CN113171335A

CN113171335A - Polianthes tuberosa polysaccharide

Info

Publication number: CN113171335A
Application number: CN202010011943.XA
Authority: CN
Inventors: 田咏轩
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-01-08
Filing date: 2020-01-08
Publication date: 2021-07-27

Abstract

Described herein is a composition for skin care products having moisturizing and anti-aging promoting functions, which comprises acidic polysaccharides and basic amino acids produced by in vitro culture of tuberose cells. The composition has antioxidant, skin moistening, antiaging, skin hydration degree increasing, skin roughness improving, and wrinkle smoothing effects. The preparation process of the composition includes regulating pH of the culture liquid of tuberose cell to acidity, filtering to purify, mixing the culture liquid with alcohol, separating out polysaccharide, re-dissolving the polysaccharide in water and mixing with basic amino acid to obtain the composition.

Description

Polianthes tuberosa polysaccharide

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a composition for skin care and application thereof in cosmetics.

Background

In the development and production of cosmetics, natural functional plant components have the advantages of natural sources, natural degradation, high market acceptance degree and the like. The production of natural plant components generally involves collecting artificially cultivated or naturally growing plant tissues and then extracting them. The plants cultivated in the field or facility are easily affected by factors such as soil, climate, plant diseases and insect pests, and the product quality of each cultivation batch is different, which is not beneficial to the standardization of the product quality. In order to reduce the influence of external factors and improve the yield and quality stability, an in vitro culture technology of plant cells has been developed, cells at specific parts on a plant body are separated and grow in an artificially prepared culture medium, and the plant cells are enabled to synthesize a large amount of required components by regulating and controlling the conditions of the components of the culture medium and the like.

Flowers of tuberose (Poliantha tuberosa L.) have a strong aroma and are generally used as spice plants to extract essences. The petal cells can generate extracellular polysaccharide composed of monosaccharide such as mannose, arabinose, galactose, glucuronic acid, etc. in specific culture medium, and the molecular weight of the extracellular polysaccharide is rich in hydrophilic hydroxyl, so that the extracellular polysaccharide has good moisture retention property, and can be used as skin moisture retention component in cosmetics. As a high molecular substance, the exopolysaccharide enables the culture solution to have higher viscosity, components such as cell fragments and the like are not easy to remove, the appearance of the exopolysaccharide extracting solution is relatively turbid, and the exopolysaccharide extracting solution is not beneficial to the application of the exopolysaccharide in cosmetics. Under neutral pH, the acid groups on the extracellular polysaccharide are in a dissociated state and have negative charges, polysaccharide molecular chains repel each other due to the same charges and are highly extended in water, so that the culture solution has high viscosity, and turbid substances such as cell debris and the like are not easy to remove. The development of low-cost and efficient culture and treatment technology can promote the application of the exopolysaccharide in cosmetics.

Good skin moisturizing ability is beneficial to keeping the skin in good condition, and the skin moisturizing ability is generally determined by factors such as stratum corneum lipid barrier, natural moisturizing factor content and the like. The natural moisturizing factor in the keratinocytes consists of urea, organic acid salt, amino acid, saccharide and the like, and the concentration of the natural moisturizing factor can be improved by the degradation of enzymes in the skin through the amino acid supplementation, so that the skin moisturizing is improved. Amino acid is also a raw material for synthesizing various extracellular matrix proteins and has a certain anti-aging effect.

Disclosure of Invention

The invention aims to provide an in vitro culture production method of tuberose exopolysaccharide and a composition for skin care based on the technology.

The technical scheme adopted by the invention comprises the following steps: optimizing the in vitro culture condition of synthesizing extracellular polysaccharide by tuberose cells, separating process of extracellular polysaccharide, and composition of extracellular polysaccharide and basic amino acid. For the in vitro culture of tuberose cells for the synthesis of exopolysaccharides, optimization is carried out on the basis of commonly used plant cell culture media, such as White medium (White, 1963), Gnmhorg medium (Gnmhorg et al, 1968), LS medium (Linsmnler and Skoog, 1965), and the like.

Exopolysaccharides, which are secondary metabolites of cells, are generally produced in large quantities under specific culture conditions, and can be targeted by optimizing the composition of the culture medium.

