New application of coptis polysaccharide in preparation of medicine for preventing and treating candida albicans infectious diseases

Abstract

The invention belongs to the technical field of candida albicans treatment medicines, and particularly relates to a novel application of coptis chinensis polysaccharide in preparation of medicines for preventing and treating candida albicans infectious diseases. In a Candida albicans infected oropharyngeal candidiasis mouse model for the first time, it is found that after 50 mg / kg and 100 mg / kg of coptis polysaccharide are used for treatment for four days, the fungal loading capacity and inflammatory response in mouse tongue tissue are reduced, and the cure rate of mice is increased. It is shown that the coptis chinensis polysaccharide has a treatment effect on the oropharyngeal candidiasis, infected by candida albicans, of mice. Compared with antibiotic drugs, the coptis polysaccharide is an effective component of traditional Chinese medicine coptis, is not easy to generate drug resistance, has small toxic and side effects, is low in price, and has a treatment effect on mice with oropharyngeal candidiasis, so that the coptis polysaccharide can replace the antibiotic drugs and becomes a new drug for treating oropharyngeal candidiasis.

Description

Technical Field

[0001] This invention belongs to the field of therapeutic drugs for Candida albicans, and specifically relates to a novel use of Coptis chinensis polysaccharide in the preparation of drugs for the prevention and treatment of Candida albicans infectious diseases. Background Technology

[0002] Candida albicans is an opportunistic pathogenic yeast belonging to the genus Candida in the subphylum Deuteromycetes of the kingdom Fungi. It is widely distributed in nature and on the mucous membranes of the human body, including skin, mouth, digestive tract, and vagina, and is a component of the normal human flora. Its morphology combines a yeast phase (round or oval single-celled organism) and a hyphal phase (pseudomycetes or fungal hyphae). The yeast phase facilitates adhesion to mucous membrane surfaces and colonization, while the hyphal phase can invade tissues and cause inflammation. It produces virulence factors such as adhesins, phospholipases, and proteases, enhancing its adhesion and invasion capabilities to host cells and disrupting tissue barriers. It possesses multidrug resistance potential and can evade drug action through mechanisms such as gene mutation and biofilm formation, posing a challenge to clinical treatment.

[0003] Oropharyngeal candidiasis (OPC) is a mucosal candidiasis caused by Candida albicans infection of the oral and pharyngeal mucosa, and is one of the most common superficial fungal infections in clinical practice. After Candida albicans adheres to the epithelial cells of the oral mucosa, it proliferates to form a biofilm, releases virulence factors, and triggers an inflammatory response in the mucosa, manifesting as symptoms such as mucosal congestion, leukoplakia, pseudomembranes, and erosion. Weakened immunity (such as in HIV patients and cancer patients undergoing chemotherapy), oral mucosal damage (such as from oral surgery or dentures), and the abuse of broad-spectrum antibiotics leading to oral flora imbalance can all weaken the inhibitory effect on Candida albicans. The incidence rate in HIV patients can reach 50%-90%, and it is often the first symptom. It not only causes discomfort such as oral pain, difficulty swallowing, and abnormal taste, but in severe cases, it can spread to the esophagus causing esophageal candidiasis, and even spread through the bloodstream to cause systemic candidiasis, endangering life; at the same time, it reduces the patient's quality of life and increases the medical burden.

