Orchid polypeptide as well as preparation method and application thereof

Abstract

The invention discloses orchid polypeptide and a preparation method and application thereof, and belongs to the technical field of skin care products. The amino acid sequences of the polypeptide are as follows: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and EQ ID NO. 6. According to the orchid polypeptide and the preparation method thereof, the raw materials of the orchid polypeptide are derived from natural plants, the orchid polypeptide is cultured in a callus culture mode, no other organic solvent is introduced in the extraction process, clear amino acid identification is performed on the orchid polypeptide, and gene sequencing verification and in-vitro efficacy comparison with commercially available synthetic polypeptide show that the orchid polypeptide has a good application prospect. The composition has the advantages of being green, natural, high in safety, clear in effective component and high in efficiency, and is easily applied to cosmetics.

Description

Technical Field

[0001] The present invention belongs to the technical field of skin care products, and specifically relates to an orchid polypeptide, a preparation method thereof, and uses thereof. Background Art

[0002] Bioactive peptides refer to small peptide segments with biological activities. They are usually composed of dozens of amino acid residues and are connected by specific peptide bonds. These peptide segments play important physiological functions in organisms, such as regulating immunity, promoting wound healing, and inhibiting inflammatory responses. Compared with proteins, bioactive peptides have smaller molecular weights, higher bioavailability, and lower toxic and side effects. This makes their applications in the cosmetic field more extensive and effective. Bioactive peptides can enter the skin through various channels, including the epidermis, dermis, and subcutaneous tissue, and bind to receptors on skin cells, thereby exerting their biological effects.

[0003] However, most of the active peptides currently used in the cosmetic field are still artificially synthesized. There are substances of short peptides naturally present in plant cells, which are used for the regulation of the plant's own life activities, such as resisting external stimuli such as free radicals. Such plant polypeptides can naturally help the human skin achieve antioxidant and anti-inflammatory effects.

[0004] The Orchidaceae family is one of the largest families of angiosperms, with approximately 7,360 genera and 28,000 species globally, distributed all over the world, mainly produced in Asia and South America. It not only has high ornamental value and ecological value but also has rich medicinal value. Phenanthrenes are the main chemical substances of orchid plants. Existing research on orchid plants mostly focuses on phenanthrene substances, but there are also some rare plant proteins in orchids. For example, it was found in the article "The HSP70 Gene Family of the Genus Calanthe in Orchidaceae and Its Adaptive Evolution" that the genus Calanthe contains 47 HSP70 gene proteins. However, the structure of plant proteins is relatively large and cannot enter the skin smoothly, so there are still certain limitations in their applications in the cosmetic field. Summary of the Invention

[0005] Aiming at the above problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for an orchid polypeptide, a preparation method thereof, and uses thereof. By culturing orchid callus and extracting orchid polypeptide, it is found that the orchid polypeptide has a good promoting effect on collagen, elastin, and barrier protein, and has a strong anti-aging effect.

[0006] The described orchid polypeptide is characterized in that the amino acid sequence of the polypeptide is SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, EQ ID NO.6.

[0007] The described method for preparing an orchid polypeptide is characterized by the following steps:

[0008] 1) Select an orchid plant, induce plant callus, and then place it in a culture medium for cultivation to obtain orchid callus;

[0009] 2) Subject the orchid callus to pre-freezing, freeze-drying, and pulverization in sequence to obtain orchid callus powder;

[0010] 3) Mix the orchid callus powder with a buffer solution containing NaCl and perform freeze-thaw treatment to obtain an intermediate;

[0011] 4) Place the intermediate in an environment of -5 to 5 °C for 5 to 20 min, and then perform heat treatment and centrifugation in sequence to obtain a supernatant;

[0012] 5) Take the supernatant after centrifugation, separate it using an ultrafiltration centrifuge tube, collect the retained liquid and perform freeze-drying to obtain orchid polypeptide.

