CN113750018B

CN113750018B - Plant composition and preparation method and application thereof

Info

Publication number: CN113750018B
Application number: CN202110433276.9A
Authority: CN
Inventors: 邱显荣; 刘有停; 吕永博; 苏牧楠; 杨素珍; 李燕; 韩婷婷; 王晓娜
Original assignee: Shandong Furida Biological Co ltd; Nutri Woods Bio Tech Beijing Co ltd
Current assignee: Shandong Furida Biological Co ltd; Nutri Woods Bio Tech Beijing Co ltd
Priority date: 2021-04-22
Filing date: 2021-04-22
Publication date: 2022-10-11
Anticipated expiration: 2041-04-22
Also published as: CN113750018A

Abstract

The invention discloses a plant composition and a preparation method and application thereof, and belongs to the field of plant extraction research. The plant composition comprises dendrobium officinale, rhizoma polygonati, tuberose and lucid ganoderma and is prepared by a bidirectional fermentation process. Compared with the traditional water extraction method, the plant composition prepared by the bidirectional fermentation process has high content of active ingredient polysaccharide. The plant composition achieves a good anti-aging effect by inhibiting non-enzymatic glycosylation and inhibiting clock gene expression, achieves a good whitening effect by inhibiting tyrosinase activity, and can achieve double effects of anti-aging and whitening by adding the plant composition into cosmetics and/or skin care products.

Description

Plant composition and preparation method and application thereof

Technical Field

The invention relates to the field of plant extraction research, and particularly relates to a plant composition and a preparation method and application thereof.

Background

The bidirectional fermentation of traditional Chinese medicine is a new high-tech technology for preparing traditional Chinese medicine by combining modern microbiological engineering and absorbing microecological research results on the basis of inheriting a fermentation method in traditional Chinese medicine processing science. The 'bidirectional fermentation' is characterized in that traditional Chinese medicinal materials or herb residues with certain active ingredients are used as a medicinal substrate to replace a traditional nutrient substrate, and optimized strains are added into the medicinal substrate for microbial transformation, so that a fermented composition formed by the medicinal materials or herb residues is called medicinal mycoplasm. The bidirectional property is realized in that the medicinal matrix provides nutrition required by fungi, and simultaneously is influenced by enzymes in the fungi to change self tissues and components to generate new flavor functions. Therefore, the microbial fermentation of the traditional Chinese medicine is environment-friendly, high in extraction efficiency and good in effect, can be applied to multiple industries such as food and medicines, but is less in application in cosmetics, can provide a way for expanding new components and new effects in the field of cosmetic raw materials, and has a very wide application prospect.

A regulator of clock gene expression amount and its preparation method are disclosed in patent CN 1078931A, and the regulator is herba Dendrobii extract. The dendrobium extract is found to promote the expression of Clock gene for the first time. However, studies (grand ocean BMAL1/CLOCK regulation of keratinocyte apoptosis, DNA damage and immunity under UVB irradiation [ D ] tianjin university, 2018.) have shown that (1) small RNA-mediated silencing of CLOCK genes can promote keratinocyte proliferation and induce differentiation of primary keratinocytes under normal culture conditions without UV irradiation; (2) clock gene silencing can block UVB-induced apoptosis of primary keratinocytes and HaCat cells; (3) in keratinocytes, silencing Clock is effective in inhibiting UVB-induced high expression of cytokines; (4) in primary keratinocytes, clock gene silencing had a strong inhibitory effect on UVB-induced increase in p53 protein amount and phosphorylation. In conclusion, the promotion of Clock can cause adverse reaction on skin, inhibit the expression of Clock gene, and effectively reduce keratinocyte apoptosis and resist the adverse reaction of UVB on organisms.

Aiming at the fact that the single dendrobium extract promotes the expression of Clock gene and the adverse reaction on skin, it is necessary to develop a plant composition which has high active ingredient content, can inhibit non-enzymatic glycosylation reaction, inhibit the expression of Clock gene and inhibit the activity of tyrosinase, and has the effects of resisting aging and whitening skin.

Disclosure of Invention

Based on the defects of the prior art, the invention provides the plant composition which has high active matter content, can inhibit non-enzymatic glycosylation reaction, inhibit clock gene expression and tyrosinase activity, and has the effects of resisting aging and whitening.

The technical problem to be solved by the invention is realized by the following technical scheme:

in a first aspect, the present invention provides a plant composition comprising the following components: dendrobium officinale, rhizoma polygonati, tuberose and lucid ganoderma.

According to the invention, the plant composition is prepared by a bidirectional fermentation process.

According to the invention, the addition amount of dendrobium officinale in the plant composition is 1-10% by weight of the bidirectional fermentation medium.

According to the invention, the addition amount of rhizoma polygonati in the plant composition is 0.01-1% by weight of the bidirectional fermentation medium.

According to the invention, the tuberose is added into the plant composition in an amount of 0.01-1% by weight of the bidirectional fermentation medium.

According to the invention, the plant composition contains 5.11-5.98mg/mL of polysaccharide and 0.11-0.23mg/mL of flavone.

According to some embodiments of the invention, the plant composition has a polysaccharide content of 5.23 to 5.98mg/mL and a flavone content of 0.13 to 0.23mg/mL.

According to the invention, the plant composition is prepared by a preparation method comprising:

(1) pretreatment of raw materials: pulverizing herba Dendrobii, rhizoma Polygonati, and tuberose respectively, and sieving;

(2) activating the ganoderma lucidum: preparing a ganoderma lucidum culture medium, inoculating ganoderma lucidum, and culturing to obtain ganoderma lucidum seed liquid;

(3) preparing a bidirectional fermentation culture medium;

(4) inoculation and fermentation: inoculating the lucid ganoderma seed liquid into a bidirectional fermentation culture medium for culture;

(5) post-treatment of fermentation liquor: sterilizing and filtering.

According to the invention, the activated ganoderma lucidum culture medium in the step (2) of the plant combination preparation method is 0.5-5% of starch, 0.1-5% of yeast powder, 0.1-2% of sodium chloride, 0.01-0.1% of calcium carbonate and water added to 100%.

According to the invention, the culture temperature of the ganoderma lucidum is 25-35 ℃.

According to the invention, the culture speed of the ganoderma lucidum is 50-300rpm.

According to the invention, the culture time of the ganoderma lucidum is 50-80h.

According to the invention, in the step (3) of the preparation method of the plant combination, the bidirectional fermentation medium comprises 0.5-5% of starch, 0.1-5% of yeast powder, 0.1-2% of sodium chloride, 0.01-0.1% of calcium carbonate, 1-10% of dendrobium officinale, 0.01-1% of rhizoma polygonati, 0.01-1% of tuberose and water to 100%.

According to some embodiments of the present invention, in the step (3) of the method for preparing the plant composition, the bidirectional fermentation medium comprises 0.5-5% of starch, 0.1-5% of yeast powder, 0.1-2% of sodium chloride, 0.01-0.1% of calcium carbonate, 1-2% of dendrobium officinale, 0.01-0.2% of rhizoma polygonati, 0.01-0.2% of tuberose, and water to 100%.

According to the invention, the temperature of the bidirectional fermentation culture in the step (3) is 25-35 ℃.

