Application of delta-junifolene in defending against cold and preventing freezing

Abstract

The invention discloses application of delta-junifolene in defending against cold and preventing freezing. According to the present invention, the delta-juniene has characteristics of strong skin frostbite resistance, high safety, aromatic odor and the like, and the research results show that the delta-juniene can down-regulate the cold sensitivity of mice under the transdermal drug delivery route, such that the preference temperature of the mice is generally reduced; compared with beta-caryophyllene, after a mouse is frostbitten, the skin structure of the mouse which is pre-smeared with the delta-juniene is clear and complete, the injury of frostbite is remarkably relieved, the wound healing rate can be increased by smearing 1%-10% of the delta-juniene on a frostbitten wound of the mouse, it is indicated that the delta-juniene has the effects of defending against cold, preventing freezing and promoting frostbite healing, and meanwhile, the effect is remarkable. The alpha, delta-juniper directly acts on the skin without obvious skin irritation, is high in safety in practical application, has obvious stratum corneum residence capability, can be widely applied to external preparations and skin care product formulas, and provides protection for people working in a cold environment.

Description

Technical Field

[0001] The present invention belongs to the field of life and medical sciences, and relates to the application of delta-cadinene in protecting against cold and freezing, and specifically relates to the application of delta-cadinene in protecting against cold and freezing through a transdermal external application route. Background Art

[0002] The skin is the largest organ in humans and animals, primarily protecting the body from toxins, microorganisms, and other potential harms. Humans have evolved mechanisms to cope with excessive heat, such as sweating, but are unable to cope with overwhelming cold. Frostbite can occur when the skin is exposed to cold temperatures. This condition most commonly affects exposed body parts, including the hands, feet, cheeks, ears, and nose. People exposed to low temperatures, especially high-altitude climbers, soldiers fighting in cold regions, and scientific researchers, are particularly susceptible to frostbite if they are unable to effectively protect themselves from the cold. Localized frostbite causes the skin to become pale, cold, numb, or numb, making it difficult to distinguish the depth of the injury. Frostbite is categorized as either non-freezing frostbite or freezing frostbite, depending on whether the tissue has frozen. Frostbite can be divided into four grades based on the severity of the injury after rewarming and thawing, ranging from superficial Grades I and II frostbite, which heals spontaneously, to severe, gangrenous frostbite, which can ultimately lead to functional impairment or disability. Therefore, if the anti-cold and anti-freeze ingredients can be applied externally and directly on the skin, it will greatly improve the army's mobile combat efficiency in cold areas, reduce the physical burden of ice and snow sports and polar operators, and bring convenience to people's work and life in cold seasons. Summary of the Invention

[0003] Purpose of the invention: In response to the current situation of insufficient cold-resistant and frostbite-preventing compounds, the present invention provides the use of δ-cadinene in cold-resistant and frostbite-preventing. The present invention is the first to propose a new use of δ-cadinene for cold-resistant and frostbite-preventing through transdermal external application, and the effect is significant in cold-resistant and frostbite-preventing.

[0004] Technical solution: In order to achieve the above-mentioned purpose, the δ-cadinene or natural substances containing δ-cadinene of the present invention are used in cold protection and frost prevention.

[0005] Application of the δ-cadinene or a natural substance containing δ-cadinene in the preparation of a cold-resistant and antifreeze agent or medicine.

[0006] The invention discloses an application of the delta-cadinene or a natural substance containing delta-cadinene in the preparation of a cold protection agent or a cold-proof skin care product for keeping out cold and freezing.

[0007] Preferably, the natural substance containing δ-juniperene is pink pepper, ylang ylang, cypress and the like.

[0008] The δ-cadinene is used in cold protection and frost prevention by significantly reducing the thermal preference temperature.

[0009] The δ-cadinene is used in cold protection and frostbite prevention by alleviating or improving the damage to skin structure caused by frostbite.

[0010] The δ-cadinene is used in cold protection and frost prevention by inhibiting TRPM8 to change thermal preference behavior.

[0011] Among them, the δ-cadinene is used for transdermal administration in protecting against cold and freezing.

[0012] The cold-resistant and antifreeze composition of the present invention comprises delta-cadinene as the sole cold-resistant and antifreeze component or in combination with other cold-resistant and antifreeze components, and also comprises raw materials, auxiliary materials or carriers required for the preparation.

[0013] The dosage form of the composition includes emulsion, microemulsion, gel, solution, tincture, film, ointment, cream or patch.

