Novel uses of peony leaves or their extracts and hydrogels containing peony leaves or their extracts

Abstract

This invention provides the use of peony leaf or its extract in the preparation of a medicament for treating vaginitis. This invention also provides the use of peony leaf extract in combination with carboxymethyl chitosan in the preparation of an antibacterial drug, wherein the peony leaf extract is a methanol extract of peony leaves. This invention further provides a hydrogel loaded with peony leaf extract, prepared from carboxymethyl chitosan (CMCS) and peony leaf extract as raw materials, wherein the peony leaf extract is a methanol extract of peony leaves, and the mass ratio of peony leaf extract to carboxymethyl chitosan (CMCS) is (5-20):(100-150); wherein it contains 25-35 mg / mL of total flavonoids and 195-205 mg / mL of total polyphenols. This invention combines peony leaf extract (PLE) and carboxymethyl chitosan (CMCS) to endow the hydrogel with excellent antibacterial and antioxidant capabilities, effectively treating vaginitis caused by various bacterial infections. It avoids the drug resistance that arises from antibiotic treatment, providing a new drug delivery strategy for the safe and effective treatment of vaginitis.

Description

Technical Field

[0001] This invention relates to novel uses of peony leaves or their extracts, as well as hydrogels containing peony leaves or their extracts, and belongs to the pharmaceutical field. Background Technology

[0002] Vaginitis is a lifelong concern for women, often caused by an imbalance in the vaginal microbiota. A healthy woman's vaginal flora consists of aerobic and anaerobic bacteria, primarily lactobacilli. When pathogenic bacteria outnumber the healthy flora, an imbalance occurs, leading to vaginitis. Mixed vaginitis is caused by bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, Beta-streptococcus, Escherichia coli, and Candida albicans. Incomplete treatment can lead to deep uterine infections or premature birth in pregnant women. Frequent and prolonged use of broad-spectrum antibiotics can cause vaginal flora imbalance, secondary infections, and drug tolerance.

[0003] Incomplete treatment of mixed vaginitis can lead to deep uterine infection or premature birth in pregnant women. Frequent and prolonged use of broad-spectrum antibiotics can cause vaginal flora imbalance, secondary infections, and drug tolerance. Current treatments for aerobic vaginitis (AV) and vulvovaginitis (VVC) only treat a single type of vaginitis and have led to drug tolerance, resulting in vaginal flora imbalance and increased recurrence rates. Aerobic vaginitis (AV) is caused by bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, Beta-streptococcus, and Escherichia coli. Incomplete treatment can lead to deep uterine infection or premature birth in pregnant women. Frequent and prolonged use of broad-spectrum antibiotics can cause vaginal flora imbalance, secondary infections, and drug tolerance. Vulvovaginitis (VVC) is a common fungal vulvovaginal inflammatory disease, usually caused by Candida albicans. Approximately 75% of women worldwide suffer from this disease, which has a high recurrence rate. Currently, the four most widely used antifungal drugs for treating Candida vaginitis are clotrimazole, fluconazole, miconazole, and nystatin. However, some of these medications can only inhibit fungal growth, not kill them. Furthermore, long-term use can damage normal vaginal cells and tissues, further exacerbating vaginal flora imbalance, pH disturbances, recurrent infections, and recurrence, thus significantly increasing the difficulty of clinical treatment. There is an urgent need to explore methods for treating candidal vaginitis that can regulate the vaginal microenvironment.

[0004] Hydrogels are hydrophilic polymers with high water absorption and a three-dimensional network structure. When hydrogels swell in water or biofluids, they can absorb and retain large amounts of water without dissolving, a structure very similar to the natural extracellular matrix. Their moist and soft surface greatly reduces irritation upon contact with human tissues, giving hydrogels excellent tissue affinity and biocompatibility. Therefore, hydrogels are widely used in the biomedical field as an ideal material. There are many types and classification methods for hydrogels. Based on the source of raw materials, they can be divided into three main categories: natural polymer hydrogels, synthetic polymer hydrogels, and composite polymer hydrogels. Based on the cross-linking method, they can be divided into physically cross-linked, self-assembled, and chemically cross-linked hydrogels.

[0005] Carboxymethyl chitosan (CMCS) is a natural polysaccharide that is non-toxic and possesses good biodegradability, blood compatibility, and antibacterial properties, making it widely used in the medical field. However, due to the high solubility of CMCS, the degree of polymerization of standalone CMCS polymers is too weak, thus weakening their antibacterial and antioxidant properties. Therefore, CMCS is not suitable for use alone. To improve the functionality of this hydrogel, bioactive substances that enhance its polymerization properties and possess antibacterial and antioxidant properties were added.

[0006] Peony leaves originate from the leaves of *Paeonia rockii* Andr. and *Paeonia szechuanica* Fang, both belonging to the genus *Paeonia* in the family Ranunculaceae. *Paeonia szechuanica* Fang is a species endemic to China, found in northwestern Sichuan (Maerkang), growing wild on mountain slopes, riverside meadows, or in forests at altitudes of 2400–3100 m. *Paeonia szechuanica* is closely related to *Paeonia rockii*, differing only in leaf lobes, hairiness, and flower disc morphology. Its root bark can also be used as "peony bark." Modern research has found that the non-medicinal part of peony—the leaf—has antioxidant, antibacterial, lipid-lowering, and vasoactivating effects, and is used to treat gynecological and cardiovascular diseases. (Xue J, Li T, Wang S, et al. Elucidation of the mechanism of reflowering in tree peony (Paeonia suffruticosa) 'Zi Luo Lan' by defoliation and gibberellic acid application[J]. Plant Physiology and Biochemistry, 2018, 132: 571-578.; Honarvar Nazari M, Shihab MS, Cao L, et al. A peony-leaves-derived liquid corrosion inhibitor: protecting carbon steel from NaCl[J]. Green Chemistry Letters and Reviews, 2017, 10(4): 359-379. Wang Z, Zhu C, Liu S, et al. Comprehensive metabolic profile analysis of the root bark of different species of tree peonies (Paeonia suffruticosa) 'Zi Luo Lan' by defoliation and gibberellic acid application[J]. Plant Physiology and Biochemistry, 2018, 132: 571-578.; Honorvar Nazari M, Shihab MS, Cao L, et al. A peony-leaves-derived liquid corrosion inhibitor: protecting carbon steel from NaCl[J]. Green Chemistry Letters and Reviews, 2017, 10(4): 359-379. Wang Z, Zhu C, Liu S, et al. Comprehensive metabolic profile analysis of the root bark of different species of tree peonies (Paeonia suffruticosa) Sect. Moutan)[J]. Phytochemistry, 2019, 163: 118-125. Gao Zhiqing, et al., Research progress and application of peony leaves, Yunnan Chemical Industry, November 2021, Vol. 48, No. 11). Application No. 200610167661.9, Invention title: A total flavonoid extract of peony leaves and its preparation method and uses, discloses a total flavonoid extract of peony leaves isolated and purified from a new medicinal part of peony leaves and its preparation method. Specifically, the total flavonoid content in the peony leaf total flavonoid extract is 50% to 95%.The application of total flavonoid extract from peony leaves in the preparation of anti-inflammatory drugs is also disclosed. Application No. 201510763809.4, invention title: A method for extracting effective components from peony leaves and the resulting product, discloses a method for extracting effective components from peony leaves and the resulting product. The method involves: washing peony leaves, adding salt and kneading; after kneading, microwave drying at 40-50℃ until the moisture content is below 10%; after drying, freezing at -15℃ for 5-10 minutes, then freezing at -5℃ for 20-30 minutes; placing the frozen peony leaves in a heated container, compacting them, keeping them at 50-60℃ for 0.5-1 hour, then heating to boiling, collecting the distillate, which is the peony leaf aqueous extract. This invention is simple, easy to implement, clean, and efficient. The pretreatment method of adding salt and kneading-microwave drying-low temperature freezing allows for the extraction of more effective components. Experimental verification shows that the obtained peony leaf aqueous extract has good antibacterial and anti-inflammatory effects and can be used as a raw material for anti-inflammatory drugs.

