Nicotiana benthamiana callus extract as well as preparation method and cosmetics thereof

Abstract

The invention relates to the field of cosmetics, in particular to a nicotiana benthamiana callus extract, a preparation method thereof and cosmetics. The method comprises the following steps: inoculating an induction medium with young leaves of nicotiana benthamiana as explants, and inducing the explants to form calluses; the induction culture medium is prepared from 1.0 to 3.0 mg / L of 6-benayl aminopurine; 0.2 to 1.0 mg / L of naphthylacetic acid; inoculating the callus on a subculture medium for multiplication culture; the subculture medium is prepared from the following components: 0.5 to 2.0 mg / L of 6-benayl aminopurine, 0.0 to 1.0 mg / L of naphthylacetic acid and 0.0 to 2.0 mg / L of 2, 4-dichlorphenoxyacetic acid; and extracting the calluses of the nicotiana benthamiana after multiplication culture by using water as a solvent according to a material-liquid ratio of 1: (5-20). According to the method, the Bensi tobacco callus with high dedifferentiation degree, good loosening degree and high growth speed is obtained, and the callus extract with the DPPH removal rate reaching 85% or above is obtained through extraction.

Description

Technical Field

[0001] The present application relates to the field of cosmetics, and in particular, to a Nicotiana benthamiana callus extract, a preparation method thereof, and cosmetics. Background Art

[0002] At present, there is no application of Nicotiana benthamiana callus in the field of cosmetics. Summary of the invention

[0003] The purpose of the embodiments of the present application is to provide a Nicotiana benthamiana callus extract, a preparation method thereof, and a cosmetic.

[0004] In a first aspect, the present application provides a method for preparing a Nicotiana benthamiana callus extract, comprising:

[0005] Young leaves of Nicotiana benthamiana were used as explants and inoculated on induction medium to induce the explants to form callus tissue.

[0006] The induction medium includes: 6-benzylaminopurine 1.0 mg / L-3.0 mg / L; naphthaleneacetic acid 0.2 mg / L-1.0 mg / L;

[0007] The callus tissue is inoculated on a subculture medium for proliferation culture;

[0008] The subculture medium includes: 6-benzylaminopurine 0.5 mg / L-2.0 mg / L, naphthylacetic acid 0.0 mg / L-1.0 mg / L, 2,4-dichlorophenoxyacetic acid 0.0 mg / L-2.0 mg / L;

[0009] The Nicotiana benthamiana callus tissue after proliferation and culture is extracted with water as solvent, and the solid-liquid ratio is 1:(5-20).

[0010] The present application scheme can obtain Nicotiana benthamiana callus with a high degree of dedifferentiation, good looseness and fast growth rate, and extract Nicotiana benthamiana callus extract with a DPPH scavenging rate of more than 85%; the Nicotiana benthamiana callus extract is applied to cosmetics and an in vitro efficacy evaluation is performed, which shows that the Nicotiana benthamiana callus extract has certain effects of improving BJ cell survival rate (firming), promoting cell scratch healing (repair), and scavenging ABTS / DPPH free radicals (anti-oxidation) at the tested concentration, laying a foundation for its application in anti-aging cosmetics.

[0011] Furthermore, the present application is the first to extract Nicotiana benthamiana callus to obtain Nicotiana benthamiana callus extract, and the preparation method is short in time and low in cost, and there is no organic solvent residue, and the solvent used is water, which is highly safe and has high extraction efficiency. The present application is the first to apply Nicotiana benthamiana callus extract to anti-aging cosmetics, which has antioxidant, repairing, and firming effects. There has been no development of "Nicotiana benthamiana callus" in the art, let alone extraction of it, and its extract is used in cosmetics, so it has great application value.

[0012] In other embodiments of the present application, the above-mentioned induction culture medium includes: 1.0 mg / L-2.0 mg / L of 6-benzylaminopurine; 0.2 mg / L-1.0 mg / L of naphthaleneacetic acid.

[0013] In other embodiments of the present application, the subculture culture medium includes: 0.5 mg / L-1.0 mg / L 6-benzylaminopurine, 0.5 mg / L-1.0 mg / L naphthylacetic acid, and 0.0 mg / L-1.0 mg / L 2,4-dichlorophenoxyacetic acid.

[0014] In other embodiments of the present application, the Nicotiana benthamiana callus after proliferation and culture is extracted with water as a solvent, including: extracting with a solid-liquid ratio of 1:(5-10).

[0015] In other embodiments of the present application, the Nicotiana benthamiana callus after proliferation and culture is extracted using water as a solvent, comprising:

[0016] A high-voltage pulse electric field with a field strength of 30 kV / m-50 kV / m is used for extraction; the number of pulses of the high-voltage pulse electric field is 60 times-120 times.

[0017] In other embodiments of the present application, the Nicotiana benthamiana callus after proliferation and culture is extracted using water as a solvent, further comprising:

[0018] After the extraction is completed, centrifuge at 6000rpm-12000rpm for 5min-15min and take the supernatant.

