Dendrobium plant callus filtrate as well as preparation method and application thereof

Abstract

The invention provides dendrobium plant callus filtrate as well as a preparation method and application thereof, and belongs to the technical field of plant extracts. The preparation method of the dendrobium plant callus filtrate comprises the following steps: (1) performing dedifferentiation culture on dendrobium stems to obtain dendrobium calluses; and (2) carrying out propagation culture on the dendrobium callus obtained in the step (1), crushing, carrying out solid-liquid separation, and taking a liquid part to obtain dendrobium plant callus filtrate. According to the method disclosed by the invention, a group of propagation culture media capable of obviously improving the propagation coefficient and quality of the dendrobium callus are obtained by screening and optimizing the propagation culture media during dendrobium callus culture, and a basis is provided for efficient culture of the dendrobium callus.

Description

Technical Field

[0001] The invention relates to the technical field of plant extracts, and in particular to a dendrobium plant callus filtrate and a preparation method and application thereof. Background Art

[0002] Ultraviolet radiation, minor skin damage caused by daily environment, redness caused by skin sensitivity, acne (redness and swelling caused by inflammation), etc. can cause the skin's red blood cells to rupture, release hemoglobin, and cause skin cells to release inflammatory signals, accelerating the inflammatory cascade reaction, reducing the number of cells, producing tissue cavitation, and degrading the extracellular matrix, and the skin shows signs of aging. It can be seen that it is of great significance to provide a product that can protect skin cells and resist skin aging. Summary of the invention

[0003] The purpose of the present invention is to provide a dendrobium plant callus filtrate and a preparation method and application thereof, in order to provide a theoretical basis for studying a product with high safety and capable of protecting skin cells and resisting cell aging.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for preparing a dendrobium plant callus filtrate, comprising the following steps:

[0006] (1) dedifferentiating and culturing young stems of Dendrobium officinale to obtain Dendrobium officinale callus;

[0007] (2) The dendrobium callus obtained in step (1) is crushed after propagation culture, and the solid and liquid are separated, and the liquid portion is taken to obtain the dendrobium plant callus filtrate.

[0008] Preferably, the culture medium used in the dedifferentiation culture is a 2 / 3MS culture medium containing 25-35 g / L maltose, 0.4-0.6 mg / L 6-BA, 1.3-1.7 mg / L KT and 0.04-0.06 mg / L NAA, and the pH of the culture medium is 5.5-6.0;

[0009] The dedifferentiation culture is carried out at a temperature of 23 to 27° C. for 15 to 25 days in a dark environment.

[0010] Preferably, the culture medium also includes 5.0-6.0 g / L agar.

[0011] Preferably, the culture medium used in the propagation culture is a 2 / 3MS culture medium containing 0.4-0.6 mg / L 6-BA, 0.04-0.06 mg / L NAA, 25-35 g / L sucrose and 5-7 g / L phytagel, and the pH of the culture medium is 5.5-6.0.

[0012] Preferably, the illumination intensity of the expansion culture is 20 to 30 μmol·m -2 ·S -1 , cultured alternately under 10-14h light and 10-14h dark conditions, the temperature during culture under light conditions is 20-24°C, and the temperature during culture under dark conditions is 16-20°C; the humidity during culture is 60-80%.

[0013] Preferably, 1,3-propylene glycol, fructose and water are added to the obtained Dendrobium plant callus filtrate to prepare an extract;

[0014] The mass ratio of 1,3-propylene glycol, fructose and water is 2.5-3.5:0.8-1.2:5-7;

[0015] The mass volume ratio of the mixture of the dendrobium callus tissue and 1,3-propylene glycol, fructose and water is 160-240 mg: 800-1200 mL.

[0016] The invention provides a dendrobium plant callus filtrate prepared according to the method.

[0017] The invention provides an application of the dendrobium plant callus filtrate in preparing cosmetics.

