Use of a nephrolepis extract in the preparation of a medicine for preventing calcium oxalate kidney stones

Abstract

This invention discloses the application of *Nephrolepis cordifolia* extract in the preparation of drugs for preventing calcium oxalate kidney stones. The extract is an aqueous or ethanolic extract of the tuber of *Nephrolepis cordifolia* (monkey egg), preferably an aqueous extract. In vitro experiments show that the extract can inhibit the growth and aggregation of calcium oxalate crystals, promote the conversion of calcium oxalate monohydrate to calcium oxalate dihydrate with lower adhesion, and inhibit the crystal nucleation process. In a glyoxylate-induced kidney stone mouse model, the extract can reduce kidney crystal deposition, alleviate histopathological damage, and improve abnormal serum and urine biochemical indicators; its protective mechanism is related to reducing oxidative stress and downregulating the expression of crystal adhesion-related proteins CD44 and OPN. This invention also provides a pharmaceutical composition containing the extract and a method for in vitro evaluation of its inhibitory effect on calcium oxalate crystallization. This invention confirms that *Nephrolepis cordifolia* extract can effectively prevent the formation of calcium oxalate kidney stones by regulating the crystallization process, oxidative stress, and crystal retention mechanisms through multiple targets, and has good pharmaceutical prospects.

Description

Technical Field

[0001] This invention relates to the field of natural medicines and pharmaceutical technology, specifically to the use of a Nephrolepis cordifolia extract in the preparation of a medicament or pharmaceutical composition for the prevention and / or treatment of calcium oxalate kidney stones. Background Technology

[0002] Kidney stones (also known as nephrolithiasis) are a common urinary system disease with an increasing global burden, and recurrence remains a major clinical challenge. Calcium oxalate (CaOx) stones account for approximately 70%-80% of kidney stones, primarily existing in two crystal forms: calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD). Calcium oxalate monohydrate crystals adhere strongly to renal epithelial cells, easily remaining in the kidney and forming a stone core; while calcium oxalate dihydrate crystals have weaker adhesion and are more easily excreted in urine. Therefore, regulating the crystallization process of calcium oxalate and promoting the conversion of calcium oxalate monohydrate to calcium oxalate dihydrate is considered an effective strategy for preventing stone formation. Current clinical treatments mainly include extracorporeal shock wave lithotripsy (ESWL) and endoscopic lithotripsy. These methods can effectively remove existing stones but cannot fully prevent recurrence. Statistics show that about half of patients experience stone recurrence within five years of receiving current treatment. Furthermore, surgical intervention may cause related kidney damage, and repeated treatments impose a significant economic burden and physical and psychological suffering on patients. Therefore, there is an urgent need to develop safe and effective prevention strategies for early CaOx crystallization and crystal retention.

[0003] Kidney stone formation involves multiple interconnected pathological processes. Oxidative stress is considered a key mediator linking calcium oxalate crystal exposure to renal epithelial damage; excessive reactive oxygen species damage renal tubular epithelial cells, creating favorable conditions for crystal adhesion and retention. Simultaneously, molecules such as CD44 and osteopontin play important roles in the adhesion of the crystal to the surface of renal tubular epithelial cells, and their upregulation is closely related to crystal retention. However, current techniques mostly focus on intervening in single aspects of stone formation, lacking multi-target synergistic regulation of the crystallization process, oxidative stress, adhesion protein expression, and the in vivo biochemical environment.

[0004] Mounting evidence suggests that plant-derived compounds can inhibit CaOx crystallization, regulate crystal morphology, reduce crystal aggregation, and mitigate oxidative and inflammatory damage to kidney tissue caused by crystals. For example, it has been reported that extracts from the leaves of *Quercus dentatus* can inhibit CaOx crystallization, promote the conversion of COM to COD, and alleviate kidney damage through the OPN / CD44 and NLRP3 pathways. Studies have shown that degraded soybean polysaccharides can inhibit CaOx crystal aggregation and repair oxidatively damaged renal epithelial cells. These studies indicate that plant-derived materials may act on multiple pathological processes in the formation of CaOx kidney stones, rather than affecting only a single stage of stone formation. However, many traditional medicinal plants with a long history of folk applications still lack sufficient research, and their potential anti-stone activity needs systematic evaluation.

[0005] Nephrolepis cordifolia, also known as tuberous sword fern, monkey egg, sparrow egg, phoenix egg, centipede grass, and stone yellow skin, is a perennial fern widely distributed in tropical and subtropical regions. In traditional medicine, it has been used to treat jaundice, rheumatism, urinary tract infections, fever, cough, and edema, and is generally considered suitable for long-term use. Previous phytochemical studies have shown that Nephrolepis cordifolia contains flavonoids, phenolic compounds, polysaccharides, terpenoids, and alkaloids, which may endow it with antioxidant, anti-inflammatory, diuretic, and antibacterial activities. These properties are closely related to the prevention of CaOx stones, as stone formation is closely associated with oxidative stress, epithelial damage, inflammation, crystal adhesion, and urinary homeostasis disturbances. However, whether Nephrolepis cordifolia can exert its preventive effect on kidney stones through multiple targets, such as inhibiting calcium oxalate crystallization, promoting the conversion of calcium oxalate monohydrate to calcium oxalate dihydrate, reducing oxidative stress damage, and downregulating the expression of crystal adhesion-related markers, has not been previously reported in research. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to provide the use of a *Nephrolepis cordifolia* extract in the preparation of a medicament for the prevention and / or treatment of calcium oxalate kidney stones. This extract can prevent the formation of calcium oxalate kidney stones by regulating the calcium oxalate crystallization process, oxidative stress, and crystal retention mechanisms.

