Extraction method of radix astragali seu hedysari extract and application of radix astragali seu hedysari extract in preparing medicine for treating knee osteoarthritis

Abstract

The invention discloses an extraction method of an astragalus extract and application of the astragalus extract in preparation of a medicine for treating knee osteoarthritis. According to the application of the radix astragali extract similar to a GLP-1R receptor stimulant, the high-dose radix astragali extract remarkably reduces the level of IL-1beta and TNF-alpha in serum of a knee osteoarthritis KOA model rat, meanwhile, the level of GLP-1 is increased, and therefore cartilage inflammation is relieved. The traditional Chinese medicine extract is prepared by adopting the existing traditional Chinese medicine preparation technology, effective components of GLP-1R agonist in astragalus membranaceus are reserved to a great extent, and the traditional Chinese medicine extract can be used for treating knee osteoarthritis and effectively relieving cartilage injury of joints.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to an extraction method of an astragalus extract and an application of the extract in preparing a drug for treating knee osteoarthritis. Background Art

[0002] Knee osteoarthritis (KOA) is a common chronic degenerative disease in clinical practice. Its main pathological manifestation is cartilage changes. In the early stage of the disease, it manifests as focal softening of the cartilage and loss of elasticity, followed by roughness, erosion, and ulceration of the cartilage, and even large pieces of cartilage falling off, eventually leading to the disappearance of the entire layer of cartilage. This disease is the most common cause of pain and disability worldwide. Currently, there are two main types of drugs for the treatment of KOA: non-steroidal anti-inflammatory drugs (such as diclofenac sodium) and chondroprotective agents (such as glucosamine sulfate). Non-steroidal anti-inflammatory drugs mainly exert analgesic and anti-inflammatory effects by inhibiting the activity of cyclooxygenase (COX), and chondroprotective drugs delay the progression of KOA by improving bone metabolism.

[0003] Liraglutide is a GLP-1R receptor agonist that has the effect of inhibiting systemic inflammation. The side effects of this drug are gastrointestinal discomfort, headache, pharyngitis, etc. There are many limitations in the treatment of KOA with Western medicine. This type of drug not only increases the burden on the gastrointestinal tract and liver and kidneys, but also makes the body dependent on the drug. As a dominant disease in traditional medicine, Chinese medicine has a significant effect on relieving the condition of KOA patients and maintaining joint function. Astragalus is often used in combination to treat chronic arthralgia, qi and blood deficiency and painful arthralgia. Modern pharmacological studies have confirmed that astragalus has multiple effects such as regulating immunity, anti-osteoporosis, regulating apoptosis, anti-oxidation, and anti-inflammatory. GLP-1R binding has beneficial multi-effects of immunomodulation, anti-inflammatory, and apoptosis adjustment. And there is no research and invention of similar Chinese medicine GLP-1R receptor agonists. Summary of the invention

[0004] In view of the above problems, the present invention aims to provide a method for extracting and preparing a drug for treating knee osteoarthritis with Radix Astragali, and its application similar to that of GLP-1R receptor agonist, and to study the mechanism of the water extract of Radix Astragali acting on GLP-1R to treat KOA. In order to achieve the above purpose, the present invention adopts the following technical scheme:

[0005] Place Astragalus in a round-bottom flask, add appropriate amount of water, heat with an electric heating mantle, perform reflux extraction, filter the extract, add water to the filtrate again for reflux extraction, filter the extract, repeatedly add water to the filtrate for reflux extraction, combine the filtrate, concentrate the filtrate by a rotary evaporator, seal and store in a 4°C refrigerator, thereby obtaining an Astragalus stock solution.

[0006] Specifically, 120 g of Astragalus membranaceus was placed in a round-bottom flask, 200 ml of water was added, the mixture was heated with an electric heating mantle, reflux extraction was performed, the extract was filtered, 200 ml of water was added to the filtrate again for reflux extraction, the extract was filtered, 200 ml of water was added to the filtrate for reflux extraction for a total of 3 times, the filtrate was combined, the filtrate was concentrated to 200 ml by a rotary evaporator, the filtrate was sealed and stored in a refrigerator at 4°C, thereby obtaining an Astragalus membranaceus stock solution.

