A plant lactobacillus plantarum ccfm1351 postbiotic for preventing hair loss and protecting hair based on multi-target
The metabiotic prepared by Lactobacillus plantarum CCFM1351 regulates the gene and protein expression of dermal papilla cells in hair follicles, solving the problem of side effects of existing hair loss drugs and achieving safe and effective hair loss prevention and hair care.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIANGNAN UNIV
- Filing Date
- 2023-11-22
- Publication Date
- 2026-05-29
AI Technical Summary
Existing hair loss treatments such as minoxidil and finasteride have side effects, and there is a need to find a safe and effective natural substance that can regulate multiple targets to achieve the effect of preventing hair loss and protecting hair.
The metabiotic prepared using *Lactobacillus plantarum* CCFM1351 promotes hair follicle growth by regulating the expression of genes and proteins related to dermal papilla cells, including inhibiting BAX expression, promoting VEGF and IGF-1 expression, inhibiting TGF-β2 and DKK-1 expression, promoting Cyclin D1 expression, and enhancing the cell migration effect of *Platycladus orientalis* leaves.
The metabiotic prepared from Lactobacillus plantarum CCFM1351 can significantly promote hair follicle growth, increase hair growth rate, regulate multiple targets to achieve anti-hair loss and hair care effects, and has no obvious side effects.
Smart Images
Figure CN117625455B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a postbiotic prepared from a plant lactobacillus CCFM1351 that achieves anti-hair loss and hair care effects based on multiple targets, belonging to the fields of microbial technology and pharmaceutical technology. Background Technology
[0002] Hair plays various roles in the human body, including protecting the head and maintaining its temperature. Hair loss is defined as a decrease in hair density, and recently, multiple factors, including genetics, stress, diet, and nutritional imbalances, have been linked to it. While hair loss is not life-threatening, it can affect an individual's aesthetics, social activities, and quality of life.
[0003] Hair formation follows a regular cycle. The hair growth cycle consists of three phases: anagen (growth phase), catagen (transitional phase), and telogen (resting phase). The dermal papilla releases cytokines, causing the hair follicle to enter the anagen phase. After the anagen phase, the hair follicle enters the catagen phase, where metabolic activity within the hair follicle cells decreases significantly. After several weeks, the hair follicle enters the telogen phase. Thus, a single hair follicle completes a full cycle. Therefore, research on hair formation and loss should also consider factors related to hair growth and the hair cycle. Hair follicles are composed of dermal cells (DPCs) and epithelial cells. DPCs play a crucial role in follicle proliferation and differentiation, as well as controlling each phase of the hair cycle. Furthermore, DPCs secrete various substances to promote and maintain hair follicle growth and development, including insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), apoptosis-related proteins Bcl2, and BAX.
[0004] To date, non-surgical treatments for hair loss approved by the U.S. Food and Drug Administration (FDA) include minoxidil and finasteride. Minoxidil is converted into minoxidil sulfate by sulfonyltransferases present in the scalp, thereby promoting hair follicle cell growth and reducing hair loss. Finasteride is a 5α-reductase inhibitor that blocks the conversion of testosterone to dihydrotestosterone (DHT), thus causing androgenetic alopecia. However, these drugs have side effects for patients. Therefore, it is necessary to identify new, safe, and effective substances (derived from natural materials) to treat hair loss. Summary of the Invention
[0005] The technical problem to be solved by this invention is to provide a plant-derived Lactiplantibacillus plantarum that can regulate multiple targets to achieve anti-hair loss and hair care effects.
[0006] This invention provides a strain of Lactiplantibacillus plantarum CCFM1351, which was deposited on October 25, 2023, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 63921), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0007] The Lactiplantibacillus plantarum CCFM1351 was isolated from the feces of healthy human beings and has the following characteristics:
[0008] The colonies of Lactiplantibacillus plantarum CCFM1351, after being inoculated on MRS solid medium and cultured for 48 hours, are generally milky white, smooth, raised, and round with a diameter of 0.5-2 mm.
[0009] The Lactiplantibacillus plantarum CCFM1351 is a Gram-positive, facultative anaerobic bacterium that thrives in warm temperatures, with an optimal growth temperature of 35–40°C and an optimal growth pH of 6.0–7.0.
[0010] The present invention provides a composition containing *Lactobacillus plantarum* CCFM1351, or containing a metabiotic prepared from *Lactobacillus plantarum* CCFM1351.
[0011] In one embodiment, the metabiotic includes fermentation supernatant, cell lysate, and / or fermentation broth.
[0012] In one embodiment, the metabiotic is obtained by inoculating the above-mentioned *Lactobacillus plantarum* CCFM1351 into MRS medium, culturing the bacterial solution, and then subjecting it to heat treatment and lysis.
