Method for evaluating anti-light aging effectiveness of bioactive component

Abstract

The invention discloses a screening method for verifying photoaging resistance effectiveness of bioactive components. Comprising the following steps: by adopting a H2O2 damage model of HaCaT cells, detecting the cell migration rate of active ingredients, and / or detecting the expression level of senescence-related genes p21, p16 and p53, and / or detecting the expression level of senescence-related protein SIRT1; the photoaging resistance effectiveness of the active ingredients is further evaluated by detecting the expression and positioning of autophagy related proteins LC3 and p62, and compared with a traditional model and method, the photoaging resistance effectiveness of the active ingredients is more stable, accurate and efficient.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for evaluating the effectiveness of bioactive ingredients in resisting photoaging. Background Technology

[0002] As the largest organ in the human body, the skin is in direct contact with the external environment and is the first line of defense against external damage. Its aging characteristics are mainly degenerative changes in structure and function, manifested as wrinkles, dry skin, reduced barrier integrity and thinning of the epidermis, and it is mainly divided into natural aging and photoaging.

[0003] Ultraviolet radiation is a major cause of photoaging in the skin. Its mechanism involves both direct DNA damage and oxidative stress. When damage exceeds the cell's self-repair threshold, the p53 / p21 signaling pathway is activated, triggering cell cycle arrest until repair is complete. If damage continues to accumulate, it may induce mitochondrial-dependent apoptosis, accompanied by an NF-κB-mediated inflammatory response, forming a persistent inflammatory microenvironment. SIRT1, as a NAD+ receptor antagonist... + SIRT1-dependent deacetylases play a central role in maintaining cellular homeostasis. They can dynamically regulate antioxidant stress responses, repair DNA damage, and modulate autophagy. Their activity is significantly correlated with the progression and severity of skin aging. Furthermore, UV-induced photoaging is closely related to the imbalance of the SIRT1-autophagy axis. SIRT1 can rescue apoptosis by deacetyling target proteins such as p53 and FOXO3a. Regarding autophagy as a defense mechanism, SIRT1 can form autophagosomes to clear damaged organelles and misfolded proteins, helping damaged cells re-enter the cell cycle. However, UV radiation may cause excessive cell damage, leading to impaired autophagic flux, abnormal accumulation of autophagy-related proteins such as LC3-II, and excessive consumption of the autophagy substrate p62. This ultimately exacerbates cellular homeostasis imbalance, hindering self-repair and leading to aging or apoptosis. Studies have reported a vicious cycle between autophagy imbalance and downregulation of SIRT1 expression levels; excessive autophagy can accelerate SIRT1 degradation and exacerbate skin damage.

[0004] Polydeoxyribonucleotide (PDRN) is a linear DNA fragment extracted from male salmon sperm cells, with a molecular weight ranging from 50 to 1500 kDa. Due to its 98% base sequence similarity to human DNA, it exhibits excellent biocompatibility and low immunogenicity. PDRN and its derivatives have been successfully applied in various tissue repair fields, and studies have shown that these substances can alleviate abnormal skin texture and excessive melanin deposition caused by ultraviolet radiation exposure. These biological effects make it an important functional component for implantable medical aesthetic products. However, research on its mechanisms and effectiveness in alleviating photoaging is scarce, which hinders its further development in the field of anti-photoaging. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a screening method for verifying the effectiveness of bioactive ingredients in resisting photoaging.

[0006] According to an embodiment of the present invention, the method specifically includes the following steps:

[0007] (1) Seed HaCaT cells into a culture dish and culture them in cell culture medium until HaCaT cells adhere and grow.

[0008] (2) Add a complete culture medium mixture containing H2O2 to the culture vessel and culture.

[0009] (3) Add bioactive ingredients to the culture vessel.

[0010] According to an embodiment of the present invention, in step (1), the cell culture medium is MEM-EBSS complete medium containing 10% (volume fraction) FBS (fetal bovine serum); the culture conditions are: cells are cultured at 37°C with 5% CO2 and saturated humidity.

