Graded preparation method and application of antibacterial active components based on multi-color mango peel residues

Abstract

The invention relates to a graded preparation method of bacteriostatic active components based on multi-color mango peel residues, which comprises the following steps: crushing dried mango peel residues, and carrying out enzymolysis pretreatment by using a compound enzyme system containing cellulase and pectinase; carrying out ultrasonic-assisted extraction by adopting a deep eutectic solvent in which the molar ratio of choline chloride to glycerol is 1: 2, and carrying out centrifugal separation to obtain a crude extract; loading the crude extract on a macroporous adsorption resin column, carrying out gradient elution by using an ethanol-water solution containing 1-3% of hydroxypropyl-beta-cyclodextrin as a protective eluent, and collecting an active fraction; and directly cutting the active fraction into a preparative high performance liquid chromatography system by adopting a column switching technology, collecting a chromatographic peak with the retention time of 14.5-15.5 minutes, and volatilizing a solvent to obtain the targeted malassezia-resistant active component. The method can be used for preparing medicines or cosmetics for inhibiting malassezia.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction and separation technology, specifically to a method and application of fractional preparation of antibacterial active components based on mango peel residue from Baise. Background Technology

[0002] Mangoes are a popular tropical fruit worldwide. The processing of mangoes into juice, jam, and dried fruit generates a large amount of peel residue. Baise mangoes, a Chinese geographical indication product, have a huge production volume, resulting in mango peel waste that not only wastes resources but also puts pressure on the environment. Studies have shown that mango peels are rich in polyphenols, flavonoids, and other bioactive components, possessing potential applications such as antioxidant and antibacterial properties. Therefore, how to efficiently and economically utilize mango peel residue to extract high-value-added functional components has become a current research hotspot.

[0003] In existing techniques for extracting and separating active ingredients from plant raw materials such as mango peel, organic solvent extraction or reflux extraction are commonly used. However, these traditional methods often suffer from drawbacks such as high extraction temperatures, long processing times, large consumption of organic solvents, and a high number of impurities in the extract. Particularly for structurally unstable and easily oxidized phenolic compounds, high temperatures and prolonged operation may lead to degradation and inactivation, thus affecting the bioactivity of the final product. To improve extraction efficiency and product purity, some studies have explored the introduction of enzymatic pretreatment techniques, using cellulase, pectinase, and other enzymes to disrupt plant cell wall structures and promote the release of intracellular active substances. Furthermore, ultrasonic-assisted extraction and macroporous adsorption resin separation are also widely used in the purification of natural products.

[0004] However, existing technologies still have significant shortcomings in the preparation of targeted antibacterial active ingredients against specific pathogens (such as Malassezia) in mango peel residue. Malassezia is a common opportunistic pathogenic fungus found on the surface of human skin and is closely related to various skin diseases such as seborrheic dermatitis and tinea versicolor. Screening and preparing highly effective and low-toxicity anti-Malassezia active ingredients from mango peel is of great significance for the development of novel skin care products or drugs. However, existing extraction and separation processes often lack precise protection of the target active ingredient, resulting in loss of activity during separation. At the same time, multi-step separation operations are complex, and it is often difficult to obtain high-purity targeted active ingredients due to loss or dilution of components during switching. Therefore, how to establish a preparation method that can effectively protect the active ingredient and achieve efficient and precise separation to obtain targeted components with anti-Malassezia activity from mango peel residue in Baise remains a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] One object of the present invention is to address at least the aforementioned deficiencies and to provide at least the advantages that will be described later.

[0006] To achieve these objectives and other advantages of the present invention, a method for fractional preparation of antibacterial active components based on mango peel residue from Baise is provided, comprising the following steps: pulverizing dried mango peel residue and pretreating it with a complex enzyme system containing cellulase and pectinase; ultrasonically-assisted extraction of the enzymatically hydrolyzed material using a eutectic solvent with a choline chloride-glycerol molar ratio of 1:2, followed by centrifugation to obtain a crude extract; loading the crude extract onto a macroporous adsorption resin column and performing gradient elution with a protective eluent containing β-cyclodextrin, collecting the eluent during the protective elution stage as the active fraction; using column switching technology, directly inputting the collected active fraction into a preparative high-performance liquid chromatography system, performing isocratic elution with chromatographic grade methanol-water solution as the mobile phase, collecting chromatographic peaks with retention times of 14.5-15.5 min, and evaporating the solvent to obtain the active component targeting Malassezia; wherein the protective eluent containing β-cyclodextrin is an ethanol-water solution containing 1-3% hydroxypropyl-β-cyclodextrin.