[0013] The described method for preparing an orchid polypeptide is characterized in that in step 1): the orchid plant is a Calanthe plant, a Bletilla sinensis var. chinensis plant, or a Bletilla striata plant.

[0014] The described method for preparing an orchid polypeptide is characterized in that in step 2): the pre-freezing treatment temperature is -40 to -20 °C, and the pre-freezing treatment time is 12 to 36 h; preferably, the pre-freezing treatment temperature is -30 to -35 °C, and the pre-freezing treatment time is 20 to 25 h.

[0015] The described method for preparing an orchid polypeptide is characterized in that in step 2): the cold trap temperature for freeze-drying is -100 to -60 °C, the freezing chamber temperature for freeze-drying is -45 °C to 35 °C, the vacuum degree for freeze-drying is 0 to 10 Pa, and the freeze-drying time is 36 to 72 h; preferably, the cold trap temperature for freeze-drying is -90 to -70 °C, the freezing chamber temperature for freeze-drying is -20 °C to 25 °C, the vacuum degree for freeze-drying is 2 to 8 Pa, and the freeze-drying time is 45 to 60 h; more preferably, the cold trap temperature for freeze-drying is -80 to -75 °C, the freezing chamber temperature for freeze-drying is 0 °C to 10 °C, the vacuum degree for freeze-drying is 4 to 6 Pa, and the freeze-drying time is 50 to 55 h.

[0016] The described method for preparing an orchid polypeptide is characterized in that in step 3): the material-liquid ratio of orchid callus powder mixed with a buffer solution containing NaCl is 1:5 to 10, preferably 1:6 to 8; the buffer solution is PBS, and the final concentration of NaCl in the buffer solution is 100 to 200 mM, preferably 120 to 160 mM; in the freeze-thaw treatment, the freezing stage uses liquid nitrogen freezing, the melting stage in the freeze-thaw treatment uses a temperature of 35 to 40 °C, and the number of times of the freeze-thaw treatment is 2 to 4 times.

[0017] The described method for preparing an orchid polypeptide is characterized in that in step 4): the intermediate is placed in an environment of 0 to 2 °C for 10 to 15 min; the heat treatment is carried out by boiling water bath, and the time of the heat treatment is 5 to 15 min, preferably 8 to 10 min; the centrifugation time is 10 to 30 min, preferably 15 to 20 min; the centrifugation speed is 2000 to 6000 g, preferably 3000 to 5000 g; the centrifugation temperature is 3 to 5 °C.

[0018] The described method for preparing an orchid polypeptide is characterized in that in step 5): the cut-off molecular weight of the ultrafiltration tube is 3KD.

[0019] The application of the orchid polypeptide in cosmetics, especially in cosmetics with anti-aging effects.

[0020] The promoting effect of the orchid polypeptide on collagen, elastin, and barrier protein, especially on type XVII collagen, type VI collagen, type V collagen, type IV collagen, type III collagen, type I collagen, elastin, endopilin, fibrin, keratin, tight junction protein, SIRT2, and SIRT6. The orchid polypeptide prepared by the present invention has a good promoting effect on genes such as type XVII collagen, type VI collagen, type V collagen, type IV collagen, type III collagen, type I collagen, elastin, endopilin, fibrin, keratin, tight junction protein, SIRT2, and SIRT6, and has a strong anti-aging effect.

[0021] The above method for preparing an orchid polypeptide uses natural plants as raw materials, is cultivated by callus culture, no other organic solvents are introduced during the extraction process, and the amino acids of the orchid polypeptide are clearly identified. After being verified by gene sequencing and compared with the in vitro efficacy of commercially available synthetic polypeptides, it has the advantages of being green, natural, highly safe, with clear active ingredients, and high efficiency, and is easy to be applied in cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a diagram showing the promoting effect of the calanthe orchid polypeptide prepared in Example 1 of the present invention on longevity genes;

