Preparation method and application of liposome of monkey ear ring polyphenol

Abstract

This invention discloses a method for preparing monkey earring polyphenol liposomes and their applications, belonging to the field of traditional Chinese medicine pharmaceutical technology. A method for preparing monkey earring polyphenol liposomes includes the step of dissolving soybean lecithin and cholesterol in anhydrous ethanol and then adding monkey earring polyphenol. The mass ratio of soybean lecithin to cholesterol is 5:1. The mass ratio of soybean lecithin to monkey earring polyphenol is 15:1. The monkey earring polyphenol liposomes prepared by this invention have an encapsulation efficiency of 89.31% and a drug loading of 4.61%, exhibiting good flowability and stability. These monkey earring polyphenol liposomes also possess antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, and Salmonella paratyphi B. The monkey earring polyphenol liposomes prepared by this invention can achieve the effects of increasing solubility and improving the bioavailability of monkey earrings.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine pharmaceutical technology, and in particular to the preparation method and application of monkey ear ring polyphenol liposomes. Background Technology

[0002] Pithecellobium clypearia Benth is a medicinal plant widely distributed in tropical Asia, including southern coastal provinces of China, Vietnam, Malaysia, Indonesia, the Philippines, and India. Polyphenols are important secondary metabolites in Pithecellobium clypearia, and can be classified into phenolic acids, flavonoids, and non-flavonoids based on their chemical structure. Polyphenols are believed to possess antioxidant, antibacterial, anti-inflammatory, anticancer, and antidiabetic properties. However, the easy degradation, insolubility in water, and high toxicity of Pithecellobium clypearia polyphenols limit the widespread application of polyphenolic drugs.

[0003] Liposomes are tiny vesicles that, as nanocarriers, possess outstanding biocompatibility. They have a phospholipid bilayer structure similar to biological membranes, enabling efficient drug delivery, improved drug bioavailability, controlled drug release rates, and reduced drug toxicity. They can also be used to encapsulate drugs that are poorly soluble in water. Therefore, monkey earring polyphenols can be prepared into monkey earring polyphenol liposomes (PCBPL-Lips) to further increase solubility and improve bioavailability. However, no publicly available method for preparing monkey earring polyphenol liposomes has been found in the current technology. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing monkey earring polyphenol liposomes and their applications, thereby solving the problems existing in the prior art. The PCBPL-Lips prepared by the method of this invention have an encapsulation efficiency of 89.31% and a drug loading of 4.61%, exhibiting good flowability and stability.

[0005] To address the above problems, the present invention provides the following solution:

[0006] Technical Solution 1: A method for preparing monkey earring polyphenol liposomes, comprising the step of dissolving soybean lecithin and cholesterol in anhydrous ethanol and then adding monkey earring polyphenol.

[0007] Furthermore, the mass ratio of the soybean lecithin to the cholesterol is 5:1.

[0008] Furthermore, the mass ratio of the soybean lecithin to the monkey ear ring polyphenol is 15:1.

[0009] Technical Solution 2: The monkey earring polyphenol liposomes prepared by the aforementioned preparation method.

[0010] Technical Solution 3: The application of the described monkey earring polyphenol liposomes in the preparation of antibacterial drugs.

[0011] Furthermore, the antibacterial activity includes resistance to Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, and Salmonella paratyphi B.

[0012] Technical Solution 4: A method for improving the stability of the monkey earring polyphenol liposomes, comprising placing the monkey earring polyphenol liposomes in an environment of 4°C.

[0013] Furthermore, the method includes placing the monkey earring polyphenol liposomes in a neutral or slightly acidic environment.

[0014] Furthermore, the pH of the acidic environment is 5.

[0015] Further, this includes placing the monkey earring polyphenol liposomes in a solution of 0-250 mM NaCl.

[0016] The monkey earring polyphenol raw material prepared by this invention contains a large amount of phenolic acids, mainly catechins and gallic acids.

[0017] The present invention discloses the following technical effects:

[0018] (1) This invention uses a thin-film dispersion method with monkey ear rings as the active pharmaceutical ingredient and different proportions of phospholipids and cholesterol to prepare polyphenol liposomes (PCBPL-Lips). The effects of the film material ratio and the lipid-to-pharmaceutical ratio on the performance of PCBPL-Lips were investigated, as well as the tolerance and long-term stability of PCBPL-Lips under different environments. This invention also investigated the stability of the prepared monkey ear ring polyphenol liposomes under pH, ionic strength, temperature, and long-term storage conditions. The results showed that PCBPL-Lips are more stable in neutral or acidic environments, possibly because the phenolic acids in the polyphenols react in an alkaline environment, making the solution system unstable. The liposomes showed high stability in 0-250 mM NaCl solutions; higher ion concentrations caused liposome flocculation, resulting in reduced encapsulation efficiency and larger particle size. Five temperatures were tested on the liposomes, and PCBPL-Lips showed stability at all five temperatures. In terms of long-term stability, within 30 days, the liposomes were more stable at 4°C than at 25°C, indicating that lower temperatures are beneficial for long-term storage.