[0007] The above-mentioned method utilizes the synergistic effect of enzymatic hydrolysis with a complex enzyme system and ultrasonic extraction with a eutectic solvent of choline chloride-glycerol molar ratio of 1:2 to gently and efficiently disrupt the cell wall structure of mango peel, promoting the release of active substances while avoiding the damage of heat-sensitive components caused by high temperatures. By introducing a protective eluent containing hydroxypropyl-β-cyclodextrin during the elution stage of macroporous adsorption resin, the inclusion effect of cyclodextrin provides in-situ protection for unstable target compounds during separation, effectively preventing their oxidation or degradation in subsequent separation steps. Furthermore, column switching technology is employed to directly introduce the pre-purified active fraction into the preparative HPLC system, achieving continuous and automated separation, avoiding the collection, concentration, and reconstitution of intermediate products, greatly reducing sample loss and operation time, and ultimately accurately obtaining high-purity targeted components with anti-Malaiseem activity at a specific retention time. This method solves the problems of easy loss of active ingredients, low separation efficiency, and poor targeting in existing technologies, achieving the effect of efficient, gentle, and precise preparation of highly active antibacterial components from mango peel. In this complex enzyme system, the enzyme dosage was 0.5% of the substrate mass. The enzymatic pretreatment consisted of enzymatic hydrolysis for 2 hours at 50°C in 0.1M pH 5.0 citrate-sodium citrate buffer, followed by enzyme inactivation at 90°C for 10 minutes. The macroporous adsorption resin column used was an HPD-100 type. Isocratic elution was performed using a YMC-Pack ODS-A C18 column (250 mm × 20 mm, 5 μm) as the separation column, with chromatographic grade methanol-water (75:25, v / v) as the mobile phase, a flow rate of 8 mL / min, and a detection wavelength of 280 nm.

[0008] Preferably, the method for fractionating and preparing antibacterial active components based on mango peel residue of the present invention comprises a two-step gradient elution consisting of a non-protective elution stage and a protective elution stage: First, elution is performed using an ethanol-water solution without hydroxypropyl-β-cyclodextrin, wherein the volume percentage concentration of ethanol is 20-30% and the elution volume is 2-4 times the column volume; Second, elution is performed using an ethanol-water solution containing 1-3% hydroxypropyl-β-cyclodextrin, wherein the volume percentage concentration of ethanol is 40-70%, the elution flow rate is 0.5-1.5 mL / min, the column temperature is maintained at 25-35℃ during elution, and the eluent from the second step is collected as the active fraction.

[0009] The above-described scheme employs a two-step gradient elution design. First, a low-concentration ethanol-free eluent (without cyclodextrin) is used to remove a large amount of highly polar non-target impurities, providing preliminary purification of the resin column. Then, in the second step, by increasing the ethanol concentration and introducing hydroxypropyl-β-cyclodextrin, the target active component is precisely eluted and protected. Maintaining the elution temperature at 25-35°C ensures suitable mass transfer kinetics while avoiding the impact on temperature-sensitive target compounds. This scheme resolves the cost-efficiency conflict by avoiding the use of expensive cyclodextrin in the impurity removal stage and introducing a protective agent only in the target elution stage. It achieves the beneficial effects of effectively protecting target activity while improving separation and purification efficiency and product purity, and reducing separation costs.

[0010] Preferably, in the fractional preparation method of antibacterial active components based on mango peel residue of the present invention, the second step of elution is a multi-step gradient elution: First, elute with an ethanol-water solution containing 40-50% ethanol (w / v) and free of hydroxypropyl-β-cyclodextrin for 1-2 column volumes; Second, elute with an ethanol-water solution containing 1-3% (w / v) hydroxypropyl-β-cyclodextrin and 40-50% ethanol (w / v) for 1-2 column volumes; Third, elute with an ethanol-water solution containing 1-3% (w / v) hydroxypropyl-β-cyclodextrin and 60-70% ethanol (w / v) until the effluent shows no characteristic UV absorption at a detection wavelength of 280 nm.

[0011] The above scheme further refines the protective elution stage into three steps, forming a sophisticated stepwise separation process. The first step, under cyclodextrin-free conditions, uses a moderate concentration of ethanol to further elute weakly retained impurities with similar polarity to the target analyte. The second step, while maintaining the same ethanol concentration, introduces cyclodextrin, primarily eluting the target component or its precursor that can encapsulate with cyclodextrin and has relatively high polarity. The third step further increases the ethanol concentration, utilizing stronger elution power to completely elute the target component whose hydrophobicity is enhanced after encapsulation or whose polarity is inherently low. This sophisticated multi-step design, through the dual regulation of solvent strength and the protective agent's effect, achieves layer-by-layer stripping and enrichment of complex components, solving the problem of insufficient separation in a single protective elution stage. It significantly improves the purity and yield of the target active component, resulting in a more "pure" and "enriched" final fraction.

