A combined drug for treating acne

Abstract

The present invention discloses a combined drug for treating acne, which contains bletilla stilbene components and patchouli essential oil for simultaneous or separate administration. By combining bletilla stilbene components and patchouli essential oil in a specific ratio, the present invention significantly inhibits Propionibacterium acnes and acne inflammation, thereby playing a role in treating acne. Moreover, the effect is better than that of either bletilla stilbene components or patchouli essential oil used alone, and is more effective than adapalene gel commonly used at present, showing a synergistic effect and having practical popularization and application value.

Description

Technical Field

[0001] The present invention specifically relates to a combined drug for treating acne. Background Art

[0002] Acne is a chronic inflammatory skin disease of the pilosebaceous unit, mainly affecting the face, back and chest, characterized by the formation of comedones, inflammatory papules, pustules, nodules or cysts. Severe forms of acne can lead to disfigurement and scarring, resulting in emotional distress and a decline in quality of life. Modern medicine believes that the pathogenesis of acne is related to excessive sebum secretion of sebaceous glands, abnormal keratinization of the pilosebaceous duct, imbalance of the skin microbiome, and release of inflammatory mediators. Propionibacterium acnes, as the main pathogen causing acne, inhabits the hair follicles and sebaceous glands of the skin, has the ability to regulate the proliferation of keratinocytes, secretes virulence enzymes involved in sebum degradation (lipase) and tissue damage (hyaluronidase), and triggers or exacerbates the inflammatory response by activating keratinocytes, sebocytes and monocytes to produce pro-inflammatory cytokines. At present, commonly used anti-acne drugs include retinoids, antibiotics, benzoyl peroxide, salicylic acid and hormones, and these drugs can produce varying degrees of adverse reactions, such as skin irritation, skin dryness, organ damage and bacterial drug resistance.

[0003] Traditional Chinese medicine believes that acne belongs to the category of "facial pustules" and "wind acne of the lung". It has unique advantages in the treatment of acne, with the characteristics of small adverse reactions, low recurrence rate and long-lasting curative effect. Bletilla striata is the dried tuber of the orchid plant Bletilla striata (Thunb.) Reichb.f., which has the effects of arresting bleeding and promoting granulation. Its clinical applications of "whitening, removing acne and making the skin more radiant" are recorded in "Supplement to the Thousand Golden Remedies", "Taiping Holy Prescriptions" and "Forbidden Prescriptions of the Lu Mansion", etc., and it has a certain curative effect in the treatment of skin diseases. Modern research shows that stilbene components (dibenzyl and phenanthrene) of Bletilla striata are one of the important active components of Bletilla striata, with various pharmacological activities such as anti-inflammatory, antibacterial and anti-tumor, and have good development value in beauty and skin care. Pogostemon oil is the main active ingredient of Pogostemon cablin, with various pharmacological activities such as anti-inflammatory, antibacterial and antioxidant, and is widely used in the manufacture of soaps, perfumes, body lotions and detergents. Some studies have shown that pogostemon essential oil can effectively control skin infections and odors in patients with ulcers, skin tears, skin abrasions and pressure sores, and shorten the healing time. It can also maintain the structural integrity of the skin irradiated by ultraviolet rays and prevent photoaging and recurrence of skin lesions. At present, the research on stilbene components of Bletilla striata mostly focuses on anti-tumor, and there is no relevant research and report on the combination of stilbene components of Bletilla striata and pogostemon oil in the treatment of acne. Summary of the Invention

[0004] To solve the above problems, the present invention provides the use of stilbene components of Bletilla striata combined with pogostemon oil in the preparation of a drug for treating acne;

[0005] Furthermore, the bletilla stilbene component is the substance obtained by purifying the ethanol extract of bletilla striata using column chromatography;

[0006] The patchouli essential oil is an organic substance extracted from pogostemon cablin using steam distillation.

[0007] Furthermore, the bletilla stilbene component is the substance obtained by concentrating the eluate eluted with 95% ethanol after loading the 65% ethanol extract of bletilla striata onto a chromatography column.

