Chrysanthemum micranthum stamen extract as well as extraction method and application thereof

Abstract

The invention discloses a rhododendron micranthum stamen extract and an extraction method and application thereof, the rhododendron micranthum stamen extract raw material is rhododendron micranthum stamen, and the rhododendron micranthum stamen extract is obtained through the steps of processing, reflux extraction, vacuum concentration, freeze drying and the like. The rhododendron micranthum stamen extract is a natural plant extract, has small toxic and side effects, is convenient to carry and use, has anti-inflammatory and antibacterial effects on skin, and takes effect quickly.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an extract of the stamens of Rhododendron molle, its extraction method, and its application. Background Technology

[0002] As the largest organ in the human body, the skin is the first line of defense against external pathogens and the maintenance of internal homeostasis. In daily life, the skin is susceptible to infections from microorganisms (bacteria, fungi, etc.), physical and chemical irritants, and the activation of inflammatory factors, leading to various skin diseases such as acne, eczema, dermatitis, and skin infections. These diseases not only cause symptoms such as itching, redness, pain, and impaired barrier function, but in severe cases, they can also lead to systemic infections, affecting the patient's health and quality of life. Currently, commonly used medications in clinical practice are mainly topical Western medicine preparations. These preparations work by inhibiting or killing pathogenic microorganisms and reducing inflammatory responses, playing an important role in the treatment of skin diseases. Commonly used topical Western antibacterial agents include preparations containing chlorhexidine, povidone-iodine, triclosan, miconazole nitrate, and phenols, which can achieve antibacterial and anti-inflammatory effects to a certain extent, providing options for the clinical treatment of skin diseases.

[0003] However, existing topical antibacterial agents may cause various adverse reactions in clinical use, mainly including the following categories: First, contact dermatitis is the most common; for example, preparations containing chlorhexidine may cause erythema, itching, desquamation, and contact eczema. Second, they may induce skin irritation; for example, high concentrations of povidone-iodine (>5%) can easily cause burning sensations and dryness and peeling, especially in children with fragile skin barriers. Third, there are issues with drug resistance; long-term use of triclosan has led to the emergence of drug-resistant strains of Staphylococcus aureus, and the US FDA banned the sale of bath products containing this ingredient in 2016. There are also special cases showing that misuse of miconazole nitrate cream on large wounds may lead to systemic absorption and abnormal liver function (see the 2019 case report in the *Journal of Adverse Drug Reactions*). Furthermore, alcohol-containing phenolic antibacterial agents may disrupt the skin's microecological balance; a 2020 study showed that continuous use for two weeks reduced skin commensal bacteria by 40%.

[0004] Given the adverse reactions that are easily caused by Western medicine preparations, it is necessary to design safe and effective topical skin medications that can quickly exert antibacterial and anti-inflammatory effects to meet the needs of prevention and treatment of various skin infections and inflammatory diseases. Summary of the Invention

[0005] One of the objectives of this invention is to provide an extract of Rhododendron molle stamens that is safe, effective, has few toxic side effects, takes effect rapidly, is convenient to use in clinical treatment, and is easy for patients to carry and use.

[0006] The second objective of this invention is to provide a method for extracting the stamen extract of Rhododendron molle, which can be used to prepare the stamen extract of Rhododendron molle.

[0007] The third objective of this invention is to provide an application of the extract of Rhododendron molle flower stamen, which can be used for anti-inflammatory and antibacterial effects on the skin, with significant effects.

[0008] The first objective of this invention is achieved by the following technical solution: An extract of Rhododendron molle stamens, the raw material of which is Rhododendron molle stamens.

[0009] Preferably, the extract of Rhododendron molle stamens is prepared by the following method: taking Rhododendron molle stamens before the full bloom period as raw material, processing the Rhododendron molle stamens and extracting them by reflux with an ethanol aqueous solution to obtain an extract, concentrating the extract under reduced pressure to obtain a paste, freeze-drying the paste, and pulverizing the dried material to obtain the Rhododendron molle stamens extract.

[0010] The second objective of this invention is achieved by the following technical solution: The preparation method of extract from the stamens of Rhododendron molle includes the following steps: S1. Harvest the stamens of Rhododendron molle before its peak blooming period, and process the stigma of the stamens of Rhododendron molle to obtain processed Rhododendron molle stamens. S2. Extract the prepared stamens of Rhododendron simsii using solvent reflux to obtain an extract; S3. The extract is concentrated under reduced pressure to obtain an extract paste; S4. Freeze-dry the extract to obtain a dried product; S5. After pulverizing the obtained dried material, the extract of Rhododendron molle stamens is obtained.