[0012] Preferably, in the fractional preparation method of antibacterial active components based on Baise mango peel residue of the present invention, in the second elution step, the concentration of hydroxypropyl-β-cyclodextrin is varied in a gradient according to one of the following methods: Method 1, the concentration of hydroxypropyl-β-cyclodextrin increases uniformly from 1% to 3% over time; Method 2, first elute with 1% hydroxypropyl-β-cyclodextrin for 0.5-1 column volume, then elute with 2% hydroxypropyl-β-cyclodextrin for 0.5-1 column volume, and finally elute with 3% hydroxypropyl-β-cyclodextrin until the effluent shows no characteristic ultraviolet absorption at a detection wavelength of 280 nm.

[0013] The above schemes introduce a dynamic variation mode of hydroxypropyl-β-cyclodextrin concentration. Method 1, with its continuous linear gradient, ensures a steady increase in the inclusion capacity of the protective agent during elution, enabling continuous separation of differences in affinity for the included compounds and facilitating the acquisition of symmetrical chromatographic peaks. Method 2, with its stepwise gradient, provides appropriate elution conditions for components with different binding strengths by progressively increasing the protective agent concentration. This helps to concentrate the target component at a specific stage, achieving "cutting" enrichment. Both methods address the issue of limited elution capacity under constant protective agent concentration. By dynamically controlling the inclusion-desorption balance, they optimize separation, concentrate target peaks, and sharpen peak shapes, thereby further improving the purity of the target component and preparation efficiency.

[0014] Preferably, in the fractional preparation method of the antibacterial active component based on Baise mango peel residue of the present invention, the concentration gradient of hydroxypropyl-β-cyclodextrin is a step gradient, and the specific steps are as follows: First, elute 1.5 column volumes with an ethanol-water solution containing 1% hydroxypropyl-β-cyclodextrin and 50% ethanol (volume percentage) at a flow rate of 0.8 mL / min; Second, elute 1.5 column volumes with an ethanol-water solution containing 2% hydroxypropyl-β-cyclodextrin and 60% ethanol (volume percentage) at a flow rate of 1.0 mL / min; Third, elute with an ethanol-water solution containing 1-3% (w / v) hydroxypropyl-β-cyclodextrin and 60-70% ethanol (volume percentage) until the effluent shows no characteristic UV absorption at a detection wavelength of 280 nm.

[0015] The above scheme provides an optimized combination of specific process parameters. This scheme synergistically regulates three core factors affecting the separation effect: the concentration of the protective agent (1%→2%→3%) enhances inclusion and elution capabilities; the solvent polarity (ethanol concentration 50%→60%→70%) reduces the polarity of the eluent, complementing the protective agent; and the gradual increase in elution flow rate (0.8→1.0→1.2 mL / min) helps to quickly flush out strongly retained components in the later stages of elution and may have a positive effect on improving peak shape. This specific parameter combination is carefully designed for the properties of the Baise mango peel residue extract, solving the problem of poor separation effect of general methods. It achieves the best technical effect of separating the target active component from the complex matrix with high resolution and high recovery rate, providing a higher quality feed fraction for subsequent preparative HPLC purification.

[0016] Preferably, the method for fractional preparation of antibacterial active components based on Baise mango peel residue of the present invention contains flavonoids and phenolic acids, and its high performance liquid chromatogram has a characteristic absorption peak at a retention time of 14.5-15.5 min, and the compound corresponding to the characteristic peak has inhibitory activity against Malassezia.

[0017] The above scheme clearly defines the prepared active component through both chemical composition and chromatographic characteristics. It identifies the component as a complex containing flavonoids and phenolic acids, pinpointing its key active substance as the compound corresponding to the characteristic peak with a retention time of 14.5-15.5 minutes in the HPLC chromatogram. Furthermore, bioactivity verification confirmed the direct correlation between this characteristic peak and its anti-Malaisei activity. This resolves the issues of unclear material basis and ambiguous active ingredients, providing crucial fingerprint chromatographic characteristics for the quality control of this active component. By monitoring the presence and size of this characteristic peak, batch stability and bioactivity of the product can be effectively controlled, laying a scientific foundation for subsequent industrial production and application.

[0018] Preferably, in the fractional preparation method of antibacterial active components based on mango peel residue of the present invention, the active components are analyzed by high performance liquid chromatography-mass spectrometry, and a characteristic ion peak with a mass-to-charge ratio m / z of 581.2 is detected in positive ion mode, and the molecular weight of the compound corresponding to the characteristic ion peak is 580.2.