[0011] According to an embodiment of the present invention, in step (2), the preparation method of the complete culture medium mixture containing H2O2 is as follows: dilute a 20-40% H2O2 aqueous solution with water to a 200-300 mM H2O2 aqueous solution, and then mix the diluted H2O2 with the complete culture medium at a mass ratio of 1:800-1200; in some embodiments, the preparation method further includes sterilization using a 0.22 μm filter membrane. In some embodiments, the concentration of the water-diluted H2O2 aqueous solution can be selected from 200, 250, or 300 mM; in some embodiments, the mass ratio of the water-diluted H2O2 aqueous solution to the complete culture medium is 1:800, 1:900, 1:1000, 1:1100, or 1:1200.

[0012] According to an embodiment of the present invention, the preparation method of the mixed culture medium solution containing H2O2 is as follows: first, dilute 30% H2O2 aqueous solution with sterile water for injection to 250mM H2O2 aqueous solution, then mix 250mM H2O2 with the complete culture medium at a mass ratio of 1:1000 and filter through a 0.22μm filter membrane for sterilization.

[0013] According to an embodiment of the present invention, the final concentration of H2O2 in the complete culture medium mixture containing H2O2 is 200-300 μM, preferably 250 μM.

[0014] According to an embodiment of the present invention, the concentration of the bioactive ingredient is 500-1200 μg / mL, preferably 600-1000 μg / mL, for example 600, 700, 800, 900, or 1000 μg / mL.

[0015] According to an embodiment of the present invention, the screening method includes step (4): testing the cell migration rate of bioactive components, and / or detecting the expression levels of aging-related genes p21, p16, and p53, and / or detecting the expression level of aging-related protein SIRT1; and / or detecting the expression and localization of autophagy-related proteins LC3 and p62.

[0016] According to an embodiment of the present invention, in step (1), the HaCaT cell seeding concentration is 0.5 × 10⁻⁶. 6 cells / mL to 1.5 × 10⁻⁶ 6 Cells / mL, preferably 0.8 × 10⁻⁶. 6 cells / mL to 1.2 × 10⁻⁶ 6 cells / mL, for example, 1×10 6 per mL.

[0017] According to an embodiment of the present invention, the culture vessel is a six-well plate.

[0018] According to an embodiment of the present invention, the HaCaT cells are added to the complete culture medium mixture containing H2O2 after they adhere to the culture vessel to cover 70-80% of the bottom area of ​​the culture vessel.

[0019] According to an embodiment of the present invention, in step (2), the culture time is 16-48 hours, for example, 16, 24, 32, or 48 hours; according to an embodiment of the present invention, the HaCaT cells are human cells derived from human epidermis and are human immortalized keratinocytes.

[0020] According to an embodiment of the present invention, the method includes the following steps: seeding HaCaT cells into a six-well plate and culturing them until the HaCaT cells adhere and grow, then adding a complete culture medium mixture containing H2O2 with a final concentration of 250 μM H2O2 to the six-well plate, and continuing to culture for 24 hours.

[0021] According to embodiments of the present invention, the bioactive ingredient is selected from natural products or nucleic acid raw materials with photoaging potential; in some embodiments, the bioactive ingredient is selected from substances that regulate SIRT1 expression levels; in some embodiments, the bioactive ingredient is selected from PDRN and its derivatives; in some embodiments, the PDRN and its derivatives include a series of nucleic acid raw materials such as PDRN, PN, and DNA zinc.

[0022] PDRN, short for polydeoxyribonucleotide, is the sodium salt of DNA. PN is a product of secondary refining of PDRN. To adapt to different application scenarios, various modification methods and derivatives have emerged on the market. For example, PDRN modified with polyethylene glycol (PEG) can increase its molecular weight to over 2000 kDa, forming a sustained-release system suitable for scenarios requiring long-term effects. PDRN modified with thiophosphate can enhance its nuclease resistance and prolong its half-life, making it more suitable for treating chronic wound healing.