[0007] Although there are literature reports on the activity of peony leaves, they are rarely developed and utilized, and are usually incinerated as waste, resulting in low utilization, environmental pollution, and resource waste. Currently, there are no reports of peony leaves being used for vaginitis, nor are there any reports of peony leaf extracts being used in specific formulations. Summary of the Invention

[0008] The technical solution of the present invention provides a new use for peony leaves or their extracts and a hydrogel containing peony leaves or their extracts.

[0009] This invention provides the use of peony leaves or extracts thereof in the preparation of medicaments for treating vaginitis.

[0010] The medication mentioned is for treating mixed vaginitis.

[0011] The medication mentioned is for treating aerobic vaginitis and vulvovaginitis.

[0012] The present invention also provides the use of peony leaves or extracts thereof in the preparation of medicaments with anti-Candida albicans properties.

[0013] The peony leaf extract is a methanol extract of peony leaves.

[0014] The peony leaf methanol extract contains gallic acid 55-65 mg / mL, paeoniflorin 35-45 mg / mL, kaempferol 15-25 mg / mL, quercetin 25-35 mg / mL and isorhamnetin 15-25 mg / mL; it also contains total flavonoids 170-180 mg / mL and total polyphenols 640-650 mg / mL.

[0015] More preferably, the peony leaf methanol extract contains 197.267 mg / mL of total polyphenols and 31.599 mg / mL of total flavonoids.

[0016] The detection method for the peony leaf extract is as follows:

[0017] a. Take peony leaf extract and dissolve it in methanol to prepare peony leaf extract solution;

[0018] b. High-performance liquid chromatography (HPLC) was used for detection. The chromatographic conditions were as follows: SunFire™ C18 column (100 mm × 2.1 mm, 1.7 μm); mobile phase A: 0.2% phosphoric acid water; mobile phase B: acetonitrile; elution gradient as follows: 0–10 min, 2% B; 10–15 min, 2%–15% B; 15–25 min, 15%–16% B; 25–35 min, 16%–17% B; 35–40 min, 17%–18% B; 40–50 min, 18%–20% B; 50–60 min, 20%–25% B; 60–70 min, 25%–40% B; flow rate 0.3 mL / min; injection chamber temperature 25 °C; column temperature 30 °C; injection volume 10 μL.

[0019] This invention provides the use of peony leaf extract in combination with carboxymethyl chitosan in the preparation of antibacterial drugs, wherein the peony leaf extract is a methanol extract of peony leaves.

[0020] This invention provides the use of peony leaf extract in combination with carboxymethyl chitosan in the preparation of a drug for treating vaginitis, wherein the peony leaf extract is a methanol extract of peony leaves.

[0021] This invention provides a hydrogel loaded with peony leaf extract, which is prepared from carboxymethyl chitosan (CMCS) and peony leaf extract as raw materials. The peony leaf extract is a methanol extract of peony leaves, and the mass ratio of peony leaf extract to carboxymethyl chitosan (CMCS) is (5-20):(100-200). It contains 25-35 mg / mL of total flavonoids and 195-205 mg / mL of total polyphenols.

[0022] More preferably, the mass ratio of the peony leaf extract to carboxymethyl chitosan (CMCS) is 15:100.

[0023] The present invention also provides the use of hydrogels loaded with peony leaf extract in the preparation of antibacterial drugs, drugs for treating vaginitis, or drugs with antioxidant effects.

[0024] The inventors discovered that peony leaf extract has certain antibacterial effects against Gram-negative bacteria, Gram-positive bacteria, and fungi. However, peony leaf extract has high solubility; it dissolves rapidly in water or other organisms, failing to retain its medicinal properties. Furthermore, it has a short residence time at the lesion site, resulting in minimal drug absorption. This weakens the antioxidant and antibacterial properties of peony leaf extract. Therefore, peony leaf extract is not suitable for use alone. Thus, developing and utilizing this abundant peony leaf resource in conjunction with CMCS to create hydrogels as a treatment material for aerobic vaginitis and vulvovaginitis is a meaningful scientific research endeavor.

[0025] The peony leaf extract of this invention exhibits certain antibacterial effects against Gram-negative bacteria, Gram-positive bacteria, and fungi. However, the peony leaf extract has high solubility, dissolving rapidly in water or organisms without retaining its medicinal properties. Furthermore, it has a short residence time at the lesion site, resulting in minimal drug absorption. This weakens the antioxidant and antibacterial properties of the peony leaf extract. Therefore, the peony leaf extract is not suitable for use alone. Carboxymethyl chitosan (CMCS) is a natural polysaccharide that is non-toxic, possesses good biodegradability, blood compatibility, and antibacterial properties, and is widely used in the medical field. However, due to the high solubility of CMCS, the degree of polymerization of standalone CMCS polymers is too weak, thus weakening its antibacterial and antioxidant properties. Therefore, CMCS is not suitable for use alone.

[0026] This invention combines peony leaf extract (PLE) and carboxymethyl chitosan (CMCS) to develop a novel natural antibacterial and antioxidant hydrogel (hereinafter referred to as: CP hydrogel). Based on a mixing process, a one-step mixing process is used to construct CMCS / PLE self-assembled hydrogels with different PLE contents. The mechanical force introduced during stirring promotes effective collisions between carboxymethyl chitosan and PLE molecules, accelerating the molecular self-assembly process. The amino groups of carboxymethyl chitosan and the hydroxyl groups of PLE collide and combine during stirring, thereby endowing the hydrogel with excellent antibacterial and antioxidant capabilities.

[0027] The CP hydrogel exhibits antibacterial activity against both *Avianthromyces avianus* (AV) and *VVC* (VVC) pathogens. The CP hydrogel was characterized by FTIR spectroscopy, X-ray diffraction (XRD), and scanning electron microscopy (SEM). SEM micrographs revealed a three-dimensional porous network structure within the hydrogel. The swelling behavior and degradation properties of the CP hydrogel were also investigated; the swelling ratio and weight loss rate gradually decreased with increasing PLE content. The antioxidant properties of the hydrogel were demonstrated by its scavenging rate of DPPH and ABTS free radicals. It was found that the scavenging rate of DPPH and ABTS free radicals gradually increased with increasing PLE concentration, reaching a maximum of 90%, indicating that the CP hydrogel possesses good antioxidant properties. A novel hydrogel material prepared by combining peony leaf extract and carboxymethyl chitosan showed good antibacterial effects in in vitro antibacterial experiments against Staphylococcus aureus, drug-resistant Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Candida albicans, and β-streptococci. The antibacterial rate reached 99% when the peony leaf content was increased to 2 wt%. In an in vivo mouse model of vaginitis, the peony leaf extract hydrogel inhibited vaginal flora by 94.74%, while reducing vaginal edema, congestion, and increased secretions, improving vaginal tissue inflammatory infiltration, and reducing the expression of inflammatory factors IL-1β and IL-6, effectively treating vaginitis caused by multiple bacterial infections. This approach avoids the drug resistance that can result from antibiotic treatment and provides a new drug delivery strategy for the safe and effective treatment of vaginitis. Attached Figure Description

[0028] Figure 1 Structural diagram and determination of PLE content: A. Structural diagram of the article; B. High performance liquid chromatogram of PLE at wavelength 254 nm; 1. Gallic acid; 2. Paeoniflorin; 3. Kaempferol; 4. Quercetin; 5. Isorhamnetin; C. Chemical structural formula of the standard.