[0023] Figure 2 Promotion effect diagram of the Calanthe discolor orchid polypeptide prepared in Example 1 of the present invention on skin barrier protein-related genes;

[0024] Figure 3 Promotion effect diagram of the Calanthe discolor orchid polypeptide prepared in Example 1 of the present invention on collagen-related genes;

[0025] Figure 4 Promotion effect diagram of the Bletilla sinensis orchid polypeptide prepared in Example 2 of the present invention on longevity genes;

[0026] Figure 5 Promotion effect diagram of the Bletilla sinensis orchid polypeptide prepared in Example 2 of the present invention on skin barrier protein-related genes;

[0027] Figure 6 Promotion effect diagram of the Bletilla sinensis orchid polypeptide prepared in Example 2 of the present invention on collagen-related genes;

[0028] Figure 7 Influence diagram of the Calanthe discolor orchid polypeptide prepared in Example 1 of the present invention and other commercially available polypeptides on collagen and elastin genes. Detailed implementation manners

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0030] Example 1 Calanthe discolor callus culture and polypeptide extraction

[0031] Select Calanthe discolor plants (species: Orchidaceae, Latin name: Calanthe discolor), induce plant callus, and then place it in a culture medium for cultivation (the culture medium consists of 2 g / L MS medium, 6 g / L agar, 0.5 mg / L 6-BA, and 0.5 mg / L NAA) to obtain Calanthe discolor callus;

[0032] Take the Calanthe discolor callus and pre-freeze it in a constant temperature refrigerator at -40°C for 12 h, then put it into a vacuum freeze dryer for drying. The cold trap temperature is -100°C, the freezing chamber temperature is -45°C - 35°C, the vacuum degree is 0 - 10 Pa, and the vacuum freezing is 36 h. During the 36 h drying process, the freeze-drying program adopted is as follows:

[0033] Program 1 (1 h, -45 °C, vacuum 0), Program 2 (0.5 h, -45 °C, vacuum 0), Program 3 (0.5 h, -45 °C, vacuum 0 - 10 Pa), Program 4 (0.5 h, -30 °C, vacuum 0 - 10 Pa), Program 5 (1 h, -10 °C, vacuum 0 - 10 Pa), Program 6 (5 h, 0 °C, vacuum 0 - 10 Pa), Program 7 (4 h, 5 °C, vacuum 0 - 10 Pa), Program 8 (3 h, 10 °C, vacuum 0 - 10 Pa), Program 9 (5 h, 15 °C, vacuum 0 - 10 Pa), Program 10 (5 h, 20 °C, vacuum 0 - 10 Pa), Program 11 (5 h, 25 °C, vacuum 0 - 10 Pa), Program 12 (5 h, 30 °C, vacuum 0 - 10 Pa), Program 13 (5 h, 30 °C, vacuum 0 - 10 Pa), Program 14 (7.5 h, 35 °C, vacuum 0 - 10 Pa).

[0034] Use liquid nitrogen to pre-cool the mortar and pestle 3 times, grind the freeze-dried Calanthe discolor callus into powder, then put it into a pre-cooled 50 ml centrifuge tube, and add the 1X PBS solution containing 100 mM NaCl pre-cooled at 4 °C according to the solid-liquid ratio of 1:5. Mix well using a vortex oscillator, and perform freeze-thaw treatment 2 times (freeze in liquid nitrogen for 45 s and dissolve at 40 °C for 2 min 30 s). After the treatment, let it stand on ice for 16 min, then perform boiling water bath treatment for 5 min. After cooling to room temperature, centrifuge for 10 min (centrifugation conditions: 2000 g, 3 °C).

[0035] Take the supernatant after centrifugation, separate it using a 3KD ultrafiltration centrifuge tube, collect the retained liquid for freeze-drying, and the freeze-drying program and parameters are the same as above to obtain Calanthe discolor orchid polypeptide.