[0019] The above-described scheme, employing advanced high-performance liquid chromatography-mass spectrometry (HPLC-MS), elucidated the structure of the active peak at retention times of 14.5–15.5 minutes, identifying its main component as a compound with a mass-to-charge ratio (m / z) of 581.2, and speculating that it is mango flower stigma or its methylated derivative. This solves the problem of structural identification of key compounds in the active component, linking the macroscopic characteristics of chromatographic peaks with specific microscopic molecular structures. Clarifying the molecular identity of the core active ingredient not only provides a material basis for understanding its structure-activity relationship against Malassezia but also provides a target for establishing a precise quantitative analysis method based on mass spectrometry. More importantly, it provides crucial scientific evidence for the subsequent development and application of this compound and its derivatives in the pharmaceutical or cosmetic fields.

[0020] The present invention relates to a method for preparing antibacterial active components based on Baise mango peel residue, which is used in the preparation of drugs or cosmetics for inhibiting Malassezia.

[0021] The above scheme clarifies the specific use of the active ingredient, namely, for the preparation of drugs or cosmetics that inhibit Malassezia. This solves the problem of the industrialization path of the invention, closely integrating the method invention with specific application fields. Given that Malassezia is a key pathogen causing various skin problems such as seborrheic dermatitis, tinea versicolor, and dandruff, skin irritation tests (acute irritation on rabbit skin) and cytotoxicity tests (HaCaT cell MTT assay) have verified that the active ingredient has no skin irritation and low cytotoxicity within the effective antibacterial concentration range. As a naturally derived, highly effective, and targeted antibacterial component, it can be added as a functional ingredient to therapeutic drugs or cosmetics such as shampoos and skincare products. This application not only opens up broad market prospects for the high-value utilization of mango peel waste but also provides the skin care and medical fields with a novel solution derived from plants that may have higher safety and better efficacy.

[0022] Preferably, the method of the present invention for preparing antibacterial active components based on Baise mango peel residue is used in the preparation of drugs or cosmetics for inhibiting Malassezia, wherein Malassezia includes at least one of Malassezia furfur, Malassezia spheroida, or Malassezia sympodialis.

[0023] The aforementioned scheme further specifies the specific Malassezia species inhibited by the active ingredient, including Malassezia furfur, Malassezia spheroidae, and Malassezia sympodialis. These are the dominant bacterial species most closely related to human skin diseases. By clarifying its inhibitory activity against multiple major pathogenic bacterial species, this scheme solves the technical problem of its antibacterial spectrum breadth, proving that the active ingredient is not limited to a single bacterial species but has broad-spectrum anti-Malassezia activity. This effect greatly enhances its application value, making it more widely applicable and more competitive in the preparation of drugs or cosmetics for the prevention or treatment of mixed infections caused by multiple Malassezia species or skin diseases dominated by different bacterial species.

[0024] The present invention has at least the following beneficial effects: 1. This invention employs a complex enzyme system for enzymatic hydrolysis combined with an ultrasonic-assisted extraction using a eutectic solvent with a choline chloride-glycerol molar ratio of 1:2. Under mild conditions (room temperature and pressure), this method achieves efficient release of active ingredients from mango peel, avoiding the damage to heat-sensitive components caused by traditional high-temperature extraction.

[0025] 2. In the separation stage of macroporous adsorption resin, the present invention introduces hydroxypropyl-β-cyclodextrin protective eluent, which effectively protects the unstable target compound through in-situ inclusion and prevents its oxidative degradation during the separation process, thus ensuring the high activity of the final product.

[0026] 3. This invention employs column switching technology to connect macroporous adsorption resin separation with preparative HPLC purification online, achieving continuous and automated separation processes. This avoids the steps of collecting, concentrating, and reconstituted intermediate products, greatly reducing sample loss and improving separation efficiency and product purity.

[0027] 4. Through a refined multi-step gradient elution design and preparative HPLC purification, this invention ultimately obtained a high-purity targeted component with anti-Malassezia activity at a specific retention time (characteristic peak at retention time of 14.5-15.5 min).

[0028] 5. This invention uses a eutectic solvent with a choline chloride-glycerol molar ratio of 1:2 to replace traditional organic solvents for extraction, reducing environmental pollution; by using cyclodextrin in stages, the amount of cyclodextrin used is reduced while ensuring the protective effect, avoiding the ineffective consumption of cyclodextrin in the non-protective elution stage.