[0011] Preferably, in step S1, the processing step includes: rinsing the stigma of Rhododendron molle and steaming it at 100°C for 2 hours, then drying it at 60°C until the moisture content is ≤10%, to obtain the processed Rhododendron molle product.

[0012] Preferably, in step S2, the solvent is an aqueous ethanol solution with a concentration of 40-80% (mL / mL); the ratio of the processed *Zhaoshan Baihua* stamen product to the aqueous ethanol solution is 1g:(8-12)mL.

[0013] Preferably, in step S2, the extraction time for a single reflux extraction is 90-150 min, and the number of extractions is 1-3.

[0014] The third objective of this invention is achieved by the following technical solution: The extract of Rhododendron molle flower stamen has significant anti-inflammatory effects in the preparation of drugs and / or skin care products for skin anti-inflammation.

[0015] The application of Rhododendron simsii flower stamen extract in the preparation of drugs and / or skin care products for skin antibacterial use, targeting bacteria including but not limited to Pseudomonas aeruginosa, Candida albicans and Staphylococcus aureus.

[0016] Beneficial effects: This invention provides an extract of Rhododendron molle stamens, its extraction method, and its application. The extract is obtained by alcohol extraction, concentration, and drying of Rhododendron molle stamens as raw material. It is a natural plant extract with low toxicity and side effects, is easy to carry and use, and has anti-inflammatory and antibacterial effects on the skin with rapid results. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0019] Figure 1 The image shows the skin lesions of the model group mice on day 3 of treatment in Example 4. Figure 2 The image shows the skin lesions of the model group mice in Example 4 on day 13 of treatment. Figure 3 The image shows the skin lesions of mice in the positive control group treated with the Western medicine on day 3 of Example 4. Figure 4 The image shows the skin lesions of the positive control mice in Example 4 on day 13 of treatment. Figure 5 The skin lesions of mice in the Rhododendron simsii stem and leaf extract group on day 3 of treatment in Example 4; Figure 6 The skin lesions of mice in the Rhododendron simsii stem and leaf extract group on day 13 of treatment in Example 4; Figure 7 The skin lesions of mice in the Rhododendron simsii flower stamen extract group on day 3 of treatment in Example 4; Figure 8 The skin lesions of mice in the Rhododendron simsii flower stamen extract group on day 13 of treatment in Example 4; Figure 9 Microscopic images of mouse skin tissue sections from each group in Example 4; Figure 10 The tumor necrosis factor α (TNF-α) levels in each group in Example 4; Figure 11The interleukin-1β (IL-1B) levels in each group in Example 4; Figure 12 The interferon-γ (IFN-γ) levels in each group in Example 4; Figure 13 The levels of interleukin-2 (IL-2) in each group in Example 4; Figure 14 The interleukin-4 (IL-4) levels in each group in Example 4; Figure 15 The antibody used in WB in Example 5; Figure 16 These are electrophoresis images of each group in Example 5; Figure 17 The antibacterial zone effect of the extract group of Rhododendron molle stem and leaf in Example 6; wherein: (a) is Pseudomonas aeruginosa, (b) is Candida albicans, and (c) is Staphylococcus aureus; Figure 18 The antibacterial zone effect of the extract group of Rhododendron molle in Example 6; wherein: (a) is Pseudomonas aeruginosa, (b) is Candida albicans, and (c) is Staphylococcus aureus. Figure 19 The antibacterial zone effect of eucalyptol in Example 7; wherein: (a) is Pseudomonas aeruginosa, (b) is Candida albicans, and (c) is Staphylococcus aureus; Figure 20 The antibacterial zone effect of ursolic acid in Example 7; wherein: (a) is Pseudomonas aeruginosa, (b) is Candida albicans, and (c) is Staphylococcus aureus; Figure 21 The inhibition zone effect of α-terpineol in Example 7; wherein: (a) is Pseudomonas aeruginosa, (b) is Candida albicans, and (c) is Staphylococcus aureus. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1 An extract of Rhododendron molle stamens, the raw material of which is Rhododendron molle stamens. The extract is obtained by processing Rhododendron molle stamens through alcohol extraction, vacuum concentration, freeze drying, and pulverization.

[0022] Example 2 The preparation method of the extract of Rhododendron molle includes the following steps (1)-(4).

