Application of extract of Callicarpa nudiflora in the fight against methicillin-resistant Staphylococcus aureus

Abstract

This invention discloses the application of *Callicarpa nudiflora* extract in the fight against methicillin-resistant *Staphylococcus aureus* (MRSA). The extract was obtained by adsorption using macroporous resin. The active components were identified based on their anti-MRSA activity. The chemical composition of the active components was analyzed using UPLC-Q-TOF-MS / MS, identifying a total of 43 compounds. Furthermore, the antibacterial activity, bactericidal effect, and biofilm formation of the active components against MRSA T144 were evaluated, verifying that the active components exhibit good antibacterial activity even at low concentrations (≤64 μg / mL).

Description

Technical Field

[0001] This invention relates to the application of extracts of Callicarpa nudiflora in the treatment of methicillin-resistant Staphylococcus aureus (MRSA), and belongs to the field of anti-MRSA. Background Technology

[0002] Staphylococcus aureus is an important zoonotic pathogen and the leading cause of bacterial mortality worldwide. Methicillin-resistant Staphylococcus aureus (MRSA) is known as a "superbug" due to its multidrug resistance, high detection rate, and high mortality rate, posing a great challenge to public health security.

[0003] Statistics show that MRSA accounts for over 30% of clinical Staphylococcus aureus isolates globally, exhibiting a clear trend of cross-resistance. my country is a major producer and consumer of antibiotics, and also a region severely affected by antibiotic abuse and bacterial resistance. According to data from the China Antimicrobial Resistance Surveillance Network, 500,114 Staphylococcus aureus strains were isolated clinically in 2023, accounting for 32.9% of Gram-positive bacteria, ranking third among all isolated strains. The national average detection rate of MRSA was 29.1%. Therefore, the search and development of novel anti-MRSA drugs is urgently needed.

[0004] Natural products, with their unique advantages such as rich structural diversity, broad biological activity, and low risk of drug resistance, have long been an important source for antibacterial drug development. As an important source of natural products, traditional Chinese medicine and its active ingredients have shown great potential in antibacterial activity, especially against drug-resistant bacteria. Flavonoids, terpenes, alkaloids, and polyphenols have strong anti-MRSA activity.

[0005] Nude Beautyberry ( Callicarpa nudiflora Hook.et Arn. As a traditional Li medicine belonging to the genus *Callicarpa* of the Verbenaceae family, its dried leaves have a long history of medicinal use. They are neutral in nature, bitter and slightly pungent in taste, and possess anti-inflammatory, detoxifying, blood-activating, and hemostatic effects. In Hainan, they are commonly used by the Li people to treat suppurative inflammation and respiratory infections. The chemical composition of *Callicarpa nudiflora* is complex and diverse. Currently, more than 300 compounds have been isolated and identified from the plant, mainly including flavonoids, terpenes, and phenylpropanoids. Among these, terpenes and flavonoids have attracted widespread attention due to their excellent pharmacological activity.

[0006] Extracts of Callicarpa nudiflora have been reported to have certain inhibitory effects on various bacterial strains, but the specific antibacterial components of Callicarpa nudiflora are unclear, and there is an urgent need for a highly efficient method for preparing Callicarpa nudiflora extract. Summary of the Invention

[0007] Purpose of the invention: The purpose of this invention is to provide the application of Callicarpa nudiflora extract in the treatment of methicillin-resistant Staphylococcus aureus.

[0008] Technical solution: The present invention provides the use of Callicarpa nudiflora extract in the preparation of drugs or pharmaceutical compositions for the treatment of methicillin-resistant Staphylococcus aureus.

[0009] The extract of Callicarpa nudiflora contains one or more of the following compounds: diterpenoids, flavonoids, triterpenoids, phenylpropanoids, sesquiterpenoids, or fatty acids.

[0010] The extract of *Callicarpa nudiflora* contains p-hydroxycinnamic acid, iris flavin, santalin, apigenin, kaempferol, 3,7,11-trimethyl-2,6,10-dodecanetrienal, rosin-8,11,13,15-tetraen-18-ol, eicosapentaenoic acid, pevidone, alfalfa extract, hydroxyasiatic acid, argentin, dehydrorosin acid, retinaldehyde, jatropha resin B, gibberellin, calcipoene A, glycyrrhetinic acid, dimethoxyflavone, asiatic acid, cystatin C, mesotrol, citrinin, 2-methyl-2-propylethyl{2-[(2-hydroxyethyl)amino]cyclohexyl}carbamate, nandrolone, and hydrocortisone. One or more of the following: (10E,12E)-9-oxo-10,12-octadecadienoic acid, purpuric acid M4, nudiflorene L, 7β-hydroxydehydrorosinic acid, steviol, hawthorn acid, pomocyanic acid, medanone, hederamenoate, retinoic acid, 15-oxo-11Z,13E-eicosadienoic acid, 7α-hydroxysantalipic acid, linolenic acid, oleanolic acid, 3-hydroxyhexadecanoic acid, oleamide, or 12-hydroxystearic acid.

[0011] The structural formula of the compound (the MRSA-resistant portion of Callicarpa nudiflora) is as follows: , Among them, the compound serial number and Figure 1 , 2 The serial numbers in the chromatograms of the anti-MRSA fraction of *Callicarpa nudiflora* correspond to those in the chromatograms.