[0007] To achieve the above objectives, the present invention provides the following technical solution: In one embodiment, the present invention provides the use of a pteridium bream extract in the preparation of a medicament for the prevention and / or treatment of calcium oxalate kidney stones.

[0008] Further, the *Nephrolepis cordifolia* extract is an aqueous extract or an ethanolic extract of *Nephrolepis cordifolia*. Preferably, the *Nephrolepis cordifolia* extract is an aqueous extract of *Nephrolepis cordifolia* tubers.

[0009] Furthermore, the Nephrolepis extract is prepared by the following method: the Nephrolepis tubers are dried, pulverized, and then soaked and extracted at room temperature using water or 60% ethanol as solvents. The extracts are combined, concentrated to a crude drug concentration of 0.5 g / mL, and the supernatant is collected by centrifugation.

[0010] Furthermore, the drug has one or more of the following uses: Inhibit the nucleation and / or growth processes of calcium oxalate crystals; Promotes the conversion of calcium oxalate monohydrate (COM) to calcium oxalate dihydrate (COD); Reduce oxidative stress in renal tubular epithelial cells; Maintaining the migration ability of renal tubular epithelial cells; It can reduce glyoxylic acid-induced renal histopathological damage and crystal deposition. Reduce the expression levels of CD44 and / or osteopontin (OPN) in kidney tissue; Improve oxidative stress markers in kidney tissue, including increasing levels of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and / or decreasing levels of malondialdehyde (MDA). Improve abnormal serum and / or urine biochemical parameters associated with kidney stones, including reducing the levels of one or more of the following in serum: blood urea nitrogen (BUN), creatinine, uric acid, calcium, phosphorus, alanine aminotransferase (ALT), and aspartate aminotransferase (AST), and / or reducing the levels of calcium, phosphorus, oxalate, and / or creatinine in urine.

[0011] Furthermore, the aqueous extract of the Nephrolepis tuber, at a concentration in the range of 0.02-0.5 g / mL, can promote the transformation of calcium oxalate crystals from calcium oxalate monohydrate to calcium oxalate dihydrate (can transform the morphology of calcium oxalate crystals from hexagonal aggregates to tetragonal bipyramidal, prismatic, or plate-like morphologies).

[0012] In another embodiment, the present invention provides a pharmaceutical composition for the prevention and / or treatment of calcium oxalate kidney stones, comprising the extract of *Nephrolepis cordifolia* as described above.

[0013] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier or excipient.

[0014] Furthermore, the dosage form of the pharmaceutical composition is an oral preparation, which includes tablets, capsules, granules, oral liquids, or suspensions.

[0015] In another embodiment, the present invention provides a method for in vitro evaluation or screening of the inhibitory effect of any of the above-described pteris extracts on calcium oxalate crystallization, comprising the following steps: (a) Add the test extract of Nephrolepis cordifolia to a crystallization system containing calcium ions and oxalate ions to form a test solution; (b) Monitor the change in conductivity of the test solution over time in real time; (c) The change in conductivity was compared with a control system without the addition of pteris extract; If the conductivity of the test solution decreases more slowly than that of the control system, or if a longer induction period occurs, it indicates that the extract of Nephrolepis cordifolia can inhibit the nucleation and / or growth process of calcium oxalate crystals.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) Regulation of calcium oxalate crystallization: In vitro experimental results showed that the extract of Nephrolepis cordifolia could inhibit the growth and aggregation of calcium oxalate crystals. Specifically, the extract of Nephrolepis cordifolia could promote the conversion of calcium oxalate monohydrate (COM) to calcium oxalate dihydrate (COD), which has lower adhesion. In the concentration range of 0.02-0.5 g / mL, both the aqueous extract and the ethanol extract of Nephrolepis cordifolia could promote the transformation of crystal morphology from hexagonal aggregates to tetragonal bipyramidal, prismatic or plate-like morphologies.

[0017] (2) Reduced renal tissue pathological damage: In a glyoxylate-induced mouse model, the extract of Nephrolepis cordifolia can reduce renal crystal deposition and alleviate histopathological damage such as renal tubular dilation and inflammatory cell infiltration.

[0018] (3) Improvement of oxidative stress indicators: In vivo experimental results showed that the extract of Nephrolepis cordifolia can increase the levels of superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) in kidney tissue and reduce the level of malondialdehyde (MDA).

[0019] (4) Downregulation of crystal adhesion-related protein expression: Immunohistochemical results showed that the extract of Nephrolepis cordifolia could reduce the expression levels of CD44 and osteopontin (OPN) in kidney tissue.

[0020] (5) Improvement of serum and urine biochemical indicators: Nephrolepis extract can improve abnormal serum and urine biochemical indicators related to kidney stones, including reducing the levels of blood urea nitrogen (BUN), creatinine, uric acid, calcium, phosphorus, ALT and AST in serum, as well as reducing the levels of calcium, phosphorus, oxalate and creatinine in urine.