[0023] (1) Preparation of materials: Before the full bloom, the stamens of Rhododendron molle are picked. The stigmas of the Rhododendron molle are processed and the processed products are packed into self-sealing bags and refrigerated for later use. The processing of the stigmas adopts the steaming method in the 2025 edition of the Chinese Pharmacopoeia. The steps are as follows: the stigmas of the fresh flowers (the nectaries and nectar need to be removed) are rinsed with clean water, layered in a steamer, steamed at 100℃ for 2 hours, and then dried at 60℃ until the moisture content is ≤10% to obtain the processed Rhododendron molle stamens. According to the test, the degradation rate of leucotoxins in the processed Rhododendron molle stamens is >78% and the flavonoid retention rate is >85%. (2) Extraction: 200 g of processed Rhododendron molle stamens were refluxed with 1600 mL of 65% ethanol aqueous solution (single extraction time 120 min, extraction times 2) to obtain the extract; (3) Concentration: Add 500 mL of extract to a 1000 mL rotary flask and concentrate under reduced pressure. The cooling water circulation temperature is maintained at around -10℃, the rotary flask rotation speed is 60 r / min, the pressure of the circulating water vacuum pump is 0.10 MPa, and the water bath temperature is 50℃. After most of the ethanol is recovered, the water bath temperature is adjusted to 70℃ and concentrated under reduced pressure is continued until the relative density in the rotary flask device is 1.10 g / mL. The concentration of the extract solution under reduced pressure is completed, and the extract is obtained. (4) Drying: Add the above extract to a 500 mL drying bottle and freeze dry. Lower the temperature of the pre-freezing bath to about -40°C. Fix the drying bottle containing the liquid to be frozen to the rotating shaft with a buckle and adjust it to a suitable height. Cover the liquid surface with the cap (to prevent alcohol evaporation or splashing). Turn on the rotary switch. When the liquid is completely frozen and evenly adhered to the bottle wall, turn off the rotary switch to stop the rotation and remove the drying bottle. At the same time, start the freeze dryer to cool to -80°C and reduce the pressure to below 30 Pa. Hang the pre-frozen drying bottle on the hanging arm and freeze dry for 36 h. Then remove the drying bottle and crush the dried material to obtain the extract of Rhododendron molle.

[0024] In step (2), the specific parameters were obtained from the following experiments; all experiments were repeated 3 times or more, and the average value of the results was taken.

[0025] ① Extraction Steps - Single-Factor Influence Experimental Study The single-factor experiment on the extraction steps of Rhododendron simsii stamens mainly investigated the ratio of material to solvent, different solvent ratios, and extraction time. Group experiments were conducted for different levels of these influencing factors, and the yield of extract obtained from different groups was compared (extract yield = extract weight / weight of processed Rhododendron simsii stamens × 100%) to evaluate the advantages and disadvantages and to preliminarily screen the scope of the extraction steps for different factors. The factor design of the single-factor experiment on the extraction steps is shown in Table 1. The weight of the processed Rhododendron simsii stamens in a single group was 200 g.

[0026] Table 1. Single-factor extraction experimental design table

[0027] Extraction experiments were conducted to investigate the factors affecting the material-liquid ratio. The design and results are shown in Table 2.

[0028] Table 2. Experimental Factors Affecting the Feed-to-Liquid Ratio

[0029] Extraction experiments were conducted to investigate the factors affecting the concentration of ethanol-water solution. The design and results are shown in Table 3.

[0030] Table 3. Experimental Factors Affecting the Concentration of Ethanol Aqueous Solution

[0031] The extraction experiments were conducted to investigate the factors affecting extraction time. The design and results are shown in Table 4.

[0032] Table 4. Experimental Factors Affecting Extraction Time

[0033] ② Extraction Steps - Orthogonal Optimization Process Study By analyzing and optimizing the results of the above single-factor influence experiments, a more suitable scope of investigation was selected. Three levels were set for each of the four influencing factors: material-liquid ratio, ethanol concentration, extraction time, and number of extractions. Orthogonal optimization experiments were conducted using an orthogonal optimization design table. The yield of each group of extracts was assigned a score, and the experimental results were analyzed by variance analysis. The design of the orthogonal optimization influencing factors is shown in Table 5, and the implementation of the orthogonal optimization experiment is shown in Table 6.

[0034] Table 5 Orthogonal Optimization Design Table

[0035] Table 6 Orthogonal Optimization Experiment Table

[0036] Summary: Through the orthogonal optimization experiments on the extraction steps described above, different extract yields were obtained in 9 groups of extraction experiments. The extract yields were assigned scores, and the quality of different groups was evaluated. Statistical analysis of the data was performed using statistical software, and an analysis of variance was conducted. The T-value was less than 0.002, indicating statistical significance. The influencing factors were determined to be B>A>C>D, where D is the error term. The optimal extraction parameters were: a ratio of Rhododendron molle flower stamens to solvent of 1:8, an ethanol-water solution concentration of 65%, a single extraction time of 120 min, and two extractions.