[0012] The preparation method of the extract of Callicarpa nudiflora includes the following steps: (1) Extract the crude powder of dried leaves of Callicarpa nudiflora to obtain a total extract; concentrate the total extract to remove ethanol and then filter and concentrate to obtain crude extract of Callicarpa nudiflora; (2) Prepare the crude extract of Callicarpa nudiflora described in step (1) into a loading solution, add it into a macroporous resin column at a uniform speed, allow it to stand for adsorption, remove impurities, and elute with 30-90% ethanol in a gradient manner. Collect the eluent to obtain the extract of 70% fraction.

[0013] In step (1), the reagent used for extraction is a 70-85% ethanol solution; the ratio of the dried leaf powder of Callicarpa nudiflora to the 70-85% ethanol solution is 1:10-15 (g / mL).

[0014] In some embodiments, the reagent used for extraction in step (1) is a 70%, 80%, or 85% ethanol solution; the ratio of the crude powder of dried leaves of Callicarpa nudiflora to the reagent used for extraction is 1:10, 1:12, or 1:15 (g / mL).

[0015] The extraction method described in step (1) is heating reflux extraction, ultrasonic extraction, or cold soaking.

[0016] In step (2), the flow rate of the sample loaded onto the macroporous resin column is 1~3 BV / h.

[0017] The macroporous resin pretreatment in step (2) is as follows: Take an appropriate amount of HP20 macroporous adsorption resin and soak it in 95% ethanol for 24 h, changing the ethanol 3 times during this period to ensure that the resin is fully swollen. Use wet packing (column volume BV=10 L), first wash with 95% ethanol at a flow rate of 3 BV / h until the effluent and water are mixed at a ratio of 1:5 (v / v) without turbidity, and then wash with ultrapure water at a flow rate of 3 BV / h until the effluent has no alcohol odor.

[0018] The column loading treatment of macroporous resin in step (2) is as follows: dissolve it in ultrapure water to prepare a loading solution of 0.5 g / mL, and load it onto the pretreated HP20 macroporous resin column at a flow rate of 2 BV / h. After loading, allow it to stand for 2 h to fully bind the components.

[0019] The elution treatment of the macroporous resin in step (2) is as follows: First, elute with 4 BV of ultrapure water at a flow rate of 3 BV / h to remove water-soluble impurities such as polysaccharides and inorganic salts; then, elute sequentially with 30%, 50%, 70%, and 90% (v / v) ethanol solutions of 4 BV at a flow rate of 3 BV / h, collect the eluent of each ethanol gradient, condense under reduced pressure, and freeze-dry to obtain the extract powder.

[0020] The extract of *Callicarpa nudiflora* obtained in the above steps was subjected to in vitro screening for anti-MRSA activity at 30%, 50%, 70%, and 90% ethanol fractions. MSSA ATCC29213 and MRSA T144 were used as screening strains. The MIC values ​​of each fraction were determined using the microbroth dilution method. The activity test results are as follows: The MIC values ​​for MSSA and MRSA in the 30% ethanol eluent are both >128 μg / mL and >128 μg / mL, respectively. The MIC values ​​for MSSA and MRSA in the 50% ethanol eluent are both >128 μg / mL and >128 μg / mL, respectively. The MIC values ​​for MSSA and MRSA in the 70% ethanol eluent are both >128 μg / mL and 64 μg / mL, respectively. The MIC values ​​for MSSA and MRSA in the 90% ethanol eluent are both >128 μg / mL. The MIC value for MSSA in oxacillin (positive control) is <0.25 μg / mL and 16 μg / mL, respectively. The MIC value for MSSA in vancomycin hydrochloride (positive control) is 0.5 μg / mL and 0.5 μg / mL, respectively.

[0021] Low concentrations of ethanol (30%, 50%) mainly elute highly polar components (such as polysaccharides and saponins), while high concentrations of ethanol (90%) mainly elute weakly polar components (such as flavonoid aglycones and terpenes). Components eluted by 70% ethanol fall into the moderate polarity range. The results of this invention show that anti-MRSA activity only occurs in the 70% ethanol elution region, indicating that the chemical components in *Callicarpa nudiflora* that exert anti-MRSA activity have moderate polarity characteristics; they are neither water-insoluble (eluted by 30%–50% ethanol) nor highly lipid-soluble (eluted by 90% ethanol).

[0022] The concentration of the extract of Callicarpa nudiflora ranged from 64 to 256 μg / mL.

[0023] Preferably, the concentration of the extract of Callicarpa nudiflora is 128~256 μg / mL.

[0024] More preferably, the concentration of the extract of Callicarpa nudiflora is 192~256 μg / mL.

[0025] The present invention also provides the use of Callicarpa nudiflora extract in the preparation of medicaments or pharmaceutical compositions for bacterial diseases caused by Staphylococcus aureus infection.

[0026] The bacterial diseases mentioned include one or more of the following: inflammatory swelling, acute infectious hepatitis, or internal or external bleeding.

[0027] The present invention also provides a drug or drug composition, characterized in that it contains extract of Callicarpa nudiflora.

