CN102585024A - Isaria feline crude polysaccharide, and preparation method and application thereof - Google Patents

Abstract

The invention discloses Isaria feline crude polysaccharide and a preparation method and application thereof in the preparation of medicaments for treating chronic renal failure. The preparation method of the Isaria feline crude polysaccharide comprises the steps of adding distilled water into hypha powder, wherein the weight of the distilled water is 3-5 times that of the hypha powder, then extracting in a boiling water bath for 70-110 minutes, and centrifuging for 4-6 minutes at a rotating speed being 3000 rounds/minutes; continuously extracting three times; combining extracted liquid supernatant, and concentrating to be 800-1000ml in a water bath way; and adding 3-4 times 95% ethanol, mixing uniformly and fully, and precipitating overnight so as to obtain precipitate, namely Isaria feline crude polysaccharide. A certain dosage of Isaria feline crude polysaccharide can obviously reduce levels of creatinine (CRE) and urea nitrogen (BUN) in renal failure rat blood serum, and therefore Isaria feline crude polysaccharide has a good protection effect on pathological harms to the kidney, and can partially improving an anemia symptom in chronic renal failure progressive disease. The Isaria feline crude polysaccharide can improve renal functionsand has obvious treat effect on the chronic renal failure.

Description

I. catalis crude polysaccharide and its preparation method and application

technical field

The invention relates to a fungal extract, in particular to a IFP crude polysaccharide (IFP) and a preparation method thereof, as well as the application of the IFP in the preparation of a medicine for treating chronic renal failure.

Background technique

Isaria felina (Isaria felina) is a strain isolated and cultured from the fruiting bodies of natural Cordyceps sinensis, and belongs to the class Deuteromycetes, the order Amuliacelia, and the genus Isaria felina. It was identified as Isaria felina (DC.: Fr) Fr by the Institute of Microbiology, Chinese Academy of Sciences. This strain has been preserved by the General Microbiology Center of the China Microbiological Culture Collection Management Committee (preservation number: CGMCC NO.0706), and has obtained a national invention patent ( ZL02103669.1).

Deffieux Gerard et al. (J Antibiot (Tokyo). 1981, 34 (10): 1261-1265.) extracted and separated from Isaria felina the insecticides Isariins B, C and D of the ring carboxyphenolic acid peptide structure, and to Isariins The structures of B, C and D were identified (J Antibiot (Tokyo). 1981, 34(10): 1266-1270.). Guo Yongxia isolated and purified the cyclic depsipeptide isarfelin with antifungal effect from the mycelium of Isarfelinus catalis, analyzed the physical and chemical properties and identified the structure of isarfelin, and found that isarfelin has the activity of inhibiting bacteria and resisting fungi (Guo Yongxia. Purification and Characterization of Antifungal Polypeptides from I. catalis and Tricholoma gigantea: [Doctoral Dissertation]. Beijing: China Agricultural University, 2005). Ikumoto et al. found that the leachate of Cordyceps sinensis, Cordyceps militaris and I. catissima had a negative contractile effect on the isolated atrium, and at the same time had an inhibitory effect on the contractile response of the electrically stimulated ileum and the coagulation of human platelets (Journal of the Pharmaceutical Society of Japan, 1991 , 111(9):504-509).

In our research on I. cats, I found that I. cats mycelium powder has a good protective effect on acute renal injury caused by cisplatin, and applied for a patent. The patent application number is 201110175421.4. After the research on the extraction of crude polysaccharides from I. catalis and its treatment of chronic renal failure, it was found that the crude polysaccharides in I. catifolia also have a renal protective effect and have a good therapeutic effect on chronic renal failure. At present, there is no research report on the treatment of chronic renal failure with I. felis crude polysaccharide.

Contents of the invention

The object of the present invention is to provide a kind of I. felis crude polysaccharide and the application of the polysaccharide in the preparation of medicine for treating chronic renal failure.

The present invention relates to I. catalis is isolated and cultivated from natural Cordyceps sinensis fruiting bodies. The strain is preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee (abbreviated as CGMCC), and the preservation number is CGMCC NO.0706.

The I. catalis mycelium used in the present invention can be obtained by cultivating the method of the patent No. ZL02103669.1.

A kind of IFP crude polysaccharide (IFP) provided by the invention is obtained by the following method: get mycelium powder, add 3-5 times the distilled water of mycelium powder weight, extract in boiling water bath for 70-110min, 3000r/min rotating speed Centrifuge for 4-6min; add distilled water 3-5 times the weight of mycelium powder to the sediment, extract in a boiling water bath for 20-40min, centrifuge at 3000r/min for 4-6min; continue adding 3-5 times to the sediment Distilled water with the weight of mycelium powder, extracted in a boiling water bath for 20-40min, centrifuged at 3000r/min for 4-6min; combined the supernatants extracted three times, concentrated in a water bath to 800-1000mL; added 3-4 times of 95% ethanol, mixed thoroughly Mix well and precipitate overnight; take the precipitate and dry it at 70°C for later use.