[0014] As a preference, other cold-resistant and antifreeze ingredients include urea, vitamin E, glycerin, etc.

[0015] The cold-resistant and antifreeze composition of the present invention is used in preparing a cold-resistant and antifreeze reagent or medicine.

[0016] The cold-proof and antifreeze composition of the present invention is used in preparing a cold-proof and antifreeze cold-protective agent or a cold-proof skin care product.

[0017] Preferably, the δ-cadinene is commercial δ-cadinene, such as commercial δ-cadinene with CAS NO of 483-76-1.

[0018] Furthermore, the δ-cadinene significantly reduces the thermal preference temperature under the transdermal administration route and is significantly better than β-caryophyllene.

[0019] Furthermore, the δ-cadinene can reduce the damage to the skin structure caused by frostbite after being pre-acted on the skin, and is significantly better than β-caryophyllene.

[0020] Furthermore, the δ-cadinene has a good intradermal retention effect and is significantly better than β-caryophyllene.

[0021] Furthermore, the δ-cadinene has a significant inhibitory effect on TRPM8, which is comparable to that of β-caryophyllene.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0023] The present invention provides a novel use of δ-cadinene for cold protection via transdermal topical application. Studies have shown that transdermal administration of δ-cadinene reduces cold sensitivity in mice and humans, demonstrating a trend toward lower preferred temperatures. Under the same modeling conditions, the δ-cadinene pre-applied group experienced minimal skin frostbite damage, with clearer and more intact skin structure. Direct application of δ-cadinene to the skin did not cause significant irritation, demonstrating high safety in practical applications. δ-cadinene can be used to protect against cold and provide protection for people working in cold weather. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The protective effect of δ-cadinene on frostbitten skin, (a): 1% δ-cadinene, (b): 1% β-caryophyllene, (c): blank solvent;

[0025] Figure 2 Comparison of the intradermal distribution of δ-cadinene and β-caryophyllene (n=6), (A): stratum corneum, (B) intradermal;

[0026] Figure 3 This is a diagram showing the inhibitory effects of β-caryophyllene and δ-cadinene on TRPM8 channels;

[0027] Figure 4 This is a graph analyzing the inhibitory effects of β-caryophyllene and δ-cadinene on TRPM8 channels. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with specific embodiments, which are only used to explain the present invention and are not to be construed as limiting the present invention. Those skilled in the art will appreciate that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and purpose of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

[0029] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified.

[0030] The δ-cadinene CAS NO: 483-76-1 and β-caryophyllene CAS NO: 87-44-5 in the embodiments of the present invention have a purity of more than 95%.

[0031] In the embodiments of the present invention, 0.1%, 1%, 5% and 10% δ-cadinene or β-caryophyllene represent concentrations of 1 mg / mL, 10 mg / mL, 50 mg / mL and 100 mg / mL, respectively.

[0032] Example 1

[0033] Effects of transdermal application of δ-cadinene on temperature preference in mice

[0034] The preferred temperature (PT) of 8-week-old C57BL6 / J mice weighing 20-25 g was measured using a TGR thermal preference tester (temperature range set at 5-40°C) before and after drug administration. Mice were randomly divided into experimental, blank, and positive control groups. The experimental groups were given varying concentrations of δ-cadinene (in isopropyl alcohol:propylene glycol (7:3, v / v) as a solvent), the blank control group was given isopropyl alcohol:propylene glycol (7:3, v / v) as a solvent, and the positive control group was given varying concentrations of β-caryophyllene (in isopropyl alcohol:propylene glycol (7:3, v / v) as a solvent). In a dark and quiet environment, mice were placed in the tester to adapt to the environment for 15 minutes, and then the drug administration operation was simulated. The mice were released after 2 minutes, and the PT value was calculated every 5 minutes after being released. The data within 30 minutes were recorded as the baseline value before drug administration. After each group of mice adapted for 15 minutes, the corresponding drug solution was applied (20 μL / mouse forepaw and 30 μL / mouse hind paw). The mice were released after 2 minutes, and the PT value was calculated every 5 minutes after being released. The data within 30 minutes were recorded as the post-dose value.

[0035] According to the formula △PT=PT 给药前 -PT 给药前后 The △PT change curve over time was drawn to evaluate the cold-resistant effect of drugs in different groups. If △PT>0, it means that after drug intervention, the preferred temperature of mice shifted to low temperature; if △PT<0, it means that after drug intervention, the preferred temperature of mice changed to high temperature.