According to the invention, the rotation speed of the bidirectional fermentation culture in the step (3) is 50-300rpm.

According to the invention, the bidirectional fermentation culture time in the step (3) is 1-7d.

According to the invention, the pH value of the bidirectional fermentation culture in the step (3) is 3.5-7.5.

According to some embodiments of the invention, the temperature of the bidirectional fermentation in step (3) is 30 ℃.

According to some embodiments of the invention, the rotation speed of the bidirectional fermentation culture in the step (3) is 150rpm.

According to some embodiments of the invention, the bidirectional fermentation in step (3) is carried out for 3d.

According to some embodiments of the invention, the pH of the bidirectional fermentation culture in step (3) is 5.5.

According to the invention, the inoculation amount of the ganoderma lucidum seed solution in the step (4) in the plant combination preparation method is 1-15%.

According to some embodiments of the present invention, the inoculation amount of the ganoderma lucidum seed solution in step (4) of the plant combination preparation method is 10%.

According to the present invention, the sterilization means in step (5) of the method for preparing a plant composition is a conventional sterilization means in the art.

According to the present invention, the sterilization mode in step (5) of the method for preparing a plant composition is high-temperature sterilization.

According to the present invention, the sterilization temperature in the step (5) of the method for preparing a plant composition is 115 to 121 ℃.

According to the invention, the high-temperature sterilization condition in the step (5) in the preparation method of the plant combination is that the sterilization time is 10-60min.

According to the invention, the preparation of the ganoderma lucidum culture medium in the step (2) in the plant combination preparation method further comprises subpackaging and sterilization.

According to the invention, in the preparation method of the plant combination, the ganoderma lucidum culture medium in the step (2) is subpackaged in a 250mL triangular flask, and the liquid filling amount of each flask is 50-100mL.

According to the invention, the sterilization mode of the ganoderma lucidum culture medium in the step (2) in the plant combination preparation method is a conventional sterilization mode in the field.

According to the invention, the ganoderma lucidum culture medium in the step (2) in the plant combination preparation method is sterilized at high temperature.

According to the invention, the high-temperature sterilization temperature of the ganoderma lucidum culture medium in the step (2) in the plant combination preparation method is 115-121 ℃.

According to the invention, the high-temperature sterilization time of the ganoderma lucidum culture medium in the step (2) in the plant combination preparation method is 10-60min.

According to the invention, the preparation of the bidirectional fermentation medium in the step (3) in the plant combination preparation method further comprises subpackaging and sterilizing.

According to the invention, in the preparation method of the plant combination, the bidirectional fermentation medium in the step (3) is subpackaged in 500mL triangular flasks, and the liquid filling amount of each flask is 50-200mL.

According to the invention, the sterilization mode of the bidirectional fermentation medium in the step (3) in the plant combination preparation method is a conventional sterilization mode in the field.

According to the invention, the bidirectional fermentation medium in step (3) of the plant combination preparation method is sterilized at high temperature.

According to the invention, the high temperature sterilization temperature of the bidirectional fermentation medium in the step (3) in the plant combination preparation method is 115-121 ℃.

According to the invention, the bidirectional fermentation medium in the step (3) in the plant combination preparation method is sterilized at high temperature for 10-60min.

According to the invention, the post-treatment of the fermentation liquor in step (5) in the preparation method of the plant composition further comprises the addition of auxiliary materials after sterilization.

According to the invention, in the plant combination preparation method, the auxiliary materials added in the fermentation liquor post-treatment in the step (5) comprise glycerol, pentanediol and hexanediol.

According to the invention, the auxiliary materials added in the post-treatment of the fermentation liquor in the step (5) in the preparation method of the plant combination comprise 10-50% of glycerol, 0.1-5% of pentanediol and 0.1-5% of hexanediol by weight of bidirectional fermentation liquor.

According to the invention, the post-treatment of the fermentation liquor in step (5) in the preparation method of the plant composition further comprises the sterilization after the addition of auxiliary materials.

According to the invention, in the preparation method of the plant combination, the fermentation liquor in the step (5) is subjected to post-treatment, auxiliary materials are added, and the sterilization temperature is 70-100 ℃.

According to the invention, in the preparation method of the plant combination, the fermentation liquor in the step (5) is subjected to post-treatment, auxiliary materials are added, and the sterilization time is 20-60min.

According to the invention, in the pretreatment of the raw material in the step (1) in the plant combination preparation method, the dendrobium officinale kimura et migo is crushed and sieved by a sieve of 80-120 meshes, and the parts below the sieve of 80 meshes and above the sieve of 120 meshes are taken for standby.

According to the invention, in the pretreatment of the raw materials in the step (1) in the preparation method of the plant combination, the rhizoma polygonati is crushed and sieved by a 10-25-mesh sieve, and the parts below the 10-mesh sieve and above the 25-mesh sieve are taken for standby.

According to the invention, in the preparation method of the plant combination, the tuberose in the pretreatment of the raw material in the step (1) is crushed and sieved by a sieve with 10-25 meshes, and the parts below the sieve with 10 meshes and above the sieve with 25 meshes are taken for later use.

In a second aspect, the present invention provides a method for preparing a plant composition according to the first aspect of the present invention.

According to the invention, the preparation method comprises the following steps:

(2) activating the ganoderma lucidum: adding water to 100% of starch 0.5-5%, yeast powder 0.1-5%, sodium chloride 0.1-2%, and calcium carbonate 0.01-0.1%, mixing, packaging, and sterilizing to obtain Ganoderma culture medium; selecting Ganoderma mycelia, inoculating to culture medium, and culturing at 25-35 deg.C and 50-300rpm for 50-80 hr to obtain Ganoderma seed solution;

(3) preparing a bidirectional fermentation culture medium: mixing starch 0.5-5% and yeast powder 0.1-5%; 0.1-2% of sodium chloride, 0.01-0.1% of calcium carbonate, 1-10% of dendrobium officinale, 0.01-1% of rhizoma polygonati, 0.01-1% of tuberose, and water which is added to 100% are mixed, subpackaged and sterilized to prepare a bidirectional fermentation culture medium;

(4) inoculation and fermentation: inoculating the ganoderma lucidum seed liquid in the step (2) into a fermentation culture medium according to the inoculation amount of 1-15%, and then culturing for 1-7d at the temperature of 25-35 ℃ and at the rpm of 50-300;

(5) post-treatment of fermentation liquor: sterilizing and filtering.

According to some embodiments of the present invention, in the step (3) of the method for preparing the plant composition, the bidirectional fermentation medium comprises 0.5-5% of starch, 0.1-5% of yeast powder, 0.1-2% of sodium chloride, 0.01-0.1% of calcium carbonate, 1-2% of dendrobium officinale, 0.01-0.2% of rhizoma polygonati, 0.01-0.2% of tuberose and water to 100%.

According to some embodiments of the invention, the pH of the bidirectional fermentative culture in step (3) is 5.5.

According to some embodiments of the invention, the bidirectional fermentation in step (3) is carried out for a period of 3 days.

According to the invention, the inoculation amount of the ganoderma lucidum seed liquid in the step (4) in the plant combination preparation method is 1-15%.