[0019] In other embodiments of the present application, the induction culture medium includes: 4.5 g / L to 5.0 g / L MS powder, 25 g / L-35 g / L sucrose, and 7.0 g / L-8.0 g / L agar; and the pH value of the culture medium is 5.8-5.9.

[0020] In other embodiments of the present application, the subculture culture medium further includes: 4.5 g / L to 5.0 g / L MS powder, 25 g / L-35 g / L sucrose, and 7.0-8.0 g / L agar; the pH value of the culture medium is 5.8-5.9.

[0021] In a second aspect, the present application provides a Nicotiana benthamiana callus extract, which is prepared by the preparation method of the Nicotiana benthamiana callus extract provided in the first aspect.

[0022] In a third aspect, the present application provides a cosmetic comprising the Nicotiana benthamiana callus extract provided in the second aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 To evaluate the in vitro efficacy of Nicotiana benthamiana callus extract (biochemical method);

[0025] Figure 2 To evaluate the in vitro efficacy of Nicotiana benthamiana callus extract (cell method);

[0026] Figure 3 Effects of Nicotiana benthamiana callus extract on cell scratch healing. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments.

[0028] Therefore, the following detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0029] The present application provides a method for preparing a Nicotiana benthamiana callus extract, comprising:

[0030] Young leaves of Nicotiana benthamiana were used as explants and inoculated on induction medium to induce the explants to form callus tissue.

[0031] The induction medium includes: 6-benzylaminopurine 1.0 mg / L-3.0 mg / L; naphthaleneacetic acid 0.2 mg / L-1.0 mg / L;

[0032] The callus tissue is inoculated on a subculture medium for proliferation culture;

[0033] The subculture medium includes: 6-benzylaminopurine 0.5 mg / L-2.0 mg / L, naphthylacetic acid 0.0 mg / L-1.0 mg / L, 2,4-dichlorophenoxyacetic acid 0.0 mg / L-2.0 mg / L;

[0034] The Nicotiana benthamiana callus tissue after proliferation and culture is extracted with water as solvent, and the solid-liquid ratio is 1:(5-20).

[0035] In the above technical scheme, the above preparation method can be used to effectively prepare the Nicotiana benthamiana callus extract; thus, it can be applied to cosmetics. The culture medium provided in the present application has high induction efficiency, and the obtained Nicotiana benthamiana callus has a high degree of dedifferentiation, good looseness, and fast growth rate. The present application is the first to extract Nicotiana benthamiana callus to obtain Nicotiana benthamiana callus extract, and the preparation method is time-consuming and low-cost. At the same time, there is no organic solvent residue, and the solvent used is water, which is highly safe and has high extraction efficiency. The present application is the first to apply the Nicotiana benthamiana callus extract to anti-aging cosmetics, which has antioxidant, repairing, and firming effects. There has been no development of "Nicotibacterium benthamiana callus" in the art, let alone extraction of it, and its extract is applied to cosmetics, so it has great application value.

[0036] Further optionally, illustratively, in some embodiments of the present application, young leaves of Nicotiana benthamiana are used as explants and inoculated on an induction medium to induce the explants to form callus tissue;

[0037] The induction medium includes: 6-benzylaminopurine 1.0 mg / L, 1.1 mg / L, 1.2 mg / L, 1.5 mg / L, 1.8 mg / L, 2.0 mg / L, 2.1 mg / L, 2.2 mg / L, 2.3 mg / L, 2.4 mg / L, 2.5 mg / L, 2.6 mg / L, 2.7 mg / L, 2.8 mg / L, 2.9 mg / L, 3.0 mg / L or the range between any two of the foregoing values; naphthylacetic acid 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1.0 mg / L or the range between any two of the foregoing values.

[0038] Further optionally, illustratively, in some embodiments of the present application, the callus is inoculated on a subculture medium for proliferation culture to obtain vigorously growing Nicotiana benthamiana callus;

[0039] Subculture medium includes: 6-benzylaminopurine 0.5mg / L, 0.6mg / L, 0.7mg / L, 0.8mg / L, 0.9mg / L, 1.0mg / L, 1.1mg / L, 1.2mg / L, 1.3mg / L, 1.4mg / L, 1.5mg / L, 1.6mg / L, 1.7mg / L, 1.8mg / L, 1.9mg / L, 2.0mg / L or the range between any two of the foregoing values; naphthylacetic acid 0.0mg / L, 0.01mg / L, 0.05mg / L, 0.1mg / L, 0.2mg / L, 0.3mg / L, 0.4mg / L, 0.5 0.0mg / L, 0.01mg / L, 0.05mg / L, 0.1mg / L, 0.2mg / L, 0.3mg / L, 0.4mg / L, 0.5mg / L, 0.6mg / L, 0.7mg / L, 0.8mg / L, 0.9mg / L, 1.0mg / L or the range between any two of the foregoing values; 2,4-dichlorophenoxyacetic acid 0.0mg / L, 0.01mg / L, 0.05mg / L, 0.1mg / L, 0.2mg / L, 0.3mg / L, 0.4mg / L, 0.5mg / L, 0.6mg / L, 0.7mg / L, 0.8mg / L, 0.9mg / L, 1.0mg / L, 1.2mg / L, 1.5mg / L, 1.8mg / L, 2.0mg / L or the range between any two of the foregoing values.

