Xanthoceraside and preparation method and application thereof

Abstract

This invention discloses a *Xanthoceras sorbifolium* leaf extract, its preparation method, and its application. The preparation method includes the following steps: (1) drying the *Xanthoceras sorbifolium* leaves, pulverizing them, and sieving them; (2) hot-extracting the sieved *Xanthoceras sorbifolium* leaf powder with ethanol to obtain an ethanol extract of *Xanthoceras sorbifolium* leaves; (3) concentrating the ethanol extract of *Xanthoceras sorbifolium* leaves and freeze-drying it to obtain the *Xanthoceras sorbifolium* leaf extract. This invention uses a simple ethanol hot extraction method to obtain the *Xanthoceras sorbifolium* leaf extract. The *Xanthoceras sorbifolium* leaf extract obtained by this invention has significant inhibitory activity against xanthine oxidase.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an extract of *Sapindus mukorossi* leaves, its preparation method, and its application. Background Technology

[0002] Hyperuricemia (HUA) is a metabolic disorder affecting organs such as the liver, intestines, and kidneys, caused by the continuous accumulation of uric acid in the blood. In recent years, the incidence of hyperuricemia has risen sharply, with a significant trend towards affecting younger people, seriously endangering human health. The pathological characteristic of hyperuricemia is a serum uric acid (UA) concentration exceeding the physiological solubility threshold. According to international standards, hyperuricemia is diagnosed when a fasting serum uric acid concentration exceeds 416.4 μmol / L for men and 356.9 μmol / L for women. The imbalance between uric acid secretion and excretion is the main contributing factor to hyperuricemia. Lowering serum uric acid levels is the primary method for preventing and treating hyperuricemia. Xanthine oxidase (XOD), a key enzyme in purine metabolism, catalyzes the oxidation of xanthine and hypoxanthine in the body to uric acid, while simultaneously generating peroxide free radicals, making it an important target for the prevention and treatment of hyperuricemia. Xanthine oxidase inhibitors have attracted much attention due to their effectiveness in lowering uric acid levels. Allopurinol and febuxostat are commonly used xanthine oxidase inhibitors in clinical practice. While they show good clinical performance, they frequently cause serious side effects, limiting their clinical application to some extent. Therefore, the search for novel, highly effective, and low-toxicity xanthine oxidase inhibitors remains a hot research topic in the pharmaceutical field. Compared to Western medicine, traditional Chinese medicine has advantages such as wide availability, mild effects, high safety, low toxicity, broad pharmacological effects, improvement of secondary symptoms, and suitability for long-term use, making it highly promising for development and utilization. Currently, dietary regulation of human health is gaining popularity, and medicinal and edible plants are receiving unprecedented attention, providing more options for developing drugs for the prevention and treatment of hyperuricemia and functional health foods.

[0003] In recent years, numerous studies have focused on the inhibitory effects of extracts from traditional Chinese medicine on xanthine oxidase, but there are few reports on the inhibitory effects of *Xanthine saxifrage* leaf extract on xanthine oxidase activity. *Xanthine saxifrage* ( Xanthoceras sorbifoliumXanthoceras sorbifolium Bunge is a small tree or deciduous tree belonging to the genus Xanthoceras in the family Sapindaceae. It was first recorded in "Jiuhuang Bencao" (in 1406 AD) and was called "Xanthoceras flower". It is a rare woody oil and medicinal plant unique to China and is widely distributed in North China, Northeast China, and Northwest China. In 2023, the seeds and leaves of Xanthoceras sorbifolium Bunge were approved as new food raw materials. Modern research shows that the leaves of Xanthoceras sorbifolium Bunge contain components such as saponins, flavonoids, coumarins, sugars, amino acids, and trace elements, and have effects such as antioxidant, antibacterial, antiviral, anti-inflammatory, lipid-lowering, blood pressure-lowering, and immune regulation, with high edible and medicinal values and worthy of development and utilization. Summary of the Invention

[0004] The primary object of the present invention is to provide an extract of Xanthoceras sorbifolium Bunge leaves with the effect of inhibiting xanthine oxidase and a preparation method thereof, and this extract of Xanthoceras sorbifolium Bunge leaves can be used to develop health products or drugs with the effect of inhibiting xanthine oxidase.

[0005] The present invention is achieved through the following technical solutions.

[0006] A preparation method of an extract of Xanthoceras sorbifolium Bunge leaves includes the following steps: [[ID=I2]]

[0007] (1) Dry the Xanthoceras sorbifolium Bunge leaves, crush them, and then sieve them.

[0008] (2) Use ethanol to conduct hot extraction on the sieved Xanthoceras sorbifolium Bunge leaf powder to obtain an ethanol extract of Xanthoceras sorbifolium Bunge leaves.

[0009] (3) Concentrate the ethanol extract of Xanthoceras sorbifolium Bunge leaves and then freeze-dry it to obtain an extract of Xanthoceras sorbifolium Bunge leaves.

[0010] Further, in step (1), the sieving is through a 50 - 80 mesh sieve.