[0029] Figure 2 A comparison chart of the screening of hydrogel matrices for this invention;

[0030] Figure 3 Hydrogel structure characterization: A is the SEM image of CMCS, CP1, CP2, CP3 and CP4; B is the FTIR spectrum of CMCS, PLE and CP4 hydrogels; C is the FTIR spectrum of CP1, CP2, CP3 and CP4; D is the XRD spectrum of CMCS, PLE and CP4 hydrogels; E is the XRD spectrum of CP1, CP2, CP3 and CP4.

[0031] Figure 4The structural properties of the hydrogels were characterized as follows: A represents the G′ and G″ values ​​of the sample; B represents the swelling ratio of the CMCS and CP hydrogels; C represents the in vitro drug release via PLE; and D represents the degradation rate of the CMCS and CP hydrogels. Statistical analysis: *P<0.01; **P<0.01; ***P<0.001; ****P<0.0001;

[0032] Figure 5 Characterization of the antioxidant and safety properties of hydrogels: A represents the DPPH free radical scavenging rate (wt%) of CMCS and CP hydrogels; B represents the ABTS free radical scavenging rate (wt%) of CMCS and CP hydrogels; C represents the hemolysis rate of CMCS, PLE and CP hydrogels; and D represents the cytotoxicity (wt%) of CMCS and CP hydrogels.

[0033] Figure 6 Hydrogel antibacterial experiment: Survival charts and inhibition rates of various bacterial species after incubation with CMCS and CP hydrogels at 37℃ for 16 hours on agar plates. A: Staphylococcus aureus; B: methicillin-resistant Staphylococcus aureus (MRSA); C: Staphylococcus epidermidis; D: Escherichia coli; E: Candida albicans; F: β-Streptococcus; G: Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis; H: Escherichia coli, Candida albicans, and β-Streptococcus. Statistical analysis: *P<0.1; **P<0.01; ***P<0.001; ****P<0.0001;

[0034] Figure 7 Histological analysis of mouse vaginal images: A) Mouse vaginal image; B) Mouse vaginal tissue pathological analysis section, CIL-6 and TNF-α.

[0035] Figure 8 Analysis of vaginal flora in mice: A. Plate count of vaginal wash in mice on days 0, 3, and 8 after administration; B. Colony count of vaginal wash in mice on days 0, 3, and 8 after administration; C. Antibacterial rate in mice on days 3 and 8 after administration.

[0036] Figure 9Vaginal wash flora 16Sr gene sequencing map; A. Mean relative percentage abundance of the top 10 phyla; B. Mean relative percentage abundance at the genus level; C. Pie chart of phylum-level community analysis; D. Circos plot at the genus level; E. Ace index at the OUT level; F. Vaginal community richness; G. Vaginal community diversity; H. Relative richness of Vagococcus; I. Relative richness of Lactobacillus Beijerinck; J. Relative richness of Escherichia Castellani and Chalmers; K. PCoA analysis; L. PCA analysis (Statistical analysis: *P<0.01; **P<0.01; ***P<0.001; ****P<0.0001). Detailed Implementation

[0037] Example 1: Preparation of Peony Leaf Extract of the Present Invention

[0038] After freeze-drying, peony leaves were pulverized using a grinder and passed through a 65-mesh sieve (Pharmacopoeia). 100g of the pulverized leaves were weighed and added to 1000ml of methanol in a 1L conical flask. The leaves were ultrasonically extracted for 1 hour each time. After filtration three times, the three filtrates were combined and the methanol solution was evaporated using a vacuum rotary evaporator to obtain peony leaf extract PLE.

[0039] Example 2: Preparation of Peony Leaf Extract Hydrogel of the Present Invention

[0040] Carboxymethyl chitosan (CMCS) and peony leaf extract (PLE) solution were mixed and stirred until homogeneous, then centrifuged to remove foam, and allowed to stand at room temperature for 48 hours to prepare hydrogels. After preparation, hydrogels were prepared by grouping peony leaf extract concentrations of 0.5 wt%, 1 wt%, 1.5 wt%, and 2 wt% and carboxymethyl chitosan concentration of 10 wt% into groups CP1, CP2, CP3, and CP4, and stored in sealed bags at 4°C. Pure CMCS (as a control) was also prepared using the same method. The amounts of carboxymethyl chitosan (CMCS) and peony leaf extract (PLE) were as follows: peony leaf extract: 5 mg, 10 mg, 15 mg, and 20 mg; carboxymethyl chitosan: 110 mg.

[0041] Example 3: Quality detection method for peony leaf extract of the present invention

[0042] The components in peony leaf extract (PLE) were analyzed by high performance liquid chromatography. The prepared peony leaf extract was accurately weighed and dissolved in methanol to a concentration of 5 mg / mL. One mL of the 5 mg / mL peony leaf extract solution was mixed with 1 mL of methanol reagent, shaken thoroughly, and incubated at 37°C for 30 minutes. The reference standard was prepared using the PLE method. Chromatographic conditions: The column was a SunFire™ C18 (100 mm × 2.1 mm, 1.7 μm); the mobile phase was set as follows: Mobile phase A: 0.2% phosphoric acid water; Mobile phase B: acetonitrile; Elution gradient: 0–10 min, 2% B; 10–15 min, 2%–15% B; 15–25 min, 15%–16% B; 25–35 min, 16%–17% B; 35–40 min, 17%–18% B; 40–50 min, 18%–20% B; 50–60 min, 20%–25% B; 60–70 min, 25%–40% B; Flow rate: 0.3 mL / min; Injector temperature: 25 °C; Column temperature: 30 °C; Injection volume: 10 μL. The total flavonoids and total phenols in PLE were measured using a UV spectrophotometer.

[0043] Hydrogels were prepared using a free radical mixing method, such as... Figure 1 A. The hydrogel formed by combining CMCS and PLE is brownish-red, and the color gradually deepens with increasing PLE concentration. Studies have shown that the mixture of carboxymethyl chitosan (CMCS) and PLE forms hydrogen bonds. PLE contains polyphenols and flavonoids, and these polyphenols react with CMCS, stabilizing the CMCS hydrogel structure. The higher the PLE content, the more stable the structure. The content of PLE was determined using high-performance liquid chromatography (HPLC). Figure 1 B. Five standards—gallic acid, paeoniflorin, kaempferol, quercetin, and isorhamnetin—were selected to determine their content in PLE. The results showed that their contents in PLE were in the ranges of 55–65, 35–45, 15–25, 25–35, and 15–25 mg / mL, respectively. Simultaneously, a UV spectrophotometer was used to determine the contents of total flavonoids and total polyphenols in PLE and CP1, CP2, CP3, and CP4. The results showed that the total flavonoid content was in the ranges of 170–180, 0–10, 8–15, 15–25, and 25–35 mg / mL, respectively, and the total polyphenol content was in the ranges of 640–650, 75–80, 100–110, 155–165, and 195–205 mg / mL, respectively. Because the concentration of PLE increases sequentially from CP1 to CP4, the contents of total polyphenols and total flavonoids also increase sequentially, indicating that CP4 has the highest contents of total polyphenols and total flavonoids. Its total polyphenol and total flavonoid contents are 197.267 mg / mL and 31.599 mg / mL, respectively.