[0018] Preferably, the cosmetics are cosmetics with moisturizing, anti-wrinkle, firming, soothing, repairing and anti-oxidation effects.

[0019] The present invention also provides a cosmetic with moisturizing, anti-wrinkle, firming, soothing, repairing and anti-oxidation effects, wherein the cosmetic contains the dendrobium plant callus filtrate.

[0020] The present invention provides a dendrobium plant callus filtrate and a preparation method thereof, wherein a group of culture mediums that can significantly improve the proliferation coefficient and quality of dendrobium callus are obtained by screening and optimizing the culture medium for the cultivation of dendrobium callus, thereby providing a basis for the efficient cultivation of dendrobium callus. The present invention crushes the cultivated dendrobium callus to obtain the dendrobium plant callus filtrate.

[0021] The present invention also tests the moisturizing ability, flavonoid content, elastin inhibition rate, firming and anti-wrinkle ability, density and carbon and water non-volatile matter, pesticide residue and heavy metal content, soothing effect, repair effect, light absorption characteristics, skin irritation, skin allergy and acute eye irritation of the prepared Dendrobium plant callus tissue filtrate, and finds that the Dendrobium plant callus tissue filtrate prepared by the present invention has good moisturizing, anti-wrinkle, firming, soothing, repairing and antioxidant effects on the skin, and has high safety, and can be used for the preparation of functional cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0023] Figure 1 The young stem of Dendrobium officinale (left) and the cultured callus of Dendrobium officinale (right) in Example 1;

[0024] Figure 2 The Dendrobium callus obtained by the propagation culture in Example 1 is shown from left to right in the states of 0 day, 15 day, 30 day, 45 day and 60 day of culture;

[0025] Figure 3 The growth of Dendrobium callus obtained by culturing for 60 days using 4 groups of different propagation medium in Example 1;

[0026] Figure 4 The results of measuring the moisture retention capacity of the Dendrobium plant callus filtrate in Example 2;

[0027] Figure 5 The results are as follows for determining the flavonoid content of the Dendrobium plant callus filtrate in Example 2;

[0028] Figure 6 The results of the determination of the elastin inhibition rate of the Dendrobium plant callus filtrate in Example 2;

[0029] Figure 7 The results of the determination of the firming and anti-wrinkle ability of the Dendrobium plant callus filtrate in Example 2;

[0030] Figure 8 The results of the determination of the density and carbon non-volatiles of the Dendrobium plant callus filtrate in Example 2;

[0031] Fig. 9 The results of the determination of the pesticide residue and heavy metal content in the filtrate of the Dendrobium plant callus in Example 2;

[0032] Fig.10 The results of the soothing effect of the Dendrobium plant callus filtrate in Example 2 are as follows;

[0033] Fig.11 The results of the determination of the repairing effect of the Dendrobium plant callus filtrate in Example 2;

[0034] Fig.12 The results of measuring the light absorption characteristics of the Dendrobium plant callus filtrate in Example 2;

[0035] Fig.13 The results of the determination of the skin irritation of the Dendrobium plant callus filtrate in Example 2;

[0036] Fig.14 The results of the determination of skin allergy of the Dendrobium plant callus filtrate in Example 2;

[0037] Fig.15 The following are the results of the determination of the acute eye irritation of the Dendrobium plant callus filtrate in Example 2. DETAILED DESCRIPTION

[0038] The present invention provides a method for preparing a dendrobium plant callus filtrate, comprising the following steps:

[0039] (1) dedifferentiating and culturing young stems of Dendrobium officinale to obtain Dendrobium officinale callus;

[0040] (2) The dendrobium callus obtained in step (1) is crushed after propagation culture, and the solid and liquid are separated, and the liquid portion is taken to obtain the dendrobium plant callus filtrate.