[0036] The experimental results are shown in Table 1. Starting 5 minutes after administration, the preferred temperature of mice in both the 1% and 10% δ-cadinene groups shifted toward lower temperatures. This shift was most pronounced between 10 and 20 minutes in the δ-cadinene group. At 15 minutes, the 0.1%, 1%, and 10% δ-cadinene groups all showed significant differences from the blank solvent control group. This demonstrates that after application of δ-cadinene, the preferred temperature of mice shifted toward lower temperatures compared to before administration. This also suggests that δ-cadinene can reduce mice's sensitivity to cold and provide protection against cold. Furthermore, after 25 minutes, the ΔPT values of mice approached 0, indicating that the cold-protective effect of δ-cadinene is reversible and does not cause irreversible damage to the mice's sensory systems. The ΔPT values of mice in the blank group fluctuated around 0, indicating that 50% ethanol application had no cold-protective effect, eliminating the possibility that solvent evaporation may have influenced the animals' preferred temperature choices. Compared with the blank control group, the cold-resistant effect of the 1% β-caryophyllene group showed significant difference only at 15 minutes, and compared with the δ-cadinene group with the same concentration, the △PT was significantly reduced at 5-20 minutes, indicating that its cold-resistant effect was not as good as δ-cadinene.

[0037] Table 1 Effects of topical application of different concentrations of δ-cadinene and β-caryophyllene on the thermal preference temperature (△PT) of mice (n=5)

[0038]

[0039] Compared with the blank control group: *P<0.05, **P<0.01, ***P<0.001

[0040] Compared with the 1% β-caryophyllene group: #P<0.05, ##P<0.01.

[0041] Example 2

[0042] Effect of δ-cadinene on protecting rats from frostbite by topical application

[0043] 24 hours in advance, the back hair of male SD rats weighing (200 ± 20) g was shaved. After hair removal with a depilatory cream, the skin was cleaned with a cotton swab. A blank control group (isopropyl alcohol:propylene glycol (7:3, v / v)), a 1% δ-cadinene group (isopropyl alcohol:propylene glycol (7:3, v / v) as solvent), and a 1% β-caryophyllene group (isopropyl alcohol:propylene glycol (7:3, v / v) as solvent) were set up on the marked dosing area on the back. The dosing volume was 50 μL and the dosing area was 1.5 cm. 2 After 10 minutes of action, a metal rod (1 cm in diameter) immersed in liquid nitrogen for 20 minutes was tightly attached to the skin of the administration site for 4 seconds. The skin tissue of the administration site was obtained 72 hours after frostbite, and HE pathological sections were performed using 4% paraformaldehyde fixative.

[0044] Histopathological sections ( Figure 1 ) As can be seen, skin tissue sections treated with 1% δ-cadinene showed a clear, intact structure, encompassing both the stratum corneum and dermis, with a dense texture. In contrast, the boundaries between the epidermis and dermis were blurred in the 1% β-caryophyllene group and the blank control (solvent) group. Furthermore, the blank control group had a dermis filled with cracks and a loose structure, exhibiting the most severe frostbite. This suggests that pre-treatment with 1% δ-cadinene has a protective effect against frostbite.

[0045] Example 3

[0046] Effect of δ-cadinene on frostbite in rats through transdermal application

[0047] A blank control (isopropanol:propylene glycol (7:3, v / v)) group, a 0.1% δ-cadinene group, a 1% δ-cadinene group, and a 1% β-caryophyllene group (using isopropanol:propylene glycol (7:3, v / v) as the solvent) were set up. The frostbite model and drug administration methods were the same as in Example 2. Drug administration began 24 hours after frostbite and continued once daily for 5 days. The wound size of each group of animals was measured with a vernier caliper and photographed. The images were analyzed and the wound area was calculated using Image J software.

[0048] The wound healing rate was calculated according to the formula: Wound healing rate (%) = [1-(wound area on the nth day / wound area on the 1st day)] × 100%. The results are shown in Table 2. It can be seen that on the 3rd and 6th days after administration, the wound healing rates of the 1% and 10% δ-cadinene treatment groups were significantly better than those of the blank control group, while the 1% β-caryophyllene group did not show a significant wound healing rate. On the 6th day after administration, the 1% δ-cadinene treatment group was significantly better than the 1% β-caryophyllene group (P < 0.01).