[0044] Example 4: Screening of the hydrogel matrix of the present invention

[0045] This study prepared hydrogels by combining sodium alginate, gelatin, and carboxymethyl chitosan (CMCS) with peony leaf extract (PLE), highlighting the advantages of CMCS. All hydrogels were prepared using a one-pot mixing method. The preparation method for sodium alginate-peony leaf extract hydrogel was as follows: sodium alginate and peony leaf extract solution were mixed and stirred until homogeneous, then centrifuged to remove foam. The preparation method for gelatin-peony leaf extract hydrogel was as follows: gelatin and peony leaf extract were mixed and stirred in a 60℃ water bath until dissolved, then cooled. The preparation method for CMCS-peony leaf extract hydrogel was as follows: CMCS and peony leaf extract (PLE) solution were mixed and stirred until homogeneous, then centrifuged to remove foam, and allowed to stand at room temperature for 48 hours. The amounts of each raw material were: PLE: 150 mg, CMCS: 1 g, sodium alginate: 1 g, gelatin: 1 g.

[0046] Results analysis:

[0047] like Figure 2 As shown, the first row shows the appearance of hydrogels. From the appearance of hydrogels with three different matrices, it can be seen that the hydrogels with gelatin as the matrix and carboxymethyl chitosan as the matrix are blocky hydrogels, which are easy to store and administer. However, the hydrogel with sodium alginate as the matrix is ​​a viscous semi-solid hydrogel, which is inconvenient for storage, carrying and administration. Figure 2 The second row shows the adhesive properties of hydrogels with different matrices. The figure shows that the gelatin-PLE hydrogel has no adhesive properties, while the sodium alginate-PLE hydrogel and CMCS-PLE hydrogel both exhibit good adhesiveness. Furthermore, the CMCS-PLE hydrogel in this study is a self-assembled hydrogel, which is simpler than chemically synthesized hydrogels. Chemically synthesized hydrogels require the addition of chemical adhesives, which is not environmentally friendly, while carboxymethyl chitosan can be dissolved in water. In conclusion, using CMCS as the hydrogel matrix in this study is convenient for storage, easy to carry, convenient for drug administration, and environmentally friendly.

[0048] The following specific experiments demonstrate the beneficial effects of the present invention.

[0049] Experimental Example 1: Hydrogel Performance and Pharmacodynamic Tests of Peony Leaf Extract of the Present Invention

[0050] I. Experimental Methods:

[0051] 1. Fourier Transform Infrared Spectroscopy (FTIR)

[0052] Using FTIR at 400cm -1 Up to 4000cm -1Spectra of PLE, CMCS, and CP hydrogels were acquired in a regional sampling area to analyze their chemical structures and interactions between components. 5 mg and 500 mg of KBr particles from each sample were ground into powder in a mortar, and then a small amount was compressed into tablets. The background was KBr particles, and the scanning area was 400 cm⁻¹. -1 Up to 4000cm -1 A total of 32 scans were performed, with a design resolution of 4cm.

[0053] 2. X-ray diffraction (XRD) test of materials and hydrogels

[0054] The crystal morphology of PLE, CMCS, and CP hydrogels was investigated using an X-ray diffractometer (Ultima IV, Rigaku, Japan). Each sample was ground into powder, placed on slides, and inserted into the instrument. The required conditions were set to 40 kV, 40 mA, and X-ray radiation of Cu (=1.54). The scanning range for each sample was 5° to 50°, the scanning rate was 1° per minute, and the continuous scanning range was 20°.

[0055] 3. Observation of hydrogel using scanning electron microscopy (SEM)

[0056] The freeze-dried sample was fixed onto a circular conductive carrier platform using double-sided conductive tape. After a 30-second gold sputtering process, its morphology was observed using a scanning electron microscope at an accelerating voltage of 20 kV.

[0057] 4. Rheological investigation of hydrogels

[0058] For rheological testing, hydrogel samples were prepared with a diameter of 1 cm and a thickness of 4 mm. The rheological behavior of different hydrogel samples under different conditions was measured using a rheometer. Specific experiments were conducted under oscillating angular frequency sweep testing. The oscillating angular frequency sweep testing was performed at 25 ± 0.1 °C and a constant strain of 10%, measuring the storage modulus (G') and loss modulus (G”) of the hydrogels at angular frequencies ranging from 0.1 to 100 rad / s. Samples included CP1, CP2, CP3, CP4, and CMCS.

[0059] 5. Swelling ratio of hydrogels

[0060] The swelling ratio of hydrogels was characterized by monitoring mass changes during incubation at 37°C. Hydrogels (6 x 6 x 5 mm, n = 3) were dried for 24 hours and designated M0. Various hydrogels were immersed in PBS and placed in an incubator at 37°C. At regular intervals, the swollen hydrogels were removed with tissue paper to absorb surface moisture, and weighed immediately (Ms). The swelling ratio of the hydrogels was calculated using the following formula.

[0061] Swelling rate (%) = (Ms – M0) / M0 × 100%

[0062] 6. In vitro degradation of hydrogels

[0063] To monitor the degradation rate of CMCS, CP1, 2, 3, and 4 hydrogels, each test sample (1 x 1 cm) was prepared. 2 Incubate in a container containing 10 mL of PBS solution (pH 7.4), then add 15 mg / mL lysozyme solution (similar to lysozyme solution in human serum). Incubate the mixture at 37°C for 8 h. After degradation, collect the sample from the culture medium, rinse with distilled water, and dry to constant weight in a vacuum drying oven at 45°C. Each experiment was performed in triplicate, and the weight loss (%) was calculated according to the following equation.

[0064] (W0-W t )) / W0×100

[0065] Where W0 is the initial weight of the sample, W t This is the final weight after degradation.

[0066] 7. In vitro release study

[0067] PLE release was conducted in simulated vaginal fluid at pH 5.0. Different concentrations of PLE hydrogel were added to 50 mL centrifuge tubes containing 30 mL of simulated vaginal fluid. The tubes were then placed at 37°C and shaken at 100 rpm for 0 to 7 days. During this period, 1 mL of the supernatant was replaced with an equal volume of fresh simulated vaginal fluid, and 250 μL of Folin-Ciocalteau reagent was added to the 1 mL supernatant. The mixture was stirred and incubated in the dark for 5 minutes, followed by the addition of 750 μL of anhydrous sodium carbonate (20% w / v) and incubation in the dark at room temperature for 2 hours. The absorbance was then measured at 765 nm using a UV spectrophotometer. The final values ​​were used to obtain the cumulative release curve of the sample based on a standard curve of gallic acid aqueous solution (concentration range: 0-30 mg / L).

[0068] 8. Evaluation of the blood compatibility of hydrogels

[0069] Red blood cells were obtained from 5 mL of fresh SD rat blood. After centrifugation at 3500 rpm for 5 min, the cells were washed 5 times with sterile PBS after centrifugation. Then, 1 mL of the original blood was diluted to 10 mL with PBS to obtain a 10% red blood cell-PBS solution. Another 1 mL of the 10% red blood cell-PBS solution was then diluted to 5 mL to obtain a 2% red blood cell-PBS solution. Next, 2 mL of the 2% red blood cell-PBS solution was added to a centrifuge tube, followed by 5 mg of each test sample. The tube was then incubated at 37°C (100 rpm) for 2 h, centrifuged at 3500 rpm for 5 min, and 100 μL of the supernatant from each sample was transferred to a 96-well plate. The PBS-treated group and the pure water-treated group served as negative and positive controls, respectively. The absorbance of the samples was measured at 545 nm. The formula for calculating the hemolysis rate is as follows:

[0070] Hemolysis rate (%) = (Mas – Man) / (Map – Man) × 100%

[0071] Wherein, Mas, Man, and Map are the absorbance values ​​of the sample, negative control, and positive control, respectively. Each experiment was performed in triplicate.