[0019] (2) This invention characterizes the polyphenol liposomes of monkey ear rings (PCBPL-Lips) to understand their material composition and morphological characteristics, laying the foundation for subsequent experiments. The PCBPL-Lips were characterized and analyzed, revealing a liposome particle size of 124 nm, a dispersion index of 0.3519, and an average potential of -31.42 mV. The encapsulation efficiency of the PCBPL-Lips prepared according to the optimal formulation was 89.31%, and the drug loading was 4.61%. Rheological analysis showed that PCBPL-Lips possessed good flowability and stability. The apparent viscosity decreased with increasing shear rate, approaching 0 after 50 s⁻¹, exhibiting non-Newtonian pseudoplastic behavior. Liquid chromatography-mass spectrometry analysis showed that the monkey ear ring polyphenol raw material prepared in this invention contained a large amount of phenolic acids, mainly catechins and gallic acids.

[0020] (3) This invention evaluates the in vitro activity and safety of monkey earring polyphenol liposomes through in vitro antibacterial experiments, acute cellular toxicity tests, and blood compatibility tests. This invention tested the antibacterial activity and safety of PCBPL-Lips, finding that monkey earring polyphenols were active against five types of bacteria, with stronger activity against Gram-positive bacteria than against Gram-negative bacteria. Scanning electron microscopy revealed that the drug caused bacterial cell wall lysis, leakage of contents, and cell death. The safety of PCBPL-Lips was assessed through acute cytotoxicity and erythrocyte hemolysis rate. The PCBPL-Lips prepared by this invention are safe at certain concentrations. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Standard curve of polyphenols in monkey earrings;

[0023] Figure 2 The effect of film material ratio on the encapsulation efficiency and drug loading of PCBPL-Lips;

[0024] Figure 3 The effect of lipid-to-drug ratio on encapsulation efficiency and drug loading of PCBPL-Lips;

[0025] Figure 4 The effect of different pH values ​​on PCBPL-Lips particle size and encapsulation efficiency;

[0026] Figure 5The effect of different temperatures on PCBPL-Lips particle size and encapsulation efficiency;

[0027] Figure 6 The effect of different ionic strengths on the particle size and encapsulation efficiency of PCBPL-Lips;

[0028] Figure 7 The effect of 30 days of storage at 4℃ and 25℃ on the encapsulation efficiency of PCBPL-Lips;

[0029] Figure 8 The effect of 30 days of storage at 4℃ and 25℃ on the particle size of PCBPL-Lips;

[0030] Figure 9 The potential distribution diagram of PCBPL-Lips;

[0031] Figure 10 Visual representations of blank liposomes and PCBPL-Lips;

[0032] Figure 11 Scanning electron microscope image of PCBPL-Lips;

[0033] Figure 12 For the rheology of PCBPL-Lips;

[0034] Figure 13 Scanning electron microscope images of Staphylococcus aureus treated with sterile water or PCBPL; where a1 (1 μm) and a2 (500 nm) are Staphylococcus aureus treated with sterile water; a3 (1 μm) and a4 (500 nm) are Staphylococcus aureus treated with PCBPL.

[0035] Figure 14 Scanning electron microscope images of Escherichia coli treated with sterile water or PCBPL; b1 (1 μm) and b2 (500 nm) are Escherichia coli treated with sterile water; b3 (1 μm) and b4 (500 nm) are Escherichia coli treated with PCBPL.

[0036] Figure 15 Cell viability of PCBPL-Lips;

[0037] Figure 16 The hemolysis rate of PCBPL-Lips. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0042] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0043] Example 1: Preparation of monkey earring polyphenol liposomes

[0044] 1. Instruments

[0045] The experimental instruments are shown in Table 1.

[0046] Table 1 Instrument Models and Manufacturers

[0047] name model factory Thermostatic magnetic stirrer 524G Shanghai Meiyingpu Co., Ltd. Rotary evaporator RE-52AA Shanghai Yarong Biochemical Instrument Factory Ultrasonic cell disruptor SCIENTZ-ⅡD Ningbo Xinzhi Biotechnology Co., Ltd. Multi-angle particle size and high-sensitivity Zeta potential analyzer NanoBrookOmni Brookhaven Company, USA

[0048] 2. Experimental Methods

[0049] 2.1 Preparation of monkey earring polyphenol liposomes (PCBPL-Lips)

[0050] Soybean lecithin and cholesterol (both purchased from Shanghai Maclean Biotechnology Co., Ltd.) were weighed and dissolved in anhydrous ethanol. The solution was then sonicated (for 5 minutes at 40 kHz) until completely dissolved. Monkey ear loop polyphenol solution was slowly added (for preparation: monkey ear loop polyphenol powder was weighed and dissolved in anhydrous ethanol. The anhydrous ethanol was evaporated under reduced pressure in a rotary evaporator at 45°C, forming a thin film on the wall of the evaporator flask. An equal volume of purified water was then added, and the evaporation was continued for 10 minutes. Finally, the solution was hydrated for 30 minutes and then processed in an ultrasonic cell disruptor (for 2 minutes at 25 kHz). The mixture was then filtered through 0.45 μm and 0.22 μm microporous membranes to obtain monkey ear loop polyphenol liposomes.

