A kind of external Chinese medicine compound essential oil for scleroderma

Abstract

The invention provides a topical Chinese medicinal compound essential oil for scleroderma, and relates to the technical field of topical Chinese medicinal essential oil pharmaceuticals. The invention follows the principle of TCM syndrome differentiation and treatment, and rationally combines the ingredients to form the Chinese medicinal compound essential oil with wormwood essential oil, peony bark essential oil, salvia miltiorrhiza essential oil, prickly ash essential oil, cnidium monnieri essential oil, clove essential oil, angelica essential oil, and borneol essential oil. The Chinese medicinal compound essential oil has good therapeutic effects of promoting blood circulation and removing blood stasis, warming the meridians and dispelling cold, reducing swelling and relieving pain, and clearing away heat and detoxifying.

Description

Technical Field

[0001] The invention relates to the technical field of external Chinese medicinal essential oil pharmaceutical preparations, and in particular to an external Chinese medicinal compound essential oil for scleroderma. Background Art

[0002] Scleroderma is a connective tissue disease that mainly affects the skin and mucosal tissues and is characterized by fibrosis and sclerosis. It includes two types: localized scleroderma (LS) and systemic scleroderma (SS). Scleroderma is an isolated disease with unknown etiology and complex pathogenesis. Autoimmune changes and fibrosis are the prominent features of the disease. Systemic sclerosis is a chronic and unpredictable process that affects multiple organs. In addition to varying degrees of skin involvement and Raynaud's phenomenon, interstitial lung disease, extensive microvascular disease, intestinal pathology and cardiovascular complications also occur from time to time. The symptoms of patients with advanced scleroderma are usually accompanied by pulmonary hypertension, pulmonary fibrosis and small intestinal dysfunction. The survey results of relevant organizations show that the current incidence of scleroderma is second only to rheumatoid arthritis and systemic lupus erythematosus, but due to the combined effects of autoimmunity, tissue fibrosis and vascular lesions, its mortality rate is higher than that of patients with rheumatoid arthritis and systemic lupus erythematosus. The pathogenesis of scleroderma is still unclear and may be related to multiple factors such as gender, genetics, external environment, systemic immune abnormalities, etc. In recent years, there have been many reports on the research on the treatment of scleroderma with traditional Chinese medicine.

[0003] Western medicine treatment of scleroderma is mainly based on drug control, supplemented by other physical therapies. However, due to its long course of disease, many types of drugs, and potential hazards, patients have low compliance with treatment, and it is difficult to persist in the long term, and the treatment effect is also average. Traditional Chinese medicine has a good clinical effect on the treatment of scleroderma. Studies have shown that early use of traditional Chinese medicine treatment combined with traditional Chinese medicine physical therapy and rehabilitation methods has a significant effect on promoting the prognosis of scleroderma patients and can significantly improve the quality of life of patients in the later stage.

[0004] However, there are certain limitations in the treatment of scleroderma with traditional Chinese medicine, such as the long onset time of traditional Chinese medicine, and the effect is not obvious in a short time. Most of the traditional Chinese medicine dosage forms used to treat scleroderma are mainly decoctions, the dosage forms are relatively single, and the medication is slightly cumbersome. Pure natural Chinese herbal medicine plants can be extracted and refined into Chinese medicine essential oils through modern technology. Chinese medicine essential oils have the advantages of high active ingredient content, fast efficacy, strong penetration, etc., and are used to develop many medicinal preparations. There are thousands of Chinese herbal medicines extracted and refined from pure natural Chinese herbal medicine plant essential oils. my country has a vast territory, superior natural conditions, and extremely rich varieties; Chinese medicine compound essential oils based on classic ancient prescriptions or compatibility prescriptions have the advantages of strong pertinence and significant therapeutic effects; Chinese medicine compound essential oils focus on the efficacy of medicinal properties, can directly enter the meridian product, directly enter the meridians through the skin, nourish the internal organs, and the effective ingredients are absorbed by small molecules to achieve better therapeutic effects. Based on this, the present invention provides a kind of external compound essential oil that can take effect quickly and has obvious therapeutic effects. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of the deficiencies in the prior art, the present invention provides an external Chinese medicinal compound essential oil for scleroderma.

[0007] (II) Technical solution

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0009] The invention provides a topical Chinese medicinal compound essential oil for scleroderma. The Chinese medicinal compound essential oil for scleroderma comprises the following raw materials in parts by weight: 5-15 parts of wormwood essential oil, 5-15 parts of peony bark essential oil, 5-15 parts of salvia miltiorrhiza essential oil, 3-10 parts of zanthoxylum essential oil, 3-10 parts of cnidium monnieri essential oil, 3-10 parts of clove essential oil, 3-10 parts of angelica essential oil and 3-10 parts of borneol essential oil.