[0072] 9. Cytotoxicity

[0073] This study used the CCK-8 assay to detect the cytotoxicity of CMCS, CMCSPLE1, CMCSPLE2, CMCSPLE3, and CMCSPLE4 hydrogels against L929 fibroblasts. Cells were cultured in 10 ml of DMEM low-glucose complete medium prepared with 10% FBS and 1% penicillin-streptomycin antibiotics, under aseptic conditions of 37°C and 5% CO2. All hydrogel samples were immersed in 6 cm water according to the Chinese Pharmacopoeia standard method (GB / T 16886.5-2017). 2 In DMEM at / ml, the cells were cultured at 37℃ for 24 h. The extract was then diluted with culture medium, and hydrogel samples of 0.05, 0.5, 1, and 2 mg / ml were collected. 100 μL of cell suspension was added to each well of a 96-well plate. Finally, 10 μL of CCK-8 solution was added to each well, and the plates were incubated at 37℃ for 2 h. The optical density was measured at 450 nm using enzyme-linked immunosorbent assay (OD). Using the above techniques, background experiments were performed, and the cell viability of the samples was calculated using the following formula:

[0074] Cell viability (%) = (ODs - ODb) / (ODn - ODb) × 100%

[0075] The ODS, ODn, and ODb values ​​above represent the optical density values ​​of the sample group, negative control group, and background culture medium, respectively. The experiment was performed in five replicates per sample.

[0076] 10. Hydrogel Antioxidant Experiment

[0077] 10.1 In vitro DPPH free radical scavenging rate

[0078] The antioxidant efficiency of the hydrogels was determined using the DPPH free radical scavenging method. The procedure was as follows: 2 ml of hydrogel sample solutions (0.05, 0.075, 0.1, and 0.2 mg / ml) were dispersed in 2 ml of DPPH-ethanol solution. The mixture was stirred and incubated in the dark for half an hour. Next, the absorbance of DPPH at 517 nm was measured using a UV-Vis spectrophotometer. The degradation rate of DPPH was calculated using the following formula:

[0079] DPPH clearance rate (%) = (MAb - MAh) / MAb × 100%

[0080] MAb and MAh were the blank control group (DPPH + ethanol) and the hydrogel sample (DPPH + ethanol + hydrogel solution), respectively, and were repeated three times.

[0081] 10.2 In vitro ABTS free radical scavenging rate

[0082] 0.3844 g of ABTS reagent and 0.0667 g of potassium persulfate were weighed into water and dissolved in deionized water to a final volume of 100 mL, preparing a 0.007 M ABTS solution. This solution was then thoroughly mixed with a 2.45 mM potassium persulfate solution at a 1:1 volume ratio and reacted in the dark for 12–16 h to obtain an ABTS stock solution. Before use, the absorbance of the mixture at 734 nm was measured to be 0.7 using anhydrous ethanol. 20 μL of the ethanol extract of the membrane was added to 180 μL of the diluted solution, and the mixture was incubated in the dark for 5 min. The absorbance of the solution at 734 nm was measured using a microplate reader. The ABTS free radical scavenging rate was calculated using the following formula.

[0083] ABTS radical scavenging rate (%) = (MAb - MAh) / MAb × 100%

[0084] Where MAb represents the blank absorbance and MAh represents the sample absorbance, repeated three times.

[0085] 11. Antibacterial activity of hydrogel

[0086] The antibacterial effect of the hydrogel was evaluated using colony counting and liquid culture tests. The antibacterial activity of the hydrogel against Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae (B-streptococcus), and Escherichia coli (causative agents of aerobic vaginitis (AV)), Candida albicans (causative agent of vulvovaginitis (VVC)), and drug-resistant Staphylococcus aureus were tested. Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and drug-resistant Staphylococcus aureus were cultured on nutrient agar, B-streptococcus on blood agar, and Candida albicans on Sabouraud dextrose agar. Colonies of these bacteria were picked using a sterile inoculation loop and inoculated into 4 ml of sterile liquid culture medium. The culture was evenly dispersed and then placed in a constant temperature shaker at 37°C and shaken at 200 rpm for 6 hours. Then, sterilized EP tubes were used for serial dilution. 2 ml of each diluted bacterial suspension was taken and the OD value of the bacterial suspension was measured at 600 nm using a UV spectrophotometer. The concentration of the bacterial suspension was determined based on the fitted curve of the OD value. 10 μl of bacterial suspension was then taken from each sample and diluted with 1 ml of PBS, repeating this process three times. 100 μl of the diluted solution was then plated onto an agar plate and incubated for 16-18 hours. Once colonies had formed, the viable colonies on the plate were photographed.

[0087] 12. Mouse vaginitis treatment experiment

[0088] First, mice were divided into four groups to establish the pseudoestrus cycle: normal group, model group, positive group, PLE group, and CP4 group. Except for the normal group, mice in the other groups were subcutaneously injected with estradiol benzoate (2 mg / mL) to establish pseudoestrus, once every 2 days for 6 consecutive days. On day 7, mice in pseudoestrus were vaginally injected with a mixed culture of Candida albicans and Escherichia coli, once daily. After successful modeling, the vagina was repeatedly washed with sterile PBS (20 μl), and the solution was pipetted 5 times to obtain vaginal wash fluid. This was used for quantitative analysis of Candida albicans and Escherichia coli viability on MRS agar plates. Then, drugs (clotrimazole suppositories, PLE, CP4) were administered vaginally. Vaginal wash fluid was collected three times on days 0, 3, and 8 of drug administration and stored in cryovials. Finally, mice in each group were euthanized under diethyl ether anesthesia, and the vagina, heart, liver, spleen, lungs, and kidneys were collected and fixed with paraformaldehyde (4%). These tissues were dissected, analyzed with H&E staining, and imaged using an optical microscope.

[0089] II. Experimental Results:

[0090] 1. Structural analysis of hydrogels

[0091] SEM observation of the cross-section of the prepared hydrogel showed that the binding of CMCS and PLE altered the microstructure of the CP hydrogel cross-section, such as... Figure 3A. Pure CMCS has fewer, larger, and unevenly distributed pores in its cross-section. After adding PLE, the CP hydrogel exhibits a more numerous, smaller, denser, and more uniformly distributed microporous structure, making it more stable than the pure CMCS hydrogel. These results indicate a significant interaction between CMCS and PLE, effectively enhancing the hydrogel's structure and properties. Furthermore, due to the relatively large pore size of CMCS, PLE can fill the hydrogel network. These results suggest the possible formation of intermolecular hydrogen bonds between CMCS and PLE. It was also found that with increasing PLE concentration, the pore size of the CP hydrogel gradually decreases and the degree of polymerization increases. In summary, this hydrogel possesses a three-dimensional network porous structure.