[0051] 2.2 Determination of encapsulation efficiency of monkey earring polyphenol liposomes (PCBPL-Lips)

[0052] 2.2.1 Drawing the Standard Curve

[0053] Prepare a 0.1 mg / mL PCBPL standard solution using 50% ethanol. Pipette 0.2, 0.4, 0.6, 0.8, and 1.0 mL of each solution to prepare solutions with concentrations of 0.02, 0.04, 0.06, 0.08, and 0.1 mg / mL, respectively. Pipette 1 mL of each solution and determine the concentration using the Folin-Ciocalteu method at 765 nm. Plot a standard curve.

[0054] 2.2.2 Determination of encapsulation efficiency by ultrafiltration centrifugation method

[0055] The encapsulation efficiency of PCBPL-Lips was determined using ultrafiltration centrifuge tubes. 1 ml of liposomes was placed in an ultrafiltration centrifuge tube and centrifuged at 5500 rpm for 20 min. The lower layer of liquid in the outer tube was collected as the free polyphenols from the liposomes. Simultaneously, 1 ml of liposomes was added to 4 mL of anhydrous ethanol and centrifuged at 7500 rpm for 10 min to obtain the total polyphenols from the liposomes. The encapsulation efficiency (EE)% was then measured. Encapsulation efficiency (EE)% = (1 - (A - A0 / A)) × 100, where A0 is the free polyphenol content and A is the total polyphenol content from the liposomes.

[0056] 2.3 Optimization of PCBPL-Lips Preparation Conditions

[0057] 2.3.1 Single-factor experiment

[0058] Liposomes were prepared with a fixed soybean lecithin to cholesterol mass ratio of 5:1 and a water bath temperature of 50℃. Different soybean lecithin to PCBPL mass ratios (w / w) of 10:1, 15:1, 20:1, 25:1, and 30:1 were used to analyze the encapsulation efficiency and drug loading of PCBPL-Lips. Liposomes were also prepared with a fixed soybean lecithin to PCBPL mass ratio of 15:1 and a water bath temperature of 50℃. Different soybean lecithin to cholesterol mass ratios (w / w) of 1:3, 1:5, 1:7, 1:9, and 1:11 were used to analyze the encapsulation efficiency and drug loading of PCBPL-Lips.

[0059] 2.3.2 Stability assessment of PCBPL-Lips

[0060] (1) Temperature stability

[0061] The PCBPL-Lips prepared with the optimal formulation were placed in centrifuge tubes and sampled at 4, 25, 37, 45, and 60°C for 4 hours to determine the particle size and encapsulation efficiency.

[0062] (2) pH strength

[0063] The PCBPL-Lips prepared with the optimal formulation were placed in centrifuge tubes, and samples were taken at pH 3, 5, 7, 9, and 11 for particle size and encapsulation efficiency determination.

[0064] (3) Ionic strength

[0065] The PCBPL-Lips prepared with the optimal formulation were placed in centrifuge tubes. Different concentrations of NaCl (0, 50, 100, 250 and 500 mM) were mixed with an equal volume of PCBPL-Lips, and then reacted at room temperature for 2 h. Samples were taken for particle size and encapsulation efficiency determination.

[0066] (4) Storage time

[0067] The prepared PCBPL-Lips were stored at 4℃ and 25℃, and samples were taken at 0, 3, 5, 7, 14 and 21℃ for particle size and encapsulation efficiency determination.

[0068] 3. Results and Discussion

[0069] 3.1 Polyphenol Standard Curve

[0070] Plotting different concentrations of monkey earring polyphenol solution on the x-axis and the corresponding absorbance values ​​on the y-axis, the polyphenol standard curve was obtained as y = 0.0853x + 0.0239, R0. 2 =0.999, used for determining the polyphenol encapsulation efficiency in PCBPL-Lips. The standard curve for monkey earring polyphenols is shown below. Figure 1 .

[0071] 3.2 Results of Single-Factor Analysis

[0072] 3.2.1 Effect of film material ratio on encapsulation efficiency and drug loading of PCBPL-Lips

[0073] With other conditions remaining unchanged, by Figure 2 It can be seen that the encapsulation efficiency and drug loading of PCBPL-Lips are affected by different membrane material ratios. As the membrane material ratio decreases, the encapsulation efficiency gradually increases and the drug loading gradually increases. Considering both the encapsulation efficiency and drug loading, the optimal mass ratio of soybean phospholipids to cholesterol is 5:1.

[0074] 3.2.2 Effect of lipid-to-drug ratio on encapsulation efficiency and drug loading of PCBPL-Lips

[0075] like Figure 3 As shown, the mass ratio of soybean lecithin to PCBPL gradually increases the encapsulation efficiency of PCBPL-Lips while gradually decreasing the drug loading. Considering the encapsulation efficiency, drug loading, and material loss, a ratio of 15:1 is selected.

[0076] 3.3 Stability assessment of PCBPL-Lips

[0077] The widespread use of liposomes necessitates testing their stability. The effect of pH on the stability of PCBPL-Lips is as follows: Figure 4 As shown in the figure, when particle size is used as an indicator, the particle size at pH=7 is the smallest, at 131 nm. When encapsulation efficiency is used as an indicator, the difference between pH5 and pH7 is not significant, indicating that liposomes are more stable in neutral or slightly acidic environments. Strongly alkaline environments can cause liposomes to hydrolyze, leading to leakage of encapsulated polyphenols and loss of their original efficacy.