[0004] Treatment for oropharyngeal candidiasis primarily involves antifungal medications. Currently, clinically used drugs mainly include azoles, polyenes, and echinocandins. Azole drugs, developed and marketed gradually since the 1970s, are currently first-line treatments for oropharyngeal candidiasis, with advantages including good oral absorption, strong tolerability, and a broad antibacterial spectrum. Azole drugs inhibit key enzymes in fungal cell membrane synthesis, blocking the synthesis of ergosterol, a major component of the cell membrane, leading to increased fungal cell membrane permeability, cell rupture, and death. Fluconazole is the first-line azole for mild to moderate OPC. Polyene antifungals were among the earliest antifungal drugs used clinically and are widely used in topical formulations for mucosal infections. They bind directly to ergosterol on the fungal cell membrane, forming transmembrane channels, causing cell membrane permeability disturbances, leakage of intracellular substances, and fungal cell death. Representative drugs include nystatin and amphotericin B. Echinocandins are a new type of antifungal drug developed after the 1990s. They target azole-resistant strains or severe infections by inhibiting key enzymes in fungal cell wall synthesis, leading to cell wall defects, loss of morphology, and rupture and death under osmotic pressure. They also have low toxicity to humans, thus filling a gap in the treatment of drug-resistant strains. A representative drug is caspofungin, commonly used for moderate to severe oropharyngeal candidiasis or fluconazole-resistant cases. However, while polyene antibiotics have clear antifungal effects, they are highly irritating to the mucous membranes. Echinocandins have significant bactericidal effects against Candida, but intravenous administration is limited, and they are expensive, making azoles still the first-line antifungal drugs in clinical practice.

[0005] However, with the long-term use of azole drugs in oral mucosal infections, the detection rate of drug-resistant Candida albicans strains has been increasing year by year. The emergence of multidrug-resistant strains, exhibiting cross-resistance to multiple azole drugs such as fluconazole and itraconazole, poses a significant challenge to the treatment of refractory infections. The resistance rate to fluconazole, a first-line drug, has increased significantly; in HIV patients and those on long-term antibiotic or hormone therapy, the resistance rate can reach 10%-30%, and is even higher in some severely ill patients. Infections caused by drug-resistant strains lead to decreased efficacy of conventional drugs, increased risk of adverse reactions, and higher disease recurrence rates, severely impacting patients' quality of life and causing considerable inconvenience. Especially with the increase in patients undergoing radiotherapy and chemotherapy, those on long-term glucocorticoid therapy, and those with weakened immune systems, the incidence of oropharyngeal candidiasis continues to rise, making the clinical need for antifungal drugs increasingly urgent. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a novel use of Coptis chinensis polysaccharide in the preparation of drugs for the prevention and treatment of Candida albicans infections. This invention is the first to discover that Coptis chinensis polysaccharide exhibits excellent therapeutic effects against oropharyngeal candidiasis caused by Candida albicans infection, with low likelihood of developing drug resistance and minimal toxic side effects, making it a promising new drug for the treatment of oropharyngeal candidiasis.

[0007] On the one hand, the present invention provides the use of Coptis chinensis polysaccharide in the preparation of medicaments for the prevention and / or treatment of diseases caused by Candida albicans infection.

[0008] In some implementations, depending on the site and severity of infection, diseases caused by Candida albicans include, but are not limited to: oral candidiasis, candidal vaginitis, candidal paronychia, skin diseases such as chronic mucocutaneous candidiasis, and gastrointestinal candidiasis, preferably oropharyngeal candidiasis. In the treatment of this disease, Coptis chinensis polysaccharide can reduce damage to the tongue surface by improving the inflammatory response of the tongue tissue and reducing the Candida albicans load on the tongue tissue.