[0041] In the present invention, the culture medium used in the dedifferentiation culture is a 2 / 3MS culture medium containing 25-35 g / L maltose, 0.4-0.6 mg / L 6-BA, 1.3-1.7 mg / L KT and 0.04-0.06 mg / L NAA, preferably a 2 / 3MS culture medium containing 28-32 g / L maltose, 0.45-0.55 mg / L 6-BA, 1.4-1.6 mg / L KT and 0.045-0.055 mg / L NAA, and more preferably a 2 / 3MS culture medium containing 30 g / L maltose, 0.5 mg / L 6-BA, 1.5 mg / L KT and 0.05 mg / L NAA; the pH of the culture medium is 5.5-6.0, preferably 5.6-5.9, and more preferably 5.8;

[0042] The dedifferentiation culture temperature is 23-27°C, preferably 24-26°C, more preferably 25°C, the time is 15-25 days, preferably 18-22 days, more preferably 19-21 days, more preferably 19 days, and the culture is carried out in dark conditions.

[0043] In the present invention, the culture medium further comprises 5.0-6.0 g / L of agar, preferably 5.2-5.8 g / L of agar, further preferably 5.4-5.6 g / L of agar, and more preferably 5.5 g / L of agar.

[0044] In the present invention, the culture medium used for the propagation culture is a 2 / 3MS culture medium containing 0.4-0.6 mg / L 6-BA, 0.04-0.06 mg / L NAA, 25-35 g / L sucrose and 5-7 g / L plant gel, preferably a 2 / 3MS culture medium containing 0.45-0.55 mg / L 6-BA, 0.045-0.055 mg / L NAA, 28-32 g / L sucrose and 5.5-6.5 g / L plant gel, and more preferably a 2 / 3MS culture medium containing 0.5 mg / L 6-BA, 0.05 mg / L NAA, 30 g / L sucrose and 6 g / L plant gel; the pH of the culture medium is 5.5-6.0, preferably 5.6-5.9, and more preferably 5.8.

[0045] In the present invention, the illumination intensity of the expansion culture is 20 to 30 μmol·m -2 ·S -1 , preferably 22 to 28 μmol·m -2 ·S -1 , more preferably 24 to 26 μmol·m -2 ·S -1 , more preferably 25 μmol·m -2 ·S -1 ; Alternately cultivate under 10-14h light and 10-14h dark conditions, preferably alternately cultivate under 11-13h light and 11-13h dark conditions, and further preferably alternately cultivate under 12h light and 12h dark conditions; the temperature during cultivation under light conditions is 20-24°C, preferably 21-23°C, and further preferably 22°C, and the temperature during cultivation under dark conditions is 16-20°C, preferably 17-19°C, and further preferably 18°C; the humidity during cultivation is 60-80%, preferably 65-75%, and further preferably 70%.

[0046] In the present invention, 1,3-propylene glycol, fructose and water are added to the obtained Dendrobium plant callus filtrate to prepare an extract;

[0047] The mass ratio of 1,3-propylene glycol, fructose and water is 2.5-3.5:0.8-1.2:5-7, preferably 2.8-3.2:0.9-1.1:5.5-6.5, and more preferably 3:1:6;

[0048] The mass volume ratio of the mixture of the dendrobium callus tissue and 1,3-propylene glycol, fructose and water is 160-240 mg:800-1200 mL, preferably 180-220 mg:900-1100 mL, and more preferably 200 mg:1000 mL.

[0049] The invention provides a dendrobium plant callus filtrate prepared according to the method.

[0050] The invention provides an application of the dendrobium plant callus filtrate in preparing cosmetics.

[0051] In the present invention, the cosmetics are cosmetics with moisturizing, anti-wrinkle, firming, soothing, repairing and anti-oxidation effects.

[0052] The present invention also provides a cosmetic with moisturizing, anti-wrinkle, firming, soothing, repairing and anti-oxidation effects, wherein the cosmetic contains the dendrobium plant callus filtrate.