[0021] (6) Providing a basis for active ingredients: LC-MS analysis of the ethanol extract of Nephrolepis cordifolia preliminarily identified several active ingredients, mainly glycosides, including: 1,3-dihydroxy-2-hydroxymethylanthraquinone-3-O-β-D-xylanose (1→6)-β-D-glucopyranoside (18.35% by mass), Nelumboroside A (13.15% by mass), and 14-deoxyandrographolide-19β-glucoside (11.20% by mass). The biological activities of these compounds are mainly antioxidant, anti-inflammatory, and immunomodulatory, providing a chemical basis for the biological activities of the above-mentioned Nephrolepis cordifolia extract. Attached Figure Description

[0022] Figure 1 A schematic diagram illustrating the mechanism by which Nephrolepis externa extract inhibits calcium oxalate kidney stones.

[0023] Figure 2 The effects of Nephrolepis extract on calcium oxalate crystallization and growth kinetics; Figure 2 middle: The image above shows scanning electron microscope (SEM) images of calcium oxalate crystals formed in the presence of different concentrations (0, 0.02, 0.1, 0.5 g / mL) of ethanol extract from Nephrolepis cordifolia. The figure shows SEM images of calcium oxalate crystals formed in the presence of different concentrations (0, 0.02, 0.1, 0.5 g / mL) of water extract from Nephrolepis cordifolia. The il plot shows the conductivity kinetics curves in the presence of different concentrations (0, 0.02, 0.1, 0.5 g / mL) of Nephrolepis expansa aqueous extract.

[0024] Figure 3 To investigate the cell compatibility, antioxidant activity and migration-related effects of Nephrolepis extract in HEK293T cells; Figure 3 middle: Figure a shows the cell viability of HEK293T cells after treatment with different concentrations of Nephrolepis extract; Figure b shows the effect of Nephrolepis extract on the level of reactive oxygen species (ROS) in HEK293T cells under oxidative stress (100 μM H2O2). Figure c shows the quantitative analysis of the relative migration distance of HEK293T cells after treatment with different extracts.

[0025] Figure 4 The effects of Nephrolepis extract on renal histopathology, crystal deposition, and expression of crystal adhesion-related markers in a glyoxylate-induced renal calculi model mouse model; Figure 4 middle: Figure a shows a representative histological image of a kidney section stained with hematoxylin and eosin (H&E) and von Kossa. Figure b shows the immunohistochemical staining results of CD44 and osteopontin (OPN) in kidney tissue; Figure c shows the quantitative analysis of the CD44 positive region; Figure d shows the quantitative analysis of the OPN positive region.

[0026] Figure 5 The effect of Nephrolepis extract on oxidative stress in a glyoxylate-induced kidney stone model mouse; Figure 5 middle: Figure a shows the level of superoxide dismutase (SOD) in kidney tissue; Figure b shows the level of catalase (CAT) in kidney tissue; Figure c shows the level of glutathione (GSH) in kidney tissue; Figure d shows the level of malondialdehyde (MDA) in kidney tissue.

[0027] Figure 6 The effects of Nephrolepis externa extract on serum and urine biochemical parameters in a glyoxylate-induced kidney stone model mouse. Figure 6 middle: The figure shows serum biochemical parameters: blood urea nitrogen (BUN, a), creatinine (b), uric acid (c), calcium (d), phosphorus (e), magnesium (Mg²⁺, f), alanine aminotransferase (ALT, g), and aspartate aminotransferase (AST, h). The il diagram shows urine biochemical parameters: calcium (i), creatinine (j), phosphorus (k), and oxalate (l).

[0028] Figure 7 This is a table of components identified from the ethanol extract of Nephrolepis cordifolia in positive ion mode based on LC-MS analysis.

[0029] Figure 8 This is a table of components identified from the ethanol extract of *Nephrolepis cordifolia* under negative ion mode based on LC-MS analysis. Detailed Implementation

[0030] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The described embodiments are merely some embodiments of the present invention, and 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. Example 1: Preparation of Nephrolepis externa extract

[0031] Fresh Nephrolepis tuber samples were washed, air-dried, and sliced, then dried in a 60°C hot air oven and subsequently pulverized using a mechanical grinder. 100 g of the powder was placed in a stoppered 1000 mL Erlenmeyer flask and extracted three times at room temperature by reflux with 500 mL of 60% ethanol or by water bath extraction with ultrapure water. After filtering the mixture, the residue was extracted again with another 500 mL of 60% ethanol or ultrapure water under the same conditions for 7 days, followed by filtration. The combined filtrates were concentrated to obtain an ethanol extract or an aqueous extract with a crude drug concentration of 0.5 g / mL. The extract was centrifuged at 10,000 rpm for 20 min, and the supernatant was collected and stored at low temperature for later use.

[0032] Example 2: Effect of Nephrolepis externa extract on the morphology of calcium oxalate crystals (SEM observation)

[0033] A CaOx crystallization model was established by mixing 32 mL of a 5 mmol / L CaCl2 solution. In a clean beaker, 32 mL of a 5 mmol / L Na2C2O4 solution and 136 mL of double-distilled water were added to achieve a final ion concentration of [Ca...]. 2+ ]=[C2O4 2- =0.8 mmol / L. Three hydrophilic glass slides were placed in each beaker. To evaluate the effect of the Nephrolepis extract, the double-distilled water in the above system was replaced with water or ethanol extracts at different crude drug concentrations (0.02, 0.1, and 0.5 g / mL). After 5 days, the glass slides were removed, air-dried at room temperature, and the morphology of calcium oxalate crystals was observed using a scanning electron microscope (SEM).

