CN112472720A

CN112472720A - Application of deer tail in preparation of products for treating gout and hyperuricemia

Info

Publication number: CN112472720A
Application number: CN202011434479.1A
Authority: CN
Inventors: 孙印石; 李志满
Original assignee: Institute Special Animal and Plant Sciences CAAS
Current assignee: Institute Special Animal and Plant Sciences CAAS
Priority date: 2020-12-10
Filing date: 2020-12-10
Publication date: 2021-03-12

Abstract

The invention discloses application of deer tails in preparing products for treating gout and hyperuricemia, belonging to the field of medicines. The invention aims to effectively treat and relieve gout symptoms. The invention provides application of deer tails in preparing products for treating gout and hyperuricemia, the deer tails are cleaned and cut into pieces, the pieces are placed in an electric heating constant temperature blast drying oven to be dried and crushed, then the pieces are added into water to be heated and refluxed at 100 ℃ to obtain water extract, then the supernatant is centrifuged, the supernatant is taken, the residues are repeatedly extracted for 2 times, the 3 times of supernatants are combined, and the deer tail water extract is obtained after concentration and drying. Pharmacological experiment results show that the deer tail aqueous extract has the effect of resisting gouty arthritis by reducing the ankle joint swelling rate of rats and reducing the levels of IL-1, IL-6 and TNF-alpha inflammatory factors in serum, and has the effect of improving hyperuricemia by inhibiting uric acid, creatinine and urea nitrogen in the serum of mice with hyperuricemia.

Description

Application of deer tail in preparation of products for treating gout and hyperuricemia

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of deer tails in preparation of products for treating gout and hyperuricemia.

Background

Along with the improvement of living standard of people, the hyperuricemia is a metabolic disease which causes the synthesis of uric acid in the body to be increased or the excretion to be reduced due to the disorder of purine metabolic system in the body, and the clinical manifestations of the hyperuricemia are that the blood urate is higher than 420 mu mol/L for men and higher than 360 mu mol/L for women. Hyperuricemia and acute gouty arthritis are different stages of gout occurrence and development. When the content of uric acid exceeds the maximum solubility in human bodies, uric acid is deposited on joints and surrounding tissues in the form of urate crystals, so that acute gouty arthritis, tophus, joint deformity and the like are induced. According to the Chinese expert consensus on hyperuricemia and gout treatment in 2013, the morbidity of hyperuricemia in China is increased year by year, the morbidity is as high as 10%, and even the morbidity in coastal and economically developed areas is as high as 20%. The incidence of hyperuricemia tends to be younger, the incidence rate of male is generally higher than that of female, the 2004 survey result shows that the incidence rate of gout in adult male is 6.1%, the incidence rate in female is only 1%, and the incidence time of female is obviously later than that of male. In addition, hyperuricemia is closely related to hypertension, dyslipidemia, type II diabetes, metabolic syndrome, kidney and cardiovascular diseases. The hyperuricemia has the characteristics of long disease period, difficulty in radical treatment and the like, and the uric acid reducing medicine which is clinically taken for a long time has the problems of large toxic and side effects, high price and the like, so that a natural product which is safe and effective and has high cost performance and is beneficial to treating the hyperuricemia needs to be found.

The deer tail is tail of Cervidae animal such as Cervus Nippon Temminck or Cervus Elaphus L, and comprises caudal vertebra, muscle tissue, adipose tissue, caudal gland, connective tissue and tail skin, has special tissue structure and important biological function, and can regulate and control multiple behaviors of deer body by secreting special substances through caudal gland. The deer tail is a traditional Chinese medicine in China, and has the effects of strengthening waist and tonifying kidney, replenishing vital essence and tonifying deficiency, enhancing immunity, eliminating fatigue, promoting blood circulation, treating rheumatism and the like. The unique tissue structure of deer tail is characterized by the tail gland and its histological features. The glandular changes in the area of the coccygeal gland include changes in the sebaceous and apocrine sweat glands, which are developed with much apocrine sweat and thickest around the ventral caudal apex. The deer tail tissue structure is also unique in that it has a mechanism of action similar to that of a gallbladder. Ancient languages: the deer is gallbladder-free and the tail is substituted. The tissue structure of the deer tail is like a perfect filtering system, can decompose redundant fat through blood circulation, has protective effect on the liver of the deer body, and is a mechanism similar to a gall bladder which is not possessed by other animals.