[0037] Example 3 A scale-up experiment was conducted on the preparation method of the extract of Rhododendron molle from Example 2. Three parallel experiments were set up. In each group, 2 kg of processed Rhododendron molle stamens were weighed and extracted with 16 L of 65% ethanol aqueous solution. The extraction was performed twice for 120 min each time, and the weight loss was made up. After the extract cooled, the two extracts were combined and concentrated under reduced pressure. The concentrated extract was then freeze-dried, and the dried material was pulverized to obtain the Rhododendron molle stamen extract. The yield of the Rhododendron molle stamen extract was calculated (yield of Rhododendron molle stamen extract = weight of Rhododendron molle stamen extract / weight of processed Rhododendron molle stamens × 100%). Three batches of experiments were conducted (n=3). Three batches of scale-up stability experiments were conducted to verify the stability of the preparation process of the Rhododendron molle extract of the present invention. The Rhododendron molle extract produced by this preparation method can provide an extract material basis for the subsequent preparation processes of ointments, granules, liniments, plasters, gels, tablets and capsules, and can be industrialized. In addition, the prepared Rhododendron molle extract is in powder form, which is convenient to carry.

[0038] Table 7. Scale-up Stability Experiment Table for Extraction Steps

[0039] Example 4 Pharmacodynamic experiments were conducted on the extract of *Solanum nigrum* stamens.

[0040] I. Experimental Basis 1. Study subjects: Thirty healthy SPF-grade Kunming mice, half male and half female, less than one month old. They were fed under standard conditions.

[0041] 2. Establishment of a rat eczema model: Hair was removed from the abdomen of rats, and 100 μL of 7% DNCB solution was applied to the abdominal skin to sensitize them. On days 6, 9, 12, and 15, 25 μL of 0.5% DNCB solution was applied to the left auricle.

[0042] 3. Treatment grouping: The model rats were randomly divided into 5 groups: model group, positive control drug group, Rhododendron molle stem and leaf extract group, Rhododendron molle flower stamen extract group and blank group, with 6 rats in each group. Treatment started 24 hours after stimulation and lasted for 15 days as one course of treatment. Model group: 1.5 mL of physiological saline was applied to the dorsal side of mice in a single application; Positive control group (Western medicine group): Hydrocortisone butyrate ointment was applied topically, 1.5 mL in a single application to the dorsal side of mice; Rhododendron molle stem and leaf extract group: Rhododendron molle stem and leaf extract (raw materials were branches, stems, and leaves of Rhododendron molle, and the rest of the preparation method was the same as that of Rhododendron molle flower stamen extract group) was diluted and dissolved with physiological saline to obtain a concentration of 25 mg / mL, and 1.5 mL in a single application was applied to the dorsal side of mice; Rhododendron molle flower stamen extract group: Rhododendron molle flower stamen extract was diluted and dissolved with physiological saline to obtain a concentration of 25 mg / mL, and 1.5 mL in a single application was applied to the dorsal side of mice; Blank group: 1.5 mL of physiological saline was applied to the dorsal side of mice in a single application.

[0043] II. Results Analysis 1. Toxicity analysis The extract of Rhododendron simsii flower stamens is for external use only and is not intended for oral administration. In animal experiments, no signs of toxicity were observed in mice after administration to the nervous, cardiovascular, or digestive systems, indicating that the extract of Rhododendron simsii flower stamens, when administered at the above dosage, has no toxic effects.

[0044] 2. Skin lesions in mice On days 3, 9, and 13 of treatment, the skin lesions and skin inflammation scores of mice in the model group, positive control group (Western medicine group), group treated with Rhododendron molle stem and leaf extract, and group treated with Rhododendron molle flower stamen extract were recorded; photographic records are shown below. Figure 1-8 As shown in the figure (6 mice in each group, only a portion is shown in the figure); the skin inflammation scoring rules are shown in Table 8 below, and the scoring results (the average score of the six mice in each group) are shown in Table 9 below.

[0045] Table 8 Scoring Rules for Skin Inflammation in Mice

[0046] Table 9. Skin Inflammation Scores for Mice in Each Group

[0047] Combined with Table 9, Figure 1-8 It is evident that, compared to the model group, the mice in the Rhododendron simsii flower stamen extract group, the Rhododendron simsii stem and leaf extract group, and the positive control Western medicine group all showed significant improvement in the erythema and edema on their backs. Among them, the mice in the Rhododendron simsii flower stamen extract group showed a significantly better therapeutic effect than the other groups, and the effect was more pronounced.