[0009] In some implementation schemes, Coptis chinensis refers to a plant belonging to the genus Coptis in the family Ranunculaceae, whose dried rhizome has been used as a traditional Chinese medicine for over two thousand years. Coptis chinensis contains various active ingredients, including alkaloids (such as berberine), polysaccharides, and flavonoids. Coptis chinensis polysaccharide is a water-soluble heteropolysaccharide extracted from the rhizome of the medicinal herb Coptis chinensis. It is mainly composed of monosaccharides such as glucose, galactose, and arabinose, possessing a specific molecular weight and branched structure, and is the core active ingredient of Coptis chinensis besides alkaloids. The preparation method of Coptis chinensis polysaccharide includes: extracting Coptis chinensis with water to obtain an extract; concentrating the extract to obtain a concentrate; adding an alcohol solvent to the concentrate for alcohol precipitation, followed by solid-liquid separation and collection of the liquid to obtain a crude polysaccharide solution; and purifying the crude polysaccharide solution to obtain Coptis chinensis polysaccharide. Preferably, after removing the roots of Coptis chinensis, it is soaked in cold water overnight, and then decocted and extracted 1-3 times. The material-to-liquid ratio for each extraction is 1kg:5-7L, for example, 1kg:5L, 1kg:6L, or 1kg:7L. The extraction temperature is 90-100℃, for example, 90℃, 95℃, or 100℃, and the extraction time is 1-3 hours, for example, 1 hour, 1.5 hours, 2 hours, 2.5 hours, or 3 hours. Preferably, the alcohol solvent for alcohol precipitation is a C1-C3 lower alcohol, preferably ethanol, and the concentration of the alcohol solvent is 80-90% v / v, for example, 80% v / v, 85% v / v, or 90% v / v. The volume ratio of the concentrated liquid to the alcohol solvent is 1:7-9, for example, 1:7, 1:8, or 1:9. Preferably, conventional deproteinization and dialysis can be used for purification. For example, the sevage method (chloroform and n-butanol in a volume ratio of 3 to 5:1) can be used to remove proteins from the crude polysaccharide, and the polysaccharide solution can be transferred into a dialysis bag and dialyzed with flowing water for 40 to 50 hours, with a molecular weight cutoff of 3500 Da.

[0010] In some embodiments, the drug of the present invention is a preparation made of an effective amount of Coptis chinensis polysaccharide as the main or only active ingredient and pharmaceutically acceptable excipients. The dosage form of the excipients and the preparation is not specifically limited. The dosage form of the preparation can be any pharmaceutically acceptable dosage form, including but not limited to any one of solution, granules, tablets, capsules, pills or powders, preferably a solution. For example, Coptis chinensis polysaccharide is mixed and dissolved in pure water to prepare a solution of 50~100μg / mL.

[0011] In some embodiments, to achieve the intended therapeutic effect and enhance the antibacterial efficacy, the drug of the present invention can be administered using any known method of administration, including but not limited to oral administration and injection administration, preferably oral administration. The dosage, depending on the desired antibacterial effect, the nature and severity of the disease to be treated, the individual circumstances of the patient or animal, the route of administration, and the dosage form, can be 50-100 mg / kg / day in mice. -1 The dosage range is converted to human dosage. Furthermore, the Coptis chinensis polysaccharide of this invention can be taken alone or in combination with other therapeutic or symptomatic drugs. When the Coptis chinensis polysaccharide of this invention is used in combination with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

