Application of aloe-emodin in treating or preventing acne

Abstract

The invention discloses an application of aloe-emodin in preparation of medicines, foods or health-care foods for treating, preventing or improving acnes.

Description

Technical Field

[0001] The present invention relates to the fields of traditional Chinese medicine, food, and health food, and particularly to the use of aloe-emodin in the treatment or prevention of acne. Background Art

[0002] Acne, also known as pimples, comedones, and acne vulgaris, is a chronic inflammatory skin disease that commonly occurs in adolescents and affects the hair follicles and sebaceous glands of the face. Due to the complex etiology of acne, its pathogenesis has not been fully elucidated. Although there are many treatment methods, no ideal and recognized treatment plan has been found so far. Therefore, it is urgent to actively explore suitable products for treating acne.

[0003] Clinically, it is treated by methods such as anti-inflammatory and bactericidal, adjusting hormone levels, and removing cutin. These treatment methods have a relatively quick onset of effect, but require a long treatment time and are prone to recurrence after stopping the medication. With the development needs of the era of great health and the change of the disease spectrum, the pathogenesis and treatment methods of acne have become research hotspots.

[0004] Since the pathogenesis of acne has not been fully elucidated, existing drugs mainly target improving the keratinization of the hair follicle sebaceous gland duct, dissolving comedones, and anti-inflammation, such as retinoids, hormones, and antibacterial drugs. Most drugs have relatively large side effects and often treat the symptoms rather than the cause. Traditional Chinese medicine has diverse means in the treatment of acne. Clinically, the traditional Chinese medicine treatment principles for acne include dispersing lung heat, clearing the bowels and promoting diuresis, purging heart fire, cooling blood and removing stasis, soothing the liver and regulating qi, softening hardness and dissipating nodules, dredging the spleen, removing dampness and detoxifying, nourishing the kidney and nourishing yin, and cooling blood and promoting blood circulation. Generally speaking, clinicians still mainly use methods such as clearing heat and detoxifying, cooling blood and promoting blood circulation in the treatment. More clinicians choose a comprehensive therapy of internal and external treatment. At present, there are also many methods for external treatment of acne in traditional Chinese medicine, including traditional Chinese medicine masks, traditional Chinese medicine fumigation, acupuncture point treatment, acupoint injection, pricking blood and cupping, bloodletting, fire needle, and auricular point therapy, etc., and they are comprehensively applied to improve the curative effect. Summary of the Invention

[0005] One or more embodiments of the present application provide the use of aloe-emodin in the preparation of drugs, foods, or health foods for treating or preventing acne.

[0006] One or more embodiments of the present application provide the use of aloe-emodin in the preparation of drugs, foods, or health foods for improving acne.

[0007] In one or more embodiments, the aloe-emodin is obtained by extraction and separation.

[0008] In one or more embodiments, the aloe-emodin is obtained by extraction and separation from Aloe barbadensis Miller or Rheum palmatum L.

[0009] In one or more embodiments, the aloe-emodin is obtained by chemical synthesis.

[0010] In one or more embodiments, the purity of the aloe-emodin is equal to or greater than 95% by weight, such as 95% by weight or 98% by weight.

[0011] In one or more embodiments, common experimental animals, rats, were selected as the research subjects. According to the literature reports, Propionibacterium acnes was injected into the auricles of rats and oleic acid was applied topically to replicate the acne model. The aloe-emodin was used to intragastrically intervene in the establishment of the acne model by prophylactic administration, and the effect of aloe-emodin on acne in the auricles of rats was observed.

[0012] In one or more embodiments, aloe-emodin improves the clinical symptoms of acne, reduces skin inflammatory reactions, and regulates blood flow to improve the repair of subcutaneous vascular injuries.

[0013] In one or more embodiments, the aloe-emodin is prepared into an oral preparation with a dose of 200 mg / kg body weight for rats (equivalent to 33.3 mg / kg body weight per day for adults).

[0014] As the main effective ingredient of aloe and processed rhubarb, which are raw materials that can be used in health foods in the List of Articles that Can Be Used in Health Foods in the Notice of the Ministry of Health on Further Standardizing the Management of Raw Materials for Health Foods (No. 51

[2002] of the Ministry of Health of the People's Republic of China, Annex 2), aloe-emodin has anti-tumor, memory-improving, anti-inflammatory, anti-pathogenic microorganism, cell autophagy and apoptosis-regulating effects in modern research.

