Use of Morinda officinalis polysaccharide in preparing products for improving renal fibrosis

Abstract

The present invention relates to the field of pharmaceutical technology, and more particularly to the use of Morinda officinalis polysaccharide in preparing a product for improving renal fibrosis. The preparation method of the Morinda officinalis polysaccharide comprises the following steps: (1) extracting Morinda officinalis from water, filtering, and obtaining a supernatant; (2) subjecting the supernatant to alcohol precipitation to obtain a precipitate; and (3) dialyzing and removing protein from the precipitate. The Morinda officinalis polysaccharide has a significant therapeutic effect in improving renal fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to use of Morinda officinalis polysaccharide in preparing a product for improving renal fibrosis. Background Art

[0002] Renal interstitial fibrosis (RIF) is a hallmark and ultimate outcome of various progressive chronic kidney diseases (CKD). CKD is considered a major cause of renal failure. Renal fibrosis is caused by the accumulation of connective tissue in the kidneys. This process gradually disrupts the structure and function of the renal tubules, glomeruli, and interstitium, thereby affecting the normal functioning of the kidneys.

[0003] In-depth research into the pathophysiological mechanisms of renal infarction (RIF) in recent years has revealed that the immune response plays a crucial role in the development and progression of renal infarction (RIF). Modulating the immune response may be a key pathway for altering the course of RIF. Imbalanced immune regulation is closely associated with the development of RIF. In CKD, persistent immune activation leads to massive inflammatory cell infiltration, renal parenchymal cell damage, or fibroblast activation, promoting persistent renal fibrosis and ultimately, progression to chronic renal failure. During the course of RIF, the immune response is often abnormally activated, resulting in significant changes in the renal immune environment. This persistent activation of the immune response is caused by pathological inflammation and is closely associated with the involvement of multiple immune cells in the kidney. For example, the infiltration and persistent activation of immune cells such as T cells, B cells, macrophages, and dendritic cells promotes the exacerbation of the local inflammatory response. Immune cell infiltration and the cytokines and chemokines they secrete further exacerbate renal parenchymal cell damage and dysfunction. Therefore, modulating the renal immune response has become a potential therapeutic target for altering the course of RIF.

[0004] Morinda officinalis is the dried root of Morinda officinalis, a plant of the Rubiaceae family. It is pungent, sweet, and slightly warm. It enters the kidney meridian and has the effects of tonifying the kidney and promoting yang, dispersing wind and dispelling cold and dampness. Morinda officinalis polysaccharide is one of the main active ingredients of Morinda officinalis. The existing technology (Liang Limin, Xu Yong. Research Progress of Morinda officinalis Polysaccharide in Southern Medicinal Plants [J]. Food Industry Science and Technology, 2011(8):3.) discloses that Morinda officinalis polysaccharide has significant biological activities in many aspects, such as anti-oxidation, hypoglycemic, immunomodulation, and anti-osteoporosis. Among them, Morinda officinalis polysaccharide can significantly increase the thymus index and spleen index of immunosuppressed mice, enhance macrophage phagocytosis, and promote lymphocyte transformation. Its mechanism involves regulating the balance of Th1 / Th2 cells and inhibiting the secretion of pro-inflammatory factors, thereby alleviating the inflammatory response of liver tissue.

[0005] However, the prior art does not disclose the use of Morinda officinalis polysaccharide in improving renal fibrosis. Summary of the Invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a use of Morinda officinalis polysaccharide in the preparation of a product for improving renal fibrosis. Morinda officinalis polysaccharide has a significant therapeutic effect in improving renal fibrosis.

[0007] The present invention is achieved through the following technical solutions:

[0008] The present invention provides use of Morinda officinalis polysaccharide in preparing a product for improving renal fibrosis.

[0009] As an embodiment of the present invention, the preparation method of Morinda officinalis polysaccharide comprises the following steps:

[0010] (1) Add Morinda officinalis to water for extraction, filter, and obtain the supernatant;

[0011] (2) performing alcohol precipitation on the supernatant to obtain a precipitate;

[0012] (3) The precipitate is dialyzed and protein removed to obtain the product.

[0013] As a preferred embodiment of the present invention, the extraction process parameters in step (1) are: reflux extraction at 72-90 °C for 1-2 h.