[0040] Furthermore, in some embodiments of the present application, the vigorously growing Nicotiana benthamiana callus is extracted using water as a solvent, and the solid-liquid ratio is 1:5, 1:6, 1:8, 1:10, 1:12, 1:15, 1:18, 1:19, 1:20 or a range between any two of the foregoing values.

[0041] Further optionally, in some embodiments of the present application, the induction culture medium comprises: 1.0 mg / L-2.0 mg / L 6-benzylaminopurine; 0.2 mg / L-1.0 mg / L naphthylacetic acid.

[0042] In the above technical solution, the induction culture medium is provided to include: 1.0 mg / L-2.0 mg / L 6-benzylaminopurine; 0.2 mg / L-1.0 mg / L naphthaleneacetic acid; which is more conducive to inducing callus tissue with a softer texture.

[0043] Illustratively, in some embodiments of the present application, the induction medium includes: 6-benzylaminopurine 1.0 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L, 1.5 mg / L, 1.6 mg / L, 1.7 mg / L, 1.8 mg / L, 1.9 mg / L, 2.0 mg / L or the range between any two of the foregoing values; naphthylacetic acid 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1.0 mg / L or the range between any two of the foregoing values.

[0044] Further optionally, in some embodiments of the present application, the subculture culture medium includes: 6-benzylaminopurine 0.5 mg / L-1.0 mg / L, naphthylacetic acid 0.5 mg / L-1.0 mg / L, and 2,4-dichlorophenoxyacetic acid 0.0 mg / L-1.0 mg / L.

[0045] In the above technical scheme, by setting the subculture culture medium including: 6-benzylaminopurine 0.5mg / L-1.0mg / L, naphthaleneacetic acid 0.5mg / L-1.0mg / L, 2,4-dichlorophenoxyacetic acid 0.0mg / L-1.0mg / L; it is more conducive to improving the growth rate of callus tissue and obtaining vigorously growing Nicotiana benthamiana callus tissue.

[0046] Illustratively, in some embodiments of the present application, the subculture culture medium includes: 6-benzylaminopurine 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1.0 mg / L or a range between any two of the foregoing values; naphthylacetic acid 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1.0 mg / L or a range between any two of the foregoing values; 2,4-dichlorophenoxyacetic acid 0.0 mg / L, 0.01 mg / L, 0.05 mg / L, 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1.0 mg / L or a range between any two of the foregoing values.

[0047] Furthermore, in some embodiments of the present application, the vigorously growing Nicotiana benthamiana callus is extracted using water as a solvent, including: extracting with a solid-liquid ratio of 1:(5-10).

[0048] In the above technical scheme, by setting the vigorously growing Nicotiana benthamiana callus to be extracted with water as solvent, including: extracting with a solid-liquid ratio of 1:(5-10), it is further beneficial to obtain Nicotiana benthamiana callus extract with higher DPPH removal rate.

[0049] Illustratively, in some embodiments of the present application, vigorously growing Nicotiana benthamiana callus is extracted using water as a solvent, including: extracting with a solid-liquid ratio of 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or a range between any two of the foregoing values.

[0050] Further, in some embodiments of the present application, the vigorously growing Nicotiana benthamiana callus is extracted using water as a solvent, comprising:

[0051] A high-voltage pulse electric field with a field strength of 30 kV / m-50 kV / m is used for extraction; the number of pulses of the high-voltage pulse electric field is 60 times-120 times.

[0052] In the above technical scheme, a high-voltage pulse electric field with a field strength of 30kV / m-50kV / m is used for extraction; the number of pulses of the high-voltage pulse electric field is 60-120 times, and water is used as a solvent for extraction of vigorously growing Nicotiana benthamiana callus; this is conducive to extracting Nicotiana benthamiana callus extract.

[0053] For example, in some embodiments of the present application, the vigorously growing Nicotiana benthamiana callus is extracted using water as a solvent, comprising:

[0054] The field strength is 30kV / m, 31kV / m, 32kV / m, 33kV / m, 34kV / m, 35kV / m, 36kV / m, 37kV / m, 38kV / m, 39kV / m, 40kV / m, 41kV / m, 42kV / m, 43kV / m, 44kV / m, 45kV / m, 46kV / m, 47kV / m, 48k The extraction is carried out by a high-voltage pulse electric field of 49 kV / m, 50 kV / m or a range between any two of the foregoing values; the number of pulses of the high-voltage pulse electric field is 60, 61, 62, 65, 68, 70, 72, 75, 78, 80, 85, 90, 95, 100, 105, 110, 115 or 120 times.

[0055] Further, in some embodiments of the present application, the vigorously growing Nicotiana benthamiana callus is extracted using water as a solvent, further comprising:

[0056] After the extraction is completed, centrifuge at 6000rpm-12000rpm for 5min-15min and take the supernatant.