[0048] 3. HE staining and pathological changes Fifteen days after treatment, the mice were sacrificed. Skin tissue from the backs of the mice in the model group, the positive control group (medicated drug group), the extract group of Rhododendron simsii stem and leaf group, and the extract group of Rhododendron simsii flower stamen was collected, fixed, and subjected to histopathological examination. The experimental steps are as follows: 1) Dewaxing paraffin sections to water: Place the sections in xylene I for 15 min, xylene II for 15 min, xylene III for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% ethanol for 5 min, and 85% ethanol for 5 min, then wash with tap water; 2) Staining: First, stain the sections with hematoxylin solution for 1-2 min, wash with tap water, differentiate with differentiation solution, wash with tap water, blue back solution, and rinse with running water; then stain with eosin solution for 2-3 min. 3) Dehydration and mounting: After staining, the sections are sequentially immersed in anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - anhydrous ethanol III for 5 min - xylene I for 5 min - xylene II for 5 min for clearing, and then mounted with neutral resin; 5) Perform microscopic examination / panoramic scanning on the slides and analyze them, including but not limited to congestion, ecchymosis, hemorrhage, edema, degeneration, necrosis, hyperplasia, fibrosis, and degree of damage repair. Image typical lesion sites and mark them with arrows in the images.

[0049] The inspection results are as follows Figure 9 As shown (scale bar 100 μm), in the model group mice, no obvious abscesses were observed in the stratum corneum of the skin tissue. Extensive epidermal and dermal necrosis was observed (black arrows), with visible hair follicle (purple arrows) and sebaceous gland necrosis (cyan-green arrows). Numerous inflammatory cell infiltrations were observed in the damaged areas (red arrows). The number of collagen fibers in the dermis was reduced, and the dermis was relatively thick. A small amount of inflammatory cell infiltration was observed in the subcutaneous tissue, with no other obvious abnormalities. In the positive control group mice treated with the western medicine, abscesses were observed in the stratum corneum of the skin tissue (green arrows), with extensive epidermal and superficial dermal necrosis, and localized thinning or loss of the epidermis (yellow arrows). The number of collagen fibers in the dermis was reduced (blue arrows), and a small amount of inflammatory cell infiltration was observed in the damaged areas. The dermis was relatively thick, and no obvious hair follicles, sebaceous glands, or other appendages were observed in large areas of the dermis. The subcutaneous tissue structure was clear, with no other obvious abnormalities. In mice treated with Rhododendron simsii stem and leaf extract, abscesses were observed in the stratum corneum of the skin tissue (green arrows). Small areas of epidermal and superficial dermal necrosis were also observed, with significant local epidermal thickening and a marked increase in the number of squamous epithelial cells (yellow arrows). Small areas of collagen fiber count in the dermis were reduced, and numerous inflammatory cell infiltrations were observed in the damaged area (red arrows). The dermis was relatively thick, and no obvious hair follicles, sebaceous glands, or other appendages were observed in the small areas of the dermis. The subcutaneous tissue structure was clear, and no other obvious abnormalities were observed.

[0050] and Figure 9 The results showed that no obvious abscesses were observed in the stratum corneum of the skin tissue of mice in the Rhododendron simsii flower stamen extract group, the number of squamous epithelium on the surface was not significantly increased, the dermis was relatively thick, the structures of appendages such as hair follicles and sebaceous glands were clear, the subcutaneous tissue structure was clear, and no other obvious abnormalities were observed.

[0051] Therefore, combined with histopathological analysis, it can be seen that the extract of Rhododendron simsii flower stamen has a significant therapeutic effect on mice with eczema, and no adverse reactions such as redness, necrosis, or inflammation of the mouse skin were caused after 15 days of treatment, indicating that it is safe and effective.

[0052] 4. ELISA detection of inflammatory factor levels The levels of inflammatory factors, including tumor necrosis factor α (TNF-α) ELISA kit, interleukin 1β (IL-1B) ELISA kit, interleukin γ (IFN-γ) ELISA kit, interleukin 2 (IL-2) ELISA kit, and interleukin 4 (IL-4) ELISA kit, were measured in the serum of mice in each group using the ELISA kits manufactured by Wuhan Feien Biotechnology Co., Ltd., according to the ELISA kit steps.

[0053] The results are as follows Figure 10-14 As shown, after 15 days of treatment, the levels of inflammatory factors in mice in the Rhododendron molle flower stamen extract group were lower, close to those in the control group. The positive control group (Western medicine) and the Rhododendron molle stem and leaf extract group showed some anti-inflammatory effects, but their effects were lower than those in the Rhododendron molle flower stamen extract group.

[0054] In summary, based on animal model observation and evaluation, the extract group of Rhododendron simsii flower stamens showed significant differences compared with the model group, and was superior to the positive control western medicine group and the Rhododendron simsii stem and leaf extract group, indicating that its therapeutic effect was good.

[0055] Example 5 In vitro cell experiments were conducted to verify the anti-inflammatory effect of the extract of Rhododendron molle.