[0010] Furthermore, the Chinese medicine compound essential oil comprises the following raw materials in parts by weight: 8-12 parts of wormwood essential oil, 8-12 parts of peony bark essential oil, 8-12 parts of salvia miltiorrhiza essential oil, 5-8 parts of Zanthoxylum essential oil, 5-8 parts of Cnidium monnieri essential oil, 5-8 parts of clove essential oil, 5-8 parts of angelica essential oil, and 5-8 parts of borneol essential oil.

[0011] Furthermore, the Chinese medicine compound essential oil comprises the following raw materials in parts by weight: 9 parts of wormwood essential oil, 9 parts of peony bark essential oil, 9 parts of salvia miltiorrhiza essential oil, 6 parts of Zanthoxylum essential oil, 6 parts of Cnidium monnieri essential oil, 6 parts of clove essential oil, 6 parts of angelica essential oil, and 6 parts of borneol essential oil.

[0012] Specifically, the single essential oils of the above-mentioned ingredients can be obtained by direct purchase, or by extracting them from the original plants using conventional essential oil extraction methods, wherein the conventional essential oil extraction methods include but are not limited to pressing extraction, steam distillation extraction, supercritical carbon dioxide extraction, etc.

[0013] Preferably, the wormwood essential oil, peony bark essential oil and salvia miltiorrhiza essential oil are respectively made from wormwood, peony bark and salvia miltiorrhiza as raw materials, and the volatile oil is extracted by supercritical carbon dioxide method, and the distillation concentration is controlled at more than 99%; the Zanthoxylum bungeanum essential oil is made from Zanthoxylum bungeanum as raw material, and the volatile oil is extracted by pressing method, and the distillation concentration is controlled at more than 99%; the Cnidium monnieri essential oil, clove essential oil, Angelica sinensis essential oil and borneol essential oil are respectively made from Cnidium monnieri, clove, Angelica sinensis and borneol as raw materials, and the volatile oil is extracted by steam distillation method, and the distillation concentration is controlled at more than 99%.

[0014] The present invention also provides the use of the Chinese herbal compound essential oil for preparing an external preparation for treating scleroderma. Specifically, medically acceptable excipients are added to the Chinese herbal compound essential oil to prepare an external preparation usable by the human body, including but not limited to creams, liniments, sprays, and tinctures.

[0015] Furthermore, the effective content of the traditional Chinese medicine compound essential oil in the external preparation for treating scleroderma is 1-10%.

[0016] (III) Beneficial effects

[0017] Scleroderma belongs to the category of "skin arthralgia" in traditional Chinese medicine. Its etiology is caused by the invasion of the skin by wind and cold evil, which damages the qi and blood over time, leading to qi stagnation and blood stasis, blocked meridians, and skin malnutrition. Therefore, the treatment of this disease is based on the principles of replenishing qi and blood, promoting blood circulation, dredge meridians, nourishing blood and moisturizing the skin. The present invention follows the principle of TCM syndrome differentiation and treatment, and rationally combines the essential oils to form the Chinese medicine compound essential oil with wormwood essential oil, peony bark essential oil, salvia miltiorrhiza essential oil, prickly ash essential oil, cnidium monnieri essential oil, clove essential oil, angelica essential oil, and borneol essential oil. In the formula, mugwort is the main medicine for warming the meridians and stopping bleeding, dispelling cold and relieving pain, and its fragrance penetrates the skin and opens the orifices; peony bark and salvia miltiorrhiza are the assistant medicines to strengthen the effect of promoting blood circulation, and angelica sinensis replenishes blood and promotes blood circulation without damaging the blood; cloves, zanthoxylum bungeanum and cnidium monnieri are used as auxiliary medicines to strengthen the spleen and warm the kidneys, replenishing blood and warming yang, supporting yang qi, regulating the nutrient and defensive systems, warming yang and replenishing blood, and resolving cold stagnation to treat the symptoms; borneol clears heat and relieves pain, and is used for exogenous cold and dampness, long-term heat accumulation, and diseases caused by blocked collaterals; it has good therapeutic effects of promoting blood circulation and removing stasis, warming the meridians and dispelling cold, reducing swelling and relieving pain, clearing away heat and detoxifying.