[0057] For example, in some embodiments of the present application, the vigorously growing Nicotiana benthamiana callus is extracted using water as a solvent, and further comprises:

[0058] After the extraction is completed, the mixture is centrifuged at 6000 rpm, 6500 rpm, 7000 rpm, 7500 rpm, 8000 rpm, 8500 rpm, 9000 rpm, 9500 rpm, 10000 rpm, 10500 rpm, 11000 rpm, 12000 rpm or in the range between any two of the aforementioned values; and the speed is centrifuged for 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min or in the range between any two of the aforementioned values, and the supernatant is taken.

[0059] Exemplarily, in some embodiments of the present application, take vigorous Nicotiana benthamiana callus and homogenize. Take pure water as solvent, and the solid-liquid ratio is 1:(5-20). Use high-voltage pulse electric field (PEF) extraction technology to extract Nicotiana benthamiana callus, the field intensity of the high-voltage pulse electric field is 30kV / m-50kV / m, and the pulse number of the high-voltage pulse electric field is 60 times-120 times. After the extraction is completed, 6000rpm-12000rpm high-speed centrifugation 5min-15min, take the supernatant and it is Nicotiana benthamiana callus extract.

[0060] Furthermore, in some embodiments of the present application, the induction culture medium also includes: MS powder 4.5g / L-5.0g / L, sucrose 25g / L-35g / L, agar 7.0g / L-8.0g / L; the pH value of the culture medium is 5.8-5.9.

[0061] Illustratively, in some embodiments of the present application, the induction medium also includes: MS powder 4.5 g / L, 4.6 g / L, 4.7 g / L, 4.8 g / L, 4.9 g / L, 5.0 g / L or the range between any two of the foregoing values; sucrose 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L or the range between any two of the foregoing values; agar 7.0 g / L, 7.1 g / L, 7.2 g / L, 7.3 g / L, 7.4 g / L, 7.5 g / L, 7.6 g / L, 7.7 g / L, 7.8 g / L, 7.9 g / L, 8.0 g / L or the range between any two of the foregoing values.

[0062] For example, in some embodiments of the present application, the induction of callus tissue includes: using a sterile seedling of Nicotiana benthamiana cultured for 10-20 days under long-day light as a material, cutting off its complete young leaves as explants and inoculating them on an induction medium, and switching to long-day light culture after dark culture for 3 days. The induction medium contains the following components: MS powder 4.74g / L, sucrose 25g / L-35g / L, 6-benzylaminopurine (6-BA) 1.0mg / L-3.0mg / L, naphthaleneacetic acid (NAA) 0.2mg / L-1.0mg / L and agar 7.0-8.0g / L; the pH value of the culture medium is 5.8-5.9.

[0063] Furthermore, in some embodiments of the present application, the subculture culture medium also includes: 4.5 g / L to 5.0 g / L MS powder, 25 g / L-35 g / L sucrose, and 7.0-8.0 g / L agar; and the pH value of the culture medium is 5.8-5.9.

[0064] Illustratively, in some embodiments of the present application, the subculture culture medium also includes: MS powder 4.5g / L, 4.6g / L, 4.7g / L, 4.8g / L, 4.9g / L, 5.0g / L or the range between any two of the foregoing values; sucrose 25g / L, 26g / L, 27g / L, 28g / L, 29g / L, 30g / L, 31g / L, 32g / L, 33g / L, 34g / L, 35g / L or the range between any two of the foregoing values; agar 7.0g / L, 7.1g / L, 7.2g / L, 7.3g / L, 7.4g / L, 7.5g / L, 7.6g / L, 7.7g / L, 7.8g / L, 7.9g / L, 8.0g / L or the range between any two of the foregoing values.

[0065] For example, in some embodiments of the present application, callus tissue begins to form about 10 days after explant inoculation. The induced Nicotiana benthamiana callus tissue with good growth status is cut into small pieces and inoculated on a subculture medium for proliferation culture (if inoculated on the original induction medium for subculture proliferation, the callus tissue gradually differentiates and sprouts, and the effect is poor).

[0066] Further optionally, in some embodiments of the present application, the subculture culture medium contains the following components: MS powder 4.74 g / L, sucrose 30 g / L, 6-benzylaminopurine (6-BA) 0.5 mg / L-2.0 mg / L, naphthylacetic acid (NAA) 0.0 mg / L-1.0 mg / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 0.0 mg / L-2.0 mg / L and agar 7.0 g / L-8.0 g / L; the pH value of the culture medium is 5.8-5.9.

[0067] Further optionally, in some embodiments of the present application, the size of the callus block is preferably (0.5 cm-1.0 cm)×(0.5 cm-1.0 cm)×(0.5 cm-1.0 cm), and the inoculation amount is preferably (8-12) / 50 cm 2 The subculture period is 10-20 days. The specific induction conditions of the present invention can obtain Nicotiana benthamiana callus with loose structure and vigorous growth.

[0068] In some embodiments of the present application, a method for preparing a Nicotiana benthamiana callus extract further includes: preparing Nicotiana benthamiana young leaves.