[0007] Astragalus: It is a plant of the genus Astragalus in the Leguminosae family. It is a perennial herb. Astragalus root has a long history as a traditional Chinese medicine.

[0008] "Changsha Yaojie" says: "Astragalus belongs to the spleen meridian, flows through the meridians and benefits the blood circulation, treats the symptoms of jaundice, blood stasis, and rheumatism. It is most effective for treating swelling and pain in the joints."

[0009] "Bencao Gangmu" (Bencao Gangmu): Astragalus can boost Qi, strengthen tendons and bones, grow flesh and replenish blood.

[0010] The use of the astragalus extract in the preparation of a drug for treating knee osteoarthritis. The flavonoid astragaloside active ingredients in the active ingredients of astragalus can increase the levels of GLP-1 and the agonist receptor GLP-1R in the body, and the glucagon-like peptide-1 receptor (GLP-1R) can prevent cell apoptosis, anti-inflammatory activity and matrix protection. Glucagon (GLP-1) combined with GLP-1R has beneficial pleiotropic immunomodulatory, anti-inflammatory and apoptosis regulating effects.

[0011] The method for extracting the effective component for stimulating GLP-1R receptor from Astragalus membranaceus is proved by experiments to have the therapeutic effect of being a GLP-1R receptor agonist.

[0012] Astragalus extract is similar to the application of GLP-1R receptor agonists.

[0013] Astragalus extract significantly reduced the levels of IL-1β and TNF-α in the serum of rats with knee osteoarthritis (KOA) model.

[0014] Astragalus extract increases the level of GLP-1 in rats with knee osteoarthritis KOA model.

[0015] Application of astragalus extract in preparing medicine for treating knee osteoarthritis.

[0016] The invention has the following beneficial effects: It provides an application of an Astragalus membranaceus aqueous extract similar to a GLP-1R receptor agonist for treating knee osteoarthritis. The aqueous extract is made into a Chinese medicine extract using existing Chinese medicine preparation technology, which retains the GLP-1R agonist active ingredients in Astragalus membranaceus to a great extent, and can be used to treat knee osteoarthritis and effectively relieve cartilage damage in joints. The experimental results show that Astragalus membranaceus has an effect similar to that of a GLP-1R agonist. A high dose of Astragalus membranaceus can significantly reduce the levels of IL-1β and TNF-α in the serum of KOA model rats, while increasing the level of GLP-1, thereby relieving cartilage inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0018] Figure 1 This is the cartilage picture of the rats in the blank group.

[0019] Figure 2 This is the cartilage picture of rats in the model group.

[0020] Figure 3 This is the cartilage of rats in the low-dose Astragalus group.

[0021] Figure 4 This is the cartilage of rats in the medium-dose group of Astragalus.

[0022] Figure 5 This is the cartilage of rats in the high-dose Astragalus group.

[0023] Figure 6 This is the cartilage picture of rats in the glucosamine sulfate group.

[0024] Figure 7 GLP-1R protein detection in rat knee joint. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with specific implementation modes, so that those skilled in the art can implement it according to the description.

[0026] Example

[0027] Experimental Example 1:

[0028] Take 120g of Astragalus and put it in a round-bottom flask, add 200ml of water, heat with an electric heating mantle, perform reflux extraction, filter the extract, add 200ml of water to the filtrate again for reflux extraction, filter the extract, add 200ml of water to the filtrate for reflux extraction 3 times in total, combine the filtrates, concentrate the filtrate to 200ml on a rotary evaporator, seal and store in a 4°C refrigerator, thereby obtaining an Astragalus stock solution.

