Application of cinnamomum longepaniculatum extract in preparation of medicine for promoting skin wound healing and treating eczema

Abstract

The invention discloses application of a cinnamomum longepaniculatum extract in preparation of a medicine for promoting skin wound healing and treating eczema, belongs to the technical field of biological medicines, and aims to provide application of delta-terpilenol, alpha-terpilenol or eucalyptus oil in the cinnamomum longepaniculatum extract in preparation of the medicine for promoting skin wound healing or treating eczema. The application depends on two brand-new action mechanisms of promoting keratinocyte migration / proliferation to accelerate reepithelization and inhibiting inflammation through a bidirectional immunoregulation mechanism based on the cinnamomum longepaniculatum compound instead of known antibacterial, analgesic or anti-inflammatory activity in the prior art; and a theoretical basis is laid for the use of the cinnamomum longepaniculatum compound in the new application field.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, in particular to the application of oil camphor extract in the preparation of drugs for promoting skin wound healing and treating eczema. BACKGROUND

[0002] The oil camphor extract generally refers to the plant volatile oil extracted from the leaves or branches of oil camphor by water vapor distillation, ethanol reflux extraction or solvent extraction method. The main components include eucalyptol, terpineol, cinnamic aldehyde, camphor, etc. Among them, eucalyptol is the main component, which has antibacterial, analgesic and anti-inflammatory activities, and can be widely used in cosmetics, food, medicine and aerospace fields.

[0003] In the field of cosmetics, Chinese patent CN109157426A discloses a skin care agent containing 4-terpineol, which is composed of 99% 4-terpineol and other components: polyoxyethylene sorbitan monolaurate, butanediol, glycerol, dipotassium glycyrrhizinate, LUJ20 mixture, dendrobium extract, cationic hyaluronic acid sodium, LUJ200 mixture, chlorella extract, p-hydroxyacetophenone, 1,2-hexanediol, LUJ04 mixture, D-panthenol, lemon grass extract, cinnamon extract, thyme extract and water. As a skin care agent, it can avoid diseases and irritation caused by harmful bacteria and pathogens on the skin, relieve skin aging, clean and prevent harmful bacteria from multiplying, keep the skin healthy, moist, bright and white, and meet the skin cleaning and hygiene needs. In the field of food and medicine, Chinese patent CN119039993A also discloses an antioxidant composition containing oil camphor extract, which is composed of 1,8-eucalyptol, alpha-terpineol and cinnamic aldehyde in a mass ratio of 1:1:1. It has the strongest scavenging ability for DPPH free radicals and ABTS+ free radicals.

[0004] Currently, the treatment drugs for wound healing usually use erythromycin ointment, mupirocin ointment, povidone-iodine solution, etc. These drugs can prevent bacterial infection of the wound, thereby promoting wound healing. However, they need to be used under the guidance of a doctor, and blind use may cause discomfort. In addition, for skin eczema, anti-infective drugs, hormone drugs and antiallergic drugs are usually used for treatment. However, due to different mechanisms of action of different drugs, treatment still needs to be guided by a doctor. Therefore, it is very important to study the mechanism of plant extracts (such as oil camphor extract) in promoting skin wound healing and treating eczema for the safe application of skin wound treatment drugs. SUMMARY

[0005] The application aims to provide an application of an extract of Cinnamomum oleiferum in preparing a medicament for promoting skin wound healing and treating eczema, which is not based on the antibacterial, analgesic or anti-inflammatory activity known in the prior art, but is based on two new mechanisms of action of the Cinnamomum oleiferum compound, i.e., promoting keratinocyte migration / proliferation to accelerate re-epithelialization and inhibiting inflammation through a bidirectional immune regulation mechanism, thereby laying a theoretical foundation for the use of the Cinnamomum oleiferum compound in a new application field.

[0006] One of the technical solutions of the application is that the extract of Cinnamomum oleiferum is applied in preparing a medicament for promoting skin wound healing, and delta-terpinenol, alpha-terpinenol or eucalyptus oil in the extract of Cinnamomum oleiferum is used for preparing the medicament for promoting skin wound healing.

[0007] The skin wound includes a burn wound or a scald wound.

[0008] The effective amount of the delta-terpinenol in the medicament is greater than or equal to 10 mg / g.

[0009] The effective amount of the alpha-terpinenol in the medicament is greater than or equal to 10 mg / g.

[0010] The effective amount of the eucalyptus oil in the medicament is greater than or equal to 10 mg / g.