[0034] The results showed that under extract-free conditions (such as...) Figure 2 As shown in Figure a), CaC2O4 crystals exhibit a typical COM morphology, appearing as slender hexagonal aggregates with an average diagonal length of approximately 5.11 micrometers. Treatment with different concentrations of Nephrolepis effica ethanol extract significantly promoted the conversion of COM to COD (e.g., Figure 2 (See diagram BD). At an extract concentration of 0.02 g / mL (e.g., ... Figure 2 As shown in Figure b), irregular plate-like and tetragonal bipyramidal COD crystals coexist (e.g. Figure 2 (As shown in Figure c); at a concentration of 0.1 g / mL, tetragonal bipyramidal and prismatic COD crystals predominate; at a concentration of 0.5 g / mL (as shown in Figure c), the dominance is due to the presence of tetragonal bipyramidal and prismatic COD crystals. Figure 2 As shown in Figure d), thin, plate-like COD crystals were mainly observed. The ethanol extract inhibited the formation of COD crystals in a concentration-dependent manner and favored the formation of less adherent COD crystals.

[0035] Without the addition of water extracts (such as Figure 2As shown in Figure e), the crystals mainly exhibit the characteristic COD morphology, namely slender hexagonal aggregates, with an average diagonal length of approximately 6.07 µM for the hexagonal cross-section. After treatment with 0.02 g / mL water extract, typical tetragonal bipyramidal COD crystals were observed (e.g., Figure 2 (As shown in Figure f); when the concentration was increased to 0.1 g / mL, rod-shaped and prismatic COD crystals were formed (e.g., ...). Figure 2 (As shown in Figure g); while at a concentration of 0.5 g / mL, the crystals mainly exhibit smaller rod-like or plate-like structures (e.g., ...). Figure 2 (As shown in Figure h). Similar to the ethanol extract, the water extract inhibited COM formation and promoted a COD-dominant crystallization process. Furthermore, with increasing water extract concentration, the crystals gradually became smaller and fewer in number.

[0036] Overall, both the ethanol and water extracts of Nephrolepis cordifolia effectively modulate the in vitro CaC2O4 crystallization process by inhibiting COM formation, promoting the conversion of COM to COD, and reducing the number of crystals. Since COD crystals have lower adhesion and are more easily expelled than COM crystals, this crystal phase regulation helps reduce crystal retention and the risk of stone formation. Example 3: Effect of Nephrolepis externa extract on calcium oxalate crystallization kinetics (conductivity method)

[0037] The crystallization kinetics of calcium oxalate were monitored by measuring the solution conductivity. 16 mL of a 5 mmol / L CaCl2 solution and 68 mL of ultrapure water were added to a 100 mL beaker, followed by the rapid addition of 16 mL of a 5 mmol / L Na2C2O4 solution. The conductivity of the solution was recorded immediately and monitored every 30 seconds under continuous stirring. To evaluate the effect of *Nephrolepis cordifolia*, the ultrapure water was replaced with water extracts at crude drug concentrations of 0.02, 0.1, and 0.5 g / mL, and the conductivity was measured under the same conditions.

[0038] The results show that: Figure 2 As shown in the figure, in the control system, the conductivity drops sharply within the first 6 minutes and gradually reaches equilibrium at about 11 minutes, indicating that Ca 2+ and C2O4 2- Rapid binding and rapid crystal nucleation. In contrast, the conductivity curve decreased more slowly and for a longer period after the addition of the water extract of Nephrolepis cordifolia. At a concentration of 0.02 g / mL (e.g. Figure 2As shown in Figure i), the delayed decrease in conductivity indicates that the extract inhibits the initial nucleation process; while at higher concentrations of 0.10 and 0.50 g / mL, the curves show a longer induction period and recurring shoulder-like features during the decrease, indicating a more significant inhibitory effect on nucleation and subsequent crystal growth. This concentration-dependent inhibition can be attributed to the active ingredient in the water extract and Ca... 2+ Interacting with or adsorbing onto the crystal surface reduces free Ca. 2+ The availability of the extract interferes with the formation of CaOx crystal nuclei and hinders further crystal growth. Combined with scanning electron microscopy (SEM) observations, conductivity data further confirm that the aqueous extract of Nephrolepis cordifolia regulates the crystallization process of CaOx by delaying the nucleation process and inhibiting crystal growth in a concentration-dependent manner. Example 4: Evaluation of the cell compatibility of Nephrolepis extract

[0039] Human embryonic kidney 293T (HEK293T) cells were purchased from ATCC and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. The cultures were maintained in a humidified incubator at 37°C and 5% CO2. After 72 h of culture, cells were isolated from the culture plates using trypsin and centrifuged at 1000 rpm for 4 min. The supernatant was discarded after centrifugation, and the cells were washed with PBS. The cells were then resuspended in DMEM and seeded at a density of 30,000 cells per well in 96-well tissue culture plates.

[0040] HEK293T cells were seeded at a density of 30,000 cells per well in 96-well cell culture plates and cultured for 24 hours. Subsequently, *Nephrolepis cordifolia* extract (ethanol / water extract of the tuber) was diluted with DMEM to different concentrations (0.0625, 0.125, 0.25, 0.5, 1.0 mg / mL) and added to each well (100 μL per well), and cultured for another 24 hours. After washing the cells with 100 μL of DPBS in each well, 100 μL of Alamar Blue working solution (DPBS solution containing 10% Alamar Blue) was added to each well, and the cells were incubated for 1 hour under standard cell culture conditions. Fluorescence signals were recorded using a SpectraMax M3 microplate reader at an excitation wavelength of 570 nm and an emission wavelength of 590 nm. Untreated control cells were used to normalize fluorescence values, which are expressed as 100% viability.