[0008] Even further, the chromatography column is preferably a polyamide column.

[0009] Furthermore, the drug is a drug for inhibiting acne inflammation.

[0010] Furthermore, the drug is a drug for inhibiting propionibacterium acnes.

[0011] Furthermore, the mass ratio of the bletilla stilbene component to the patchouli essential oil is 1-2:1-2, preferably 2:1.

[0012] The present invention also provides a combined drug for treating acne, which contains the bletilla stilbene component and the patchouli essential oil for simultaneous or separate administration.

[0013] Furthermore, the mass ratio of the bletilla stilbene component to the patchouli essential oil is 1-2:1-2.

[0014] Even further, the mass ratio of the bletilla stilbene component to the patchouli essential oil is 2:1.

[0015] The present invention also provides a composition for treating acne, which is an oral preparation prepared by adding pharmaceutically acceptable excipients using the bletilla stilbene component and the patchouli essential oil as active ingredients.

[0016] Furthermore, it is characterized in that the mass ratio of the bletilla stilbene component to the patchouli essential oil is 1-2:1-2.

[0017] Even further, the mass ratio of the bletilla stilbene component to the patchouli essential oil is 2:1.

[0018] Even further, the oral preparation is a granule, powder, pill, capsule or solution.

[0019] Finally, the present invention provides a preparation method of the aforementioned composition, which includes the following steps:

[0020] Weigh the bletilla stilbene component and the patchouli essential oil according to the ratio, and then add pharmaceutically common excipients or auxiliary components, and mix them evenly to obtain the product.

[0021] The combined drug for treating acne of the present invention significantly inhibits Propionibacterium acnes and acne inflammation by combining the bletilla striata and stilbene components with patchouli oil in a specific ratio, thereby playing a role in treating acne, and the effect is better than that of using either the bletilla striata and stilbene components or the patchouli oil alone, and the efficacy is better than the currently commonly used adapalene gel, playing a synergistic effect, and having practical promotion and application value.

[0022] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0023] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Effects of Bletilla striata and Patchouli oil 2:1 on changes in auricular lesions in mice

[0025] Figure 2 Effects of Bletilla striata components and Patchouli oil 2:1 on pathological changes of mouse ear tissue (HE staining)

[0026] Figure 3 Effects of Bletilla striata and Patchouli oil 2:1 on the growth of Propionibacterium acnes DETAILED DESCRIPTION

[0027] The raw materials and equipment used in the specific implementation of the present invention can be obtained through commercial purchase, among which Bletilla striata and Patchouli were purchased from Sichuan Province Chinese Medicine Pieces Co., Ltd.

[0028] Example 1 Pharmaceutical composition of the present invention

[0029] ① Preparation of Bletilla striata stilbene components

[0030] Take the medicinal material of Bletilla striata and grind it into coarse powder. Reflux extract it with 8 times the volume of 65% ethanol for 3 times, 1 hour each time. Combine the extracts and load them on a polyamide column (80-120 mesh, diameter-to-height ratio 1:5). Elute with 6 times the column volume of 95% ethanol. Concentrate the eluate to obtain the stilbene components of Bletilla striata. Calculated by the paste yield, the stilbene component yield is 0.4%.

[0031] ②Preparation of patchouli oil

[0032] Patchouli was taken without crushing or soaking, and water was directly added to it for extraction by steam distillation for 8 hours, with an oil yield of 0.90%.

[0033] ③Drug preparation

[0034] Mix the Bletilla striata resveratrol components and Pogostemon cablin oil in a mass ratio of 1:1, and add pharmaceutically acceptable excipients to obtain the product.

[0035] Example 2 The pharmaceutical composition of the present invention

[0036] ① Preparation of Bletilla striata resveratrol components

[0037] Same as Example 1

[0038] ② Preparation of Pogostemon cablin oil

[0039] Same as Example 1

[0040] ③ Preparation of the drug

[0041] Mix the Bletilla striata resveratrol components and Pogostemon cablin oil in a mass ratio of 1:2, and add pharmaceutically acceptable excipients to obtain the product.