[0018] The results of animal experiments show that the topical application of the Chinese herbal compound essential oil of the present invention has a good therapeutic effect on scleroderma model mice, and can significantly reduce the level of inflammatory factors in mouse serum and the levels of TGF-β, CTGF, and VEGF in skin tissue homogenate, reduce the content of hydroxyproline in skin tissue, improve skin fibrosis, improve skin pathological changes, and reduce the thickness of the skin dermis. The clinical efficacy results show that the combination of the Chinese herbal compound essential oil of the present invention and Western medicine can significantly improve the therapeutic effect of localized scleroderma, and its cure rate can reach 85.71%, which is significantly higher than the cure rate of the control group (41.07%). The Chinese herbal compound essential oil of the present invention can significantly improve the patient's skin hardness, joint pain, and promote the improvement and recovery of clinical symptoms such as joint function. The Chinese herbal compound essential oil of the present invention can achieve the treatment of both the symptoms and the root cause of scleroderma. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The effect of angelica flower essential oil on inflammatory indicators of scleroderma in mice: IL-6 (A), IL-10 (B), IL-17 (C), and TNF-α (D); Note: *P<0.05, **P<0.01 compared with the model group.

[0020] Figure 2 The effect of angelica flower essential oil on the levels of TGF-β (A), CTGF (B), and VEGF (C) in scleroderma mice; Note: *P<0.05, **P<0.01 compared with the model group.

[0021] Figure 3 Effects of angelica flower essential oil on hydroxyproline content in scleroderma mice (A) and skin fibrosis in scleroderma mice (B); Note: *P<0.05, **P<0.01 compared with the model group.

[0022] Figure 4 The effect of angelica flower essential oil on dermal thickness in mice with scleroderma; Note: *P<0.05, **P<0.01 compared with the model group. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Example 1

[0025] A topical Chinese medicinal compound essential oil for scleroderma, the essential oil consisting of the following components in parts by volume: 5 mL of wormwood essential oil, 5 mL of peony bark essential oil, 5 mL of salvia miltiorrhiza essential oil, 3 mL of zanthoxylum essential oil, 3 mL of cnidium monnieri essential oil, 3 mL of clove essential oil, 3 mL of angelica essential oil, and 3 mL of borneol essential oil.

[0026] The above-mentioned unilateral essential oils are all commercially available.

[0027] Medically acceptable excipients are added to the traditional Chinese medicine compound essential oil to prepare external preparations usable by humans, including creams, liniments, sprays or tinctures, etc.; wherein the effective content of the traditional Chinese medicine compound essential oil in the external preparation is 5%.

[0028] Example 2

[0029] A topical Chinese medicinal compound essential oil for scleroderma, the essential oil consisting of the following components in parts by volume: 15 mL of wormwood essential oil, 15 mL of peony bark essential oil, 15 mL of salvia miltiorrhiza essential oil, 10 mL of zanthoxylum essential oil, 10 mL of cnidium monnieri essential oil, 10 mL of clove essential oil, 10 mL of angelica essential oil, and 10 mL of borneol essential oil.

[0030] Example 3

[0031] A topical Chinese medicinal compound essential oil for scleroderma, the essential oil being composed of the following components in parts by volume: 12 mL of wormwood essential oil, 8 mL of peony bark essential oil, 10 mL of salvia miltiorrhiza essential oil, 4 mL of zanthoxylum essential oil, 6 mL of cnidium monnieri essential oil, 4 mL of clove essential oil, 8 mL of angelica essential oil, and 5 mL of borneol essential oil.

[0032] Example 4

[0033] A topical Chinese medicinal compound essential oil for scleroderma, the essential oil consisting of the following components in parts by volume: 8 mL of wormwood essential oil, 8 mL of peony bark essential oil, 8 mL of salvia miltiorrhiza essential oil, 5 mL of zanthoxylum essential oil, 5 mL of cnidium monnieri essential oil, 5 mL of clove essential oil, 5 mL of angelica essential oil, and 5 mL of borneol essential oil.

[0034] Example 5

[0035] A topical Chinese medicinal compound essential oil for scleroderma, the essential oil being composed of the following components in parts by volume: 12 mL of wormwood essential oil, 12 mL of peony bark essential oil, 12 mL of salvia miltiorrhiza essential oil, 8 mL of zanthoxylum essential oil, 8 mL of cnidium monnieri essential oil, 8 mL of clove essential oil, 8 mL of angelica essential oil, and 8 mL of borneol essential oil.

[0036] Example 6

[0037] A topical Chinese medicinal compound essential oil for scleroderma, the essential oil consisting of the following components in parts by volume: 9 mL of wormwood essential oil, 9 mL of peony bark essential oil, 9 mL of salvia miltiorrhiza essential oil, 6 mL of zanthoxylum essential oil, 6 mL of cnidium monnieri essential oil, 6 mL of clove essential oil, 6 mL of angelica essential oil, and 6 mL of borneol essential oil.

[0038] Test Example 1

[0039] 1 Materials and methods

[0040] 1.1 Experimental animals and groups

[0041] A total of 105 female BALB / c SPF mice aged 6-9 weeks and weighing (19-20) g were selected. The mice were adaptively fed for one week and randomly divided into 7 groups, with 15 mice in each group, namely blank group, model group, and test groups 1-5.