As is known, all deer bodies are treasure, and the most studied antler in the current genus is deer antler, and then products such as deer skin glue, deer sinew, deer blood, deer penis and the like are gradually developed, but the research on deer tails is less, and the deer tail products are difficult to see in the market. The deer tails in ancient China are used as famous and precious tonic tonics and are highly advocated. However, the current research on deer tails is few and lacks systematicness, and the pharmacological effects thereof are to be explored.

Disclosure of Invention

The invention aims to solve the problem of how to effectively treat and relieve gout symptoms, and provides application of deer tails in preparing products for treating gout or hyperuricemia in order to solve the technical problems.

The invention also provides application of the deer tail in preparing a product for assisting in treating gout or hyperuricemia.

The invention also provides application of the deer tail in preparing health-care products for relieving gout or hyperuricemia.

The invention also provides application of the deer tail in preparing food for relieving gout or hyperuricemia.

Further defined, the processing method of the deer tail comprises the following steps:

(1) taking sika deer tails, cleaning the sika deer tails, cutting into pieces, then placing the cut sika deer tails in an electric heating constant-temperature air-blast drying oven for drying at 40 ℃, and then crushing and drying to obtain the sika deer tails;

(2) mixing and soaking the crushed sika deer tails obtained in the step (1) with water according to the mass ratio of 1:8-1:10, and performing heating reflux extraction at 100 ℃ for 1-1.5 hours to obtain a water extract;

(3) centrifuging the water extract obtained in step (2) at 4500r/min for 10-15min, collecting supernatant, extracting the residue under reflux for 2 times, mixing the supernatant extractive solutions for 3 times, concentrating, and drying to obtain water extract of cauda Cervi.

Further defined, the gout is a disease caused by acute gouty arthritis.

The product is further limited to be in any one of tablets, capsules, granules, powder and liquid preparations.

The health care product is further limited to be in any one of tablets, capsules, granules, powder and liquid preparations.

The food can be prepared into any one of tablets, capsules, granules, powder and liquid preparations.

Has the advantages that: in an acute gouty arthritis experiment of a rat, the acute gouty arthritis is induced by injecting MSU crystals into ankle joints of the rat, the circumferences of the ankle joints of the rat are measured at 12h, 24h and 48h after sodium urate is injected, from the result of the swelling degree of the ankle joints, the cauda cervi extract can inhibit swelling of the ankle joints caused by MSU, the cauda cervi extract can reduce the content of IL-1, IL-6 and TNF-alpha in serum, the cauda cervi extract has the effect of resisting the gouty arthritis, and the cauda cervi extract can reduce the content of uric acid, creatinine and urea nitrogen in the serum of the mouse, so that the cauda cervi extract can reduce the content of blood uric acid and has the capability of improving hyperuricemia.

Drawings

FIG. 1 shows the effect of deer tail extract on the ankle swelling rate of rats with acute gouty arthritis

Wherein, # p, as compared to the normal group<0.01，#p<0.05; comparison with model group<0.05，**p<0.01, the abscissa is the group and the ordinate is the swelling rate;

FIG. 2 shows the effect of deer tail extract on IL-1 content in serum of rat with acute gouty arthritis

n-6), wherein, in contrast to the normal group, # p<0.05; in contrast to the model group,*p<0.05, the abscissa is the group and the ordinate is the IL-1 content in serum;

FIG. 3 shows the effect of deer tail extract on IL-6 content in serum of rat with acute gouty arthritis (II)

n ═ 6), in which, ## p contrasts with normal groups<0.01; comparison with model group<0.05，**p<0.01; the abscissa is the group and the ordinate is the IL-6 content in serum;