[0078] The effect of temperature on stability, such as Figure 5 As shown, the particle size and encapsulation efficiency did not change significantly between 4℃ and 37℃, but the particle size gradually increased with increasing temperature. This indicates that rising temperature disrupts the internal system of the liposome, leading to the aggregation of phospholipid molecules and thus increasing particle size.

[0079] Ionic strength affects liposome aggregation. Figure 6 As shown, within the NaCl concentration range of 0-250 nM, the encapsulation efficiency of PCBPL-Lips did not decrease significantly, and the particle size did not increase significantly. This indicates that PCBPL-Lips is stable in NaCl solutions of a certain concentration. The storage stability of PCBPL-Lips was evaluated by comparing the changes in particle size and encapsulation efficiency after 30 days of storage at 4℃ and 25℃.

[0080] from Figure 7 and Figure 8It can be seen that the particle size of PCBPL-Lips gradually increases and the encapsulation efficiency gradually decreases over time. Compared with 25℃, PCBPL-Lips at 4℃ are significantly more stable. This indicates that with increasing time, the liposome system undergoes flocculation, and particles adhere to each other, forming larger particles, leading to decreased stability.

[0081] This invention uses soybean lecithin and cholesterol as membrane materials and monkey ear loop polyphenol as the active ingredient to prepare monkey ear loop polyphenol liposomes. The effects of membrane material ratio and lipid-to-drug ratio on the encapsulation efficiency and drug loading of monkey ear loop polyphenol liposomes were investigated. Based on the comprehensive experimental results, the optimal preparation process of monkey ear loop polyphenol liposomes was found to be a soybean lecithin:cholesterol ratio of 5:1 and a soybean lecithin:PCBPL ratio of 15:1 (optimal formulation).

[0082] 4. Preparation of monkey earring polyphenol liposomes according to the optimal formulation

[0083] Weigh 15g of soybean lecithin and 3g of cholesterol (both purchased from Shanghai Maclean Biotechnology Co., Ltd.), dissolve them in anhydrous ethanol, and sonicate (for 5 minutes at 40 kHz) until completely dissolved. Slowly add 1g of monkey ear fungus polyphenol solution (preparation of monkey ear fungus polyphenol solution: weigh monkey ear fungus polyphenol powder, dissolve it in anhydrous ethanol, evaporate the anhydrous ethanol under reduced pressure in a rotary evaporator at 45°C, forming a thin film on the wall of the rotary evaporator flask, then add an equal volume of purified water, continue rotary evaporation for 10 minutes, and finally hydrate for 30 minutes. Then process in an ultrasonic cell disruptor (for 2 minutes at 25 kHz), and filter by extrusion using 0.45μm and 0.22μm microporous membranes respectively to obtain monkey ear fungus polyphenol liposomes.

[0084] The stability of the prepared monkey ear loop polyphenol liposomes (PCBPL-Lips) was investigated under conditions of pH, ionic strength, temperature, and long-term storage. The results showed that PCBPL-Lips were more stable in neutral or acidic environments, possibly because the phenolic acids in the polyphenols react in alkaline environments, making the solution system unstable. The liposomes exhibited high stability in solutions ranging from 0-250 mM NaCl; higher ionic concentrations caused liposome flocculation, resulting in reduced encapsulation efficiency and increased particle size. The liposomes were tested at five temperatures, and PCBPL-Lips showed stability at all five. Regarding long-term stability, within 30 days, the liposomes were more stable at 4°C than at 25°C, indicating that lower temperatures are beneficial for long-term storage.

[0085] Example 2 Characterization of monkey earring polyphenol liposomes

[0086] 1. Experimental Methods

[0087] 1.1 Characterization of PCBPL-Lips

[0088] 1.2 Particle size and potential

[0089] PCBPL-Lips were diluted tenfold with purified water, and their particle size and potential were determined using a ZETA nanoparticle potential analyzer (DelsaNano C, Beckman Coulter, USA).

[0090] 1.3 Observation of appearance and morphology

[0091] The blank liposomes (prepared by weighing soybean lecithin and cholesterol (both purchased from Shanghai Maclean Biotechnology Co., Ltd.), dissolving them in anhydrous ethanol, sonicating (for 5 min at 40 kHz) until completely dissolved, evaporating the anhydrous ethanol under reduced pressure in a rotary evaporator at 45°C to form a thin film on the wall of the evaporation flask, then adding an equal volume of purified water, continuing rotary evaporation for 10 min, finally hydrating for 30 min, and then processing in an ultrasonic cell disruptor (for 2 min at 25 kHz), and then filtering by squeezing using 0.45 μm and 0.22 μm microporous membranes respectively) and the monkey earring polyphenol liposomes prepared according to the optimal formulation in Section 4 of Example 1 were observed for their morphology and color by the naked eye.

[0092] 1.4 Scanning electron microscopy observation

[0093] The freeze-dried PCBPL-Lips powder was extracted, sputter-coated with gold, and observed under a scanning electron microscope (Zeiss Sigma 300, Germany).