[0012] The beneficial effects of this invention are as follows: This invention, for the first time, demonstrated in a mouse model of oropharyngeal candidiasis caused by Candida albicans that treatment with 50 mg / kg and 100 mg / kg of Coptis chinensis polysaccharide for four days reduced fungal load and inflammatory response in the tongue tissue of mice, and increased the cure rate. This indicates that Coptis chinensis polysaccharide has a therapeutic effect on oropharyngeal candidiasis caused by Candida albicans in mice. Compared with antibiotics, Coptis chinensis polysaccharide is an effective component of the traditional Chinese medicine Coptis chinensis, less prone to drug resistance, has fewer toxic side effects, is inexpensive, and has a therapeutic effect on oropharyngeal candidiasis in mice. Therefore, Coptis chinensis polysaccharide may replace antibiotics as a new drug for the treatment of oropharyngeal candidiasis. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1A UPLC-MS / MS spectra of metabolites of Coptis chinensis polysaccharide (substances 1-14); Figure 1B UPLC-MS / MS spectra of metabolites of Coptis chinensis polysaccharide (substances 15-16). Figure 2A The overall experimental flowchart for each group of mice; Figure 2B Fungal smears were prepared from the tongue tissue of mice on days 1-5 after treatment with Coptis chinensis polysaccharide. Figure 2C Image of mouse tongue tissue on day 5 after treatment with Coptis chinensis polysaccharide; Figure 2D The colonization rate of fungi on the tongue surface of mice in each group; Figure 2E Results of tongue tissue damage in each group of mice; Figure 2F The results of tongue histological scoring for each group of mice; Figure 2G Photographs of HE-stained tongue tissue from each group of mice; Figure 3A The results of ELISA detection of IL-1β content in the tongue tissue of mice in each group; Figure 3B The results of ELISA detection of TNF-α content in the tongue tissue of mice in each group; Figure 3C Results of ELISA detection of IL-6 content in the tongue tissue of mice in each group; Figure 3D Results of ELISA detection of IL-10 content in the tongue tissue of mice in each group; Figure 3E The results of ELISA detection of IL-17A content in the tongue tissue of mice in each group; Figure 3F Results of ELISA detection of CXCL1 content in tongue tissue of mice in each group; Figure 3G Results of ELISA detection of CCL2 content in the tongue tissue of mice in each group; Figure 3H To detect PI3K mRNA levels using RT-qPCR; Figure 3I To detect AKT mRNA levels using RT-qPCR. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Example 1: Preparation of Coptis chinensis polysaccharide Take 1 kg of Coptis chinensis, remove the roots, and soak in 6 L of water overnight. The next day, decoct the soaked herbs for 2 hours, starting from a gentle boil and maintaining a gentle boil. After decoction, filter through gauze to obtain filtrate and residue. The residue is then soaked in water and decocted again; this process is repeated three times. Filter again, combine the filtrates, and concentrate the combined filtrate to 1 L using a water bath; at this point, it will be viscous. Add 8 L of 95% v / v ethanol to soak and remove pigments. After soaking, pour off the supernatant and continue adding ethanol to the precipitate. Repeat this process three times until the supernatant becomes lighter in color. After removing the pigments, continue soaking the precipitate in 8 L of 80% v / v ethanol; repeat this process twice to obtain crude polysaccharide. Use the Sevage method (chloroform to n-butanol volume ratio 4:1) to remove protein from the crude polysaccharide. After the protein was removed, the polysaccharide solution was transferred into a dialysis bag and dialyzed with flowing water for 48 hours to obtain purified Coptis chinensis polysaccharide.

[0017] Example 2 Metabolomics Analysis of Coptis chinensis Polysaccharides 1. Dry sample extraction (1) The Coptis chinensis polysaccharide prepared in Example 1 was placed in a freeze dryer and freeze-dried under vacuum for 63 hours; (2) Grind the powder using a grinder (30 Hz, 1.5 minutes); (3) Weigh 30 mg of sample powder using an electronic balance, add 1500 μL of 70% methanol-water internal standard extraction solution pre-cooled at -20 ℃. The internal standard extraction solution is prepared by dissolving 1 mg of standard (2-chlorophenylalanine; purity: 98%) in 1 mL of 70% methanol-water to prepare a 1000 μg / mL standard stock solution. The 1000 μg / mL standard stock solution is further diluted with 70% methanol to a 250 μg / mL internal standard extraction solution. (4) Vortex once every 30 minutes, each lasting 30 seconds, for a total of 6 vortices; (5) After centrifugation (12000 rpm, 3 minutes), aspirate the supernatant, filter the sample with a microporous membrane (0.22 μm pore size), and store it in a sample vial for UPLC-MS / MS analysis.

[0018] 2. Chromatographic and mass spectrometric acquisition conditions The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (UPLC) and tandem mass spectrometry (MS / MS).

[0019] 2.1 Liquid phase conditions mainly include (1) Column: Agilent SB-C18 1.8 µm, 2.1 mm × 100 mm; (2) Mobile phase: Phase A is ultrapure water (with 0.1% formic acid added), and Phase B is acetonitrile (with 0.1% formic acid added). (3) Elution gradient: The proportion of phase B is 5% at 0.00 min, and increases linearly to 95% within 9.00 min and is maintained at 95% for 1 min. From 10.00 to 11.00 min, the proportion of phase B decreases to 5% and is balanced at 5% until 14 min. (4) Flow rate 0.35 mL / min; column temperature 40°C; injection volume 2 μL.