[0092] 2. FTIR analysis

[0093] The molecular interactions between CMCS and PLE were analyzed using FTIR spectroscopy. Figure 3 B and Figure 3 As shown in C, the Fourier transform infrared spectrum of carboxymethyl chitosan (CMCS) is at 3418 cm⁻¹. -1 There is an absorption peak at 2938 cm⁻¹, which is the stretching peak of the OH / -NH bond. -1 and 1329cm -1 The spectral bands at 1609 cm⁻¹ represent the stretching and bending of CH₄, respectively. -1 and 1421cm -1 Strong absorption peaks appeared at 1075 cm⁻¹, representing the symmetric and asymmetric stretching vibration absorption peaks of the COO-bond, respectively. The spectral band appeared at 1075 cm⁻¹. -1 There is a strong absorption peak at 3418 cm⁻¹, which is the stretching peak of the CN bond. Therefore, all hydrogel samples show similar absorption peaks at this location. -1 The broad peak flattens with increasing PLE concentration, indicating that the -OH groups of the polyphenols and the -OH / -NH groups of CMCS interact, reducing the stretching of free -OH / -NH groups. Therefore, hydrogen bonds may form between the two compounds, promoting their interaction through physical reactions. The interaction between the polyphenols and CMCS may lead to changes in the internal bonds between some functional groups in the compounds. This results in the peak at 2938 cm⁻¹. -1 and 1609cm -1 The frequency band becomes difficult to identify with increasing extract concentration. Furthermore, with increasing PLE concentration, the 1075 cm⁻¹ frequency band becomes less identifiable. -1 The peak value shifted to 1084 cm. -1 The peak value of COC under symmetrical stretching is approximately 1075 cm. -1 Furthermore, it was noted that no additional polyphenol peaks were observed in the FTIR spectrum of the CP hydrogel after the addition of the extract, which may be due to the low content of the extract in the hydrogel.

[0094] 3. XRD Analysis

[0095] like Figure 3 As shown in Figures D and E, CMCS exhibits a distinct crystallization peak at 20° in XRD, while PLE shows no obvious peak, indicating that PLE lacks crystallinity. With increasing PLE concentration, its diffraction peak becomes increasingly flat, indicating a decrease in the crystallinity of the hydrogel. When the PLE concentration increases to 2 wt% (CP4), the diffraction peak almost disappears. Therefore, the addition of polyphenols may interfere with the formation of an ordered crystal structure in the CMCS matrix. Notably, the degradability of the hydrogel largely depends on the crystallinity within its structure. The low crystallinity exhibited in the CP hydrogel may contribute to a higher degradation rate.

[0096] 4. Rheological analysis of hydrogels

[0097] The mechanical properties of hydrogels were determined using a rheometer. The content of poly(ethylene glycol) (PLE) is a major factor affecting the rheological properties of hydrogels. Oscillating sweep frequency measurements were used to verify the rheological properties of the hydrogels. Figure 4 The results of the oscillation angular frequency sweep test show that the G′ and G″ of the hydrogel change with frequency under different degrees of crosslinking. The G′ of the hydrogel is always about one order of magnitude higher than G″, indicating that the hydrogel is in an elastic solid gel state. Moreover, the G′ value of the hydrogel increases with the increase of PLE content, which is the same as the SEM results, indicating that the increase of crosslinking degree helps to improve the mechanical properties of the hydrogel.

[0098] 5. Swelling rate analysis of hydrogels

[0099] The swelling capacity of hydrogels is closely related to the material structure. After drying the hydrogel samples, they were placed in PBS solution, and the initial sample weight (M0) and the weight of the hydrogel after a preset swelling time were recorded. The weights of each sample (M0, M0, M0, M0, M0, M0, M1, M2, M3, M4, M5, M6, M7, M8, M9, M10, M11, M12, M13, M14, M15, M16, M17, M18, M19 ...9, M19, M10, M19, M19, M10, M19, M19, M10, M19, M19, M10, M19, M10, M19, M10, M19, M10 1-7 ).like Figure 4 Sample B showed a significant increase in swelling rate within 50 minutes, which gradually stabilized after 60 minutes. Furthermore, the swelling rate decreased with increasing PLE concentration, because the key intermolecular forces of the hydrogel become stronger with increasing PLE concentration, resulting in a more stable structure. CMCS hydrogels have more hydrophilic groups (-COOH, -OH, and -NH2) than CMCS / PLE hydrogels, making it easier for water molecules to enter the hydrogel network. At pH 7.4, the swelling rate of the sample was between 300% and 400%, exhibiting a significantly high water absorption rate.

[0100] 6. In vitro drug release of PLE

[0101] To investigate the release rate of PLE in the vaginal environment, the release rate of PLE in an artificially simulated vaginal fluid was measured in vitro. Figure 4 As shown in Figure C, PLE and CP hydrogels with different concentrations of PLE exhibited rapid release behavior in simulated vaginal fluid, reaching over 90% within 12 hours. It can be seen that the release rate of PLE alone immediately reached 90%, with CP1 showing the fastest release rate and CP4 showing a slower release rate, consistent with SEM and swelling results. After 12 hours, the release rate gradually decreased. However, due to the higher PLE content in CP4, the release rates of CP3 and CP4 remained relatively stable after 12 hours, while the release rates of CP1 and CP2 decreased significantly. Therefore, CP4 showed the highest total release after 7 days. Combining the SEM and swelling degradation results, it can be concluded that CP4 has a more stable structure and the highest total PLE release.

[0102] 7. In vitro degradation rate analysis of hydrogels

[0103] In vitro degradation tests on the hydrogel: lysozyme hydrolysis revealed that CMCS possesses numerous hydrophilic groups (-COOH, -OH, and -NH2), making it susceptible to lysozyme degradation. For example... Figure 4 D. The weight loss rate of pure CMCS is higher than 65%. With the increase of PLE concentration, the weight loss rates of CP1, CP2, CP3, and CP4 are in the range of 60-65%, 55-60%, 50-55%, and 45-50%, respectively. This indicates that with the increase of PLE concentration, the degree of crosslinking gradually increases and the weight loss rate gradually decreases.

[0104] 8. Hemolysis rate analysis of hydrogels

[0105] Biocompatibility of materials is crucial before their application in biomedicine. Hemolysis testing is also a simple method for assessing the blood compatibility of biomaterials. For example... Figure 5 As shown in Figure C, after co-incubating the CMCS and various hydrogels with erythrocyte suspension according to the operating requirements, the hemolysis rates of each group were similar to those of the PBS-treated group, all below 4%, showing a significant difference compared to the water-treated group. These results indicate that the prepared precursor material and composite hydrogel both possess good erythrocyte compatibility, do not cause hemolysis of erythrocytes, and are blood-compatible materials that can be safely applied in the biomedical field.

[0106] 9. Cytotoxicity analysis

[0107] Good cell compatibility is essential for the design of hydrogel materials. The effect of hydrogels on L929 cell viability was assessed using the CCK-8 cytotoxicity assay. The principle of the CCK-8 assay is that, in the presence of an electron-coupled reagent, the tetrazolium salt WST8 can be reduced by dehydrogenases in mitochondria, producing an orange-yellow formazan dye that is positively correlated with the number of viable cells in the culture medium. The number of viable cells was then dynamically quantified using a colorimetric method for biosafety assessment. Figure 5 As shown in Figure D, the cell viability of each hydrogel group was good, with the highest cell survival rate approaching 100%, indicating that the composite hydrogel is non-toxic. Overall, the cell survival rate of all hydrogel groups exceeded 80%, visually demonstrating that these hydrogels have good biocompatibility.