The added amount of said 95% ethanol is preferably 3.75 times of the supernatant volume after concentration.

The application of the present invention in the preparation of the medicament for treating chronic renal failure: the crude polysaccharide of I. felis is used in animal experiments. The experimental results showed that after continuous intragastric administration of adenine to rats for 30 days (100 mg·kg ^-1 ·d ^-1 ), the levels of CRE and BUN in serum increased significantly, and the levels of CRE and BUN in the serum of rats after intragastric administration of I. The levels of CRE and BUN in serum decreased significantly; IFP could partially correct the common anemia in the progression of chronic renal failure; the rats in the IFP model group had severe renal lesions, while the pathological changes in the rats in the drug treatment group were mild.

The results of experimental research show that the crude polysaccharide of I. catalis has a good therapeutic effect on chronic renal failure in rats caused by adenine, and it has a good therapeutic effect on chronic renal failure at a dosage of no more than 50 mg/kg. The effect is that it is an anti-renal failure drug with good development prospects.

Description of drawings

Figure 1: Pathological picture of kidney in normal group (HE staining, ×100).

Figure 2: Pathological picture of kidney in the model group (HE staining, ×100)

Figure 3: Kidney pathology of low-dose IFP administration group (HE staining, ×100)

Detailed ways

1. Experimental materials and methods

1.1 Experimental animals

SPF (specific pathogen free) grade SD rats, male, 220-240g, 7-8 weeks old, were purchased from the Experimental Animal Center of the Academy of Military Medical Sciences of the Chinese People's Liberation Army, license number: SCXK-(Army) 2007-004. The breeding temperature was 20°C-22°C, 12h/12h lighting, free drinking water, and fed sterile full-price nutritional pellet feed (Beijing Ke'ao Xieli Feed Co., Ltd.).

1.2 Cultivation of I. catalis mycelium

Medium composition: peptone 0.6%, yeast extract 1.2%, sucrose 2.4%, MgSO ₄ 0.05%, KH ₂ PO ₄ 0.1% and saturated NaHCO ₃ solution.

Cultivation method: Inoculate 0.5g strains into a 3000mL Erlenmeyer flask with 500mL culture medium, seal it with kraft paper, and cultivate it on a shaker at 180r/min at a temperature of 26.5°C. Collect the fermentation broth after 2d-3d, and put the fermentation broth in Centrifuge at 2000r/min for 5min to obtain mycelium. The obtained mycelium was dried and pulverized for the following experiments.

1.3 Extraction of IFP crude polysaccharide (IFP)

Take 300g mycelium powder, add 1200mL distilled water, extract in boiling water bath for 90min, centrifuge at 3000r/min for 5min; continue to add 1200mL distilled water to the precipitate, extract in boiling water bath for 30min, centrifuge at 3000r/min for 5min; continue to add 1200mL distilled water in the precipitate, Extract in boiling water bath for 30 minutes, centrifuge at 3000r/min for 5 minutes; combine the supernatants extracted three times, and concentrate in water bath to 900mL; add 3.75 times of 95% ethanol, mix well, and precipitate overnight; take the precipitate and dry it at 70°C for later use.

1.4 Animal grouping and modeling

After purchasing 40 SD rats, they were adaptively fed for one week, weighed, and randomly divided into 5 groups according to body weight, namely normal control group, adenine model group, high-dose IFP administration group, middle-dose IFP administration group, IFP was administered to the low-dose group, with 8 animals in each group.

Except for the normal control group, rats in other groups were given adenine 100 mg/kg by intragastric administration once a day for 30 consecutive days. Blood was collected from the tip of the tail to detect the levels of BUN and CRE in serum to verify whether the modeling was successful.

1.5 Administration method and materials

After the modeling was completed, the IFP administration group started drug treatment, and the dosage was determined according to the ratio of the occupied body surface area per kilogram of body weight. 50mg/kg, the intragastric administration volume was 5mL/kg, and the blank control group and the model control group were given the same amount of distilled water. The administration was carried out for 60 days, and the rats were given free access to food and water during the administration.

One day before the end of administration, the rats were transferred to metabolic cages for feeding, and 24-hour urine was collected to measure the contents of urine protein and creatinine.