[0041] The results show that: Figure 3As shown in Figure a, within the test concentration range of 0.0625–1.0 mg / mL, neither the ethanol extract nor the water extract of *Nephrolepis cordifolia* significantly affected cell viability, which remained above 90%, indicating that the extract has good cell compatibility. The biological effects of the extract were further evaluated in HEK293T cells. Example 5: Scavenging effect of Nephrolepis cordifolia extract on intracellular reactive oxygen species (ROS)

[0042] HEK293T cells were seeded at a density of 30,000 cells per well in 96-well plates and cultured for 24 hours. Following the kit instructions, cells were first stimulated with 100 μM hydrogen peroxide for 1 hour to induce oxidative stress, followed by treatment with 1.0 mg / mL Nephrolepis externa extract for 1 hour. The ROS kit was then diluted 1:1000 with serum-free basal medium. After removing the cell culture medium, an appropriate amount of the diluted ROS kit solution was added, and the cells were incubated for 30 minutes. Cells were washed with DPBS to remove excess probes, and intracellular ROS levels were immediately measured using a SpectraMax M3 microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0043] The results showed that under oxidative stress induced by 100 μM hydrogen peroxide, the ROS fluorescence intensity in the positive control group was significantly increased, while the ROS accumulation was significantly reduced after pretreatment with either the ethanol or water extract of *Nephrolepis cordifolia* (e.g., *Nephrolepis cordifolia*). Figure 2 As shown in Figure b), both extracts exhibited comparable antioxidant activity, indicating that *Nephrolepis cordifolia* can alleviate oxidative stress within renal epithelial cells. The reduction in reactive oxygen species (ROS) levels provides cellular-level evidence that *Nephrolepis cordifolia* extract has a protective effect against oxidative damage associated with calcium oxy-oxidative kidney stones. Example 6: Effects of Nephrolepis externa extract on cell migration ability (cell scratch assay)

[0044] To assess whether the extract from *Nephrolepis cordifolia* tubers affected cell growth and migration, a cell scratch assay was performed. HEK293T cells were seeded at a density of 1,500,000 cells per well in 12-well plates and cultured for 24 hours until confluence. A linear scratch was made on the monolayer of cells using a sterile 200 μL pipette tip. The plates were washed twice with 1 mL of DMEM solution to remove cell debris, followed by the addition of 1.0 mg / mL *Nephrolepis cordifolia* extract solution. The distance between the scratch edges at 0 hours was recorded using an Olympus IX81 fluorescence microscope (denoted as d0). After another 24 hours of culture, the distance between the scratch edges was measured again and recorded as d1. The relative migration distance (scratch closure degree) was calculated using the following formula: Relative migration distance (%) = [(d0- d1) / d0] × 100%.

[0045] The results show that: Figure 3 Chinese C diagram and Figure 3 As shown in Figure d, there was no significant difference in relative migration distance between cells treated with Nephrolepis extract and control cells, indicating that neither the ethanol extract (EE) nor the water extract (WE) of Nephrolepis extract impaired cell migration or closure ability.

[0046] The results of Examples 2-6 indicate that the Nephrolepis externa extract is biocompatible, can reduce intracellular oxidative stress, and maintain the repair capacity of epithelial cells. Combined with the results of crystallization experiments, these cellular findings suggest that the extract can not only reduce the risk of CaOx stone formation by regulating crystal formation, but also play a role in protecting renal epithelial cells from oxidative damage. Example 7: Establishment and administration of a glyoxylate-induced mouse model of kidney stones

[0047] Although *Nephrolepis externa* extract inhibits CaOx crystallization and alleviates oxidative stress in vitro, in vivo assessment is necessary to determine whether these effects are reflected in a complex physiological environment. Unlike in vitro crystallization systems, in vivo kidney stone formation involves crystal deposition, renal tubular epithelial damage, inflammatory responses, and crystal retention within the kidney tissue. Therefore, this study established a glyoxylate-induced kidney stone mouse model to further evaluate the preventive effects of *Nephrolepis externa* extract in vivo.

[0048] Male SPF-grade C57BL / 6 mice (8 weeks old, weighing 19-23g, purchased from Chongqing Tengxin Biotechnology Co., Ltd., all animal experiments were conducted in accordance with the guidelines of the Animal Ethics Committee of Anshun University) were used. Two mice per cage were housed in a controlled environment (temperature 25±1℃, 12-hour light / dark cycle) with free access to food and water. After a one-week acclimatization period, the mice were randomly divided into four groups (n=6 per group): control group (Ctrl), glyoxylate-induced kidney stone group (Gly), glyoxylate combined with ethanol extract group (Gly+EE), and glyoxylate combined with water extract group (Gly+WE). The plant extract was administered orally via gavage for 14 days. Kidney stones were induced by intraperitoneal injection of glyoxylate (100 mg / kg / day, dissolved in PBS, pH 7.4) for 7 consecutive days. Animals in the control group received an equal volume of PBS solution. Gavage of the plant extract and injection of glyoxylate were performed at 9:00 AM and 4:00 PM, respectively. The day after the last gavage, the mice were anesthetized, given cardiac perfusion, and then sacrificed. The kidneys were collected for further analysis. Example 8: Effects of Nephrolepis externa extract on renal histopathology and crystal deposition

[0049] Using the four groups of kidneys collected in Example 7 as materials, left kidney tissue from each group was fixed in 4% paraformaldehyde for 24 hours, then embedded in paraffin. Four-micrometer thick sections were cut and stained using hematoxylin-eosin (H&E) and von Kossa staining methods, respectively. The stained sections were observed under a microscope (CX43, Olympus, Tokyo, Japan). The amount of calcium oxalate monohydrate (COM) crystals deposited in the von Kossa stained sections was quantitatively analyzed using ImageJ software (National Institutes of Health, USA).