[0056] NF-κB is a key regulator of gene expression in many cellular processes, such as inflammation, immunity, differentiation, proliferation, and apoptosis. Many methods exist for detecting the NF-κB signaling pathway in cells; we chose Western blotting to detect the activity of nuclear transcription factor κB (NF-κB). Samples typically have low phosphorylation levels or are not expressed at all. In such cases, physical or chemical stimulation and induction can be used. LPS stimulation activates the NF-κB signaling pathway, and these inflammation-related transcription factors induce the synthesis of pro-inflammatory mediators and increased expression of surface proteins.

[0057] 1. Cells used in the experiment: Raw264.7 (mouse mononuclear macrophage leukemia cells) is one of the commonly used inflammatory cell models, and NF-κB is closely related to inflammation.

[0058] 2. Experimental setup (using 6-well plates, 2 mL of culture medium per well): Blank group: No processing; LPS group: LPS with a final concentration of 1 μg / mL was prepared using basal DMEM medium, which served as the blank control group; Drug A: The extract of Rhododendron molle was diluted and dissolved using LPS to obtain an extract solution with a final concentration of 1 mg / mL, which was used as drug A; 33.3 μL of drug A was mixed with 1967 μL of basal DMEM medium; Drug B: Dexamethasone was diluted and dissolved using LPS to obtain an extract solution with a final concentration of 1 mg / mL, which was used as drug B; 33.3 μL of drug B was mixed with 1967 μL of basal DMEM medium.

[0059] 3. Experimental steps: (1) Each group was seeded with raw264.7 cells in 6-well plates, with 4.5 x 10 cells per well. 6 Cells were cultured for 10 hours.

[0060] (2) Discard the original culture medium, wash with PBS, and then add drugs A and B prepared with basic DMEM culture medium. The blank group and LPS group only add basic DMEM culture medium.

[0061] (3) After 14 h of drug treatment, the culture medium was discarded, the well was washed with PBS, and each well was treated with basic DMEM culture medium containing 1 μg / mL LPS for 30 min.

[0062] (4) Protein extraction ① Extracting proteins from lysed cells a. Discard the culture medium and place the cells on ice; b. Add 300 μL of RIPA lysis buffer (containing 1 mM PMSF and phosphatase inhibitor) to each centrifuge tube and lyse for 30 min, shaking and mixing for 1 min every 10 min during the process; c. Centrifuge at 13,000 rpm for 30 min at 4°C, and collect the protein-rich supernatant into a new EP tube.

[0063] (5) Western blot detection of protein expression a. Take 20 μg of purified protein sample and internal control protein solution and mix them with 6 µL of 6× Protein Buffer, heat at 95℃ for 5 min, and place on ice; b. Prepare the separating gel and stacking gel according to the instructions of the SDS-PAGE gel preparation kit. Add the prepared 12% separating gel to 2 cm from the edge of the glass plate, fill it with anhydrous ethanol, let it stand at room temperature for 30 min, pour out the anhydrous ethanol, fill it with stacking gel, insert the comb, and wait for 30 min. c. Install the gel running apparatus correctly, pour 1×Running Buffer into the electrophoresis tank, add the protein marker and the cooked sample into the sample well, set the voltage to 80 V to run the sample out of the stacking gel, change the voltage to 120 V until the bromophenol blue reaches the edge of the gel; d. Cut two PVDF membranes of appropriate size, activate them in methanol for 1 min, place them in 1×Transfer Buffer, and assemble the transfer apparatus in the following order: black base plate, rough sponge, filter paper, gel, PVDF membrane, filter paper, rough sponge, white base plate. Place the membrane in the transfer tank, in 1×Transfer Buffer, at 4℃ and 350 mA for 50 min. e. Prepare 5% skim milk powder using 1×TBST, and incubate the PVDF membrane in the blocking solution on a shaker at room temperature for 1 h; f. Dilute Flag antibody at 1:5000 with 1×TBST and GAPDH antibody at 1:50000. Place the large PVDF membrane with purified protein into the Flag antibody dilution solution and the PVDF membrane with internal control protein into the GAPDH antibody dilution solution. Incubate overnight at 4°C. g. Wash the membrane 3 times with 1×TBST, 10 min each time.

[0064] h. Dilute goat anti-mouse fluorescent secondary antibody with 1×TBST at a ratio of 1:50000 and incubate on a shaker at room temperature for 1 h; I. Membrane washing: Wash the membrane 3 times with 1×TBST, 10 min each time; j. Observation results using a dual-color infrared laser imager (Odyssey CLX).

[0065] The antibodies used in WB, such as Figure 15 As shown. The results of the gel running are as follows. Figure 16 As shown.