[0013] In one embodiment, the heat treatment is performed at 60–70°C for 25–35 minutes.
[0014] In one embodiment, the fermentation supernatant is the supernatant obtained by centrifuging the above-mentioned fermentation broth.
[0015] In one embodiment, the method for preparing the cell lysate is to homogenize the heat-treated fermentation broth under high pressure and centrifuge it to obtain the cell lysate.
[0016] In one embodiment, the post-generic can be dried into powder or used directly by various drying methods such as vacuum drying, spray drying, vacuum freeze drying, and fluidized bed drying.
[0017] This invention provides the application of the above-mentioned Lactiplantibacillus plantarum CCFM1351 in the preparation of hair care and anti-hair loss products.
[0018] In one embodiment, the application method includes, but is not limited to, topical application or oral administration.
[0019] This invention provides the use of the composition in the preparation of medicaments for the prevention and / or treatment of hair loss.
[0020] The present invention also provides food, health products, pharmaceuticals or cosmetics containing the aforementioned *Lactobacillus plantarum* CCFM1351 postbiotic.
[0021] In one embodiment, the food product includes the above-described composition and conventional excipients.
[0022] In one embodiment, the health product includes the above-described composition and conventional excipients.
[0023] In one embodiment, the pharmaceutical product comprises the above-described composition, a drug carrier, and / or pharmaceutical excipients.
[0024] In one embodiment, the pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, and release inhibitors.
[0025] In one embodiment, the cosmetic comprises the above-described composition, matrix ingredients, and / or conventional excipients.
[0026] In one embodiment, the matrix raw materials include oil-based raw materials, wax-based raw materials, synthetic oil-based raw materials, powder-based raw materials, gel-based raw materials, coagulants, and surfactants.
[0027] In one embodiment, the conventional excipients include one or more of the following: moisturizers, whitening agents, flavoring agents, adhesives, lubricants, preservatives, film-forming agents, antioxidants, emulsifiers, and cosmetic nutritional additives.
[0028] Beneficial effects:
[0029] This invention screened and obtained a strain of Lactiplantibacillus plantarum, CCFM1351. The metabiotic prepared from this Lactiplantibacillus plantarum CCFM1351 has the ability to regulate multiple targets to achieve anti-hair loss and hair care effects, specifically manifested in:
[0030] (1) Regulation of the expression of genes and proteins related to dermal papillary cells (HDPCs):
[0031] Inhibition of BAX expression decreased mRNA expression by 44.6%; promotion of VEGF expression increased secretion by 41.0%; inhibition of TGF-β2 expression decreased mRNA expression by 80.0%; inhibition of DKK-1 expression decreased secretion by 40.8%; promotion of Cyclin D1 expression increased expression by 1.43-fold; and promotion of IGF-1 expression increased expression by 9.12-fold.
[0032] (2) Promotes the migration of dermal papillary cells (HDPC) in hair follicles, increasing the migration rate by 2.13 times;
[0033] (3) Promotes hair growth in mice;
[0034] (4) Promotes the effect of Platycladus orientalis leaves on dermal papillary cells (HDPCs):
[0035] To enhance the effect of Platycladus orientalis leaf on cell migration, compared with the direct addition of Platycladus orientalis leaf extract, the fermentation supernatant after co-fermentation of Lactobacillus plantarum CCFM1351 and Platycladus orientalis leaf extract increased the migration rate of HDPC cells by 1.63 times.
[0036] The effect of Platycladus orientalis leaves on promoting cell gene expression was enhanced. Compared with the direct addition of Platycladus orientalis leaf extract, the fermentation supernatant after co-fermentation of Lactobacillus plantarum CCFM1351 and Platycladus orientalis leaf extract increased the expression of Cyclin D1 in HDPC cells by 2.70 times and the expression of IGF-1 by 17.54 times.
[0037] Therefore, the metabiotic prepared from Lactiplantibacillus plantarum CCFM1351 has great application potential in the preparation of anti-hair loss and hair care products.
[0038] Preservation of biological materials
[0039] A strain of Lactiplantibacillus plantarum, CCFM1351, taxonomically named Lactiplantibacillus plantarum, was deposited on October 25, 2023, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 63921), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description
[0040] Figure 1 Effects of the metagener CCFM1351 on HDPC cell proliferation;
[0041] Figure 2 Effects of the post-biotic CCFM1351 on the expression of TGF-β2 mRNA and BAX mRNA in HDPC cells;
[0042] Figure 3 Effects of the metagener CCFM1351 on VEGF and DKK-1 expression in HDPC cells;
[0043] Figure 4 Effects of the post-biotic CCFM1351 on mouse hair;
[0044] Figure 5 Effects of CCFM1351 arborvitae leaf fermentation supernatant on HDPC cell migration;
[0045] Figure 6 Effects of CCFM1351 arborvitae leaf fermentation supernatant on Cyclin D1 mRNA and IGF-1 mRNA expression in HDPC cells.