Firstly, the type and concentration of sugars, the synthesis of exopolysaccharides requires large amounts of monosaccharides as building blocks and large amounts of energy, the source of which is also provided by sugars under non-photosynthetic conditions. In the LS liquid culture medium, sucrose, glucose and fructose are compared, and the fact that the glucose and the fructose are reducing sugars and are easy to brown in the culture process is found, so that the LS liquid culture medium has a certain inhibition effect on cells, the color of the culture solution is dark, and the post-treatment difficulty is improved. Sucrose is a non-reducing disaccharide, and browning is slight during the culture, so sucrose is selected as the saccharide in the culture medium. In terms of concentration, experiments show that the yield of the exopolysaccharide and the sucrose concentration are positively correlated within the range of 1-5% (W/V), but the yield is improved by a limited range after the yield exceeds 3%, and the sucrose concentration is determined to be 3% (W/V) by comprehensively considering factors such as cost, efficiency and the like.

Secondly, the concentrations of 2, 4-Dichlorophenoxyacetic acid (2, 4-Dichlorophenoxyacetic acid, commonly known as 24D), 1-naphthylacetic acid (1-naphthalene acetic acid) and 6-benzylaminopterin (6-benzyl aminopterine) are studied, the concentration ranges are 1-10-4-1-10-6 mol/L, 1-10-4-1-10-6 mol/L and 1-10-5-1-10-7 mol/L respectively, when the three plant hormones are used together at the concentrations of 1-10-5 mol/L, 1-10-5 mol/L and 1-10-6 mol/L, the yield of the exopolysaccharides can reach 4.0-6.0 g/L, and when the plant hormones are not used, the yield of the exopolysaccharides is only 1.4-2.2 g/L. The existence of exopolysaccharide makes the culture solution have higher viscosity, and opaque impurities such as cell debris are not easy to remove when suspended in the culture solution with higher viscosity. According to monosaccharide composition and properties of the exopolysaccharide, the pH value of the culture solution is adjusted to be acidic after the culture technology, so that acidic groups in molecules of the exopolysaccharide are in an uncharged non-dissociation state, the extension degree of molecular chains is reduced due to hydrophobic interaction of a sugar ring structure, the viscosity of the culture solution is further reduced, and turbid substances such as cell fragments and the like are easily separated to obtain the culture solution with a transparent appearance. Acids that may be used include hydrochloric acid, citric acid, acetic acid, malic acid, and the like. The hydrochloric acid has strong acidity and low price, but the hydrochloric acid belongs to a controlled medicine, has strong corrosivity, influences the environment and the human health during operation, and has corrosivity to equipment due to chloride ions in the hydrochloric acid, so the hydrochloric acid is not used, and the organic acid is used instead. The factors such as cost, smell and the like are comprehensively considered in the organic acid, and the citric acid is finally selected. After the culture is finished, the pH of the culture solution is 6.0-7.0, the viscosity is 900-1200 mPa.s measured by using a rotational viscometer, the pH is reduced to be within the range of 2.0-5.0 after 3-5 g of citric acid is added into each liter of the culture solution, the viscosity of the culture solution is 240-380 mPa.s when the pH is 5.0, the viscosity of the culture solution is 70-140 mPa.s when the pH is 4.0, the viscosity of the culture solution is 20-70 mPa.s when the pH is 3.0, and the reduction of the viscosity enables the culture solution to be more easily subjected to filtration or centrifugation treatment. Adjusting the pH of the culture solution to 3.0, adding 0.2% (W/V) of activated carbon and 2% (W/V) of diatomite as a decolorizing agent and a filtering aid, and performing suction filtration to obtain an approximately colorless and transparent filtrate.

