effect on skin cells, which is not beneficial to the repair and the renewal of the skin cells.

In addition, repair and renewal of skin cells helps to further slow down skin aging.

Therefore, it is highly desirable to provide a cell culture composition that not only can help to proliferate skin-like cells, but also can help to improve the anti-aging ability of the skin.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a composition for culturing pluripotent cells, a preparation method and application thereof, wherein the composition is beneficial to the proliferation of skin cells and the repair and renewal of the skin cells. The composition also facilitates culture differentiation of stem cells. The composition is applied to cosmetics or medical products, and is helpful for improving the anti-aging capability of the skin.

The invention conception of the invention is as follows: the composition contains snow lotus extract, ginkgo leaf extract and calendula extract, and the three substances have synergistic interaction, so that the composition obviously improves the proliferation capacity of skin cells and is further beneficial to repair and update the skin cells. The composition also has antioxidant effect and can be used for delaying skin aging. The composition can be applied to cosmetics or medical products, and can remarkably improve the anti-aging capability of the skin.

In a first aspect of the invention, a pluripotent cell culture composition is provided.

Specifically, the composition for culturing the pluripotent cells comprises saussurea involucrate extract, ginkgo leaf extract and calendula officinalis extract.

Further preferably, the composition further comprises yam extract. The addition of the yam extract is helpful for further improving the proliferation capacity of the composition for promoting skin cells.

Preferably, the composition further comprises at least one of amino acids, salts, vitamins, minerals, sugars, fatty acids, and solvents. These components are conventional components of cell culture media.

Preferably, the amino acids include at least three of arginine, lysine, leucine, isoleucine, threonine, valine, phenylalanine, histidine, serine, glycine, cysteine, proline, methionine, tryptophan, glutamic acid, aspartic acid, or alanine.

Preferably, the salts include sodium salt, potassium salt, calcium salt, magnesium salt, zinc salt and copper salt.

Further preferably, the sodium salt comprises sodium chloride.

Further preferably, the potassium salt comprises potassium chloride.

Further preferably, the calcium salt includes at least one of calcium chloride, calcium carbonate, and calcium pantothenate.

Further preferably, the magnesium salt comprises magnesium sulfate and/or magnesium chloride.

Further preferably, the zinc salt comprises zinc sulfate and/or zinc chloride.

Further preferably, the copper salt comprises copper sulfate.

Preferably, the composition further comprises a phosphate buffered saline solution. The pH of the composition is preferably 7.2-7.4, and phosphate buffered saline may be added depending on the pH.

Preferably, the vitamins include vitamin A, vitamin B, vitamin C and vitamin E.

Preferably, the sugar comprises glucose.

Preferably, the fatty acids are linoleic acid and linolenic acid.

Preferably, the minerals include iron sulfate and selenium sulfate.

Preferably, the solvent comprises water and/or glycerol.

Preferably, the composition further comprises carnosine and 5, 7-dihydroxyflavone. The carnosine and the 5, 7-dihydroxyflavone are added simultaneously, and the combination of the carnosine and the 5, 7-dihydroxyflavone can obviously improve the anti-skin-aging capacity of the composition by being matched with the snow lotus extract, the ginkgo leaf extract and the calendula extract.

Preferably, the composition comprises, by weight, 5-18 parts of saussurea involucrate extract, 1-6 parts of ginkgo leaf extract and 3-16 parts of calendula officinalis extract.

Preferably, the composition comprises, by weight, 5-15 parts of saussurea involucrate extract, 1-5 parts of ginkgo leaf extract and 3-15 parts of calendula officinalis extract.

Preferably, the composition further comprises 10-25 parts by weight of yam extract; further preferably, the composition further comprises 10-20 parts by weight of Chinese yam extract.

Preferably, the composition further comprises, by weight, 5-20 parts of amino acid, 5-15 parts of salt, 5-25 parts of vitamin, 0.01-0.5 part of mineral, 20-50 parts of sugar, 20-70 parts of fatty acid and 80-300 parts of solvent.

More preferably, the composition further comprises 8-15 parts by weight of amino acid, 8-12 parts by weight of salt, 10-20 parts by weight of vitamin, 0.01-0.5 part by weight of mineral, 40-50 parts by weight of sugar, 40-70 parts by weight of fatty acid and 100-200 parts by weight of water.

Preferably, the composition further comprises 0.1-2 parts of carnosine and 0.1-3 parts of 5, 7-dihydroxyflavone by weight; further preferably, the composition further comprises 0.5-1 part of carnosine and 0.5-2 parts of 5, 7-dihydroxyflavone according to parts by weight.