[0046] "*" indicates a statistically significant difference from the control group (P<0.05), "**" indicates a statistically significant difference from the control group (P<0.01), and "***" indicates an extremely statistically significant difference from the control group (P<0.001). Detailed Implementation
[0047] The present invention will be further described below with reference to specific embodiments.
[0048] The strain *Lactobacillus plantarum* FHNMY13M5 involved in the following examples was a strain screened by the laboratory of the Food Biotechnology Center of Jiangnan University.
[0049] The culture media involved in the following examples are as follows:
[0050] PBS buffer solution ( / L): Sodium chloride 8.0g, potassium chloride 0.2g, disodium hydrogen phosphate 1.44g, potassium dihydrogen phosphate 0.24g, adjust pH to 7.4. Autoclave at 115℃ for 20min.
[0051] MRS liquid culture medium ( / L): anhydrous glucose 20.0g, beef extract 10.0g, peptone 10.0g, yeast extract 5.0g, dipotassium hydrogen phosphate trihydrate 2.6g, anhydrous sodium acetate 2.0g, diammonium hydrogen citrate 2.0g, magnesium sulfate heptahydrate 0.5g, manganese sulfate monohydrate 0.25g, Tween 80 1.0mL. Autoclave at 115℃ for 20min.
[0052] For MRS solid medium, add 1.5% agar to the prepared liquid medium and autoclave at 115°C for 20 minutes.
[0053] Arborvitae leaf fermentation medium ( / L): 50g Arborvitae leaf extract, 10g yeast powder, adjust pH to 6.0-6.2. Autoclave at 115℃ for 20min.
[0054] Cell culture medium: 89% (v / v) DMEM medium, 10% fetal bovine serum, 1×10 5 U / L penicillin, 100mg / L streptomycin.
[0055] Example 1: Screening and identification of Lactobacillus plantarum
[0056] 1. Screening
[0057] The samples were derived from feces of healthy individuals. After pretreatment, the samples were stored in 30% glycerol at -80°C. After thawing, 0.2 mL of the sample was diluted in 1.8 mL of sterile physiological saline to obtain 10... -1 Dilute the solution, then take 0.5 mL of 10 -1 The diluent was added to 4.5 mL of physiological saline to obtain 10. -2 Diluents were prepared by repeating this process to obtain different concentrations of diluents. Suitable gradient diluents were then plated onto MRS solid medium and incubated at 37°C for 48 hours. Typical colonies of *Lactobacillus plantarum* were picked and streaked onto MRS solid medium for purification. Single colonies were then transferred to MRS liquid medium and incubated at 37°C for 18 hours. The culture was preserved with 30% glycerol to obtain *Lactobacillus plantarum* CCFM1351. Typical colonies of *Lactobacillus plantarum* are white, round, smooth, and raised.
[0058] 2. Identification
[0059] The genome of strain CCFM1351 was extracted, and the 16S rDNA of strain CCFM1351 was amplified and sequenced (performed by Suzhou Genewiz Biotechnology Co., Ltd.). The nucleotide sequence of the amplified 16S rDNA of CCFM1351 was compared with the nucleic acid sequence in NCBI. The results showed that the strain was Lactiplantibacillus plantarum and named Lactiplantibacillus plantarum CCFM1351.
[0060] Example 2: Effect of Lactobacillus plantarum CCFM1351 on HDPC cell proliferation
[0061] 1. Cell resuscitation and culture
[0062] First, retrieve the frozen human dermal papillary cells (HDPCs), rapidly thaw them in a 37°C water bath, then centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and use a solution containing 10% fetal bovine serum and 1×10⁻⁶... 5 Cells were cultured in DMEM high-glucose medium containing U / L penicillin and 100 mg / L streptomycin at 37°C in a cell culture incubator with 5% CO2. When the cells adhered and reached confluence of more than 80%, they were digested and passaged using trypsin containing 0.25% EDTA, and cells in the logarithmic growth phase were used for experiments.