[0069] In some embodiments of the present application, preparing Nicotiana benthamiana young leaves comprises: sterilizing Nicotiana benthamiana seeds, sowing them on MS solid culture medium, culturing them in the dark, and switching to long-day culture after germination.

[0070] Exemplarily, in some embodiments of the present application, preparing young leaves of Nicotiana benthamiana includes: selecting full Nicotiana benthamiana seeds, surface sterilizing with ethanol for 10s-30s in a clean bench, washing away the ethanol with sterile water, then sterilizing with sodium hypochlorite solution for 8min-15min, and washing with sterile water 4-5 times. The washed Nicotiana benthamiana seeds are soaked in sterile water overnight, sown on a 1 / 2MS solid culture medium without any growth regulator the next day, cultured in the dark, and switched to long-day culture after germination.

[0071] Further optionally, the long-day culture conditions are: temperature 20°C to 30°C, relative humidity 50% to 60%, light intensity 1500lux to 3000lux, and light time 12h / d to 18h / d.

[0072] Exemplarily, in some embodiments of the present application, preparing young leaves of Nicotiana benthamiana includes: selecting full Nicotiana benthamiana seeds, surface sterilizing with ethanol for 10s, 12s, 15s, 20s, 25s or 30s in a clean bench, washing away the ethanol with sterile water, and then sterilizing with sodium hypochlorite solution for 8min, 9min, 10min, 11min, 12min, 13min, 14min or 15min, and rinsing with sterile water 4-5 times. The rinsed Nicotiana benthamiana seeds are soaked in sterile water overnight, sown on a 1 / 2MS solid culture medium without any growth regulator the next day, cultured in the dark, and switched to long-day culture after germination.

[0073] Exemplarily, long-day culture conditions are: temperature 20°C, 22°C, 25°C, 28°C or 30°C, relative humidity 50%, 52%, 55%, 58% or 60%, light intensity 1500lux, 1800lux, 2000lux, 2500lux, 2800lux or 3000lux, and light time 12h / d, 13h / d, 14h / d, 15h / d, 16h / d, 17h / d or 18h / d.

[0074] Some embodiments of the present application also provide a Nicotiana benthamiana callus extract, which is prepared by the preparation method of the Nicotiana benthamiana callus extract provided in any of the aforementioned embodiments.

[0075] Some embodiments of the present application also provide a cosmetic comprising the Nicotiana benthamiana callus extract provided in the aforementioned embodiments.

[0076] The features and performance of the present application are further described in detail below in conjunction with the embodiments:

[0077] Example 1

[0078] Induction of Nicotiana benthamiana callus:

[0079] (1) Seed disinfection and sowing: Select full Nicotiana benthamiana seeds, disinfect the surface with 75% ethanol for 15 seconds in a clean bench, wash off the ethanol with sterile water, disinfect with 5% sodium hypochlorite solution for 10 minutes, and rinse with sterile water 5 times. Soak the rinsed Nicotiana benthamiana seeds in sterile water overnight, sow on 1 / 2MS solid culture medium without any growth regulator the next day, culture in the dark, and switch to long-day culture after germination. Long-day culture conditions: temperature 25°C, relative humidity 60%, light intensity 2000lux, light time 16h / d.

[0080] (2) Callus induction: Sterile seedlings of Nicotiana benthamiana cultured for 15 days under long-day light were used as materials. The intact young leaves were cut off and inoculated on the induction medium (120 inoculations). After 3 days of dark culture, they were switched to long-day culture. The callus rate was 78.3%.

[0081] The induction culture medium comprises the following components: 4.74 g / L MS powder, 30 g / L sucrose, 1.0 mg / L 6-benzylaminopurine (6-BA), 0.2 mg / L naphthaleneacetic acid (NAA) and 7.5 g / L agar; the pH value of the culture medium is 5.8-5.9.

[0082] Embodiment 2-7

[0083] The difference from Example 1 is that the composition of the induction medium is different, see Table 1 for details.

[0084] Table 1 Effects of different culture media on callus formation of Nicotiana benthamiana

[0085]

[0086]

[0087] The above table tests the formation of Nicotiana benthamiana callus under different hormone concentrations and ratios. In each induction medium, the callus induction rate of Example 4 is the highest, which is 98.3%, while the callus induction rate of Example 1 is the lowest, which is 78.3%. From the growth of callus, increasing the concentration of NAA is conducive to inducing softer callus, while the callus induced by higher concentrations of 6-BA is yellow-brown in color and easily browned and dead. Based on the callus induction rate and callus growth, Example 4, i.e. 2.0 mg / L 6-BA+0.2 mg / L NAA, is selected as the best medium for inducing Nicotiana benthamiana callus.

[0088] Example 8

[0089] The Nicotiana benthamiana callus tissue with good growth status obtained by induction in Example 4 was inoculated on the subculture medium for subculture proliferation culture:

[0090] The Nicotiana benthamiana callus tissue with good growth status obtained by induction in the above Example 4 was cut into small pieces (0.5 cm×0.5 cm×0.5 cm) and inoculated on the subculture medium for subculture proliferation culture. The inoculation amount was 10 / 50 cm 2 The subculture cycle is 14 days.