[0014] As a preferred embodiment of the present invention, the extraction in step (1) is performed 1-3 times, and the supernatants obtained each time are combined.

[0015] As a preferred embodiment of the present invention, the mass ratio of Morinda officinalis to water in step (1) is 1:0.5-2.

[0016] As a preferred embodiment of the present invention, after filtering in step (1), an extract is obtained, and the extract is centrifuged to obtain a supernatant.

[0017] More preferably, the centrifugation parameters are: 3000-5000 rpm for 5-20 min.

[0018] Particularly preferably, step (1) includes the following steps:

[0019] Add 1 times the amount of water to extract Morinda officinalis 1-3 times, reflux extraction at 72-90 ℃ for 1-2 hours each time, filter and obtain the supernatant.

[0020] As a preferred embodiment of the present invention, the specific operation of the alcohol precipitation in step (2) is: adding ethanol to an alcohol content of 75-85%, letting it stand, and obtaining a precipitate.

[0021] Further preferably, the parameters of the standing in step (2) are: standing at 1-10°C for 12-24 hours.

[0022] As a preferred embodiment of the present invention, the dialysis in step (3) is performed using water with a molecular weight cut-off of 3.5 kDa.

[0023] As a preferred embodiment of the present invention, the protein removal method in step (3) is the Savage method.

[0024] As a preferred embodiment of the present invention, step (3) includes: washing, dialyzing, decolorizing, deproteinizing, and drying the precipitate to obtain Morinda officinalis polysaccharide.

[0025] Further preferably, the reagent used for washing is 85-95% ethanol.

[0026] Further preferably, the decolorization is carried out using activated carbon.

[0027] Further preferably, the drying method is freeze-drying, and the freeze-dried powder is off-white.

[0028] As an embodiment of the present invention, the polysaccharide content of the Morinda officinalis polysaccharide is greater than 85%, and more preferably greater than or equal to 94%.

[0029] The beneficial effects of the present invention are:

[0030] The Morinda officinalis polysaccharide prepared by the present invention has a significant therapeutic effect in improving renal fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the infrared spectrum of the Morinda officinalis polysaccharide prepared in Example 1.

[0032] Figure 2 This is the glucose standard curve drawn for Test Example 1 of the present invention.

[0033] Figure 3 The weight growth trend and renal index of each group of mice in Test Example 2; among them, A is the weight growth trend from the beginning of modeling to sampling; B is the weight change of mice at the time of sampling compared with the beginning of modeling; C is the renal index at the time of sampling; in the figure, ns represents no significant difference between the groups; ** represents a significant difference compared with the RIF model group, p < 0.01; *** represents a significant difference compared with the RIF model group, p < 0.001.

[0034] Figure 4Serum biochemical indicators of mice in each group of test example 2; ns in the figure represents no significant difference between the groups; * represents a significant difference compared with the RIF model group, p < 0.05; *** represents a significant difference compared with the RIF model group, p < 0.001.

[0035] Figure 5 The urine biochemical indicators of mice in each group of test example 2; in the figure, * represents a significant difference compared with the RIF model group, p < 0.05; ** represents a significant difference compared with the RIF model group, p < 0.01; *** represents a significant difference compared with the RIF model group, p < 0.001.

[0036] Figure 6 The histomorphology and pathological staining results of each group of mice in Test Example 2 are shown. DETAILED DESCRIPTION

[0037] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0038] Example 1 Preparation of Morinda officinalis polysaccharide

[0039] Ultrapure water (100 g) and Morinda officinalis (100 g) slices were mixed and extracted under reflux at 90°C for 2 h. The extracted solution was centrifuged at 4000 rpm for 20 min, and the supernatant was harvested. This process was repeated for a second extraction and centrifugation. After multiple filtrations, the supernatants were combined. The resulting mixture was treated with anhydrous ethanol to an 80% alcohol content and allowed to stand at 4°C for 24 h to precipitate the crude polysaccharide.

[0040] The crude polysaccharide was washed twice with 95% ethanol, then dialyzed against distilled water (using a 3.5 kDa molecular weight cutoff), decolorized using activated carbon, and deproteinized using the Savage method (Sevage reagent (n-butanol:chloroform volume ratio of 1:4) and 500 mg / ml polysaccharide solution volume ratio of 1:2, shaken for 20 minutes, centrifuged at 3000 rpm for 3 minutes, and the supernatant was retained) to obtain purified polysaccharide. The purified polysaccharide was lyophilized to an off-white powder, obtaining Morinda officinalis polysaccharide. The infrared spectrum of Morinda officinalis polysaccharide is shown in Figure 2. Figure 1 shown.