[0011] Further, in step (2), the ethanol is ethanol with a volume fraction of 50 - 80%.

[0012] Further, in step (2), the material-liquid ratio of the Xanthoceras sorbifolium Bunge leaf powder to ethanol is 1:10 - 1:30 g / mL.

[0013] Further, in step (2), the hot extraction is: extract at a temperature of 50 - 80 °C for 1.5 - 6 h, repeat three times, and combine the filtrates.

[0014] Further, in step (3), the concentration is to concentrate to a density relative to water of 1.15 - 1.30.

[0015] An extract of Xanthoceras sorbifolium Bunge leaves obtained by the preparation method described in any one of the above.

[0016] Further, the extract of Xanthoceras sorbifolium Bunge leaves has the activity of inhibiting xanthine oxidase.

[0017] Furthermore, the inhibition of xanthine oxidase activity is manifested in in vitro inhibition of xanthine oxidase activity, and the in vitro inhibition of xanthine oxidase activity is detected by spectrophotometry.

[0018] Furthermore, the inhibition of xanthine oxidase activity is manifested by an IC50 value of 2.83 mg / mL for the inhibition of xanthine oxidase activity.

[0019] The *Xanthoceras sorbifolium* leaf extract can be used in the preparation of health foods or medicines that inhibit xanthine oxidase.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] (1) The advantages of the preparation method of the leaf extract of Xanthoceras sorbifolium in this invention are that the process is simple, the operation is convenient, the technology is easy to master, the energy consumption is low, the solvent can be recycled and reused, it is green and environmentally friendly, the production cost is low, and it is easy to realize large-scale industrial production.

[0022] (2) The leaf extract of *Sapindus mukorossi* prepared in this invention has good inhibitory activity against xanthine oxidase and can be further used in health foods or medicines with uric acid-lowering effects.

[0023] (3) The leaves of the Chinese saxifrage of the present invention are a new food raw material with high safety, and are widely distributed and readily available in my country. They have a very good prospect for development and utilization as a xanthine oxidase inhibitor. Attached Figure Description

[0024] Figure 1 This is a graph showing the inhibitory activity of allopurinol on xanthine oxidase.

[0025] Figure 2 The graph shows the inhibitory activity curve of Xanthoceras sorbifolium leaf extract against xanthine oxidase. Detailed Implementation

[0026] The present invention is further illustrated below by way of examples, but the present invention is not limited to the scope of the examples described. Experimental methods in the following examples that do not specify specific conditions are all in accordance with conventional methods and conditions or selected in accordance with the product instructions. The raw materials in the following examples are all commercially available.

[0027] Example 1

[0028] Extraction of Sapindus mukorossi leaf extract

[0029] The leaves of *Xanthoceras sorbifolium* were dried, pulverized, and passed through an 80-mesh sieve. 0.5 kg of the sieved *Xanthoceras sorbifolium* leaf powder was extracted with 15 L of 50% ethanol at 50°C for 6 h, repeated three times. The filtrates were combined to obtain an ethanol extract of *Xanthoceras sorbifolium* leaves. The ethanol extract was concentrated to a density relative to water of 1.30 and then freeze-dried to obtain 101 g of *Xanthoceras sorbifolium* leaf extract, with a yield of 20.2%.

[0030] Example 2

[0031] Extraction of Sapindus mukorossi leaf extract

[0032] The leaves of *Xanthoceras sorbifolium* were dried, pulverized, and passed through a 50-mesh sieve. 0.5 kg of the sieved *Xanthoceras sorbifolium* leaf powder was extracted with 5 L of 80% ethanol at 70°C for 3 h. This process was repeated three times, and the filtrates were combined to obtain the ethanol extract of *Xanthoceras sorbifolium* leaves. The ethanol extract of *Xanthoceras sorbifolium* leaves was concentrated to a density relative to water of 1.15 and then freeze-dried to obtain 109 g of *Xanthoceras sorbifolium* leaf extract, with a yield of 21.8%.

[0033] Example 3

[0034] Extraction of Sapindus mukorossi leaf extract

[0035] The leaves of *Xanthoceras sorbifolium* were dried, pulverized, and passed through a 65-mesh sieve. 0.5 kg of the sieved *Xanthoceras sorbifolium* leaf powder was extracted with 10 L of 70% ethanol at 80°C for 1.5 h, repeated three times. The filtrates were combined to obtain the ethanol extract of *Xanthoceras sorbifolium* leaves. The ethanol extract was concentrated to a density relative to water of 1.20 and then freeze-dried to obtain 125 g of *Xanthoceras sorbifolium* leaf extract, with a yield of 25.0%.

[0036] Example 4

[0037] Inhibitory activity of Xanthoceras sorbifolium leaf extract against xanthine oxidase

[0038] 1. Preparation of experimental samples and reagents

[0039] Xanthoceras sorbifolium leaf extract solutions: The Xanthoceras sorbifolium leaf extract prepared by the method described in Example 3 was formulated into Xanthoceras sorbifolium leaf extract solutions with concentrations of 1.67, 2.22, 3.33, 3.70, and 4.17 mg / mL, respectively.