[0042] 1.2 Animal model preparation

[0043] The model group and test groups 1-5 were used to prepare animal models of scleroderma according to the reference method. The specific method was as follows: 2×2 cm of hair was shaved on the back of the mice, and 0.3 mL of hypochlorous acid (HCLO) solution was subcutaneously injected into the shaved area on the back of the mice except for the blank group, once a day for 6 consecutive weeks. The concentration of hypochlorous acid (HCLO) solution was 166 μL of sodium hypochlorite solution + 11.1 mL of potassium dihydrogen phosphate solution.

[0044] 1.3 Treatment methods

[0045] At the same time as modeling, each group was given corresponding drug intervention. Among them, the blank group did not undergo any modeling and did not receive any treatment; the model group did not undergo any treatment; the experimental groups 1-5 were given different formulas of Chinese herbal compound essential oils to be applied externally on the back, with a dose of 5 μL of the corresponding Chinese herbal compound essential oil evenly applied to each mouse in each group. After modeling every day, each medication group was given the corresponding drug once a day for 6 consecutive weeks. After the end of the medication, the samples were collected and the relevant indicators were tested. The formulas of the Chinese herbal compound essential oils in the experimental groups 1-5 are as follows:

[0046] Experimental group 1: Artemisia argyi essential oil 9mL, Paeonia suffruticosa essential oil 9mL, Salvia miltiorrhiza essential oil 9mL, Zanthoxylum bungeanum essential oil 6mL, Cnidium monnieri essential oil 6mL, Clove essential oil 6mL, Angelica sinensis essential oil 6mL, Borneol essential oil 6mL.

[0047] Experimental group 2: 9 mL of salvia miltiorrhiza essential oil, 6 mL of Zanthoxylum bungeanum essential oil, 6 mL of Cnidium monnieri essential oil, 6 mL of clove essential oil, 6 mL of Angelica sinensis essential oil, and 6 mL of borneol essential oil.

[0048] Experimental group 3: Artemisia argyi essential oil 9mL, Paeonia suffruticosa essential oil 9mL, Salvia miltiorrhiza essential oil 9mL, Cnidium monnieri essential oil 6mL, Clove essential oil 6mL, and Angelica sinensis essential oil 6mL.

[0049] Experimental group 4: Artemisia argyi essential oil 9mL, Paeonia suffruticosa essential oil 9mL, Zanthoxylum bungeanum essential oil 6mL, Cnidium monnieri essential oil 6mL, Clove essential oil 6mL, Borneol essential oil 6mL.

[0050] Experimental group 5: Artemisia argyi essential oil 9mL, Paeonia suffruticosa essential oil 9mL, Salvia miltiorrhiza essential oil 9mL, Zanthoxylum essential oil 6mL, Angelica sinensis essential oil 6mL, Borneol essential oil 6mL.

[0051] The above-mentioned unilateral essential oils are all commercially available.

[0052] 1.4 Detection indicators

[0053] Effects on skin pathology and dermal thickness in scleroderma model mice

[0054] The back skin of the mouse was fixed with 4% paraformaldehyde solution, embedded in paraffin and sliced, stained with hematoxylin-eosin (HE), the nucleus turned blue-purple, the cytoplasm turned red, and the pathological changes of the mouse skin were observed. The thickness of the mouse dermis was measured using digital pathology software, and 3 points were selected for each mouse skin for measurement and the average value was taken. Semi-quantitative analysis of mouse skin pathology was performed: "-" The structure of each layer of the skin is intact, and the cells are arranged regularly and neatly; "+" The skin is slightly thickened and the fibrous hyperplasia is mild; "++" The dermis of the skin is significantly thickened, the fibrous tissue proliferates a lot, and a large area of ​​subdermal fat tissue is invaded.

[0055] 1.5 Data processing

[0056] Graphpad Prism 8.2.0 statistical software was used for statistical analysis. The comparison between the two groups of data was performed using the t-test, P < 0.05 indicated a statistically significant difference, P < 0.01 indicated a significant statistical difference, and all data were expressed as mean ± standard deviation. The rank data were analyzed by Radit test and graphed using Graphpad Prism 8.2.0 software.

[0057] 2 Results

[0058] 2.1 Effects of different formulas of Chinese herbal essential oils on skin pathology and dermal thickness in scleroderma model mice

[0059] As shown in the results in Table 1, compared with the blank group, the dermal thickness of the mice in the model group was significantly increased (P < 0.01). Compared with the model group, the dermal thickness of the mice in test groups 1-5 decreased to varying degrees, among which the dermal thickness of the mice in test groups 1 and 3 was significantly reduced compared with the model group (P < 0.01), and the reduction in dermal thickness of the mice in test group 1 was greater than that in test group 3; the dermal thickness of the mice in test groups 2 and test groups 4-5 was significantly reduced (P < 0.05).