[0042] Example 3 The pharmaceutical composition of the present invention

[0043] ① Preparation of Bletilla striata resveratrol components

[0044] Same as Example 1

[0045] ② Preparation of Pogostemon cablin oil

[0046] Same as Example 1

[0047] ③ Preparation of the drug

[0048] Mix the Bletilla striata resveratrol components and Pogostemon cablin oil in a mass ratio of 2:1, and add pharmaceutically acceptable excipients to obtain the product.

[0049] The beneficial effects of the present invention are illustrated by the following test examples.

[0050] Test Example 1 Evaluation of the anti-inflammatory activities of Bletilla striata extract, Pogostemon cablin extract and their combinations in different ratios 1 Experimental materials

[0051] 1.1 Animals

[0052] 88 SPF-grade male KM mice, with a body weight of (20 ± 1) g, provided by Chengdu Dashuo Experimental Animal Co., Ltd., and the experimental animal production license number is SCXK (Sichuan) 2020-030. The animals were raised in an environment with a temperature of 20°C to 25°C and a relative humidity of 40% to 70%, and they could drink water and eat freely.

[0053] 1.2 Drugs and reagents

[0054] The aqueous extract of Bletilla striata (Bletilla striata was decocted with 8 times the amount of water for 3 times, each extraction for 2 hours, and the collected aqueous extract was concentrated into an extract), the aqueous extract of Pogostemon cablin (Pogostemon cablin was decocted with 8 times the amount of water for 3 times, each extraction for 2 hours, and the collected aqueous extract was concentrated into an extract), Bletilla striata stilbenoids (prepared according to Example 1), and Pogostemon cablin essential oil (prepared according to Example 1) were all provided by the Chemistry Laboratory of Chengdu University of Traditional Chinese Medicine; Compound Dexamethasone Acetate Cream (Hubei Ketian Pharmaceutical Co., Ltd., batch number: 220505); xylene (Chengdu Kelong Chemical Co., Ltd., batch number: 2022012101); polyethylene glycol 400 (Chengdu Kelong Chemical Co., Ltd., batch number: 2021110801); sodium carboxymethylcellulose (Shanghai Yuanye Bio-Technology Co., Ltd., batch number: J07HS180136).

[0055] 1.3 Instruments

[0056] ME204E electronic balance (Mettler-Toledo Instruments Co., Ltd.); UPH-II-20T pure water instrument (Sichuan Youpu Ultra-Pure Technology Co., Ltd.); KQ5200DE type numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.).

[0057] 2 Experimental methods

[0058] 2.1 Drug preparation

[0059] Weigh an appropriate amount of the aqueous extract of Bletilla striata, the aqueous extract of Pogostemon cablin, and the 1:1 combination of the aqueous extract of Bletilla striata and the aqueous extract of Pogostemon cablin, dissolve them with normal saline, add the sodium carboxymethylcellulose solution matrix, and mix the drug and the matrix thoroughly to obtain a drug concentration of 0.38%. Weigh an appropriate amount of Bletilla striata stilbenoids, Pogostemon cablin essential oil, the 1:1 combination of Bletilla striata stilbenoids and Pogostemon cablin essential oil, the 1:2 combination of Bletilla striata stilbenoids and Pogostemon cablin essential oil, and the 2:1 combination of Bletilla striata stilbenoids and Pogostemon cablin essential oil, dissolve the drug with polyethylene glycol 400. After the drug is dissolved, add the sodium carboxymethylcellulose solution matrix, and mix the drug and the matrix thoroughly to obtain a drug concentration of 0.38%. At the same time, prepare a blank matrix without drugs.

