Uniflower rhaponticum ointment as well as preparation method and application thereof

Abstract

The invention discloses a rhaponticum uniflorum ointment as well as a preparation method and application thereof, and relates to the technical field of medicines, and the ointment is prepared from rhaponticum uniflorum powder, glycerol, distilled water, stearic acid, white vaseline, wool fat, liquid paraffin, glycerin monostearate and an emulsifier. The rhaponticum uniflorum ointment disclosed by the invention is reasonable in formula, simple in preparation method and obvious in acute trauma treatment effect.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a louboutin ointment and a preparation method and application thereof. Background Art

[0002] Rhaponticum uniflorum (L.) DC., also known as Hongguleh-Zhuri in Mongolian medicine, is the dried flower of the Asteraceae plant Rhaponticum uniflorum (L.) DC. It is widely distributed in northern my country, covering the Northwest, North China, Northeast China and other regions.

[0003] The flowers of the elm are cool in nature and bitter in taste. They have the effects of "killing stickiness", detoxification, and clearing away heat. As a Mongolian medicine, the ingredients of the elm are relatively many and complex, including flavonoids, polysaccharides, γ-lactones, saponins, volatile oils, alkaloids, and plant ecdysone, so its pharmacological effects are also relatively extensive.

[0004] Relevant literature reports that the flower of smilax china has pharmacological effects such as antiviral, anti-tumor, analgesia, immune regulation, hypoglycemic, liver protection, and treatment of brain damage and myocardial ischemia. In their clinical studies, researchers also found that the main active ingredients of the flower of smilax china are flavonoids, which have anti-inflammatory and antioxidant pharmacological activities, and have certain research value for anti-inflammatory and antioxidant treatments.

[0005] At present, the quality control and extraction research of Mongolian medicine Eleutherodactyla odorata has taken shape, but few of its pharmaceutical preparations are on the market, which leads to difficulties in its clinical application. Based on this, it is very meaningful to develop a pharmaceutical preparation containing Eleutherodactyla odorata extract. Summary of the invention

[0006] In view of the above problems, the present invention provides a lonicera japonica ointment and a preparation method and application thereof.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The invention provides a scutellaria baicalensis ointment, which is composed of scutellaria baicalensis powder, glycerin, distilled water, stearic acid, white vaseline, lanolin, liquid paraffin, glyceryl monostearate, and an emulsifier; The mass ratio of the smilax glabra powder, glycerin, distilled water, stearic acid, white vaseline, lanolin, glyceryl monostearate, and emulsifier is 2:5:29:6:0.5:2.5:1.8:0.2; The volume ratio of water to liquid paraffin is 29:3.

[0008] Furthermore, the emulsifier is triethanolamine.

[0009] The present invention provides a method for preparing a scutellaria baicalensis ointment, which specifically comprises the following steps: S1: Put the powder of Eleutherodactyla sphaerocephala in a bottle, add 50% ethanol, weigh, and ultrasonicate for 60 minutes. After ultrasonication, defat with petroleum ether, filter with suction, and put the filtrate into a rotary evaporator and evaporate to an extract to obtain Eleutherodactyla sphaerocephala extract; S2: mixing stearic acid, white vaseline, lanolin, liquid paraffin, and glyceryl monostearate to obtain an oil phase; S3: mixing glycerol and distilled water to obtain an aqueous phase; S4: Heat the oil phase and the water phase separately, add the eloud extract to the oil phase, add the emulsifier to the water phase, and then slowly add the oil phase to the water phase while stirring, and after mixing evenly, continue stirring and emulsifying to obtain a mixed matrix; put the mixed matrix into a mortar and grind it finely, and after cooling, spread it on the surface of a sterile container, place it under ultraviolet light, and obtain an eloud ointment.

[0010] Furthermore, in the step S1, the ultrasonic frequency is 40 kHz, the power is 100%, and the specification is 500W; the density of the Eleutherodactyla scabra extract is 1.00-1.10 g / mL.

[0011] Furthermore, in step S4, the heating temperature is 80° C., the emulsification time is 10 min, and the irradiation time under the ultraviolet lamp is 1 h.

[0012] The invention also provides an application of the loulouhua ointment in preparing a product for treating acute trauma.

[0013] Compared with the prior art, the present invention has the following beneficial effects: In the present invention, a novel loulouhua ointment is prepared by mixing the Mongolian medicine loulouhua extract with auxiliary materials; the loulouhua ointment of the present invention is convenient to use, has a high absorption rate, has no side effects, and has good skin affinity; according to the results of pharmacodynamics research and pathological sections, the ointment has obvious effects on the treatment of acute trauma, can effectively inhibit the occurrence of wound inflammation, and promote its rapid healing, while the results of non-targeted metabolomics research reveal its mechanism of action for treating acute trauma rats. Based on this, the loulouhua ointment of the present invention has a reasonable formula, a simple preparation method, and obvious effects on the treatment of acute trauma, and has good application prospects and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The wound healing conditions of rats in the MO, BO and RL groups after dressing change 4 days of the present invention; Figure 2 It is a bar graph of wound healing rates of MO group, BO group and RL group of the present invention; Figure 3 The pathological morphological changes of wound healing tissues in MO group, BO group, RL group and N group on the 4th day of the present invention; Figure 4It is a bar graph of the levels of inflammatory factors and growth factors in the wound tissue of mice of the present invention; Figure 5 It is the PCA score graph of the combined positive and negative ion modes of the present invention; Figure 6 is the OPLS-DA score diagram of the present invention combining positive and negative ion modes, wherein, Figure 6 A is the OPLS-DA score diagram of the combined positive and negative ion modes of MO.vs.N. Figure 6 B is the OPLS-DA score diagram of the merged positive and negative ion modes of RL.vs.MO; Figure 7 is the OPLS-DA permutation test diagram of the present invention combining positive and negative ion modes, wherein, Figure 7 A is the OPLS-DA permutation test diagram of MO.vs.N in the combined positive and negative ion modes. Figure 7 B is the OPLS-DA permutation test diagram of the combined positive and negative ion modes of RL.vs.MO; Figure 8 is a volcano plot of the present invention combining positive and negative ion modes, wherein, Figure 8 A is the volcano plot of the combined positive and negative ion modes of MO.vs.N. Figure 8 B is the volcano plot of the merged positive and negative ion modes of RL.vs.MO; Fig. 9 is the Pathway Impact diagram of each comparison group of the present invention, wherein, Fig. 9 A is the PathwayImpact diagram of MO.vs.N, Fig. 9 B is the pathway impact diagram of RL.vs.MO. DETAILED DESCRIPTION