[0094] 1.5 Determination of rheological properties

[0095] The rheological properties of PCBPL as a function of shear rate were investigated using a DHR-3 rheometer (TA Instruments, Inc., USA) at 25°C with shear rates ranging from 0.01 to 200 s⁻¹.

[0096] 1.6 PCBPL Composition Analysis

[0097] A Thermo Scientific Ultimate 3000 liquid chromatography-mass spectrometry system equipped with a Q Exactive Orbitrap and an electrospray ionization source was used. 5 μL of sample was injected into a Hypersil Gold C18 column at 40 °C. 0.1% formic acid aqueous solution (A) and methanol (B) were used as eluents, and the liquid chromatography flow rate was set to 250 μL / min. The gradient elution program for monkey ear fungus polyphenols is shown in Table 2. The mass spectrometric signal of the analyte was obtained using negative ion ionization with an injection voltage of -2.5 kV. The capillary and auxiliary gas heater temperatures were set to 320 °C and 350 °C, respectively. The first mass spectrometric signal was obtained at 70,000 fwhm, with a target MS / MS scan resolution of 175,000 fwhm, a separation width of 0.4 m / z, and an m / z scan range of 50–750.

[0098] Table 2 Gradient elution program for polyphenols in monkey earrings

[0099] Time (min) Mobile phase A: 0.1% formic acid aqueous solution (%) Mobile phase B: Methanol (%) 0-2 98-80 2-20 2-10 80-5 20-95 10-16 5 95 16-20 98 2

[0100] 2. Results and Discussion

[0101] 2.1 Characterization Analysis of PCBPL-Lips

[0102] Encapsulation efficiency represents the percentage of PCBPL in the total drug content after entering the liposomes, and is one of the important indicators for evaluating liposome quality, reflecting the efficacy of the liposomes. The PCBPL-Lips prepared according to the optimal formulation (see Section 4 of Example 1) have an encapsulation efficiency of 89.31% and a drug loading of 4.61%, indicating that the prepared PCBPL-Lips have good performance. The particle size of the PCBPL-Lips is concentrated between 89-158 nm, with an average particle size of 124.51 nm and a polydispersity index (PDI) of 0.3519. Most liposomes prepared by thin-film dispersion have a particle size greater than 100 nm, thus proving that the PCBPL-Lips prepared in this invention meet the requirements. The polydispersity index is often used to indicate the uniformity of particle size distribution in a suspension; the smaller the polydispersity index, the more uniform the particle size distribution. Generally, a PDI < 0.4 is considered acceptable; the prepared PCBPL-Lips have a polydispersity index less than 0.4, indicating uniform dispersion. The value of the zeta potential is related to the stability of the solution dispersion; the higher the absolute value, the more stable the system. The average potential of PCBPL-Lips is -31.42mV, and the potential is above 20mV, indicating that PCBPL-Lips have good stability. The potential distribution diagram of PCBPL-Lips is shown below. Figure 9 .

[0103] 2.2 PCBPL-Lips Morphology Analysis

[0104] Depend on Figure 10 It can be seen that the blank liposomes are milky white, while the solution turns reddish-brown after the addition of PCBPL. This is because the PCBPL is encapsulated by the liposomes. The darkening of the solution's color, the decrease in transparency, and the appearance of turbidity indicate that the liposome particle size increases after the addition of PCBPL.

[0105] 2.3 Scanning electron microscopy observation of PCBPL-Lips

[0106] like Figure 11 As shown, PCBPL-Lips are spherical, with the outer liposomes encapsulating the internal PCBPL, and are relatively uniformly dispersed. Scanning electron microscopy images reveal that the liposomes are spherical and fused together, with PCBPL fully encapsulated within the phospholipid bilayer.

[0107] 2.4 Analysis of Rheological Properties of PCBPL-Lips

[0108] from Figure 12 It can be seen that within the shear rate range of 0.1-200 s⁻¹, the higher the shear rate of PCBPL-Lips, the lower its apparent viscosity. Before the shear rate reaches 50 s⁻¹, the apparent viscosity decreases significantly, but when the shear rate exceeds 50 s⁻¹, the apparent viscosity approaches zero. With increasing shear rate, the viscosity of PCBPL-Lips consistently decreases, exhibiting typical non-Newtonian pseudoplastic behavior, showing shear-thinning behavior.

[0109] 2.5 PCBPL Composition Analysis

[0110] Liquid chromatography-mass spectrometry analysis revealed that PCBPL contains the following main substances: gallic acid, methyl gallate, gallocatechin, epicatechin acid, syringic acid, epigallocatechin gallate, 5,5'-dehydrodisvanillic acid, myricetin, L-epicatechin gallate, quercetin, 7,4'-digalloyltertrolofran, and 15,16-dihydroxyoctadec-9Z,12Z-dienoic acid (see Table 3).

[0111] Table 3 shows the results of LC-MS analysis of the composition in PCBPL.