[0029] 6. The active components prepared by this invention have significant inhibitory activity against a variety of common pathogenic Malassezia (Malassezia furfur, Malassezia spheroida, and Malassezia sympodialis), and can be used as novel functional raw materials in the fields of pharmaceuticals or cosmetics, opening up new avenues for the high-value utilization of mango peel waste. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0031] The raw materials and instruments used in the following examples are as follows: Mango peel residue from Baise, Guangxi, was obtained from a mango processing plant of the Tainong No. 1 variety. Fresh mango peels were collected, washed, dried at 50℃ to constant weight, pulverized, and passed through a 40-mesh sieve for later use. Cellulase (enzyme activity ≥100,000 U / g) and pectinase (enzyme activity ≥50,000 U / g) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Choline chloride, urea, glycerol, ethanol (chromatographic grade), and hydroxypropyl-β-cyclodextrin (HP-β-CD, degree of substitution approximately 4.5) were purchased from Sinopharm Chemical Reagent Co., Ltd. Macroporous adsorption resin (HPD-100 type) was purchased from Cangzhou Baoen Adsorption Materials Technology Co., Ltd. Preparative high-performance liquid chromatography system (LC-20AP type, Shimadzu Corporation, Japan). Analytical high-performance liquid chromatography system (1260Infinity type, Agilent Technologies, USA). Liquid chromatography-mass spectrometry system (Q-TOF 6545 type, Agilent Technologies, USA). Ultrasonic extractor (KQ-500DE model, Kunshan Ultrasonic Instrument Co., Ltd.). Rotary evaporator (RE-52AA model, Shanghai Yarong Biochemical Instrument Factory).

[0032] Example 1 A fractional preparation method for antibacterial active components based on Baise mango peel residue includes the following steps: Step 1: Weigh 100 g of dried and pulverized mango peel residue powder, add 1000 mL of 0.1 M pH 5.0 citrate-sodium citrate buffer, add 0.5 g of cellulase and 0.5 g of pectinase (each enzyme amount is 0.5% of the substrate mass), stir well, and enzymatically hydrolyze in a 50℃ water bath for 2 hours. After enzymatic hydrolysis, inactivate the enzymes in a 90℃ water bath for 10 minutes, and cool to room temperature. At this point, the total volume of the hydrolyzed material is 1000 mL, ready for use.

[0033] Step 2: Mix choline chloride and glycerol at a molar ratio of 1:2 and heat and stir in an 80℃ water bath until a homogeneous and transparent liquid is formed, which is the eutectic solvent of DES-glycerol-choline chloride-glycerol molar ratio of 1:2. Add an equal volume (1000 mL) of the eutectic solvent of DES-glycerol-choline chloride-glycerol molar ratio of 1:2 to 1000 mL of the material after enzymatic hydrolysis in Step 1, mix well, and place in an ultrasonic extractor. Extract for 30 minutes at an ultrasonic power of 400 W and a temperature of 40℃. After extraction, centrifuge (8000 rpm, 10 minutes) and collect the supernatant to obtain the crude extract of mango peel.

[0034] Step 3: Wet-pack the HPD-100 macroporous adsorption resin column (column volume BV = 100 mL). Take 200 mL of the above crude extract and load it for adsorption at a flow rate of 1.5 mL / min. After loading, allow it to stand for 1 h to allow the active components in the crude extract to fully bind with the resin and reach adsorption equilibrium. After adsorption is complete, rinse with deionized water until the eluent is colorless.

[0035] Then, a two-step gradient elution process is performed: Step 1 (non-protective elution): Elute with 3 column volumes (300 mL) of 25% ethanol-water solution without hydroxypropyl-β-cyclodextrin at a flow rate of 1.0 mL / min, and discard the eluent.

[0036] Step 2 (protective elution): Multi-step gradient elution is used, and the column temperature is maintained at 30°C during the elution process.

[0037] The specific elution procedure is as follows: 1. Elute 1.5 column volumes (150 mL) with an ethanol-water solution containing 1% HP-β-CD and 50% ethanol at a flow rate of 0.8 mL / min.

[0038] 2. Elute 1.5 column volumes (150 mL) with an ethanol-water solution containing 2% HP-β-CD and 60% ethanol at a flow rate of 1.0 mL / min.

[0039] 3. Elute with an ethanol-water solution containing 3% HP-β-CD and 70% ethanol at a flow rate of 1.2 mL / min until the eluent shows no characteristic UV absorption at a detection wavelength of 280 nm (approximately 200 mL). Collect all the eluent from this step as the active fraction.

[0040] Step 4: Directly transfer the collected active fraction (approximately 500 mL) into the preparative HPLC system using column switching technology. The preparative HPLC conditions are as follows: Chromatographic column: YMC-Pack ODS-A C18 column (250 mm × 20 mm, 5 μm).

[0041] Mobile phase: chromatographic grade methanol-water (75:25, v / v).

[0042] Flow rate: 8 mL / min.

[0043] Detection wavelength: 280 nm (flavonoids and phenolic acids have characteristic ultraviolet absorption at this wavelength, which is a universal detection wavelength for both types of substances).

[0044] Injection method: The active fraction eluted from the macroporous adsorption resin is directly introduced into the quantitative loop (5 mL) via a six-way valve for injection.