[0108] 10. Antioxidant analysis of hydrogels

[0109] Under pathological conditions, free radicals generated under excessive oxidative stress are chemically reactive and highly active in the body, participating in a series of chain reactions that cause lipid peroxidation on cell membranes, leading to cell damage, primarily affecting lipids and cell membranes, proteins and enzymes, nucleic acids and chromosomes, and sugar molecules. This study uses the scavenging rate of DPPH and ABTS free radicals to explain the antioxidant activity of CP hydrogels. Figure 5 As shown in Figure A, it can be seen that the highest PLE concentration of CP4 can achieve a DPPH free radical scavenging rate of over 70%. The CMCS gel's DPPH free radical scavenging rate is less than 10%. The higher the PLE concentration, the higher the DPPH free radical scavenging rate, indicating that a higher PLE concentration results in a better antioxidant effect. Figure 5 As shown in Figure B, the ABTS radical scavenging rate reached 90% at the highest CP4 concentration. This indicates that the lower the PLE concentration, the lower the ABTS radical scavenging rate. The CMCS hydrogel's ABTS radical scavenging rate was no greater than 10%. Higher PLE concentrations resulted in better antioxidant effects. In conclusion, this hydrogel exhibits excellent antioxidant properties.

[0110] 11. Antibacterial activity analysis

[0111] Vaginitis is mainly caused by an imbalance in the vaginal microbiota. A healthy woman's vaginal flora consists of aerobic and anaerobic bacteria, primarily lactobacilli. When pathogenic bacteria outnumber the healthy flora, an imbalance occurs, leading to vaginitis. Aerobic vaginitis (AV) is caused by bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae (β-streptococci), Escherichia coli, Pseudomonas aeruginosa, and Enterococcus faecalis. Vulvovaginitis (VVC) is caused by Candida albicans. This study mainly conducted antibacterial experiments on Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Escherichia coli, Candida albicans, and drug-resistant Staphylococcus aureus. CMCS has good antibacterial properties, water solubility, and non-toxicity. The -NH2 in CMCS interacts with bacterial cell wall acid and lipopolysaccharides, achieving antibacterial effect by disrupting the bacterial cell wall. The main components of PLE are polyphenols and flavonoids. Polyphenols exert their antibacterial effect by disrupting bacterial cell walls, while flavonoids achieve their antibacterial effect by damaging bacterial cell membranes. Figure 6 Compared to the control group, all other groups showed a certain antibacterial effect. Figure 6 The bacteria listed in the diagram (A, B, C, D, E, F) are, in order, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Candida albicans, and Beta-Streptococcus. Visual observation shows that both CMCS and CP hydrogels have antibacterial effects against these bacteria. Figure 6 As can be seen from G and H, the antibacterial rate of CMCS is significantly lower than that of PLE. Higher PLE concentrations result in a higher antibacterial rate against the above-mentioned bacterial colonies. CP4 showed an antibacterial rate exceeding 90% against all bacteria. This indicates that CP4 hydrogel has a good antibacterial effect against various pathogens causing vaginitis, laying the foundation for the treatment of vaginitis.

[0112] 12. In vivo antibacterial effect of CP hydrogel on vulvovaginitis and candidal vaginitis. To further evaluate its potential as a local antibacterial agent, the in vivo antibacterial effect of CP4 against vaginitis induced by a mixture of Candida albicans and Escherichia coli was evaluated, with clotrimazole suppositories used as a positive control for comparison. First, mice were subcutaneously injected daily with estradiol benzoate injection (2 mg / mL) to establish a pseudoestrus period. Then, a mixed bacterial solution of Candida albicans and Escherichia coli was injected intravaginally to establish a mixed bacterial vaginosis model. After successful modeling, as... Figure 7 On day 0, mice were observed to have redness and swelling of the vulva with a large amount of purulent discharge. The medication was then administered once daily for 7 consecutive days. Finally, vaginal wash fluid was collected for colony counting and microbial composition analysis. On day 8, the mice were euthanized, and their vaginas and internal organs were collected for pathological analysis. Figure 7A. In the model group, no significant changes were observed visually on days 3 and 8. The positive control group and PLE group showed no significant changes on day 3 of administration, but the swelling decreased significantly on day 8, although it was still significantly more swollen than in the normal group. The CP4 group showed significant changes on day 3, and by day 8, it had recovered to a level similar to the normal group. Histological staining analysis of vaginal tissue (…) Figure 7 B) In the model group, vaginal mucosal shedding was observed, with epithelial inflammatory cell infiltration and increased inflammatory factors in the muscle layer. Compared to the model group, the positive and PLE groups showed less mucosal hyperplasia and reduced epithelial inflammatory cell infiltration, but this was not significant. In contrast, the CP4 group showed significant mucosal hyperplasia and a significant reduction in epithelial inflammatory cell infiltration. This indicates that CP4 promotes vaginal mucosal repair. Histochemical analysis showed that after 7 days of continuous vaginal administration in each treatment group, major organs such as the heart, liver, spleen, lungs, and kidneys were not affected. Figure 7 C). To investigate the effect of CP4 on inflammation caused by a mixed bacterial culture of Candida albicans and Escherichia coli, the secretion of il-6 and TNF-α in vaginal tissue cells of mice in each group was measured. Figure 7 In mice treated with clotrimazole, PLE, and CP4, the production of IL-6 and TNF-α was significantly reduced, indicating that clotrimazole, PLE, and CP4 can all inhibit inflammation caused by mixed Candida albicans and Escherichia coli infections. However, the reduction in IL-6 and TNF-α was more pronounced in the CP4 group, with levels approaching those of the normal group. This suggests that CP4 has a better inhibitory effect on inflammation caused by mixed Candida albicans and Escherichia coli infections. Figure 8 A. Vaginal wash fluid was counted using the plate count method. It can be seen that the colony count in the model group did not change significantly on day 8. However, after 7 days of treatment with clotrimazole, PLE, and CP4, the colony count decreased significantly. Nevertheless, the positive and PLE groups still showed significant differences compared to the normal group, while the colony count in the CP4 group decreased to near the normal group. This indicates that CP4 is more effective in treating vaginitis caused by mixed Candida albicans and Escherichia coli infections. CP4 can significantly reduce the risk of recurrence of mixed vaginitis.

[0113] Evidence suggests that vaginitis is associated with vaginal microbiome dysbiosis, characterized by reduced biodiversity, a decreased proportion of Firmicutes, and an increased proportion of Proteobacteria. Therefore, this study investigated whether CP4 treatment could better modulate the composition of the vaginal microbiota in mice than clotrimazole treatment using Sobs and Shannon indices derived from 16S rRNA gene sequencing. Analysis of vaginal wash samples showed that CP4 treatment significantly improved fungal and bacterial diversity in mice with vaginitis compared to clotrimazole, as evidenced by an increase in the number of unique operational taxa. Figure 9A healthy woman's vaginal flora consists of a combination of aerobic and anaerobic bacteria, primarily lactobacilli. When pathogenic bacteria outnumber the healthy flora, an imbalance occurs, leading to vaginitis. In this study, the lactobacilli were *Lactobacillus* (a member of the Firmicutes phylum), while the *Escherichia coli* causing the dysbiosis were *Enterobacter* (a member of the Proteobacteria phylum). Sequencing analysis of the bacterial 16S rRNA genes revealed that, after 8 days of treatment, the vaginal bacterial richness and diversity in the CP4 group mice had returned to normal levels, and the bacterial composition was also close to that of normal mice. Figure 9 Further analysis at the phylum level showed that CP4 treatment significantly reduced the relative abundance of Proteobacteria and increased the relative abundance of Firmicutes, reshaping a healthier vaginal microbiota. At the genus level, the relative abundance of Lactobacillus significantly increased after CP4 administration (K, L). Figure 9 I), while the relative abundance of Enterobacteriaceae was significantly reduced ( Figure 9 Lactobacilli promote vaginal homeostasis and prevent the colonization and growth of harmful microorganisms. To further understand the effects of clotrimazole, PLE, and CP4 on vaginal microbiome function, the PIRCUSt2 pipeline based on 16S rRNA gene sequencing data was used to predict the vaginal bacterial functional profile. CP4 counteracted the decrease in vaginal bacterial evenness and changes in β-diversity caused by mixed Candida albicans and Escherichia coli. Figure 9 This invention focuses on the effects of CP4 on changes in the bacterial functional spectrum induced by a mixture of Candida albicans and Escherichia coli. To this end, it was observed that the estimated bacterial functional spectrum of diseased mice was separate from that of healthy mice in the control group, indicating that CP4 treatment induced significant changes in the vaginal bacterial functional spectrum. Notably, the functional spectrum of diseased mice treated with CP4 differed from that of diseased animals but was similar to that of healthy mice. Figure 9 (K, L), which is consistent with the hydrogel's ability to counteract bacterial dysbiosis. In conclusion, compared with clotrimazole, CP4 can increase the richness and diversity of the vaginal microbiota, effectively treat vaginitis, reduce recurrence rate, and shows promise for the treatment and prevention of vaginal dysbiosis.