[0112]

[0113] This invention characterized PCBPL-Lips, revealing a liposome particle size of 124 nm, a dispersion index of 0.3519, and an average potential of -31.42 mV. The PCBPL-Lips prepared according to the optimal formulation exhibited an encapsulation efficiency of 89.31% and a drug loading of 4.61%. Rheological analysis demonstrated that PCBPL-Lips possessed good flowability and stability. The apparent viscosity decreased with increasing shear rate, reaching a minimum at 50 s⁻¹. -1The value then approaches 0, exhibiting non-Newtonian pseudoplastic behavior. Liquid chromatography-mass spectrometry analysis revealed that the monkey earring polyphenol raw material contains a large amount of phenolic acids, primarily catechins and gallic acids.

[0114] Example 3: In vitro activity and safety evaluation of monkey earring polyphenol liposomes

[0115] Monkey earring polyphenols possess excellent antibacterial activity, but they are easily degraded, metabolized rapidly in vivo, and have high toxicity. These drawbacks limit the widespread application of polyphenol drugs. Monkey earring liposomes reduce their degradation rate and toxicity, but the prepared liposomes may trigger immunogenic reactions, causing adverse effects on the organism. This invention evaluates the in vitro activity and safety of monkey earring polyphenol liposomes through in vitro antibacterial experiments, acute cellular toxicity tests, and blood compatibility experiments.

[0116] 1. Materials and Instruments

[0117] Nutritional broth, hydrolyzed casein peptone broth, and agar powder were all purchased from Guangzhou Huankai Microbial Technology Co., Ltd., and levofloxacin hydrochloride was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. The experimental strains and their suppliers are shown in Table 4.

[0118] Table 4 Experimental bacterial strains

[0119] name Specification factory Staphylococcus aureus AR Guangzhou Huankai Microbial Technology Co., Ltd. Bacillus subtilis AR Guangzhou Huankai Microbial Technology Co., Ltd. Pseudomonas aeruginosa AR Guangzhou Huankai Microbial Technology Co., Ltd. E. coli AR Guangzhou Huankai Microbial Technology Co., Ltd. Salmonella paratyphi B AR Guangzhou Huankai Microbial Technology Co., Ltd.

[0120] The experimental instruments are shown in Table 5.

[0121] Table 5 Main Instruments

[0122] name model factory Mold incubator MJX-150BL Huanghua Feisifu Experimental Instrument Co., Ltd. Clean bench SW-CJ-IBU Shanghai Shunyu Hengping Scientific Instruments Co., Ltd. Portable autoclave SYQ-DSK-280B Shanghai Shenan Medical Equipment Factory pneumatic shaking bed KYC-100C Shanghai Fuma Experimental Equipment Co., Ltd.

[0123] 2. Experimental Methods

[0124] 2.1 Preparation of culture medium and resuscitation and activation of bacterial strains

[0125] Nutrient broth (NB) and Mueller-Hinton agar (MHA) contain the essential nutrients required for the growth and reproduction of most bacteria and are commonly used for bacterial resuscitation, activation, and preservation. Subsequent antimicrobial experiments use Mueller-Hinton broth (MHB). Under heating conditions, the agar and hydrolyzed casein peptone (MH) broth powder in the Erlenmeyer flask are stirred until completely dissolved, then autoclaved at 121°C for 20 minutes. After cooling to 50°C, the mixture is poured into sterile petri dishes for use in subsequent experiments.

[0126] On a clean bench, four single colonies stored in glycerol cryovials were picked using an inoculation loop and placed into NB liquid medium. These four liquid media were then cultured using an aerodynamic shaker at 120 rpm for 8-10 hours. The temperature was set to 37°C. After the medium became turbid, it was stored at 4°C for later use. Before the experiment, the liquid medium was suspended in sterile physiological saline and diluted with sterile physiological saline to prepare a bacterial suspension with a turbidity of 0.5 McFarland, approximately 10⁻⁶. 8 CFU / mL, then dilute the bacterial suspension to 10. 5 ~10 6 CFU / mL for subsequent experiments.

[0127] 2.2 Determination of inhibition diameter

[0128] The antibacterial efficacy of PCBPL and PCBPL-Lips was evaluated using the filter paper diffusion method. The concentration of the test bacterial strains was diluted to 1×10⁻⁶ with sterile physiological saline. 6 CFU / mL, and 50 μL of the diluted bacterial suspension was evenly spread onto Mueller-Hinton agar (MHA) using a sterile swab. Filter paper discs (6 mm) were immersed in the sample solution for 2 h, then blotted dry on the filter paper and placed on the agar surface. The plates were incubated at 37°C for 24 h. The diameter of the inhibition zone was used to measure the antibacterial activity of the drug. A positive control was levofloxacin (30 μg / mL). All measurements were repeated in triplicate.

[0129] 2.3 Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

[0130] The samples were diluted from 8 mg / mL to 0.03125 mg / mL using a two-fold dilution method. Then, 100 μL of sample solutions of different concentrations were mixed with 50 μL of bacterial suspension (approximately 1 × 10⁻⁶) in a 96-well plate. 5 Mix (CFU / mL). Incubate at 37°C for 20 h, then add 25 μL of 0.2 g / L iodonitrotetrazole violet (INT) to each well and incubate at 37°C for 6 h. The sample concentration corresponding to the first well where obvious discoloration begins is the MIC. Take 50 μL from all wells in the 96-well plate that have not yet shown discoloration, spread it evenly on solid culture medium, and incubate at 37°C for 24 h. Observe whether there is bacterial growth in the culture dish. The lowest sample concentration corresponding to the culture dish with no bacterial growth is the MBC.