FIG. 4 shows the effect of deer tail extract on the TNF-alpha content in serum of rat with acute gouty arthritis ((

n-6), wherein, in contrast to the normal group, # p<0.05; comparison with model group<0.01, the abscissa is the group and the ordinate is the content of TNF-alpha in serum;

FIG. 5 shows the effect of deer tail extract on the uric acid content in the serum of mice with hyperuricemia: (

n-8), wherein, # # # p is compared with the normal group<0.001, control with model group<0.001, the horizontal axis is the group, and the vertical axis is the content of uric acid in serum;

FIG. 6 shows the effect of deer tail extract on the creatinine content in serum of mice with hyperuricemia: (

n-8), wherein, # # # p is compared with the normal group<0.001, control with model group,. about.p<0.05，***p<0.001, the horizontal axis is the group, and the vertical axis is the creatinine content in the serum;

FIG. 7 shows the effect of deer tail extract on the content of urea nitrogen in serum of mice with hyperuricemia

n-8), wherein, # # # p is compared with the normal group<0.001, control with model group<0.05，***p<0.001, group on abscissa, ordinateIs the urea nitrogen content in the serum.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

1. Experimental animals: SPF-class male Kunming mice and SPF-class male SD rats were purchased from Liaoning Biotechnology Ltd, and were manufactured under license number SCXK (Liao) 2015-0001.

2. Experimental materials: the Cervus Nippon Temminck tail is from Jilin Shuangyang Luxiang; potassium oxonate was purchased from Sigma Aldrich (Shanghai) trade, Inc.; allopurinol was purchased from sigma aldrich trade ltd; colchicine was purchased from sigma aldrich trade ltd; sodium urate was purchased from sigma aldrich trade ltd; the uric acid determination kit, the Creatinine (CR) determination reagent and the urea nitrogen (BUN) determination kit are purchased from Nanjing to build a bioengineering institute; formalin was purchased from beijing solibao reagent; IL-1Elisa kit, IL-6Elisa kit and TNF-alpha Elisa kit are available from Andy Gene reagents, Inc.

3. Experimental equipment: an Epoch2 type microplate reader was purchased from Botryn instruments, Inc. USA; the DHG-9123A electric heating constant temperature air-blast drying oven is purchased from Shanghai sperm macro experimental equipment Co., Ltd; the CPA225D electric heating constant temperature water bath kettle is purchased from Yongguang medical instrument factory in Beijing; MS204S electronic analytical balance was purchased from MettlerToledo, Switzerland; the ultra-low temperature centrifuge was purchased from Saimer Feishell science Co.

Example 1.

A method for processing deer tails comprises the following steps: the method comprises the following specific steps:

(1) taking sika deer tails, cleaning the sika deer tails, cutting into pieces, then placing the cut sika deer tails in an electric heating constant-temperature air-blast drying oven for drying at 40 ℃, and then crushing the sika deer tails;

(2) mixing the crushed sika deer tails obtained in the step (1) with water according to a mass ratio of 1:10 mixing and soaking, and then heating and refluxing the mixed solution at 100 ℃ for 1 hour to obtain a water extract;

(3) centrifuging the water extract obtained in step (2) at 4500r/min for 10min, collecting supernatant, extracting the residue under reflux for 2 times, mixing the supernatant and the supernatant for 3 times, concentrating, and drying to obtain deer tail water extract with extraction rate of 15.79%.

The following experiments were used to verify the experimental effect:

the first experiment method comprises the following steps:

1. animal feeding: SPF grade male Kunming mice 48 (20 + -2 g), SPF grade male SD rats 24 (200 + -20 g) were housed: illuminating for 12h/d, raising in an environment with the temperature (22 +/-3 ℃) and the relative humidity (55 +/-15%), and feeding water freely.

2. Preparing a deer tail extract: referring to example 1, the deer tail extract was obtained. The deer tail extract contains nucleoside, biogenic amine, protein, polysaccharide and amino acid as main components.