[0036] Example 2: Callus culture and polypeptide extraction of Bletilla sinensis

[0037] Select Bletilla sinensis plants (species: Orchidaceae, Latin name: Mengzia foliosa), induce plant callus, and then place it in a culture medium for cultivation (the culture medium consists of 2 g / L MS medium, 6 g / L agar, 0.5 mg / L 6-BA, and 0.5 mg / L NAA) to obtain Bletilla sinensis callus;

[0038] Take the Bletilla sinensis callus and pre-freeze it in a -20 °C constant temperature refrigerator for 36 h, then put it into a vacuum freeze-dryer for drying. The cold trap temperature is -60 °C, the freezing chamber temperature is -45 °C - 35 °C, the vacuum is 0 - 10 Pa, and the vacuum freezing is for 72 h. During the 72 h drying process, the following freeze-drying program is adopted:

[0039] Program 1 (1 h, -45 °C, vacuum 0), Program 2 (0.5 h, -45 °C, vacuum 0), Program 3 (0.5 h, -45 °C, vacuum 0 - 10 Pa), Program 4 (0.5 h, -30 °C, vacuum 0 - 10 Pa), Program 5 (1 h, -10 °C, vacuum 0 - 10 Pa), Program 6 (5 h, 0 °C, vacuum 0 - 10 Pa), Program 7 (4 h, 5 °C, vacuum 0 - 10 Pa), Program 8 (3 h, 10 °C, vacuum 0 - 10 Pa), Program 9 (5 h, 15 °C, vacuum 0 - 10 Pa), Program 10 (5 h, 20 °C, vacuum 0 - 10 Pa), Program 11 (5 h, 25 °C, vacuum 0 - 10 Pa), Program 12 (5 h, 30 °C, vacuum 0 - 10 Pa), Program 13 (5 h, 30 °C, vacuum 0 - 10 Pa), Program 14 (7.5 h, 35 °C, vacuum 0 - 10 Pa).

[0040] Use liquid nitrogen to pre-cool the mortar and pestle 3 times. Grind the freeze-dried Bletilla striata and callus into powder, then transfer it into a pre-cooled 50 ml centrifuge tube. Add the 1X PBS solution containing 200 mM NaCl pre-cooled at 4 °C according to the solid-liquid ratio of 1:10. Mix well using a vortex oscillator, and perform repeated freeze-thaw treatment 4 times (freeze in liquid nitrogen for 45 s and dissolve at 35 °C for 2 min 30 s). After the treatment, let it stand on ice for 16 min, then perform a boiling water bath treatment for 15 min. After cooling to room temperature, centrifuge for 30 min (centrifugation conditions: 6000 g, 5 °C).

[0041] Take the supernatant after centrifugation, separate it using a 3KD ultrafiltration centrifuge tube, collect the retained liquid for freeze-drying, and the freeze-drying program and parameters are the same as above to obtain Bletilla striata and orchid polypeptide.

[0042] Comparative Example 1

[0043] Commercially available synthetic polypeptides: palmitoyl tripeptide-1, palmitoyl tripeptide-5, palmitoyl pentapeptide-4, palmitoyl hexapeptide-12, acetyl tetrapeptide-8.

[0044] The present invention will be further described below through corresponding test data and experimental data.

[0045] I. Identification of orchid polypeptide:

[0046] Select the amino acid sequence as the test index, and use the multi-stage mass spectrometry identification (LC-MS / MS) method to identify the polypeptide sequences in the Calanthe orchid polypeptide obtained in Example 1 and the Bletilla striata and orchid polypeptide obtained in Example 2;

[0047] The dissolved Calanthe discolor orchid polypeptide and Bletilla sinensis were loaded onto a nano Viper C18 pre-column (3μm, 100A) in appropriate volumes and rinsed with 20μL for desalting. The liquid phase system was an Easy-nLC1200 nano liquid phase system (ThermoFisher, USA). After the test samples were desalted and retained on the pre-column, they were separated by the analytical column. The specifications of the analytical column were C18 reversed-phase chromatographic column (Acclaim Pep Map RSLC, 75μm×25cm C18-2μm ).