[0011] Another technical solution of the application is that the extract of Cinnamomum oleiferum is applied in preparing a medicament for treating eczema, and delta-terpinenol, alpha-terpinenol or eucalyptus oil in the extract of Cinnamomum oleiferum is used for preparing the medicament for treating eczema.

[0012] The effective amount of the delta-terpinenol in the medicament is greater than or equal to 0.2 mg / g.

[0013] The effective amount of the alpha-terpinenol in the medicament is greater than or equal to 0.2 mg / g.

[0014] The effective amount of the eucalyptus oil in the medicament is greater than or equal to 0.2 mg / g.

[0015] The medicament includes a cream, a gel, a spray, a liniment, a lotion or a tincture.

[0016] It should be noted that the effective amount of the Cinnamomum camphora extract in promoting skin wound healing and treating eczema is significantly different. This is mainly due to the differences in drug delivery environment and core pathological mechanisms between the two application scenarios: for skin wounds (such as burns and scalds), the drug needs to penetrate the eschar and act on the deep tissues to promote re-epithelialization, and the wound environment is complex, so a higher local drug concentration (≥10 mg / g) is required to ensure efficacy; for eczema, the main pathological manifestations are superficial inflammation and immune disorders, the drug targets the epidermis and immune cells, and the safety and long-term tolerance of drug use need to be considered, so a lower concentration (≥0.2 mg / g) can effectively exert its anti-inflammatory and immune regulation effects. This dose differentiation design based on different treatment goals is reasonably expected and understood by those skilled in the art.

[0017] Compared with the prior art, the present application has the following advantages and beneficial effects: (1) As a natural plant-derived active ingredient, the Cinnamomum camphora extract exhibits better biocompatibility, lower potential side effect risk, and is more easily accepted and used in clinical and daily care compared to chemically synthesized drugs or potent hormone preparations.

[0018] (2) The Cinnamomum camphora compound has the characteristics of multi-target and comprehensive regulation, and not only has the dual functions of anti-inflammatory and immune regulation, but also can inhibit inflammation while promoting tissue repair, showing the synergistic advantage of "anti-inflammatory and repair at the same time". In addition, the ingredient also has certain antibacterial ability, which helps to prevent and control wound infection and provides a more favorable microenvironment for the healing process.

[0019] (3) In promoting skin wound healing, the Cinnamomum camphora compound not only accelerates wound closure, but also improves healing quality, has certain anti-fibrosis potential, and helps to reduce scar formation and pigmentation; in the treatment of eczema, the ingredient shows strong anti-inflammatory effect and has regulating function on the body's immune balance, showing potential for treating chronic inflammatory skin diseases.

[0020] (4) Through cell experiments and animal model experiments, it has been confirmed that the selected Cinnamomum camphora compound has good effects in promoting skin wound healing and relieving eczema-like dermatitis.

[0021] In summary, the present application discloses the new use of Cinnamomum camphora extract in skin wound healing and eczema treatment, and its mechanism of action is not limited to known antibacterial, analgesic or anti-inflammatory activity, but promotes keratinocyte migration / proliferation to accelerate re-epithelialization, and inhibits inflammation and participates in tissue repair through bidirectional immune regulation, thereby playing a therapeutic role, providing new scientific basis for the clinical application of Cinnamomum camphora extract. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Effect of different concentrations of Cinnamomum osmophloeum compounds on cell viability.

[0023] Figure 2 Effect of Cinnamomum osmophloeum compounds on HaCaT cell migration.

[0024] Figure 3 Effect of Cinnamomum osmophloeum compounds on Raw264.7 cell inflammatory factors and TGF-β mRNA expression.

[0025] Figure 4 Mouse scald burn modeling.

[0026] Figure 5 Wound healing of mice administered with δ-Terpinene and α-Terpinene.

[0027] Figure 6 Wound healing of mice administered with Eucalyptus oil and silver sulfadiazine.

[0028] Figure 7 Wound healing of mice in the model group Figure 8 Skin condition of mice for safety evaluation.

[0029] Figure 9 Wound healing of mice administered with δ-Terpinene and α-Terpinene.

[0030] Figure 10 Wound healing of mice administered with Eucalyptus oil and silver sulfadiazine.

[0031] Figure 11 Wound healing of mice in the model group

[0032] Figure 12 Wound paper weight of mice in each group before and after administration and calculated wound healing rate.

[0033] Figure 13 Flowchart of mouse eczema treatment experiment.

[0034] Figure 14 List of organs of each mouse after dissection.

[0035] Figure 15 Average value of organs after weighing in each group. DETAILED DESCRIPTION

[0036] The invention purpose, technical solutions and beneficial effects will be further described in detail below.