In a second aspect, the invention provides a method for preparing a pluripotent cell culture composition.

Specifically, the preparation method of the composition for culturing the pluripotent cells comprises the following steps:

taking the raw material components, stirring and mixing to obtain the composition.

Preferably, the preparation method of the composition comprises the following steps:

mixing the saussurea involucrate extract, the ginkgo leaf extract and the calendula officinalis extract with a part of solvent to prepare a mixture A, then mixing amino acid, salt, vitamin, mineral, sugar and fatty acid with the rest of solvent to prepare a mixture B, and mixing the mixture A, the mixture B and the rest of components to prepare the composition.

A third aspect of the invention provides the use of a composition for pluripotent cell culture as described above.

The application of the composition for culturing the pluripotent cells in preparing cosmetics or medical products.

A cosmetic comprises the above composition and adjuvants. The auxiliary material is a substance commonly used in cosmetics, such as a preservative.

Preferably, the cosmetic comprises a face cleaning lotion, a cosmetic water, a spray, an essence, a facial mask, a gel, a beauty freeze-dried powder, a solvent, an emulsion and a cream.

A medical product comprises the composition and medical auxiliary materials. The medical auxiliary materials are conventional substances in the field.

The medical product comprises medical freeze-dried powder, a solvent, a dressing, an ointment and a cold compress patch.

Compared with the prior art, the invention has the following beneficial effects:

(1) the composition contains snow lotus extract, ginkgo leaf extract and calendula extract, and the three substances have synergistic interaction, so that the composition obviously improves the proliferation capacity of skin cells and is further beneficial to repair and update the skin cells. The composition also has antioxidant effect and can be used for delaying skin aging.

(2) The composition further comprises yam extract. The addition of the yam extract is helpful for further improving the proliferation capacity of the composition for promoting skin cells. The composition further comprises carnosine and 5, 7-dihydroxyflavone. The carnosine and the 5, 7-dihydroxyflavone are added simultaneously, and the combination of the carnosine and the 5, 7-dihydroxyflavone can obviously improve the anti-skin-aging capacity of the composition by being matched with the snow lotus extract, the ginkgo leaf extract and the calendula extract.

(3) The composition can be applied to cosmetics or medical products, and can remarkably improve the anti-aging capability of the skin.

Detailed Description

In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are not intended to limit the scope of the claimed invention.

The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.

Example 1: preparation of the composition

The composition for culturing the pluripotent cells comprises, by weight, 6 parts of saussurea involucrate extract, 2 parts of ginkgo leaf extract, 4 parts of calendula extract, 6 parts of amino acid (0.5 part of arginine, 0.5 part of lysine, 0.5 part of leucine, 0.5 part of isoleucine, 0.5 part of threonine, 0.5 part of valine, 0.5 part of phenylalanine, 0.5 part of histidine, 0.5 part of serine, 0.5 part of glycine, 0.5 part of cysteine, 0.5 part of aspartic acid), 5 parts of salt (3.35 parts of sodium chloride, 0.5 part of potassium chloride, 0.5 part of calcium pantothenate, 0.5 part of magnesium sulfate, 0.1 part of zinc sulfate, 0.05 part of copper sulfate), 5 parts of vitamin (vitamin A2 parts, vitamin B1 parts, vitamin C1 parts, vitamin E1 parts), 0.11 parts of mineral matter (0.1 part of ferric sulfate, 0.01 part of selenium sulfate), 40 parts of glucose, 60 parts of fatty acid (30 parts of linoleic acid), and a solvent (130 parts of water) (water solvent), 20 parts of glycerin).

The preparation method of the composition for culturing the pluripotent cells comprises the following steps:

mixing herba Saussureae Involueratae extract, folium Ginkgo extract, and herba Sidae Rhombifoliae extract with partial solvent (60 parts of water and 10 parts of glycerol) to obtain mixture A, mixing amino acids, salts, vitamins, minerals, glucose, and fatty acids with the rest solvent to obtain mixture B, and mixing mixture A and mixture B to obtain the composition.