According to the present invention, the sterilization means in step (5) of the method for preparing a plant composition is a sterilization means that is conventional in the art.

According to the invention, the high-temperature sterilization condition in the step (5) in the plant combination preparation method is that the sterilization time is 10-60min.

According to the invention, the high-temperature sterilization temperature of the ganoderma lucidum culture medium in the step (2) in the preparation method of the plant combination is 115-121 ℃.

According to the invention, the high temperature sterilization temperature of the bidirectional fermentation medium in the step (3) in the preparation method of the plant combination is 115-121 ℃.

According to the invention, in the preparation method of the plant combination, the fermentation liquor post-treatment addition auxiliary materials in the step (5) comprise glycerol, pentanediol and hexanediol.

According to the invention, in the plant combination preparation method, the fermentation liquor is post-treated in the step (5), auxiliary materials are added, and the sterilization temperature is 70-100 ℃.

According to the invention, in the pretreatment of the raw materials in the step (1) in the preparation method of the plant combination, the dendrobium officinale kimura et migo is crushed and sieved by a sieve of 80-120 meshes, and the parts below the sieve of 80 meshes and above the sieve of 120 meshes are taken for standby.

According to the invention, in the preparation method of the plant combination, in the pretreatment of the raw material in the step (1), the tuberose is crushed and sieved by a sieve with 10-25 meshes, and the part under the sieve with 10 meshes and the part on the sieve with 25 meshes are taken for standby.

In a third aspect, the present invention provides the use of a plant composition according to the first aspect of the present invention or a plant composition prepared by a method for preparing a plant composition according to the second aspect of the present invention in cosmetics and/or toiletries.

According to the invention, the plant composition is applied to the preparation of skin care products and/or cosmetics with anti-aging and/or whitening effects.

According to the present invention, the cosmetic is not particularly limited in kind or form, and may be, for example, a mask, a cream, a face lotion, a serum or a milky lotion.

The invention has the beneficial effects that:

1. the plant composition is prepared by a bidirectional fermentation process, the dendrobium officinale, the polygonatum and the tuberose are subjected to low-temperature fermentation by adopting the fungus lucid ganoderma which is homologous in medicine and food, the dendrobium officinale, the polygonatum and the tuberose are subjected to deep fermentation decomposition by the fungus into a micromolecule carbon source by utilizing a strong enzyme system of the fungus for decomposing substances such as cellulose, the utilization rate of active ingredients of plant raw materials is increased, and the content of polysaccharide which is the active ingredient of the plant composition is improved.

2. The plant composition achieves a good anti-aging effect by inhibiting non-enzymatic glycosylation and inhibiting Clock gene expression, achieves a good whitening effect by inhibiting tyrosinase activity, and can achieve double effects of anti-aging and whitening by adding the plant composition into cosmetics and/or skin care products.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the following will be briefly described.

FIG. 1 is a graph showing the results of the expression of the gene by Clock.

Detailed Description

The invention is further illustrated below with reference to specific examples, to which, however, the invention is not restricted.

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the experimental materials and reagents are commercially available, unless otherwise specified.

The names of the main raw materials, suppliers/manufacturers and production places of the invention are shown in table 1:

TABLE 1 raw materials and sources

Name of raw materials	Supplier/producer	Producing area
			DendrobiumDendrobium officinale Kimura et Migo	Kunming plant institute of Chinese academy of sciences	Yunnan province
Polygonatum sibiricum	Beijing Qiancao Chinese medicinal drink Co Ltd	Hebei river
			Tuberose	Yunnan flowers Co Ltd	Yunnan province
Ganoderma lucidum	China center for preservation and management of industrial microbial strains	China
			Hexanediol	De Zhi Xin (Shanghai) Co., ltd	Germany
Pentanediol	De Zhi Xin (Shanghai) Co., ltd	Germany

Example 1

(1) Pretreatment of raw materials: crushing Dendrobium officinale, sieving with a 80-mesh sieve, respectively crushing rhizoma Polygonati and tuberose, and sieving with a 10-mesh sieve;

(2) activating the ganoderma lucidum: 2% of starch, 1.5% of yeast powder, 0.3% of sodium chloride, 0.02% of calcium carbonate, and 100% of pure water, preparing a ganoderma lucidum culture medium, and subpackaging in 250mL conical flasks, wherein the liquid filling amount of each flask is 100mL. Respectively placing the culture medium in a sterilization pot, sterilizing at 121 deg.C for 20min, and cooling in a super clean bench after sterilization; selecting mycelium from the ganoderma lucidum slant, and inoculating the mycelium into a ganoderma lucidum culture medium; culturing the inoculated culture medium in a constant-temperature shaking table at the temperature of 30 ℃ and the rotation speed of the shaking table of 150rpm for 3d to obtain ganoderma lucidum seed liquid;

(3) preparing a bidirectional fermentation culture medium: according to the mixture ratio of the raw materials in the table 2, 2% of starch, 1.5% of yeast powder, 0.3% of sodium chloride and 0.02% of calcium carbonate are added, water is added to 100%, a bidirectional fermentation culture medium is prepared, the initial pH value is 5.5, the bidirectional fermentation culture medium is subpackaged in 250mL conical flasks, the liquid filling amount of each flask is 100mL, and each culture medium is 3 flasks. Respectively placing the two-way fermentation culture medium in a sterilization pot, sterilizing at 121 deg.C for 20min, and cooling in a super clean bench after sterilization;

(4) inoculation and fermentation: inoculating the lucid ganoderma seed liquid according to the inoculation amount of 10 percent, then placing the culture medium in a constant-temperature shaking table for culture at the culture temperature of 30 ℃ and the shaking table rotating speed of 150rpm for 3d, wherein each group of samples is 3 bottles;

(5) post-treatment of fermentation liquor: and after the fermentation is finished, sterilizing the fermentation liquor for 20min at 121 ℃. And (4) filtering the fermentation liquor after sterilization, and detecting the content of polysaccharide and flavone in the filtrate.

TABLE 2 optimization of the amount of Chinese medicinal materials added

Sample numbering	Dendrobium officinale (%)	Sealwort (%)	Tuberose (%)
				Sample 1	1	0.1	0.05
Sample 2	1	0.2	0.1
				Sample 3	1	0.4	0.2
Sample No. 4	2	0.1	0.1
				Sample No. 5	2	0.2	0.2
Sample No. 6	2	0.4	0.05
				Sample 7	4	0.1	0.2
Sample 8	4	0.2	0.05
				Sample 9	4	0.4	0.1

Example 2

(3) preparing a bidirectional fermentation culture medium: 2% of dendrobium officinale, 0.2% of rhizoma polygonati, 0.2% of tuberose, 2% of starch, 1.5% of yeast powder, 0.3% of sodium chloride, 0.02% of calcium carbonate, and 100% of water is added to prepare a bidirectional fermentation culture medium, the initial pH value is 5.5, the bidirectional fermentation culture medium is subpackaged in 250mL conical flasks, the liquid filling amount of each flask is 100mL, and each culture medium is 3 flasks. Respectively placing the two-way fermentation culture medium in a sterilization pot, sterilizing at 121 deg.C for 20min, and cooling in a super clean bench after sterilization;

(4) inoculation and fermentation: inoculating lucid ganoderma seed liquid according to the inoculation amount (2%, 5%, 10% and 15%) in the table 3, then placing the culture medium in a constant-temperature shaking table for culturing for 3d at the culture temperature of 30 ℃ and the rotation speed of the shaking table of 150rpm, and culturing each group of samples in 3 bottles;

(5) and (3) post-treatment of fermentation liquor: and after the fermentation is finished, sterilizing the fermentation liquor for 20min at 121 ℃. And (4) filtering the fermentation liquor after sterilization, and detecting the content of polysaccharide and flavone in the filtrate.