[0015] In order to make the purpose and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0016] The instruments, reagents, materials, etc. involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0017] In this embodiment, experimental animals: 25 male and female SD rats, 10 in each group, 8-10 weeks old, 180-220 g in weight, purchased from Beijing Sibeifu Experimental Animal Center, animal production license number: SCXK (Beijing) 2030-0003; animals were fed ad libitum and kept in an SPF animal room; experiments were conducted after 1 week of adaptive feeding.

[0018] Main instruments: Vanquishcore high-performance liquid chromatograph (ThermoFisher, USA); QExactivePlus mass spectrometer (ThermoScientific, USA); NexeraX2LC-30AD ultra-high-pressure liquid chromatograph (Shimadzu, Japan); HypersilGold-C18 chromatographic column (ThermoFisher, USA); ACQUITY Rising LCHSST3 chromatographic column (Waters, USA); AB-135S analytical balance (1 / 100,000, MettlerToledo, Switzerland); AL240 analytical balance (1 / 10,000, MettlerToledo, Switzerland); KQ-5200KDE high-power digital control ultrasonic instrument (Jiangsu Kunshan Ultrasonic Instrument Co., Ltd.); KQ-500DE ultrasonic instrument (Kunshan Ultrasonic Instrument Factory); HJ-3 digital constant temperature magnetic stirrer (Changzhou Guohua Electric Co., Ltd.); SHB-ⅢA circulating water multi-purpose vacuum pump (Shanghai Ailan Instrument Co., Ltd.); RE-52 rotary evaporator (Shanghai Ailan Instrument Co., Ltd.); digital display constant temperature water bath HH-6 (Shanghai Ailan Instrument Co., Ltd. Company); H-1650R desktop high-speed refrigerated centrifuge (Changsha Xiangyi Centrifuge Instrument Co., Ltd.); DM2000 fluorescence microscope (Leica, Germany); RM2135 paraffin ultrathin sectioner (Leica, Germany); EG1150C paraffin embedding machine (Leica, Germany); AutostainerXL automatic staining machine (Leica, Germany); EP tubes and gun tips (Axygen, USA); Biortron ultrasonic system (Diagenode, Japan); Concentratorplus vacuum centrifugal concentrator (Eppendorf, Germany); Centrifuge5424R centrifuge (Eppendorf, Germany).

[0019] Medicinal materials and reagents: A total of 10 batches of scutellaria baicalensis were collected from different origins and identified as Qizhou scutellaria baicalensis by Associate Professor Zhang Huiwen of the Pharmacy Teaching and Research Section of Inner Mongolia Medical University. Rhaponticum uniflorum Dried inflorescence of (L.) DC.; Chlorogenic acid reference substance (batch number B20782, purity ≧98%), quercetin reference substance (batch number B20526, purity ≧98%), quercetin reference substance (batch number B20527, purity ≧98%), and apigenin reference substance (batch number B20981, purity ≧98%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Acetonitrile, phosphoric acid, methanol, ammonia water, and formic acid were of chromatographic grade; ultrapure water (self-made in the laboratory); propylene glycol (Tianjin Jindong Tianzheng Fine Chemical Reagent Factory, 20190518), lanolin (Tianjin Zhiyuan Chemical Reagent Co., Ltd., 20230201802), vaseline (Tianjin Yongsheng Fine Chemical Co., Ltd., 20180301), liquid paraffin (Shandong Dexinkang Medical Technology Co., Ltd., 230503); stearic acid (Tianjin Dengfeng Chemical Reagent Factory, 20230702); glyceryl monostearate (Tianjin Lianzhong Chemical Reagent Co., Ltd., 20230731), triethanolamine (Tianjin Yongsheng Fine Chemical Co., Ltd., 20180301), and stearic acid (Tianjin Dengfeng Chemical Reagent Factory, 20230702). Sheng Fine Chemical Co., Ltd., 20190805); Veetin Hair Removal Cream (Reckitt Benckiser (China) Investment Co., Ltd., 2888174); Chloral hydrate (Shanghai Aladdin Biochemical Technology Co., Ltd., C104202); Normal saline (Beijing Prilai Gene Technology Co., Ltd., B1219); 4% paraformaldehyde universal tissue fixative (China Biosharp Company, BL539A); Hematoxylin and eosin (HE) staining kit (Biosharp Biotechnology Co., Ltd., C0105); IL-6, TNF-α, and EGF kits were all purchased from Quanzhou Ruixin Biotechnology Co., Ltd.

[0020] Example 1 This embodiment provides a scutellaria baicalensis ointment, which is composed of scutellaria baicalensis powder, glycerin, distilled water, stearic acid, white vaseline, lanolin, liquid paraffin, glyceryl monostearate, and triethanolamine; Among them, the mass ratio of eloeda powder, glycerin, distilled water, stearic acid, white vaseline, lanolin, glyceryl monostearate and emulsifier is 2:5:29:6:0.5:2.5:1.8:0.2; the volume ratio of water to liquid paraffin is 29:3.

