A polysaccharide with protective effects against kidney damage

Abstract

The present invention provides a polysaccharide that has a protective effect on kidney damage. The polysaccharide of the present invention is obtained from the receptacle of Podocarpus carylus or Podocarpus brevifolia, plants of the Podocarpaceae family, after ultrasonic extraction, distilled water reheating extraction, reduced pressure concentration, ethanol precipitation, and freeze-drying. According to content determination, the mass percentage of the polysaccharide of the present invention is greater than or equal to 50% in terms of glucose. The polysaccharide is composed of 7 monosaccharides, including mannose, rhamnose, glucose, galactose, xylose, arabinose and L-fucose. In vivo experiments on mice have confirmed that the polysaccharide of the present invention has a good protective effect on kidney damage caused by cisplatin. The polysaccharide of the present invention can be used to prepare medicines with a protective effect on kidney damage.

Description

Technical Field

[0001] The invention belongs to the technical fields of natural medicinal chemistry, traditional Chinese medicine chemistry, pharmacology and toxicology, and particularly relates to a plant polysaccharide having a protective effect on kidney damage. Background Art

[0002] Kidney injury is damage to renal tissue caused by various bodily and external factors. Clinical symptoms often include a sharp decline in renal function, elevated serum creatinine, and decreased urine output. Severe cases can lead to shock and infection, leading to critical illness. Numerous factors can cause kidney injury, including: violent injuries such as collisions and falls; iatrogenic injuries such as extracorporeal shock wave lithotripsy; pathogenic injuries such as diabetic nephropathy and hypertensive nephropathy; and drug-induced injuries such as platinum-based antitumor drugs and high-dose nonsteroidal anti-inflammatory drugs. With the continued rise in the incidence of hypertension, diabetes, and malignant tumors, the incidence of hypertension- and diabetes-related kidney injury and drug-induced kidney injury is also increasing annually. However, there are currently few clinically effective protective agents or therapeutic drugs for kidney injury. Antihypertensive drugs that act on the renin-angiotensin-aldosterone system, such as captopril, valsartan, and irbesartan, can improve renal blood circulation and promote renal function recovery, and are therefore used to treat mild kidney injury. However, these drugs may have adverse effects on the cardiovascular system. Severe renal injury requires treatment with glucocorticoids, such as prednisone. However, hormonal therapy can cause serious adverse reactions. Furthermore, some traditional Chinese medicines with kidney-tonifying properties, such as Zhuangyao Jianshen Pills and Liuwei Dihuang Pills, are also used to treat renal injury. Therefore, the clinical use of drugs for the treatment of renal injury is limited. Therefore, the discovery of drugs with effective protective and therapeutic effects on renal injury is of great scientific value.

[0003] Podocarpus receptacle polysaccharide is a polysaccharide extracted from the receptacles of Podocarpus macrophyllus (Thunb.) D. Don or Podocarpus macrophyllus (Thunb.) D. Don var. maki (Sieb.) Endl., plants of the Podocarpaceae family. The receptacles and seeds of Podocarpus are components of the traditional Chinese medicine Semen Podocarpi. The "Compendium of Chinese Materia Medica" states that Podocarpus receptacle has the effects of promoting blood circulation, relieving pain, and tonifying the kidneys and lungs. In folk medicine, Podocarpus receptacle is used as a component of traditional Chinese medicine formulas to improve blood circulation in the brain. Therefore, Podocarpus receptacle polysaccharide is likely to have certain preventive and therapeutic effects on kidney damage. However, no research has been reported on the chemical composition of Podocarpus receptacle polysaccharides or their therapeutic or protective effects on kidney damage.

[0004] The following two invention patents related to Podocarpus receptacle polysaccharide have been published in China.

[0005] Huang Zengqiong (CN2019100509349) disclosed the use of Podocarpus macrophylla polysaccharide in the preparation of medicines or health foods for preventing and treating liver cancer.

[0006] Huang Zengqiong (CN2020113914088) disclosed the composition of Podocarpus macrophylla polysaccharide and its use in immune regulation function.

[0007] In addition, the following three domestic invention patents related to Podocarpus fruit extract have been disclosed.

[0008] Zeng Fanyan (CN2021104463996) disclosed a composition with anti-nasopharyngeal carcinoma effect, which is composed of two raw materials: Podocarpus fruit volatile oil and Podocarpus fruit total flavonoids.

[0009] Huang Zengqiong (CN201610460262) disclosed a method for efficiently extracting and separating high-purity ligustrazine from Podocarpus seeds.

[0010] Zhang Mingdong (CN201110095609) discloses a method for producing a Podocarpus fruit extract and its products. This patent application protects the preparation method of the Podocarpus fruit ethanol extract concentrate and its application in the food industry.