TABLE 3 fermentation inoculum size optimization

Sample numbering	Inoculum size (%)
		Sample 10	2
Sample 11	5
		Sample 12	10
Sample 13	15

Example 3

(1) Pretreatment of raw materials: pulverizing herba Dendrobii, sieving with 80 mesh sieve, pulverizing rhizoma Polygonati and tuberose respectively, and sieving with 10 mesh sieve;

(2) activating the ganoderma lucidum: 2% of starch, 1.5% of yeast powder, 0.3% of sodium chloride, 0.02% of calcium carbonate, and 100% of pure water, preparing a ganoderma lucidum culture medium, and subpackaging in 250mL conical flasks, wherein the liquid filling amount of each flask is 100mL. Respectively placing the culture medium in a sterilization pot, sterilizing at 121 deg.C for 20min, and cooling in a super clean bench after sterilization; selecting mycelium from the ganoderma lucidum slant, and inoculating the mycelium into a ganoderma lucidum culture medium; culturing the inoculated culture medium in a constant temperature shaking table at the culture temperature of 30 ℃ and the rotation speed of the shaking table of 150rpm for 3d to obtain ganoderma lucidum seed liquid;

(4) inoculation and fermentation: inoculating the ganoderma lucidum seed liquid according to the inoculation amount of 10%, and then placing the culture medium in a constant-temperature shaking table for culture at the culture temperature of 30 ℃ and the rotation speed of the shaking table of 150rpm for 1d, 3d, 5d and 7d, wherein each group of samples comprises 3 bottles;

TABLE 4 fermentation time optimization

Sample numbering	Fermentation time (d)
		Sample 14	1
Sample 15	3
		Sample 16	5
Sample 17	7

Example 4

(4) inoculation and fermentation: inoculating the ganoderma lucidum seed liquid according to the inoculation amount of 10%, then placing the culture medium in a constant-temperature shaking table for culturing at the culture temperature of 28 ℃, 30 ℃ and 35 ℃ at the shaking table rotating speed of 150rpm for 3d, wherein each group of samples comprises 3 bottles;

(5) and (3) post-treatment of fermentation liquor: and (4) after the fermentation is finished, sterilizing the fermentation liquor for 20min at 121 ℃. And (4) filtering the fermentation liquor after sterilization, and detecting the content of polysaccharide and flavone in the filtrate.

TABLE 5 fermentation temperature optimization

Sample numbering	Fermentation temperature (. Degree.C.)
		Sample 18	28
Sample 19	30
		Sample 20	35

Example 5

(3) preparing a bidirectional fermentation culture medium: 2% of dendrobium officinale, 0.2% of rhizoma polygonati, 0.2% of tuberose, 2% of starch, 1.5% of yeast powder, 0.3% of sodium chloride and 0.02% of calcium carbonate, adding water to 100%, preparing a bidirectional fermentation culture medium, adjusting the pH to 3.5 by using citric acid, adjusting the pH to 7.5 by using sodium hydroxide, and adjusting the pH of the culture medium to 5.5 in a natural state. The medium was dispensed into 250mL conical flasks, each containing 100mL of the medium, 3 flasks for each medium. Respectively placing the two-way fermentation culture medium in a sterilization pot, sterilizing at 121 deg.C for 20min, and cooling in a super clean bench after sterilization;

TABLE 6 fermentation initial pH optimization

Sample numbering	pH value of fermentation
		Sample 21	3.5
Sample 22	5.5
		Sample 23	7.5

Example 6

(5) post-treatment of fermentation liquor: after fermentation, sterilizing the fermentation broth at 121 ℃ for 20min, cooling to normal temperature, fine-filtering, adding 30% glycerol, 1% hexanediol and 2% pentanediol, sterilizing at 95 ℃ for 40min, and cooling to obtain the product of example 6.

Example 7

(3) preparing a traditional Chinese medicine bidirectional fermentation culture medium: 1% of dendrobium officinale, 0.01% of rhizoma polygonati, 0.01% of tuberose, 2% of starch, 1.5% of yeast powder, 0.3% of sodium chloride, 0.02% of calcium carbonate, and 100% of water are added to prepare a traditional Chinese medicine bidirectional fermentation culture medium, the initial pH value is 5.5, the traditional Chinese medicine bidirectional fermentation culture medium is subpackaged in 250mL conical flasks, the liquid filling amount of each flask is 100mL, and each culture medium is 3 flasks. Respectively placing the two-way fermentation culture medium of the traditional Chinese medicine in a sterilization pot, sterilizing at 121 ℃ for 20min, and after sterilization, placing in a super clean bench for cooling for later use;

(4) inoculation and fermentation: inoculating lucid Ganoderma seed liquid according to the inoculation amount of 10%, then placing the culture medium in a constant-temperature shaking table for culturing at the culture temperature of 30 ℃ and the rotation speed of the shaking table of 150rpm for 3d, wherein each group of samples comprises 3 bottles;

(5) and (3) post-treatment of fermentation liquor: after the fermentation is finished, sterilizing the fermentation liquor at 121 ℃ for 20min, cooling to normal temperature, finely filtering, adding 30% of glycerol, 1% of hexanediol and 2% of pentanediol, sterilizing at 95 ℃ for 40min, and cooling to obtain the product of example 7.

Example 8

(3) preparing a traditional Chinese medicine bidirectional fermentation culture medium: 10% of dendrobium officinale, 1% of polygonatum sibiricum, 1% of tuberose, 2% of starch, 1.5% of yeast powder, 0.3% of sodium chloride, 0.02% of calcium carbonate, and adding water to 100% to prepare a traditional Chinese medicine bidirectional fermentation culture medium, wherein the initial pH value is 5.5, the traditional Chinese medicine bidirectional fermentation culture medium is respectively packaged in 250mL conical flasks, the liquid loading amount of each flask is 100mL, and each culture medium is 3 flasks. Respectively placing the two-way fermentation culture medium of the traditional Chinese medicine in a sterilization pot, sterilizing at 121 ℃ for 20min, and after the sterilization is finished, placing the two-way fermentation culture medium in a super clean bench for cooling for later use;

(5) and (3) post-treatment of fermentation liquor: after the fermentation is finished, sterilizing the fermentation liquor at 121 ℃ for 20min, cooling to normal temperature, finely filtering, adding 30% of glycerol, 1% of hexanediol and 2% of pentanediol, sterilizing at 95 ℃ for 40min, and cooling to obtain the product of example 8.

