Application of chaenomeles speciosa extract polyphenol in preparation of medicine for treating osteoarthritis

Abstract

This invention provides the application of wrinkled papaya polyphenols in the preparation of drugs for treating osteoarthritis. The wrinkled papaya polyphenols are used in the preparation of drugs that reduce the release of pro-inflammatory cytokines and the inflammatory mediator NO in primary chondrocytes, and / or reduce the mRNA expression of inflammation-related enzymes, and / or inhibit the phosphorylation of proteins related to the NF-κB signaling pathway. To test and clarify the anti-inflammatory effect of wrinkled papaya polyphenols, an ADTC5 primary chondrocyte inflammation model was constructed for observation. The results showed that wrinkled papaya polyphenols significantly inhibited IL-1β-induced inflammation in ADTC5 primary chondrocytes, effectively reduced the secretion of pro-inflammatory cytokines (IL-6 and TNF-α), inhibited the release of the inflammatory mediator NO, downregulated the mRNA expression of inflammatory enzymes (iNOS and Cox-2), and reduced the phosphorylation of IκBα and p65 proteins.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, specifically to the application of polyphenols extracted from wrinkled papaya in the preparation of drugs for treating osteoarthritis. Background Technology

[0002] Osteoarthritis (OA) is a chronic degenerative joint disease with complex etiologies. Its main pathological feature is the destruction and degeneration of articular cartilage, and its main clinical symptoms are joint pain and limited movement. It commonly affects joints such as the knee, hip, hand, and foot, and is prevalent in obese elderly individuals, posing a significant threat to the health of middle-aged and elderly people. The onset of this disease is mainly related to age, obesity, and genetic factors. The pathogenesis of OA is not yet fully understood, but the main cause is inflammatory damage to articular cartilage cells, specifically manifested as high secretion of pro-inflammatory cytokines, high expression of inflammatory enzymes, and high phosphorylation of proteins related to the NF-κB signaling pathway. Therefore, there is ample reason to believe that reducing inflammatory damage to articular cartilage cells while simultaneously exerting a chondrocyte-protective effect significantly enhances the efficacy of preventing or treating osteoarthritis.

[0003] Currently, common clinical treatments for osteoarthritis include surgical and non-surgical approaches. Non-surgical treatment primarily involves medication to relieve symptoms, but there is no specific cure. Common medications for osteoarthritis include nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and anesthetics / sedatives. While these medications are widely used clinically, long-term use can lead to significant side effects and impair bodily function. Therefore, a long-term, non-toxic medication is needed to replace these classic drugs in the clinical treatment of osteoarthritis.

[0004] Papaya, belonging to the genus Chaenomeles in the Rosaceae family, includes varieties such as wrinkled-skin papaya, smooth-skin papaya, and hairy-leaved papaya. All of these varieties are widely cultivated in my country, primarily in the central regions such as Hubei and Anhui provinces, and have a long history of cultivation. Wrinkled-skin papaya, also known as Zi papaya, is the dried fruit of the Chaenomeles speciosa. It has a fragrant and sour taste, is slightly cold in nature, and contains various active ingredients such as polyphenols, flavonoids, polysaccharides, saponins, organic acids, and terpenoids. Polyphenols are the most effective active ingredient in papaya, and the polyphenol content in wrinkled-skin papaya is significantly higher than in other species. Therefore, wrinkled-skin papaya is often used as the highest quality raw material for extracting papaya polyphenols. Wrinkled-skin papaya is associated with the spleen, lung, liver, and kidney meridians. Its polyphenol extract has the effects of resolving dampness, eliminating phlegm, relieving pain, reducing swelling, and relaxing muscles in traditional Chinese medicine. Furthermore, as a natural plant extract, its polyphenol extract has relatively few toxic side effects, low drug residues, and high safety. To date, there are no reports on the prevention or treatment of osteoarthritis using wrinkled-skin papaya extract polyphenols. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides the application of wrinkled papaya polyphenols in the preparation of drugs for treating osteoarthritis. Wrinkled papaya polyphenols have a good inhibitory effect on osteoarthritis and can be used to prepare drugs for treating osteoarthritis.

[0006] The technical solution of the present invention is the application of wrinkled papaya polyphenols in the preparation of drugs for treating osteoarthritis.