[0048] The concentration gradient of the mobile phase was 5% of mobile phase B (80% acetonitrile, 0.1% formic acid) within 0 - 30 min; then it was gradually increased to 40% according to a 5% concentration gradient.

[0049] The mass spectrometry used a Thermo Fisher Q Exactive system (Thermo Fisher, USA) combined with a nano spray Nano Flex ion source (Thermo Fisher, USA). The spray voltage was 1.9 kV, and the heating temperature of the ion transfer tube was 275°C. The scanning mode was in the data-dependent acquisition working mode (DDA, Data Dependent Analysis). The resolution of the first-level mass spectrometry scan was 70000, the scanning range was 350 - 2000 m / z, and the maximum injection time was 100 ms. Each DDA cycle collected a maximum of 20 secondary spectra with a charge of 2 + to 5 + and the maximum injection time of the secondary mass spectrometry ions was 50 ms. The collision cell energy (high-energy collision-induced dissociation, HCD) was set to 28 eV, and the dynamic exclusion was set to 25 seconds.

[0050] The original raw data files collected by mass spectrometry were processed and retrieved through the PEAKS Studio 8.5 (Bioinformatics Solutions Inc. Waterloo, Canada) software, and the identification results are shown in Table 1 and Table 2.

[0051] Table 1 Amino acid sequence list of Calanthe discolor orchid polypeptide prepared in Example 1

[0052] Serial number Amino acid sequence SEQ ID NO.1 GYSFTTTAER SEQ ID NO.2 SROGIPLITGRFDSLEOLDEFSKSF SEQ ID NO.3 VAPEEHPVLLTEAPLNPK

[0053] Table 2 Amino acid sequence list of Bletilla sinensis orchid polypeptide prepared in Example 2

[0054] Serial number Amino acid sequence SEQ ID NO.4 AVFPSIVGRPR SEQ ID NO.5 LGEHNIDVLEGNEOFINAAK SEQ ID NO.6 VATVSLPR

[0055] II. Gene sequencing:

[0056] Test 1. The calanthe polypeptide was obtained as prepared in Example 1. The specific test process is as described below:

[0057] The sample orchid polypeptide solution was diluted into different concentration gradients using DMEM basal medium, and the culture medium with appropriate concentration was screened as the test sample. Take HaCaT cells in good growth state and inoculate them into a 6-well culture plate at a density of 1×10 5 cells per well and culture overnight (16 h) at 37 °C in a 5% CO 2 environment.

[0058] Remove the waste culture medium from the cell culture plate cultured overnight, and make corresponding treatments according to the settings of the control group and the sample group (experimental group: 2 mL of DMEM culture medium containing 50 ppm orchid polypeptide; 2 mL of DMEM culture medium containing 10 ppm orchid polypeptide; blank control group: 2 mL of DMEM culture medium). Each treatment was set with 3 replicates, and different treatments were added to co-incubate with the cells for 24 h.

[0059] Remove the medium of the cells after the above incubation, collect the cells, add lysis buffer, extract the total cellular RNA, and perform RNA-seq sequencing after detecting the RNA concentration and purity.

[0060] The results are shown in Figure 1 、 Figure 2 、 Figure 3 .

[0061] The Sirtuin protein family, as a class of highly conserved and NAD+-dependent protein deacetylases, plays a crucial role in organisms. They are widely involved in and regulate life activities such as oxidative stress response and cell senescence. As Figure 1 shown, compared with the blank control, the calanthe polypeptide increased the expression levels of SIRT2 and SIRT6 genes by 22.34% and 33.87% respectively. The calanthe polypeptide can increase the expression of SIRT family genes, delay the process of cell senescence, and has a certain anti-aging effect.