3. Establishing a rat acute gouty arthritis model: healthy SPF male SD rats were randomly divided into 4 groups of 6 rats each, namely a normal group, a model group, a colchicine positive control group and a deer tail group (200 mg/kg).

Adopting a gastric lavage administration mode, wherein the dose of colchicine in the positive control group is 0.3mg/kg, the administration volume is 10mL/kg, the gavage is carried out once a day for 8 days, after the gavage is carried out for 1h on the 5 th day, 25g/L of sodium urate crystal solution is injected into the ankle joint cavity of the rat, and the injection volume is 200 mu L;

performing intragastric administration on the normal group with the same amount of normal saline, wherein the intragastric administration volume is 10mL/kg, and the intragastric administration is performed once a day for 8 days;

the equivalent physiological saline of the model group is perfused with stomach, the perfusion volume is 10mL/kg, the total time is 8 days, after the perfusion for 1h on the 5 th day, 25g/L sodium urate crystal solution is injected into the ankle joint cavity of the rat, and the injection volume is 200 mu L;

intragastric feeding 200mg/kg of cauda Cervi extract in cauda Cervi group for 8 days, and injecting 25g/L sodium urate crystal solution into ankle joint cavity of rat after intragastric feeding for 1h on day 5, with injection volume of 200 μ L;

1h after the administration on the 8 th day, blood is taken from the heart of rats in each group by anesthesia, the whole blood is kept stand for 30min, then is centrifuged for 10min at 3000r/min, and the supernatant is taken and stored at-80 ℃.

4. The method for measuring the knee joint swelling rate of the rat comprises the following steps: after injecting sodium urate crystals into the knee joint of the mouse, measuring the circumferences of the right knee joint of 0h, 12h, 24h and 48h by a vernier caliper, wherein the swelling degree (%) is the change degree of the circumference.

5. Method for measuring inflammatory factors in rat serum: the IL-1, IL-6 and TNF-alpha content in mouse serum was determined according to the instructions of the Elisa kit.

6. Establishing a hyperuricemia model: the 48 healthy SPF-level Kunming male mice were randomly divided into 6 groups of 8 mice each, namely a normal group, a model group, an allopurinol group (a positive drug for treating hyperuricemia, 5mg/kg), a low, medium and high dosage deer tail group (100, 200, 300mg/kg groups)

After the allopurinol group starts to take 250mg/kg potassium oxonate orally at 9 am every day, the allopurinol is administrated by gastric lavage at 10 am, the administration volume is 10mL/kg, and the administration lasts for 7 days continuously;

normal group is perfused with normal saline, the volume is 10mL/kg, and the continuous perfusion lasts for 7 days;

after the deer tail group starts to take 250mg/kg oteracil potassium salt orally at 9 am every day, 10 am of the deer tail group is gavaged with 100, 200 and 300mg/kg of deer tail extract, and the medicine is continuously taken for 7 d;

after the model group starts to take 250mg/kg of oteracil potassium salt orally at 9 am every day, the normal saline solution for gastric gavage is injected at 10 am, the volume is 10mL/kg, and the time is continuously 7 days;

after the 7 th intragastric administration for 1h, blood is taken from eyeballs of each group of mice, the mice are killed by dislocation of cervical vertebrae, livers are dissected and taken out, bilateral kidneys are weighed, middle leaves and unilateral kidneys of the livers are taken and stored in formalin, and the rest liver tissues and the kidneys are stored in a refrigerator at the temperature of-80 ℃. Standing whole blood for 30min, centrifuging at 3000r/min for 10min, and separating serum.

7. The method for measuring uric acid, creatinine and urea nitrogen in mouse serum comprises the following steps: the content of uric acid, creatinine and urea nitrogen in mouse serum is detected according to the specification of the Nanjing constructed kit.