[0060] As shown in the results of Table 2, the model group showed significant pathological changes in skin tissue compared with the blank group by Ridit test (P<0.01). Compared with the model group, both test group 1 and test group 3 could significantly improve the pathological changes in mouse skin tissue (P<0.01), and the improvement degree of test group 1 was more advantageous than that of test group 3; test group 2 and test groups 4-5 could significantly improve the pathological changes in mouse skin tissue (P<0.05).

[0061] Table 1 Effects of different formulas of Chinese herbal essential oils on dermal thickness in scleroderma model mice

[0062] Grouping Dosage Dermis thickness (μm) Blank Group — 125.05±10.07 Model Group — 238.51±5.53## Test Group 1 <![CDATA[5μL / cm 2 ]]> 143.62±5.95** Test Group 2 <![CDATA[5μL / cm 2 ]]> 204.98±10.15* Experiment 3 groups <![CDATA[5μL / cm 2 ]]> 183.37±10.00** Test 4 groups <![CDATA[5μL / cm 2 ]]> 194.94±15.21* Test 5 groups <![CDATA[5μL / cm 2 ]]> 202.88±15.91*

[0063] Note: Compared with the blank group, # p<0.05, ## p<0.01; compared with the model group, *p<0.05, **p<0.01.

[0064] Table 2 Effects of different formulas of Chinese herbal essential oils on the skin tissue morphology of scleroderma model mice

[0065] Grouping Dosage Quantity(n) — + ++ P Blank Group — 15 15 0 0 — Model Group — 15 0 1 14 ## Test Group 1 <![CDATA[5μL / cm 2 ]]> 15 12 2 1 ** Test Group 2 <![CDATA[5μL / cm 2 ]]> 15 4 7 4 * Experiment 3 groups <![CDATA[5μL / cm 2 ]]> 15 7 5 2 ** Test 4 groups <![CDATA[5μL / cm 2 ]]> 15 5 8 3 * Test 5 groups <![CDATA[5μL / cm 2 ]]> 15 4 8 3 *

[0066] Note: Compared with the blank group, # p<0.05, ## p<0.01; compared with the model group, *p<0.05, **p<0.01.

[0067] 3 Conclusion

[0068] The above results show that the Chinese herbal compound essential oil of the present invention has a significant improvement effect on the skin tissue morphology of scleroderma model mice, can significantly reduce the dermis thickness of model mice, and has a significant therapeutic effect on scleroderma model mice. In addition, the various drugs in the Chinese herbal compound essential oil of the present invention work synergistically, and the lack of any component cannot achieve the good therapeutic effect of the Chinese herbal compound essential oil of the present invention on scleroderma model mice.

[0069] Test Example 2

[0070] 1 Materials and methods

[0071] 1.1 Experimental animals and groups

[0072] Sixty female BALB / c SPF mice aged 6-9 weeks and weighing (19-20) g were selected. The mice were adaptively fed for one week and randomly divided into 5 groups, with 12 mice in each group, namely, blank group, model group, penicillamine group, high-dose group (10 μL) of angelica flower essential oil, and low-dose group (5 μL).

[0073] 1.2 Animal model preparation

[0074] Except for the blank group, the other groups of mice were used to prepare scleroderma animal models according to the reference method. The specific method was as follows: 2×2 cm of hair was shaved on the back of the mice, and 0.3 mL of hypochlorous acid (HCLO) solution was subcutaneously injected into the shaved area on the back of the mice except the blank group, once a day for 6 consecutive weeks. The concentration of hypochlorous acid (HCLO) solution was 166 μL sodium hypochlorite solution + 11.1 mL potassium dihydrogen phosphate solution.

[0075] 1.3 Treatment methods

[0076] At the same time as modeling, each group was given corresponding drug intervention. Among them, the blank group was not modeled and no treatment was performed; the model group was modeled and no treatment was performed; the penicillamine group was gavaged with a concentration of 125mg / Kg penicillamine solution, and the dose was 1mL / 100g. When the ginseng flower essential oil high-dose group, each mouse was evenly smeared with 10μL of the Chinese medicine compound essential oil of the formula described in Example 1; when the ginseng flower essential oil low-dose group, each mouse was evenly smeared with 5μL of the Chinese medicine compound essential oil of the formula described in Example 1; each medication group was given the corresponding drug after modeling every day, once a day, for 6 consecutive weeks. After the end of the medication, the samples were collected and the relevant indicators were tested.