[0007] The first objective of this invention is to provide the application of wrinkled papaya polyphenols in the preparation of drugs for treating osteoarthritis.

[0008] The application of the wrinkled papaya polyphenols in the preparation of drugs that reduce the release of pro-inflammatory cytokines and the inflammatory mediator NO in primary chondrocytes, and / or reduce the mRNA expression of inflammation-related enzymes, and / or inhibit the phosphorylation of proteins related to the NF-κB signaling pathway.

[0009] The pro-inflammatory cytokines mentioned are TNF-α and IL-1, the inflammation-related enzymes are iNOS and Cox-2, and the NF-κB signaling pathway-related proteins are IκBα and p65.

[0010] The wrinkled papaya polyphenols mentioned above are obtained by extracting wrinkled papaya powder through ethanol solution soaking, drying, and then enzymatic hydrolysis with cellulase.

[0011] The ethanol solution has a mass concentration of 95% or higher.

[0012] The amount of cellulase added is 1.0~2.0% of the mass of the dry powder obtained after ethanol extraction of wrinkled papaya polyphenols; the enzymatic hydrolysis temperature is 50-60℃, the enzymatic hydrolysis pH is 4.5-6.0, and the enzymatic hydrolysis time is 2-4h.

[0013] A second object of the present invention is to provide a medicament for treating osteoarthritis, wherein the medicament uses papaya polyphenols as the active ingredient and contains all pharmaceutically acceptable excipients or carriers.

[0014] Furthermore, the drug dosage form is often a suspension, capsule, tablet, pill, powder, injection, aerosol, or other pharmaceutically acceptable dosage form. Generally, oral medications are often solid preparations such as capsules, tablets, pills, and powders, or liquid preparations such as suspensions and syrups; nasal inhalation medications are often gaseous preparations such as aerosols; intravenous and intramuscular injections are often liquid preparations such as suspensions and injections.

[0015] The present invention has the following advantages:

[0016] IL-1β, TNF-α, and IL-6 are common inflammatory factors, and IL-1β has been reported to be highly expressed in animals and humans with osteoarthritis. IL-1β can stimulate the high secretion of pro-inflammatory cytokines (TNF-α and IL-6) and the inflammatory mediator NO in primary chondrocyte ADTC5 cells, and upregulate the mRNA expression of inflammatory enzymes (iNOS and Cox-2), while promoting the phosphorylation of IκBα and p65 proteins, causing inflammatory damage to primary chondrocytes, thereby establishing an in vitro osteoarthritis model. Therefore, we used IL-1β to stimulate primary chondrocyte ADTC5 cells to construct an in vitro osteoarthritis model.

[0017] Currently, common clinical treatments for osteoarthritis include surgical and non-surgical approaches. Non-surgical treatment primarily involves medication to relieve symptoms, but there is no specific cure. Common medications for osteoarthritis include nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and anesthetics / sedatives. While these medications are widely used clinically, long-term use can lead to significant side effects and impair bodily function. Therefore, a long-term, non-toxic drug is needed to replace classic medications for treating osteoarthritis. Current research confirms that *Papaya pudding* polyphenols, a novel drug with no long-term toxicity and drug residues, exerts a good anti-inflammatory effect by reducing the release of pro-inflammatory cytokines (IL-6 and TNF-α) and the inflammatory mediator NO in ADTC5 primary chondrocytes, downregulating the mRNA expression of inflammatory enzymes (iNOS and Cox-2), and inhibiting the phosphorylation of IκBα and p65 proteins, thereby further alleviating osteoarthritis symptoms.

[0018] To date, there are no reports on the use of polyphenols from wrinkled papaya extract in the prevention or treatment of osteoarthritis.

[0019] To test and clarify the anti-inflammatory effects of papaya polyphenols, this invention constructed an ADTC5 primary chondrocyte inflammation model for observation. The results showed that papaya polyphenols significantly inhibited IL-1β-induced inflammation in ADTC5 primary chondrocytes, effectively reducing the secretion of pro-inflammatory cytokines (IL-6 and TNF-α), inhibiting the release of the inflammatory mediator NO, downregulating the mRNA expression of inflammatory enzymes (iNOS and Cox-2), and reducing the phosphorylation of IκBα and p65 proteins. Attached Figure Description

[0020] Figure 1 This is the hydrogen spectrum of the polyphenols extracted from the wrinkled papaya extract obtained in Example 1.