[0011] None of the above patents disclose the Podocarpus receptacle polysaccharide described in the present invention, nor do they disclose that the Podocarpus receptacle polysaccharide has a protective effect on renal damage. Although the invention patents (CN2019100509349) and (CN2020113914088) disclose the preparation method, composition and use of Podocarpus fruit polysaccharide in the prevention and treatment of liver cancer and immunoregulation. However, the extraction and separation method of the Podocarpus receptacle polysaccharide described in the present invention is different from the extraction and separation method of the disclosed invention patent, and the purity and monosaccharide composition of the obtained polysaccharide are also different. These differences make the polysaccharide described in the present invention have different biological activities from the polysaccharides described in the disclosed invention patents. Therefore, whether the Podocarpus receptacle polysaccharide described in the present invention has a protective effect on renal damage and how effective it is, professionals in this field cannot predict. Summary of the Invention

[0012] The present invention provides a plant polysaccharide having a protective effect on kidney damage.

[0013] The Podocarpus receptacle polysaccharide having a protective effect on kidney damage of the present invention has the following characteristics:

[0014] (1) The preparation method of the polysaccharide is as follows: take dried Podocarpus receptacle, add purified water 5 times the mass of the receptacle, extract with ultrasound, ultrasonic power 100W, water temperature 45°C, ultrasound for 30 minutes, then extract in a water bath at 60-75°C for 1 hour, filter, extract the residue again 3 times by the same method, combine the 3 filtrates, concentrate under reduced pressure in a water bath at 60°C to a volume of 80-100 ml, add an equal volume of 95% ethanol, place in a refrigerator at 4°C for 24 hours, collect the precipitate, rinse the precipitate 3 times with anhydrous ethanol, and freeze-dry to obtain Podocarpus receptacle polysaccharide;

[0015] (2) The mass percentage of the polysaccharide as glucose is greater than or equal to 50%;

[0016] (3) The mass percentage of protein in the polysaccharide is less than or equal to 0.05%;

[0017] (4) The polysaccharide is composed of seven monosaccharides: mannose, rhamnose, glucose, galactose, xylose, arabinose and L-fucose.

[0018] The podocarpus receptacle polysaccharide having a protective effect on kidney damage of the present invention is characterized in that the kidney damage is caused by metal platinum anti-tumor drugs.

[0019] The podocarpus receptacle polysaccharide of the present invention can be used to prepare medicines with kidney damage protective effects, wherein the amount of the podocarpus receptacle polysaccharide used is 20-30% by weight of the auxiliary materials.

[0020] In vivo animal experiments have confirmed that the Podocarpus receptacle polysaccharide of the present invention has a good protective effect on cisplatin-induced renal damage in mice and can significantly reduce renal damage caused by the metal platinum anti-tumor drug cisplatin.

[0021] The following will further describe the content of the present invention in detail with specific embodiments, but the scope of protection claimed by the present invention is not limited to the following embodiments. All technologies realized based on the above content of the present invention belong to the scope of the present invention. DETAILED DESCRIPTION

[0022] Example 1

[0023] Take 200g of dried Podocarpus receptacle, add 1000ml of distilled water, heat to 45°C, set the ultrasonic power to 100W, and perform ultrasonic extraction for 30min. Transfer the extract and receptacle to a round-bottom flask, extract in a 65°C water bath for 1 hour, filter, take the filtrate, and extract the residue twice in the same way. Combine the filtrates three times and concentrate under reduced pressure in a 60°C water bath. When the volume of the concentrate is 100ml, take it out, cool it, add 100ml of 95% ethanol, and place it in a 4°C refrigerator for 24 hours. Filter, collect the precipitate, rinse the precipitate three times with anhydrous ethanol, and freeze-dry to obtain Podocarpus receptacle polysaccharide.

[0024] An appropriate amount of polysaccharide was taken and the mass percentage of the polysaccharide was determined by the phenol-sulfuric acid method. The mass percentage of the polysaccharide was 50.56% based on glucose. An appropriate amount of polysaccharide was taken and the mass percentage of the protein in the polysaccharide was determined by the Coomassie brilliant blue method. The result showed that the mass percentage of protein was 0.03%.

[0025] Liquid chromatography tandem mass spectrometry (LC-MS) was used to determine the monosaccharide composition of the Podocarpus receptacle polysaccharide. The results showed that the polysaccharide was composed of seven monosaccharides: mannose, rhamnose, glucose, galactose, xylose, arabinose, and L-fucose.

[0026] Example 2

[0027] Take 100g of dried Podocarpus receptacle, add 500ml of distilled water, heat to 45°C, set the ultrasonic power to 100W, and perform ultrasonic extraction for 30min. Transfer the extract and receptacle to a round-bottom flask, extract in a 75°C water bath for 1 hour, filter, take the filtrate, and extract the residue twice in the same way. Combine the three filtrates, concentrate under reduced pressure in a 60°C water bath, and when the volume of the concentrate is 80ml, take it out, cool, add 80ml of 95% ethanol, and place it in a 4°C refrigerator for 24 hours. Filter, collect the precipitate, rinse the precipitate three times with anhydrous ethanol, and freeze-dry to obtain Podocarpus receptacle polysaccharide.