[0023] Beneficial effects

[0024] The inventors discovered that, compared to HDF cells, HaCaT cells are more suitable for evaluating the effects of bioactive components on anti-photoaging (e.g., promoting cell migration) and for verifying the anti-photoaging effects of active components related to the SIRT1-autophagy axis pathway / mechanism. Furthermore, compared to traditional UVB irradiation-induced cell senescence models, the model used in this invention overcomes the uncertainty inherent in in vitro cell senescence models due to the complexity of UVB irradiation doses, providing a more stable and efficient method for verifying the anti-photoaging effectiveness of active components and screening for drugs with anti-photoaging effects. Attached Figure Description

[0025] Figure 1 This illustrates the cell migration of UVB / H2O2-damaged HaCaT cells after 12 h and 24 h of PDRN treatment.

[0026] Figure 2 This illustrates the cell migration of PDRN-treated UVB / H2O2-damaged HDF cells after 12 h and 24 h.

[0027] Figure 3The expression levels of Sirt1, p53, p21, p16, and MMP1 on mRNA after HaCaT was treated with 250 μM H2O2 for 24 hours following classical photoaging-induced UVB irradiation: (A) Statistical results of SIRT1 gene expression; (B) Statistical results of p53 gene expression; (C) Statistical results of p21 gene expression; (D) Statistical results of p16 gene expression; (E) Statistical results of MMP1 gene expression.

[0028] Figure 4 The diagram shows that after HaCaT cells were treated with UVB irradiation and 250 μM H2O2 for 24 hours following classical photoaging, the protein expression of SIRT1 and p62 in the H2O2-damaged group was significantly lower than that in the control group, while the levels of p53 and LC3II / I were higher. The above trends were reversed after PDRN intervention, but the sensitivity of the UVB-treated group to cell-related regulatory proteins was not as high as that in the H2O2-damaged group.

[0029] Figure 5 This illustrates that after HaCaT cells were treated with UVB irradiation and 250 μM H2O2 for 24 hours following classical photoaging-induced autophagy, the number of autophagy sites in the nucleus increased, while the number of autophagy sites decreased significantly after PDRN intervention. Detailed Implementation

[0030] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0031] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0032] Experimental materials and reagents

[0033] HaCaT cells were purchased from the Center for Biological Resources Conservation and Research, Chinese Academy of Sciences (BCRC, GCA, China) and cultured in MEM-EBSS (Gibco) medium containing 10% fetal bovine serum (FBS) (HyClone). HDF cells were purchased from Boxi Biotechnology and cultured in low-glucose DMEM (Cytiva) medium containing 10% fetal bovine serum (FBS). The antibodies used in this study included: anti-GAPDH (TA-08, ZSGB-BIO), anti-SIRT1 (8469S, Cellsignaling), anti-LC3 (L7543, Sigma-Aldrich), and anti-p62 (PM045, MBL). Anti-rabbit and anti-mouse secondary antibodies were purchased from Zhongshan Jinqiao.

[0034] Example 1. Screening study of in vitro experimental cell lines

[0035] Cell migration ability detection

[0036] HaCaT cells in the logarithmic growth phase were digested with trypsin and then prepared into a 1×10⁻⁶ concentration using complete culture medium. 6 Cells were cultured at a concentration of 2 mL / mL and seeded into 6-well plates. After cross-mixing, the cells were incubated in a cell culture incubator for 24 h. Once the cells adhered, a sterilized ruler was placed above the wells of the plate, and a vertical line was drawn between the well and the ruler using a 1 mL pipette. The supernatant was completely discarded, and the cells were gently rinsed twice with PBS. Then, the cells were treated with UVB or H2O2. Target culture medium with a serum concentration of 2% (800 μg / mL PDRN was dissolved in the treatment group) was added to each group. The cells were divided into six groups: control group, PDRN group (without UVB and H2O2 treatment), UVB group, UVB+PDRN group, H2O2 group, and H2O2+PDRN group. Cell images were taken under a microscope at 0 h, 12 h, and 24 h, and cell migration rate was calculated at different time points.