[0066] The results showed that, by establishing an LPS-induced Raw264.7 (mouse mononuclear macrophage leukemia cell) model, LPS-stimulated phosphorylated proteins increased, while the addition of drugs A and B (including extract of Rhododendron molle and dexamethasone) inhibited the expression of phosphorylated nuclear transcription factor κB (NF-κB) proteins. This indicates that Rhododendron molle extract can inhibit the P65 pro-inflammatory signaling pathway in LPS-stimulated RAW 264.7 cells, thus clarifying its anti-inflammatory mechanism.

[0067] Example 6 The antibacterial effect of the extract of *Smilax glabra* was tested using the paper disc diffusion method.

[0068] Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans were cultured to the logarithmic growth phase to obtain 1×10⁻⁶ of each of the three bacteria. 7 The test bacterial suspensions were prepared at cfu / mL. Bacterial culture dishes were prepared (Pseudomonas aeruginosa: LB medium; Staphylococcus aureus: TSA medium; Candida albicans: SDA medium). Using a sterile pipette, 0.1 mL of the test bacterial suspensions for Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans were respectively added to the culture dishes. The bacterial suspensions were then evenly spread on the surface of the culture medium using a sterile spreader, and allowed to stand for 10 min.

[0069] The following were set up: a blank control group: sterile round filter paper was soaked in sterile water for 8 hours and then autoclaved to obtain blank control filter paper discs; a positive control group: sterile round filter paper was soaked in 50 mg / mL ciprofloxacin for 8 hours and then autoclaved to obtain positive control filter paper discs; experimental groups: 50 mg of Rhododendron molle stem and leaf extract and Rhododendron molle flower stamen extract were dissolved in 1 mL of sterile water, filtered through a 0.22 μm filter membrane into new centrifuge tubes, sterile round filter paper was soaked in the centrifuge tubes for 8 hours, and then autoclaved to obtain Rhododendron molle stem and leaf extract drug-containing filter paper discs and Rhododendron molle flower stamen extract drug-containing filter paper discs; these were prepared for use.

[0070] Blank control filter paper, positive control filter paper, filter paper containing extracts from the stems and leaves of Rhododendron simsii, and filter paper containing extracts from the stamens of Rhododendron simsii were respectively attached to the surface of the culture medium of different bacteria. The culture dishes were covered and three replicates were made for each type of bacteria. The plates were incubated at 37°C for 24 h. The presence or absence of inhibition zones around the filter paper discs was observed, and the diameter of the inhibition zones was measured (if the diameter of the inhibition zone is <7 mm, the filter paper has no antibacterial effect; if it is ≥7 mm, the filter paper has an antibacterial effect).

[0071] The results are shown in Table 10 and Figure 17 ,18 (The positive control group is located above the filter paper, the Rhododendron molle stem and leaf extract group / Rhododendron molle flower stamen extract group is located in the lower left, and the blank control group is located in the lower right.) Using the paper disc method to construct inhibition zones, it was found that the Rhododendron molle flower stamen extract had inhibitory effects on Pseudomonas aeruginosa, Candida albicans, and Staphylococcus aureus. The Rhododendron molle stem and leaf extract group showed inhibitory effects on Staphylococcus aureus, but poor inhibitory effects on the other two bacteria. Furthermore, by comparing the diameter of the inhibition zones, it was clear that the Rhododendron molle flower stamen extract had a better inhibitory effect on Staphylococcus aureus than the stem and leaf extract. Therefore, the Rhododendron molle flower stamen extract has good antibacterial effects and can be used to prepare antibacterial drugs and / or skin care products.

[0072] Table 10. Diameter of the inhibition zone of extracts from the stems, leaves, and flowers of Rhododendron simsii.

[0073] Example 7 The active ingredients of the extract of Rhododendron molle from Example 1 were determined by liquid chromatography. An Agilent 1290 Infinity II mass spectrometer was used. The chromatographic column was a Waters ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.7 μm), and the mobile phase was acetonitrile (LC-MS grade) and ultrapure water (containing 0.1% formic acid or 0.1% acetic acid). Sample preparation: 0.2 g of the extract of Rhododendron molle from Example 1 was added to 5 mL of 70% methanol (containing 0.1% formic acid), and the mixture was ultrasonically extracted for 30 min (40 kHz, 50 °C). The mixture was then centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm organic filter membrane. The constituent compounds of the extract were determined by comparison with relevant literature, ion fragmentation in databases (PubChem, METLIN), and standard comparisons. The results are shown in Table 11. It can be seen that the extract of Rhododendron molle contains effective ingredients such as eucalyptol, ursolic acid, α-terpineol, myricetin, quercetin, hyperoside, chlorogenic acid, gallic acid, and kaempferol.