24 hours after the last administration, the rats were weighed, and the rats that had been fasted for 12 hours were anesthetized with 20% urethane (urethane) at a dose of 5 mL/kg. Use ordinary serum tubes and EDTA anticoagulant tubes to collect blood from the abdominal aorta, part of the EDTA anticoagulant blood for routine blood testing, centrifuge the other part of the EDTA anticoagulant blood, separate the plasma, and store it at -80°C for testing; centrifuge blood in ordinary serum tubes , the serum was collected and stored at -80°C for testing. The left and right kidneys were dissected and weighed. Take the left kidney, peel off the outer membrane, fix it in 4% formaldehyde solution, and wait for the test.

1.6 Observation and detection items and methods

1.6.1 Observe the general living conditions and food and water intake of rats during the modeling process and drug administration process.

1.6.2 Regularly collect blood from the tip of the tail of the rats to measure the levels of serum CRE and BUN: creatinine kit and blood urea nitrogen kit are used for detection.

1.6.3 Determination of biochemical indicators such as CRE, BUN, UA, GLU, CK, and LDH in rat serum: use a fully automatic biochemical analyzer for detection.

1.6.4 Determination of RBC, HGB, HCT, MCV and other blood routines in rats: automatic blood cell analyzer is used for detection.

1.6.5 Kidney pathological examination: the left kidney was removed, the outer membrane was removed, fixed in 4% formaldehyde solution, routinely dehydrated, cleared, embedded in paraffin, sectioned, and stained with HE, and the pathological changes of the kidney were observed under a microscope.

1.6.6 Statistical analysis

表示，采用SPSS17.0软件进行数据统计分析，组间均数比较采用单因素方差分析，两组间比较采用最小显著差异法(LSD)，取α＝0.05为检验水准。Experimental data with

Said that SPSS17.0 software was used for statistical analysis of data, the comparison of means between groups was carried out by one-way analysis of variance, and the comparison between two groups was carried out by least significant difference method (LSD), and α=0.05 was taken as the test level.

2. Results

2.1 The general state of the rats during the experiment

Compared with the rats in the normal control group, after 3-5 days of gavage with adenine, the rats in the model group began to show reduced food intake, increased urine output, and decreased activity. After about a week, the rats began to have sparse, dull fur, and weight loss. slow growth. One animal died during the modeling process. During the administration, 4 animals died, including 1 animal in the model group, 1 animal in the high-dose IFP group, and 2 animals in the middle-dose group.

2.2 Changes of animal serum BUN and CRE during the modeling period

The results in Table 1 and Table 2 show that compared with the normal control group, BUN and CRE in the modeling group were significantly increased (P<0.05), indicating that the modeling was successful.

Table 1 Changes of serum BUN in rats during the modeling period

Note: ^* means there is a significant difference compared with the normal control group (P<0.05).

Table 2 Changes of serum CRE in rats during the modeling period

Note: ^* means there is a significant difference compared with the normal control group (P<0.05).

2.3 Changes in renal function indicators of rats after drug treatment

After 60 days of treatment, the BUN and CRE in the model group decreased, but were still significantly higher than those in the blank control group (P<0.05), and the UA in the model group was also significantly higher than that in the blank control group (P<0.05). Compared with the blank control group, there was a significant decrease (P<0.05); BUN and CRE in the low-dose IFP administration group were significantly reduced compared with the model group, which was statistically significant (P<0.05), and the low-dose IFP administration group Compared with the model group, UA decreased to a certain extent, and the endogenous creatinine clearance rate in the low-dose IFP administration group was significantly higher than that in the model group (P<0.05). It shows that IFP can reduce BUN, CRE, and UA in rats with renal failure at a certain dose, improve the clearance rate of endogenous creatinine in rats with renal failure, and play a role in protecting the kidneys (see Table 3).

Table 3 Effect of IFP on renal function indicators in rats with chronic renal failure

^* means there is a significant difference compared with the normal control group (P<0.05); ^△ means there is a significant difference compared with the model group (P<0.05).

2.4 Changes of RBC, HGB and HCT in rats after treatment

Compared with the normal control group, the RBC, HGB, and HCT of rats in the model group decreased significantly (P<0.05), and the above indicators in the low-dose IFP administration group increased significantly compared with the model group (P<0.05). It shows that IFP can partially correct the common anemia problem in the progression of chronic renal failure.

Table 4 The effect of IFP on RBC, HGB, HCT of rats with adenine chronic renal failure

^* means there is a significant difference compared with the normal control group (P<0.05); ^△ means there is a significant difference compared with the model group (P<0.05).