[0021] The preparation method of the louluhua ointment specifically comprises the following steps: S1: Place the powder of Eleutherodactyla sphaerocephala in a bottle, add 50% ethanol, weigh, and ultrasonicate for 60 min (frequency is 40 kHz, power is 100%, specification is 500 W). After ultrasonication, defat the powder with petroleum ether, filter the powder with suction, and place the filtrate in a rotary evaporator and evaporate it to an extract to obtain an Eleutherodactyla sphaerocephala extract with a density of 1.00 g / mL. S2: mixing stearic acid, white vaseline, lanolin, liquid paraffin, and glyceryl monostearate to obtain an oil phase; S3: mixing glycerol and distilled water to obtain an aqueous phase; S4: Heat the oil phase and the water phase to 80°C respectively, add the radix ophiopogonis extract to the oil phase, add the emulsifier to the water phase, then slowly add the oil phase to the water phase, stirring while adding, and after mixing evenly, continue stirring and emulsifying for 10 minutes to obtain a mixed matrix; grind the mixed matrix in a mortar and grind it finely, after cooling, spread it on the plane of a sterile container, place it under ultraviolet light for 1 hour, and obtain a radix ophiopogonis ointment. The ointment is used in the preparation of products for treating acute skin injuries.

[0022] Example 2 The difference from Example 1 is that the density of the extract of Elaeagnus scoparia is 1.05 g / mL.

[0023] Example 3 The difference from Example 1 is that the density of the extract of Elaeagnus scoparia is 1.10 g / mL.

[0024] Experimental Example 1 Preparation of Lophatherum odoratum ointment 1. Preparation and identification of ophiopogon japonicus extract (1) Accurately weigh 1.0 g of Eleutherodactyla sphaerocephala powder and place it in a bottle. Add 125 mL of 50% ethanol, weigh it, and ultrasonicate it for 60 min (frequency is 40 kHz, power is 100%, specification is 500 W). After ultrasonication, defat it with petroleum ether, filter it, and place the filtrate into a rotary evaporator and evaporate it to an extract to obtain an Eleutherodactyla sphaerocephala extract with a density of 1.00-1.10 g / mL.

[0025] To identify the flavonoid compounds in the extract, a certain amount of the extract of the smilax china flower was weighed, heated, and dissolved in an appropriate amount of distilled water to prepare a 0.1 g / mL smilax china flower extract test solution, and the flavonoid compounds were identified: ① Reaction with ammonia water: Take 5 mL of the solution to be tested of the loblolly ointment, place it in a test tube, add a small amount of ammonia water, and observe the phenomenon of the solution; ②Reaction with 4% NaOH: Take 5 mL of the test solution of Luhua ointment, put it in a test tube, add a small amount of 4% NaOH, and observe the phenomenon; ③Reaction with concentrated sulfuric acid: Take 5 mL of the test solution of Luhua ointment, put it in a test tube, add a small amount of concentrated sulfuric acid, and observe the phenomenon; ④ Reaction with hydrochloric acid-zinc powder: Take 5 mL of the solution to be tested of the loblolly ointment, put it in a test tube, add a small amount of zinc powder, then add a few drops of concentrated hydrochloric acid, place it in a water bath and heat it for 1 minute, and observe the phenomenon; ⑤ Reaction with hydrochloric acid-magnesium powder: Take 5mL of the test solution of Luhua ointment, put it in a test tube, add a small amount of magnesium powder, then add a few drops of concentrated hydrochloric acid, place it in a water bath and heat for 1 minute, and observe the phenomenon.

[0026] Table 1 Identification of flavonoids

[0027] (2) The HPLC content of extracts of 10 different batches and origins of the medicinal material of Lophatherum gracile was determined by the existing method; the medicinal materials from the best origin were selected to prepare Lophatherum gracile ointment.

[0028] Table 2 Sample content determination

[0029] It can be seen from the above table that the contents of the four components of the medicinal materials of the three origins of S1, S5 and S10 are relatively stable, among which the extract of S1 contains fewer impurities and insoluble components, so the medicinal materials of the medicinal materials of the three origins of S1 are selected for the preparation of ointment.

[0030] 2. Matrix type screening The prepared oily base and emulsion base are evaluated based on the appearance, color and spreadability of the ointment to determine the ointment base.

[0031] prescription: (1) O / W type matrix: Eleutherodactyla scabra extract (including Eleutherodactyla scabra powder 1.0 g); glycerin 5.0 g, distilled water 29 mL; stearic acid 5 g, white vaseline 0.5 g, lanolin 2.5 g, liquid paraffin 3 mL, glyceryl monostearate 1.5 g; emulsifier: triethanolamine 0.2 g; (2) Oily matrix: Eleutherodactylus odoratus extract (containing Eleutherodactylus odoratus powder 1.0 g); liquid paraffin 5 mL, white vaseline 10 g.

[0032] Table 3 Evaluation results of ointments with different matrix types

[0033] As shown in Table 3, the appearance, color and spreadability of the O / W type matrix ointment are better than those of the oily matrix, so the O / W type matrix was selected to prepare the louluhua ointment.

[0034] 3. Orthogonal experimental design to optimize the prescription of Lophatherum ointment Consult the literature and select formulation ingredients that do not react with the main drug; Oil phase: white petrolatum, stearic acid, liquid paraffin, lanolin, glyceryl monostearate; Aqueous phase: glycerol, water; Emulsifier: triethanolamine; According to the research content, L9 (3 3 )Orthogonal experiment.