[0063] 2. Preparation of post-biotics from *Lactobacillus plantarum* CCFM1351
[0064] All strains were preserved in 30% glycerol at -80°C before activation. A small amount of *Lactobacillus plantarum* CCFM1351 bacterial suspension was streaked onto MRS solid medium using a sterile inoculation loop and incubated at 37°C for 48 h. Single colonies were then picked and inoculated into MRS liquid medium and incubated at 37°C for 18 h. Finally, a 2% inoculation was carried out into 200 ml of MRS liquid medium, and the culture was continued at 37°C for another 18 h to obtain 2.5 × 10⁻⁶ cells / year. 9 CFU / ml bacterial culture.
[0065] The bacterial culture was heat-treated at 65℃ for 30 min, centrifuged (8000g, 4℃, 15 min), and the supernatant was collected and freeze-dried to obtain *Lactobacillus plantarum* CCFM1351 supernatant freeze-dried powder (CCFM1351-S) for later use. The resulting bacterial sludge was homogenized under high pressure (800–1200 MPa, 3 times) to obtain bacterial cell lysates, which were then freeze-dried to obtain *Lactobacillus plantarum* CCFM1351 bacterial cell lysate freeze-dried powder (CCFM1351-N) for later use. The preparation methods for *Lactobacillus plantarum* FHNMY13M5 supernatant freeze-dried powder (FHNMY13M5-S) and bacterial cell lysate freeze-dried powder (FHNMY13M5-N) were the same.
[0066] In cell experiments, the lyophilized supernatant and lyophilized cell lysate were resuspended separately using fresh culture medium. The concentration of the resuspended *Lactobacillus plantarum* CCFM1351 and *Lactobacillus plantarum* FHNMY13M5 supernatants was determined by fermentation to 1.3 × 10⁻⁶. 8 The concentration of postbiotics prepared from bacterial suspensions at CFU / ml was comparable; the concentration of *Lactobacillus plantarum* CCFM1351 and *Lactobacillus plantarum* FHNMY13M5 cell lysates after resuspension was comparable to that obtained after fermentation to 7.8 × 10⁻⁶. 7 The concentration of metabiotics prepared from bacterial cultures with CFU / ml is equivalent.
[0067] 3. Effects of metabiotics prepared from *Lactobacillus plantarum* CCFM1351 on HDPC cell proliferation
[0068] HDPC cells in the logarithmic growth phase were digested, counted, and the cell suspension was adjusted to 1×10⁶. 5 100 μl of the sample was seeded per well in a 96-well plate at a density of 1 / mL, with the outermost ring filled with PBS solution to prevent edge effects. The plates were then incubated in a 5% CO2 incubator at 37°C for 24 h. Blank, control, and treatment groups were set up.
[0069] Control group: Contains only cell culture medium and does not contain HDPC cells;
[0070] Control group: Contains both cell culture medium and HDPC cells, without the addition of post-biotics;
[0071] Yangshen group: contains both cell culture medium and HDPC cells, with 0.1μm minoxidil added;
[0072] Treatment group: Contains both cell culture medium and HDPC cells, with added metagenes; the amount of metagenes added follows the dilution instructions in step 2 above. The supernatants of *Lactobacillus plantarum* CCFM1351 and *Lactobacillus plantarum* FHNMY13M5 are named CCFM1351-S and FHNMY13M5-S, respectively; the cell lysates of *Lactobacillus plantarum* CCFM1351 and *Lactobacillus plantarum* FHNMY13M5 are named CCFM1351-N and FHNMY13M5-N, respectively.
[0073] Cells treated in each group were incubated overnight at 37°C. After incubation, the supernatant was discarded, and freshly prepared MTT working solution was added. Cells were then incubated at 37°C in the dark. After 4 hours, the supernatant was discarded, and 100 μl of dimethyl sulfoxide was added to each well. The cells were shaken to dissolve and crystallize for 5 minutes. The absorbance (OD value) was read at 490 nm using a microplate reader. The cell proliferation rate was calculated using the following formula: Cell proliferation rate (%) = (OD value of treatment group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.
[0074] Effects on cell proliferation, such as Figure 1 As shown, compared with the control group (cell proliferation rate of 100%), the cell proliferation rates of Minoxidil, Lactobacillus plantarum CCFM1351-S, CCFM1351-N, FHNMY13M5-S, and FHNMY13M5-N were 97%, 109%, 93%, 106%, and 101%, respectively, with no significant effect on HDPC cell proliferation. This concentration can be selected for subsequent cell experiments.
[0075] Example 3: *Lactobacillus plantarum* CCFM1351 promotes the expression of anti-hair loss and hair care-related genes and proteins in HDPC cells. 1. Effects of the post-biotic prepared from *Lactobacillus plantarum* CCFM1351 on the expression of TGF-β2 mRNA and BAX mRNA in HDPC cells.