Second, experimental results

1. Influence of deer tail extract on ankle joint swelling of rats with acute gouty arthritis

After injecting sodium urate crystals for 12h, 24h and 48h into the normal group of rats, the model group, the positive control group and the deer tail group, measuring the knee joint circumferences of the rats, and the results are shown in figure 1, compared with the normal group, the swelling degree of the right ankle joints of the rats in the model group is remarkably increased (p is less than 0.01, and p is less than 0.05), which indicates that the acute gouty arthritis model of the rats is successful. Deer tail and colchicine positive drugs significantly inhibited ankle swelling at 12h and 48h (p < 0.05); at 48h, colchicine significantly inhibited the degree of ankle swelling in rats (p < 0.01). At 24h, neither group significantly inhibited the swelling caused by sodium urate crystals.

2. Influence of deer tail extract on IL-1 in serum of rat with acute gouty arthritis

After injecting sodium urate crystals into a normal group of rats, a model group, a positive control group and a deer tail group for 12h, 24h and 48h, detecting IL-1 in the serum of the rats by adopting an Elisa kit, wherein the result is shown in figure 2, and compared with the normal group, the content of IL-1 in the serum of the rats in the model group is obviously increased (p is less than 0.05) after injecting the sodium urate crystals, which indicates that the acute gouty arthritis model of the rats is successful. Compared with the model group, the deer tail extract and the positive group can obviously reduce the IL-1 content (p <0.05) in the serum of rats. The deer tail extract is shown to be capable of reducing the IL-1 content in the serum of rats with acute gouty arthritis.

3. Influence of deer tail extract on IL-6 in serum of rat with acute gouty arthritis

After injecting sodium urate crystals for 12h, 24h and 48h into a normal group of rats, a model group, a positive control group and a deer tail group, detecting IL-6 in the serum of the rats by adopting an Elisa kit, wherein the result is shown in figure 3, and compared with the normal group, the content of IL-6 in the serum of the rats is remarkably increased (p is less than 0.01), which indicates that the acute gouty arthritis model of the rats is successful. Compared with a model group, the deer tail extract can obviously reduce the IL-6 content in the serum of a rat (p is less than 0.05, p is less than 0.01), and a positive group can extremely obviously reduce the IL-6 level in the serum of the rat. The deer tail extract is shown to be capable of reducing the IL-6 content in the serum of rats with acute gouty arthritis.

4. Influence of deer tail extract on TNF-alpha in serum of rat with acute gouty arthritis

After injecting sodium urate crystals for 12h, 24h and 48h into a normal group of rats, a model group, a positive control group and a deer tail group, detecting TNF-alpha in rat serum by adopting an Elisa kit, wherein the result is shown in figure 4, compared with the normal group, the content of the TNF-alpha in the rat serum of the model group is remarkably increased (p is less than 0.01) after injecting the sodium urate crystals, and the success of the rat acute gouty arthritis model is shown. Compared with the model group, the deer tail extract and the positive drug remarkably reduce the content of TNF-alpha in the serum of rats (p < 0.05). The deer tail extract is shown to be capable of reducing the content of TNF-alpha in the serum of rats with acute gouty arthritis.

In the acute gouty arthritis experiment of rats, the effect of the drug on acute gouty arthritis is evaluated by inducing acute gouty arthritis by injecting MSU (monosodium urate) crystals into ankle joints of rats. The circumferences of ankle joints of rats were measured at 12h, 24h and 48h after the injection of sodium urate, and the degree of swelling of ankle joints was calculated. From the results of the swelling degree of the ankle joint, it was suggested that the deer tail extract can inhibit MSU-induced swelling of the ankle joint. The pathogenesis of gout is closely related to inflammation, and sodium urate crystals stimulate synovial cells, monocytes, macrophages and neutrophils and produce inflammatory factors such as IL-1 beta, IL-6 and TNF-alpha. The results show that the deer tail extract can reduce the content of IL-1, IL-6 and TNF-alpha in serum, and the deer tail extract has the function of resisting gouty arthritis.