Example 2: preparation of the composition

The composition for culturing the pluripotent cells comprises, by weight, 12 parts of saussurea involucrate extract, 4 parts of ginkgo leaf extract, 3 parts of calendula extract, 10 parts of amino acid (arginine 1 part, lysine 1 part, leucine 1 part, isoleucine 1 part, threonine 1 part, valine 0.5 part, phenylalanine 0.5 part, histidine 0.5 part, serine 0.5 part, glycine 0.5 part, cysteine 0.5 part, aspartic acid 0.5 part, proline 0.5 part, methionine 0.5 part, tryptophan 0.5 part), 6.65 parts of salt (sodium chloride 3 parts, potassium chloride 1 part, calcium pantothenate 2 parts, magnesium sulfate 0.5 part, zinc sulfate 0.1 part, copper sulfate 0.05 part), 8 parts of vitamin (vitamin A3 parts, vitamin B2 parts, vitamin C2 parts, vitamin E1 parts), 0.11 parts of mineral matter (iron sulfate 0.1 part, selenium sulfate 0.01 part), 40 parts of glucose 40 parts, and 50 parts of fatty acid (linoleic acid 20 parts), and the like, 30 parts of linolenic acid) and 200 parts of solvent (180 parts of water and 20 parts of glycerol).

mixing herba Saussureae Involueratae extract, folium Ginkgo extract, and herba Sidae Rhombifoliae extract with part of solvent (80 parts of water and 10 parts of glycerol) to obtain mixture A, mixing amino acids, salts, vitamins, minerals, glucose, and fatty acids with the rest of solvent to obtain mixture B, and mixing mixture A and mixture B to obtain the composition.

Example 3: preparation of the composition

Compared with example 1, example 3 is different in that the composition further contains 15 parts of yam extract, and the rest components and preparation method are the same as example 1.

Example 4: preparation of the composition

Compared with example 2, the difference of example 4 is that the composition also contains 0.5 part of carnosine and 1 part of 5, 7-dihydroxyflavone, and the rest components and the preparation method are the same as example 2.

Example 5: preparation of the composition

Compared with example 4, example 5 is different in that the composition contains 1 part of carnosine but does not contain 5, 7-dihydroxyflavone, and the rest components and preparation method are the same as example 4.

Example 6: preparation of the composition

Example 6 is different from example 4 in that the composition contains 1.5 parts of 5, 7-dihydroxyflavone but does not contain carnosine, and the rest of the components and the preparation method are the same as example 4.

Example 7: preparation of the composition

Compared with the embodiment 1, the embodiment 7 is characterized in that the composition comprises 4 parts of saussurea involucrate extract, 7 parts of ginkgo leaf extract and 1 part of calendula extract, and the rest components and the preparation method are the same as the embodiment 1.

Comparative example 1

Compared with example 1, comparative example 1 is different in that calendula officinalis extract is replaced with chamomile extract, and the rest of the components and the preparation method are the same as example 1.

Comparative example 2

Compared with example 2, comparative example 2 is different in that the composition does not contain the calendula extract and the saussurea involucrate extract, and the rest components and the preparation method are the same as example 2.

Comparative example 3

Compared with example 2, comparative example 3 is different in that the composition does not contain saussurea involucrate extract and ginkgo biloba leaf extract, and the rest components and preparation method are the same as example 2.

Comparative example 4

Compared with example 2, comparative example 4 is different in that the composition does not contain the calendula extract and the ginkgo biloba extract, and the rest of the components and the preparation method are the same as example 2.

Product effectiveness testing

1. Skin cell proliferation effect test

HaCaT cells in logarithmic growth phase at 5X 10⁴one/mL of the cells were seeded in 96-well plates at 37 ℃ in CO₂The cells were cultured in an incubator for 24 hours (the culture medium used for the culture was a conventional DMEM medium), and the same amount of the composition (referred to as a sample to be tested) prepared in examples 1, 2, 3, and 7 and comparative examples 1 to 4 was added thereto, respectively, to treat the cells for 24 hours and 48 hours, and then the effect of the sample to be tested on the cell proliferation was examined by the CCK-8 (a detection reagent for cell proliferation and cytotoxicity). In addition, recombinant bovine basic fibroblast growth factor bFGF (which is known to have a promoting effect on skin cell proliferation) was used as a positive control group under the same conditions. The HaCaT cell viability results are shown in table 1.

Table 1: results of cell viability

	24-hour cell survival (%)	Cell viability at 48 hours (%)
			Example 1	130	139
Example 2	131	140
			Example 3	135	148
Example 7	128	136
			Comparative example 1	118	123
Comparative example 2	101	106
			Comparative example 3	104	110
Comparative example 4	106	113
			Positive control group	101	103

As can be seen from Table 1, the compositions prepared in examples 1, 2, 3 and 7 have a significant promoting effect on the proliferation of HaCaT cells, which is much greater than that of comparative examples 1 to 4 and the positive control group. Particularly, from the results of comparative examples 2-4 and example 2, it can be seen that the use of the saussurea involucrate extract, the ginkgo leaf extract and the calendula officinalis extract in the composition of the invention has the effect of obviously promoting the proliferation of HaCaT cells, namely the saussurea involucrate extract, the ginkgo leaf extract and the calendula officinalis extract have the synergistic effect.