[0060] 2.2 Grouping, drug administration, and model establishment

[0061] 88 SPF-grade KM mice were randomly divided into 11 groups according to body weight: blank group, model group, positive group (Compound Dexamethasone Acetate Cream), aqueous extract of Bletilla striata group, aqueous extract of Pogostemon cablin group, 1:1 group of aqueous extract of Bletilla striata - aqueous extract of Pogostemon cablin, Bletilla striata stilbenoids group, Pogostemon cablin essential oil group, 1:1 group of Bletilla striata stilbenoids - Pogostemon cablin essential oil, 1:2 group of Bletilla striata stilbenoids - Pogostemon cablin essential oil, 2:1 group of Bletilla striata stilbenoids - Pogostemon cablin essential oil, with 8 mice in each group. The blank group and the model group were smeared with an equal volume of blank matrix, the mice in the positive group were smeared with 0.04 mg / cm 2 ·d, and the mice in the remaining drug groups were smeared with 0.34 mg / cm 2· d, twice a day, for 3 consecutive days. 30 minutes after administration on the 4th day, except for the blank group, 40 μL of xylene was applied to the right ear of each mouse in the other groups. 30 minutes after inflammation induction, the mice were sacrificed by cervical dislocation. Both ears were cut off, and ear pieces of the same part on the left and right ears were taken with a punch with a diameter of 8 mm, weighed immediately, the weights of the left and right ear pieces of the mice were recorded, and the ear swelling degree and ear swelling inhibition rate were calculated. Ear swelling degree (mg) = weight of right ear piece - weight of left ear piece, ear swelling inhibition rate (%) = (average swelling degree of model group - average swelling degree of administration group) / average swelling degree of model group × 100%.

[0062] 3. Statistical methods

[0063] Statistical analysis was performed using SPSS 26.0 software. Measurement data were expressed as mean ± standard deviation For comparison among multiple groups, one-way ANOVA was used. P < 0.05 indicated that the difference was statistically significant.

[0064] 4. Experimental results

[0065] Compared with the model group, the aqueous extracts of Bletilla striata and Pogostemon cablin did not significantly reduce the ear swelling degree of mice caused by xylene. The positive group, Bletilla striata stilbenoids and Pogostemon cablin oil could significantly reduce the ear swelling degree of mice caused by xylene (P < 0.01). It can be seen that the anti-inflammatory effects of the extracts from different parts of Bletilla striata and Pogostemon cablin were better than those of the aqueous extracts. The anti-inflammatory effects of the combinations of Bletilla striata extracts and Pogostemon cablin extracts at different ratios were better than those used alone. Among them, the combinations of Bletilla striata stilbenoids - Pogostemon cablin oil (1:1, 1:2, 2:1) could reduce the ear swelling degree of mice caused by xylene to varying degrees (P < 0.01), and the ear swelling inhibition rates were ranked from high to low as follows: Bletilla striata stilbenoids - Pogostemon cablin oil 2:1 group > Bletilla striata stilbenoids - Pogostemon cablin oil 1:2 group > Bletilla striata stilbenoids - Pogostemon cablin oil 1:1 group. It can be seen that the combination of Bletilla striata stilbenoids - Pogostemon cablin oil 2:1 had better anti-inflammatory effects and significant effects, laying a foundation for subsequent anti-acne pharmacodynamic studies. The results are shown in Table 1.

[0066] Table 1 Effects of extracts from Bletilla striata and Pogostemon cablin and their different ratios on ear swelling in mice caused by xylene( n = 8)

[0067]

[0068]

[0069] Note: Compared with the blank group, *P < 0.05, **P < 0.01; compared with the model group, # P < 0.05, ## P < 0.01

[0070] Experimental example 2: Study on the effect of the combination of Bletilla striata stilbenoids and Pogostemon cablin oil 2:1 on the acne inflammation model in mice 1. Experimental materials

[0071] 1.1 Animals

[0072] Sixty SPF-grade male ICR mice, with a body weight of (30 ± 2) g, were provided by Chengdu Dashuo Laboratory Animal Co., Ltd., and the experimental animal production license number was SCXK (Sichuan) 2020-030. The animals were housed in an environment with a temperature of 20°C to 25°C and a relative humidity of 40% to 70%, and they could drink water and eat freely. The experimental animal ethics approval number was: 2022-66.