Example 9

(3) preparing a traditional Chinese medicine bidirectional fermentation culture medium: 10% of dendrobium officinale, 1% of rhizoma polygonati, 0.01% of tuberose, 2% of starch, 1.5% of yeast powder, 0.3% of sodium chloride, 0.02% of calcium carbonate, and adding water to 100% to prepare a traditional Chinese medicine bidirectional fermentation culture medium, wherein the initial pH value is 5.5, the traditional Chinese medicine bidirectional fermentation culture medium is subpackaged in 250mL conical flasks, the liquid filling amount of each flask is 100mL, and each culture medium is 3 flasks. Respectively placing the two-way fermentation culture medium of the traditional Chinese medicine in a sterilization pot, sterilizing at 121 ℃ for 20min, and after the sterilization is finished, placing the two-way fermentation culture medium in a super clean bench for cooling for later use;

(5) post-treatment of fermentation liquor: after the fermentation is finished, sterilizing the fermentation liquor at 121 ℃ for 20min, cooling to normal temperature, finely filtering, adding 30% of glycerol, 1% of hexanediol and 2% of pentanediol, sterilizing at 95 ℃ for 40min, and cooling to obtain the product of example 9.

Comparative example 1

2% of dendrobium officinale, 0.2% of polygonatum sibiricum and 0.2% of tuberose, adding water to 100%, extracting for 2 hours at 80 ℃, filtering, and detecting the content of polysaccharide and flavone in the filtrate.

Comparative example 2

(1) Pretreatment of raw materials: respectively crushing rhizoma Polygonati and tuberose, and sieving with 10 mesh sieve;

(3) preparing a bidirectional fermentation culture medium: 0.2% of polygonatum sibiricum, 0.2% of tuberose, 2% of starch, 1.5% of yeast powder, 0.3% of sodium chloride, 0.02% of calcium carbonate, and 100% of water is added to prepare a bidirectional fermentation culture medium, the initial pH value is 5.5, the bidirectional fermentation culture medium is respectively filled into 250mL conical flasks, the liquid filling amount of each flask is 100mL, and each culture medium is 3 flasks. Respectively placing the two-way fermentation culture medium in a sterilization pot, sterilizing at 121 deg.C for 20min, and cooling in a super clean bench after sterilization;

(5) post-treatment of fermentation liquor: and (4) after the fermentation is finished, sterilizing the fermentation liquor for 20min at 121 ℃. And after sterilization, filtering the fermentation liquor, and detecting the contents of polysaccharide and flavone in the filtrate.

Comparative example 3

(3) preparing a bidirectional fermentation culture medium: 0.5% of dendrobium officinale, 0.2% of rhizoma polygonati, 0.2% of tuberose, 2% of starch, 1.5% of yeast powder, 0.3% of sodium chloride and 0.02% of calcium carbonate, adding water to 100% to prepare a bidirectional fermentation culture medium, wherein the initial pH value is 5.5, the culture medium is subpackaged in 250mL conical flasks, the liquid filling amount of each flask is 100mL, and each culture medium is 3 flasks. Respectively placing the two-way fermentation culture medium in a sterilization pot, sterilizing at 121 deg.C for 20min, and cooling in a super clean bench after sterilization;

(5) and (3) post-treatment of fermentation liquor: and (4) after the fermentation is finished, sterilizing the fermentation liquor for 20min at 121 ℃. And after sterilization, filtering the fermentation liquor, and detecting the contents of polysaccharide and flavone in the filtrate.

Comparative example 4

(3) preparing a bidirectional fermentation culture medium: 0.2% of rhizoma polygonati, 0.2% of tuberose, 2% of starch, 1.5% of yeast powder, 0.3% of sodium chloride, 0.02% of calcium carbonate, and adding water to 100% to prepare a bidirectional fermentation culture medium, wherein the initial pH value is 5.5, the bidirectional fermentation culture medium is subpackaged in 250mL conical flasks, the liquid filling amount of each flask is 100mL, and each culture medium is 3 flasks. Respectively placing the two-way fermentation culture medium in a sterilization pot, sterilizing at 121 deg.C for 20min, and cooling in a super clean bench after sterilization;

(5) post-treatment of fermentation liquor: and (4) after the fermentation is finished, sterilizing the fermentation liquor for 20min at 121 ℃. After sterilization, the fermentation broth was filtered, 30% glycerol, 1% hexanediol, 2% pentanediol were added, sterilized at 95 ℃ for 40min, and cooled to obtain comparative example 4.

Comparative example 5

(5) post-treatment of fermentation liquor: and after the fermentation is finished, sterilizing the fermentation liquor for 20min at 121 ℃. After sterilization, the fermentation broth was filtered, 30% glycerol, 1% hexylene glycol, 2% pentanediol were added, and the mixture was quenched at 95 ℃ for 40min, and cooled to obtain comparative example 5.

Comparative example 6

(1) Pretreatment of raw materials: crushing the dendrobium officinale and sieving the crushed dendrobium officinale with a 80-mesh sieve;

(3) preparing a bidirectional fermentation culture medium: 2% of dendrobium officinale, 2% of starch, 1.5% of yeast powder, 0.3% of sodium chloride, 0.02% of calcium carbonate, and adding water to 100% to prepare a bidirectional fermentation culture medium with an initial pH of 5.5, wherein the two-way fermentation culture medium is subpackaged in 250mL conical bottles, the liquid filling amount of each bottle is 100mL, and each culture medium is 3 bottles. Respectively placing the two-way fermentation culture medium in a sterilization pot, sterilizing at 121 deg.C for 20min, and cooling in a super clean bench after sterilization;

(5) post-treatment of fermentation liquor: and after the fermentation is finished, sterilizing the fermentation liquor for 20min at 121 ℃. After sterilization, the fermentation broth was filtered, 30% glycerol, 1% hexylene glycol, 2% pentanediol were added, and the mixture was quenched at 95 ℃ for 40min, and cooled to obtain comparative example 6.

Comparative example 7

(1) Pretreatment of raw materials: crushing the dendrobium officinale, sieving the crushed dendrobium officinale with a 80-mesh sieve, crushing the polygonatum sibiricum, and sieving the crushed polygonatum sibiricum with a 10-mesh sieve;

(3) preparing a bidirectional fermentation culture medium: 2% of dendrobium officinale, 0.2% of rhizoma polygonati, 2% of starch, 1.5% of yeast powder, 0.3% of sodium chloride, 0.02% of calcium carbonate, and adding water to 100% to prepare a bidirectional fermentation culture medium, wherein the initial pH value is 5.5, the bidirectional fermentation culture medium is subpackaged in 250mL conical bottles, the liquid filling amount of each bottle is 100mL, and each culture medium is 3 bottles. Respectively placing the two-way fermentation culture medium in a sterilization pot, sterilizing at 121 deg.C for 20min, and cooling in a super clean bench after sterilization;

(5) post-treatment of fermentation liquor: and after the fermentation is finished, sterilizing the fermentation liquor for 20min at 121 ℃. After sterilization, the fermentation broth was filtered, 30% glycerol, 1% hexylene glycol, 2% pentanediol were added, and the mixture was quenched at 95 ℃ for 40min, and cooled to obtain comparative example 7.