[0037] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the claimed invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0038] The specific implementation of the present invention will be described below with reference to the embodiments. Of course, the scope of protection of the present invention is not limited to the following embodiments.

[0039] Example 1: Cellular virulence test This embodiment involves detecting the cytotoxicity of δ-terpineol, α-terpineol, and eucalyptus oil on Raw264.7 macrophages and HacaT keratinocytes at different concentration gradients to determine their safe concentration range.

[0040] The specific experimental procedure is as follows: 1) Raw264.7 and HacaT cells were seeded in 96-well plates overnight (n=4).

[0041] 2) Use a pipette to remove the culture medium and add freshly prepared culture medium with or without the compound and a control group containing 0.3% Triton.

[0042] 3) After 24 hours, discard the culture medium, add 100 μL of MTT diluted 1:10 with the culture medium, and incubate at 37°C for 4 hours. Discard the culture medium, add 100 μL of DMSO to each well, and incubate at 37°C with a shaker for 30 minutes to fully dissolve. Measure the absorbance at 490 nm using a microplate reader.

[0043] 4) Groups: Blank, 10, 20, 40, 80, 120, 150, 200, 300 ug / ml δ-terpineol (α-terpineol, eucalyptus oil), 0.3% triton.

[0044] See the experimental results. Figure 1 . Figure 1 In Figure A, δ-terpineol affects the survival rate of Raw264.7 cells; Figures B through D show the effects of δ-terpineol, α-terpineol, and eucalyptus oil on the survival rate of HacaT cells, respectively.

[0045] Depend on Figure 1 Analysis showed that, compared with Blank, Raw264.7 and HacaT cells cultured for 48 hours with different concentrations of δ-terpineol, α-terpineol, and eucalyptus oil did not exhibit significant cytotoxicity, with only slight cytotoxicity observed at 300 μg / mL. The cell survival percentage was higher than 100% at moderate concentrations of δ-terpineol, α-terpineol, and eucalyptus oil, demonstrating that these substances were non-toxic to cells at the tested concentrations and may have some effect in promoting cell metabolism or proliferation.

[0046] Example 2: Cell Scratch Assay This embodiment uses a cell scratch assay to evaluate the effects of δ-terpineol, α-terpineol, and eucalyptus oil on the migration ability of HacaT cells at different time points (0 to 48 hours), and uses scratch healing rate for quantitative analysis.

[0047] The specific experimental procedure is as follows: 1) Seed HacaT cells in 6-well plates overnight. Mark the scratched area and the reference line for taking pictures on the bottom of the plate to ensure that the pictures are taken from the same location each time.

[0048] 2) Remove the well plate from the incubator and, using a 200μL sterile pipette tip, draw a straight line steadily along the previously drawn reference line, perpendicular to the bottom of the plate.

[0049] 3) Wash the plate 3 times with PBS to remove the exfoliated cell debris. Change the medium: add the prepared low serum medium (containing the treatment drug).

[0050] 4) Take photos and return the plate to the CO2 incubator for further incubation. Remove the plate at the predetermined time points (0, 12, 24, 36, 48h) and take photos at the exact same marked locations.

[0051] 5) Evaluation Method: Image analysis software (ImageJ) was used for analysis. Mobility was calculated. Wound confluence % = [(A0 - A] t ) / A0] × 100% A0 = Scratch area at 0 hours A t = Scratch area in t hours See the experimental results. Figure 2 . Figure 2 Image A shows a microscopic image of scratch healing in HaCaT cells after treatment with δ-terpineol, α-terpineol, and eucalyptus oil; Image B shows the migration rate of cells in each group 48 hours after treatment.

[0052] Depend on Figure 2 Analysis showed that, compared with Blank, HacaT cells treated with δ-terpineol, α-terpineol, and eucalyptus oil exhibited a significant increase in migration rate in a time-dependent manner. After 48 hours, the migration rate of cells treated with camphor oil extract reached approximately 50%. With increasing time, eucalyptus oil was more effective than α-terpineol and δ-terpineol in promoting HacaT migration. However, compared to the Blank group, all three treatments demonstrated that δ-terpineol and the other two camphor oil extracts possessed some ability to promote skin wound healing.

[0053] Example 3: Real-time fluorescent quantitative PCR This example analyzes the effects of δ-terpineol, α-terpineol and eucalyptus oil on the mRNA expression levels of inflammatory factors IL-1β, IL-6 and TNF-α of Raw264.7 cells induced by LPS by real-time fluorescent quantitative PCR technology.