[0076] The preparation methods and concentrations of *Lactobacillus plantarum* CCFM1351 and *Lactobacillus plantarum* FHNMY13M5 postbiotics are the same as in Example 2.
[0077] HDPC cells in the logarithmic growth phase were digested, counted, and the cell suspension was adjusted to 1.5 × 10⁻⁶. 6 Cells were seeded at a density of 2 mL / well in a 6-well plate and cultured overnight in a 5% CO2 incubator at 37°C until cell attachment. The old culture medium was discarded, and the cells were washed three times with PBS. The control and treatment groups were set up as in Example 2.
[0078] Add 2 ml of each of CCFM1351-S, CCFM1351-N, FHNMY13M5-S, and FHNMY13M5-N to a 6-well plate and incubate overnight at 37°C. Perform triple replicates for each sample. For detection, wash cells twice with pre-cooled PBS, add 1 ml of Trizol, and repeatedly pipette. Add 200 μl of chloroform, mix thoroughly, and incubate for 10 min. Centrifuge at 12000 rpm for 15 min at 4°C, collect the supernatant to a new enzyme-free centrifuge tube, add 0.8 times the volume of isopropanol, invert to mix, and incubate for 10 min. Centrifuge at 12000 rpm for 10 min at 4°C. Discard the supernatant, add 1 ml of 75% ethanol, and centrifuge at 7500 rpm for 5 min at 4°C. Repeat twice. Collect the white precipitate at the bottom of the tube; this is the sample RNA. After synthesizing cDNA using a reverse transcription kit, real-time quantitative polymerase chain reaction (RT-qPCR) was performed according to the instructions for SYBR Green fluorescent dye. The internal control was GAPDH, and the primers are shown in Table 1.
[0079] Table 1 Primer sequences
[0080]
[0081]
[0082] Depend on Figure 2 The results showed that, with the expression levels of TGF-β2 mRNA and BAX mRNA in the control group as 1, the relative expression levels of TGF-β2 mRNA and BAX mRNA in *Lactobacillus plantarum* CCFM1351-S were 0.94 and 1.30, respectively; the relative expression levels of TGF-β2 mRNA and BAX mRNA in CCFM1351-N were 0.20 and 0.55, respectively; while the relative expression levels of TGF-β2 mRNA and BAX mRNA in *Lactobacillus plantarum* FHNMY13M5-S were 0.64 and 0.67, respectively; and the relative expression levels of TGF-β2 mRNA and BAX mRNA in FHNMY13M5-N were 1.51 and 1.54, respectively. These results indicate that the bacterial lysate prepared from *Lactobacillus plantarum* CCFM1351 significantly inhibited the expression of TGF-β2 mRNA and BAX mRNA in HDPC cells.
[0083] TGF-β is involved in multiple signaling pathways during hair follicle development, including regulating epithelial and stromal components, and plays a crucial role in hair follicle development, cell differentiation, extracellular matrix formation, and angiogenesis. TGF-β can be divided into three subtypes: TGF-β1, TGF-β2, and TGF-β3. TGF-β2 promotes the transition of hair follicles to the catagen phase by inducing apoptosis, and plays a negative regulatory role in hair follicle growth and cyclical changes. Apoptosis and premature termination of hair follicle growth are key factors in hair loss. In the presence of apoptotic stimuli, the expression of the pro-apoptotic protein Bax increases, binding to the anti-apoptotic protein Bcl-2 to release Bax / Bak to avoid inhibition. Free Bax and Bak form oligomers, leading to the release of cytochrome C from the mitochondrial intermembrane space into the cytoplasm through the formation of a channel in the outer mitochondrial membrane. The released cytochrome C activates the caspase cascade to induce apoptosis.
[0084] Therefore, it can be seen that the metabiotic prepared by *Lactobacillus plantarum* CCFM1351 has the potential to prevent / improve hair loss by regulating the expression of TGF-β2 mRNA and BAX mRNA, inhibiting apoptosis, preventing hair follicles from transitioning to the regression phase.
[0085] 2. Effects of metabiotics prepared from *Lactobacillus plantarum* CCFM1351 on VEGF and DKK-1 expression in HDPC cells.
[0086] The preparation methods and concentrations of *Lactobacillus plantarum* CCFM1351 and *Lactobacillus plantarum* FHNMY13M5 postbiotics are the same as in Example 2.
[0087] HDPC cells in the logarithmic growth phase were digested, counted, and the cell suspension was adjusted to 1.5 × 10⁻⁶. 6 Cells were seeded at a density of 2 mL / well in a 6-well plate and cultured overnight in a 5% CO2 incubator at 37°C until cell attachment. The old culture medium was discarded, and the cells were washed three times with PBS. The control and treatment groups were set up as in Example 2.