[0041] Test Example 1 Determination of polysaccharide content

[0042] 1. Drawing of glucose standard curve

[0043] Take 10 mg of glucose reference substance, accurately weigh it, dissolve it in distilled water, and then transfer it to a 100 ml volumetric flask and make up to volume to prepare a glucose reference substance stock solution with a concentration of 0.100 mg / mL.

[0044] The prepared glucose reference stock solution was diluted to the following concentration series: 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.08 mg / mL, 0.10 mg / mL, then 1.0 mL was drawn into a test tube, 1.0 mL of 5% phenol reagent and 5.0 mL of 98% concentrated sulfuric acid were added, shaken and boiled for 30 min. The absorbance (A) was measured at 490 nm using an ultraviolet spectrophotometer, and a standard curve was drawn with the glucose concentration (C) as the horizontal axis and A as the vertical axis. Figure 2 shown.

[0045] 2. Determination of polysaccharide content

[0046] Accurately weigh approximately 0.2 g (B) of the Morinda officinalis polysaccharide prepared in Example 1 into a 100 mL volumetric flask. Dissolve it in distilled water and dilute to the mark. Remove 10 mL and transfer it to the 100 mL volumetric flask. Dilute to the mark again with distilled water. Then, pipette 1.0 mL into a test tube. Add 1.0 mL of 5% phenol reagent and 5.0 mL of concentrated sulfuric acid, shake well, and boil for 30 minutes. Measure the absorbance (A1) at 490 nm using a UV spectrophotometer. Substitute the A1 value into the linear regression equation of the standard curve to obtain the polysaccharide concentration C1.

[0047] The absorbance A1 was measured three times and the average value was 1.406. It was substituted into the linear regression equation of the standard curve to obtain the polysaccharide concentration C1 of 0.1882 mg / mL. The polysaccharide content of Morinda officinalis polysaccharide was calculated to be 94.1%.

[0048] Test Example 2: Improvement of Renal Fibrosis Test

[0049] 1. Experimental Animals

[0050] Forty C57BL / 6J mice were purchased from Guangdong Ruige Biotechnology Co., Ltd. Mice were housed in an SPF environment at the Animal Experimental Center of Guangdong Pharmaceutical University with a 12-h light / dark cycle, a temperature of 20–25°C, and a humidity of 60 ± 5%. Animals had free access to food and water. All mice were acclimated to a diet for 1 week before the experiment. The experimental protocol and procedures were approved by the Animal Experimentation Ethics Committee of Guangdong Pharmaceutical University and complied with the Guide for the Care and Use of Laboratory Animals.

[0051] 2. Drug-induced renal fibrosis (RIF) and treatment

[0052] Forty mice were randomly divided into four groups, each with 10 mice. One group served as a healthy control group and was fed a normal diet. The other three groups of 30 mice served as model mice and were pre-fed with a diet containing 0.25% adenine for 5 weeks. This diet was then switched to a 0.1% adenine diet until the 11th week. Treatment began in the 6th week: one group served as a renal fibrosis (RIF) model group and was given water for 6 weeks; one group served as a losartan positive control group and was given losartan for 6 weeks; and one group served as a Morinda officinalis polysaccharide (MOPs) group and was given Morinda officinalis polysaccharide prepared in Example 1 for 6 weeks.

[0053] The specific groups are as follows:

[0054] (1) Renal fibrosis (RIF) model group (RIF): adenine feed, oral administration of distilled water (100 μL / D, D represents day);

[0055] (2) Healthy control group (Control, Ctrl): normal diet, oral administration of distilled water (100 μL / D);

[0056] (3) Losartan drug positive control group (LOS-Postive Control, LOS): adenine feed, oral administration of losartan aqueous solution 100 μL / D (losartan aqueous solution concentration 30 mg / kg);

[0057] (4) Morinda officinalis polysaccharide group (MOPs): adenine feed, oral administration of MOPs aqueous solution 100 μL / D (MOPs aqueous solution concentration was 200 mg / kg).