[0050] The results show that: Figure 4 As shown in Figure a, the kidney sections of the control group exhibited normal kidney structure, with intact renal tubules, orderly arranged epithelial cells, and no obvious tissue damage observed in H&E staining. Von Kossa staining was also negative. The model group (Gly) displayed typical pathological features of kidney stone-related renal injury, including renal tubular dilatation, inflammatory cell infiltration, connective tissue hyperplasia, focal fibrosis, epithelial cell necrosis, and extensive black calcium deposition within the renal tubular lumen. These findings confirmed the successful establishment of the glyoxylate-induced kidney stone model. The above pathological changes were significantly reduced in the treatment groups (Gly+EE and Gly+WE). In the Gly+WE group, renal tubular dilatation, inflammatory infiltration, and fibrosis were significantly reduced, and the renal tubular structure was largely preserved. Von Kossa staining showed almost no detectable calcium deposition, indicating that the water extract had a strong inhibitory effect on renal crystal accumulation. Compared with the untreated Gly group, the ethanol extract also reduced tissue damage and crystal deposition, although residual pathological changes and calcium deposition were still more pronounced than in the Gly+WE group. Overall, both extracts were able to combat glyoxylate-induced kidney damage, with the water extract being more effective in reducing crystal deposition and protecting kidney tissue structure. Example 9: Effects of Nephrolepis externa extract on CD44 and osteopontin (OPN) expression

[0051] Using the four groups of kidneys collected in Example 7 as materials, the left kidney tissue of each group was fixed in 4% paraformaldehyde for 24 hours and then embedded in paraffin. 4-micrometer thick sections were cut, and the kidney sections were subjected to immunohistochemical staining for CD44 and osteopontin (OPN). ImageJ software was used to perform quantitative analysis on the positive areas.

[0052] The results show that: Figure 4 As shown in Figure b, strong CD44 immunoreactivity was observed in the Gly group, particularly in tubular regions containing crystal deposits. Both WE and EE treatments significantly reduced CD44 staining intensity (e.g., Figure 4 Figure b in the middle and Figure 4(See Figure c). OPN expression also showed a similar pattern: OPN staining intensity was significantly enhanced in the Gly group, while OPN immunoreactivity was significantly weakened in the extract-treated group. The decrease in CD44 and OPN was more pronounced in the Gly+WE group, which is consistent with its stronger effect on crystal deposition and tissue protection.

[0053] The results of Examples 8 and 9 indicate that the extract of *Nephrolepis cordifolia* (especially the aqueous extract) can reduce renal crystal deposition in a glyoxylate-induced kidney stone model mouse, alleviate histopathological damage, and decrease the expression of crystal adhesion-related markers. These findings provide in vivo evidence that the extract can both reduce crystal load and improve renal tissue conditions conducive to crystal retention. Example 10: Effects of Nephrolepis externa extract on oxidative stress indices in kidney tissue

[0054] Using the four kidneys collected in Example 7 as materials, the right kidney tissue blocks of each group were homogenized in PBS using an ultrasound processor. Oxidative stress markers (including malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and catalase (CAT)) were detected according to the manufacturer's instructions (Servicebio, Wuhan, China).

[0055] The results show that: Figure 5 As shown, compared with the control group, glyoxylic acid administration significantly disrupted the redox balance of the kidneys: the levels of total superoxide dismutase (T-SOD), catalase (CAT), and glutathione (GSH) in the kidneys were significantly reduced, while the level of malondialdehyde (MDA) was significantly increased. These changes indicate that the antioxidant defense function of kidney tissue is impaired and lipid peroxidation is enhanced during glyoxylic acid-induced kidney stone formation. Treatment with *Gynostemma pentaphyllum* extract alleviated these oxidative damages to varying degrees. Compared with the untreated Glycyrrhiza uralensis group, both the ethanol extract (Gly+EE) and the water extract (Gly+WE) significantly restored renal T-SOD and CAT levels and reduced MDA accumulation. The water extract showed a stronger antioxidant effect, particularly in restoring T-SOD and CAT activity and inhibiting MDA elevation. In contrast, the recovery of GSH was relatively limited, with only partial improvement observed after extract treatment.

[0056] In summary, extracts from Nephrolepis cordifolia (especially the aqueous extract) can alleviate oxidative stress in glyoxylate-induced kidney stones. The consistency between the oxidative stress regulation, reduced expression of adhesion markers, and decreased crystal deposition supports a protective mechanism that works by mitigating crystal-induced oxidative damage and inhibiting crystal retention. Example 11: Effects of Nephrolepis externa extract on serum and urine biochemical parameters

[0057] To further determine whether improvements at the histological and molecular levels are accompanied by functional recovery, serum and urinary biochemical parameters were assessed. These indicators reflect renal function, metabolic balance, and stone-forming risk during the kidney stone formation process.