[0054] The specific experimental process is as follows: 1) RAW264.7 was plated in a twelve-well plate overnight, and then was stimulated with compounds (150 μg / mL). After 4 h, LPS (1 μg / mL) was added for 24 h.

[0055] 2) The RAW cell precipitate was collected from the twelve-well plate, 500 μL of trizol was added, and the vortex was mixed. After standing for 5 min, centrifugation was performed at 12000 rpm at 4°C for 10 min, and the supernatant was taken.

[0056] 3) 200 μL of chloroform was added, and the mixture was mixed thoroughly up and down. After standing for 5 min, centrifugation was performed at 12000 rpm at 4°C for 15 min, 200 ul of supernatant was taken, 200 μL of cold isopropanol was added, and the mixture was mixed gently up and down. After standing for 10 min, centrifugation was performed at 12000 rpm at 4°C for 10 min, and the white precipitate was left. 70% ethanol (15 ml) was prepared with enzyme-free water, each tube was washed with 1 mL of 70% ethanol, centrifugation was performed at 12000 rpm at 4°C for 5 min, and the ethanol was dried. The white solid was added with 20 μL of enzyme-free water to dissolve the precipitate.

[0057] 4) About 100 ng of RNA was reversely transcribed into cDNA in a 20 μL reaction using the PrimeScript™ RT kit.

[0058] 5) Q-PCR experiment was performed using the TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) kit.

[0059] Table 1: Primer sequences

[0060] The experimental results are shown in Figure 3 . Figure 3 A to C are the effects of eucalyptus oil, α-terpineol and δ-terpineol on the mRNA expression of IL-6, IL-1β and TNF-α under LPS induction; D and E are the effects of the three compounds on the mRNA expression of IL-6, IL-1β, TNF-α and TGF-β under normal conditions.

[0061] From Figure 3The analysis shows that LPS can significantly induce the mRNA expression of IL-6, IL-1β, TNF-α, indicating that the modeling is successful. Dexamethasone (DXM) can effectively inhibit the gene expression of these inflammatory factors induced by LPS, and eucalyptus oil, α-pinene, δ-pinene also have certain inhibitory effect on the gene expression of inflammatory cytokines induced by LPS, although the effect is lower than that of DXM, but still can show certain anti-inflammatory potential; δ-pinene has obvious inhibitory effect on pro-inflammatory factors (IL-1b, TNF-α), and also has certain inhibitory effect on TGF-β, indicating that δ-pinene may directly act on the upstream link of inflammatory cascade, and its effect does not depend on immunosuppression, has better safety, indicating that δ-pinene has good application prospect in the treatment of eczema.

[0062] Example 4: Mouse in vivo scald burn experiment In this example, a mouse deep Ⅱ degree scald model is established to evaluate the promoting effect of δ-pinene and other oil camphor extract creams on wound healing after 21 days of continuous administration, and sulfadiazine silver cream is used as a positive control.

[0063] The specific experimental process is as follows: Select adult male mice weighing 18-22 g, after depilation with depilatory cream, inject a solution of chloral hydrate.

[0064] 10g size weight is placed in a metal bath heated to 200C and contacted with the exposed skin of the mouse for 8s.

[0065] The wound is scabbed the day after scalding, and administration of the preparation (δ-pinene or α-pinene, eucalyptus oil cream) is started on the third day. After 21 days of continuous administration, the wound size is observed.

[0066] The positive control uses sulfadiazine silver.

[0067] Evaluation method: Nagelschmidt method is used to determine the healing area of the wound, i.e. weighing paper is covered on the scalded wound on the back of the mouse, the wound edge is drawn on the transparent weighing paper, the wound shape is cut out, and its weight is weighed, and the healing rate is calculated according to the following formula:

[0068] The main groups are as follows:

[0069] The experimental results are shown in Figures 4 to 12 .

[0070] Among them, Figure 4Burn modeling condition (modeling condition: the first day modeling wound is shallow, after one day, 10g weight is burned on alcohol lamp for 10s, then contact with the original wound for 5s, the next day to see if the modeling is successful); Figure 5 Wound healing condition of mice administered with δ-terpinene and α-terpinene; Figure 6 Wound healing condition of mice administered with eucalyptus oil and silver sulfadiazine; Figure 7 Wound healing condition of mice in the model group; Figure 8 Skin condition of mice for safety evaluation (the figure is the wound healing condition after 10 days of administration), from left to right in the figure are No. 1: δ-terpinene, No. 2: α-terpinene, No. 3: eucalyptus oil; Figure 9 Wound healing condition of mice administered with δ-terpinene and α-terpinene; Figure 10 Wound healing condition of mice administered with eucalyptus oil and silver sulfadiazine; Figure 11 Wound healing condition of mice in the model group (the figure is the wound healing condition after 21 days of administration); Figure 12 Wound healing rate of mice in each group before and after administration.