[0091] The subculture medium contains the following components: 4.74 g / L MS powder, 30 g / L sucrose, 0.5 mg / L 6-benzylaminopurine (6-BA), 0.5 mg / L naphthaleneacetic acid (NAA), 0.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), and 7.5 g / L agar; the pH value of the medium is 5.8-5.9. See Table 2 for details.

[0092] Examples 9-13

[0093] The difference from Example 8 is that the composition of the subculture medium is different, see Table 2 for details.

[0094] Table 2 Effects of different culture media on subculture of Nicotiana benthamiana callus

[0095]

[0096] From the above table, it can be seen that the callus tissues of Examples 8 and 13 grew slowly, while the callus tissues of Examples 11 and 12 grew poorly. After comprehensive comparison, Example 9, i.e., 0.5 mg / L 6-BA + 1.0 mg / L NAA, was selected as the best medium for subculture of Nicotiana benthamiana callus tissues.

[0097] Embodiment 14

[0098] Preparation of Nicotiana benthamiana callus extract:

[0099] Take the vigorous growing Nicotiana benthamiana callus obtained in the above Example 9 and homogenize it. Use pure water as the solvent for extraction, and the solid-liquid ratio is 1:5. Use high-voltage pulsed electric field (PEF) extraction technology to extract Nicotiana benthamiana callus, the field strength of the high-voltage pulsed electric field is 40kV / m, and the number of pulses of the high-voltage pulsed electric field is 60 times. After the extraction is completed, high-speed centrifugation is performed at 10000rpm for 10 minutes, and the supernatant is taken as the Nicotiana benthamiana callus extract. See Table 3 for details.

[0100] Example 15-Example 21

[0101] The difference from Example 14 is that the extraction process parameters for Nicotiana benthamiana callus are different, see Table 3 for details.

[0102] The DPPH scavenging rate of the Nicotiana benthamiana callus extracts prepared in Examples 14-21 was tested, and the testing method was as follows:

[0103] Referring to the content of standard T / GDCA 021-2023 "Chemical Evaluation Method for Evaluation of Antioxidant Activity of Cosmetics", the DPPH radical scavenging rate of each embodiment and comparative example was calculated.

[0104] The test results are shown in Table 3.

[0105] Table 3

[0106]

[0107] It can be seen from the above table that the DPPH scavenging rate of the Nicotiana benthamiana callus extract prepared in each embodiment can reach more than 85%, which shows that the Nicotiana benthamiana callus extract prepared by the preparation method of the present application has an excellent DPPH scavenging effect. Therefore, applying it to cosmetics can effectively improve the antioxidant, repairing, and firming effects of the cosmetics.

[0108] Further, according to the data in the above table, taking into account the DPPH removal rate, extraction time and extraction cost, Example 20 is the optimal extraction condition, that is, pure water is used as the solvent, the solid-liquid ratio is 1:10, the field strength of the high-voltage pulse electric field is 40 kV / m, and the number of pulses is 100 times.

[0109] Application Example 1

[0110] Provided is a Nicotiana benthamiana callus extract aqueous solution, which comprises:

[0111] The Nicotiana benthamiana callus extract prepared in the above Example 20 was added to water in an amount of 5% by mass.

[0112] Application Examples 2-3

[0113] The difference from Application Example 1 is that the amount of Nicotiana benthamiana callus extract added is different, see Table 4 for details.

[0114] Negative control group: water.

[0115] Table 4

[0116] Group Addition amount of Nicotiana benthamiana callus extract Application Example 1 5％ Application Example 2 10％ Application Example 3 20％ Negative control /

[0117]

Performance test

[0118] 1. The in vitro efficacy of each of the above application examples was evaluated to evaluate the in vitro efficacy of Nicotiana benthamiana callus extract.

[0119] (1) The biochemical test method is as follows:

[0120] ① ABTS free radical scavenging test: Referring to the content of standard T / GDCA 021-2023 "Chemical Evaluation Method for Evaluation of Antioxidant Activity of Cosmetics", the ABTS free radical scavenging rate of each embodiment and comparative example was calculated.

[0121] ②DPPH free radical scavenging test: Referring to the content of standard T / GDCA021-2023 "Chemical Evaluation Method for Evaluation of Antioxidant Activity of Cosmetics", the DPPH free radical scavenging rate of each embodiment and comparative example was calculated.

[0122] ③ Tyrosinase inhibition test: Referring to the content of standard T / GDCA 006-2021 "Test method for inhibition of tyrosinase activity by cosmetic raw materials (in vitro method)", the tyrosinase inhibition rate of each embodiment and comparative example was calculated.