The polysaccharide can be precipitated as solid after mixing the polysaccharide aqueous solution with alcohols with multiple volumes, such as ethanol, and the like, which is a common method for precipitating the polysaccharide from the polysaccharide solution. Mixing the filtrate obtained in the method with 80% -95% ethanol with the volume of 1-3 times that of the filtrate to obtain white flocculent extracellular polysaccharide, fishing out the white flocculent extracellular polysaccharide and squeezing the white flocculent extracellular polysaccharide until no liquid flows out, wherein the white flocculent extracellular polysaccharide is wet solid polysaccharide. Sampling and measuring the solid content (dry matter content), and calculating the dry matter weight of the exopolysaccharide according to the weight of the wet solid polysaccharide and the solid content. The wet solid polysaccharide is re-dissolved in a certain amount of water to make the concentration of the polysaccharide in the water solution reach a set value, generally within the range of 1-5%. Adding basic amino acids such as arginine, histidine, lysine, ornithine, etc., or mixture thereof into the solution. The addition of basic amino acid can neutralize hydrogen ion carried by exopolysaccharide, so that pH of final composition is close to neutral, and the final composition is easy to use in cosmetic preparation. The addition of the basic amino acid can also improve the functionality of the composition and better play the roles of moisturizing the skin and resisting aging. And determining the adding weight of the basic amino acid according to the dry matter weight of the exopolysaccharide, adding 0.05-0.5 g of the basic amino acid into each gram of the dry matter of the exopolysaccharide, stirring and dissolving to obtain a solution with the pH close to neutrality, or adjusting the pH to a specific range according to the requirement.

Detailed Description

Example 1

According to the instructions, the MS or LS culture medium dry powder purchased from the market is reconstituted by distilled water or deionized water, 2, 4-dichlorophenoxyacetic acid and 1-naphthylacetic acid are pre-dissolved by a small amount of absolute ethyl alcohol, 6-benzylaminopterin is pre-dissolved by a small amount of 0.1N NaOH solution, the mixture is added into the reconstituted liquid culture medium to ensure that the concentration of the mixture is 1 x 10 to 5mol/L, 1 x 10 to 5mol/L and 1 x 10 to 6mol/L, 3 percent (W/V) of sucrose is added and stirred for dissolution, and the pH is adjusted to be within the range of 6.0 to 7.0 by NaOH. Transferring the prepared culture solution into a fermentation tank, heating to 118 ℃, sterilizing for 15 minutes, cooling to 25-30 ℃, inoculating the cultured tuberose petal cell liquid culture, wherein the inoculation amount is 1% (V/V), culturing at the constant temperature of 26 +/-1 ℃ without illumination, the stirring rotation speed of the fermentation tank is 80 r/min, the ventilation of the fermentation tank is 30L/min, and placing the fermentation tank after culturing for 15-30 days to collect the culture solution. Adding anhydrous citric acid into the culture solution at a ratio of 4 g/L under stirring to ensure that the pH value is about 3.0, adding 0.2% (W/V) of powdered activated carbon and 2% (W/V) of 100-mesh diatomite, stirring uniformly, standing for 1-2 hours, stirring uniformly, and pouring into a suction filtration funnel for suction filtration. The filtrate can be concentrated by using vacuum evaporation equipment, so that the volume is reduced to 1/3-1/2 of the original volume, the ethanol dosage in the alcohol precipitation is reduced, and the alcohol precipitation can also be directly carried out without concentration. Pouring the filtrate into 95% ethanol with 2 times of the volume of the filtrate, stirring and mixing, separating white flocculent polysaccharide, separating the precipitate by using filter cloth, squeezing until no liquid flows out to obtain wet solid polysaccharide, weighing, sampling, drying at 60-90 ℃ by using a rapid moisture tester to determine the dry matter content, and multiplying the weight by the dry matter content to obtain the dry matter weight of the polysaccharide. Putting the wet solid polysaccharide into water preheated to 70-90 ℃, stirring and dissolving, adding 0.2g of arginine into each gram of extracellular polysaccharide dry matter, stirring and dissolving to ensure that the pH is 6.0-7.0, supplementing water to ensure that the extracellular polysaccharide concentration is 3.0% (W/V), adding a proper amount of preservative after slight cooling, and cooling to obtain the composition for cosmetics.

Example 2

Example 1 was followed except that 0.2g of arginine was replaced with 0.178g of histidine.

Example 3

Example 1 was followed except that 0.2g of arginine was replaced with 0.168g of lysine.

Example 4

Example 1 was followed except that 0.2g of arginine was replaced with 0.152g of ornithine.

Example 5

Example 1 was followed except that 0.2g of arginine was replaced with a mixture of 0.1g of arginine and 0.089 g of histidine.

Example 6

Example 1 was followed except that 0.2g of arginine was replaced with 0.046g of sodium hydroxide.