[0131] 2.4 Ultrastructure of bacteria

[0132] Combining scanning electron microscopy (SEM) with this method allows for a more comprehensive view of the surface and internal morphology of bacteria. Specifically, an 8 mg / mL sample is placed in a 1×10⁻⁶ micrometer on a plate. 8 CFU / mL *E. coli* and *Staphylococcus aureus* were mixed and cultured at 37°C for 10 h. The mixture was centrifuged at 6000 rpm for 5 min, and the supernatant was discarded. The bacteria were washed three times with 0.1 mM pH 7.4 PBS, and 1 mL of 4% glutaraldehyde solution was added. The mixture was then incubated overnight. The supernatant was removed, the bacteria were washed, and 2% glutaraldehyde was added. The mixture was then incubated for 1 h. The bacteria were dehydrated in a gradient manner with 20%, 50%, 80%, and 100% ethanol, respectively, followed by dehydration with tert-butanol instead of ethanol. Untreated *E. coli* and *Staphylococcus aureus* served as negative controls. The treated bacteria were dried, sputter-coated with gold, and observed under a scanning electron microscope.

[0133] Preparation of 2.52% red blood cell suspension

[0134] SPF-grade male C57BL / 6 mice, 20±3g, were purchased from the Guangdong Provincial Medical Experimental Animal Center. Blood was collected from the abdominal aorta of the mice and placed in centrifuge tubes containing prepared anticoagulant. The cells were centrifuged at 1200 rpm for 10 min at 4℃. The supernatant was discarded, and 5-10 times the amount of physiological saline was added to the precipitate. The mixture was shaken well and washed repeatedly 3-4 times until the supernatant was no longer red. 2 mL of red blood cells were taken and diluted with physiological saline to 10 mL to obtain a 2% red blood cell suspension.

[0135] 2.6 Red blood cell hemolysis test

[0136] Take clean test tubes, label them, and add 0.5 mL of PCBPL-Lips (60, 100, 200, 300, 400 μg / mL) to each tube. Mix well with physiological saline, then add 0.5 mL of 2% erythrocyte suspension to each tube. Incubate in a 37°C water bath for 1 hour, then centrifuge at 3000 rpm for 5 minutes. Add 200 μL of supernatant to a 96-well plate and measure the absorbance at 545 nm. Use distilled water as a positive control and physiological saline as a negative control. Calculate the hemolysis rate using the following formula:

[0137] Hemolysis rate (%) = (A-A0 / A1-A0)*100

[0138] Where A is the absorbance of PCBPL-Lips, A0 is the absorbance of the negative control, and A1 is the absorbance of the positive control. A hemolysis rate >5% indicates the presence of hemolysis.

[0139] 2.7 PCBPL-Lips Cytotoxicity Assay

[0140] 100 μL of logarithmically growing RAW264.7 mouse macrophages were seeded into 96-well plates at a concentration of 1 × 10⁻⁶. 5Cells were cultured per well at 37°C for 24 hours in a CO2 incubator before drug administration and testing. Different concentrations of PCBPL-Lips solution (200, 150, 100, 75, 50, 25 μg / mL) were added and cultured under the same conditions for 24 hours. The original culture medium was removed, and fresh culture medium containing CCK-8 was added for 4 hours of incubation. The absorbance was then measured at 450 nm. Cell culture medium without PCBPL-Lips was used as a control, and culture medium without cells was used as a blank group. Results are expressed as X±S of cell viability in three independent experiments. Cell viability was calculated using the following formula:

[0141] Cell viability (%) = (A-A0 / A1-A0)*100

[0142] A represents the absorbance of PCBPL-Lips, A1 represents the absorbance of the control group, and A0 represents the absorbance of the blank group.

[0143] Each experiment was conducted at least three times, and results are expressed as mean ± standard deviation (SD). SPSS 26 software was used for regression analysis of the data. P < 0.05 was considered statistically significant.

[0144] 3. Results and Discussion

[0145] 3.1 Determination of inhibition zone

[0146] The antibacterial activity of monkey ear ring polyphenol (PCBPL) and polyphenol liposomes (PCBPL-Lips) can be preliminarily determined by the size of the inhibition zone. The results show that both PCBPL and PCBPL-Lips exhibit good antibacterial activity, indicating that the antibacterial activity was not reduced after encapsulation with liposomes, thus maintaining the activity of PCBPL. The antibacterial activities of PCBPL, PCBPL-Lips, and levofloxacin are shown in Table 6.

[0147] Table 6. Antibacterial activity of PCBPL, PCBPL-Lips and levofloxacin

[0148]

[0149] 3.2 Minimum inhibitory concentration and minimum bactericidal concentration

[0150] MIC and MBC are the lowest concentrations at which a drug inhibits and kills the growth of pathogens within 24 hours. These are important indicators of the antibacterial activity of a substance. In this invention, the MIC and MBC results of PCBPL and PCBPL-Lips are shown in Table 7. The MIC / MBC against Pseudomonas aeruginosa, Escherichia coli, and Salmonella typhi B were 0.5 / 2 mg / mL, against Bacillus subtilis were 0.125 / 0.5 mg / mL, and against Staphylococcus aureus were 0.0625 / 0.5 mg / mL. Based on these results, it can be concluded that the antibacterial activity of PCBPL is similar to that of PCBPL-Lips, and its antibacterial effect against Gram-positive bacteria is better than that against Gram-negative bacteria, indicating that PCBPL is well encapsulated in liposomes.