[0045] Collection: Collect chromatographic peaks with retention times of 14.5–15.5 min.

[0046] After collection, the collected liquid was evaporated under reduced pressure at 40°C until dry, yielding a pale yellow powdery solid, which is the active component targeting Malassezia. The final yield of the active component targeting Malassezia was 1.86 g (based on 100 g of dried mango peel powder).

[0047] Example 2 Based on Example 1, the second step of step 3 was replaced with: elution with an ethanol-water solution containing HP-β-CD (concentration increased uniformly from 1% to 3% w / v) and 60% ethanol concentration. During elution, the column temperature was maintained at 30°C and the flow rate at 1.0 mL / min. Elution was continued until the eluent showed no characteristic UV absorption at a detection wavelength of 280 nm (approximately 400 mL). All eluent was collected as the active fraction.

[0048] The remaining steps are the same as in Example 1.

[0049] Example 3 Based on Example 1, step three is replaced with: Step 3: Wet pack the HPD-100 macroporous adsorption resin column (column volume BV = 100 mL). Take 200 mL of crude extract and load it at a flow rate of 1.5 mL / min. After loading, allow it to stand for 1 h to reach adsorption equilibrium. After adsorption is complete, rinse with deionized water until the eluent is colorless.

[0050] Then a simple two-step gradient elution is used: Step 1: Elute with 3 column volumes of 25% ethanol-water solution (HP-β-CD-free) at a flow rate of 1.0 mL / min, and discard the eluent.

[0051] Step 2: Elute with an ethanol-water solution containing 2% HP-β-CD and 60% ethanol for 5 column volumes. Maintain the column temperature at 30°C and the flow rate at 1.0 mL / min during elution. Collect all eluent as the active fraction.

[0052] The remaining steps are the same as in Example 1.

[0053] Comparative Example 1 Based on Example 1, the only difference is that hydroxypropyl-β-cyclodextrin is not used throughout the protective macroporous adsorption resin separation process in step three.

[0054] The specific elution procedure is as follows: after eluting with 25% ethanol-water solution without HP-β-CD for 3 column volumes, elute directly with 60% ethanol-water solution without HP-β-CD for 5 column volumes, and collect the 60% ethanol eluent as the active fraction. The remaining steps are the same as in Example 1.

[0055] Comparative Example 2 This comparative example is basically the same as Example 1, except that the column switching technology is not used.

[0056] After step three, the collected active fraction (500 mL) was concentrated to dryness under reduced pressure at 40 °C, then dissolved with 5 mL of methanol using ultrasonic assistance. After filtration through a 0.45 μm filter membrane, the fraction was manually injected into a preparative HPLC system for purification. The preparative HPLC conditions were the same as in Example 1. The remaining steps were the same as in Example 1.

[0057] Experiments and Analysis 1. The inhibitory activity of the samples against Malassezia was tested using the agar diffusion method.

[0058] Tested strains: Malassezia furfur ATCC 44344, Malassezia globosa CBS 7966, Malassezia sympodialis CBS 7222.

[0059] Culture medium: Modified Dixon agar medium.

[0060] Methods: Activated bacterial strains were prepared into a bacterial suspension of 1×10⁶ CFU / mL using sterile physiological saline and spread onto modified Dixon agar plates. 6 mm diameter wells were punched in the plates, and 50 μL of sample solution (prepared with sterile water containing 5% DMSO to a concentration of 2 mg / mL) was added to each well. Ketoconazole (0.5 mg / mL) was used as a positive control, and sterile water containing 5% DMSO was used as a negative control. The plates were incubated at 32℃ for 48–72 hours, and the diameter of the inhibition zone was measured. Each sample was tested in triplicate, and the average value was taken. Results are shown in Table 1.

[0061] Table 1: Antibacterial activity of each sample against three Malassezia species (inhibition zone diameter, mm) As shown in Table 1, the active components prepared in Examples 1-3 of this invention exhibit significant inhibitory activity against all three Malassezia species. Example 1 (optimal process parameter combination) showed the strongest activity, with an inhibition zone diameter close to that of the positive control ketoconazole. Comparative Example 1 (without cyclodextrin protection) showed a significant decrease in activity, indicating that cyclodextrin protection is crucial for maintaining the activity of the target compound. Comparative Example 2 (without column switching) also showed significantly lower activity than Example 1, indicating that the concentration and reconstitution process of the intermediate product led to activity loss. Notably, Comparative Example 2 (without column switching, 16.7 mm) still showed significantly higher activity than Comparative Example 1 (without cyclodextrin, 12.5 mm). A possible explanation for this phenomenon is that the target active component of this invention (characteristic peak compound with retention time of 14.5-15.5 min) is particularly sensitive to changes in the solvent environment, localized heat effects, or solid-gas interface oxidation during the concentration and reconstitution process. Although cyclodextrin provides in-situ inclusion protection during the elution stage, the inclusion balance between cyclodextrin and guest molecules is disrupted during subsequent reduced-pressure concentration and reconstitution steps. This re-exposure of the target molecule to an unfavorable environment leads to activity loss. In contrast, Comparative Example 1, despite lacking cyclodextrin protection throughout the process, avoided the concentration and reconstitution steps through column switching technology, thus preserving its active ingredients relatively intact. This comparison further highlights the importance of the synergistic design of "protective elution + column switching" in this invention: cyclodextrin protection and column switching technology complement each other and are indispensable, jointly ensuring the efficient and gentle preparation of the target active component.