[0062] The skin barrier is the first line of defense against external adverse factors, and its function mainly depends on the formation of the stratum corneum and tight junctions. The keratinocyte differentiation proteins in the stratum corneum include fibrin (FBLNI), involucrin (IVL), transglutaminase 2 of keratinocytes (TGM2), keratin (KRTI6), etc. They and the tight junction protein (CLDN4) in the stratum corneum and granular layer are key proteins for maintaining the skin barrier. As Figure 2As shown, compared with the blank control group, the Calanthe discolor orchid polypeptide increased the gene expression levels of transglutaminase (TGM2), involucrin (IVL), fibrin (FBLNI), keratin (KRTI6), and claudin-4 (CLDN4) by 137.48%, 193.13%, 43.84%, 41.89%, and 67.51%, respectively. The Calanthe discolor orchid polypeptide can enhance skin barrier proteins, playing a barrier protection role and achieving the effects of protection and anti-aging in the stratum corneum.

[0063] Collagen is an important protein that supports the skin structure and plays an important role in skin firmness and elasticity. Matrix metalloproteinases (MMPs) are important proteases that cleave collagen. Inhibiting MMPs can further protect the collagen in the skin. As Figure 3 shown, compared with the blank control, the Calanthe discolor orchid polypeptide increased the expression levels of COL17A1, COL6A1, COL5A1, COL4A1, and COLIA1 by 43.78%, 112.68%, 53.30%, 32.91%, and 27.79%, respectively, and decreased matrix metalloproteinase 13 (MMP13), matrix metalloproteinase 7 (MMP7), and matrix metalloproteinase 1 (MMP1) by 81.4%, 44.73%, and 52.79%. The Calanthe discolor orchid polypeptide can increase skin collagen and reduce the content of matrix metalloproteinases, playing a role in protecting and promoting collagen and having a certain anti-aging effect.

[0064] Test 2. The Bletilla sinensis var. albo-marginata orchid polypeptide was prepared as in Example 2. The specific test process was the same as in Test 1, and the results are shown in Figure 4 、 Figure 5 、 Figure 6 。

[0065] As Figure 4 shown, compared with the blank control, the Bletilla sinensis var. albo-marginata orchid polypeptide increased the gene expression levels of SIRT2 and SIRT6 by 18.67% and 103.64%, respectively. The Bletilla sinensis var. albo-marginata orchid polypeptide can increase the expression of SIRT family genes, delay the process of cell aging, and has a certain anti-aging effect.

[0066] As Figure 5 shown, compared with the blank control group, the Bletilla sinensis var. albo-marginata orchid polypeptide increased the gene expression levels of transglutaminase (TGM2), involucrin (IVL), filaggrin 1 (FLG1-AS1), and claudin-4 (CLDN4) by 32.15%, 187.10%, 43.59%, and 49.73%, respectively. The Bletilla sinensis var. albo-marginata orchid polypeptide can enhance skin barrier proteins, playing a barrier protection role and achieving the effects of protection and anti-aging in the stratum corneum.

[0067] As Figure 6As shown, compared with the blank control, the polypeptides from Bletilla striata and Cymbidium can increase the expression levels of COL17A1, COL12A1, and COL4A1 by 30.79%, 61.63%, 53.30%, and 29.57% respectively, and decrease matrix metalloproteinase 13 (MMP13) and matrix metalloproteinase 1 (MMP1) by 74.51% and 54.11%. The polypeptides from Bletilla striata and Cymbidium can increase skin collagen and reduce the content of matrix metalloproteinases, playing a role in protecting and promoting collagen, and having a certain anti-aging effect.