[0021] Figure 2 This is a graph showing the effect of polyphenols from the wrinkled papaya extract on cell viability in Example 2.

[0022] Figure 3This is a graph showing the effect of different concentrations of IL-1β on NO release from ADTC5 cells in Example 3.

[0023] Figure 4 This is a graph showing the effect of polyphenols extracted from wrinkled papaya on IL-1β-induced NO release from ADTC5 cells in Example 4; among them, compared with the blank control group, ####P<0.0001; compared with the model group, *P<0.05, ****P<0.0001.

[0024] Figure 5 This is a graph showing the effect of polyphenols extracted from wrinkled papaya on IL-1β-induced TNF-α release from ADTC5 cells in Example 5; where, compared with the blank control group, ####P<0.0001; compared with the model group, ****P<0.0001.

[0025] Figure 6 This is a graph showing the effect of polyphenols from the wrinkled papaya extract on IL-1β-induced IL-6 release from ADTC5 cells in Example 5; among them, compared with the blank control group, ####P<0.0001; compared with the model group, ***P<0.005, ****P<0.0001.

[0026] Figure 7 This is a graph showing the effect of polyphenols from the extract of Papaya pudding on IL-1β-induced iNOS mRNA expression in ADTC5 cells in Example 6; among them, compared with the blank control group, ####P<0.0001; compared with the model group, ***P<0.005, ****P<0.0001.

[0027] Figure 8 This is a graph showing the effect of polyphenols from the extract of Papaya pudding on IL-1β-induced Cox-2 mRNA expression in ADTC5 cells in Example 6; where, compared with the blank control group, ####P<0.0001; compared with the model group, **P<0.01, ****P<0.0001.

[0028] Figure 9 This is a graph showing the results of the analysis of the effect of polyphenols from the wrinkled papaya extract on the phosphorylated p65 (p-p65) protein level induced by IL-1β in ADTC5 cells in Example 7; among them, compared with the blank control group, ####P<0.0001; compared with the model group, ***P<0.005, ****P<0.0001.

[0029] Figure 10This is a graph showing the results of the analysis of the effect of polyphenols from the wrinkled papaya extract on the level of phosphorylated IκBα (p-IκBα) protein induced by IL-1β in ADTC5 cells in Example 7; among them, compared with the blank control group, ####P<0.0001; compared with the model group, **P<0.01, ****P<0.0001. Detailed Implementation

[0030] The present invention will be described in detail below with reference to the embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the present invention.

[0031] Example 1: Preparation and Extraction of Polyphenols from Wrinkled-Skin Papaya

[0032] Preparation of wrinkled papaya powder: Wrinkled papayas harvested from Changyang County, Yichang City, Hubei Province were dried at 40℃ for 12 hours in an electric heating drying oven, then pulverized by a pulverizer and passed through a 50-mesh sieve to obtain wrinkled papaya powder for later use.

[0033] Degreasing of wrinkled papaya powder: Take 50.00 g of wrinkled papaya powder, add 95% ethanol at a material-to-liquid ratio of 1:10, reflux for 2 h, filter to remove ethanol, dry and weigh to obtain 35.0 g of degreased wrinkled papaya powder.

[0034] Enzymatic extraction of polyphenols from wrinkled papaya: Weigh 5.0 g of the defatted wrinkled papaya powder, add distilled water at a material-to-liquid ratio of 1:30, adjust the pH to 5, add 1.5% cellulase, and enzymatically hydrolyze at 52°C for 2.5 h. Then raise the temperature to 80°C to inactivate the enzyme, cool, and filter to obtain the filtrate. The obtained product is the wrinkled papaya polyphenol extract (and the following examples were performed). The polyphenol content obtained by this method was 150.48 mg / g, with a yield of 15.05%. Figure 1 The extracted polyphenol 1H NMR spectrum is shown below. The chemical formula of the polyphenol extracted in this example is verified as follows:

[0035]

[0036] Formula 1.