[0035] Scoring Rules: Centrifugation test: Take 1g of Lophatherum ointment and place it in a centrifuge tube. Centrifuge it at 3000r / min for 30min to observe whether it is stratified. The full score for this item is 3 points; (no stratification: 3 points; a small amount of stratification: 2 points; partial stratification: 1 point; stratification: 0 points) Heat and cold resistance test: Place a certain amount of ointment in a 55℃ constant temperature box and a refrigerator at -15℃ for 6 hours, and then observe the color and property changes of the ointment, and whether there is mildew and oil-water separation. The full score for this item is 3 points. (3 points if there is no mildew, oil-water and color change after heating or freezing; 2 points if there is no mildew but a small amount of oil-water separation and the color does not change; 1 point if there is no mildew but some oil-water separation and the color changes; 0 points if there is mildew and a large amount of oil-water separation and the color changes) Appearance: The ointment is evaluated based on its color uniformity, fineness, and spreadability; the maximum score for this item is 3 points. (Uniform color, fine paste, and easy to spread: 3 points; uniform color but with a small amount of particles: 2 points; uneven color, granularity, and difficult to spread: 1 point; the ointment is not formed: 0 points) Table 4 Orthogonal experiment level table L9 (3 3 )

[0036] Table 5 Orthogonal experimental design table for formula optimization of Louluhua ointment

[0037] Table 6 Scoring results of each group

[0038] Table 7 Results of variance analysis of orthogonal test of Louluhua ointment

[0039] The results of the orthogonal experiment for formula optimization of louluhua ointment are shown in Tables 5 and 6. The effects of the three matrices on the quality of the ointment are C>B>A (triethanolamine>glyceryl monostearate>stearic acid). The optimal ointment matrix is ​​A3B3C3, i.e. 6 g stearic acid, 1.8 g glyceryl monostearate, and 0.2 g triethanolamine.

[0040] 4. Screening of emulsification temperature When the cream was prepared by the emulsification method, the appearance and centrifugal stratification (centrifugation at 4000r / min for 30min) were used as indicators to investigate the effects of different emulsification temperatures (70℃, 80℃, 90℃) on the stability of the matrix.

[0041] Table 8 Emulsification temperature test results

[0042] It can be seen from Table 8 that when the emulsification temperature is 80°C, the quality of the prepared ointment is optimal, so the emulsification temperature is set to 80°C.

[0043] 5. Screening of emulsification time When the cream was prepared by the emulsification method, the appearance and centrifugation (centrifugation at 4000r / min for 30min) stratification were used as indicators to investigate the effects of different emulsification times (5min, 10min, 15min) on the stability of the matrix.

[0044] Table 9 Emulsification time test results

[0045] It can be seen from Table 9 that when the emulsification time is 10 min, the prepared ointment is fine and uniform, without stratification, and easy to apply, so the emulsification time is set to 10 min.

[0046] 6. Investigation on the amount of smilax china extract added After confirming the optimal matrix, the effects of different drug loading ratios on the quality of the ointment matrix were investigated using appearance, properties, coating properties, stability, and pH as indicators.

[0047] Table 10 Results of the investigation on the amount of extract added to the smilax china

[0048] It can be seen from Table 10 that when the mass of the Elaeagnus scoparia powder used for extraction is 2.0 g, the quality of the prepared ointment is optimal.

[0049] 7. Best process verification test The best process verification test was carried out based on the best prescription ratio and preparation process obtained under the above experiment. A total of three ointments were prepared to test the best process.

[0050] Based on the above content: the best process conditions for Lophatherum ointment are: Lophatherum gracile extract (containing Lophatherum gracile powder 2.0 g); Aqueous phase: 5.0 g glycerol, 29 mL distilled water; Oil phase: stearic acid 6g, white vaseline 0.5g, lanolin 2.5g, liquid paraffin 3mL, glyceryl monostearate 1.8g; Emulsifier: triethanolamine 0.2g; emulsification temperature 80℃, emulsification time 10min; Heat the oil phase and the water phase to 80°C respectively, add the eloud flower extract to the oil phase, add the emulsifier triethanolamine to the water phase, slowly add the oil phase to the water phase, stirring while adding, mix evenly, stir for about 10 minutes, take out the mixed matrix and put it into a mortar and grind it finely. After cooling, spread the ointment on the flat surface of a sterile container, place it under ultraviolet light for 1 hour, and obtain the eloud flower ointment.

[0051] Table 11 Best process validation scoring results

[0052] As can be seen from Table 11, three repeated experiments proved that the ointment matrix prepared by the "optimal process" has a uniform and fine texture, good stability, easy coating, good ductility, and no separation and stratification phenomenon, indicating that the process results are reliable.

[0053] Experimental Example 2 Quality Evaluation of Lophatherum odoratum Ointment 1. Centrifugation experiment Centrifugation experiment: Take 1 g of the Lophatherum ointment and place it in a centrifuge tube. Centrifuge it at 2500, 3000, and 4000 r / min for 30 min.

[0054] The results showed that 1 g of the Lophatherum ointment was placed in a centrifuge tube and centrifuged at 2500, 3000, and 4000 r / min for 30 min, and no stratification occurred.

[0055] 2. Heat and cold resistance test To simulate low temperature, human skin temperature and high temperature conditions, the loofah ointment was placed in a refrigerator at -15°C, an oven at 37°C, and an oven at 55°C for 12h, 24h, and 48h, and the phenomena were observed.

[0056] The results showed that the loofah ointment was placed in a refrigerator at -15°C, an oven at 37°C, and an oven at 55°C for 12h, 24h, and 48h, and observed, and no stratification, discoloration, or mold was observed.

[0057] 3. Determination of pH Accurately weigh 3 portions of 1.0 g of Lophatherum odoratum ointment, dilute with 20 mL of distilled water, heat and stir, transfer to a round-bottom flask after melting, heat and reflux for 2 h, filter, take the filtrate, and determine the pH value.

[0058] The pH value was measured with a pH meter (according to the 2020 edition of the "Chinese Pharmacopoeia", the pH value of ointments is generally controlled at a pH of 4.4~8.3), and the pH was between 6.0 and 7.0, which met the requirements.