[0073] 1.2 Drugs and Reagents

[0074] Bletilla and Stilbene and Pogostemon oil were provided by the Chemistry Laboratory of Chengdu University of Traditional Chinese Medicine; adapalene gel (Wuhan Nuoan Pharmaceutical Co., Ltd., batch number: 20221103); polyethylene glycol 400 (Chengdu Kelong Chemical Co., Ltd., batch number: 2021110801); sodium carboxymethylcellulose (Shanghai Yuanye Bio-Technology Co., Ltd., batch number: J07HS180136); mouse IL-6 cytokine detection kit (Chengdu Novo Bio-Technology Co., Ltd., batch number: MR230110, product number: R0204); mouse IFN-β and IL-18 cytokine detection kits (Chengdu Novo Bio-Technology Co., Ltd., batch number: MR230110, product number: R0202).

[0075] 1.3 Instruments

[0076] HR40-ⅡA2 laminar flow hood (Qingdao Haier Biomedical Co., Ltd.); DH124L electrothermal constant temperature incubator (Tianjin Test Instrument Co., Ltd.); ME204E electronic balance (Mettler-Toledo Instruments Co., Ltd.); KQ5200DE digital control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); UPH-II-20T pure water instrument (Sichuan Youpu Ultra-Pure Technology Co., Ltd.); Allegra X-30R centrifuge (BeckMan).

[0077] 2 Experimental Methods

[0078] 2.1 Drug Preparation

[0079] Weigh the drugs according to the ratio of Bletilla and Stilbene - Pogostemon oil of 2:1. First, dissolve them in polyethylene glycol 400, and then add the sodium carboxymethylcellulose solution matrix. Mix the drugs and the matrix thoroughly to make the total concentrations of the low, medium, and high doses of the Bletilla and Stilbene - Pogostemon oil 2:1 combination be 0.19%, 0.38%, and 0.75% respectively. At the same time, prepare a blank matrix without drugs.

[0080] 2.2 Grouping, Modeling and Drug Administration

[0081] Sixty SPF male ICR mice were randomly divided into 6 groups according to body weight: blank group, model group, positive group (adapalene gel), low, medium, and high dose groups of the combination of bletilla and stilbene-patchouli oil at a ratio of 2:1, with 10 mice in each group. Except for the mice in the blank group that were injected with 20 μL of normal saline intradermally into the right ear, the mice in the other groups were injected with 20 μL of Propionibacterium acnes bacterium solution intradermally into the right auricle, once a day for 5 consecutive days of modeling. It was found that the right auricles of the mice were significantly red, swollen, and thickened, and some were desquamated and crusted, indicating that the acne model of the mice was successfully constructed. The blank group and the model group were smeared with blank matrix, the positive group was smeared with 0.06 mg / cm 2 ·d, the low dose group was smeared with 0.17 mg / cm 2 ·d, the medium dose group was smeared with 0.34 mg / cm 2 ·d, the high dose group was smeared with 0.68 mg / cm 2 ·d, twice a day for 7 consecutive days.

[0082] 2.3 Specimen collection

[0083] After the last administration, the mice were fasted but allowed to drink water for 24 h, blood was taken from the orbital cavity, centrifuged at 3500 rpm for 15 min at 4 °C, and the serum was collected. After blood collection from the orbital cavity, the right ear tissues of the mice in each group were cut. Part of the right ear tissue samples were fixed in 4% paraformaldehyde, and the remaining right ear tissues were stored in a -80 °C refrigerator for later use.

[0084] 2.4 Changes in skin lesions of the right auricle of mice

[0085] The skin inflammation and redness and swelling of the right auricles of the mice in each group were observed macroscopically. The thickness of the right auricles of the mice before and after modeling and after drug administration and treatment was measured using a vernier caliper, and the auricle swelling degree was calculated. The auricle swelling degree (mm) = the thickness of the auricle after the last administration - the thickness of the auricle before modeling.