[0028] An appropriate amount of polysaccharide was taken and the mass percentage of the polysaccharide was determined by the phenol-sulfuric acid method. The mass percentage of the polysaccharide was 51.30% based on glucose. An appropriate amount of polysaccharide was taken and the mass percentage of the protein in the polysaccharide was determined by the Coomassie brilliant blue method. The result showed that the mass percentage of protein was 0.05%.

[0029] Liquid chromatography tandem mass spectrometry (LC-MS) was used to determine the monosaccharide composition of the Podocarpus receptacle polysaccharide. The results showed that the polysaccharide was composed of seven monosaccharides: mannose, rhamnose, glucose, galactose, xylose, arabinose, and L-fucose.

[0030] Example 3

[0031] Protective effect of Podocarpus receptacle polysaccharide on cisplatin-induced renal injury in mice

[0032] 1. Establishment of Renal Injury Model

[0033] Adult male SPF Kunming mice weighing (20±2) g were randomly divided into six groups: blank group, model group, polysaccharide group, and cisplatin plus polysaccharide low-, medium-, and high-dose groups. The blank and model groups received intraperitoneal injections of normal saline; the polysaccharide (SPP) group received intraperitoneal injections of 480 mg / ml of Podocarpus receptacle polysaccharide; and the cisplatin plus SPP low-, medium-, and high-dose groups received SPP at doses of 120, 240, and 480 mg / ml, respectively. Each group received medication once daily for 14 consecutive days, and the body weight, activity, food intake, and excretion of the mice were recorded daily. On day 14, all groups, except the blank and polysaccharide groups, received intraperitoneal injections of 25 mg / kg of cisplatin. The mice were harvested on day 17, having been deprived of food and water for 12 hours prior to harvesting. Blood samples were collected by eye extraction, and mice were sacrificed by cervical dislocation. During autopsy, both kidneys were observed and quickly removed, washed twice with saline, dried on filter paper, and weighed and recorded. Finally, the left and right kidney tissues were fixed with 4% paraformaldehyde and stored at -80°C for biochemical analysis.

[0034] 2. Determination of serum and renal tissue biochemical indicators and observation of renal tissue pathological sections

[0035] After refrigerating blood samples overnight at 4°C, centrifuge at 3000 rpm for 10 minutes at 4°C. Carefully collect the upper serum layer, aliquot into multiple tubes, and store frozen at -20°C. This was used to measure renal function indicators such as serum creatinine and blood urea nitrogen, as well as serum levels of IL-1β, IL-6, IL-10, and TNF-α. To prepare a mouse kidney homogenate, transfer 0.5 g of kidney tissue into an EP tube and add 4.5 mL of normal saline. Place the EP tube in a tissue homogenizer and repeatedly grind to produce a 10% kidney tissue homogenate. Centrifuge at 3000 rpm for 10 minutes, collect the supernatant, and store in a refrigerator at 4°C. Glutathione peroxidase, malondialdehyde, and superoxide dismutase activities were measured.

[0036] All experimental data were processed using SPSS 17.0 software. The experimental results were expressed as mean ± standard deviation. One-way analysis of variance was used to analyze the differences between the groups. The differences were considered statistically significant when P < 0.05.

[0037] 3. Results

[0038] 3.1 Effects of Podocarpus receptacle polysaccharide on serum biochemical parameters in mice

[0039] As shown in Table 1, compared with the blank group, serum creatinine (Cr) and blood urea nitrogen (BUN) levels in the model group mice were significantly increased (P < 0.01), indicating that cisplatin injection induced renal damage and confirming successful model establishment. Compared with the model group, Cr and BUN levels in the SPP group were significantly decreased (P < 0.01). Cr levels were significantly reduced in the cisplatin + SPP medium- and low-dose groups (P < 0.05), and BUN levels were significantly reduced in the cisplatin + SPP low-dose group (P < 0.05). This suggests that SPP can alleviate cisplatin-induced renal damage.

[0040] Table 1 Effects of SPP on serum creatinine and urea nitrogen levels in mice (x±s, n=8)

[0041]

[0042] Compared with the blank group: ▲ P<0.05, ▲▲ P<0.01; compared with the model group: * P<0.05, ** P<0.01.