[0037] The control group received no UVB or H2O2 treatment and no PDRN intervention; the PDRN group received no UVB or H2O2 treatment but received PDRN intervention; and the UVB group received UVB irradiation treatment (HaCaT cell line irradiation energy was 300 mJ / cm²). 2 The irradiation dose of the HDF cell line was 1800 mJ / cm². 2 The following groups were treated with different treatments: UVB irradiation and PDRN intervention (HaCaT cell line treated at 800 μg / mL, HDF cell line treated at 1000 μg / mL); H2O2 group treated with hydrogen peroxide, i.e., cells were treated with a mixture of 250 μM H2O2 and complete culture medium (the H2O2-containing mixed culture medium was prepared as follows: first, 30% H2O2 aqueous solution was diluted with sterile water for injection to 250 mM H2O2 aqueous solution, then 250 mM H2O2 was mixed with the complete culture medium at a mass ratio of 1:1000 and filtered through a 0.22 μm filter for sterilization. The treatment refers to adding a final concentration of 250 μH2O2 and continuing to culture for 24 hours); H2O2+PDRN group treated with hydrogen peroxide and drug intervention, i.e., cells were treated with a mixture of 250 μM H2O2 and complete culture medium (HaCaT cell line treated at 800 μg / mL, HDF cell line treated at 1000 μg / mL); H2O2 group treated with hydrogen peroxide and drug intervention, i.e., cells were treated with a mixture of 250 μM H2O2 and complete culture medium (HDF cell line treated at 1000 μg / mL, HDF cell line treated at 1000 μg / mL, HDF cell line treated at 1000 μM H2O2 and ... The cells were treated with a complete culture medium mixture containing H2O2 and PDRN (HaCaT cell line concentration: 800 μg / mL; HDF cell line concentration: 1000 μg / mL).

[0038] The steps for HDF cell migration detection are the same as above. The culture medium used is low-glucose DMEM medium, and the concentration of PDRN used is 1000 μg / mL.

[0039] Experimental Results and Analysis: Figure 1 and 2 The results of cell migration assays for HaCaT and HDF cell lines are shown. The results indicate that PDRN promotes migration in both healthy and damaged HaCaT cells, but its effect is more pronounced in UVB / H2O2-damaged cells. PDRN promotes migration in damaged HDF cells, but does not promote migration in healthy HDF cells, and shows a tendency to inhibit cell migration at the 24-hour time point.

[0040] Promoting cell migration has great application value in the fields of skin regeneration and tissue repair. The above experiment, by comparing the biological response characteristics of HaCaT and HDF cells in a photoaging model, shows that PDRN exhibits a more significant repair sensitivity to HaCaT in a photodamage model.

[0041] Example 2: Study on different HaCaT cell senescence models

[0042] 2.1 Cell Culture: HaCaT cells were cultured in MEM-EBSS complete medium containing 10% (v / v) FBS (fetal bovine serum) at 37°C with 5% CO2 and saturated humidity. Cells in the logarithmic growth phase were digested with trypsin containing 0.25% EDTA and incubated at 37°C for 6-8 min. Once the cell margins were confirmed to have retracted under a microscope, three times the volume of complete medium was added to terminate the digestion. After digestion, the cells were gently pipetted to the inner wall of the culture flask to promote complete cell detachment. The cells were centrifuged at 1000 rpm for 5 min at room temperature, the supernatant was discarded, and the cells were resuspended in MEM-EBSS complete medium to achieve a cell concentration of 1×10⁶ cells / mL. 6 Prepare a cell suspension of 2 mL / well and seed it into a six-well plate. Incubate the plate at 37°C, 5% CO2 and saturated humidity for 24 hours until the cells adhere and grow to a density of about 80% covering the bottom of the culture dish. Then discard the old culture medium.

[0043] 2.2 Experimental Grouping: Based on the cell culture samples from each well, the samples were randomly divided into a control group, a UVB irradiation group, and an H2O2 damage group for the experiments. In the H2O2 damage group, a complete culture medium mixture containing 250 μM H2O2 was added for treatment (the preparation method of the H2O2-containing mixed culture medium solution is as follows: first, dilute a 30% H2O2 aqueous solution with sterile water for injection to a 250 mM H2O2 aqueous solution, then mix the 250 mM H2O2 with the complete culture medium at a mass ratio of 1:1000, and then filter through a 0.22 μm filter membrane for sterilization. The treatment refers to adding a final concentration of 250 μH2O2 and continuing to incubate for 24 hours). Subsequent experiments were then conducted. The control group received no treatment. The UVB irradiation group was treated as follows:

[0044] The petri dish was irradiated under a UVB lamp (λ = 313 nm) with an irradiation dose of 300 mJ / cm². 2 (Cells are approximately 10cm from the lamp, irradiation time is approximately 5 minutes). Because UVB lamps experience functional degradation over time, they must be preheated for 10 minutes before use until the irradiation value stabilizes. Use a radiometer to confirm that the measured value at the irradiated area is consistently 1mW / cm². 2 Only then can experiments be carried out. After irradiation, discard the PBS, add MEM-EBSS complete medium containing the target concentration of 800 μg / mL PDRN, and continue culturing in a cell culture incubator at 37°C with 5% CO2 for 24 hours before proceeding with subsequent experiments.

[0045] (II) Indicator Testing Methods

[0046] 1) Quantitative Real-Time PCR

[0047] Quantitative real-time PCR was used to detect the expression levels of the target gene among different groups. The specific procedure was as follows: After cell treatment, 1 mL of pre-chilled TRIzol lysis buffer was added to each well of a 6-well plate. After complete lysis at room temperature for 5 minutes, the cells were transferred to new sterile, enzyme-free EP tubes. 200 μL of chloroform was added to each tube, and the mixture was manually inverted until the solution turned pinkish-white. The mixture was incubated at room temperature for 5 minutes to promote layering. Centrifugation was performed at 12000 rpm at 4°C for 15 minutes. 500 μL of the upper aqueous phase was transferred to a new EP tube, and an equal volume of pre-chilled isopropanol was added. The mixture was gently inverted for 30 seconds and incubated at room temperature for 10 minutes to allow RNA to aggregate into a flocculent precipitate. The precipitate was then centrifuged again at 12000 rpm at 4°C for 10 minutes. A white or transparent precipitate was visible at the bottom of the tube. The precipitate was washed with pre-chilled 75% ethanol prepared with DEPC water and air-dried. After the precipitate was fully dried, RNase-free H2O was added to dissolve it, yielding the extracted RNA. After the extracted RNA was transcribed into cDNA using a reverse transcription kit, the expression levels of Sirt1, p53, p21, p16, and MMP1 on mRNA were detected by real-time quantitative PCR. The expression levels were calculated using the relative quantification 2-ΔΔCt method and normalized to the GAPDH amplification value as the baseline.

[0048] 2) Western Blot

[0049] Cells were treated with different methods, and the relative expression levels of relevant proteins were determined by Western blotting. The specific steps were as follows: Cells were washed twice with PBS, then lysed with RIPA buffer (containing 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS), and a mixture of 1 mM EDTA and protease inhibitor (Roche 1183617001) was added. After lysis on ice for 30 minutes, the cells were centrifuged at 15000g and 4°C for 15 minutes. The supernatant was quantified, and bromophenol blue solution was added. The mixture was heated to 100°C and boiled for 15 minutes. The samples were separated using 4% to 12% PAGE Bis-Tris gels and then transferred to PVDF membranes. After blocking with 5% milk blocking buffer for 1 hour, the cells were incubated overnight at 4°C with SIRT1 antibody. The membrane was washed three times with TBS-T (0.05% Tween) and then incubated with a secondary antibody diluted 1:1000. The membrane was visualized using a Tanon-5200 chemiluminescence system.

[0050] 3) Immunofluorescence detection

[0051] Cells were treated with different methods, and the expression of the target protein was observed using immunofluorescence staining. The specific procedure was as follows: Cells were fixed in PBS with 4% paraformaldehyde for 10 minutes, infiltrated in PBS with 0.2% Triton X-100 for 5 minutes, and then blocked with 3% BSA for 1 hour. Subsequently, the cell samples were co-incubated overnight at 4°C with primary antibodies against the specific antigen in blocking buffer. The samples were washed three times with PBS and then incubated at room temperature for one hour with specific Alexa Fluor 488 / 555 / 647 secondary antibodies (Zhongshan Jinqiao). Subsequently, slides were mounted using mounting medium containing DAPI. Images were taken using an Olympus confocal microscope equipped with a 60×Oil objective lens.