Comparative example 8

(1) Pretreatment of raw materials: crushing the dendrobium officinale, sieving with a 80-mesh sieve, respectively crushing the tuberose, and sieving with a 10-mesh sieve;

(3) preparing a bidirectional fermentation culture medium: 2% of dendrobium officinale, 0.2% of tuberose, 2% of starch, 1.5% of yeast powder, 0.3% of sodium chloride, 0.02% of calcium carbonate, and adding water to 100% to prepare a bidirectional fermentation culture medium, wherein the initial pH value is 5.5, the bidirectional fermentation culture medium is subpackaged in 250mL conical flasks, the liquid filling amount of each flask is 100mL, and each culture medium is 3 flasks. Respectively placing the two-way fermentation culture medium in a sterilization pot, sterilizing at 121 deg.C for 20min, and cooling in a super clean bench after sterilization;

(5) and (3) post-treatment of fermentation liquor: and (4) after the fermentation is finished, sterilizing the fermentation liquor for 20min at 121 ℃. After sterilization, the fermentation broth was filtered, 30% glycerol, 1% hexylene glycol, 2% pentanediol were added, the mixture was quenched at 95 ℃ for 40min, and the comparative example 8 was obtained after cooling.

Comparative example 9

(1) Pretreatment of raw materials: pulverizing rhizoma Polygonati, and sieving with 10 mesh sieve;

(3) preparing a bidirectional fermentation culture medium: 0.2% of rhizoma polygonati, 2% of starch, 1.5% of yeast powder, 0.3% of sodium chloride and 0.02% of calcium carbonate, adding water to 100%, preparing a bidirectional fermentation culture medium with an initial pH of 5.5, and subpackaging in 250mL conical flasks, wherein the liquid filling amount of each flask is 100mL, and each culture medium is 3 flasks. Respectively placing the two-way fermentation culture medium in a sterilization pot, sterilizing at 121 deg.C for 20min, and cooling in a super clean bench after sterilization;

(5) post-treatment of fermentation liquor: and (4) after the fermentation is finished, sterilizing the fermentation liquor for 20min at 121 ℃. After sterilization, the fermentation broth was filtered, 30% glycerol, 1% hexylene glycol, 2% pentanediol were added, and the mixture was quenched at 95 ℃ for 40min, and cooled to obtain comparative example 9.

Comparative example 10

(1) Pretreatment of raw materials: grinding tuberose, and sieving with a 10-mesh sieve;

(3) preparing a bidirectional fermentation culture medium: 0.2% of tuberose, 2% of starch, 1.5% of yeast powder, 0.3% of sodium chloride, 0.02% of calcium carbonate, and adding water to 100% to prepare a bidirectional fermentation culture medium, wherein the initial pH value is 5.5, the bidirectional fermentation culture medium is subpackaged in 250mL conical flasks, the liquid filling amount of each flask is 100mL, and each culture medium is 3 flasks. Respectively placing the two-way fermentation culture medium in a sterilization pot, sterilizing at 121 deg.C for 20min, and cooling in a super clean bench after sterilization;

(5) and (3) post-treatment of fermentation liquor: and (4) after the fermentation is finished, sterilizing the fermentation liquor for 20min at 121 ℃. After sterilization, the fermentation broth was filtered, 30% glycerol, 1% hexylene glycol, 2% pentanediol were added, the mixture was quenched at 95 ℃ for 40min, and the comparative example 10 was obtained after cooling.

1. Active substance content detection

(1) Total flavone detection method

Using NaNO ₂ -Al(NO ₃ ) ₃ And (3) testing the flavone content in the sample by a colorimetric method, referring to a spectrophotometry colorimetric method for determining the total flavone content in the propolis in GB/T20574-2006.

(2) Polysaccharide detection method

Precipitating with 95% ethanol, dissolving the precipitate, and detecting polysaccharide content by phenol-sulfuric acid method, referring to Aloe juice and powder for cosmetic QB/T2488-2006.

2. Non-enzymatic glycosylation assay

One of the main characteristics of skin aging is yellowing of the skin and the appearance of age spots, mainly due to lipid peroxidation and non-enzymatic glycosylation reactions. The non-enzymatic glycosylation reaction in organisms refers to that under the condition of no enzyme catalysis, the aldehyde group or ketone group of reducing sugar and the amino group of macromolecules such as protein and the like undergo Maillard (Maillard) reaction to generate yellow brown glycosylation end products (AGEs).

By inhibiting the nonenzymatic glycosylation of proteins, the inhibitory effect of substances on the deposition of brown pigment can be reflected. The specific test method is as follows:

a0.5 mol/L bovine serum albumin solution (BCA) and a 20mg/mL fructose solution were mixed in equal volumes to obtain a BCA-fructose reaction solution. The reagents were added sequentially in the order of Table 7 and mixed by vortexing after addition. After incubation for 5 days at 37 ℃ in the dark, the fluorescence intensity was measured at an excitation wavelength of 370nm and an emission wavelength of 440nm. The inhibition degree of the sample to be tested on the non-enzymatic glycosylation reaction is calculated according to the Fluorescence intensity (RFU, relative Fluorescence Unit), and is shown in the formula (1).

TABLE 7 grouping and addition of non-enzymatic glycosylation test experiments

Group of	Sample to be tested (μ L)	PBS buffer (mu L)	BCA-fructose reaction (μ L)
				Test group/Positive control	+	-	+
Negative control	-	+	+
				Blank group	+	+	-

3. Inhibition of tyrosinase assay

Skin pigmentation is caused by the accumulation of melanin in the epidermis, and excessive melanin synthesis causes skin pigmentation, formation of spots, and dullness of the skin. Generally, the higher the tyrosinase activity, the more melanin synthesis. By inhibiting tyrosinase activity, the rate of melanin synthesis can be controlled. The whitening effect of the substances applied to cosmetics was evaluated by the tyrosinase activity inhibition rate, and the experimental reaction system is shown in table 8. After the sample addition, incubation was carried out at 25 ℃ for 30min, and the absorbance was measured at 475 nm.

TABLE 8 tyrosinase reaction System

Test tube numbering	PBS buffer (μ L)	L-tyrosine (u L)	sample/Positive control (μ L)	Tyrosinase (μ L)
					A	+	+	-	+
B	+	+	-	-
					C	+	+	+	-
D	+	+	+	-

The tyrosinase inhibition rate is calculated according to the formula (2):

in the formula (2): A. b, C and D are respectively a negative group light absorption value, a negative control group blank light absorption value, a sample group/positive group light absorption value and a sample group/positive group blank light absorption value.

4. Clock gene clock expression assay

In nature, all life activities from single cells to higher animals and plants and human beings have a periodic life activity phenomenon which runs according to a certain rule, and is called biological rhythm. It is now believed that a time control mechanism system, called a biological clock system, widely exists in natural organisms to control and regulate various rhythmic activities and maintain periodic oscillations in the internal environment of the organism. The molecular elements of biological clocks are composed of a series of core Clock components, including genes such as Clock, bmal1, per, cry, tim, etc., and their related protein products. UVB-induced DNA damage repair responses are also reported to exhibit clock-dependent rhythmic changes. In addition, circadian clocks also participate in regulating multiple aspects of skin homeostasis such as: hair growth, cell proliferation, stem cell function, carcinogenesis, aging, immunity, and the like.