[0123] ④Collagenase inhibition test

[0124] The substance produced by collagenase acting on FALGPA absorbs at 335nm. The activity of collagenase is investigated by monitoring the absorbance value at 335nm. Using FALGPA as a substrate, the collagenase inhibition effect of a single sample is determined by spectrophotometry. The experimental design is shown in Table 5 below:

[0125] Table 5

[0126]

[0127] Calculation of inhibition rate:

[0128]

[0129] Where:

[0130] A 0 :Absorbance value of negative control group (mean)

[0131] A 1 :Absorbance value of negative blank group (mean)

[0132] B 0 : Absorbance value of sample reaction group

[0133] B 1 : Absorbance value of sample blank group

[0134] The test results are shown in the attached manual. Figure 1 Among them, Figure 1 A in the figure shows the scavenging effect of Nicotiana benthamiana callus extract on ABTS free radicals; B shows the scavenging effect of Nicotiana benthamiana callus extract on DPPH free radicals; C shows the inhibitory effect of Nicotiana benthamiana callus extract on tyrosinase; D shows the inhibitory effect of Nicotiana benthamiana callus extract on collagenase. Multiple comparison analysis was performed using the Tukey method, and lowercase letters indicate significant differences (n=3; P<0.05).

[0135] From the instruction manual Figure 1 It can be seen that: under the experimental conditions, when the concentration of Nicotiana benthamiana callus extract was 5%, 10% and 20%, its scavenging rate for ABTS and DPPH free radicals increased linearly, and the sample solution at each concentration point had a significant scavenging effect on ABTS and DPPH free radicals ( Figure 1 A, B). When the concentration of Nicotiana benthamiana callus extract was 10%, it had a significant inhibitory effect on tyrosinase, and when the concentration of Nicotiana benthamiana callus extract was 10% and 20%, it had a significant inhibitory effect on collagenase ( Figure 1 C,D).

[0136] (2) The cell method test method is as follows:

[0137] BJ cells in the logarithmic growth phase were inoculated into a 96-well plate at a density of 2.5*10^5 cells / well and cultured in an incubator for 24 hours. The sample was diluted to the corresponding concentration with serum-free medium and added to a 96-well plate. Three replicate wells were set up, and a positive control group (10% FBS) and a blank control group (2% FBS) were also set up. The culture was continued for 24 hours, and the cell morphology and quantity were observed regularly under a microscope. After 48 hours, CCK-8 was diluted 10 times with serum-free medium, 100 μL of CCK-8 diluent was added to each well, and then incubated in a 37°C incubator for 0.5 hours. The absorbance value was detected with an enzyme marker at a wavelength of 450nm.

[0138]

[0139] Where:

[0140] OD 试验 : Absorbance value of test well or solvent control well

[0141] OD 空白 : Absorbance value of blank well

[0142] RAW 264.7 (mouse macrophages) cells in the logarithmic growth phase were inoculated into 24-well plates with an inoculation density of 4*10^5 cells / well and cultured in an incubator for 24 hours. After the cells adhered to the wall, the culture medium was aspirated and 0.5 mL of the following samples were added: ① Blank control group: 2% serum cultured cells for 24 hours; ② Positive control group: 0.2 mg / mL dexamethasone + 2 μg / mL LPS acted on cells for 24 hours; ③ LPS inflammation model group: 1 μg / mL LPS continuously stimulated RAW 264.7 cells for 24 hours; ④ Sample group: Dilute the sample to the corresponding concentration solution with 2 μg / mL LPS to act on cells for 24 hours. Then use a pipette to aspirate the supernatant in the well plate into a centrifuge tube, centrifuge at 1000r / min for 5 minutes, discard the precipitate and aspirate the supernatant, dilute the supernatant 50 times, and use the TNF-ɑ inflammatory factor detection kit to detect the change in the amount of inflammatory factors in the supernatant. The detection method is strictly in accordance with the instructions on the detection kit.

[0143] The test results are shown in the attached manual. Figure 2 Among them, Figure 2 Figure A shows the effect of Nicotiana benthamiana callus extract on the survival rate of human skin fibroblasts (BJ cells); Figure 2 B in the figure shows the effect of Nicotiana benthamiana callus extract on inflammatory factor TNF-ɑ. Multiple comparison analysis was performed using Tukey's method, and lowercase letters indicate significant differences (n=3; P<0.05).

[0144] From the instruction manual Figure 2 It can be seen that: Cytotoxicity test ( Figure 2 A) showed that 5% and 10% of Nicotiana benthamiana callus extracts were safe for human skin fibroblasts (BJ cells) (cell survival rates were > 100%), and when the concentration of Nicotiana benthamiana callus extract was 10%, it significantly promoted the survival rate of BJ cells. Cell anti-inflammatory tests showed that compared with the inflammatory model group, the concentration of inflammatory factor TNF-ɑ was slightly reduced after treatment with 10% Nicotiana benthamiana callus extract, but it did not reach a significant difference level ( Figure 2 B).

[0145] (3) Effects of Nicotiana benthamiana callus extract on cell scratch healing

[0146] The cell scratch healing test method is as follows:

[0147] BJ cells in the logarithmic growth phase were inoculated into a 24-well plate at a density of 2.5*10^5 cells / well. They were placed in a 37°C carbon dioxide incubator for 24 hours. When the cells were almost fully grown, a cross was made in the 24-well plate with a pipette tip, and then the plate was washed once with PBS. The sample was diluted with culture medium to the corresponding concentration and added to the wells, 500 μL was added to each well, and the plate was placed in a 37°C carbon dioxide incubator for the observation time set in the experiment. At the set time, the width of the scratch was observed and measured under a microscope and photographed.