[0059] 4. Irritation test 24 hours before the experiment, the rats' back hair was shaved, and the remaining hair was removed with a depilatory cream. After depilation, the skin was cleaned with sterile distilled water. The skin of the rats was observed after depilation. If there was no abnormality on the second day, the rats were given the drug. The SD rats were randomly divided into a single-dose group and a multiple-dose group, with 6 rats in each group, half of which were male and half were female. Single-dose group: The left and right sides of the back of the rats were smeared with the Lophatherum odoratum ointment and the blank ointment (ointment without Lophatherum odoratum extract), respectively, with the thickness of 1 mm, once, and the skin was observed for erythema and edema within 0-72 hours; Multiple-dose group: The left and right sides of the back of the rats were smeared with the Lophatherum ointment and the blank ointment, respectively. The smear thickness was controlled at 1 mm. The ointment was applied once a day for 7 days. The rat skin was cleaned 6 hours after the last application of the Lophatherum ointment and the blank ointment. The rat skin was observed after 1 hour. If there was no abnormality, the observation was continued for 7 days.

[0060] The results showed that there were no obvious allergic symptoms such as redness, edema, congestion, itching, and pain on the back skin of rats in the single-dose group and the multiple-dose group; the loofah ointment was easily applied on the back skin of rats.

[0061] 5. Particle size inspection According to the first method of General Chapter 0982 of the 2020 edition of the "Chinese Pharmacopoeia", the particle size of the three batches of prepared lonicera japonica ointment was inspected. The ointment was evenly applied on a glass slide and measured according to the particle size and particle size distribution determination method. No particles larger than 180μm should be detected.

[0062] The results showed that no particles larger than 180 μm were detected in the three batches of samples, and the particle size measurement results of the ointment were in compliance with the regulations.

[0063] 6. Physical properties The prepared ointment is stored in the air. The ointment is uniform, fine, has no odor, no rancidity, no discoloration, no hardening, no oil-water separation and other deterioration phenomena, is easy to scrub, is not easy to stain clothes, and no residue is found on animal skin.

[0064] 7. Determination of active ingredient content in Louluhua ointment Accurately weigh 10.0 g of Lophatherum odoratum ointment, add an appropriate amount of 50% ethanol, filter, dilute to 100 mL, heat and reflux for 1 h, filter again, place in a refrigerator overnight, filter the filtrate through a 0.45 μm microporous filter membrane, and take the filtrate to prepare the sample solution; Take 3 batches of radix eloupe ointment samples, prepare the test samples according to the above method, determine the content of quercetin, the main active ingredient of radix eloupe, record the peak area, and determine the content.

[0065] The results showed that the average content of quercetin in the three samples was 0.4002 mg / g, and the RSD was 1.9%.

[0066] Experimental Example 3 Evaluation of the efficacy mechanism of Lophatherum odoratum ointment in promoting wound healing in rat wound models A preliminary experiment was conducted in the early stage of the study, and the optimal dosage and sampling time were determined through a 7-day wound healing experiment.

[0067] 1. Preparation of acute wound model 10% chloral hydrate was intraperitoneally injected into the rats at 0.4 mL / 100 g body weight to induce deep sedation. The rats' back hair was shaved with a shaver, and the remaining hair was removed with a depilatory cream. The skin was cleaned and disinfected with iodine. A 10 mm diameter wound was made deep to the fascia on the left side of the rat's back with an animal-specific punch.

[0068] 2. Preparation of ointment The origin of the loulouhua was S1. The loulouhua ointment group contained 1.25 g of loulouhua powder (the quercetin content was about 0.2 mg / g). The blank ointment group did not contain any drug. The rest of the preparation process was the same as that of the loulouhua ointment group.

[0069] 3. Grouping and drug administration settings Combined with the preliminary experimental results, the optimal dosage and sample collection time were finally determined.

[0070] The rats were randomly divided into a normal group (N), a blank ointment (ointment without schisandra chinensis extract) control group (BO), a model group (MO), and a schisandra chinensis ointment group (RL containing 1.25 g of powder with a quercetin content of approximately 0.2 mg / g).

[0071] After the wound model was successfully made, the wound of the rat was cleaned to remove blood stains on the skin surface; the SD rats were then weighed and randomly divided into BO, MO, RL, and N groups according to the random block method (10 rats in each group, half male and half female). The drugs such as the louluhua ointment and the blank ointment were evenly applied to the wound of each group of rats with a sterilized cotton swab at four time points on the 1st, 2nd, 3rd, and 4th day, respectively. The dosage was about 0.1 g, the application thickness was controlled at 1 mm, and the application area was controlled at 1 cm 2 (1cm×1cm). Clean and bandage the wound surface, and raise the animals in separate cages.

[0072] 4. Observation indicators and methods The wound healing and scab formation of rats in each group were observed with the naked eye, and photos were taken. The wound diameter was recorded with a vernier caliper, and the wound area of ​​the rats was calculated (the length of the major axis and the minor axis was measured for elliptical wounds). The wound healing and healing time of each group were recorded. The formula for wound healing rate = (initial wound area - wound area on the day of measurement) / initial wound area × 100%.

[0073] Table 12 Wound healing rate

[0074] Note: * indicates P < 0.05 compared with the BO group; # Indicates P < 0.05 compared with the MO group.

[0075] Depend on Figure 1 It can be seen that compared with the MO group, the healing condition of the RL group was relatively good, no edema or exudation was found in the wound tissue, the healing time was significantly shortened, and the overall healing rate was better in the Louluhua ointment group (see Table 12 and Figure 2 ), while there was no significant difference in the overall healing between the BO group and the MO group, and the healing rate was lower than that of the RL group.

[0076] 5. Sample collection Samples were prepared 4 days after administration, with a large dose of chloral hydrate injected intraperitoneally and samples were collected after death. Skin tissue samples were removed from the wounds of rats with a scalpel. The sample size was about 1 cm × 1 cm and weighed about 1.0 g. The wound should be located in the center of the skin tissue. The skin tissue was divided into 4 equal parts, one of which was placed in a 50 mL centrifuge tube and immersed in 4% paraformaldehyde solution for histopathological observation. The remaining skin tissue was transferred to a centrifuge tube, frozen with liquid nitrogen, and then stored in a -80°C refrigerator for later use.