2.5 Kidney Pathological Observation

The kidneys in the normal group were broad bean-like, regular in shape, reddish-brown in color of normal kidney tissue, firm in texture, normal in size without swelling, and shiny; the cortex was normal reddish-brown in section, the medulla was light red in color, and the boundary between the cortex and medulla was clear ; The renal capsule is tightly combined and not easy to peel off. The kidneys of the rats in the other groups were swollen to varying degrees, especially in the model group, the swelling was the most obvious, the volume increased, the texture was soft, the capsule was not tightly combined with the kidney tissue, and it was easy to peel off. A large number of white particles were densely distributed on the surface of the kidney, and even The whole kidney is grayish white; the cut surface cortex becomes thinner, white crystal particles can be seen in the renal parenchyma, and the boundary between corticomedulla and medulla is not clear. In the low-dose IFP group, the kidneys were mostly normal reddish-brown kidney tissue and gray-white crystals, but there were more white particles and the distribution was uneven. The general appearance of the kidneys was changed the least in the low-dose IFP group.

The results of pathological sections showed that the renal tubules of the rats in the blank control group were not dilated, the renal tubular epithelium was intact, there was no edema, and no inflammatory cell infiltration was seen in the renal interstitium (Fig. 1); renal tubular dilation, renal tubular epithelial Cell degeneration and necrosis, protein casts, granular casts, a large amount of urate deposition, fibrous hyperplasia, and enlarged glomerular cysts (Figure 2). Inflammatory cell infiltration, mild urate deposition, and enlargement of the glomerular capsule, but in general, the pathological changes of the kidneys in the low-dose IFP group were milder than those in the model group (Figure 3). See Figure 1, Figure 2, and Figure 3 for details.

3. Conclusion

BUN and CRE are two main indicators reflecting renal function. CRE in the blood is mainly excreted by glomerular filtration, and the renal tubules basically do not reabsorb and excrete less. When the intake of exogenous CRE is stable, the concentration of CRE in the blood depends on the glomerular filtration. When the renal parenchyma is damaged and the glomerular filtration rate decreases to the critical point, the blood CRE concentration will increase significantly, so the measurement of blood CRE concentration can be used as an indicator of renal parenchyma damage. Sensitivity is better than BUN. BUN is the main end product of human protein metabolism. Amino acid deamination produces NH ₃ and CO ₂ , both of which synthesize urea in the liver and produce 0.3g of urea per gram of protein metabolism. Usually, the kidney is the main organ for excreting urea. Urea can be reabsorbed in all tubules after being filtered by the glomerulus. Like blood CRE, blood BUN can be within the normal range in the early stage of renal impairment. When the glomerular filtration rate drops below 50% of normal, the concentration of blood BUN increases.

UA is also one of the routine indicators for renal function monitoring. UA is the end product of purine metabolism in the nucleic acid in the body. Except for a small part of UA in the blood that is destroyed by the liver, most of it is filtered by the glomerulus. Elevated UA is also a sign of renal function damage. loss indicator.

The endogenous creatinine clearance test can reflect the glomerular filtration function and roughly estimate the number of effective nephrons, so it is a quantitative test for the determination of renal damage.

Patients with renal failure will develop symptoms of anemia, which is caused by damage to the renal parenchyma to a degree parallel to the degree of damage to renal function.

The experimental results show that IFP can reduce BUN, CRE, and UA in rats with renal failure at a certain dose, increase the clearance rate of endogenous creatinine in rats with renal failure, and protect the kidneys. It reflects the protective effect of IFP on the kidney, and IFP is beneficial to alleviate the common symptoms of anemia in the progression of chronic renal failure. In summary, IFP has a good therapeutic effect on chronic renal failure.

The therapeutic effect of IFP on chronic renal failure is not good at the dosage of 200mg/kg and 100mg/kg, but the therapeutic effect on renal failure is better at the dosage of 50mg/kg, so the dosage of IFP should be It is advisable not to exceed 50mg/kg.

Claims (3)

1. a cat Isaria Crude polysaccharides is characterized in that, makes through following method: get hypha powder, add the zero(ppm) water of 3-5 times of weight, boiling water bath extracts 70-110min, centrifugal 4-6min under the 3000r/min rotating speed; In deposition, continue to add the zero(ppm) water of people 3-5 times of hypha powder weight, boiling water bath extracts 20-40min, centrifugal 4-6min under the 3000r/min rotating speed; In deposition, continue to add the zero(ppm) water of people 3-5 times of hypha powder weight, boiling water bath extracts 20-40min, the centrifugal 4-6min of 3000r/min; Merge the supernatant of three extractions, water-bath is concentrated into 800-1000mL; Add people 3-4 95% ethanol doubly, abundant mixing, deposition is spent the night; Get deposition, in 70 ℃ of dry for standby.

2. cat Isaria Crude polysaccharides as claimed in claim 1 is characterized in that, said 95% alcoholic acid adds people's amount 3.75 times for supernatant volume after concentrating.

3. the application of cat Isaria Crude polysaccharides as claimed in claim 1 in preparation treatment chronic renal failure medicine.

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