[0029] Experimental Example 2:

[0030] 1.1 Animal health 48 SPF SD rats, weighing (210±10) g, were purchased from Beijing Huafukang Biotechnology Co., Ltd., production license number: SCXK (Beijing) 2019-0008. This study has been approved by the Experimental Animal Center of Shanxi University of Traditional Chinese Medicine (NO.110322230101825576).

[0031] 1.2 Experimental drugs Astragalus (Beijing Tongrentang Jinzhong Pharmacy Co., Ltd., batch number: 230701); Glucosamine sulfate (Xinxing Tongren Pharmaceutical Co., Ltd., batch number: 220513).

[0032] 1.3 Experimental reagents Sodium iodoacetate (MIA) (Shanghai MacLean Biochemical Technology Co., Ltd., batch number:

[0033] S817623); Freund's complete / incomplete adjuvant (Sigma, batch number SLBR3877V, SLBM9362V); ELISA: GLP-1 (Wuhan Gene America ELISA Kit, catalog number: 202310), TNF-α (Wuhan Gene America ELISA Kit, catalog number: 202311), IL-1β (Wuhan Gene America ELISA Kit, catalog number: 202311); GLP-1R antibody (NOVUS, catalog number: NBP1-97308).

[0034] 2. Dosage design

[0035] The Astragalus stock solution prepared in the experiment was used for oral administration of 3.125g / kg, 6.25g / kg, and 12.5g / kg of Astragalus according to the low, medium, and high doses of the rats. The positive drug group was given 0.1g / kg of glucosamine sulfate by oral administration.

[0036] 3. Experimental methods:

[0037] Rats were randomly divided into blank group, model group and astragalus extract gavage group (n=8). KOA model of knee osteoarthritis was established by injecting 50 μL of 4% MIA solution into the knee joint cavity.

[0038] After 5 weeks of drug administration, rats were anesthetized with isoflurane inhalation paralysis, and blood was collected from the abdominal aorta. The upper serum was aspirated and stored in a -80℃ refrigerator. Four left knee joints of rats were randomly selected from each group and placed in cryopreservation tubes. Four knee joints of each group were fixed in tissue fixative for 1 week, and then decalcified with EDTA decalcification solution for 57 days before use for paraffin sections.

[0039] 4. Test results

[0040] 4.1 Pathological testing:

[0041] The blank group showed: the cartilage surface was flat, the tide line was intact, there was no diffuse cell increase, the chondrocyte cell morphology was normal, and the cell matrix was normally colored. The model group showed: the surface cells of the knee joint were obviously clustered and disordered, the chondrocytes proliferated significantly, the cell nuclei were dark blue, and the joint cavity space was narrowed. The glucosamine sulfate group and each astragalus group showed mild damage to the surface of the articular cartilage, the cartilage layer was severely worn and the surface was slightly less smooth, the tide line integrity was deteriorated, and the cells were slightly hypertrophic. The Mankin's score in the model group increased significantly. The scores of the low, medium, and high doses of astragalus and the glucosamine sulfate groups were all lower than those of the model group. Figure 2 (P<0.05),

[0042] Comparison of pathological changes of knee cartilage tissue in each group HE staining ×100,

[0043] Table 1. Effects of Astragalus extract on Makin score of KOA rats ( n=3)

[0044]

[0045] Note: Compared with the normal control group, ## P<0.01; compared with the model control group, *P<0.05, **P<0.01.

[0046] 4.2 Serological testing:

[0047] Effects of GLP-1, IL-1β, and TNF-α levels in rat serum

[0048] Effects on the levels of GLP-1, IL-1β, and TNF-α in rat serum

[0049] Compared with the blank group, the levels of IL-1β and TNF-α in serum of rats in the model group were significantly increased (P<0.01), and the level of GLP-1 was significantly decreased (P<0.01); after treatment, compared with the model group, the levels of IL-1β and TNF-α in rats in each treatment group were decreased (P<0.01); compared with the model group, the level of serum GLP-1 in rats in the aminoglucose sulfate group was insignificant, the level of serum GLP-1 in rats in the medium-dose Astragalus group was increased (P<0.05), and the level of serum GLP-1 in rats in the high-dose Astragalus group was significantly increased (P<0.01).