[0077] 1.4 Detection indicators

[0078] 1.4.1 Effects on serum inflammatory factor levels in scleroderma model mice

[0079] After 6 weeks of administration, blood was collected from the mice through vein and centrifuged at 4000 r / min for 10 min. The supernatant was taken and the expression levels of IL-6, IL-10, IL-17 and TNF-α inflammatory factors in the mouse serum were detected by ELISA. The detection was performed according to the instructions of the ELISA kit, and the OD value was measured at a wavelength of 450 nm by an enzyme reader.

[0080] 1.4.2 Effects on the expression of TGF-β, CTGF and VEGF in the skin homogenate of scleroderma model mice

[0081] After 6 weeks of administration, the back skin of mice was collected, 0.9% sodium chloride solution was added, and the skin was ground into a 10% tissue homogenate. The skin was centrifuged at 4000 r / min for 10 min, and the supernatant was collected. The expression levels of TGF-β, CTGF and VEGF in the skin homogenate were detected by ELISA, and the OD value was measured at a wavelength of 450 nm by an enzyme marker.

[0082] 1.4.3 Effects on the content of hydroxyproline in the skin of scleroderma model mice

[0083] After 6 weeks of administration, the skin of the back of the mice was taken and hydrolyzed by alkaline hydrolysis. The hydroxyproline (HYP) assay kit was used for detection. The wavelength of the microplate reader was set to 550nm, and the absorbance was measured at this wavelength. The HYP content was calculated based on the absorbance.

[0084] 1.4.4 Effect on the skin fibrosis index of scleroderma model mice

[0085] The skin on the back of the mouse was taken for Masson staining to evaluate the fibrosis index. After staining, collagen fibers appeared blue, muscle fibers appeared red, and cell nuclei appeared black. The images of Masson-stained skin tissue sections were collected under a high-power microscope using a pathological section scanner and transferred to a computer. Three fields of view were randomly selected from the sections of each mouse, and blue (collagen fibers) was taken as positive. The skin collagen fibrosis index (the percentage of the blue area positive for collagen to the total area of ​​the tissue) was analyzed using imageJ software to evaluate the degree of fibrosis in the mouse.

[0086] 1.4.5 Effects on skin tissue morphology and dermal thickness in scleroderma model mice

[0087] The back skin of the mouse was fixed with 4% paraformaldehyde solution, embedded in paraffin and sliced, stained with hematoxylin-eosin (HE), the nucleus turned blue-purple, the cytoplasm turned red, and the pathological changes of the mouse skin were observed. The thickness of the mouse dermis was measured using digital pathology software, and 3 points were selected for each mouse skin for measurement and the average value was taken. Semi-quantitative analysis of mouse skin pathology was performed: "-" The structure of each layer of the skin is intact, and the cells are arranged regularly and neatly; "+" The skin is slightly thickened and the fibrous hyperplasia is mild; "++" The dermis of the skin is significantly thickened, the fibrous tissue proliferates a lot, and a large area of ​​subdermal fat tissue is invaded.

[0088] 1.5 Data processing

[0089] Graphpad Prism 8.2.0 statistical software was used for statistical analysis. The comparison between the two groups of data was performed using the t-test, P < 0.05 indicated a statistically significant difference, P < 0.01 indicated a significant statistical difference, and all data were expressed as mean ± standard deviation. The rank data were analyzed by Radit test and graphed using Graphpad Prism 8.2.0 software.

[0090] 2 Results

[0091] 2.1 Effects on serum IL-6, IL-10, IL-17, and TNF-α levels in scleroderma model mice

[0092] like Figure 1 As shown in the figure, compared with the blank group, the serum IL-6 ( Figure 1 A) IL-10 ( Figure 1 B) IL-17 Figure 1 C) TNF-α ( Figure 1D) levels were significantly increased (P<0.01). Compared with the model group, the serum IL-6 and IL-17 levels of the mice in the penicillamine group and the serum IL-6, IL-10, and IL-17 levels of the mice in the high-dose angelica flower essential oil group were significantly decreased (P<0.01), the serum TNF-α level of the mice in the high-dose group was significantly decreased (P<0.05), the serum IL-10 and TNF-α levels of the mice in the penicillamine group and the serum IL-6, IL-17, and TNF-α levels of the mice in the low-dose group were significantly decreased (P<0.05).

[0093] 2.2 Effects on TGF-β, CTGF, and VEGF levels in skin homogenates of scleroderma model mice

[0094] like Figure 2 As shown in the figure, compared with the blank group, the expression of TGF-β( Figure 2 A) CTGF ( Figure 2 B) VEGF ( Figure 2 Compared with the model group, the levels of TGF-β, CTGF, VEGF in the penicillamine group, high-dose group and CTGF in the low-dose group were significantly decreased (P < 0.01), and the levels of TGF-β and VEGF in the low-dose group were significantly decreased (P < 0.05).