[0037] Example 2: Detection of the effect of polyphenols from the extract of wrinkled papaya on the activity of ADTC5 cells using the CCK8 assay.

[0038] ADTC5 primary chondrocytes were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and placed in a 37°C cell culture incubator with 5% CO2. The medium was changed daily, and the cells were passaged every 1-2 days. The incubator was sporulated at a rate of 1×10⁻⁶ cells / year. 4Primary ADTC5 chondrocytes were seeded at 100 cells / well in 96-well plates, with three replicates per group. ADTC5 primary chondrocytes were treated with basal medium containing 0, 5, 10, 20, 40, 50, or 100 μg / mL of papaya polyphenol extract. After 24 h of cell culture, 10 µL of CCK-8 solution was added to the medium, and the cells were incubated at 37°C for 1 h. The absorbance was then measured at 450 nm using a microplate reader. Cell viability was calculated using the following formula:

[0039] Cell viability (%) = (OD) 实验组 -OD 空白组 / OD 对照组 -OD 空白组 )×100

[0040] ADTC5 primary chondrocytes were treated with different concentrations of papaya extract polyphenols (structural formula shown in Formula 1) for 24 hours. The CCK8 assay results are as follows: Figure 2 As shown, when the concentration of papaya polyphenols was below 40 μg / mL, the cell survival rate was above 95%, and no obvious cytotoxicity was observed; when the concentration was above 40 μg / mL, the cell survival rate decreased significantly by about 20%, and it had a strong toxic effect on ADTC5 primary chondrocytes. Figure 2 Therefore, in subsequent experiments, the polyphenols from the wrinkled papaya extract of Example 1 at concentrations of 5, 10, and 20 μg / mL were selected.

[0041] Example 3

[0042] An osteoarthritis injury model was established by treating ADTC5 primary chondrocytes with IL-1β.

[0043] With 1×10 4 ADTC5 cells were seeded per well in 96-well plates and cultured for 24 h in a 37°C cell culture incubator with 5% CO2. Three replicates were then set up for each group. ADTC5 cells were treated with different concentrations of IL-1β (3, 6, 9, 12, 15, 18, and 21 ng / ml), while the control group received an equal volume of basal medium. After 12 h of culture, NO release was measured using a Griess reagent kit. The NO production levels in each group were compared to determine the optimal induction concentration.

[0044] After treating ADTC5 primary chondrocytes with different concentrations for 12 hours, NO release was measured, and the results are as follows: Figure 3As shown, when the IL-1β concentration was below 12 ng / mL, NO release increased with increasing concentration; when the IL-1β concentration was above 12 ng / mL, excessive IL-1β damaged ADTC5 primary chondrocytes, resulting in a significant decrease in NO release. Furthermore, compared with the control group without IL-1β treatment, treatment with 12 ng / mL IL-1β significantly increased NO release from ADTC5 cells. Figure 3 Based on the above experimental results, it is demonstrated that 12 ng / mL IL-1β is the optimal induction concentration for the ADTC5 cell inflammatory injury model.

[0045] Example 4

[0046] Effects of polyphenols from wrinkled papaya extract on NO release from ADTC5 cells

[0047] With 1×10 4 Cells were seeded per well in 96-well plates and cultured for 24 h in a 37°C cell culture incubator with 5% CO2. Three replicates were set up for each group. The drug groups were treated with 5, 10, and 20 μg / mL of papaya extract polyphenols (each group also received 12 ng / mL IL-1β). The model group received 12 ng / mL IL-1β, and the control group received an equal volume of basal culture medium. After 12 h of culture, the supernatant was collected, and NO release was measured using a Griess assay kit. NO production was compared among the groups.

[0048] After treating ADTC5 cells with different concentrations of papaya polyphenols (structural formula shown in Formula 1) for 24 hours, the NO release was measured, and the results are as follows: Figure 4 As shown, compared with the blank control group, the NO release in the model group was significantly increased, indicating that the inflammatory damage model was successfully established in this experiment; compared with the model group, the NO release in the drug group showed a significant decreasing trend, and the NO release gradually decreased with the increase of the concentration of papaya polyphenols. Figure 4 The above experimental results indicate that the polyphenols in wrinkled papaya effectively inhibit IL-1β-induced NO release from ADTC5 primary chondrocytes.