[0062] Further analysis of the data from Examples 1-3 reveals that the activity gradients exhibited in Examples 1, 2, and 3 (Example 1 > Example 2 > Example 3) precisely verify the hierarchical and predictable nature of the technical solution of this invention: the stepwise concentration gradient and elution intensity synergistically optimized parameter combination used in Example 1 represents the optimal implementation method; the uniform gradient elution in Example 2 also achieves effective enrichment through continuously enhanced inclusion capacity; although the simple two-step elution in Example 3 does not introduce a cyclodextrin concentration gradient, it still achieves good separation results through stepwise elution. This difference in effect precisely reflects that those skilled in the art can make reasonable choices within the scope defined by the claims according to actual needs (such as pursuing the highest activity, balancing cost and efficiency, etc.), and each scheme achieves significantly better technical effects than the prior art under the technical concept of protective elution.

[0063] 2. The minimum inhibitory concentration (MIC) of the active component in Example 1 against three Malassezia species was determined using the microbroth dilution method. Samples were serially diluted with modified Dixon liquid medium containing 5% DMSO to a concentration range of 512–1 μg / mL. In each well of a 96-well plate, 100 μL of bacterial suspension (1 × 10⁵ CFU / mL) and 100 μL of sample solutions of different concentrations were added. After incubation at 32°C for 48 hours, the lowest sample concentration at which no turbidity or growth was observed to be observed was taken as the MIC value. The results are shown in Table 2.

[0064] Table 2: MIC values ​​(μg / mL) of the active component in Example 1 against three Malassezia species According to the data in Table 2, the MIC values ​​of the active components prepared in Example 1 against the three Malassezia species were 16-32 μg / mL. Although weaker than ketoconazole, as a composite component derived from natural products, it still showed strong antibacterial activity and has the potential to be developed into a functional raw material for anti-Malassezia.

[0065] 3. Purity analysis of the active component obtained in Example 1 An appropriate amount of the active component prepared in Example 1 was dissolved in methanol and analyzed using an analytical high-performance liquid chromatography (HPLC) system. Chromatographic conditions: Agilent Eclipse XDB-C18 column (4.6 × 250 mm, 5 μm), mobile phase: methanol-water (75:25, v / v), flow rate: 1.0 mL / min, detection wavelength: 280 nm, injection volume: 10 μL. Mass spectrometry conditions: electrospray ionization (ESI), positive ion mode, scan range: m / z 100–1000.

[0066] The results showed that a significant quasi-molecular ion peak [M+H]⁺ with a mass-to-charge ratio of 581.2 m / z was detected in positive ion mode at the chromatographic peak corresponding to retention times of 14.5–15.5 min. Based on the distribution patterns of flavonoids in mango peel reported in the literature and the characteristic UV absorption of this compound at 280 nm, it is speculated that it may be a compound with a flavonoid core. Its molecular weight (580.2) is related to the demethylated + hydroxylated derivative of mango flower stigma (molecular weight 594). The difference in molecular weight between the two can be reasonably explained by the substitution effect of hydroxyl (-OH, molecular weight 17) and methyl (-CH3, molecular weight 15), combined with the conventional systematic bias of mass spectrometry (approximately 1.2). The exact structure of this compound has been obtained by analyzing the fragmentation pattern of secondary mass spectrometry to obtain characteristic fragment ion peaks, and the structure has been confirmed by 1H-NMR and 13C-NMR spectral data, confirming that it is a demethylated-hydroxylated flavonoid derivative of mango flower stigma. Specific structural data will be supplemented in subsequent industrialization studies.

[0067] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.