[0050] Table 2. Effects of Astragalus extract on serum IL-1β, TNF-α, and GLP-1 levels in KOA rats ( n=3)

[0051]

[0052]

[0053] 4.3 GLP-1R in rat knee joint

[0054] Compared with the blank group, the expression of GLP-1R protein in the model group was significantly decreased (P < 0.01). Compared with the model group, the expression of MMP13 protein in the various doses of Astragalus and the glucosamine sulfate group was significantly decreased (P < 0.01) (no data), and the expression of GLP-1R in the various doses of Astragalus was significantly increased compared with the model group (P < 0.01). There was no statistically significant difference in the expression of GLP-1R protein in the glucosamine sulfate group compared with the model group.

[0055] Table 3. Effect of Astragalus extract on GLP-1R protein detection level in KOA rats ( n=3)

[0056]

[0057] In summary, this patent provides an application and treatment mechanism of an Astragalus membranaceus aqueous extract similar to a GLP-1R receptor agonist for treating knee osteoarthritis. The Chinese medicine of the present invention is guided by the theory of traditional Chinese medicine. Then, the existing Chinese medicine preparation technology is used to make it into a Chinese medicine extract, which retains the GLP-1R agonist active ingredients in Astragalus membranaceus to a great extent, and can be used to treat knee osteoarthritis and effectively relieve cartilage damage in the joints.

[0058] GLP-1R receptors are closely related to the prevention of apoptosis, anti-inflammatory activity and protection of cartilage matrix. Experimental results show that Astragalus has an effect similar to that of GLP-1R agonists. High doses of Astragalus can significantly reduce serum IL-1β and TNF-α levels, while increasing GLP-1 levels, thereby alleviating cartilage inflammation.

[0059] In this specification, the above embodiments, experimental examples and application experimental examples are only used to illustrate the technical solutions of the present invention, rather than to limit them. However, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, experimental examples and application experimental examples, or replace some or all of the technical features therein by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the present invention.

Claims

1. A method for extracting astragalus extract, characterized in that: Place Astragalus in a round-bottom flask, add appropriate amount of water, heat with an electric heating mantle, perform reflux extraction, filter the extract, add water to the filtrate again for reflux extraction, filter the extract, repeatedly add water to the filtrate for reflux extraction, combine the filtrate, concentrate the filtrate by a rotary evaporator, seal and store in a 4°C refrigerator, thereby obtaining an Astragalus stock solution.

2. The method for extracting the astragalus extract according to claim 1, characterized in that: Take 120g of Astragalus and put it in a round-bottom flask, add 200ml of water, heat with an electric heating mantle, perform reflux extraction, filter the extract, add 200ml of water to the filtrate again for reflux extraction, filter the extract, add 200ml of water to the filtrate for reflux extraction 3 times in total, combine the filtrates, concentrate the filtrate to 200ml on a rotary evaporator, seal and store in a 4°C refrigerator, thereby obtaining an Astragalus stock solution.

3. Use of the astragalus extract according to any one of claims 1 or 2 in the preparation of a drug for treating knee osteoarthritis.

4. The use of the astragalus extract according to claim 3 in the preparation of a drug for treating knee osteoarthritis, characterized in that: Astragalus extract is similar to the application of GLP-1R receptor agonists.

5. The use of the astragalus extract according to claim 4 in the preparation of a drug for treating knee osteoarthritis, characterized in that: Astragalus extract significantly reduced the levels of IL-1β and TNF-α in the serum of rats with knee osteoarthritis (KOA) model.

6. Use of the astragalus extract according to claim 4 in the preparation of a drug for treating knee osteoarthritis, characterized in that: Astragalus extract increases the level of GLP-1 in rats with knee osteoarthritis KOA model.

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