[0086] 2.5 Histopathological observation of the right auricle tissue of mice

[0087] The right ear tissue samples fixed in 4% paraformaldehyde were dehydrated, embedded, sectioned, and stained with hematoxylin-eosin, and then examined under a microscope and analyzed by image acquisition.

[0088] 2.6 Changes in the contents of IL-6, IL-18, and IFN-β in the serum of mice

[0089] The contents of IL-6, IL-18, and IFN-β in the serum of the mice in each group were measured by flow cytometry fluorescence method, and the experiment was carried out strictly according to the instructions of the kit.

[0090] 3 Statistical methods

[0091] SPSS 26.0 software was used for statistical analysis. Measurement data were expressed as mean ± standard deviation It is shown that one-way ANOVA was used for comparison among multiple groups, and P<0.05 indicated statistically significant differences.

[0092] 4 Experimental results

[0093] 4.1 Macroscopic observation of skin lesions in the right auricles of mice in each group

[0094] The skin color of the right auricles of mice in the blank group was light pink, with soft and smooth texture, and capillaries were clearly visible; the right auricles of mice in the model group were significantly swollen, with dry, rough texture, desquamation and crusting; compared with the model group, the redness and swelling of the right auricles of mice in the positive group and the low, medium, and high-dose groups of the combination of Bletilla and Stilbene - Pogostemon oil 2:1 were significantly improved, with softer and smoother texture and clearer capillaries. The results are shown in Figure 1 。

[0095] 4.2 Changes in the thickness of the right auricles of mice in each group and auricle swelling rate

[0096] After modeling, compared with the blank group, the auricle thickness of mice in the model group and the drug administration groups was significantly increased (P<0.01). After drug administration, compared with the model group, the positive group and the low, medium, and high-dose groups of the combination of Bletilla and Stilbene - Pogostemon oil 2:1 could improve the auricle thickness and swelling degree of mice to varying degrees (P<0.01). The results are shown in Table 2.

[0097] Table 2 Effects of the combination of Bletilla and Stilbene - Pogostemon oil 2:1 on the auricle thickness of mice ( n = 10)

[0098]

[0099] Note: Compared with the blank group, *P<0.05, **P<0.01; compared with the model group, # P<0.05, ## P<0.01.

[0100] 4.3 Pathological changes in the auricles of mice in each group

[0101] In the auricle tissues of mice in the blank group, the skin epidermis, dermis, and subcutaneous tissue structures were normal, with abundant hair follicles and sebaceous glands in the dermis, and no obvious pathological damage changes were seen; in the auricle tissues of the model group, the skin structure was damaged, the epidermis was thickened, there were local skin defects, large-scale necrosis of the subcutaneous tissue, and a large number of inflammatory cells infiltrated; compared with the model group, in the positive group and the low, medium, and high-dose groups of the combination of Bletilla and Stilbene - Pogostemon oil 2:1, the pathological damage of the auricle tissues of mice was reduced, the structures of the skin epidermis and dermis were distinguishable, and the infiltration of inflammatory cells in the dermis and subcutaneous tissue was reduced. The HE staining results are shown in Figure 2 。

[0102] 4.4 Contents of serum IL-6, IL-18, and IFN-β in mice in each group

[0103] Compared with the blank group, the contents of serum IL-6, IL-18, and IFN-β in the model group mice were significantly increased (P<0.05); compared with the model group, the contents of serum IL-6, IL-18, and IFN-β in the positive group and the low, medium, and high dose groups of the combination of Bletilla striata and Pogostemon cablin essential oil at a ratio of 2:1 were significantly decreased (P<0.05), and the results are shown in Table 3.

[0104] Table 3 Effects of the combination of Bletilla striata and Pogostemon cablin essential oil at a ratio of 2:1 on the contents of serum IL-6, IL-18, and IFN-β in mice ( n = 10)

[0105]

[0106] Note: Compared with the blank group, *P<0.05, **P<0.01; compared with the model group, # P<0.05, ## P<0.01.