[0028] The drug or drug composition can treat and / or improve bacterial diseases caused by methicillin-resistant Staphylococcus aureus.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. It clarifies the specific active site and clear material basis of anti-MRSA; 2. It has precise and efficient activity and strain selectivity; 3. It has a more comprehensive mechanism of action and is expected to break through the bottleneck of MRSA resistance. Attached Figure Description

[0030] Figure 1 Chromatogram of the MRSA-resistant portion of Callicarpa nudiflora: positive mode total ion chromatogram detected by ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry; Figure 2 Chromatogram of the MRSA-resistant fraction of Callicarpa nudiflora: total ion chromatogram in negative mode; Figure 3 The growth curves of the active components of Callicarpa nudiflora on MRSA T144 under different dilution conditions; Figure 4 The bactericidal curves of the active components with different concentrations of MIC on MRSA T144; Figure 5 The effect of active components of Callicarpa nudiflora on the biofilm formation of MRSA T144; Figure 6 The activity of active components of Callicarpa nudiflora in scavenging the mature biofilm of MRSA T144; Figure 7 Scanning electron microscopy images of MRSA cells: A is the control group; B is the crude extract treated for 6 hours. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0032] Example 1: Preparation of anti-MRSA active components

[0033] 1. Equipment and Reagents: Anhydrous ethanol (analytical grade, purity ≥ 99.5%) was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.; HP20 macroporous adsorption resin was purchased from Beijing Lvbaicao Technology Co., Ltd.; ultrapure water system was purchased from Millipore, USA; constant temperature water bath (model WB-4) was purchased from Bikman Biotechnology Co., Ltd.; rotary evaporator was purchased from Shanghai Ailang Instrument Co., Ltd.; vacuum freeze dryer was purchased from Tokyo Rika Kiki Co., Ltd.; electronic analytical balance was purchased from Mettler Toledo, Switzerland. The medicinal material described is a plant of the genus Callicarpa in the family Verbenaceae (Callicarpa). Callicarpa nudiflora Hook.et Arn. This product is listed in the 2025 edition of the Chinese Pharmacopoeia. It is produced in the Tropical Rainforest Reserve of Wuzhishan City, Hainan Province, and the batch number of the medicinal material is 20230512.

[0034] 2. Preparation of crude extract of Callicarpa nudiflora Approximately 1.5 kg of dried, coarsely powdered *Callicarpa nudiflora* leaves was weighed and extracted with 80% ethanol at 85°C under reflux for 3 hours each time, for a total of 3 extractions. The extracts from the 3 extractions were combined and filtered through gauze to obtain the total extract. This extract was concentrated until the ethanol was removed, then filtered again through gauze. The filtrate was collected and concentrated to obtain a dry extract of crude *Callicarpa nudiflora*. The ratio of dried, coarsely powdered *Callicarpa nudiflora* leaves to 80% ethanol was 1:12.

[0035] 3. Preparation of extracts of different polarities from macroporous resins of Callicarpa nudiflora 3.1 Macroporous Resin Pretreatment Take an appropriate amount of HP20 macroporous adsorption resin and soak it in 95% ethanol for 24 hours, changing the ethanol 3 times during this period to ensure that the resin is fully swollen. Pack the column using the wet method (column volume BV = 10 L), first wash with 95% ethanol at a flow rate of 3 BV / h until the effluent mixed with water at a ratio of 1:5 (v / v) is clear, and then wash with ultrapure water at a flow rate of 3 BV / h until the effluent has no alcohol odor.

[0036] 3.2 Sample loading and gradient elution Weigh 30 g of the prepared crude extract of Callicarpa nudiflora, dissolve it in ultrapure water to prepare a loading solution with a mass concentration of 0.5 g / mL, and load the entire loading solution onto a pretreated HP20 macroporous resin column at a flow rate of 2 BV / h. Preliminary experiments showed that a flow rate of 2 BV / h allowed for sufficient binding of the active ingredient with the resin, resulting in the best adsorption effect. A flow rate that was too fast could cause component leakage, while a flow rate that was too slow would prolong the experimental cycle and fail to improve adsorption efficiency, leading to incomplete elution of the anti-MRSA active component and loss of effectiveness. After loading, allow the sample to stand for 2 h to ensure complete adsorption. First, remove impurities with 4 BV ultrapure water at a flow rate of 3 BV / h. Preliminary experiments verified that this elution parameter effectively removes water-soluble impurities such as polysaccharides and inorganic salts, leaving minimal residue of the target component and achieving an elution rate of over 95%. Subsequently, a constant flow rate of 3 BV / h was used to elute sequentially with 4 BV volumes of 30%, 50%, 70%, and 90% (v / v) ethanol solutions, and the eluents from each gradient were collected in segments.

[0037] 3.3 Sample Preparation The eluents of each gradient were concentrated under reduced pressure in a rotary evaporator to recover the solvent. The concentrates were then freeze-dried under vacuum to obtain dry extracts of 30% ethanol eluent, 50% ethanol eluent, 70% ethanol eluent, and 90% ethanol eluent. The extracts were accurately weighed, sealed, and refrigerated at 4°C for later use in subsequent studies.

[0038] 4. Conclusion Preparation steps of macroporous resin extracts of different polarities of Callicarpa nudiflora: (1) Extracted by heating and reflux with 80% ethanol, and concentrated under reduced pressure by rotary evaporator to obtain total crude extract of Callicarpa nudiflora. (2) Sampled by wet loading of macroporous resin, and eluted sequentially with 30%, 50%, 70%, and 90% (v / v) ethanol solutions of 4 BV volume to obtain dry extracts of different parts.