[0058] 3. Weight change trend and kidney index

[0059] The weight growth trend of the experimental animals from the beginning of modeling to sampling, as well as the weight growth and kidney index at the time of sampling were recorded. The samples were collected on the last day of the 11th week. The results are as follows Figure 3 As shown. Figure 3 The results showed that the body weight and kidney index of the renal fibrosis (RIF) model group were significantly reduced, and treatment with Morinda officinalis polysaccharide could significantly increase the body weight and kidney index of mice. There was no significant difference in the body weight of mice in the losartan drug positive control group compared with the renal fibrosis (RIF) model group (p>0.05), but the kidney index of mice was significantly improved.

[0060] 4. Biochemical indicators

[0061] Blood samples were obtained from mice after euthanasia on the last day of week 11 and centrifuged at 3000 rpm for 15 min to obtain serum. Serum urea nitrogen biochemical indicators were measured using a biochemical indicator detection kit. The results are shown in Figure 2. Figure 4 shown.

[0062] One day before the end of the treatment cycle, 24-hour urine was collected from the mice in a metabolic cage. Biochemical indicators such as urine protein, urea nitrogen, and creatinine were measured using a biochemical indicator detection kit. Figure 5 shown.

[0063] Depend on Figure 4 and Figure 5 It can be seen that Morinda officinalis polysaccharide significantly reduced the serum urea nitrogen level, inhibited the increase of 24-hour proteinuria, and significantly enhanced the kidney's metabolic rate of urea nitrogen and creatinine.

[0064] 5. Histomorphology and pathology

[0065] Kidney and colon tissues were obtained from mice euthanized on the last day of week 11 and fixed in 4% paraformaldehyde solution at 4°C overnight. They were then embedded in paraffin and cut into 4 μm sections. Glomerular structure was observed using hematoxylin-eosin (HE) staining. Masson staining revealed the degree of renal fibrosis. Figure 6 shown.

[0066] Figure 6 In histomorphological studies, the kidneys of mice in the RIF model group showed significant atrophy and fibrosis compared to the healthy control group, while the group treated with Morinda officinalis polysaccharides showed an improvement trend. HE staining revealed glomerular atrophy in RIF mice, which was alleviated by Morinda officinalis polysaccharide treatment. Masson staining, as indicated by the arrows, showed a significant reduction in the fibrosis-positive area in the Morinda officinalis polysaccharide (MOPs)-treated group.

[0067] The above detailed description is a specific description of one feasible embodiment of the present invention. This embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not depart from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. Use of Morinda officinalis polysaccharide in preparing a product for improving renal fibrosis.

2. The use according to claim 1, characterized in that The preparation method of the Morinda officinalis polysaccharide comprises the following steps: (1) Add Morinda officinalis to water for extraction, filter, and obtain the supernatant; (2) performing alcohol precipitation on the supernatant to obtain a precipitate; (3) The precipitate is dialyzed and protein removed to obtain the product.

3. The use according to claim 2, characterized in that The extraction process parameters in step (1) are: reflux extraction at 72-90° C. for 1-2 h; and the number of extractions is 1-3 times.

4. The use according to claim 2, characterized in that In step (1), the mass ratio of Morinda officinalis to water is 1:0.5-2; after filtering, an extract is obtained, and the extract is centrifuged to obtain a supernatant.

5. The use according to claim 2, characterized in that The specific operation of the alcohol precipitation in step (2) is: adding ethanol until the alcohol content is 75-85%, letting it stand, and obtaining a precipitate.

6. The use according to claim 5, characterized in that The parameters for the standing state in step (2) are: standing state at 1-10°C for 12-24 hours.

7. The use according to claim 2, characterized in that The dialysis in step (3) is performed using water with a molecular weight cut-off of 3.5 kDa; and / or the protein removal method is the Savage method.

8. The use according to claim 2, characterized in that Step (3) includes: washing, dialyzing, decolorizing, removing protein, and drying the precipitate to obtain Morinda officinalis polysaccharide.

9. The use according to claim 8, characterized in that The washing agent is ethanol with a volume concentration of 85-95%; the decolorization is performed using activated carbon; and the drying method is freeze-drying.

10. The use according to claim 1, characterized in that The polysaccharide content of the Morinda officinalis polysaccharide is ≥94%.

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