[0148]

[0149] The test results are shown in the attached manual. Figure 3 Among them, Figure 3 Figure A shows the effect of Nicotiana benthamiana callus extract on the healing rate of BJ cells; Figure 3 B in the figure shows the comparison of the scratch of negative control cells at 0h (upper) and 24h (lower); Figure 3 C shows a comparison of cells scratched with 10% Nicotiana benthamiana callus extract at 0 h (upper) and 24 h (lower). Multiple comparison analysis was performed using Tukey's method, and lowercase letters indicate significant differences (n=3; P<0.05).

[0150] From the instruction manual Figure 3 It can be seen that after 24 hours of action of 5% and 10% Nicotiana benthamiana callus extract on cells, the cell scratch healing rates were 52.40% and 54.27%, respectively, which were significantly higher than the blank control group (48.11%), indicating that the extract at this concentration has a significant ability to promote the healing of BJ cells ( Figure 3 ).

[0151] In summary, the present application scheme can obtain Nicotiana benthamiana callus with a high degree of dedifferentiation, good looseness, and fast growth rate, and extract Nicotiana benthamiana callus extract with a DPPH scavenging rate of more than 85%; the in vitro efficacy evaluation of this Nicotiana benthamiana callus extract showed that the Nicotiana benthamiana callus extract at the tested concentration had certain effects on improving BJ cell survival rate (firming), promoting cell scratch healing (repair), ABTS / DPPH free radical scavenging (antioxidation), etc., laying a foundation for its application in anti-aging cosmetics.

[0152] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a Nicotiana benthamiana callus extract, characterized in that: include: Inoculating young leaves of Nicotiana benthamiana as explants on an induction medium to induce the explants to form callus tissue; The induction culture medium comprises: 1.0 mg / L-3.0 mg / L of 6-benzylaminopurine; 0.2 mg / L-1.0 mg / L of naphthaleneacetic acid; inoculating the callus tissue on a subculture medium for proliferation culture; The subculture culture medium comprises: 6-benzylaminopurine 0.5 mg / L-2.0 mg / L, naphthylacetic acid 0.0 mg / L-1.0 mg / L, 2,4-dichlorophenoxyacetic acid 0.0 mg / L-2.0 mg / L; The Nicotiana benthamiana callus tissue after proliferation and culture is extracted with water as solvent, and the solid-liquid ratio is 1:(5-20).

2. The method for preparing the Nicotiana benthamiana callus extract according to claim 1, characterized in that: The induction culture medium comprises: 1.0 mg / L-2.0 mg / L of 6-benzylaminopurine; and 0.2 mg / L-1.0 mg / L of naphthaleneacetic acid.

3. The method for preparing the Nicotiana benthamiana callus extract according to claim 1, characterized in that: The subculture culture medium comprises: 0.5 mg / L-1.0 mg / L of 6-benzylaminopurine, 0.5 mg / L-1.0 mg / L of naphthylacetic acid, and 0.0 mg / L-1.0 mg / L of 2,4-dichlorophenoxyacetic acid.

4. The method for preparing the Nicotiana benthamiana callus extract according to any one of claims 1 to 3, characterized in that: The Nicotiana benthamiana callus tissue after proliferation and culture is extracted with water as solvent, including: extracting with a solid-liquid ratio of 1:(5-10).

5. The method for preparing the Nicotiana benthamiana callus extract according to any one of claims 1 to 3, characterized in that: The Nicotiana benthamiana callus after proliferation and culture is extracted with water as solvent, comprising: The extraction is performed using a high-voltage pulse electric field with a field strength of 30 kV / m-50 kV / m; the number of pulses of the high-voltage pulse electric field is 60-120 times.

6. The method for preparing the Nicotiana benthamiana callus extract according to any one of claims 1 to 3, characterized in that: The Nicotiana benthamiana callus tissue after proliferation and culture is extracted with water as solvent, further comprising: After the extraction is completed, centrifuge at 6000rpm-12000rpm for 5min-15min and take the supernatant.

7. The method for preparing the Nicotiana benthamiana callus extract according to claim 1, characterized in that: The induction culture medium comprises: 4.5 g / L to 5.0 g / L MS powder, 25 g / L to 35 g / L sucrose, and 7.0 g / L to 8.0 g / L agar; and the pH value of the culture medium is 5.8 to 5.

9.

8. The method for preparing the Nicotiana benthamiana callus extract according to claim 1, characterized in that: The subculture culture medium also includes: 4.5 g / L to 5.0 g / L MS powder, 25 g / L to 35 g / L sucrose, and 7.0 to 8.0 g / L agar; and the pH value of the culture medium is 5.8 to 5.

9.

9. A Nicotiana benthamiana callus extract, characterized in that The extract is prepared by the method for preparing the Nicotiana benthamiana callus extract according to any one of claims 1 to 8.

10. A cosmetic, characterized in that: The method comprises the Nicotiana benthamiana callus extract according to claim 9.

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