[0039] Example 2: Initial screening of in vitro anti-MRSA activity 1. Materials and Methods 1.1 Materials 1.1.1 Reagents and strains Mueller-Hinton (MH) broth, LB broth, and MH agar were purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.; Staphylococcus aureus ATCC 29213 (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) T144 were purchased from the China General Microbiological Culture Collection Center and were standard quality control strains. Oxacillin for injection and vancomycin hydrochloride for injection were obtained from the China National Institutes for Food and Drug Control; 0.9% sterile physiological saline was prepared in-house, autoclaved at 121℃ for 20 min, and cooled before use; dimethyl sulfoxide (DMSO) was analytical grade (Sinopharm Chemical Reagent Co., Ltd.).

[0040] 1.1.2. Instruments and Equipment YXQ.SGH.280 high-pressure steam sterilizer (Shanghai Huaxian Medical Nuclear Instruments Co., Ltd.); GNP-9160BS-Ⅲ constant temperature incubator (Shanghai Xinmiao Medical Instrument Manufacturing Co., Ltd.); THZ-82A constant temperature shaker (Changzhou Jintan Cultural Instruments Co., Ltd., Jiangsu Province); 96-well U-shaped culture plate (Corming, USA); micropipettes (Eppendorf Life Sciences, Germany).

[0041] 1.2 Methods The minimum inhibitory concentrations (MICs) of each extract against MSSA and MRSA were determined using the micro-broth dilution method.

[0042] 1.2.1 Preparation of test sample and positive control solution Accurately weigh an appropriate amount of dried powder from each elution fraction, dissolve it in sterile DMSO to prepare a stock solution of 5120 μg / ml, and dilute it with MH bacterial broth medium before use to prepare a series of test solutions with different concentrations, ensuring that the final concentration of DMSO in each test well is ≤1%.

[0043] The positive control drugs oxacillin and vancomycin hydrochloride were prepared into a stock solution of 5120 μg / ml using sterile ultrapure water and diluted before use.

[0044] 1.2.2 Standardized preparation of standard bacterial solutions MSSA and MRSA strains frozen at -80℃ were rapidly thawed and streaked onto MH agar plates. They were then incubated at 37℃ for 18-24 h. Single colonies with consistent morphology were picked and inoculated into MH broth medium, incubated at 37℃ with shaking at 200 rpm for 12 h. The bacterial concentration was then adjusted to 0.5 McFarland turbidity (equivalent to 1×10⁻⁶). 8 (CFU / mL), then diluted with MH broth to 1×10⁻⁶. 6 CFU / mL, for later use.

[0045] 1.2.3 Determination of Minimum Inhibitory Concentration (MIC) The steps for testing the sample against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus are as follows: The experiment was conducted using 96-well plates, with a total volume of 200 μL per well.

[0046] Wells 1-10: Add 100 μL of MH broth. Add 100 μL of the 256 μg / mL test solution to well 1. Mix well and then pipette 100 μL into well 2. Perform serial dilutions up to well 10. After mixing well 10, discard 100 μL. The final concentration range of the test solution is 128 μg / mL to 0.25 μg / mL. Well 11 is a blank control: only 200 μL of MH broth was added; Well 12 is the growth control: 100 μL MH broth + 100 μL bacterial culture (excluding test sample); Add 100 μL of 1×10 to wells 1-10 and well 12. 6 The bacterial culture was prepared at a concentration of CFU / mL to achieve a final bacterial concentration of 5 × 10⁻⁶. 5 CFU / mL; Two parallel wells were set up for each sample, and a positive control was also set up (final concentration range 0.125~64 μg / mL).

[0047] The 96-well plate was placed in a 37 ℃ constant temperature incubator and incubated statically for 24 h. The bacterial growth in each well was observed with the naked eye. The lowest drug concentration at which no colonies grew was the MIC value of the sample for that bacterium.

[0048] 2. Experimental Results MSSA ATCC29213 and MRSA T144 were used as screening strains, and the MIC values ​​of each fraction were determined using the microbroth dilution method. The fraction eluted with 70% ethanol showed significant anti-MRSA activity, with a MIC of 64 μg / mL; the fractions eluted with 30%, 50%, and 90% ethanol showed no antibacterial activity at a concentration of 128 μg / mL (MIC > 128 μg / mL). The results are shown in Table 1.

[0049] Table 1. MIC results of different elution fractions of macroporous resin from *Callicarpa nudiflora* for MSSA ATCC29213 and MRSA T144.

[0050] 3. Conclusion This result indicates that the anti-MRSA active ingredient in *Callicarpa nudiflora*, after gradient elution with macroporous resin, is highly concentrated in the moderately polar region of the 70% ethanol elution fraction, while the eluates in other polar regions (30%, 50%, and 90%) contain little or no active ingredient. MSSA activity assays showed that none of the eluted fractions exhibited antibacterial activity at 128 μg / mL (MIC > 128 μg / mL). Notably, the 70% ethanol elution fraction, which exhibited significant anti-MRSA activity, also showed no inhibitory effect on MSSA, indicating that its antibacterial activity is strain-selective and exhibits stronger inhibitory activity against MRSA.