[0151] Table 7. MIC and MBC of PCBPL, PCBPL-Lips and Levofloxacin

[0152]

[0153] 3.3 Scanning electron microscopy observation results

[0154] To investigate the mechanism of action of monkey earring polyphenols on Gram-positive and Gram-negative bacteria, the morphology and surface of the target bacteria were evaluated using a SEM scanner. Staphylococcus aureus and Escherichia coli were used as representative examples. Figure 13 In the text a1-a2, the morphology of normal Staphylococcus aureus is described; its surface is smooth and intact, and it is spherical. After mixing with monkey earring polyphenols for 6 hours, from... Figure 13 In samples a3-a4, indentations, wrinkles, and folds were observed on the bacterial surface; some bacteria even showed morphological changes and damage. Figure 14 As can be seen from samples b1-b2, E. coli normally has a smooth, intact surface and is short rod-shaped. However, after 6 hours of PCBPL treatment ( Figure 14 In E. coli (b3-b4), significant surface depressions, deformation, bacterial breakage, and twisting were observed. Catechins can directly bind to the peptidoglycan of Staphylococcus aureus and induce its precipitation, thereby upregulating the peptidoglycan activity of E. coli. E. coli upregulates various defense mechanisms while downregulating metabolism and bioprotein synthesis, thus inhibiting its growth.

[0155] 3.4 Cytotoxicity assay

[0156] In vitro cytotoxicity assays are experiments that simulate the growth environment of organisms in vitro to detect the effects of drugs on cells, and are of great significance for drug evaluation. This invention uses the CCK-8 assay to test the cytotoxicity of different concentrations (200, 150, 100, 75, 50, 25 μg / mL) of PCBPL-Lips on mouse macrophage RAW264.7 cells. The results are as follows: Figure 15 As shown, when the concentration of PCBPL-Lips was 200 μg / mL, the cell viability was 49.21 ± 4.5%, and the cell viability was above 80% at concentrations of 150 μg / mL to 25 μg / mL. This indicates that the cells are safe at these concentrations, demonstrating that PCBPL-Lips has low cytotoxicity, good biocompatibility, and can be applied in biological experiments.

[0157] 3.5 Blood compatibility of PCBPL-Lips

[0158] Since intravenous injection of drugs involves direct contact with the blood, hemolysis testing is essential. Hemolysis can cause serious side effects on the liver, kidneys, and spleen. A hemolysis rate of less than 5% in the hemolysis test can be considered as having no hemolytic effect. This invention determined the hemolysis rate of different concentrations of PCBPL-Lips (60, 100, 200, 300, 400 μg / mL), and the results are as follows: Figure 16 As shown, when the concentration of PCBPL-Lips is below 400 μg / mL, the hemolysis rate is less than 5%. When the concentration is 400 μg / mL, the hemolysis rate is 7.78 ± 0.95%, indicating a slight hemolysis condition. This shows that PCBPL-Lips has good blood compatibility at the corresponding concentrations.

[0159] This invention tested the antibacterial activity and safety of PCBPL-Lips, finding that the monkey earring polyphenol was active against five types of bacteria, with stronger activity against Gram-positive bacteria than against Gram-negative bacteria. Scanning electron microscopy revealed that the drug caused bacterial cell wall lysis, leakage of contents, and cell death. The safety of PCBPL-Lips was assessed using acute cytotoxicity and erythrocyte hemolysis rate tests. It was found that PCBPL-Lips is safe at certain concentrations.

[0160] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing polyphenol liposomes from monkey earrings, characterized in that, This includes the step of dissolving soybean lecithin and cholesterol in anhydrous ethanol and then adding monkey earring polyphenols.

2. The preparation method according to claim 1, characterized in that, The mass ratio of soybean phospholipids to cholesterol is 5:

1.

3. The preparation method according to claim 1, characterized in that, The mass ratio of soybean lecithin to monkey earring polyphenol is 15:

1.

4. The monkey earring polyphenol liposomes prepared by the preparation method according to any one of claims 1-3.

5. The use of the monkey earring polyphenol liposomes according to claim 4 in the preparation of antibacterial drugs.

6. The application according to claim 5, characterized in that, The antibacterial activity includes resistance to Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, and Salmonella paratyphi B.

7. A method for improving the stability of the monkey earring polyphenol liposomes according to claim 4, characterized in that, This includes placing the monkey earring polyphenol liposomes at 4°C.

8. The method according to claim 7, characterized in that, This includes placing the monkey earring polyphenol liposomes in a neutral or slightly acidic environment.

9. The method according to claim 8, characterized in that, The slightly acidic environment has a pH of 5.

10. The method according to claim 7, characterized in that, This includes placing the monkey earring polyphenol liposomes in a 0-250 mM NaCl solution.

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