2. Skin anti-aging efficacy test

2.1. Free radical scavenging test

When external factors (such as sunlight, ultraviolet rays, polluted air and chemical agents) irritate the skin, the skin may generate hydroxyl radicals, which accelerates skin aging.

The compositions prepared in examples 1, 2, 4-7 of the present invention and comparative examples 1-4 were tested for anti-aging effect (the strength of anti-aging effect was measured by DPPH radical scavenging rate, which is 1, 1-diphenyl-2-trinitrophenylhydrazine, and the DPPH radical scavenging rate was measured by a method conventional in the art), and the results are shown in Table 2.

Table 2: anti-aging effect

	DPPH radical scavenging ratio (%)
		Example 1	95.9
Example 2	96.1
		Example 4	98.5
Example 5	96.9
		Example 6	97.1
Example 7	95.1
		Comparative example 1	89.0
Comparative example 2	72.2
		Comparative example 3	75.8
Comparative example 4	78.9

As can be seen from Table 2, the anti-aging effects of the compositions prepared in the examples of the present invention are significantly superior to those of the compositions prepared in the comparative examples. Furthermore, it can be seen from the results of examples 1-2 and 4 that the addition of carnosine and 5, 7-dihydroxyflavone significantly improves the anti-aging effect of the composition.

2.2. Skin elasticity test

50 volunteers with more wrinkles were selected, aged 30-50 years, and randomly divided into 5 groups of 10 persons each. After the volunteers cleaned their faces with the same skin cleanser, 6X 6cm was selected at the bilaterally symmetrical position of the face²As an experimental area; the compositions prepared in example 4 and comparative examples 1 to 4 were used as test samples, and 0.2g of the composition was applied to each sample in the morning and evening. Before a sample to be tested is smeared, detecting the skin elasticity of a face experiment area by using a Callegari soft plus skin tester, testing the experiment area of each volunteer for 10 times, taking an average value, and further calculating the average value of each group as a; after continuously using the sample to be tested for 3 weeks, detecting the skin elasticity of the experimental area of the face by using a Callegari soft plus skin tester, testing the experimental area of each volunteer for 10 times, taking an average value, and further calculating the average value of each group, and recording the average value as b; the result of dividing b by a is reported as elasticityAnd grading, wherein the larger the elasticity grade value is, the better the skin elasticity increasing effect of the sample to be tested is. The elasticity rating results are shown in table 3.

Table 3: elasticity rating results

	Example 4	Comparative example 1	Comparative example 2	Comparative example 3	Comparative example 4
						Elastic rating value	1.50	1.41	1.12	1.25	1.31

As can be seen from Table 3, the composition prepared in example 4 of the present invention significantly increases skin elasticity and has skin anti-aging effect.

In addition, when the sample to be detected is smeared on the face, the allergy phenomena of red and swelling and the like on the face are not found, and the composition prepared by the invention is safe and reliable.

Application example 1

A cosmetic anti-aging cream comprising the composition prepared in example 4.

Application example 2

An anti-aging medical dressing comprising the composition prepared in example 4.

The composition prepared by the embodiment of the invention has larger potential of differentiating into functional cells after the transmission times of stem cells cultured by the composition exceed 5 generations.

Claims

1. A composition is characterized by comprising saussurea involucrate extract, ginkgo leaf extract and calendula extract.

2. The composition of claim 1, wherein the composition further comprises yam extract.

3. The composition of claim 1, further comprising at least one of amino acids, salts, vitamins, minerals, sugars, fatty acids, and solvents.

4. The composition of claim 1, wherein the composition further comprises carnosine and 5, 7-dihydroxyflavone.

5. The composition as claimed in claim 1, wherein the composition comprises 5-18 parts by weight of saussurea involucrate extract, 1-6 parts by weight of ginkgo leaf extract and 3-16 parts by weight of calendula extract.

6. The composition according to claim 5, further comprising 5-20 parts by weight of amino acid, 5-15 parts by weight of salt, 5-25 parts by weight of vitamin, 0.01-0.5 part by weight of mineral, 20-50 parts by weight of sugar, 20-70 parts by weight of fatty acid, and 80-300 parts by weight of solvent.

7. A process for the preparation of a composition according to any one of claims 1 to 6, characterized in that it comprises the following steps:

8. Use of a composition according to any one of claims 1 to 6 for the preparation of a cosmetic or medical product.

9. A cosmetic comprising the composition of any one of claims 1 to 6, and an adjuvant.

10. A medical article comprising the composition of any one of claims 1-6, and a medical excipient.