[0095] 2.3 Effect of angelica flower essential oil on hydroxyproline content and fibrosis index in scleroderma mice

[0096] like Figure 3 As shown, compared with the blank group, the hydroxyproline content in the model group ( Figure 3 A) and fibrosis index ( Figure 3 B) increased significantly (P<0.01). Compared with the model group, the hydroxyproline content in the penicillamine group and the high-dose group was significantly reduced (P<0.01), and the hydroxyproline content in the low-dose group was significantly reduced (P<0.05); the fibrosis index in the penicillamine group, the high-dose group and the low-dose group was significantly reduced (P<0.01).

[0097] 2.4 Effects on the skin tissue morphology of scleroderma model mice

[0098] like Figure 4As shown in Table 3, compared with the blank group, the thickness of the dermis of the mice in the model group increased significantly (P < 0.01), compared with the model group, the thickness of the dermis in the penicillamine group and the high-dose group decreased significantly (P < 0.01), and the thickness of the dermis in the low-dose group decreased significantly (P < 0.05). As shown in Table 3, by Ridit test, compared with the blank group, the skin tissue of the model group showed significant pathological changes (P < 0.01). Compared with the model group, the high-dose group of ginseng flower essential oil and the penicillamine group could significantly improve the pathological changes of the skin tissue of mice (P < 0.01), and the low-dose group could significantly improve the pathological changes of the skin tissue of mice (P < 0.05).

[0099] Table 3 Effects of angelica flower essential oil on skin tissue morphology of scleroderma model mice (n=12)

[0100] Group Dosage Quantity(n) - + ++ P Blank Group - 12 12 0 0 ** Model Group - 12 0 2 10 - Penicillamine group 125 mg / kg 12 2 8 2 ** High-dose group of ginseng flower essential oil <![CDATA[5μL / cm 2 ]]> 12 1 9 2 ** Small dose group of ginseng flower essential oil <![CDATA[2.5μLcm 2 ]]> 12 0 8 4 *

[0101] Note: *p<0.05, **p<0.01 compared with the model group.

[0102] 3 Conclusion

[0103] External use of radix sphenantherae essential oil (the Chinese medicinal compound essential oil of the present invention) has a good therapeutic effect on scleroderma model mice, can significantly reduce the level of inflammatory factors in mouse serum and the levels of TGF-β, CTGF, and VEGF in skin tissue homogenate, reduce the content of hydroxyproline in skin tissue, improve skin fibrosis, improve skin pathological changes, and reduce the thickness of the skin dermis.

[0104] Test Example 3

[0105] 1 Clinical Data

[0106] This group of cases is a total of 112 patients with localized scleroderma who were admitted to the First Affiliated Hospital of Henan University of Traditional Chinese Medicine from January 2021 to December 2022. They were randomly divided into a control group and an observation group with 56 cases in each group. There were 24 males and 32 females in the control group, aged 21-65 years old, with an average age of (39.87±6.74) years, a course of disease of 1-10 years, and an average of (5.83±2.47) years; the skin hardness score was 10-37 points, with an average of (20.47±9.84) points. There were 28 males and 28 females in the observation group, aged 18-63 years old, with an average age of (41.22±8.85) years, a course of disease of 1-15 years, and an average of (6.41±3.05) years; the skin hardness score was 12-40 points, with an average of (22.49±11.08) points. There was no statistically significant difference in the general data between the two groups (P>0.05), and they were comparable.

[0107] Both groups met the criteria of "Chinese Medicine Disease Diagnosis and Efficacy Standards·Diagnostic Basis, Syndrome Classification, and Efficacy Evaluation of Skin Bi", and were informed and signed informed consent. Pregnancy or breastfeeding, allergy to study drugs, use of steroid drugs within 14 days before enrollment, and severe heart, brain, liver, kidney, and mental diseases were excluded.

[0108] 2 Treatment methods

[0109] Control group: oral prednisone acetate tablets (5 mg / tablet, dose 0.2 mg / kg·d); oral penicillamine tablets (125 mg / tablet, one tablet each time, twice a day).

[0110] Observation group: On the basis of the treatment of the control group, the patients were given the topical cream preparation prepared with the Chinese herbal compound essential oil described in Example 1, wherein the effective content of the Chinese herbal compound essential oil was 5%, and the cream preparation was applied to the affected area twice a day.

[0111] Both groups were treated for 6 months.

[0112] 3 Observation indicators

[0113] The skin hardness score was scored using the Steen VD scoring standard; the joint pain score was scored using the VAS scoring standard; and the joint function score was scored using the Kaham A scoring standard. The lower the score, the better the improvement.