[0049] Table 2 Effects of different concentrations of δ-cadinene and β-caryophyllene on the healing rate (%) of frostbite wounds in rats (n=5)

[0050]

[0051] Compared with the blank control group: *P<0.05, **P<0.01.

[0052] Example 4

[0053] Intradermal distribution and stratum corneum retention of δ-cadinene and β-caryophyllene

[0054] Male Sprague-Dawley rats weighing (200 ± 20) g were anesthetized, their abdominal hair shaved with a razor blade, washed with warm water, and housed overnight. 50 μL of a 5% solution of β-caryophyllene and δ-cadinene (isopropyl alcohol:propylene glycol, 7:3 v / v as solvent) and a solvent (isopropyl alcohol:propylene glycol, 7:3 v / v) were applied to the rat abdominal skin, covering an area of 3.14 cm. 2 . 90 minutes after administration, the administered skin area was carefully cleaned with a cotton swab dipped in pure water. The full-thickness skin of the rat was cut off 5 minutes, 30 minutes, and 120 minutes after cleaning the administered skin area, and the stratum corneum was separated from the living epidermis and dermis using the tape stripping method. A piece of tape was placed on the skin sample 20 times to remove the stratum corneum as a stratum corneum sample. The subcutaneous tissue of the administered skin was carefully removed as a skin sample. The stratum corneum sample and the skin sample were cut into pieces and placed in a centrifuge tube. 1 mL of methanol solution was added for ultrasonic extraction. The immersion liquid was centrifuged at 12000 r·min -1After centrifugation for 10 minutes, the supernatant was collected and quantified according to the chromatographic conditions described below. Experimental data were plotted and analyzed using GraphPad Prism 10 statistical software. Statistical analysis was performed using the t-test, with P < 0.05 considered statistically significant.

[0055] Liquid chromatography conditions for β-caryophyllene and δ-cadinene: Liquid chromatography was performed using a Waters 2695 system and a Waters C18 column (250 mm × 4.6 mm, 5 μm); the injection volume was 10 μL; the flow rate was 1 mL / min; the column temperature was maintained at 30°C; the detection wavelength was 202 nm; and the mobile phase was acetonitrile-water (95:5).

[0056] The distribution results of δ-cadinene and β-caryophyllene in the stratum corneum (A) and the epidermis (B) are shown in Figure 2. Figure 2 As shown, the results show that compared with β-caryophyllene, δ-cadinene has a more excellent and significant ability to reside in the stratum corneum, and can reside in the stratum corneum for a long time and in large quantities to form a reservoir, which is conducive to the long-term effect after transdermal administration.

[0057] Example 5

[0058] Inhibitory effects of δ-cadinene and β-caryophyllene on TRPM8 channels

[0059] HEK293T cells were seeded on a 6 mm diameter cell slide and cultured for 12 h before transient transfection of TRPM8 ion channels. Calcium ion imaging experiments were performed 48 h after transfection.

[0060] Fura-2AM was co-incubated with the cell slides at 37°C for 15 minutes. After washing the cell slides, they were observed under a calcium ion imaging fluorescence microscope and equipped with a perfusion device. Between 20s and 320s, 100μl of 50μM β-caryophyllene and δ-cadinene solutions were administered. At 320s, 100μl of 5μM menthol (Menthol), a TRPM8 agonist, was added. Changes in cell fluorescence intensity were recorded. Cells were continuously perfused with perfusion fluid between 320s and 620s. When the cell growth density reached about 80% and there were approximately 300 cells on each cell slide, β-caryophyllene, δ-cadinene, and the agonist were thoroughly rinsed to restore the fluorescence intensity before activation. At 620s, 100μl of 5μM menthol was added again to excite the cells, and changes in fluorescence intensity were recorded. The inhibitory effects of β-caryophyllene and δ-cadinene on the TRPM8 ion channel were analyzed based on the ratio of fluorescence intensities during the two activations. AMTB (N-(3-Aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide, 5 μM, 100 μL), a specific inhibitor of the TRPM8 ion channel, was used as a positive control, and the perfusate served as a blank control. All drugs used in the experiment were dissolved in DMSO as a stock solution and diluted to the target concentration in the perfusate upon use, with the DMSO content not exceeding 0.1%. Twenty representative responding cells were selected, and the inhibitory effects of δ-cadinene and β-caryophyllene on the TRPM8 ion channel were analyzed graphically based on changes in cell fluorescence intensity.