[0071] The results of this example show that (Fig. 1) Figures 4 to 12 ), each experimental group (δ-terpinene, α-terpinene, eucalyptus oil) and the positive control group all showed significant wound contraction and healing promotion effect. Importantly, at the end of the experiment (21 days), the wound healing rates of the δ-terpinene and eucalyptus oil experimental groups were comparable to that of the positive control group (silver sulfadiazine), and at all observation time points, the experimental groups all showed better healing kinetic trends than the model group. This result indicates that the oil camphor extract (δ-terpinene, α-terpinene, eucalyptus oil) itself has significant activity in promoting skin wound healing, and its therapeutic effect is not due to the cream base, but its inherent biological effect.

[0072] Example 5: Mouse eczema treatment experiment in vivo This example establishes a DNFB-induced mouse eczema model to evaluate the effects of continuous administration of oil camphor extract δ-terpinene and the like for 7 days on ear swelling and immune organ weight, to explore its effect on treating eczema.

[0073] The specific experimental process is as follows: 1) Select adult male mice weighing 18-22g, depilate with depilatory cream, then apply 2% DNFB acetone olive oil 100ul on the abdomen for sensitization.

[0074] 2) The next day, apply 50ul of 2% DNFB acetone olive oil on the mouse abdomen to enhance sensitization, and 4 hours later, apply evenly on the depilated area on the back and both sides of the ears.

[0075] 3) The back of the mice was shaved and the mice were treated with the test substance twice a day for 7 days.

[0076] 4) 30 minutes after the last administration, 30ul of 0.5% DNFB in acetone olive oil was applied to the inner and outer sides of the right ear of each mouse, and the same amount of acetone olive oil was applied to the left ear as a matrix control.

[0077] 5) The mice were sacrificed 24 hours later, the ears were punched with an 8mm puncher, and the thymus and spleen were removed and weighed.

[0078] The following table shows the experimental grouping:

[0079] The experimental results are shown in Figures 13 to 15 .

[0080] Among them, Figure 13 is the flow chart of the mouse eczema treatment experiment; Figure 14 is the organ list of each mouse after dissection (from top to bottom in the figure, the left ear, the right ear, the spleen, and the thymus); Figure 15 is the average weight of the organs in each group.

[0081] The results of this example show that Figure 14 and Figure 15 The model mice successfully showed the typical eczema characteristics of right ear swelling and increased spleen index. Compared with the model group, the δ-terpineol, α-terpineol and eucalyptus oil experimental groups could significantly inhibit the ear swelling caused by DNFB, and effectively reduce the spleen index, and the improvement degree was consistent with the trend of the positive control group. The results show that the oil camphor extract can effectively relieve the inflammatory response of eczema-like dermatitis and regulate the related immune response, thereby playing a therapeutic role.

[0082] The above is only a preferred embodiment of the present application, and does not limit the present application in any form. Any simple modification or equivalent change made on the basis of the technical essence of the present application to the above embodiment falls within the scope of protection of the present application.

Claims

1. The application of camphor oil extract in the preparation of drugs that promote skin wound healing, characterized in that: δ-terpineol, α-terpineol, or eucalyptus oil from camphor tree extract can be used to prepare drugs that promote skin wound healing.

2. The application according to claim 1, characterized in that: The skin wounds include burns or scalds.

3. The application according to claim 1, characterized in that: The effective dosage of δ-terpineol in the drug is ≥10 mg / g.

4. The application according to claim 1, characterized in that: The effective dosage of α-terpineol in the drug is ≥10 mg / g.

5. The application according to claim 1, characterized in that: The effective dosage of eucalyptus oil in the drug is ≥10mg / g.

6. The application of camphor oil extract in the preparation of eczema treatment drugs, characterized in that: δ-terpineol, α-terpineol, or eucalyptus oil from camphor tree extract can be used to prepare drugs for treating eczema.

7. The application according to claim 6, characterized in that: The effective dosage of δ-terpineol in the drug is ≥0.2 mg / g.

8. The application according to claim 6, characterized in that: The effective dosage of α-terpineol in the drug is ≥0.2 mg / g.

9. The application according to claim 6, characterized in that: The effective dosage of eucalyptus oil in the drug is ≥0.2 mg / g.

10. The application according to any one of claims 1 to 9, characterized in that: The medicine includes creams, gels, sprays, liniments, lotions, or tinctures.

Images