[0077] 6. Histopathological observation Skin tissue samples were fixed in 4% paraformaldehyde solution at room temperature for 48 h; after dehydration, the skin tissue samples were placed in a mold, and paraffin liquid was slowly dripped into the samples after they were placed. After cooling, the wax block specimens were trimmed and fixed on the microtome, and the distance between the wax block and the blade was adjusted. Continuous sections were made with a thickness of 4 μm. The wax slices were placed in a 45°C constant temperature water bath, unfolded, and the slides were quickly picked up to ensure that the wax slices were flatly attached to the slides, dried, and stained with hematoxylin-eosin (HE) dye. Tissue damage was observed under a microscope.

[0078] like Figure 3 As shown in the figure, HE staining observation showed that the epidermal structure of the wound tissue of rats in group RL was relatively complete and clear, the spinous cell layer was thicker than that in group N, and the inflammatory cell infiltration was less than that in groups MO and BO. The experimental results in this part proved that under the intervention of loofah ointment, the rat epidermis can effectively inhibit the occurrence of inflammatory response and promote epidermal repair.

[0079] 7. ELISA kit to detect inflammation and growth factors Wound tissue specimens of rats in each group were taken to prepare tissue homogenates, centrifuged at 3000r / min for 10min, and the supernatant was taken. The inflammatory factors TNF-α and IL-6 levels and the growth factor EGF level in the wound tissue were detected in strict accordance with the instructions of each ELISA kit. The statistical values ​​are expressed as mean ± standard deviation ( All data were analyzed using SPSS 26.0 statistical software. The statistical method was one-way analysis of variance, and further pairwise comparisons were performed using the LSD-t test. P < 0.05 indicated statistical significance.

[0080] Table 13 and Figure 4 The results showed that compared with the N group, the expression levels of TNF-α and IL-6 proteins in the wound healing tissue of the animals in the MO group were increased (P<0.05); compared with the MO group, the expression levels of TNF-α and IL-6 proteins in the wound healing tissue of the animals in the RL group were significantly decreased (P<0.05); compared with the N group, the expression level of EGF protein in the wound healing tissue of the animals in the MO group was significantly decreased (P<0.05), and compared with the MO group, the expression level of EGF protein in the wound healing tissue of the animals in the RL group was significantly increased (P<0.05).

[0081] Table 13 Comparison of inflammatory factors and growth factors in wound tissue of mice in each group

[0082] Note: * Indicates P < 0.05 compared with group N; # Indicates P < 0.05 compared with the MO group.

[0083] Experimental Example 4 Metabolomics Detection and Analysis 1. Sample preparation: Samples (RL, MO, and N group skin tissues) were thawed at 4°C, 50 mg of each sample was weighed, 2 medium-sized steel balls were added, and 200 μL of pre-cooled methanol-water (4:1, V:V) solution was added to mix, and the samples were homogenized and broken in a tissue disruptor at low temperature, and then 800 μL of pre-cooled methanol-water (4:1, V:V) solution was added to mix, and ultrasonicated in an ice bath for 20 min, allowed to stand at -20°C for 1 h, centrifuged at 16000r, 4°C for 20 min, and the supernatant was taken and evaporated in a high-speed vacuum concentrator centrifuge. For mass spectrometry detection, 100 μL of pre-cooled methanol-water (1:1, V:V) solution was added for re-dissolution, centrifuged at 20000r, 4°C for 15 min, and the supernatant was taken for sampling and analysis.

[0084] 2. Chromatographic conditions: The chromatographic column was ACQUITY Rising LC HSS T3 (2.1×100 mm, 1.8 µm), and the chromatographic mobile phases A and B were 0.1% formic acid in water and 1% formic acid in acetonitrile. During the entire analysis, the sample was placed in an automatic sampler at 4°C, the injection volume was 4 μL, the column temperature was 40°C, and the flow rate was 0.3 mL / min. The chromatographic gradient elution program was as follows: Table 14 Gradient elution conditions

[0085] 3. Mass spectrometry acquisition: Each sample was detected by electrospray ionization (ESI) in positive (+) and negative (-) modes. After ascending LC separation, the samples were analyzed by mass spectrometry using a QEPlus mass spectrometer (Thermo Scientific), using a HESI source for ionization. The ionization conditions were as follows: spray voltage: 3.8 kV (+) and 3.2 kV (-); capillary temperature: 320 °C (±); sheath gas: 30 Arb (±); auxiliary gas: 5 Arb (±); probe heater temperature: 350 °C (±); Mass spectrometry acquisition time: 15 min. Precursor ion scanning range: 75-1050 m / z, primary mass spectrometry resolution: 70000 m / z 200, AGC target: 3e6, primary Maximum IT: 100 ms. Secondary mass spectrometry analysis was acquired according to the following method: trigger acquisition of the secondary mass spectra of the 10 highest intensity precursor ions after each full scan, secondary mass spectrometry resolution: 17500 m / z 200, AGC target: 1e5, secondary Maximum IT: 50 ms, MS2 Activation Type: HCD, Isolation window: 2 m / z, Normalized collision energy (Setpped): 20, 30, 40.

[0086] 4. Data processing and analysis: The original data were peak aligned, retention time corrected and peak area extracted using MSDIAL software. The metabolite structures were identified by accurate mass matching (mass tolerance < 10 ppm) and secondary spectrum matching (mass tolerance < 0.01 Da), and public databases such as HMDB, Mass Bank, GNPS and the self-built Baipu metabolite standard library (BP-DB) were searched. For the extracted data, the ion peaks with missing values ​​greater than 50% within the group were deleted and excluded from subsequent statistical analysis; the total peak area of ​​positive and negative ion data was normalized, the positive and negative ion peaks were integrated, and Python software was used for pattern recognition. The data were preprocessed by Unit variance scaling (UV) before subsequent data analysis. The metabolite data were analyzed by unsupervised principal component analysis (PCA) and supervised orthogonal partial least squares discriminant analysis (OPLS-DA), and differential metabolites were screened according to VIP values ​​(VIP>1) and predefined P values ​​(P<0.05).