[0015] In one or more embodiments, aloe-emodin exhibits good efficacy in treating acne, especially in regulating hormone levels and hormone receptor levels, inhibiting inflammatory response factors and inhibiting cell apoptosis.

[0016] In one or more embodiments, aloe-emodin is obtained by extraction and separation from Aloe vera L. or Rheum palmatum L., and the monomer purity is ≥95% by weight, such as 95% by weight or 99% by weight.

[0017] In one or more embodiments, animals (such as rats) orally administered 200 mg / kg (equivalent to 33.3 mg / kg body weight per day for adults) of aloe-emodin can significantly improve the apparent symptoms of auricular acne and do not show diarrhea.

[0018] In one or more embodiments, aloe-emodin improves acne based on the traditional Chinese medicine theory of "the lung and the large intestine are interior-exteriorly related" and "the lung governs the skin and hair". Using the idea of promoting defecation to treat acne shows originality and realizes the effects of anti-inflammation and improving microcirculation. The modern scientific interpretation and application are the specific manifestations of "treating acne by promoting defecation". Description of the Drawings

[0019] Figure 1 Indicates the expression of apoptosis-related proteins in each group of rats (n = 3).

[0020] Figure 2 Indicates the expression of AR and ACC1 in auricle tissue by immunofluorescence.

[0021] Figure 3 Indicates the apparent changes in the auricles of each group of rats, where A is the control group, B is the model group, and C is the emodin group.

[0022] Figure 4 Indicates the pathological changes in the auricles of each group of rats (HE staining × 200), where A is the control group, B is the model group, and C is the emodin group.

[0023] Figure 5 Indicates the auricles of each group of rats and the laser speckle imaging diagrams, where: a1 is the color image of the control group, a2 is the black-and-white image of the control group, a3 is the laser speckle image of the control group, b1 is the color image of the model group, b2 is the black-and-white image of the model group, b3 is the laser speckle image of the model group, c1 is the color image of the emodin group, c2 is the black-and-white image of the emodin group, and c3 is the laser speckle image of the emodin group. Specific implementation mode

[0024] The following examples are exemplary descriptions of this application and do not limit the protection scope of this application.

[0025] Example

[0026] Experimental materials

[0027] 1.1 Experimental animals

[0028] Eighteen healthy male SPF-grade SD rats, weighing 200 ± 20 g. All animals were purchased in advance from Beijing Spearf Bio-Technology Co., Ltd. and raised in the animal house of Beijing University of Chinese Medicine. The laboratory temperature was 22 - 24 °C, the humidity was 50 - 70%, and the normal artificial light produced a circadian rhythm of 12 h / 12 h light and dark. They had free access to water and food, maintained a quiet environment, and were adaptively fed for 3 days, with 3 rats in each cage. Animal license number: SCXK(Beijing)2019 - 0010.

[0029] 1.2 Main experimental drugs and reagents

[0030] The main experimental drugs and reagents are shown in Table 1.

[0031] Table 1 Main experimental drugs and reagents

[0032]

[0033]

[0034]

[0035]

[0036] 1.3 Main Instruments and Equipment for Experiments

[0037] The main instruments and equipment for experiments are shown in Table 2.

[0038] Table 2 Main Instruments and Equipment for Experiments

[0039]

[0040]

[0041] 2 Experimental Methods

[0042] 2.1 Animal Grouping and Drug Administration

[0043] Eighteen SD rats were randomly divided into 3 groups of 6 rats each by the random number table method, namely the control group (Control group, Control), the model group (Model group, Model), and the aloe-emodin group (Aloe-emodin group, AE). The control group and the model group were given intragastric administration of 10 mL / kg of 5% carboxymethyl cellulose sodium (CMC-Na), and the aloe-emodin group was given intragastric administration of 200 mg / kg of aloe-emodin. The aloe-emodin was evenly suspended in CMC-Na for 14 consecutive days. On the 15th day, the rats were anesthetized with 1% sodium pentobarbital, blood was taken from the abdominal aorta, serum was separated and stored in a -80°C refrigerator. The anterior 1 / 2 of the right auricle was placed in 4% paraformaldehyde fixative, and the posterior 1 / 2 was frozen in liquid nitrogen and then stored in a -80°C refrigerator.