[0049] Example 5

[0050] Effects of polyphenols from wrinkled papaya extract on the release of pro-inflammatory cytokines from ADTC5 primary chondrocytes

[0051] With 1×10 4Cells were seeded per well in 96-well plates and cultured for 24 h in a 37°C cell culture incubator with 5% CO2. Three replicates were set up for each group. The drug groups were treated with 5, 10, and 20 μg / mL of the wrinkled papaya polyphenol extract obtained in Example 1 (each group also received 12 ng / mL IL-1β). The model group received 12 ng / mL IL-1β, and the control group received an equal volume of basal culture medium. After 12 h of culture, the supernatant was collected. The levels of TNF-α and IL-6 were determined according to the ELISA kit instructions.

[0052] After treating ADTC5 primary chondrocytes with different concentrations of papaya polyphenols (structural formula shown in Formula 1) for 24 hours, the concentrations of pro-inflammatory cytokines were measured, and the results are as follows: Figure 5 and 6 As shown, compared with the blank control group, the levels of pro-inflammatory cytokines TNF-α and IL-6 secreted by ADTC5 cells in the model group were significantly increased, indicating that the inflammatory injury model was successfully established in this experiment. Compared with the model group, the levels of pro-inflammatory cytokines secreted by ADTC5 cells in the drug group showed a significant decreasing trend. Among them, 20 μg / mL papaya polyphenols showed the most significant inhibition rate on the release of pro-inflammatory cytokines, demonstrating the most ideal anti-inflammatory effect. Figure 5 and Figure 6 Based on the above experiments, this invention demonstrates that wrinkled papaya polyphenols effectively inhibit IL-1β-induced secretion of pro-inflammatory cytokines by ADTC5 cells.

[0053] Example 6

[0054] Effects of polyphenols from wrinkled papaya extract on the expression of inflammation-related enzyme mRNA

[0055] With 1×10 4 Cells were seeded per well in 96-well plates and cultured for 24 h in a 37°C cell culture incubator with 5% CO2. Three replicates were set up for each group. The drug groups were treated with 5, 10, and 20 μg / mL of *Papaya scabra* polyphenol extract (each group also received 12 ng / mL IL-1β). The model group received 12 ng / mL IL-1β, and the control group received an equal volume of basal culture medium. After 24 h of culture, the supernatant was discarded, and the cells were collected by centrifugation at 4°C, 2000 r / min for 10 min. Total RNA was extracted using an RNA extraction kit, and cDNA was synthesized from the extracted RNA using a reverse transcription kit. The PCR reaction conditions were: 95°C pre-denaturation for 30 s, 95°C denaturation for 5 s, 60°C annealing and extension for 30 s, for 40 cycles. This invention uses GAPDH as an internal control and employs the comparative Ct method: 2 -ΔΔCT Calculate the relative expression levels of inflammation-related enzyme mRNAs.

[0056] After treating ADTC5 cells with different concentrations of polyphenols from the extract of Papaya pudding (structural formula shown in Formula 1) for 24 h, the relative expression levels of inflammatory enzyme mRNAs were detected by RT-qPCR. The results are as follows: Figure 8 and 9 Compared with the blank control group, the expression levels of the inflammatory enzymes iNOS and Cox-2 mRNA in ADTC5 cells of the model group were significantly increased. This indicates that the activity of inflammatory enzymes in ADTC5 cells was significantly increased under IL-1β stimulation, thereby promoting the development of inflammation, and fully confirming the successful construction of the inflammatory injury model of the present invention. Compared with the model group, the expression levels of the inflammatory enzymes iNOS and Cox-2 mRNA in ADTC5 cells of the drug group showed a significant downward trend. Among them, 20 μg / mL of wrinkled papaya polyphenols had the most significant inhibitory effect on the expression of inflammatory enzymes iNOS and Cox-2 mRNA, which also indicates that 20 μg / mL may be the drug concentration with the most obvious anti-inflammatory effect. Figure 7 and Figure 8 Based on the above experimental results, the polyphenols in the extract of wrinkled papaya effectively inhibited the expression of inflammatory enzyme mRNA.