[0087] (1) Principal Component Analysis (PCA): Principal component analysis is an unsupervised data analysis method that linearly combines all the metabolites originally identified to form a new set of comprehensive variables. At the same time, several comprehensive variables are selected according to the problem being analyzed so that they can reflect as much information as possible from the original variables, thereby achieving the purpose of dimensionality reduction. At the same time, principal component analysis of metabolites can also reflect the variability between and within sample groups in general. The PCA method is used to observe the overall distribution trend among all samples and find possible discrete points. PCA score diagram is shown in Figure 5 .

[0088] Figure 5 Each point in the graph represents a sample, points of different colors represent different groups, and all samples are placed within the 95% confidence interval. Figure 5 It can be concluded that the QC group of quality control samples is closely clustered, indicating the stability and repeatability of the instrumental analysis; the N and MO groups are clustered separately, with no obvious cross-overlap, and have a certain separation trend; the RL and N groups are clustered separately, with no obvious cross-overlap, and have a certain separation trend, indicating that the metabolic profile of rats has changed significantly after the intervention of Louluhua ointment and after modeling; and the MO and RL groups are clustered separately, with only a small part of the crossover, indicating that there are certain differences between the two groups.

[0089] (2) Orthogonal partial least squares discriminant analysis (OPLS-DA): Orthogonal partial least squares discriminant analysis is another supervised discriminant analysis statistical method. This method uses partial least squares regression to establish a relationship model between metabolite expression and sample category to predict the sample category. This method is modified on the basis of partial least squares discriminant analysis (PLS-DA), filtering out noise irrelevant to classification information, and improving the analytical ability and effectiveness of the model.

[0090] The OPLS-DA model of each comparison group was established. The model evaluation parameters are shown in Table 15 and the model score graph is shown in Figure 6 , permutation test diagram see Figure 7 . (R 2 X and R 2 Y represents the model's explanation rate for the X matrix and the Y matrix; Q 2 : indicates the model prediction ability; R 2 and Q 2 The closer it is to 1, the more stable and reliable the model is. 2 >0.5 indicates that the model has good predictive ability, Q 2 Less than 0.5 indicates that the model has poor prediction ability; RMSEE: root mean square error of the estimate; pre represents the number of prediction components used for modeling, ort represents the number of orthogonal components used for modeling; pR 2 Y:R 2 The p-value of Y. Table 15 OPLS-DA model parameters of each comparison group

[0091] Figure 7 To validate the OPLS-DA model, 200 permutation tests were performed. The horizontal axis in the figure represents the correlation between the random grouping Y and the original grouping Y, and the vertical axis represents R 2 and Q 2 The quality of the multivariate statistical analysis model is ultimately determined by the permutation test results. 2 The intercept of the regression line is less than 0, or the Q of 200 permutation tests is 2 The scores are lower than the original model Q 2 The score that satisfies any of the two represents that the model is very reliable, indicating that the OPLS-DA model established by the experimental data is not overfitted.

[0092] In summary, there may be differential metabolites among the MO, N, and RL groups, and further screening is necessary.

[0093] 5. Screening of differential metabolites Combining PCA and OPLS-DA metabolite differential analysis, a metabolite volcano plot was drawn, see Figure 8 ; FC≥1.5 or FC≤1 / 1.5 and P.value<0.05 were used as screening criteria. The vertical dotted lines indicate log2(1 / 1.5) and log2(1.5). The red dots in the figure represent up-regulated metabolites, and the blue dots represent down-regulated metabolites.

[0094] Combined with the KEGG database; compared with the MO group, a total of 470 differential metabolites were screened, 294 were upregulated and 176 were downregulated; compared with the RL group, a total of 191 differential metabolites were screened, 132 were upregulated and 59 were downregulated; there were 63 common differential metabolites among the three groups, including 10 glycerophospholipids, 3 fatty acids, 5 organic nitrogen compounds, 3 steroids and steroid derivatives, 2 carboximidic acids and their derivatives, 5 carboxylic acids and their derivatives, 2 linear 1,3-diarylpropanes, 2 dihydrofurans, 3 benzene and substituted derivatives, 3 adrenaline lipids, and 2 diazos; the following table shows the common significant differential metabolites among the three groups (see Table 16); Table 16 Analysis of common metabolites and differential metabolites in each group

[0095]

[0096]

[0097] 6. Metabolic pathway analysis The above differential metabolites were entered into MetaboAnalyst 5.0 online analysis platform for pathway analysis. Impact>0.2 was the condition. When the above conditions were met, it was determined to be a potential related metabolic pathway. The metabolic pathways involved in the differential metabolites between the MO and N groups were: purine metabolism, amino acid biosynthesis, aminoacyl-tRNA biosynthesis, cofactor biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, alanine, aspartate and glutamate metabolism, beta-alanine metabolism, DA amino acid metabolism, glutathione metabolism, arginine biosynthesis, 2-oxocarboxylic acid metabolism, pyrimidine metabolism, primary bile acid biosynthesis, cysteine ​​and methionine metabolism, histidine metabolism, pantothenic acid and CoA biosynthesis, glycine, serine and threonine metabolism; the metabolic pathways involved in the differential metabolites between the RL and MO groups were: sphingolipid metabolism, purine metabolism, nucleotide metabolism, cofactor biosynthesis, thiamine metabolism, tryptophan metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, glycerolipid metabolism (results see Fig. 9 ).