[0052] Experimental results: Figure 4 The results showed that the expression of related proteins in HaCaT cells after UVB irradiation and 250 μM H2O2 treatment for 24 h induced by classic photoaging were as follows: The results showed that the expression of SIRT1 and p62 proteins in the H2O2-damaged group was significantly lower than that in the control group, while the levels of p53 and LC3II / I were increased. This indicates that autophagy was overactivated and the senescence and apoptosis rates were increased. The above trends were reversed after PDRN intervention, but the sensitivity of cell-related regulatory proteins in the UVB group was not as high as that in the H2O2-damaged group.

[0053] Conclusion Analysis: Based on the above experimental results ( Figure 3 , Figure 4 , Figure 5 The H2O2 damage model constructed for HaCaT in this invention is more helpful in evaluating the effectiveness of PDRN in resisting photoaging.

[0054] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A screening method for verifying the anti-photoaging effectiveness of bioactive ingredients, characterized in that, The method specifically includes the following steps: (1) Seed HaCaT cells into a culture dish and culture them in cell culture medium until HaCaT cells adhere and grow. (2) Add a complete culture medium mixture containing H2O2 to the culture vessel and culture. (3) Add bioactive ingredients to the culture vessel; Preferably, the bioactive ingredient is selected from natural products or nucleic acid raw materials with photoaging potential; preferably, the bioactive ingredient is selected from substances that regulate SIRT1 expression levels; preferably, the concentration of the bioactive ingredient is 500-1200 μg / mL, more preferably 600-1000 μg / mL, for example 600, 700, 800, 900, 1000 μg / mL.

2. The method according to claim 1, characterized in that, The bioactive ingredient is selected from PDRN and its derivatives; preferably, the PDRN and its derivatives include nucleic acid raw materials such as PDRN, PN, and DNA zinc.

3. The method according to claim 1, characterized in that, The screening method includes step (4): testing the cell migration rate of bioactive components, and / or detecting the expression levels of aging-related genes p21, p16, and p53, and / or detecting the expression level of aging-related protein SIRT1; and / or detecting the expression and localization of autophagy-related proteins LC3 and p62.

4. The method according to any one of claims 1-3, characterized in that, In step (1), the cell culture medium is MEM-EBSS complete medium containing 10% (volume fraction) FBS (fetal bovine serum); the culture conditions are: cells are cultured at 37°C with 5% CO2 and saturated humidity.

5. The method according to any one of claims 1-3, characterized in that, In step (2), the preparation method of the complete culture medium mixture containing H2O2 is as follows: dilute 20-40% H2O2 aqueous solution with water to 200-300mM H2O2 aqueous solution, and then mix the diluted H2O2 with the complete culture medium at a mass ratio of 1:800-1200.

6. The method according to any one of claims 1-3, characterized in that, Step (2) further includes sterilization using a 0.22 μm filter membrane; preferably, the concentration of the H2O2 aqueous solution diluted with water is selected from 200, 250, or 300 mM; preferably, the mass ratio of the H2O2 aqueous solution diluted with water to the complete culture medium is 1:800, 1:900, 1:1000, 1:1100, or 1:1200.

7. The method according to any one of claims 1-3, characterized in that, The preparation method of the mixed culture medium containing H2O2 is as follows: First, dilute the 30% H2O2 aqueous solution with sterile water for injection to a 250mM H2O2 aqueous solution, then mix the 250mM H2O2 with the complete culture medium at a mass ratio of 1:1000 and filter through a 0.22μm filter membrane for sterilization.

8. The method according to any one of claims 1-3, characterized in that, The HaCaT cell seeding concentration was 0.5 × 10⁻⁶. 6 cells / mL to 1.5 × 10⁻⁶ 6 Cells / mL, preferably 0.8 × 10⁻⁶. 6 cells / mL to 1.2 × 10⁻⁶ 6 cells / mL, for example, 1×10 6 per mL.

9. The method according to any one of claims 1-3, characterized in that, After the HaCaT cells adhere to the culture dish to cover 70-80% of the bottom area, the complete culture medium mixture containing H2O2 is added.

10. The method according to any one of claims 1-3, characterized in that, In step (2), the culture time is 16-48 hours.

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