5. Influence of deer tail extract on blood uric acid content of hyperuricemia mice

The accumulation of uric acid in vivo can generate sodium urate deposition in joint cavities to cause severe painful arthritis, and the content of uric acid in the serum of a mouse is detected, and the result is shown in figure 5, compared with a normal group, the uric acid level in the serum of a hyperuricemia mouse in a model group is obviously increased (p is less than 0.001), which indicates that the mouse hyperuricemia model is successfully established. Compared with the model group, the deer tail high-dose group can remarkably reduce the content of uric acid in serum (p <0.001), and the effect is consistent with that of the positive group. The medium-dose group and the low-dose group show that the deer tail extract can reduce the content of blood uric acid and has the activity of treating hyperuricemia.

6. Influence of deer tail extract on serum creatinine content of hyperuricemia mice

The creatinine content in the mouse serum is detected, the result is shown in figure 6, and compared with the normal group, the creatinine content in the mouse serum of the model group is remarkably increased (p is less than 0.001), which indicates that the mouse hyperuricemia model is successfully established. Compared with the model group, the low-deer tail, high-dose group and positive group can obviously reduce the content of uric acid in serum (p <0.05), and the medium-dose group can obviously reduce the content of creatinine (p < 0.001). The deer tail extract can reduce the content of creatinine in serum.

7. Influence of deer tail extract on serum urea nitrogen content of hyperuricemia mice

The content of urea nitrogen in the serum of the mouse is detected, the result is shown in figure 7, compared with the normal group, the content of urea nitrogen in the mouse in the model group is obviously increased (p is less than 0.001), and the success of establishing the mouse hyperuricemia model is shown. Compared with the model group, the low and high dose deer tail group can obviously reduce the content of urea nitrogen in serum (p <0.01, p <0.001), and the effect is consistent with that of the positive group. The deer tail extract can reduce the content of blood urea nitrogen and has the activity of treating hyperuricemia.

The deer tail extract is used for researching a hyperuricemia experiment, and the potassium oxonate is used for reducing the activity of uricase, so that the metabolism and excretion of uric acid are weakened, the uric acid is increased, and a hyperuricemia model is induced. The results prove that the deer tail extract can reduce the content of uric acid, creatinine and urea nitrogen in the blood serum of a mouse. The deer tail extract can reduce the content of blood uric acid and has the capacity of treating hyperuricemia.

In conclusion, the research shows that the deer tail extract has the effect of resisting gouty arthritis by reducing the level of IL-1, IL-6 and TNF-alpha inflammatory factors. The deer tail extract has the effects of reducing uric acid, creatinine and urea nitrogen in the blood serum of a mouse with hyperuricemia and improving the hyperuricemia.

Claims

1. Application of deer tail in preparing product for treating gout or hyperuricemia is provided.

2. The deer tail is applied to the preparation of products for assisting in treating gout or hyperuricemia.

3. The deer tail is applied to the preparation of health-care products for relieving gout or hyperuricemia.

4. Application of deer tail in preparing food for relieving gout or hyperuricemia is provided.

5. The use according to any one of claims 1 to 4, wherein the deer tail is treated by the following method:

(1) taking sika deer tails, cleaning the sika deer tails, cutting into pieces, then placing the cut sika deer tails in an electric heating constant-temperature air-blast drying oven for drying at 40 ℃, and then crushing the dried sika deer tails to obtain sika deer tails;

(2) mixing and soaking the crushed sika deer tails obtained in the step (1) with water according to the mass ratio of 1:8-1:10, and then heating and refluxing the mixed solution at 100 ℃ for 1-1.5h to obtain a water extract;

6. The use according to any one of claims 1 to 4, wherein gout is a disease caused by acute gouty arthritis.

7. The use according to claim 1 or 2, wherein the product is in the form of any one of tablets, capsules, granules, powders and liquid preparations.

8. The use of claim 3, wherein the health product is in the form of any one of tablet, capsule, granule, powder and liquid.

9. The use according to claim 4, wherein the food is in the form of any one of tablets, capsules, granules, powders and liquid preparations.