[0043] 3.2 Effects of Podocarpus receptacle polysaccharide on biochemical parameters of mouse kidney tissue homogenate

[0044] As shown in Table 2, compared with the blank group, the MDA content in the renal tissue homogenate of the model group mice was significantly increased (P < 0.01), and the GSH activity was significantly decreased (P < 0.01). Although the SOD activity was decreased, the difference was not statistically significant (P > 0.05). This indicates that cisplatin can cause kidney damage by inhibiting GSH activity.

[0045] Compared with the model group, MDA levels in the SPP group and all cisplatin + SPP dose groups were significantly decreased (P < 0.05). GSH activity was significantly increased, while SOD activity was significantly decreased in all cisplatin + SPP dose groups (P < 0.05). This suggests that SPP may protect against cisplatin-induced renal injury by reducing MDA levels and increasing GSH activity.

[0046] Table 2 Effects of SPP on MDA, GSH and SOD in mouse kidney tissue (x±s, n=8)

[0047]

[0048] Compared with the blank group: ▲ P<0.05, ▲▲ P<0.01; compared with the model group: * P<0.05, ** P<0.01.

[0049] 3.3 Effects of Podocarpus polysaccharide on serum cytokines in mice

[0050] As shown in Table 3, compared with the model group, the SPP group had significantly decreased IL-1β, IL-10, and TNF-α levels (P < 0.05). Although IL-6 levels decreased, the differences were not statistically significant (P > 0.05). Compared with the model group, IL-1β levels were significantly decreased in all cisplatin + SPP dose groups (P < 0.05). IL-6 and IL-10 levels were significantly decreased in the high- and medium-dose cisplatin + SPP groups, and TNF-α levels were significantly decreased in the low-dose cisplatin + SPP group, with statistically significant differences (P < 0.01). These results suggest that SPP can protect against cisplatin-induced renal injury by reducing the levels of the inflammatory factors IL-1β and IL-6 in the serum and reducing TNF-α levels.

[0051] Table 3 Effects of SPP on the levels of IL-1β, IL-6, IL-10 and TNF-α in mouse serum (x±s, n=8)

[0052]

[0053] Compared with the model group: * P<0.05, ** P<0.01

[0054] In summary, the Podocarpus receptacle polysaccharide described in the present invention can significantly reduce the release of inflammatory factors caused by kidney damage, has a good protective effect on kidney damage caused by cisplatin, and is worthy of further development and utilization.

[0055] Example 4

[0056] Preparation of Podocarpus receptacle polysaccharide granules

[0057] Take 1 kg of Podocarpus receptacle polysaccharide with a mass percentage of 60.30%, add 2000 ml of distilled water, and heat at 60°C to dissolve to prepare a polysaccharide concentrate. Take another 5 kg of starch, add the polysaccharide concentrate while stirring, mix evenly, spray 70% ethanol, mix evenly, granulate, dry, shape the granules, and bag to obtain the product.

[0058] Example 5

[0059] Preparation of Podocarpus receptacle polysaccharide capsules

[0060] Take 200g of 70% by weight Podocarpus receptacle polysaccharide, add 500ml of distilled water, and heat at 60°C to dissolve to prepare a polysaccharide concentrate. Separately, take 500g of starch and 500g of dextrin, mix them evenly, add the polysaccharide concentrate while stirring, mix evenly, spray in 75% ethanol, mix evenly, granulate, dry, shape the granules, and fill them into hard capsules to obtain the product.

Claims

1. A podocarpus receptacle polysaccharide having a protective effect on kidney damage, characterized in that: (1) The preparation method of the polysaccharide is as follows: take dried Podocarpus receptacle, add purified water 5 times the mass of the receptacle, extract with ultrasound, ultrasonic power 100W, water temperature 45°C, ultrasound for 30 minutes, then extract in a water bath at 60-75°C for 1 hour, filter, extract the residue twice in the same way, combine the filtrates, concentrate under reduced pressure in a water bath at 60°C to a volume of 80-100 ml, add an equal volume of 95% ethanol, place in a refrigerator at 4°C for 24 hours, collect the precipitate, rinse the precipitate three times with anhydrous ethanol, and freeze-dry to obtain Podocarpus receptacle polysaccharide; (2) The mass percentage of the polysaccharide as glucose is greater than or equal to 50%; (3) The mass percentage of protein in the polysaccharide is less than or equal to 0.05%; (4) The polysaccharide is composed of seven monosaccharides: mannose, rhamnose, glucose, galactose, xylose, arabinose and L-fucose.

2. The podocarpus receptacle polysaccharide having a protective effect on kidney damage according to claim 1, characterized in that The kidney damage is caused by metal platinum anti-tumor drugs.

3. Use of the Podocarpus receptacle polysaccharide according to claim 1 in the preparation of a medicine having a protective effect against kidney damage.

4. The use of Podocarpus receptacle polysaccharide as claimed in claim 3, characterized in that In the medicine, the amount of Podocarpus receptacle polysaccharide is 20-30% by weight of the auxiliary materials.

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