[0074] Table 11 Main active ingredients of Rhododendron molle extract

[0075] The antibacterial effects of eucalyptol, ursolic acid, and α-terpineol, which have higher content among the aforementioned active ingredients, were tested using the disc diffusion method. The experimental method was the same as in Example 6. Sterile disc filter paper was soaked in sterile water and 50 mg / mL of ciprofloxacin, eucalyptol, ursolic acid, and α-terpineol, respectively, for 8 h. The mixture was then autoclaved to obtain blank control filter paper discs, positive control filter paper discs, eucalyptol filter paper discs, ursolic acid filter paper discs, and α-terpineol filter paper discs; these were then prepared for use. The prepared filter paper discs were affixed to the surface of culture media containing different bacteria (Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans), and the culture dishes were covered. Three replicates were prepared for each type of bacteria. The discs were incubated at 37°C for 24 h, and the presence and diameter of inhibition zones around the discs were observed and measured.

[0076] The results are shown in Table 12 and Figure 19-21 As shown, the antibacterial effects of eucalyptol, ursolic acid, and α-terpineol were compared with those of the extract of Rhododendron molle from Example 6. Among the active ingredients in the Rhododendron molle extract, eucalyptol showed inhibitory effects against Candida albicans and Staphylococcus aureus, but its antibacterial effect was inferior to that of the Rhododendron molle extract; it had a poor or no inhibitory effect against Pseudomonas aeruginosa. Ursolic acid showed inhibitory effects against both Pseudomonas aeruginosa and Staphylococcus aureus, but its inhibitory effect was inferior to that of the Rhododendron molle extract, as revealed by comparing the diameter of the inhibition zone; it also had a poor or no inhibitory effect against Candida albicans. α-terpineol showed inhibitory effects against Candida albicans, but its antibacterial effect was inferior to that of the Rhododendron molle extract; it also had a poor or no inhibitory effect against Pseudomonas aeruginosa and Staphylococcus aureus. It is evident that the Rhododendron molle extract prepared from Rhododendron molle raw materials achieves a good anti-inflammatory and antibacterial effect through the synergistic effect of all its constituent substances; the same effect cannot be achieved simply by superimposing known active ingredients.

[0077] Table 12 Diameter of the inhibition zones of eucalyptol, ursolic acid, and α-terpineol

[0078] 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An extract of the stamens of Rhododendron molle, characterized in that, Its raw material is the stamens of Rhododendron molle.

2. The extract of Rhododendron molle stamens according to claim 1, characterized in that, The extract of Rhododendron molle stamens is prepared by the following method: Rhododendron molle stamens before full bloom are taken as raw materials, the Rhododendron molle stamens are processed and then extracted by reflux with ethanol aqueous solution to obtain an extract, the extract is concentrated under reduced pressure to obtain a paste, the paste is freeze-dried, and the dried material is pulverized to obtain the Rhododendron molle stamen extract.

3. The method for preparing the extract of Rhododendron molle according to claim 1 or 2, characterized in that, It includes the following steps: S1. Harvest the stamens of Rhododendron molle before its peak blooming period, and process the stigma of the stamens of Rhododendron molle to obtain processed Rhododendron molle stamens. S2. Extract the prepared stamens of Rhododendron simsii using solvent reflux to obtain an extract; S3. The extract is concentrated under reduced pressure to obtain an extract paste; S4. Freeze-dry the extract to obtain a dried product; S5. After pulverizing the obtained dried material, the extract of Rhododendron molle stamens is obtained.

4. The method for preparing the extract of Rhododendron molle according to claim 3, characterized in that, In step S1, the processing steps include: rinsing the stigma of *Hylocereus undatus* and steaming it at 100°C for 2 hours, then drying it at 60°C until the moisture content is ≤10%, to obtain the processed product of *Hylocereus undatus*.

5. The method for preparing the extract of Rhododendron molle according to claim 3, characterized in that, In step S2, the solvent is an aqueous ethanol solution with a concentration of 40-80% (mL / mL); the ratio of the processed *Zhaoshan Baihua* stamen product to the aqueous ethanol solution is 1g:(8-12)mL.

6. The extraction method for the extract of Rhododendron molle stamens according to claim 3, characterized in that, In step S2, the single extraction time for reflux extraction is 90-150 min, and the number of extractions is 1-3.

7. The use of the extract of Rhododendron molle as described in claim 1 or 2 in the preparation of medicaments and / or skin care products for anti-inflammatory purposes.

8. The use of the extract of Rhododendron molle as described in claim 1 or 2 in the preparation of medicaments and / or skin care products for skin antibacterial purposes.

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