[0044] 2.2 Preparation of Rat Auricular Acne Model Induced by Propionibacterium acnes

[0045] Preparation of Propionibacterium acnes rat model: Except for the control group, each rat was injected with 0.05 mL of Propionibacterium acnes bacterial solution (bacterial solution concentration > 6×10 7 cfu / mL) into the right auricle every day, and 0.5 mL of oleic acid was applied to the injection site for 14 consecutive days to establish a Propionibacterium acnes rat acne model.

[0046] 2.3 Physical Sign Observation and Body Weight Detection

[0047] Record the body weights on the 7th and 14th days, and observe the changes in the hair color, mental state, and autonomous behavior of the rats.

[0048] 2.4 Detection of Serum Hormones and Inflammatory Response Factor Levels in Auricular Tissue Homogenate by ELISA

[0049] Take the rat serum in an -80°C refrigerator and detect T, E2, TLR2, and TLR4 in rats of each group according to the instructions of the ELISA kit to compare the differences among groups.

[0050] Take the rat auricles in an -80°C refrigerator, add physiological saline at a ratio of 1:19, grind on ice, centrifuge at 3000g to obtain the supernatant, measure the total protein according to the instructions of the total protein quantitative determination kit, and detect the expression levels of IL-1β, IL-10, and TNF-α in the auricle tissue homogenates of rats in each group according to the instructions of the ELISA kit to compare the differences among groups.

[0051] 2.5 WB detection of apoptosis factors Casp-3, Bcl-2, Bax, and Cytc

[0052] Take the rat auricles in an -80°C refrigerator, add protease inhibitor, and prepare auricle tissues on ice. Centrifuge the supernatant to 4000g and perform tests strictly according to the instructions of the kit. Extract total protein from the auricle tissues of each group and quantify the protein according to the instructions of the protein quantification kit (Nanjing Jiancheng, China). Separate the proteins by 10% SDS-PAGE, transfer them electrophoretically to a polyvinylidene fluoride membrane (PVDF), incubate with TBST containing 5% skim milk at room temperature for 1 hour, then wash with TBST 5 times, 5 minutes each time. Add diluted Casp-3 (1:1000), Bcl-2 (1:1000), Bax (1:1000), Cytc (1:1000), and β-Actin (1:5000) respectively, and incubate the membrane overnight at 4°C. The next day, wash the membrane 5 times with TBST, 5 minutes each time, and add the corresponding horseradish peroxidase-labeled secondary antibody (1:5000). Incubate the membrane at room temperature for 1 hour, then rinse it 5 times in TBST, 5 minutes each time. According to the instructions of the electrochemiluminescence (ECL) kit, these membranes are quickly exposed. ImageJ software is used for quantitative analysis of the images. Each experiment for each protein is repeated 3 times.

[0053] 2.6 Immunofluorescence detection of AR and ACC1

[0054] Repeat the 2.5 pathological staining section operation. Place the pathological section on a plastic rack, then put it into an oven at 60 °C for more than 1 h. Take it out from the 60 °C oven → Xylene IV for 15 min → Xylene V for 15 min → Absolute ethanol IV for 10 min → Absolute ethanol V for 10 min → 95% ethanol III for 10 min → 90% ethanol II for 10 min → 90% ethanol III for 5 min → 80% ethanol II for 5 min → 0.01 M PBS for 3 min × 3 times → 0.01 M citrate buffer in a microwave at high power for 2 min → low power for 5 min → cool naturally (about 1 h) / cool in a normal temperature water bath (about 15 min) → 0.01 M PBS for 3 min × 3 times → transfer to a histochemistry wet box (one box can hold 20 slides) → draw a circle with a histochemistry pen → 3% H 2 O 2 15 min → 0.01 M PBS for 3 min × 3 times → 5% BSA (bovine serum albumin, diluted with PBS) incubate at room temperature for 30 min (about 50 - 100 μl per slide) / 10% goat serum (diluted with PBS) incubate for 1 h → add the primary antibody (AR (1:500 dilution), ACC1 (1:500 dilution), diluted with PBS, about 50 - 100 μl per slide) and incubate overnight in a 4 °C refrigerator → rewarm for 30 min → 0.01 M PBS for 3 min × 3 times → add the fluorescent secondary antibody (prepared freshly before use, protect from light during preparation, about 50 - 100 μl per slide) and incubate for 1 h in the dark → 0.01 M PBS for 3 min × 3 times → add DAPI to stain the nucleus and incubate for 5 min in the dark → 0.01 M PBS for 3 min × 3 times → seal the slide with anti - quenching gel → take pictures with a fluorescence microscope.