[0088] Add 2 ml of each of CCFM1351-S, CCFM1351-N, FHNMY13M5-S, and FHNMY13M5-N to a 6-well plate and incubate overnight at 37°C. Perform triple replicates for each sample. For assay, determine the VEGF concentration in the culture supernatant according to the ELISA kit instructions.
[0089] The effects of metabiotics on VEGF and DKK-1 expression in HDPC cells are as follows: Figure 3 As shown, compared with the control group, the addition of *Lactobacillus plantarum* CCFM1351-N promoted the secretion of VEGF in HDPC cells (upregulated by 41.0%), and its promoting effect was higher than that of the *Gynostemma pentaphyllum* group (**P<0.01), with a secretion increase of 5.77%; the addition of *Lactobacillus plantarum* CCFM1351-S inhibited the secretion of DKK-1 in HDPC cells (downregulated by 40.8%).
[0090] Cultured HDPCs in vitro express VEGF at high levels. VEGF is an autocrine growth factor. VEGF mRNA is highly expressed in anagen-phase hair follicle cells but rarely in telogen-phase cells, and its expression and disappearance during the hair growth cycle are consistent with the formation and disappearance of blood vessels in the hair bulb. DKK-1 is a secreted protein and an important antagonist molecule in the Wnt signaling pathway, specifically inhibiting the classical Wnt signaling pathway and participating in embryonic development.
[0091] Therefore, the metabiotic prepared from *Lactobacillus plantarum* CCFM1351 has the potential to promote VEGF secretion, prolong the hair follicle growth phase, inhibit DKK-1 secretion, negatively regulate the Wnt signaling pathway, and thus prevent / improve hair loss.
[0092] Example 4: Lactobacillus plantarum CCFM1351 affects hair growth in mice
[0093] The preparation method of *Lactobacillus plantarum* postbiotic was the same as in Example 2, except that no lysis treatment was performed, resulting in a viable count of 2.5 × 10⁻⁶. 9 After obtaining the bacterial culture at CFU / ml, it was directly freeze-dried.
[0094] Extensive literature review has shown that C57BL / 6J mice possess melanocytes only in hair follicles, thus melanin synthesis coincides with the hair growth cycle. Therefore, monitoring the change in skin color from pink (hairless) to black (fully grown hair) is sufficient to easily characterize the hair growth cycle, and this method has been widely applied in hair loss research.
[0095] Twenty-four male C57BL / 6J mice, 6 weeks old (approximately 20g), were used for the experiment after a week of acclimatization. First, the backs of all mice were shaved using an animal hair removal device. Then, hair removal cream (purchased from the Veet official flagship store) was applied to remove all hair, covering an area of approximately 3cm x 3cm, causing the hair growth of the C57BL / 6J mice to enter the resting phase (resulting in pink skin), thus establishing the C57BL / 6J mouse alopecia model.
[0096] After 24 hours of skin stabilization, the mice in the telogen phase were randomly divided into 3 groups of 8 mice each, for a total of 24 mice. The grouping was configured as follows:
[0097] Control group: Using physiological saline as a control;
[0098] Yangshen group: Finasteride (10 mg / kg) was used as a control;
[0099] CCFM1351-D group: The mice were given a postbiotic of Lactobacillus plantarum CCFM1351 (lyophilized powder obtained by direct freeze-drying of fermentation broth and dissolved in fresh culture medium) at a dose of 500 mg / kg mouse body weight (based on the weight of lyophilized powder).
[0100] Each mouse was administered 0.2 ml / day by gavage for 3 weeks.
[0101] The skin samples from the backs of mice were recorded at 7 days, 14 days, and 21 days, and the results are as follows: Figure 4 As shown.
[0102] Depend on Figure 4 As can be seen, after 7 days of administration, the skin on the backs of mice in the control group remained pink, while the skin on the backs of mice in the CCFM1351-D group began to turn gray. After 14 days of administration, the skin on the backs of mice in the control group began to turn gray, while the black area on the backs of mice in the CCFM1351-D group significantly increased and hair shafts appeared. After 21 days of administration, compared with the control group, the skin on the backs of mice in the CCFM1351-D group was almost completely covered by hair. Therefore, oral administration of *Lactobacillus plantarum* CCFM1351 postbiotic can significantly promote hair growth in mice.