[0051] Example 3: Chemical characterization of the active components of *Callicarpa nudiflora* against MRSA 1. Materials and Methods 1.1 Equipment and Reagents Ultra-high performance liquid chromatography-high resolution quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS / MS): Equipped with a high performance liquid chromatograph, time-of-flight mass spectrometer, electrospray ionization source, C18 reversed-phase column, and guard column (all purchased from Waters). Ultrapure water was prepared using a UPH-11-20T ultrapure water production system (Chengdu Ultrapure Technology Co., Ltd.). A 0.22 μm organic phase filter membrane (Tianjin Linghang Experimental Equipment Co., Ltd.) and an ultrasonic cleaner (Shanghai Fangxu Technology Co., Ltd.) were used. Chromatographic grade acetonitrile and formic acid were purchased from Merck, Germany. 1.2 Sample Preparation Weigh 5 mg of the dry extract of the macroporous resin eluted with 70% ethanol obtained in Example 1, place it in a 2 mL volumetric flask, add 1.5 mL of chromatographic grade methanol, seal tightly, weigh, and sonicate (500 W, 40 kHz) for 30 min. After sonication, remove the flask, cool to room temperature, and make up the weight loss with chromatographic grade methanol to the mark, then shake thoroughly. Filter the above solution through a 0.22 μm microporous membrane, collect the filtrate in a brown sample vial, store at 4°C protected from light, and complete subsequent analysis within 24 h.

[0052] 1.3 Chromatographic conditions The chromatographic column was a Waters octadecylsilane-bonded silica reversed-phase column (100 mm × 2.1 mm, 1.7 μm); the guard column was a Waters C18 Guard Column (5 mm × 2.1 mm, 1.7 μm); the mobile phase consisted of phase A (water containing 0.1% (v / v) formic acid) and phase B (acetonitrile), using gradient elution: 0–10 min, 5–20% acetonitrile; 10–15 min, 20–45% acetonitrile; 15–20 min, 45–60% acetonitrile; 20–25 min, 60–80% acetonitrile; 25–30 min, 80–100% acetonitrile. The detection wavelengths were 205 nm and 254 nm. The flow rate was 0.3 mL / min. The injection volume was 1 μL. The column temperature was 25 °C.

[0053] 1.4 Mass Spectrometry Conditions: Electrospray ionization (ESI) source, simultaneously acquiring positive ion mode (ESI) + ) and negative ion mode (ESI) - Data. Mass scan range: m / z 50-1000. Capillary voltage: 3.0 kV for positive ion mode, 2.2 kV for negative ion mode. Cone voltage: 40V. Cone gas flow rate: 50 L / h (high-purity nitrogen). Ion source temperature: 120℃. Data acquisition mode: Full-ion MSE mode, simultaneously acquiring low and high collision energy data. Collision energy: Low collision energy 6 eV, high collision energy 10-40 eV linear gradient.

[0054] 2. Results As shown in Table 2 and Figures 1-2 As shown, based on the information from the precursor ion peaks and fragment ion peaks in the total ion chromatogram, a total of 43 compounds were identified, including various types of compounds such as diterpenes, flavonoids, triterpenes, phenylpropanoids, sesquiterpenes, and fatty acids. Figure 1 and Figure 2 The peaks in the middle represent compounds resolved under positive and negative modes, respectively, totaling 43 compounds. Figure 1 and Figure 2 The numbers marked in the table correspond to the serial numbers of the compounds in Table 2.

[0055] Table 2. Identification of chemical components in the active anti-MRSA fraction of Callicarpa nudiflora.

[0056]

[0057] UPLC-Q-TOF-MS / MS analysis of the active site of *Callicarpa nudiflora* against MRSA: positive ion mode (ESI) + Under negative ion mode (ESI), the chromatographic peaks are mainly distributed between 10-30 min, with the strongest response between 13-20 min, showing good ionization effects on diterpenes and methoxyflavonoids; - Although the number of peaks was relatively small, the response to polyhydroxyflavonoids, carboxytriterpenes, and fatty acids was significantly enhanced. Both modes showed stable baselines, symmetrical peak shapes, and no obvious tailing or co-elution, indicating that the chromatographic-mass spectrometry conditions were optimized appropriately.

[0058] 3. Conclusion Diterpenoids are the main components of this anti-MRSA active site, including various structural subtypes such as abirane and hemispherane. Flavonoids show good mass spectrometry response and are key active components. Triterpenoids, phenylpropanoids, and sesquiterpenoids are present in lower amounts. All these components together constitute the material basis of this site.

[0059] Example 4: Determination of the effect of the anti-MRSA active components of *Callicarpa nudiflora* on the strain 1. Materials and Methods 1.1 Materials 1.1.1 Reagents and strains Mueller-Hinton (MH) and LB broth media were purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.; methicillin-resistant Staphylococcus aureus (MRSA) T144 was purchased from the China General Microbiological Culture Collection Center and was a standard quality control strain. Oxacillin for injection and meropenem for injection (both from the China National Institutes for Food and Drug Control); dimethyl sulfoxide (DMSO) was purchased from Sinopharm Chemical Reagent Co., Ltd.; crystal violet staining solution and phosphate-buffered saline (PBS) were purchased from Beijing Solarbio Science & Technology Co., Ltd.; ethanol (analytical grade, 99% purity) and glutaraldehyde (synthetic grade, 50% purity) were purchased from Aladdin (Shanghai) Reagent Co., Ltd.