[0114] SPSS 19.0 was used to process the data, and the quantitative data were expressed as mean ± standard deviation (SD). t test was used for the expression of count data (%) and χ test was used for the expression of count data (%). 2 Test, P<0.05 was considered statistically significant.

[0115] 4 Efficacy criteria

[0116] Healed: Damaged skin returns to normal or greater than 95%.

[0117] Significantly effective: damaged skin recovered by 70% to 95%.

[0118] Effective: Damaged skin recovers 30% to 70%.

[0119] Invalid: Does not meet the “valid” criteria.

[0120] 5 Results

[0121] Table 4 Comparison of skin hardness, joint pain and joint function scores between the two groups (points, )

[0122]

[0123] Note: In the summary of scoring items of the observation group and the control group, * compared with before treatment, * P < 0.05; in each scoring item, # Compared with the control group, # P<0.05.

[0124] Table 5 Comparison of clinical efficacy between the two groups

[0125] Group example recover (n) Significantly effective (n) Effective(n) Invalid(n) Cure rate (%) Observation Group 56 19 29 6 2 85.71％ Control group 56 6 17 19 14 41.07％

[0126] The above results show that the combination of the Chinese herbal compound essential oil of the present invention and western medicine can significantly improve the therapeutic effect of localized scleroderma, and the cure rate can reach 85.71%, which is significantly higher than the cure rate of the control group (41.07%). The Chinese herbal compound essential oil of the present invention can significantly improve the patient's skin hardness, joint pain, and promote the recovery of joint function.

[0127] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Chinese medicinal compound essential oil for external use for scleroderma, characterized in that: The traditional Chinese medicine compound essential oil is composed of the following raw materials in parts by weight: 5-15 parts of wormwood essential oil, 5-15 parts of peony bark essential oil, 5-15 parts of salvia miltiorrhiza essential oil, 3-10 parts of zanthoxylum essential oil, 3-10 parts of cnidium monnieri essential oil, 3-10 parts of clove essential oil, 3-10 parts of angelica essential oil and 3-10 parts of borneol essential oil.

2. The topical Chinese medicinal compound essential oil for scleroderma according to claim 1, characterized in that: The traditional Chinese medicine compound essential oil is composed of the following raw materials in parts by weight: 8-12 parts of wormwood essential oil, 8-12 parts of peony bark essential oil, 8-12 parts of salvia miltiorrhiza essential oil, 5-8 parts of zanthoxylum essential oil, 5-8 parts of cnidium monnieri essential oil, 5-8 parts of clove essential oil, 5-8 parts of angelica essential oil and 5-8 parts of borneol essential oil.

3. The topical Chinese medicinal compound essential oil for scleroderma according to claim 1, characterized in that: The traditional Chinese medicine compound essential oil is composed of the following raw materials in parts by weight: 9 parts of wormwood essential oil, 9 parts of peony bark essential oil, 9 parts of salvia miltiorrhiza essential oil, 6 parts of zanthoxylum essential oil, 6 parts of cnidium monnieri essential oil, 6 parts of clove essential oil, 6 parts of angelica essential oil and 6 parts of borneol essential oil.

4. The external Chinese medicinal compound essential oil for scleroderma according to any one of claims 1 to 3, characterized in that: The essential oils of the above components are obtained by extracting the original plants using conventional essential oil extraction methods, including pressing extraction, steam distillation extraction, and supercritical carbon dioxide extraction.

5. The external Chinese medicinal compound essential oil for scleroderma according to claim 4, characterized in that: The wormwood essential oil, peony bark essential oil and salvia miltiorrhiza essential oil are respectively made from wormwood, peony bark and salvia miltiorrhiza as raw materials, and the volatile oil is extracted by supercritical carbon dioxide method, and the distillation concentration is controlled at more than 99%; the Zanthoxylum bungeanum essential oil is made from Zanthoxylum bungeanum as raw material, and the volatile oil is extracted by pressing method, and the distillation concentration is controlled at more than 99%; the Cnidium monnieri essential oil, clove essential oil, Angelica sinensis essential oil and borneol essential oil are respectively made from Cnidium monnieri, clove, Angelica sinensis and borneol as raw materials, and the volatile oil is extracted by steam distillation method, and the distillation concentration is controlled at more than 99%.

6. The use of the Chinese herbal compound essential oil as claimed in any one of claims 1 to 3 for preparing an external preparation for treating scleroderma, wherein a medically acceptable adjuvant is added to the Chinese herbal compound essential oil to prepare an external dosage form available to the human body, and the dosage form is an ointment, a liniment, a spray, or a tincture.

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