Clock gene Clock expression experiment, the concrete operation is as follows:

(1) cytotoxicity-based assays

The test sample is set with 8 concentrations, each concentration is set with 3 repeated holes, and meanwhile, the test is set with a negative control (culture medium), a zero setting hole (deionized water) and a positive control (culture medium containing 5% DMSO (dimethyl sulfoxide)). The assay employs a MTT activity assay to screen the maximum safe concentration for cell administration. The specific operation steps are as follows:

a. cell inoculation: by 1 × 10 ⁴ Cell/well inoculation Density cells were plated onto 96-well plates, incubators (37 ℃, 5% CO) ₂ 95% Relative Humidity (RH)) overnight.

b. Preparing liquid: test substances were prepared at different concentrations according to the concentration settings in table 9.

TABLE 9 test concentration settings

Sample name	Sample concentration (v/v)
		Bidirectional fermentation liquor	0.08%、0.16%、0.31%、0.63%、1.25%、2.5%、5%、10%

c. Administration: the administration was carried out when the plating rate of the cells in the 96-well plate reached 40-60%. Adding 200 mu L of culture solution into each hole of the solvent control group; adding 200 mu L of culture solution containing 10% DMSO into each well of the positive control group; adding 200 mu L of culture solution containing samples with corresponding concentrations into each well of the sample group; the zero-adjusted group was inoculated without cells, and only 200. Mu.L of cell culture medium was added. After completion of the administration, the 96-well plate was placed in an incubator (37 ℃ C., 5% CO) ₂ 95% RH).

d. And (3) detection: after 24h of cell incubation culture, discarding the supernatant, adding MTT working solution (0.5 mg/mL), incubating for 4h at 37 ℃ in a dark place, discarding the supernatant after the incubation is finished, adding 150 muL DMSO into each well, and reading the OD value at 490 nm.

e. Relative cell viability: according to the calculation formula:

(2) morphological examination

a. Cell inoculation: and (3) setting a sample group and a solvent control group, wherein each group is provided with two multiple holes. The cells were seeded at the corresponding seeding density in 24-well plates, incubators (37 ℃ C., 5% CO) ₂ 95% RH) overnight.

b. Preparing liquid: according to the MTT detection result, determining the morphological observation concentration of the detection sample, and preparing the working solution of the test object with different concentrations.

c. Administration: when the cell plating rate of the 24-pore plate reaches 40 percent, the medicine is administered, and the sample is obtainedAdding test substances with different concentrations into the sample group, adding cell culture solution into the solvent control group, and culturing in incubator (37 deg.C, 5% CO) ₂ 95% RH) for 24h.

d. And (3) cell observation: after incubation, the cell morphology was observed under a microscope and photographed.

(3) Gene detection

a. Cell inoculation: by 2.5X 10 ⁵ Cell/well inoculation Density cells were plated onto 6 well plates, incubators (37 ℃, 5% CO) ₂ 95% RH) overnight.

b. Administration: grouping according to the experiment of table 10, when the plating rate of the cells in the 6-well plate reaches 40-60%, the administration is carried out in groups, each well is loaded with 2mL, and each group is provided with 3 multiple wells. After completion of the administration, the 6-well plate was placed in an incubator (37 ℃ C., 5% CO) ₂ 95% RH) for 24h.

c. Collecting cells: after 24h incubation, cell supernatants were discarded, washed twice with 1 mL/well PBS, 1mL RNAioso Plus was added to each well, lysed cells were aspirated, and samples were collected.

d. And (3) gene expression detection: extracting RNA, reverse transcribing to cDNA, and fluorescent quantitative PCR detection with 2 ^-△△CT The method performs result calculation

e. And (4) analyzing results: the plots were generated using GraphPad Prism Program software and statistically analyzed between groups using t-test.

TABLE 10 test groupings

5. Analysis of test results

5.1 active ingredient content

TABLE 11 comparison of active ingredients in samples

Sample numbering	Polysaccharide content (mg/mL)	Flavone content (mg/mL)
			Sample No. 1	5.11	0.15
Sample 2	5.45	0.16
			Sample 3	5.23	0.13
Sample 4	5.83	0.18
			Sample No. 5	5.98	0.21
Sample No. 6	5.79	0.15
			Sample 7	5.38	0.12
Sample 8	5.55	0.15
			Sample 9	5.21	0.11
Sample 10	5.17	0.12
			Sample 11	5.32	0.16
Sample 12	5.89	0.19
			Sample 13	5.78	0.18
Sample 14	5.68	0.14
			Sample 15	5.85	0.18
Sample 16	5.74	0.21
			Sample 17	5.33	0.23
Sample 18	5.68	0.14
			Sample 19	5.82	0.15
Sample 20	5.74	0.17
			Sample 21	5.32	0.15
Sample 22	5.79	0.14
			Sample 23	5.13	0.11
Comparative example 1	3.11	0.11
			Comparative example 2	2.64	0.17
Comparative example 3	2.73	0.13

The polysaccharide content and the flavone content are taken as research targets, the preparation is carried out by adopting a bidirectional fermentation process, the influence of fermentation raw materials, fermentation inoculation amount (%), fermentation time (d), fermentation temperature (DEG C) and fermentation pH value on the polysaccharide content and the flavone content in the plant composition is considered, and the optimized preparation process is as follows: 1-10% of dendrobium officinale, 0.01-1% of rhizoma polygonati and 0.01-1% of tuberose by weight of the bidirectional fermentation medium; 1-15% of fermentation inoculation amount; fermenting for 1-7 days at 25-35 deg.C and pH3.5-7.5, wherein the plant composition contains polysaccharide 5.11-5.98mg/mL and flavone 0.11-0.23mg/mL.

The plant composition is prepared by a bidirectional fermentation process, the dendrobium officinale, the polygonatum and the tuberose are combined according to a specific proportion, and then medicinal and edible fungi lucid ganoderma is adopted for bidirectional fermentation, and the fungi have a strong enzyme system for decomposing cellulose and other substances and are used for deeply fermenting and decomposing the dendrobium officinale, the polygonatum and the tuberose into micromolecule carbon sources, so that the utilization rate of active ingredients of plant raw materials is increased, and the active ingredient polysaccharide of the plant composition is improved.

5.2 Non-enzymatic glycosylation assay

TABLE 12 inhibition of nonenzymatic glycosylation of the samples

Sample numbering	Non-enzymatic glycosylation inhibition ratio (%)
		2.5% example 6	73
2.5% example 7	67
		2.5% example 8	75
2.5% example 9	74
		2.5% comparative example 4	36
2.5% comparative example 5	32
		2.5% comparative example 6	24
2.5% comparative example 7	31
		2.5% comparative example 8	22
2.5% comparative example 9	24
		2.5% comparative example 10	21
0.5% Vc ethyl ether	58

The results of the non-enzymatic glycosylation test are shown in Table 12, and the plant compositions prepared in examples 6-9 of the present invention, at the same addition level (2.5%), all showed higher inhibition of non-enzymatic glycosylation than the plant compositions obtained in comparative examples 4-10 and 0.5% Vc ethyl ether (positive control), indicating that the plant compositions of the present invention were able to inhibit the non-enzymatic glycosylation better than the fermentation products obtained from any of the two-way fermentation of Ganoderma lucidum from a single source and the fermentation products obtained from any of the two combined sources. The test results prove that the dendrobium officinale, the polygonatum sibiricum and the tuberose have obvious synergistic effect after being subjected to bidirectional fermentation by the ganoderma lucidum according to a specific proportion, and compared with the combination or dosage range provided in a comparative example, the combination and proportion provided by the application can obviously increase the inhibition effect on non-enzymatic glycosylation reaction after being treated by a bidirectional fermentation process of the ganoderma lucidum.