[0060] 1.1.2. Instruments and Equipment The YXQ.SGH.280 high-pressure steam sterilizer was purchased from Shanghai Huaxian Medical Nuclear Instruments Co., Ltd.; the GNP-9160BS-Ⅲ constant temperature incubator was purchased from Shanghai Xinmiao Medical Instrument Manufacturing Co., Ltd.; the THZ-82A constant temperature shaker was purchased from Jiangsu Changzhou Jintan Cultural Instrument Co., Ltd.; the 96-well U-shaped culture plate was purchased from Corming Corporation, USA; the micropipette was purchased from Eppendorf Life Sciences, Germany; the SW-CJ-1F ultra-clean workbench was purchased from Suzhou Purification Equipment Co., Ltd.; and the PHS-3C benchtop pH meter was purchased from Shanghai Instrument & Electronics Scientific Instruments Co., Ltd.

[0061] 1.2 Experimental Methods 1.2.1 Preparation of the test solution for the macroporous resin elution fraction with 70% ethanol Accurately weigh an appropriate amount of the 70% ethanol eluent dry extract prepared in Example 1, dissolve it in sterile DMSO to prepare a 5120 μg / mL stock solution, and dilute it before the test to obtain graded concentration test solutions, controlling the final concentration of DMSO in the system to ≤1%. For the positive controls, oxacillin and meropenem were prepared with sterile ultrapure water to a stock solution of the same concentration of 5120 μg / mL, and then serially diluted to 0.125-64 μg / mL before use.

[0062] 1.2.2 Growth curve determination The frozen MRSA T144 bacterial culture was transferred to fresh MH broth medium at a ratio of 1:100 and incubated overnight in a shaker at 37 ℃ for activation. The next day, the fresh bacterial culture was taken and serially diluted at ratios of 1:100 and 1:1000.

[0063] Antimicrobial susceptibility testing was conducted using 96-well microplates. 100 μL of LB broth basal medium was added to each well, followed by 100 μL of a series of 70% ethanol solutions of the eluted fractions. The microplates were partitioned as follows: Rows A and B corresponded to 1:100 bacterial dilutions, with Row A containing different concentrations of the test sample (three replicates per group), and Row B serving as a blank control for that dilution; Rows C and D corresponded to 1:1000 bacterial dilutions, with Row C containing a series of test sample concentrations and replicates, and Row D serving as the corresponding blank control.

[0064] A multi-functional microplate reader was used to continuously measure the absorbance of each well at a wavelength of 600 nm for 16 hours, recording the real-time growth data of the strain. Based on the detection results, a growth curve of MRSA T144 strain was plotted. The results are shown below. Figure 3 .

[0065] 1.2.3 Determination of bactericidal curve The frozen MRSA T144 culture was transferred to fresh MH broth at a ratio of 1:100 and cultured overnight in a shaker at 37 ℃. The next day, the culture was diluted at a ratio of 1:1000, and the test solutions of dry extracts of different concentrations of 70% ethanol elution fractions were added and mixed well. The mixture was then placed in a constant temperature incubator at 37 ℃ for static incubation.

[0066] Samples were taken at four time points: 0 h, 4 h, 8 h, and 12 h of culture. 100 μL of bacterial suspension was taken from each time point and resuspended in sterile PBS (pH 7.2-7.4, concentration 0.01 M) for sequential 10-fold dilution. The diluted bacterial suspension was then plated onto LB agar plates and allowed to air dry before being incubated overnight at 37 ℃.

[0067] After incubation, the number of colonies on each plate was counted, and colony-forming units (CFU) were calculated to analyze the effect of the test sample on the viable count of MRSA T144. The results are shown in [Table / Reference]. Figure 4 .

[0068] 1.2.4 Assay for activity against biofilm formation inhibition First, the frozen MRSA T144 bacterial culture was transferred to fresh MH broth at a ratio of 1:100 and cultured at 37 ℃ in a shaker until the logarithmic growth phase. Then, the bacterial culture was diluted at a ratio of 1:1000 and set aside. 100 μL of LB broth was added to each well of a 96-well plate, followed by serially diluted solutions of different concentrations of 70% ethanol eluted dry extracts. The diluted bacterial culture was then inoculated, mixed thoroughly, and the 96-well plate was incubated at 37 ℃ for 36 h.

[0069] After incubation, aspirate the airborne bacterial culture from each well. Add 300 μL of PBS to each well and gently wash to remove residual airborne bacteria, taking care not to damage the biofilm formed at the bottom of the well. Repeat this washing step three times, then discard the PBS. Next, add 50 μL of methanol to each well and fix the biofilm at room temperature for 15 min. After removing the fixative, allow the 96-well plate to air dry at room temperature. Add 100 μL of 0.1% crystal violet staining solution to each well and stain in the dark for 15 min. After discarding the staining solution, gently wash three times with PBS to remove unbound dye, and allow to air dry again. Finally, add 100 μL of 33% acetic acid solution to each well and incubate at 37 ℃ for 30 min to fully dissolve the crystal violet bound to the biofilm. After the reaction, use a multi-mode microplate reader to measure the absorbance at 595 nm in each well to quantitatively evaluate the inhibitory effect of the test sample on MRSA T144 biofilm formation. The results are shown in [Figure 1]. Figure 5 .