[0107] Experimental example 3: Inhibitory effect of the combination of Bletilla striata and Pogostemon cablin essential oil at a ratio of 2:1 on Propionibacterium acnes

[0108] 1 Experimental materials

[0109] 1.1 Drugs and reagents

[0110] Both Bletilla striata and Pogostemon cablin essential oil were provided by the Chemistry Laboratory of Chengdu University of Traditional Chinese Medicine; Propionibacterium acnes (ATCC11827) was purchased from the Guangdong Provincial Microbial Culture Collection Center; AnaeroPack-Anaero anaerobic gas generating bag (Mitsubishi Gas Chemical Co., Ltd., Japan, batch number: 2088LJ-1); Brain Heart Infusion (BHI) broth medium (Beijing Solarbio Science & Technology Co., Ltd., batch number: 510V031); Agar powder (Beijing Solarbio Science & Technology Co., Ltd., batch number: 507V021); Dimethyl sulfoxide (DMSO) (Chengdu Kelong Chemical Co., Ltd., batch number: 2022051301); Triphenyltetrazolium chloride (TTC) (Sigma-Aldrich Co., batch number: BCCH3368).

[0111] 1.2 Instruments

[0112] DH124L Electric Thermostatic Incubator (Tianjin Test Instrument Co., Ltd.); HR40-IIA2 Laminar Flow Hood (Qingdao Haier Biomedical Co., Ltd.); ME204E Electronic Balance (Mettler-Toledo Instruments Co., Ltd.); UPH-II-20T Pure Water Instrument (Sichuan Youpu Ultra-pure Technology Co., Ltd.); BCD-190WDPT Refrigerator (Qingdao Haier Refrigerator Co., Ltd.); GR85DA Autoclave (Zhiwei Instruments Co., Ltd.); SpectraMax ABS Plus Microplate Reader (Shanghai MGM Molecular Instruments Co., Ltd.).

[0113] 2 Experimental Methods

[0114] 2.1 Bacterial Solution Preparation

[0115] Propionibacterium acnes was anaerobically cultured on BHI agar plates at 37 °C for 48 h. Before use, an appropriate amount of colonies was picked and the bacterial concentration was adjusted to 0.5 McFarland turbidity (1.5×10 8 CFU / mL) with normal saline.

[0116] 2.2 Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

[0117] Referring to the method of the Clinical and Laboratory Standards Institute (CLSI), the MIC of Bletilla striata extract, Pogostemon cablin extract and different proportion combinations against Propionibacterium acnes was determined by the microbroth dilution method in 96-well plates. 100 μL of BHI liquid medium was added to each well of the 96-well plate. 100 μL of the diluted extract solution was added to each well of the first column, mixed well, and diluted two-fold to the ninth column. 100 μL of Propionibacterium acnes bacterial solution (concentration of 1.5×10 6 CFU / mL) was added to each well of the first to eleventh columns. 100 μL of BHI liquid medium was added to the twelfth column. Among them, the tenth column was the 0.2% DMSO solvent control, the eleventh column was the positive control, and the twelfth column was the negative control. After anaerobic culture at 37 °C for 48 h, 30 μL of 0.5% TTC solution was added to each well and incubated for 90 min. The lowest drug concentration well that did not turn red was observed directly with the naked eye as the MIC. The liquid in the well that did not turn red was taken out and inoculated onto BHI agar plates, and the lowest drug concentration without bacterial growth was the MBC after anaerobic culture at 37 °C for 48 h.

[0118] 2.3 Effect of the 2:1 Combination of Bletilla striata Stilbenoids and Pogostemon cablin Oil on Bacterial Growth Curve

[0119] The 2:1 combination of Bletilla striata stilbenoids and Pogostemon cablin oil was added to BHI liquid medium and two-fold diluted in 96-well plates to obtain drug-containing media with different concentrations of 100 μL in each well. 100 μL of Propionibacterium acnes bacterial solution (concentration of 1.5×10 6(CFU / mL), and the final concentrations of the drug were 1 / 4 MIC, 1 / 2 MIC, MIC, and 2 MIC, respectively. At the same time, different concentration liquid medicine control groups, culture medium control groups, and solvent control groups (the final concentration of DMSO was less than 0.05%) were set up and cultured under anaerobic conditions at 37°C. Every 4 hours, the absorbance value was measured at 600 nm until 72 hours.