[0055] 2.7 HE staining to detect the auricle pathology

[0056] Fix the upper half of the right ear in 4% paraformaldehyde for 2 - 3 days → Rinse with tap water 3 times → Incubate in 0.1M PBS overnight → Trim the tissue block and place it in an embedding cassette → Incubate in 70% ethanol for 3 hours → Incubate in 80% ethanol I overnight → Incubate in 90% ethanol I for 2 hours → Incubate in 95% ethanol I for 2 hours → Incubate in 95% ethanol II for 2 hours → Incubate in absolute ethanol I for 1 hour → Incubate in absolute ethanol II for 1 hour → Incubate in absolute ethanol III for 1 hour → Incubate in xylene I for 30 minutes → Incubate in xylene II for 30 minutes → Incubate in xylene III for 30 minutes → Incubate in paraffin wax I for 1 hour → Incubate in paraffin wax II for 1 hour → Incubate in paraffin wax III for 1 hour → Embed in paraffin wax, cut sections at 3μm, place the sections on a slide rack, take the slides out of the 60°C oven → Incubate in xylene IV for 30 minutes → Incubate in xylene V for 30 minutes → Dip in absolute ethanol IV up and down 10 times → Dip in absolute ethanol V up and down 10 times → Dip in 95% ethanol III up and down 10 times → Dip in 80% ethanol II up and down 10 times → Wash with tap water for 3 minutes → Stain with hematoxylin for 5 minutes → Wash with tap water for 15 minutes → Differentiate with 1% hydrochloric acid ethanol solution for 3 seconds → Wash with tap water for 1 minute → Treat with 1% dilute ammonia water for 3 seconds → Stain with 1% eosin for 5 minutes → Dip in 80% ethanol III up and down 10 times → Incubate in 95% ethanol IV for 1 minute → Incubate in 95% ethanol V for 1 minute → Incubate in absolute ethanol VI for 2 minutes → Incubate in absolute ethanol VII for 2 minutes → Incubate in absolute ethanol VIII for 2 minutes → Incubate in xylene VI for 10 minutes → Incubate in xylene VII for 10 minutes → Incubate in xylene VIII for 10 minutes → Mount with neutral resin and observe the pathological changes of the auricles of rats in each group under a 200-fold light microscope.

[0057] 2.8 Auricle blood vessel-related indicators

[0058] Collect rat serum and measure the contents of NO and ET-1 in the serum according to the kit instructions, and measure the contents of HIF-1α and VEGF in the auricle tissue homogenate.

[0059] 2.9 Laser speckle detection of auricle blood flow

[0060] Anesthetize rats in each group by intraperitoneal injection of 1% sodium pentobarbital. First, fix in the dorsal position, flatten the right ear back of the rat, place the CCD camera of the moor FLPI-2 speckle blood flow real-time imaging system 20 - 30 cm above the vertical auricle, adjust the focus until the image is clear, set the instrument parameters to the high-resolution pixel (752×580 pixels) mode, the acquisition speed is 25 frames / s, obtain one blood flow image per second, the interval time is 1 s, the exposure time is 40 ms, continuously sample each rat for 10 - 20 s, and store them one by one.

[0061] 2.10 Data processing

[0062] Analyze using SPSS 20.0 statistical software. The measurement data of each group are expressed as It is shown that for each group of data, a normality test is performed. If each group is normal and the variances are homogeneous, a one-way ANOVA (F-test) is conducted, and at the same time, a least significant difference t-test (LSD-t) is performed for pairwise comparison. If each group is normal but the variances are not homogeneous, a Welch test (approximate F-test, F-test) is carried out, and at the same time, a Dunnett's T3 pairwise comparison is performed. If any group of data is non-normal, a Kruskal-Wallis H test (H-text) is conducted, and then pairwise comparison is carried out, with P < 0.05 taken as the statistical significance criterion.