[0068] Experiment 1: In vitro efficacy experiment

[0069] The samples to be tested are as follows: The polypeptide from Calanthe discolor is obtained by the preparation method of Example 1, and the other polypeptides are the commercially available polypeptides described in Comparative Example 1. The concentration of all samples is 10 ppm. The blank control group is cultured in serum-free medium. The specific test process is as follows:

[0070] HSF cells in the logarithmic growth phase are seeded in 6-well plates at a density of 1×10 6 cells / well and cultured in an incubator at 37°C with 5% CO 2 for 24 h;

[0071] After washing twice with PBS, the samples are added to the sample group, and serum-free medium is added to the control group. Then, they are cultured in an incubator at 37°C with 5% CO 2 for 24 h;

[0072] The supernatant is discarded, and the cells are collected after washing twice with PBS;

[0073] RNA is extracted, reverse transcribed into cDNA, and then RT-qPCR is performed.

[0074] The relative expression level of the target gene is calculated by the 2 -ΔΔct method using GAPDH as an internal reference.

[0075] The calculated values are imported into Graphpad Prism 8 for statistical analysis and graphing.

[0076] The results are shown in Figure 7 , Table 3.

[0077] Table 3 Effects of the polypeptide from Calanthe discolor and other commercially available polypeptides on collagen and elastin genes

[0078]

[0079] Type I collagen (Collagen I) is the main structural protein of the skin. Promoting its synthesis by maintaining elasticity and tension, promoting water locking, and barrier function can delay skin aging. As Figure 7As shown in Table 3, compared with the blank control group, 10 ppm of orchid polypeptide significantly increased the relative expression level of the COL1A1 gene by 69%. Among the 5 other samples, compared with the blank control group, 10 ppm of palmitoyl tripeptide-1, 10 ppm of palmitoyl tripeptide-5, and 10 ppm of palmitoyl hexapeptide-12 significantly increased the relative expression level of the COL1A1 gene by 32%, 36%, and 44%, respectively.

[0080] Type III collagen (Collagen III) has a wide distribution range. It has a high content in the skin and can maintain the elasticity and firmness of the skin. As Figure 7 As shown in Table 3, compared with the blank control group, 10 ppm of orchid polypeptide significantly increased the relative expression level of the COL3A1 gene by 55% (P<0.001). Among the 5 other samples, compared with the blank control group, 10 ppm of palmitoyl tripeptide-1, 10 ppm of palmitoyl tripeptide-5, 10 ppm of palmitoyl hexapeptide-12, and 10 ppm of acetyl tetrapeptide-8 significantly increased the relative expression level of the COL3A1 gene by 31%, 42%, 34%, and 23%, respectively.

[0081] Type V collagen (Collagen V) mainly regulates the formation and tissue arrangement of type I collagen fibers in the skin, maintains the integrity of the dermal layer structure, and plays an auxiliary role in the process of wound healing and tissue repair. As Figure 7 As shown in Table 3, compared with the blank control group, 10 ppm of orchid polypeptide significantly increased the relative expression level of the COL5A1 gene by 34%. Among the 5 other samples, compared with the blank control group, 10 ppm of palmitoyl tripeptide-1 and 10 ppm of palmitoyl tripeptide-5 significantly increased the relative expression level of the COL5A1 gene by 21% and 26%, respectively.

[0082] Elastin (ELN) is the main component of elastic fibers. Elastic fibers coexist with collagen fibers, endowing tissues with elasticity and tensile strength. The loss of elastin is the main cause of skin aging, resulting in sagging, drooping, and fine wrinkles. As Figure 7 As shown in Table 3, compared with the blank control group, 10 ppm of orchid polypeptide significantly increased the relative expression level of the ELN gene by 45% (P<0.001). Among the 5 control samples, none of them had a significant promoting effect on the relative expression of the ELN gene.

[0083] Calanthe orchids peptide has a certain promoting effect on type I, type III, type V collagen and elastin. Compared with other commercially available polypeptides, at a concentration of 10 ppm, Calanthe orchids peptide has a stronger promoting effect, and Calanthe orchids peptide has anti-aging effects.

[0084] Performing in vitro efficacy experiments with the Huabaichi and orchid polypeptides prepared in Example 2 of the present invention can also achieve the beneficial effects described in the present invention.