[0098] The invention prepares Mongolian medicine loulou flower ointment according to a disease model and preliminary experimental results; selects the type of ointment according to the appearance, color, spreadability and drug loading performance of the prepared ointment; adopts orthogonal design to optimize the dosage of key components in the loulou flower ointment prescription, and conducts a systematic evaluation on the prepared loulou flower ointment to determine whether it can be used in subsequent pharmacology and efficacy experiments.

[0099] In the efficacy study, an acute trauma model was established in rats, and the prepared loulouhua ointment was used for local administration. The wound healing was measured daily, and the wound skin tissue was collected and the ELISA kit was used to examine the inflammatory factors and growth factors. The pharmacodynamic results showed that the external application of loulouhua ointment can reduce wound edema and exudation, improve the local environment of the wound, and promote wound healing.

[0100] Non-targeted metabolomics related technologies were used to explore the mechanism of action of louluhua ointment in the treatment of acute trauma. After a large number of screening and inter-group comparisons, a total of 470 differential metabolites were screened out between the MO and N groups, and a total of 191 differential metabolites were screened out compared with the RL and MO groups. Metabolic pathway analysis was performed on the above differential metabolites. The results showed that louluhua ointment may promote the healing of acute trauma by intervening in metabolic pathways such as sphingolipid metabolism and nucleotide metabolism.

[0101] There are many studies on the role of sphingolipid metabolism in cancer and inflammation. Sphingolipids are not only important components of biological membrane structure, but also important regulators of cell transport and signal transduction. Abnormal sphingolipid metabolism is closely related to inflammation and systemic allergic reactions. Among them, phytosphingosine (PHS) is a sphingomyelin substance, which is considered to be a natural anti-inflammatory agent. Current studies have confirmed that it has anti-inflammatory effects. T. Pavicic et al. conducted in vitro and in vivo studies on its antimicrobial and inflammatory activities. The study found that PHS can effectively reduce the release of IL-1α after ultraviolet irradiation and inhibit protein kinase C activity. PHS can achieve relevant control of inflammation by inhibiting these two pathways. This study found that the low expression of PHS can be adjusted under the intervention treatment of louluhua ointment, which may play a regulatory role in the treatment of acute trauma.

[0102] Inosine is a nucleoside compound formed by the combination of hypoxanthine and ribose. It has good immunomodulatory function and shows good therapeutic effect in animal models of inflammatory injury. Lucas Liaudet et al. studied the effect of inosine on lipopolysaccharide-induced acute lung inflammation. Under the intervention of inosine, the expression of lipopolysaccharide-induced TNF-α, IL-1, IL-6 and MIP-2 was downregulated, and the expression of MIP-1 tended to be reduced, while the production of IL-4 was enhanced, further inhibiting lipopolysaccharide-induced lung inflammation. The results of metabolomics research showed that the expression trend of inosine was upregulated after the intervention of louluhua ointment, indicating that the anti-inflammatory effect of louluhua ointment is related to the upregulated expression of inosine. In summary, the ascending LC-Q-Exactive-MS / MS and non-targeted metabolomics research methods were used to study the therapeutic effect and mechanism of Lophatherum gracile in treating acute rat trauma model. According to the results of PCA and OPLS-DA, the clustering of samples in each experimental group was good, but there were certain differences in the metabolic profiles between the groups. After database processing and screening, a total of 470 differential metabolites were screened out between the MO and N groups, and a total of 191 differential metabolites were screened out compared with the RL and MO groups. There were 63 metabolites in common among the three groups. Metabolic pathway analysis was performed on the significant differential metabolism. The results showed that low-dose Lophatherum gracile ointment may be involved in the regulation of the above differential metabolites, and intervene in sphingolipid metabolism and nucleotide metabolism to promote acute wound healing.

[0103] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A loquat flower ointment, characterized in that: The ointment is composed of smilax china flower powder, glycerin, distilled water, stearic acid, white vaseline, lanolin, liquid paraffin, glyceryl monostearate, and an emulsifier; The mass ratio of the smilax glabra powder, glycerin, distilled water, stearic acid, white vaseline, lanolin, glyceryl monostearate, and emulsifier is 2:5:29:6:0.5:2.5:1.8:0.2; The volume ratio of water to liquid paraffin is 29:

3.

2. The ointment of claim 1, characterized in that: The emulsifier is triethanolamine.

3. A method for preparing the loulouhua ointment as claimed in any one of claims 1 to 2, characterized in that: The specific steps include: S1: Put the powder of Eleutherodactyla sphaerocephala in a bottle, add 50% ethanol, weigh, and ultrasonicate for 60 minutes. After ultrasonication, defat with petroleum ether, filter with suction, and put the filtrate into a rotary evaporator and evaporate to an extract to obtain Eleutherodactyla sphaerocephala extract; S2: mixing stearic acid, white vaseline, lanolin, liquid paraffin, and glyceryl monostearate to obtain an oil phase; S3: mixing glycerol and distilled water to obtain an aqueous phase; S4: Heat the oil phase and the water phase separately, add the eloud extract to the oil phase, add the emulsifier to the water phase, and then slowly add the oil phase to the water phase while stirring, and after mixing evenly, continue stirring and emulsifying to obtain a mixed matrix; put the mixed matrix into a mortar and grind it finely, and after cooling, spread it on the surface of a sterile container, place it under ultraviolet light, and obtain an eloud ointment.

4. The method for preparing the Lophatherum odoratum ointment according to claim 3, characterized in that: In the step S1, the ultrasonic frequency is 40 kHz, the power is 100%, and the specification is 500W; the density of the smilax china extract is 1.00-1.10 g / mL.

5. The method for preparing the Lophatherum odoratum ointment according to claim 3, characterized in that: In the step S4, the heating temperature is 80° C., the emulsification time is 10 min, and the irradiation time under the ultraviolet lamp is 1 h.

6. Use of the loulouhua ointment as described in any one of claims 1 to 2 in the preparation of products for treating acute trauma.

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