[0063] 3 Results

[0064] 3.1 General conditions

[0065] In the control group, the hair of the rats had normal luster, without restlessness or listlessness, the mental state was good, and the activities were normal; in the model group, the hair around the auricles was sparse, and there was often a phenomenon of scratching the auricles, which was a non-grooming behavior; in the aloe-emodin group, the body weight decreased compared with the blank group (P < 0.05), and the phenomenon of scratching the auricles decreased; in the medium-dose and high-dose groups of Yiqing, the mental state of the rats was good, the activity level decreased, the phenomenon of scratching the auricles decreased, and the body weight increased compared with the model group (P < 0.05). The stools in the high-dose group of Yiqing were slightly soft. The body weights are shown in Table 3.

[0066] Table 3 Time changes in body weights of rats in each group ( n = 6, g)

[0067]

[0068] Note: Compared with the control group a P < 0.05, compared with the model group b P < 0.05

[0069] 3.2 Serum hormone levels

[0070] Compared with the control group, the serum T content of the rats in the model group increased significantly, the E2 content decreased significantly, and A / E increased significantly (P < 0.05); compared with the model group, the serum T content of the rats in the aloe-emodin group increased significantly and A / E decreased significantly (P < 0.05). The specific results are shown in Table 4.

[0071] Table 4 Serum hormone levels of rats in each group ( n = 6)

[0072]

[0073] Note: Compared with the control group a P < 0.05, compared with the model group b P < 0.05, compared with the aloe-emodin group c P < 0.05

[0074] 3.3 Levels of inflammatory response factors in tissue homogenates

[0075] Compared with the control group, the contents of IL-1β and TNF-α in the tissue homogenates of rats in the model group were significantly increased (P < 0.05), the contents of serum TLR2 and TLR4 were significantly increased (P < 0.05), and the content of IL-10 in the tissue homogenates was significantly decreased (P < 0.05); compared with the model group, the contents of IL-1β and TNF-α in the tissue homogenates of rats in the emodin group were significantly decreased (P < 0.05), the contents of serum TLR2 and TLR4 were significantly decreased (P < 0.05), and the content of IL-10 was significantly increased (P < 0.05). The specific results are shown in Tables 5 and 6.

[0076] Table 5 Levels of inflammatory response factors in tissue homogenates of rats in each group ( n = 6, ng / g prog)

[0077]

[0078] Note: Compared with the control group a P < 0.05, compared with the model group b P < 0.05, compared with the emodin group c P < 0.05

[0079] Table 6 Levels of Toll-like receptor 2 and 4 in serum of rats in each group ( n = 6, ng / mL)

[0080]

[0081] Note: Compared with the control group a P < 0.05, compared with the model group b P < 0.05, compared with the emodin group c P < 0.05

[0082] 3.4 Expression of apoptosis-related proteins

[0083] Compared with the control group, the expression of Bcl-2 protein decreased in the model group, while the expressions of Casp-3, Bax, and Cytc increased (P < 0.05). Compared with the model group, the emodin group could increase the expression of Bcl-2 protein and decrease the expressions of Casp-3, Bax, and Cytc (P < 0.05). The results are shown in Figure 1 , note: * corresponds to P < 0.05, ** corresponds to P < 0.01.

[0084] 3.5 Expression of AR and ACC1 in auricle tissue by immunofluorescence

[0085] Compared with the control group, the expression levels of AR and ACC1 in the model group were significantly increased (P < 0.05). Compared with the model group, the expression levels of AR and ACC1 in the aloe-emodin group were significantly decreased (P < 0.05). The results are shown as Figure 2 follows, indicating the expression of AR and ACC1 in rats of each group (immunofluorescence, n = 6, ×100). Note: *P < 0.05 compared with the control group, # P < 0.05 compared with the model group.