[0114] The above experiments demonstrate that CP hydrogel has an antibacterial effect against pathogenic bacteria causing vaginitis. SEM, XRD, and FTIR methods confirmed that CMCS and PLE form a hydrogel through H-bond bonding, exhibiting a stable structure with a three-dimensional porous network, and possessing certain water absorption and degradability. With increasing PLE concentration, the CP hydrogel structure becomes increasingly stable; its swelling rate increases with increasing PLE content, while its degradation rate decreases with increasing PLE content. The antioxidant properties of CP hydrogel were explained using DPPH and ABTS, showing that higher PLE concentrations result in better antioxidant performance. Antibacterial experiments were conducted against aerobic vaginitis (AV) pathogens such as Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae (B-streptococcus), and Escherichia coli; vulvovaginitis (VVC) pathogens such as Candida albicans; and drug-resistant Staphylococcus aureus. The results showed that CMCS has a certain antibacterial effect, but the inhibition rate is low. With the addition of PLE, the inhibition rate increases with increasing PLE concentration, reaching over 90% for all bacteria at the highest PLE concentration. This invention also involved infecting the vagina of mice with a mixed bacterial solution of Candida albicans and Escherichia coli. Studies showed that CP4 had a certain therapeutic effect on vaginitis in mice infected with this mixed bacterial solution. The inhibition rate of CP4 on the vaginal flora of mice infected with this mixed bacterial solution reached 90%. This indicates that the hydrogel has a good antibacterial effect on the pathogens causing mixed vaginitis, laying the foundation for the treatment of vaginitis.

[0115] Experimental Example 2: Study on the in vitro antibacterial activity of peony leaf extract in synergy with carboxymethyl chitosan

[0116] 1. Reagent preparation

[0117] Carboxymethyl chitosan, peony leaf extract, and CP hydrogel were prepared into a solution with a concentration of 10 mg / mL (carboxymethyl chitosan: 1 mg; peony leaf extract: 0.15 mg) and stored at -20℃ for no more than 30 days.

[0118] 2. Minimum inhibitory concentration (MIC) determination

[0119] The nutrient agar dilution method was used to determine the in vitro antibacterial activity of peony leaf extract synergistically with carboxymethyl chitosan, primarily using *Escherichia coli* and *Staphylococcus aureus*. First, a small amount of *E. coli* and *S. aureus* was taken from the slant and placed in liquid culture medium, then shaken at 37°C and 200 rpm until the concentration reached 10⁸ CFU / mL, and then diluted to 10⁴ CFU / mL. In a 96-well plate, well 1 served as the negative control—200 μL of liquid culture medium; wells 2-11 served as 200 μL of antibacterial agent, serially diluted from top to bottom; and well 12 served as the positive control—200 μL of antibacterial agent. The 96-well plates were then incubated at 37°C for 18 hours; the clearest and most transparent wells represented the minimum inhibitory concentration (MIC).

[0120] 3. Determination of the FICI (Inhibitory Concentration Index)

[0121] The combined efficacy of peony extract and carboxymethyl chitosan was determined. The compound and antibacterial agent were serially diluted 2-fold in both the longitudinal (A to H) and transverse (2 to 11) directions of a two-dimensional checkerboard pattern on 96-well microplates. Based on the MIC values ​​of the compound and antibacterial agent against the tested strains, the compound was combined with the antibacterial agent at concentration gradients of 1 / 16 MIC, 1 / 8 MIC, 1 / 4 MIC, 1 / 2 MIC, MIC, 2 MIC, and 4 MIC along the y-axis from top to bottom, and along the x-axis from right to left. 100 μL of the drug solution was added to each well, followed by 100 μL of bacterial culture. Well (H, 1) with 100 μL of liquid culture medium and 100 μL of bacterial culture served as a positive control, and well (A, 9) with 200 μL of liquid culture medium served as a negative control. The plates were incubated at 37°C for 18 h. The interaction between antimicrobial drugs and compounds is determined according to the FICI (Functional Influence-Induced Compound) formula: FICI = (MIC compound in combination / MIC compound alone) + (MIC antimicrobial drug in combination / MIC antimicrobial drug alone). The interpretation criteria for FICI values ​​are: FICI ≤ 0.5 indicates a synergistic effect; 0.5 < FICI ≤ 1.0 indicates an additive effect; 1.0 < FICI ≤ 2.0 indicates an irrelevant effect; and FICI > 2.0 indicates an antagonistic effect.

[0122] Results analysis:

[0123] This study determined the interaction between peony leaf extract and carboxymethyl chitosan by testing their antibacterial properties against *Escherichia coli* and *Staphylococcus aureus*. Combined multidrug therapy is considered an effective method to address drug resistance in various bacteria in clinical practice. Due to repeated drug use, some strains become insensitive to single antibacterial agents; combining multiple drugs can induce synergistic effects to treat diseases caused by these drug-resistant bacteria. As shown in the table below, FICI tests revealed synergistic, additive, and antagonistic effects between carboxymethyl chitosan and peony leaf extract. This study, through the synergistic antibacterial effect of peony leaf extract and carboxymethyl chitosan against *Escherichia coli* and *Staphylococcus aureus*, indicated an additive effect between peony leaf extract and carboxymethyl chitosan, with FICI values ​​ranging from 0.5 to 1.0, suggesting a relatively strong synergistic effect between peony leaf extract and carboxymethyl chitosan.

[0124]

Claims

1. The use of a peony leaf extract and carboxymethyl chitosan in combination in the preparation of a medicament for treating vaginitis, wherein the peony leaf extract is a methanol extract of peony leaf; and wherein, The mass ratio of the peony leaf extract and carboxymethyl chitosan CMCS is (5-20):(100-150); the vaginitis is aerobic vaginitis or candidal vaginitis.

2. A hydrogel loaded with Paeonia leaf extract, characterized in that: It is prepared from carboxymethyl chitosan CMCS and peony leaf extract as raw materials, the peony leaf extract is peony leaf methanol extract, the mass ratio of the peony leaf extract and carboxymethyl chitosan CMCS is (5-20):(100-150); wherein, total flavonoids 25-35 mg / mL, total polyphenol 195-205 mg / mL.

3. The hydrogel of claim 2, wherein: The mass ratio of the peony leaf extract and carboxymethyl chitosan CMCS is 15:

100.

4. The use of the peony leaf extract-loaded hydrogel in the preparation of antibacterial drugs, drugs for treating vaginitis according to claim 2 or 3; the vaginitis is aerobic vaginitis or candidal vaginitis.

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