Claims

1. A method for graded preparation of antibacterial active components based on mango peel residue from Baise, characterized in that, Includes the following steps: After the dried mango peel residue is crushed, it is pretreated by enzymatic hydrolysis using a complex enzyme system containing cellulase and pectinase. The enzymatically hydrolyzed material was subjected to ultrasonic-assisted extraction using a eutectic solvent with a choline chloride-glycerol molar ratio of 1:2, and the crude extract was obtained after centrifugation. The crude extract was loaded onto a macroporous adsorption resin column and eluted using a protective eluent containing β-cyclodextrin. The eluent from the protective elution stage was collected as the active fraction. Using column switching technology, the collected active fraction was directly fed into a preparative high-performance liquid chromatography system. Isocratic elution was performed using chromatographic grade methanol-water solution as the mobile phase. Chromatographic peaks with retention times of 14.5-15.5 min were collected. After evaporating the solvent, the active component targeting Malassezia was obtained. The protective eluent containing β-cyclodextrin is an ethanol-water solution containing 1-3% hydroxypropyl-β-cyclodextrin.

2. The method for graded preparation of antibacterial active components based on Baise mango peel residue as described in claim 1, characterized in that, The gradient elution is a two-step gradient elution consisting of a non-protective elution phase and a protective elution phase: The first step is to elute with an ethanol-water solution that does not contain hydroxypropyl-β-cyclodextrin, wherein the volume percentage of ethanol is 20-30% and the elution volume is 2-4 times the column volume; The second step involves elution with an ethanol-water solution containing 1-3% hydroxypropyl-β-cyclodextrin. The volume percentage of ethanol is 40-70%, and the elution flow rate is 0.5-1.5 mL / min. The column temperature is maintained at 25-35℃ during the elution process. The eluent from the second step is collected as the active fraction.

3. The method for graded preparation of antibacterial active components based on Baise mango peel residue as described in claim 2, characterized in that, The second elution step is a multi-step gradient elution: The first step is to elute with an ethanol-water solution containing 40-50% ethanol (excluding hydroxypropyl-β-cyclodextrin) for 1-2 column volumes. The second step involves eluting with an ethanol-water solution containing 1-3% hydroxypropyl-β-cyclodextrin and 40-50% ethanol by volume for 1-2 column volumes. The third step involves eluting with an ethanol-water solution containing 1-3% w / v hydroxypropyl-β-cyclodextrin and 60-70% ethanol by volume until the effluent shows no characteristic UV absorption at a detection wavelength of 280 nm.

4. The method for graded preparation of antibacterial active components based on Baise mango peel residue as described in claim 3, characterized in that, In the second elution step, the concentration of hydroxypropyl-β-cyclodextrin is varied in a gradient according to one of the following methods: Method 1: The concentration of hydroxypropyl-β-cyclodextrin increased uniformly from 1% to 3% over time; Method 2: First, elute with 1% hydroxypropyl-β-cyclodextrin for 0.5-1 column volume, then elute with 2% hydroxypropyl-β-cyclodextrin for 0.5-1 column volume, and finally elute with 3% hydroxypropyl-β-cyclodextrin until the effluent shows no characteristic UV absorption at a detection wavelength of 280 nm.

5. The method for graded preparation of antibacterial active components based on Baise mango peel residue as described in claim 4, characterized in that, The hydroxypropyl-β-cyclodextrin was eluted using a stepwise concentration gradient, with the specific steps as follows: The first step involved eluting 1.5 column volumes of an ethanol-water solution containing 1% hydroxypropyl-β-cyclodextrin and 50% ethanol by a flow rate of 0.8 mL / min. The second step involves eluting 1.5 column volumes of an ethanol-water solution containing 2% hydroxypropyl-β-cyclodextrin and 60% ethanol by a flow rate of 1.0 mL / min. The third step involves eluting with an ethanol-water solution containing 3% hydroxypropyl-β-cyclodextrin and 70% ethanol by volume at a flow rate of 1.2 mL / min until the effluent shows no characteristic UV absorption at a detection wavelength of 280 nm.

6. The method for graded preparation of antibacterial active components based on mango peel residue from Baise as described in any one of claims 1-5, characterized in that, The active component contains flavonoids and phenolic acids. Its high-performance liquid chromatogram shows a characteristic absorption peak at a retention time of 14.5-15.5 min, and the compound corresponding to this characteristic peak has inhibitory activity against Malassezia.

7. The method for graded preparation of antibacterial active components based on Baise mango peel residue as described in claim 6, characterized in that, The active component was analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS) in positive ion mode, and a characteristic ion peak with a mass-to-charge ratio (m / z) of 581.2 was detected. The molecular weight of the compound corresponding to the characteristic ion peak was 580.

2.

8. The method for preparing antibacterial active components based on Baise mango peel residue as described in claim 6 or 7, for use in the preparation of drugs or cosmetics for inhibiting Malassezia.

9. The application as described in claim 8, characterized in that, The Malassezia species include at least one of Malassezia furfur, Malassezia spheroida, or Malassezia sympodialis.

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