[0086] 3.6 Changes in auricle thickness

[0087] The auricle tissues of rats in the control group were thin and soft, with clearly visible capillaries, smooth auricle epidermis, and no horny plugs or papules. The auricles of rats in the model group were red and purple, with thickened auricle tissues, and small, thin, black hair follicle horny plugs and raised hair follicle orifices could be seen. The thickness of the left and right auricles of rats in the control group was basically the same, and the thickness of the left ears of rats in each group was basically the same. Compared with the control group, the thickness of the right auricles of the model group and the aloe-emodin group was significantly thicker than that of the left ears (P < 0.05). Compared with the model group, the difference in the thickness of the auricles of both ears in the aloe-emodin group was significantly decreased (P < 0.05). The results are shown in Table 7, Figure 3 . Figure 3 indicating the apparent changes in the auricles of rats in each group.

[0088] Table 7 Difference in auricle thickness of rats in each group ( n = 6, mm)

[0089]

[0090] Note: Compared with the control group a P < 0.05, compared with the model group b P < 0.05

[0091] 3.7 Auricle pathology

[0092] In the control group, the stratum corneum cells were arranged densely and compactly, the boundaries of each cortex were clear, there was no infiltration of inflammatory cells, the sebaceous glands were normal, and no karyopyknosis was seen. In the model group and the low-dose Yiqing group, the spinous layer cells were significantly thickened, the volume of the sebaceous glands increased, the infundibulum of the hair follicle became pot-shaped due to the accumulation of hyperkeratotic substances, local inflammatory cell infiltration was obvious, and karyopyknosis was seen in some cells. In the aloe-emodin group, the thickening of the spinous layer cells was reduced compared with before, the volume of the sebaceous glands was increased compared with the control group but decreased compared with the model group, the accumulation of keratinized substances in the infundibulum of the hair follicle was reduced compared with the model group and the low-dose Yiqing group, there was still inflammatory cell infiltration, and karyopyknosis was reduced compared with the model group. The pathological changes of the auricle skin lesions in rats of each group are shown in Figure 4 .

[0093] 3.8 Levels of blood vessel-related indicators

[0094] Compared with the control group rats, the serum NO content of the model group rats was significantly decreased (P < 0.05), while the serum ET-1 content, and the HIF-1α and VEGF in the tissue homogenate were significantly increased (P < 0.05); compared with the model group rats, the serum NO content of the aloe-emodin group was significantly recovered (P < 0.05), and the serum ET-1 content, and the HIF-1α and VEGF in the tissue homogenate were significantly increased (P < 0.05). The results are shown in Table 8.

[0095] Table 8 Expression levels of auricular vascular factors in rats of each group ( n = 6)

[0096]

[0097] Note: Compared with the blank group a P < 0.05, compared with the model group b P < 0.05, compared with the aloe-emodin group c P < 0.05

[0098] 3.9 Results of auricular laser speckle

[0099] Compared with the control group, the blood flow velocity of the auricles of the model group rats decreased (P < 0.05). Compared with the model group, the aloe-emodin group could recover the blood flow velocity (P < 0.05). The laser speckle imaging of blood flow is shown in Figure 5 , and the blood flow volume values are shown in Table 9.

[0100] Table 9 Laser speckle blood flow analysis ( n = 6)

[0101]

[0102] Note: Compared with the blank group a P < 0.05, compared with the model group b P < 0.05.

Claims

1. Use of aloe-emodin in the preparation of a medicament, food or health food for treating or preventing acne.

2. The use according to claim 1, wherein the aloe-emodin is obtained by extraction and separation; preferably, it is obtained by extraction and separation from Aloe vera L. or Rheum palmatum L.

3. The use according to claim 1, wherein the aloe-emodin is obtained by chemical synthesis.

4. The use according to claim 2 or 3, wherein the purity of the aloe-emodin is equal to or greater than 95% by weight.

5. The use according to claim 1, wherein the aloe-emodin is prepared into an oral preparation with a dose of 33.3 mg / kg body weight per day for adults.

6. Use of aloe-emodin in the preparation of a medicament, food or health food for improving acne.

7. The use according to claim 6, wherein the aloe-emodin is obtained by extraction and separation; preferably, it is obtained by extraction and separation from Aloe vera L. or Rheum palmatum L.

8. The use according to claim 6, wherein the aloe-emodin is obtained by chemical synthesis.

9. The use according to claim 7 or 8, wherein the purity of the aloe-emodin is equal to or greater than 95% by weight.

10. The use according to claim 6, wherein the aloe-emodin is prepared into an oral preparation with a dose of 33.3 mg / kg body weight per day for adults.

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