[0085] Obviously, the above-mentioned embodiments of the present invention are merely examples for clearly explaining the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to enumerate all the implementation manners here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. An orchid polypeptide, characterized in that: The amino acid sequence of the polypeptide is SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.

6.

2. The method for preparing an orchid polypeptide according to claim 1, characterized in that The following steps are involved: 1) selecting orchid plants, inducing plant callus, and then placing them in a culture medium for cultivation to obtain orchid callus; 2) pre-freezing, freeze-drying, and crushing the orchid callus in sequence to obtain orchid callus powder; 3) mixing the orchid callus powder with a buffer solution containing NaCl, and performing freeze-thaw treatment to obtain an intermediate; 4) placing the intermediate at -5 to 5°C for 5 to 20 minutes, and then heating and centrifuging the intermediate to obtain a supernatant; 5) taking the supernatant after centrifugation, separating it with an ultrafiltration centrifuge tube, collecting the retained liquid and freeze-drying it to obtain orchid polypeptide.

3. The method for preparing an orchid polypeptide according to claim 2, characterized in that In step 1), the orchid plant is a Cymbidium orchidaceae plant, a Scutellaria baicalensis plant or a Bletilla striata plant.

4. The method for preparing an orchid polypeptide according to claim 3, characterized in that In step 2), the pre-freezing treatment temperature is -40 to -20°C, and the pre-freezing treatment time is 12 to 36 hours; preferably, the pre-freezing treatment temperature is -30 to -35°C, and the pre-freezing treatment time is 20 to 25 hours.

5. The method for preparing an orchid polypeptide according to claim 3, characterized in that In step 2), the temperature of the freeze-drying cold trap is -100 to -60°C, the temperature of the freeze-drying freezing chamber is -45°C to 35°C, the vacuum degree of freeze-drying is 0 to 10 Pa, and the freeze-drying time is 36 to 72 hours; preferably, the temperature of the freeze-drying cold trap is -90 to -70°C, the temperature of the freeze-drying freezing chamber is -20°C to 25°C, the vacuum degree of freeze-drying is 2 to 8 Pa, and the freeze-drying time is 45 to 60 hours; more preferably, the temperature of the freeze-drying cold trap is -80 to -75°C, the temperature of the freeze-drying freezing chamber is 0°C to 10°C, the vacuum degree of freeze-drying is 4 to 6 Pa, and the freeze-drying time is 50 to 55 hours.

6. The method for preparing an orchid polypeptide according to claim 3, characterized in that In step 3), the solid-liquid ratio of orchid callus powder and NaCl-containing buffer is 1:5-10, preferably 1:6-8; the buffer is PBS, and the final concentration of NaCl in the buffer is 100-200 mM, preferably 120-160 mM; liquid nitrogen is used for freezing in the freezing stage of the freeze-thaw treatment, the temperature used in the melting stage of the freeze-thaw treatment is 35-40°C, and the number of treatments of the freeze-thaw treatment is 2-4 times.

7. The method for preparing an orchid polypeptide according to claim 3, characterized in that In step 4), the intermediate is placed at 0-2°C for 10-15 min; the heating treatment is carried out in a boiling water bath for 5-15 min, preferably 8-10 min; the centrifugal treatment time is 10-30 min, preferably 15-20 min; the centrifugal treatment speed is 2000-6000 g, preferably 3000-5000 g; the centrifugal treatment temperature is 3-5°C.

8. The method for preparing an orchid polypeptide according to claim 3, characterized in that In step 5), the molecular weight cut-off of the ultrafiltration tube is 3KD.

9. Use of the orchid polypeptide as claimed in claim 1 in cosmetics, especially in cosmetics with anti-aging effects.

10. The orchid polypeptide as described in claim 1 promotes collagen, elastin, and barrier proteins, especially type XVII collagen, type VI collagen, type V collagen, type IV collagen, type III collagen, type I collagen, elastin, involucrin, fibrin, keratin, tight junction protein, SIRT2, and SIRT6.

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