[0040] Positive control solution: Accurately weigh 10 mg of allopurinol reference standard, place it in a 100 mL volumetric flask, dissolve it in 0.2 mol / L phosphate buffer (pH 7.58), and dilute to the mark. Shake well to obtain an allopurinol stock solution with a mass concentration of 0.1 mg / mL. Accurately measure an appropriate amount of the above stock solution and dilute it sequentially with the same buffer to prepare a series of allopurinol working solutions with mass concentrations of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, and 0.09 mg / mL, respectively, for later use.

[0041] 2. Experimental Procedure

[0042] 100 μL of 0.05 U / mL xanthine oxidase solution, 160 μL of 0.2 mol / L (pH=7.58) phosphate buffer, and 40 μL of Xanthoceras sorbitol leaf extract solution were added sequentially to a test tube, shaken well, and incubated at 37℃ for 5 min. Then, 100 μL of 1 mmol / L xanthine was quickly added to the test tube. After reacting for 30 min, 100 μL of 1 mol / L HCl solution was added to terminate the reaction, and the absorbance was measured at 295 nm.

[0043] 3. Calculate the inhibitory activity of XOD on *Sapindus mukorossi* leaf extract according to the formula.

[0044] Inhibition rate (%) = [(AB) - (CD)] / (AB) × 100%

[0045] A: 100 μL 0.05 U / mL xanthine oxidase + 200 μL 0.2 mol / L (pH=7.58) phosphate buffer + 100 μL 1 mmol / L xanthine.

[0046] B: 100 μL 0.2 mol / L (pH=7.58) phosphate buffer + 200 μL 0.2 mol / L (pH=7.58) phosphate buffer + 100 μL 1 mmol / L xanthine.

[0047] C: 100 μL 0.05 U / mL xanthine oxidase + 40 μL Xanthoceras sorbifolium leaf extract solution + 160 μL 0.2 mol / L (pH=7.58) phosphate buffer + 100 μL 1 mmol / L xanthine.

[0048] D: 100 μL 0.2 mol / L (pH=7.58) phosphate buffer + 40 μL Xanthoceras sorbifolium leaf extract solution + 160 μL 0.2 mol / L (pH=7.58) phosphate buffer + 100 μL 1 mmol / L xanthine.

[0049] Using allopurinol as a positive control, the inhibitory activity of different concentrations of allopurinol solutions on xanthine oxidase was determined using the same experimental method.

[0050] 4. Measurement Results

[0051] The results of the inhibitory activity of allopurinol on xanthine oxidase are as follows: Figure 1 As shown, the IC50 of allopurinol inhibits xanthine oxidase activity. 50 The value was 0.033 mg / mL. The results of the inhibitory activity of *Sapindus mukorossi* leaf extract against xanthine oxidase are as follows: Figure 2 As shown, the extract of *Xanthoceras sorbifolium* leaves exhibits significant xanthine oxidase inhibitory activity, with an IC50 value for inhibiting xanthine oxidase activity. 50 The value was 2.83 mg / mL.

Claims

1. A method for preparing an extract from the leaves of *Sapindus mukorossi*, characterized in that, Includes the following steps: (1) Dry the leaves of the Chinese tallow tree, crush them, and then sieve them; (2) The sieved Xanthoceras sorbifolium leaf powder was thermally extracted with ethanol to obtain Xanthoceras sorbifolium leaf ethanol extract; (3) The ethanol extract of *Sapindus mukorossi* leaves was concentrated and then freeze-dried to obtain *Sapindus mukorossi* leaf extract.

2. The method for preparing a *Xanthoceras sorbifolium* leaf extract according to claim 1, characterized in that, In step (1), the sieving is done through a 50-80 mesh sieve.

3. The method for preparing a *Sapindus mukorossi* leaf extract according to claim 1, characterized in that, In step (2), the ethanol is 50-80% by volume, and the ratio of the powder of *Sapindus mukorossi* leaves to ethanol is 1:10-1:30 g / mL. The hot extraction is performed at 50-80°C for 1.5-6 hours, repeated three times, and the filtrates are combined. In step (3), the concentration is performed to concentrate the powder to a density of 1.15-1.30 relative to water.

4. The *Xanthoceras sorbifolium* leaf extract obtained by the preparation method according to any one of claims 1 to 3, characterized in that, The extract of *Sapindus mukorossi* leaves has the activity of inhibiting xanthine oxidase.

5. The *Sapindus mukorossi* leaf extract according to claim 4, characterized in that, The inhibition of xanthine oxidase activity is manifested in vitro, and the in vitro inhibition of xanthine oxidase activity is detected by spectrophotometry.

6. The *Sapindus mukorossi* leaf extract according to claim 4, characterized in that, The inhibition of xanthine oxidase activity is reflected in an IC50 value of 2.83 mg / mL.

7. The application of the *Sapindus mukorossi* leaf extract according to claim 4, characterized in that, The *Xanthoceras sorbifolium* leaf extract can be used in the preparation of health foods or medicines that inhibit xanthine oxidase.

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