[0103] Example 5: Effect of Platycladus orientalis leaf fermentation supernatant prepared by Lactobacillus plantarum CCFM1351 on HDPC cell migration
[0104] All strains were preserved in 30% glycerol at -80°C before activation. A small amount of *Lactobacillus plantarum* CCFM1351 bacterial suspension was streaked onto MRS solid medium using a sterile inoculation loop and incubated at 37°C for 48 h. Single colonies were then picked and inoculated into MRS liquid medium and incubated at 37°C for 18 h. Finally, a 2% (v / v) inoculation was carried out into 200 ml of *Platycladus orientalis* leaf fermentation medium, and incubated at 37°C for another 18 h to obtain 9.5 × 10⁹ cells. 8 CFU / ml bacterial culture.
[0105] The bacterial culture was heat-treated at 65℃ for 30 min, centrifuged (8000g, 4℃, 15 min), and the supernatant was collected and freeze-dried to obtain *Lactobacillus plantarum* CCFM1351 *Platycladus orientalis* leaf fermentation supernatant freeze-dried powder (CCFM1351-CP) for later use. The preparation method for *Lactobacillus plantarum* FHNMY13M5 *Platycladus orientalis* leaf fermentation supernatant freeze-dried powder (FHNMY13M5-CP) was the same, and the concentration used was as follows. Logarithmic growth phase cells were digested and counted using HDPC, and the cell suspension was adjusted to 1.5 × 10⁻⁶. 6 Cells were seeded at a density of 2 mL / well in 6-well plates and cultured at 37°C with 5% CO2 until 90% confluence. Cells were then spread in a straight line using a 200 μL pipette tip and washed three times with PBS. Control and treatment groups were established using the same procedure.
[0106] Control group: Contains both cell culture medium and HDPC cells;
[0107] The Platycladus orientalis leaf fermentation medium group (CP) contains both cell culture medium and HDPC cells, with the cell culture medium resuspended in the Platycladus orientalis leaf fermentation medium (the Platycladus orientalis leaf fermentation medium is diluted tenfold, and the content of Platycladus orientalis leaf extract after dilution is 5mg / ml).
[0108] Treatment groups (CCFM1351-CP and FHNMY13M5-CP): The fermentation supernatant of Platycladus orientalis leaves was resuspended in cell culture medium (the resuspended fermentation supernatant of Platycladus orientalis leaves is equivalent to the fermentation supernatant obtained from fermentation medium containing 5 mg / ml Platycladus orientalis leaf extract).
[0109] Following the setup of the control and treatment groups described above, fresh culture medium was used. After incubation for 15 hours, the width of the scratches was photographed using an optical microscope as shown in the image. Figure 5 .
[0110] like Figure 5As shown, the cell migration rate was 32.63% in the control group, 42.63% in the CP group, 69.54% in the fermentation supernatant of Platycladus orientalis leaves prepared by *Lactobacillus plantarum* CCFM1351 (2.13 times that of the control group), and 23.55% in the fermentation supernatant of Platycladus orientalis leaves prepared by *Lactobacillus plantarum* FHNMY13M5. Compared with the direct addition of Platycladus orientalis leaf extract, the fermentation supernatant after co-fermentation of *Lactobacillus plantarum* CCFM1351 and Platycladus orientalis leaf extract increased the migration rate of HDPC cells by 1.63 times. Cell migration is crucial in hair follicle development, and the fermentation supernatant of Platycladus orientalis leaves prepared by *Lactobacillus plantarum* CCFM1351 significantly promoted the migration ability of HDPC cells and improved wound healing.
[0111] Example 6: Lactobacillus plantarum CCFM1351 promotes the expression of anti-hair loss and hair care related genes Cyclin D1 and IGF-1 in HDPC cells.
[0112] The preparation methods for the fermentation supernatant of *Lactobacillus plantarum* CCFM1351 and *Lactobacillus plantarum* FHNMY13M5 are the same as in Example 5.
[0113] HDPC cells in the logarithmic growth phase were digested, counted, and the cell suspension was adjusted to 1.5 × 10⁻⁶. 6 Cells were seeded at a density of 2 mL / well in a 6-well plate and cultured overnight in a 5% CO2 incubator at 37°C until cell attachment. The old culture medium was discarded, and the cells were washed three times with PBS. The control and treatment groups were set up as in Example 5.
[0114] 2 ml of CP, CCFM1351-CP, and FHNMY13M5-CP were added to 6-well plates and incubated overnight at 37°C, with three replicates per sample. For detection, cells were washed twice with pre-cooled PBS, and 1 ml of Trizol was added, followed by repeated aspiration and pipetting. 200 μl of chloroform was added, and the mixture was thoroughly mixed and allowed to stand for 10 min. The mixture was centrifuged at 12000 rpm for 15 min at 4°C, and the supernatant was collected to a new enzyme-free centrifuge tube. 0.8 volumes of isopropanol were added, and the mixture was inverted and allowed to stand for 10 min. The tube was then centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was discarded, and 1 ml of 75% ethanol was added. The tube was centrifuged at 7500 rpm for 5 min at 4°C, and this process was repeated twice. The white precipitate at the bottom of the tube was collected; this was the sample RNA. cDNA was synthesized using a reverse transcription kit, and real-time quantitative polymerase chain reaction (RT-qPCR) was performed according to the SYBR Green fluorescent dye instructions. GAPDH was used as the internal control. Primers are shown in Table 2.