[0053] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0054] Example 1

[0055] A method for preparing a dendrobium plant callus filtrate, comprising the following steps:

[0056] (1) The young stems of Dendrobium officinale germinated from the seeds (provided by the Plant Cell Engineering Research Center, Institute of Biotechnology, Shanghai Academy of Agricultural Sciences) were placed in a culture bottle filled with dedifferentiation medium, sealed with Parafilm, and cultured in the dark at 25°C for 19 days to obtain Dendrobium officinale callus. The young stems of Dendrobium officinale (left) and the cultured Dendrobium officinale callus (right) are shown in Figure 1. Figure 1 As shown;

[0057] The dedifferentiation culture medium is based on 2 / 3MS culture medium, added with 30 g / L maltose, 0.5 mg / L 6-BA, 1.5 mg / L KT and 0.05 mg / L NAA, solidified with 5.5 g / L agar powder, and has a pH of 5.8.

[0058] (2) The Dendrobium callus obtained in step (1) was transferred into a propagation medium and cultured under a light intensity of 25 μmol·m -2 ·S -1 , 12h light, 12h dark conditions, alternating culture, light culture temperature of 22 ℃, dark culture temperature of 18 ℃, culture humidity of 70%; the picture of the expanded culture of Dendrobium callus is as follows Figure 2 As shown, from left to right are the state diagrams at 0 days, 15 days, 30 days, 45 days and 60 days of cultivation;

[0059] The enrichment medium is a 2 / 3MS medium containing 0.5 mg / L 6-BA, 0.05 mg / L NAA, 30 g / L sucrose and 6 g / L Phytagel plant gel, and the pH of the medium is 5.8.

[0060] (3) The Dendrobium callus tissue expanded in step (2) is first crushed by a high-speed rotating blade at a speed of 10000 rpm for 10 seconds, and then crushed by an ultrasonic crusher (20KHz, 25°C) for 9 seconds, and then mixed with 1,3-propylene glycol, fructose and water to obtain a Dendrobium plant callus tissue filtrate; the mass ratio of 1,3-propylene glycol, fructose and water is 3:1:6, and the mass volume ratio of the 1,3-propylene glycol, fructose and water mixture to the Dendrobium callus tissue is 1000mL:200mg.

[0061] The inventors also screened the propagation culture medium used in the early stage and found that the proliferation coefficient of the callus tissue propagated by liquid culture medium was low, which was easy to cause pollution and the callus quality was also poor. Therefore, the inventors subsequently set up 4 groups of solid propagation culture media, namely:

[0062] 1) 1 / 2MS + 6-BA 0.5mg / L + NAA 0.05mg / L + sucrose 30g / L + agar powder 6g / L, pH 5.8;

[0063] 2) MS + 6-BA 0.5 mg / L + NAA 0.05 mg / L + sucrose 30 g / L + agar powder 6 g / L, pH 5.8;

[0064] 3) 2 / 3MS + 6-BA 0.5mg / L + NAA 0.05mg / L + sucrose 30g / L + agar powder 6g / L, pH 5.8;

[0065] 4) 2 / 3MS + 6-BA 0.5mg / L + NAA 0.05mg / L + sucrose 30g / L + plant gel 6g / L, pH 5.8.

[0066] The growth of Dendrobium callus obtained by culturing with the above four groups of propagation medium for 60 days is as follows Figure 3 As shown, from left to right, the callus tissues obtained using the culture medium of group 1) to group 4) are respectively shown. It can be seen that the efficiency of propagating the callus tissue of Dendrobium candidum using the culture medium of group 4) is the highest. After calculation, the weight of the callus tissues obtained using the culture medium of group 1) to group 4) is 60g, 100g, 150g and 200g respectively.

[0067] Example 2

[0068] The Xianhuyin mentioned in this embodiment is the Dendrobium plant callus filtrate prepared in Example 1.