[0020] 2.2 Mass spectrometry conditions mainly include Electrospray ionization (ESI) was performed at 500°C; the ion spray voltage (IS) was 5500 V (positive ion mode) / -4500 V (negative ion mode); ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set to 50, 60, and 25 psi, respectively, and the collision-induced ionization parameter was set to high. QQQ scans were performed using MRM mode with the collision gas (nitrogen) set to medium. The DP and CE of each MRM ion pair were optimized through further declustering potential (DP) and collision energy (CE). A specific set of MRM ion pairs was monitored at each epoch based on the metabolites eluted within each epoch.

[0021] 3. Principles of Qualitative and Quantitative Analysis of Metabolites Based on the self-built database MWDB, the substances were qualitatively identified according to secondary spectral information. Isotope signals were removed during the analysis, including those containing K. + Na + NH4 + The repeating signals, as well as the repeating signals of fragment ions that are themselves other substances with larger molecular weights.

[0022] Metabolite quantification was performed using triple quadrupole mass spectrometry in multiple reaction monitoring (MRM) mode. In MRM mode, the quadrupole first screens for precursor ions (parent ions) of the target substance, excluding ions corresponding to other molecular weight substances to initially eliminate interference. After the precursor ions are induced to ionize in the collision chamber, they break into many fragment ions. These fragment ions are then filtered by the triple quadrupole to select a characteristic fragment ion, eliminating interference from non-target ions, making the quantification more accurate and reproducible. After obtaining the metabolite mass spectrometry data of different samples, the peak areas of all chromatographic peaks are integrated, and the mass spectrometric peaks of the same metabolite in different samples are integrated and corrected.

[0023] 4. Conclusion This study performed metabolomics analysis on Coptis chinensis polysaccharide samples using a UPLC-MS / MS detection platform combined with a self-built metabolite database. A total of 1023 metabolites were detected, including 16 carbohydrate metabolites, such as... Figures 1A-1B As shown, this confirms that Coptis chinensis polysaccharide is rich in carbohydrate metabolites, and that the detection method can effectively achieve the separation and qualitative and quantitative analysis of carbohydrate substances.

[0024] Example 3: Experimental study on the therapeutic effect of Coptis chinensis polysaccharide on oropharyngeal candidiasis 1. Experimental Materials 1.1 Experimental drug: Coptis chinensis polysaccharide prepared in Example 1.

[0025] 1.2 Experimental animals: SPF-grade 8-week-old female C57BL / 6 mice, weighing 18-20g, were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd., and housed in an SPF animal room at 22±2℃ and 50%±5% humidity. They were acclimatized for 7 days before the experiment.

[0026] 1.3 Strains: Candida albicans SC5314.

[0027] 2. Experimental Methods 2.1 OPC Model Construction and Animal Grouping: On days -1, +1, and +3 after Candida albicans SC5314 infection, mice were intraperitoneally injected with 225 mg / kg cortisone acetate for immunosuppression. On the day of infection (day 0), mice were anesthetized by intraperitoneal injection of 100 mg / mL urethane, and then subjected to OPC model construction and animal grouping. 6A calcium alginate swab (cells / mL) was placed in the entire oral cavity for 75 minutes for inoculation. After successful modeling, a white, curd-like pseudomembrane appeared on the oral mucosa of mice. The pseudomembrane was difficult to peel off, and after peeling, congestion, erosion, or ulceration could be seen on the underlying mucosa; accompanied by lethargy and decreased food intake. The successfully modeled mice were randomly divided into 4 groups: model group, low-dose Coptis chinensis polysaccharide group, high-dose Coptis chinensis polysaccharide group, and positive control group, with 10 mice in each group. An equal number of normal mice were used as a blank control group.