Example 7

The compositions prepared in examples 1 to 6 were measured for radical clearance using ABTS (total antioxidant capacity assay kit method), and the specific results are shown in table 1:

TABLE 1

From the free radical scavenging data, it is seen that compositions prepared using basic amino acids, which enhance the functionality of the composition, are superior in oxidation resistance to compositions prepared using basic sodium hydroxide, and that mixtures of basic amino acids result in better oxidation resistance.

Example 8

The composition obtained in example 5 was formulated as a cosmetic moisturizer, and the formulation is shown in table 2:

TABLE 2

The active formula is smeared on the inner side of the left upper arm and the blank control is smeared on the inner side of the right upper arm of 10 volunteers with the average age of 37.5 +/-4.4, the smearing amount is 0.1 ml each time, the active formula is smeared twice a day, the water content of the skin is measured after 4 days, and the elasticity and fine lines of the skin are measured after 30 days.

The skin moisture content is measured by using a multifunctional measuring instrument Corneometer CM825 instrument of Germany couage + KHAZAKA (CK), the moisturizing effect of the product is evaluated by comparing the change of the skin moisture content before and after the moisturizing water is used, the improvement is realized by increasing 0-5 before and after the evaluation standard, the obvious improvement is realized by increasing 5-10, and the obvious improvement is realized by increasing more than 10. Skin elasticity is measured by using a multifunctional measuring instrument Corneometer CM825 instrument of the German couage + KHAZAKA (CK) company, the anti-aging effect of the product is evaluated by comparing the change of the skin elasticity value before and after the use of the moisturizing water, and the evaluation standard is that the improvement is realized by increasing 0-0.05 before and after the evaluation standard, the obvious improvement is realized by increasing 0.05-0.1, and the obvious improvement is realized by being more than 0.1. The skin fine lines are measured by using an SV600 skin wrinkle measuring instrument of Germany couage + KHAZAKA (CK) company to measure skin roughness parameters, the anti-aging effect of the product is evaluated by comparing the changes of the skin roughness parameters before and after the moisturizing lotion is used, the evaluation standard is that the skin roughness parameters are reduced by 0-5 before and after the moisturizing lotion is improved, 5-10 is obviously improved, and more than 10 is obviously improved.

The average data of volunteers before and after application of moisturizing water is compared in table 3.

TABLE 3

As can be seen from the data, the composition prepared in example 5, which contains tuberose extracellular polysaccharide and basic amino acid, has better skin moisturizing and anti-aging effects than the blank control containing only extracellular polysaccharide and no basic amino acid.

Claims

1. A composition for skin care characterized by: the composition comprises exopolysaccharides and basic amino acids produced by in vitro culture of Polianthes tuberosa L cells.

2. A process for preparing a composition according to claim 1, comprising the following features:

a, the liquid culture medium used by the tuberose cells contains 10 < -4 > -10 < -6 > mol/L of 2, 4-Dichlorophenoxyacetic acid (2, 4-Dichlorophenoxyacetic acid), 10 < -4 > -10 < -6 > mol/L of 1-naphthylacetic acid (1-naphthylacetic acid), 10 < -5 > -10 < -7 > mol/L of 6-benzylaminopterin (6-benzyl aminopterine) and 1 < -5 > (W/V) of sucrose (sucrose), and the concentration is preferably 1 < -10 > -5 > mol/L, 1 < -10 > -6 > mol/L and 3 percent respectively.

b, adjusting the pH of the liquid culture medium of the tuberose cells by using an acidic substance after the culture is finished, wherein the acidic substance can be hydrochloric acid, citric acid, acetic acid and the like, and preferably citric acid is used; the pH is adjusted to be within a range of 2 to 5, preferably within a range of 3 to 4.

And c, separating the exopolysaccharide obtained by alcohol precipitation from the liquid, weighing and measuring the dry matter content, adding the exopolysaccharide into water for dissolving again, and adding a certain amount of basic amino acid such as arginine, histidine, lysine, ornithine and the like or a mixture of the basic amino acids into the solution according to the weight of the dry matter of the exopolysaccharide. The dosage of the basic amino acid is that 0.05-0.5 g of the basic amino acid or the mixture thereof is required to be added per gram of extracellular polysaccharide dry matter.

3. Use of a composition for skin care according to claim 1 in cosmetics.