[0058] Blood samples were collected from the retroorbital sinus of the four groups of mice in Example 7. After standing for 1 hour, the samples were centrifuged at 3000 rpm for 15 minutes, and the serum was collected and stored at -80°C. The samples were thawed immediately before analysis, and the levels of serum creatinine, blood urea nitrogen (BUN), calcium, uric acid, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured using a fully automated biochemical analyzer (Chemray 800, Shenzhen, China).

[0059] In Example 7, urine samples from four groups of mice were collected via metabolic cages 24 hours later. The samples were centrifuged at 3000 rpm for 15 minutes, and the supernatant was stored at -80°C. Before analysis, the urine samples were thawed simultaneously, and the levels of urinary calcium, creatinine, phosphorus, and oxalate were measured using a urine analyzer (Urit-500B, Guilin, China).

[0060] The results show that: Figure 6 As shown in the figure, compared with the control group, glyoxylate administration led to significant disturbances in serum biochemical parameters, including marked increases in blood urea nitrogen (BUN), creatinine, uric acid, calcium, phosphorus, magnesium, ALT, and AST levels. These changes indicate impaired renal filtration function, mineral metabolism disorders, and systemic biochemical imbalances associated with kidney stones. Specifically, the increases in BUN and creatinine reflected decreased renal clearance, while the increases in uric acid, calcium, and phosphorus levels suggested a stone-promoting metabolic state. Treatment with Glycerin Bleeding Fern extract significantly improved these abnormal indicators. Compared with the untreated Gly group, both the ethanol extract (Gly+EE) and the water extract (Gly+WE) reduced serum BUN, creatinine, uric acid, calcium, phosphorus, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels. The water extract generally showed a stronger restorative effect, especially in reducing BUN, creatinine, uric acid, and AST, indicating improved renal function and relief of systemic damage. In contrast, changes in serum calcium and magnesium levels were relatively small, with no significant differences observed between the two extract treatment groups.

[0061] like Figure 6As shown in the IL figure, after administration of glyoxylate, urinary calcium, phosphorus, oxalate, and creatinine levels all significantly increased, indicating increased urinary supersaturation, creating a favorable environment for CaOx crystal formation. Hypercalciuria and hyperoxaluria are definite risk factors for CaOx kidney stone formation, while elevated urinary phosphorus levels may further promote crystal aggregation. After treatment with Nephrolepis externa extract, these stone-causing urinary abnormalities were significantly improved, suggesting that the urinary microenvironment was optimized. The water extract showed the most significant effect, especially in reducing urinary oxalate and calcium levels.

[0062] In conclusion, Nephrolepis extract, especially its aqueous extract, can not only slow the progression of kidney stones by reducing the crystal load on the kidneys, but also achieve this effect by improving kidney function, correcting stone-causing urinary tract abnormalities, and alleviating systemic metabolic disorders. Example 12: LC-MS chemical composition analysis of Nephrolepis externa extract

[0063] The 60% ethanol extract of *Nephrolepis cordifolia* was analyzed by liquid chromatography-mass spectrometry (LC-MS / MS) (Xevo G2-XSQtof, Waters Inc., Milford, Massachusetts, USA) to determine its chemical composition. A brief procedure is as follows: The dried ethanol extract was dissolved in methanol, filtered through a 0.22 µm membrane, and analyzed by UPLC-QTOF-MS / MS. Chromatographic separation was performed on an ultra-high performance liquid chromatography (UHPLC) system equipped with a reversed-phase C18 column (2.1 × 50 mm, 1.7 µm) at a column temperature maintained at 40 °C. The mobile phase consisted of solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid), with a flow rate of 0.4 mL / min. -1 The gradient elution program was as follows: 0-1 min, 5% B; 1-35 min, 5%-98% B; 35-37 min, 98% B; 37.1-40 min, reequilibrate to 5% B. Mass spectrometry was performed using a quadrupole time-of-flight (QTOF) mass spectrometer equipped with an electrospray ionization (ESI) source, capable of operating in both positive and negative ion modes. The capillary voltage was set to 2.0 kV, the ion source temperature to 110℃, the desolventization temperature to 400℃, the desolventization gas to nitrogen, and the flow rate to 800 L·h. -1Mass spectrometry data acquisition ranged from m / z 50 to 1200. MS / MS fragmentation was performed in a data-dependent acquisition mode, with collision energies gradually increasing from 20 eV to 40 eV. Preliminary identification of compounds was achieved by comparing precise mass measurements, retention times, and MS / MS fragmentation patterns with records in traditional medical library databases and existing literature. LC-MS / MS analysis was performed only on a representative sample, the ethanol extract, to provide preliminary characterization of detectable low- to medium-polarity components using ESI-MS.

[0064] (1) The component identification results of the ethanol extract of Nephrolepis cordifolia in positive ion mode based on LC-MS analysis are as follows: Figure 7 As shown.

[0065] according to Figure 7 It can be seen that by performing LC / MS positive ion mode (library search) analysis on the 60% ethanol extract (1 g / mL) of SJ, approximately (51) 75 compounds were obtained.

[0066] Further analysis of compounds with a relative content greater than 1% yielded approximately 20 (41) compounds.

[0067] It mainly consists of flavonoids, flavonoid glycosides, and terpenoids, and its biological activities are mainly antioxidant, anti-inflammatory, antibacterial, and immunomodulatory.

[0068] (2) The component identification results of the ethanol extract of Nephrolepis cordifolia under negative ion mode based on LC-MS analysis are as follows: Figure 8 As shown.

[0069] according to Figure 8 It can be seen that by performing LC / MS negative ion mode (library search) analysis on the 60% ethanol extract (1 g / mL) of SJ, approximately (147) 189 compounds were obtained.