[0115] Table 2 Primer sequences
[0116]
[0117] Depend on Figure 6 The results showed that, with the expression levels of Cyclin D1 and IGF-1 in the control group as 1, the relative expression levels of Cyclin D1 mRNA in CP, CCFM1351-CP, and FHNMY13M5-CP were 0.53, 1.43, and 1.11, respectively; and the relative expression levels of IGF-1 mRNA were 0.52, 9.12, and 2.39, respectively. These results indicate that the fermentation supernatant of Platycladus orientalis leaves prepared by *Lactobacillus plantarum* CCFM1351 significantly promoted the expression of Cyclin D1 mRNA and IGF-1 mRNA in HDPC cells. Compared with the direct addition of Platycladus orientalis leaf extract, the fermentation supernatant after co-fermentation of *Lactobacillus plantarum* CCFM1351 and Platycladus orientalis leaf extract increased the expression of Cyclin D1 in HDPC cells by 2.70-fold and the expression of IGF-1 by 17.54-fold.
[0118] The primary function of Cyclin D1 is to promote cell proliferation. Cyclin D1 binds to and activates CDK4, a cyclin-dependent kinase specific to the G1 phase. This phosphorylates the G1 phase cell cycle repressor protein (Rb), which dissociates from its bound E2F transcription factor. The E2F transcription factor then initiates transcription of genes involved in the living cell cycle, thus propelling the cell cycle from the G1 phase to the S phase. IGF-1 is a hemi-growth hormone-dependent polypeptide with multiple isoforms. IGF-1 produced within HDPC cells plays a crucial role in hair growth. Hair follicles contain abundant IGF-1-activated cells, which can improve scalp blood circulation and regulate the proliferation, migration, and differentiation of dermal and keratinocytes in the hair follicle. During the hair growth cycle, it promotes hair growth in the early anagen phase and delays the telogen and catagen phases.
[0119] Therefore, it can be seen that the fermentation supernatant of Platycladus orientalis leaves prepared by Lactobacillus plantarum CCFM1351 has the potential to affect cell proliferation and differentiation, regulate hair follicle growth cycle, and promote hair growth by promoting the expression of Cyclin D1 mRNA and IGF-1 mRNA.
[0120] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A strain of Lactiplantibacillus plantarum (CCFM1351), characterized in that, The *Lactobacillus plantarum* CCFM1351 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 63921, and the deposit date is October 25, 2023.
2. A microbial preparation containing the *Lactobacillus plantarum* CCFM1351 as described in claim 1.
3. The microbial preparation according to claim 2, characterized in that, The microbial preparation is a liquid or solid preparation containing *Lactobacillus plantarum* CCFM1351 as described in claim 1, wherein the viable count of *Lactobacillus plantarum* CCFM1351 in the microbial preparation is ≥10⁻⁶. 6 CFU / mL or ≥10 6 CFU / g.
4. A method for preparing the microbial preparation according to claim 2 or 3, characterized in that, The *Lactobacillus plantarum* CCFM1351 described in claim 1 was fermented in a culture medium.
5. A hair loss prevention and hair care medicine, characterized in that, The drug contains a live strain of *Lactobacillus plantarum* CCFM1351 as described in claim 1, lysates of *Lactobacillus plantarum* CCFM1351, or metabolites of *Lactobacillus plantarum* CCFM1351. The lysates of *Lactobacillus plantarum* CCFM1351 are obtained by inoculating *Lactobacillus plantarum* CCFM1351 into MRS medium, culturing the culture, subjecting it to heat treatment, homogenizing the heat-treated culture under high pressure, and centrifuging to obtain the lysates. The metabolites of *Lactobacillus plantarum* CCFM1351 are fermentation supernatants obtained by fermenting *Lactobacillus plantarum* CCFM1351 in *Platycladus orientalis* leaf fermentation medium, wherein the *Platycladus orientalis* leaf fermentation medium comprises 10-50 g / L of *Platycladus orientalis* leaf extract and 5-10 g / L of yeast powder.
6. The use of the *Lactobacillus plantarum* CCFM1351 as described in claim 1, or the microbial preparation as described in claim 2 or 3, in the preparation of anti-hair loss and hair care products.