[0069] In this embodiment, the prepared Dendrobium plant callus filtrate is sent to Guangzhou Jinzhi Testing Technology Co., Ltd. to test the moisturizing ability, flavonoid content, elastin inhibition rate, firming and anti-wrinkle ability, density and carbon water non-volatile matter, pesticide residue and heavy metal content, soothing effect, repair effect and light absorption characteristics of the Dendrobium plant callus filtrate (Xianhuyin (Dendrobium officinale extract)); and sent to Hunan Shanshui Testing Co., Ltd. to test the skin irritation, skin allergy and acute eye irritation of the Dendrobium plant callus filtrate.

[0070] Test results such as Figures 4 to 15 As shown, it can be seen that the Dendrobium plant callus filtrate prepared by the present invention has good moisturizing effect; can achieve a firming effect by inhibiting the activity of elastase; can significantly improve total collagen secretion, and has good anti-wrinkle and firming effects; can achieve a soothing effect by inhibiting inflammatory factors; can significantly promote cell migration, and has a repair effect; has no absorption effect on long blue light, short blue light, UVA and UVB, no total flavonoids, no agricultural residues and heavy metals, no irritation to the skin, no skin allergy, and no irritation to the eyes. It can be seen that the Dendrobium plant callus filtrate prepared by the present invention has good moisturizing, anti-wrinkle, firming, soothing and repairing effects on the skin, and has high safety, and can be used for the production of functional cosmetics.

[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a dendrobium plant callus filtrate, characterized in that: The steps include: (1) dedifferentiating and culturing young stems of Dendrobium officinale to obtain Dendrobium officinale callus; (2) The dendrobium callus obtained in step (1) is crushed after propagation culture, and the solid-liquid separation is performed, and the liquid portion is taken to obtain the dendrobium plant callus filtrate.

2. The method according to claim 1, characterized in that The culture medium used in the dedifferentiation culture is a 2 / 3MS culture medium containing 25-35 g / L maltose, 0.4-0.6 mg / L 6-BA, 1.3-1.7 mg / L KT and 0.04-0.06 mg / L NAA, and the pH of the culture medium is 5.5-6.0; The dedifferentiation culture is carried out at a temperature of 23 to 27° C. for 15 to 25 days in a dark environment.

3. The method according to claim 2, characterized in that The culture medium also includes 5.0-6.0 g / L of agar.

4. The method according to claim 3, characterized in that The culture medium used in the propagation culture is a 2 / 3MS culture medium containing 0.4-0.6 mg / L 6-BA, 0.04-0.06 mg / L NAA, 25-35 g / L sucrose and 5-7 g / L plant gel, and the pH of the culture medium is 5.5-6.

0.

5. The method according to claim 4, characterized in that The illumination intensity of the propagation culture is 20 to 30 μmol·m -2 ·S -1 , cultured alternately under 10-14h light and 10-14h dark conditions, the temperature during culture under light conditions is 20-24°C, and the temperature during culture under dark conditions is 16-20°C; the humidity during culture is 60-80%.

6. The method according to any one of claims 1 to 5, characterized in that: 1,3-propylene glycol, fructose and water are added to the obtained Dendrobium plant callus filtrate to prepare an extract; The mass ratio of 1,3-propylene glycol, fructose and water is 2.5-3.5:0.8-1.2:5-7; The mass volume ratio of the mixture of the dendrobium callus tissue and 1,3-propylene glycol, fructose and water is 160-240 mg: 800-1200 mL.

7. A Dendrobium plant callus filtrate prepared according to the method of any one of claims 3 to 6.

8. Use of the Dendrobium plant callus filtrate according to claim 7 in preparing cosmetics.

9. The use according to claim 8, characterized in that The cosmetics are cosmetics with moisturizing, anti-wrinkle, firming, soothing, repairing and anti-oxidation effects.

10. A cosmetic with moisturizing, anti-wrinkle, firming, soothing, repairing and anti-oxidation effects, characterized in that: The cosmetic comprises the Dendrobium plant callus filtrate according to claim 7.

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