[0120] 3 Experimental results

[0121] 3.1 MIC and MBC

[0122] The MIC and MBC of the combination of Bletilla striata stilbenoids and Pogostemon cablin oil at a ratio of 2:1 were 23.43 μg / mL and 93.75 μg / mL, respectively.

[0123] 3.2 Effects of the combination of Bletilla striata stilbenoids and Pogostemon cablin oil at a ratio of 2:1 on the bacterial growth curve

[0124] The growth curve of the blank group was S-shaped, and the colony growth was slow or inhibited under the action of drugs at different concentrations. It was further confirmed through the bacterial growth curve that the combination of Bletilla striata stilbenoids and Pogostemon cablin oil had a good inhibitory effect on Propionibacterium acnes. The results are shown in Figure 3 .

[0125] In summary, the combined drug for treating acne in the present invention, with the combination of Bletilla striata stilbenoids and Pogostemon cablin oil in a specific ratio, significantly inhibits Propionibacterium acnes and acne inflammation, thus playing a role in treating acne. The effect is better than that of either Bletilla striata stilbenoids or Pogostemon cablin oil used alone, and is better than the curative effect of adapalene gel commonly used at present, playing a role of synergistic enhancement.

Claims

1. Use of the bletilla stilbene components in combination with patchouli essential oil in the preparation of a medicament for treating acne, characterized in that: the bletilla stilbene components are substances obtained by concentrating the eluate eluted with 95% ethanol from the 65% ethanol extract of bletilla striata loaded onto a chromatography column; the patchouli essential oil is an organic substance extracted from pogostemon cablin by steam distillation; the mass ratio of the bletilla stilbene components to the patchouli essential oil is 1-2:1-2.

2. The use according to claim 1, characterized in that: the medicament is a medicament for inhibiting acne inflammation and / or propionibacterium acnes.

3. The use according to claim 1, characterized in that: the mass ratio of the bletilla stilbene components to the patchouli essential oil is 2:

1.

4. The use according to any one of claims 1 to 3, characterized in that: the chromatography column is a polyamide column.

5. A combined medicament for treating acne, characterized in that: the medicament is bletilla stilbene components and patchouli essential oil; the mass ratio of the bletilla stilbene components to the patchouli essential oil is 1-2:1-2; the bletilla stilbene components are substances obtained by concentrating the eluate eluted with 95% ethanol from the 65% ethanol extract of bletilla striata loaded onto a chromatography column; the patchouli essential oil is an organic substance extracted from pogostemon cablin by steam distillation.

6. The combined medicament according to claim 5, characterized in that: the mass ratio of the bletilla stilbene components to the patchouli essential oil is 2:

1.

7. A composition for treating acne, characterized in that: it is an oral preparation prepared by adding pharmaceutically acceptable excipients with bletilla stilbene components and patchouli essential oil as active ingredients; the mass ratio of the bletilla stilbene components to the patchouli essential oil is 1-2:1-2; the bletilla stilbene components are substances obtained by concentrating the eluate eluted with 95% ethanol from the 65% ethanol extract of bletilla striata loaded onto a chromatography column; the patchouli essential oil is an organic substance extracted from pogostemon cablin by steam distillation.

8. The composition according to claim 7, characterized in that: the mass ratio of the bletilla stilbene components to the patchouli essential oil is 2:

1.

9. The composition according to claim 7, characterized in that: the oral preparation is a granule, powder, pill, capsule or solution.

10. The preparation method of the composition according to any one of claims 7 to 9, characterized in that: it comprises the following steps: weigh the bletilla stilbene components and patchouli essential oil according to the ratio, and then add pharmaceutically common excipients or auxiliary components, and mix evenly to obtain.

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