[0061] The results are as follows Figure 3 and Figure 4 As shown, both β-caryophyllene and δ-cadinene have a significant inhibitory effect on the activation of TRPM8 channels, and there is no significant difference between the two. However, as shown in Example 4, compared with β-caryophyllene, most of the δ-cadinene can reside in the skin for a long time, and the ability of δ-cadinene to inhibit TRPM8 channels from changing thermal preference behavior is significantly stronger than that of β-caryophyllene.

[0062] Example 6

[0063] Comparison of skin cytotoxicity of sesquiterpenoids

[0064] Mouse skin fibroblasts (MSF) were cultured in DMEM (high glucose) medium (containing 10% fetal bovine serum and 100 U / mL penicillin-streptomycin solution) at 37°C in a 5% CO2 incubator. Logarithmic phase cells were harvested and mixed by pipetting. The MSF cell suspension was diluted with complete culture medium to a concentration of 5000 cells per well. The suspension was then pipetted evenly. 100 μL of cell suspension was added to each well (not added in the zeroing group). 100 μL of PBS was added to the periphery of each well of a 96-well plate (water evaporation is likely to occur at the periphery, so adding PBS serves as a seal). The plate was shaken up and down (5 times to maintain uniform dispersion) and incubated in a CO2 incubator for 24 hours. After the cells were cultured to adhere to the wall, the culture medium in each well was aspirated with a pipette, and 100 μL of sample solution of different concentrations was added to each well. Five replicates were set for each concentration. A blank control group (PBS), a negative control group (DMEM high glucose containing 1% DMSO) and a drug-treated group were set up. The solvent was DMEM high glucose medium containing 1% DMSO. The cells were incubated in a CO2 incubator for 24 hours. After 24 hours, the culture medium was removed, and 100 μL of 10% CCK-8 was added to each well. After incubation for 1-2.5 hours, the microplate reader was used to measure the OD value. 450nm The absorbance value (Abs) was detected at the 40 nm RT-PCR reaction site, and the cell survival rate was calculated as follows: cell survival rate = (Abs in the drug-administered well - background Abs) / (Abs in the control well - background Abs) * 100%. 50 GraphPad Prism 9 was used for calculation, see Table 3 for details.

[0065] It can be seen that the skin cytotoxicity of the common sesquiterpenoid components contained in the volatile oils of hot Chinese medicines is significantly lower than that of the common cinnamaldehyde (the main component of hot cinnamon oil). Among them, the skin cytotoxicity of δ-cadinene is equivalent to that of β-caryophyllene, but both are significantly lower than caryophyllene oxide. δ-cadinene is more suitable for making cold protectants or cold-proof skin care products for keeping warm and frostbite, with high safety and excellent effects.

[0066] Table 3 Toxicity of sesquiterpenoids to MSF cells (n=5)

[0067]

Claims

1. Application of δ-cadinene or natural substances containing δ-cadinene in cold protection and frost prevention.

2. Use of δ-cadinene or natural substances containing δ-cadinene in the preparation of cold-resistant and antifreeze reagents or medicines.

3. Use of δ-cadinene or natural substances containing δ-cadinene in the preparation of cold protection agents or cold-proof skin care products.

4. The use according to any one of claims 1 to 3, characterized in that: The delta-cadinene is used in cold protection and frost prevention by significantly reducing the thermal preference temperature.

5. The use according to any one of claims 1 to 3, characterized in that: The δ-cadinene is used in cold protection and freezing prevention by alleviating or improving the damage to skin structure caused by frostbite.

6. The use according to any one of claims 1 to 3, characterized in that: The δ-cadinene is used in cold protection and freezing prevention by inhibiting TRPM8 to change thermal preference behavior.

7. The use according to any one of claims 1 to 3, characterized in that: The application of the δ-cadinene in protecting against cold and freezing through a transdermal administration route.

8. A composition for keeping warm and preventing freezing, characterized in that: The preparation comprises δ-cadinene as the sole cold-resistant and antifreeze component or in combination with other cold-resistant and antifreeze components, and also comprises raw materials, auxiliary materials or carriers required for the preparation.

9. The composition for keeping warm and preventing freezing according to claim 8, characterized in that The dosage form of the composition preferably includes emulsion, microemulsion, gel, solution, tincture, film, ointment, cream or patch.

10. Use of the cold-resistant and antifreeze composition according to claim 8 in the preparation of a cold-resistant and antifreeze agent or medicine, or a cold-resistant and antifreeze cold-protective agent or cold-resistant skin care product.

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