[0070] 1.2.5 Determination of scavenging activity of mature biofilms MRSA T144 bacterial culture was inoculated into 96-well plates and incubated at 37 °C for 36 h to induce the formation of a mature biofilm. After incubation, airborne bacteria were aspirated from the wells, and the plates were gently washed three times with sterile PBS to thoroughly remove any unattached free bacteria. Different concentrations of the test sample solution of the elution fraction (70% ethanol) were added to the remaining biofilm, and the plates were incubated at 37 °C for 2 h. After incubation, the plates were sonicated for 10 min to elute the bacteria from the biofilm. The bacterial culture from each well was collected, serially diluted 10-fold with sterile PBS, and inoculated onto LB agar plates using the drop plate method. After overnight incubation at 37 °C, the number of colonies in each group was counted, and the colony-forming units (CFU) were calculated to quantitatively analyze the scavenging effect of the test sample on viable bacteria within the mature biofilm. The results are shown in [Figure number missing]. Figure 6 Where ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0071] 1.2.6 Observation of cell morphology Take 20 mL of methicillin-resistant Staphylococcus aureus (MRSA) bacterial suspension and treat it with the test sample solution eluted from 70% ethanol at a concentration of 4 × minimum inhibitory concentration (MIC) for 6 h. After treatment, centrifuge at 5000 r / min for 5 min at 4℃ and discard the supernatant; resuspend and wash the bacterial cells in phosphate-buffered saline (PBS), and centrifuge again at 5000 r / min for 5 min at 4℃ and discard the supernatant. Resuspend the bacterial pellet in 2.5% glutaraldehyde fixative and fix it at room temperature in the dark for 2 h, then transfer it to 4℃ for storage. A blank control group without 70% ethanol elution was also included in this experiment; the treatment conditions for the control group were exactly the same as those for the experimental group.

[0072] Sample preparation for scanning electron microscopy (SEM): The bacterial cells were dehydrated using a gradient of cold ethanol and tert-butanol, followed by critical point drying with carbon dioxide. The samples were then subjected to ion sputtering to deposit a gold film. Finally, the bacterial morphology was observed using an EVO-LS10 scanning electron microscope. Figure 7 ).

[0073] 2. Results and Analysis 2.1 Effects of the anti-MRSA active components of *Callicarpa nudiflora* on the growth curve of the strain To investigate the inhibitory effect of crude extract on the growth of MRSA T144, this experiment measured the OD of the strain within 16 h after treatment with different concentrations of the test sample. 600 Dynamic changes, results as follows Figure 3 As shown. Under a 1:100 dilution of bacterial suspension, 3× and 4× MIC (192 and 256 μg / mL) of the test sample completely inhibited bacterial proliferation, and the OD within 16 h was [not specified]. 600 No significant fluctuations were observed; 1× and 2×MIC (64 and 128 μg / mL) only inhibited the early growth of the strain, and the inhibitory effect disappeared in the later stages. This indicates that the low concentration of crude extract mainly inhibits the early proliferation of bacteria and delays the initiation of its logarithmic growth phase. In the 1:1000 dilution system of bacterial suspension, the antibacterial behavior of the crude extract was basically consistent with that of the 1:100 dilution group, and the antibacterial effect showed a clear concentration dependence.

[0074] 2.2 Effect of the anti-MRSA active components of *Callicarpa nudiflora* on the fungicidal curve of the strain MRSA T144 in the logarithmic growth phase was collected, and the bactericidal activity of the crude extract against the strain was investigated using test samples at concentrations of 1×, 2×, and 4× MIC. The results are as follows: Figure 4 As shown, the 1×MIC and 2×MIC groups (64 and 128 μg / mL) exhibited only weak bactericidal effects and limited antibacterial activity; under the 4×MIC (256 μg / mL) condition, the number of strains decreased significantly within 4 h, and no viable bacteria were detected after 12 h of culture. In summary, high concentrations of the crude extract can effectively kill MRSA T144, and its bactericidal effect is concentration-dependent, continuously increasing with the extension of the treatment time, with a significant positive correlation between the two.

[0075] 2.3 Effects of the anti-MRSA active components of *Callicarpa nudiflora* on biofilm formation in the strain The biofilm formation ability of MRSA T144 was detected by crystal violet staining. The results are shown in [Figure number missing]. Figure 5 Treatment with a gradient of 1×, 2×, 3×, and 4×MIC (64, 128, 192, and 256 μg / mL) significantly reduced biofilm formation in the strain. Analysis of bacterial growth curves indicates that the crude extract effectively inhibits biofilm formation, and this effect is not achieved by reducing bacterial numbers. The specific molecular mechanism by which it interferes with biofilm formation in MRSA T144 requires further investigation.

[0076] 2.4 Effects of the anti-MRSA active components of *Callicarpa nudiflora* on the clearance of mature biofilms by the strain The clearance effect of different concentrations of crude extract on the mature biofilm of MRSA T144 was evaluated by viable cell counting. Results are shown below. Figure 6All concentrations of crude extract could reduce the number of viable bacteria in the biofilm by 1-2 lg values, and the differences between groups were statistically significant (p<0.05), indicating that the crude extract has a certain ability to remove mature biofilms.

[0077] 2.5 Effects of the anti-MRSA active components of *Callicarpa nudiflora* on the cell morphology of the strain Under a 5000x scanning electron microscope, normal MRSA cells were observed to be regularly spherical with a smooth surface, typically clustered in grape-like patterns, exhibiting good cell viability, with some cells in the binary fission stage. Figure 7 A). After 6 h of treatment with the crude extract at 4×MIC, the morphology of MRSA cells changed significantly, with obvious shrinkage on the cell surface and disruption of the normal spherical structure, suggesting that the crude extract can act on the cell wall membrane system. Furthermore, most cells in the experimental group showed severe deformation, surface depression, and blurred cell outlines, with cell adhesion occurring due to damage to the cell wall structure. Figure 7 B). The above results indicate that the crude extract can significantly induce abnormal changes in the morphology of MRSA cells.