[0057] Example 7

[0058] Effects of polyphenols from wrinkled papaya extract on proteins related to the NF-κB signaling pathway

[0059] With 1×10 4 Cells were seeded per well in 96-well plates and cultured for 24 h in a 37°C cell culture incubator with 5% CO2. Three replicates were set up for each group. The drug groups received 5, 10, and 20 μg / mL of papaya polyphenol extract (each group also received 12 ng / mL IL-1β), the model group received 12 ng / mL IL-1β, and the control group received an equal volume of basal culture medium. After 24 h of culture, the supernatant was discarded, and cells were collected by centrifugation at 5000 r / min for 10 min at 4°C. An appropriate volume of cell lysis buffer was added to the cells, and lysis was performed at 4°C for 40 min, followed by centrifugation at 10000 r / min for 20 min at 4°C. The supernatant was collected, and the total amount of extracted protein was quantified using a BCA protein assay kit. Western blotting was then used to detect the expression levels of IκBα, p65, and phosphorylated proteins.

[0060] After treating ADTC5 cells with polyphenols extracted from wrinkled papaya (structural formula shown in Formula 1) at different concentrations for 24 h, the expression levels of proteins related to the NF-κB signaling pathway were detected by Western blotting. The results are as follows: Figure 10As shown, compared with the blank control group, the phosphorylation levels of IκBα and p65 in ADTC5 cells of the model group increased by 4.4 and 5.1 times, respectively, under IL-1β stimulation; compared with the model group, the phosphorylation levels of IκBα and p65 in ADTC5 cells of the drug group decreased significantly, with 20 μg / mL papaya polyphenols showing the most significant inhibitory effect on the phosphorylation levels of IκBα and p65. Figure 9 and Figure 10 The concentration was consistent with the optimal anti-inflammatory concentration in the above embodiments. Based on the above experiments, this invention demonstrates that the polyphenols in the extract of wrinkled papaya effectively inhibit the expression of proteins related to the NF-κB signaling pathway.

[0061] In summary, this invention provides an application of the polyphenols from the extract of *Papaya scabra* in the preparation of drugs for treating osteoarthritis. These polyphenols effectively reduce the release of pro-inflammatory cytokines (TNF-α and IL-1) and the inflammatory mediator NO from primary chondrocytes, decrease the mRNA expression of inflammation-related enzymes (iNOS and Cox-2), inhibit the phosphorylation of NF-κB signaling pathway-related proteins (IκBα and p65), alleviate chondrocyte inflammatory damage, and protect chondrocytes, thus effectively preventing and treating osteoarthritis. The polyphenols from the extract of *Papaya scabra* provided by this invention show great promise in the preparation of safe and highly active drugs for treating osteoarthritis.

[0062] The above embodiments are preferred implementations of the present invention, but the above embodiments do not limit the implementation of the present invention. Any other reasonable implementation methods that do not depart from the principles and essence of the present invention are within the protection scope of the present invention.

Claims

1. The application of wrinkled papaya polyphenol in the preparation of a drug for treating osteoarthritis, wherein the wrinkled papaya polyphenol is obtained by extracting wrinkled papaya powder with an ethanol solution, drying it, and then enzymatically hydrolyzing it with cellulase. The ethanol solution has a mass concentration of 95% or higher, and the amount of cellulase added is 1.0-2.0% of the mass of the dry powder obtained after ethanol extraction of wrinkled papaya polyphenol; the enzymatic hydrolysis temperature is 50-60℃, the enzymatic hydrolysis pH is 4.5-6.0, and the enzymatic hydrolysis time is 2-4 hours; the structural formula of wrinkled papaya polyphenol is as follows: The concentration of polyphenols in wrinkled papaya is 5-20 μg / mL.

2. The application according to claim 1, characterized in that, The application of the wrinkled papaya polyphenols in the preparation of drugs that reduce the release of pro-inflammatory cytokines and the inflammatory mediator NO in primary chondrocytes, and / or reduce the mRNA expression of inflammation-related enzymes, and / or inhibit the phosphorylation of proteins related to the NF-κB signaling pathway.

3. The application according to claim 2, characterized in that, The pro-inflammatory cytokines mentioned are TNF-α and IL-1, the inflammation-related enzymes are iNOS and Cox-2, and the NF-κB signaling pathway-related proteins are IκBα and p65.

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