5.3 tyrosinase assay

TABLE 13 tyrosinase inhibition for samples

Sample numbering	Tyrosinase inhibition (%)
		15% of example 6	63
15% example 7	59
		15% of example 8	64
15% of example 9	65
		15% comparative example 4	23
15% comparative example 5	19
		15% comparative example 6	11
15% comparative example 7	18
		15% comparative example 8	14
15% comparative example 9	13
		15% comparative example 10	12
0.075% alpha-arbutin	87

The results of the tyrosinase assay are shown in table 13, and compared with comparative examples 4-10, the plant compositions prepared in examples 6-9 of the present invention have a good tyrosinase activity inhibiting effect at the same addition amount (15%), and the tyrosinase activity inhibiting effect is lower than that of α -arbutin (positive control). The plant composition can better inhibit the activity of tyrosinase and has better whitening effect than a fermentation product obtained by any ganoderma lucidum bidirectional fermentation single raw material and a fermentation product obtained by any two combined raw materials of the ganoderma lucidum bidirectional fermentation. The test results prove that the dendrobium officinale, the polygonatum sibiricum and the tuberose have obvious synergistic effect after being subjected to bidirectional fermentation by the ganoderma lucidum according to a specific proportion, and compared with the combination or dosage range provided in a comparative example, the combination and proportion provided by the application can obviously increase the inhibition effect on the tyrosinase activity after being subjected to bidirectional fermentation process treatment by the ganoderma lucidum.

5.4 clock Gene expression assay

TABLE 14 summary of results for Clock genes

Sample name	Mean value (Mean)	Standard Deviation (SD)	Significance of difference: (p-value）
				Blank control	1.00	0.07	/
0.025% example 6	0.88	0.02	0.043*
				0.05% of example 6	0.84	0.03	0.023*
0.075% of example 6	0.77	0.04	0.007**
				0.025% comparative example 6	1.04	0.04	0.032*
0.05% comparative example 6	1.11	0.05	0.028*
				0.075% of comparative example 6	1.17	0.03	0.025*

As shown in table 14 and fig. 1, compared with the blank control, the example 6 has a good effect of inhibiting the expression of the Clock gene in the range of 0.025% to 0.075%, and the effect is significantly better than that of the comparative example 6, which shows that the plant composition of the present invention has a better effect of inhibiting the expression of the Clock gene than the fermentation product obtained by ganoderma lucidum bi-directionally fermenting dendrobium officinale. The test results prove that the dendrobium officinale, the polygonatum and the tuberose have obvious synergistic effect after being subjected to bidirectional fermentation by the ganoderma lucidum according to a specific proportion, and compared with the fermentation product of the dendrobium officinale provided in the comparative example 6, the combination and proportion of the application can obviously increase the inhibition effect on clock gene expression after being treated by the bidirectional fermentation process of the ganoderma lucidum.

According to the invention, dendrobium officinale, rhizoma polygonati and tuberose are combined in a specific ratio and then are inoculated with ganoderma lucidum to be subjected to bidirectional fermentation to obtain a fermentation product with the effects of inhibiting non-enzymatic glycosylation reaction and inhibiting Clock gene expression, and the fermentation product can also inhibit tyrosinase activity and has double effects of resisting aging and whitening.

Claims

1. A plant composition is characterized in that the addition amount of dendrobium officinale in the plant composition is 1-10%, the addition amount of rhizoma polygonati is 0.01-1%, and the addition amount of tuberose is 0.01-1% by weight of a bidirectional fermentation medium; the plant composition is prepared by a preparation method comprising the following steps:

(3) preparing a bidirectional fermentation culture medium;

(4) inoculation and fermentation: inoculating the ganoderma lucidum seed liquid obtained in the step (2) into a bidirectional fermentation culture medium for culture;

(5) post-treatment of fermentation liquor: sterilizing and filtering;

the inoculation amount of the ganoderma lucidum seed solution in the step (4) is 1-15%.

2. The botanical composition of claim 1, wherein the botanical composition has a polysaccharide content of 5.11-5.98mg/mL and a flavone content of 0.11-0.23mg/mL.

3. The plant composition according to claim 1 or 2, wherein the plant composition is prepared by the method comprising the steps of (2) adding 0.5-5% of starch, 0.1-5% of yeast powder, 0.1-2% of sodium chloride, 0.01-0.1% of calcium carbonate, and adding water to 100%; the culture conditions of the ganoderma lucidum are 25-35 ℃,50-300rpm, and the ganoderma lucidum is cultured for 50-80h.

4. The plant composition according to claim 1 or 2, wherein the bidirectional fermentation medium in step (3) of the preparation method of the plant composition is 0.5-5% of starch, 0.1-5% of yeast powder, 0.1-2% of sodium chloride, 0.01-0.1% of calcium carbonate, 1-10% of dendrobium officinale, 0.01-1% of rhizoma polygonati, 0.01-1% of tuberose and water added to 100%.

5. The plant composition according to claim 1 or 2, wherein the bidirectional fermentation culture in step (4) of the plant composition preparation method is carried out at 25-35 ℃ and 50-300rpm for 1-7 days.

6. A method of preparing a botanical composition, said method comprising the steps of:

(2) activating the ganoderma lucidum: adding 0.5-5% of starch, 0.1-5% of yeast powder, 0.1-2% of sodium chloride and 0.01-0.1% of calcium carbonate into 100% of water, mixing, subpackaging and sterilizing to prepare a ganoderma lucidum culture medium; selecting Ganoderma mycelia, inoculating to culture medium, and culturing at 25-35 deg.C and 50-300rpm for 50-80 hr to obtain Ganoderma seed solution;

(3) preparing a bidirectional fermentation culture medium: mixing starch 0.5-5% and yeast powder 0.1-5%; 0.1-2% of sodium chloride, 0.01-0.1% of calcium carbonate, 1-10% of dendrobium officinale, 0.01-1% of rhizoma polygonati, 0.01-1% of tuberose, and water added to 100%, mixing, subpackaging and sterilizing to prepare a bidirectional fermentation medium;

(4) inoculation and fermentation: inoculating the ganoderma lucidum seed liquid in the step (2) into a culture medium according to the inoculation amount of 1-15%, and then culturing for 1-7d at the temperature of 25-35 ℃ and under the condition of 50-300 rpm;

(5) and (3) post-treatment of fermentation liquor: sterilizing and fine filtering.

7. Use of the plant composition according to any one of claims 1 to 5 or prepared by the preparation method according to claim 6 for preparing a skin care product.

8. Use of the plant composition of any one of claims 1 to 5 or the plant composition prepared by the preparation method of claim 6 in preparing skin care products with anti-aging or whitening effects.