[0028] 2.2 Administration Method: Administer the medication on days +1 to +4 post-infection. All medications were administered by gavage once daily for 4 consecutive days. The low-dose and high-dose groups of Coptis chinensis polysaccharide were given 50 mg / kg and 100 mg / kg of Coptis chinensis polysaccharide, respectively. The positive control group received 10 mg / kg of fluconazole. The blank control group and model group received an equal volume of sterile distilled water. The overall experimental flowchart for each group of mice is shown below. Figure 2A As shown. Furthermore, for four days after Candida albicans infection, the surface damage of the mouse tongue mucosa was observed and photographed daily, and the fungal load in the mouse oral cavity was detected using calcium alginate swabs. On day +5 post-infection, the mice were euthanized, and tongue tissue was removed for testing of tongue surface damage, fungal load, and tissue inflammation.

[0029] 3. Experimental Results 3.1 Fungal load: The fungal load in the model group was significantly higher than that in the blank control group; the fungal load in the low-dose Coptis chinensis polysaccharide group, the high-dose Coptis chinensis polysaccharide group, and the positive control group was significantly reduced, and the reduction was dose-dependent. Figure 2B , 2D As shown.

[0030] 3.2 Pathological changes: In the model group, severe mucosal epithelial necrosis and inflammatory infiltration were observed; in the Coptis chinensis polysaccharide group, mucosal damage was reduced, and the number of fungi decreased, with more significant improvement observed in the high-dose group. Figure 2C , 2E As shown in 2F and 2G.

[0031] 3.3 Expression of inflammatory factors: The protein expression of pro-inflammatory factors IL-1β, TNF-α, IL-6, CXCL1, and IL-17A was significantly downregulated in the Coptis chinensis polysaccharide group, while the protein expression of anti-inflammatory factor IL-10 was significantly upregulated. The protein expression of macrophage-related cytokine CCL2 was significantly upregulated, and the mRNA expression of macrophage apoptosis-related genes PI3K and AKT was significantly upregulated in a dose-dependent manner. P <0.05), such as Figures 3A-3I As shown.

[0032] 4. Experimental Conclusions Coptis polysaccharide can reduce the fungal load in the oral mucosa of model animals and alleviate tissue pathological damage, thus showing excellent therapeutic effects. The overall therapeutic effect is close to that of positive drugs. It may exert its therapeutic effect by regulating the expression of inflammatory factors, inhibiting macrophage apoptosis, and promoting macrophage phagocytosis.

[0033] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of Coptis chinensis polysaccharide in the preparation of drugs for the prevention and / or treatment of diseases caused by Candida albicans infection.

2. The application according to claim 1, characterized in that, The disease described is oropharyngeal candidiasis.

3. The application according to claim 2, characterized in that, The aforementioned drug is used to improve inflammatory responses in the tongue tissue.

4. The application according to claim 2, characterized in that, The drug described is one that reduces the Candida albicans load on the tongue tissue.

5. The application according to claim 1, characterized in that, The preparation method of Coptis chinensis polysaccharide includes: extracting Coptis chinensis with water, performing solid-liquid separation after the extraction, collecting the liquid portion of the solid-liquid separation to obtain an extract; concentrating the extract to obtain a concentrate; adding an alcohol solvent to the concentrate for alcohol precipitation, performing solid-liquid separation after the alcohol precipitation, collecting the liquid portion of the solid-liquid separation to obtain a crude polysaccharide solution; and purifying the crude polysaccharide solution to obtain the Coptis chinensis polysaccharide.

6. The application according to claim 5, characterized in that, The extraction material-to-liquid ratio is 1 kg: 5-7 L, the extraction temperature is 90-100℃, and the extraction time is 1-3 h; the alcohol solvent is 80-90% v / v C1-C3 lower alcohol, and the volume ratio of the concentrate to the alcohol solvent is 1:7-9; the purification includes sequential deproteinization and dialysis.

7. The application according to claim 1, characterized in that, The drug is a preparation made with an effective amount of Coptis chinensis polysaccharide as the main or sole active ingredient and pharmaceutically acceptable excipients.

8. The application according to claim 7, characterized in that, The formulation is any pharmaceutically acceptable dosage form.

9. The application according to claim 8, characterized in that, The formulation is a solution.

10. The application according to claim 9, characterized in that, The concentration of Coptis chinensis polysaccharide in the solution is 50~100μg / mL.

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