[0070] Further analysis of compounds with a relative content greater than 1% yielded approximately 16 (34) compounds.

[0071] It is mainly composed of glycosides, and its biological activities are mainly antioxidant, anti-inflammatory, and immunomodulatory.

[0072] comprehensive Figure 7 and Figure 8It is evident that the annotated components mainly include various active ingredients, primarily glycosides. Representative compounds include 1,3-dihydroxy-2-hydroxymethylanthraquinone-3-O-β-D-xylanose (1→6)-β-D-glucopyranoside (18.35% by mass), Nelumboroside A (13.15% by mass), and 14-deoxyandrographolide-19β-glucoside (11.20% by mass). The biological activities of these compounds are mainly antioxidant, anti-inflammatory, and immunomodulatory, providing a chemical basis for the bioactivity of the aforementioned Nephrolepis extract.

[0073] As demonstrated in Examples 1 to 12, *Nephrolepis cordifolia* exhibits a preventative effect against calcium oxalate (CaOx) formation by synergistically regulating the crystallization process and the mechanism of renal tissue damage. In in vitro experiments, its extract inhibited CaOx crystal growth and promoted the conversion of crystalline intermediates (COM) into less adhesive crystalline forms (COD). In vivo experiments showed that the extract reduced renal crystal deposition, alleviated histopathological damage, and improved abnormal serum and urinary biochemical indicators associated with kidney stones. These protective effects, accompanied by a decrease in oxidative stress levels and downregulation of crystal adhesion-related markers CD44 and OPN, indicate a reduction in crystal retention in renal tissue. Notably, the protective effect of the aqueous extract was consistently superior to that of the ethanol extract. Liquid chromatography-mass spectrometry (LC-MS) analysis of the ethanol extract further revealed the presence of various glycoside bioactive components, providing preliminary chemical evidence for the observed pharmacological activities.

[0074] Other aspects of this invention that are not detailed herein are all conventional techniques known to those skilled in the art.

[0075] It should be noted that the terms “comprising,” “including,” or any other variations are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0076] The scope of protection of this invention is not limited to the technical solutions disclosed in the specific embodiments. Any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of this invention shall fall within the scope of protection of this invention.

Claims

1. Use of a pteridium bream extract in the preparation of a medicament for the prevention of calcium oxalate kidney stones.

2. The use according to claim 1, characterized in that: The Nephrolepis extract is an aqueous extract of Nephrolepis or an ethanolic extract of Nephrolepis.

3. The use according to claim 2, characterized in that: The Nephrolepis extract is an aqueous extract of Nephrolepis tubers.

4. The use according to claim 2, characterized in that: The extract of Nephrolepis cordifolia was prepared by the following method: the tubers of Nephrolepis cordifolia were dried and pulverized, and then soaked and extracted at room temperature using water or 60% ethanol as solvents. The extracts were combined and concentrated to a crude drug concentration of 0.5 g / mL. The supernatant was collected by centrifugation.

5. The use according to claim 2, characterized in that, The drug has one or more of the following uses: Inhibit the nucleation and / or growth processes of calcium oxalate crystals; Promotes the conversion of calcium oxalate monohydrate (COM) to calcium oxalate dihydrate (COD); Reduce oxidative stress in renal tubular epithelial cells; Maintaining the migration ability of renal tubular epithelial cells; It can reduce glyoxylic acid-induced renal histopathological damage and crystal deposition. Reduce the expression levels of CD44 and / or osteopontin in kidney tissue; Improve oxidative stress markers in kidney tissue, including increasing superoxide dismutase, catalase, glutathione levels and / or decreasing malondialdehyde levels; Improve abnormal serum and / or urine biochemical parameters associated with kidney stones, including reducing the levels of one or more of the following in serum: blood urea nitrogen, creatinine, uric acid, calcium, phosphorus, alanine aminotransferase, and aspartate aminotransferase, and / or reducing the levels of calcium, phosphorus, oxalate, and / or creatinine in urine.

6. The use according to claim 5, characterized in that: The aqueous extract of the Nephrolepis tuber, at a concentration in the range of 0.02-0.5 g / mL, can promote the conversion of calcium oxalate crystals from calcium oxalate monohydrate to calcium oxalate dihydrate.

7. A pharmaceutical composition for the prevention and / or treatment of calcium oxalate kidney stones, characterized in that... It contains the Nephrolepis extract as described in any one of claims 1 to 6.

8. The pharmaceutical composition according to claim 7, characterized in that: It also includes pharmaceutically acceptable carriers or excipients.

9. The pharmaceutical composition according to claim 7, characterized in that: The dosage form of the pharmaceutical composition is an oral preparation, which includes tablets, capsules, granules, oral liquids, or suspensions.

10. A method for in vitro evaluation or screening of the inhibitory effect of the extract of *Nephrolepis cordifolia* according to any one of claims 1 to 6 on calcium oxalate crystallization, characterized in that, Includes the following steps: (a) Add the test extract of Nephrolepis cordifolia to a crystallization system containing calcium ions and oxalate ions to form a test solution; (b) Monitor the change in conductivity of the test solution over time in real time; (c) The change in conductivity was compared with a control system without the addition of pteris extract; If the conductivity of the test solution decreases more slowly than that of the control system, or if a longer induction period occurs, it indicates that the extract of Nephrolepis cordifolia can inhibit the nucleation and / or growth process of calcium oxalate crystals.

Images