[0078] 3. Conclusion This invention determined the antibacterial activity, bactericidal effect, and biofilm effect of the active component of *Callicarpa nudiflora* against MRSA T144. Growth curve experiments showed that the crude extract completely inhibited bacterial growth at concentrations of 3×MIC or 4×MIC, while at concentrations of 1×MIC or 2×MIC, it mainly delayed the rapid growth phase of bacteria. Bactericidal curve experiments further revealed that the crude extract completely eliminated viable bacteria within 12 hours at a concentration of 4×MIC, exhibiting sustained and concentration-dependent bactericidal characteristics. Regarding biofilm formation, the crude extract significantly inhibited biofilm formation in the range of 1× to 4×MIC, and this effect was not solely achieved through inhibiting bacterial growth; furthermore, it reduced the number of viable bacteria within the biofilm by 1 to 2 logarithmic orders of magnitude, demonstrating a certain biofilm scavenging activity. In terms of cell morphology, the cell wall is an important structure for maintaining bacterial morphology, resisting external stress, and ensuring normal bacterial metabolism, while the cell membrane mainly participates in life activities such as substance transport and energy metabolism. The integrity of the cell membrane structure is a necessary condition for the survival and proliferation of MRSA. In this invention, the bacterial cells treated with the crude extract exhibited damage characteristics such as collapse, deformation, and adhesion, indicating that the crude extract can disrupt the structural integrity of the MRSA cell wall, causing it to lose its mechanical support function and preventing the bacterial cells from maintaining their normal morphology. This confirms that the crude extract can achieve antibacterial effect by damaging the bacterial cell membrane barrier. In summary, the crude extract exhibits good antibacterial activity even at low concentrations (≤64 μg / mL), and further separation and purification can be carried out to screen for more efficient and low-toxicity compounds.

Claims

1. The use of extracts of Callicarpa nudiflora in the preparation of drugs or pharmaceutical compositions for the treatment of methicillin-resistant Staphylococcus aureus.

2. The application according to claim 1, characterized in that, The extract of Callicarpa nudiflora contains one or more of the following compounds: diterpenoids, flavonoids, triterpenoids, phenylpropanoids, sesquiterpenoids, or fatty acids.

3. The application according to claim 1, characterized in that, The extract of *Callicarpa nudiflora* contains p-hydroxycinnamic acid, irisin, santalin, apigenin, kaempferol, 3,7,11-trimethyl-2,6,10-dodecanetrienal, rosin-8,11,13,15-tetraen-18-ol, eicosapentaenoic acid, pevidone, alfalfa extract, hydroxyasiatic acid, argentin, dehydrorosinic acid, retinaldehyde, jatropha resin B, gibberellin, calcipoene A, glycyrrhetinic acid, dimethoxyflavone, asiatic acid, cystatin C, mesotrol, citrinin, 2-methyl-2-propylethyl{2-[(2-hydroxyethyl)amino]cyclohexyl}carbamate, nandrolone, and hydrocortisone. One or more of the following: (10E,12E)-9-oxo-10,12-octadecadienoic acid, purpuric acid M4, nudiflorene L, 7β-hydroxydehydrorosinic acid, steviol, hawthorn acid, pomocyanic acid, medanone, hederamenoate, retinoic acid, 15-oxo-11Z,13E-eicosadienoic acid, 7α-hydroxysantalipic acid, linolenic acid, oleanolic acid, 3-hydroxyhexadecanoic acid, oleamide, or 12-hydroxystearic acid.

4. The application according to claim 1, characterized in that, The preparation method of the extract of Callicarpa nudiflora includes the following steps: (1) Extract the crude powder of dried leaves of Callicarpa nudiflora to obtain a total extract; concentrate the total extract to remove ethanol and then filter and concentrate to obtain crude extract of Callicarpa nudiflora; (2) Prepare the crude extract of Callicarpa nudiflora described in step (1) into a loading solution, add it into a macroporous resin column at a uniform speed, allow it to stand for adsorption, remove impurities, and elute with 30-90% ethanol in a gradient manner. Collect the eluent to obtain the extract of 70% fraction.

5. The application according to claim 4, characterized in that, The reagent used for extraction in step (1) is a 70-85% ethanol solution; the ratio of the dried leaf powder of Callicarpa nudiflora to the 70-85% ethanol solution is 1:10-15 (g / mL).

6. The application according to claim 4, characterized in that, The extraction method described in step (1) is heating reflux extraction, ultrasonic extraction, or cold soaking.

7. The application according to claim 4, characterized in that, In step (2), the flow rate for loading the sample onto the macroporous resin column is 1~3 BV / h.

8. The use of extracts of Callicarpa nudiflora in the preparation of medicaments or pharmaceutical compositions for bacterial diseases caused by Staphylococcus aureus infection.

9. The application according to claim 8, characterized in that, The bacterial diseases include one or more of the following: inflammatory swelling, acute infectious hepatitis, or internal or external bleeding.

10. A medicament or pharmaceutical composition for treating and / or improving bacterial diseases caused by methicillin-resistant Staphylococcus aureus, characterized in that, It contains extract of Callicarpa nudiflora.

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