Application of angelica oligosaccharide in preparation of medicine for preventing or treating liver and kidney injury or reproductive function disorder

Abstract

The invention relates to an application of angelica oligosaccharide in preparation of a medicine for preventing or treating liver and kidney injury or reproductive dysfunction, and belongs to the field of biomedicine.The application is characterized in that polysaccharide oligosaccharide is extracted from angelica, and the content of the polysaccharide oligosaccharide in the extracted product is 64.67 + / -1.32%; a mouse visceral organ injury model is induced by a PRV (pseudorabies virus), and the Chinese angelica oligosaccharide is used for treatment, so that the Chinese angelica oligosaccharide is found to be capable of effectively improving liver and kidney injury and reproductive function disorder caused by the PRV, has relatively good anti-inflammatory and antiviral effects, can be used as a new medicine for treating the liver and kidney injury or the reproductive function disorder, and has a good application prospect in treatment of the liver and kidney injury or the reproductive function disorder. The medicine can also be used as a pseudorabies rehabilitation medicine.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to an application of Angelica sinensis oligosaccharide in preparation of a medicine for preventing or treating liver and kidney injury or reproductive function disorder. BACKGROUND

[0002] The liver, as the core digestive and metabolic organ of the human body, plays a crucial role in the catabolism of endogenous and exogenous toxins. Not only is it responsible for the synthesis of bile, metabolism of substances such as fats and proteins, but it also plays a key role in the detoxification process. However, with the rapid development of industrial technology and the significant improvement in people's living standards, the challenges faced by the liver have also increased. Damage from toxic substances and their metabolites, deterioration of air quality, intensification of environmental pollution, excessive drinking, and drug abuse all increase the risk of chemical liver injury. Long-term or acute liver injury can further evolve into fatty liver, hepatitis, cirrhosis, and even liver cancer. At the same time, long-term or acute liver injury can lead to sex hormone metabolic disorder, HPG axis disorder, and systemic metabolic abnormalities, thereby triggering male sexual dysfunction and sperm quality decline, female menstrual disorder and infertility, and other reproductive function disorders.

[0003] Most of the liver-protecting drugs available on the market have certain limitations, such as strong side effects and relatively single efficacy. Therefore, the development of liver-protecting drugs or natural products with low toxicity and significant protective effects is of great significance for the prevention and treatment of liver injury and the reduction of the incidence and mortality of liver disease, and has become one of the hotspots in the global scientific research field. In particular, natural products are considered a powerful supplement to chemically synthesized drugs due to their low toxicity, multiple targets, and multiple pathways. These natural products effectively alleviate liver injury and protect liver tissue by improving the antioxidant stress response, anti-inflammatory response, and regulation of the intestinal microbiota. At the same time, by improving the balance of sex hormone metabolism, regulating the function of the hypothalamic-pituitary-gonadal axis (HPG axis), and reducing systemic oxidative stress, the reproductive system is protected, including improving male sperm quality and sexual function, regulating female menstrual cycles and fertility, and achieving coordinated protection of the liver and reproductive system.

[0004] Angelica sinensis, also known as dry root, horse tail root, and cloud root, is a perennial herb. The stem is erect with longitudinal grooves, about 0.4-1 m high, and has a purple color without hair. With the development of modern analytical techniques, it has been found that volatile oils, organic acids, flavonoids, coumarins, polysaccharides, amino acids, and rich nutritional components are important active ingredients of Angelica sinensis. Angelica sinensis oligosaccharide (ASO) is the main active ingredient of Angelica sinensis, which has anti-inflammatory, analgesic, immunoregulatory, anti-tumor, anti- cardiovascular disease, liver protection, neuroprotective, and antioxidant activities. SUMMARY

[0005] In order to overcome the problems in the prior art, the angelica oligosaccharide is extracted from angelica, and the tests such as the pseudorabies model are established, so that it is found that the angelica oligosaccharide has obvious regulation and treatment effects on liver and kidney injuries and reproductive function disorders caused by pseudorabies, and can be used as a new drug for treating liver and kidney injuries and reproductive function disorders.

[0006] Based on this, the first object of the present application is to provide the application of the angelica oligosaccharide in the drugs for preventing or treating liver or kidney injuries and reproductive function disorders.

[0007] Further, the liver or kidney injuries refer to the liver or kidney injuries caused by or related to the pseudorabies virus.

[0008] The second object of the present application is to provide the application of the angelica oligosaccharide in the preparation of the therapeutic drugs for treating or preventing the diseases caused by or related to the pseudorabies virus.

[0009] Further, the application of the angelica oligosaccharide in the preparation of the drugs for reducing the levels of SCR or BUN in serum or increasing the expression levels of IL-4 and IL-10.

[0010] Further, the application of the angelica oligosaccharide in the preparation of the antiviral drugs.

[0011] Further, the diseases are one or more of liver injuries, kidney injuries and reproductive function disorders.

[0012] The third object of the present application is to provide the application of the angelica oligosaccharide in the preparation of the drugs for recovering from the pseudorabies diseases.

[0013] The fourth object of the present application is to provide a drug for preventing or treating liver or kidney injuries, wherein the drug comprises the angelica oligosaccharide.

[0014] The fifth object of the present application is to provide a drug for preventing or treating the diseases induced or caused by the pseudorabies, wherein the drug comprises the angelica oligosaccharide.

[0015] The sixth object of the present application is a preparation method of the angelica oligosaccharide, comprising the following steps: S1, the dried angelica is extracted by ethanol reflux to remove the fat-soluble substances, and a residue is obtained; S2, the residue obtained in the step S1 is boiled with water and then extracted to obtain a water extract, and the insoluble components are removed by centrifugation, and the supernatant is collected; S3, the supernatant obtained in the step S2 is subjected to alcohol precipitation treatment, and the supernatant is collected after standing overnight; S4, the supernatant obtained in the step S3 is removed of ethanol to obtain a crude oligosaccharide; S5, the crude oligosaccharide obtained in the step S4 is subjected to freeze-drying treatment The beneficial effects of the present application: The research of the present application proves that angelica oligosaccharide can improve the liver injury caused by PRV, inhibit the decrease of body weight, and reduce the levels of ALT and AST, through the PRV-induced mouse liver injury model.

[0016] The research of the present application proves that angelica oligosaccharide can improve the kidney injury caused by PRV, inhibit the decrease of body weight, and reduce the levels of SCR and BUN, through the PRV-induced mouse kidney injury model.

[0017] The research of the present application proves that angelica oligosaccharide can increase the expression levels of IL-4 and IL-10, and reduce the levels of TNF-α and IL-6.

[0018] The research of the present application proves the reproductive function regulation effect of angelica oligosaccharide. Specifically, the angelica oligosaccharide significantly regulates the endocrine function disorder of the ovary induced by PRV in in vivo experiments. By regulating the hypothalamus-pituitary-ovary axis to regulate the secretion of reproductive hormones, the damage to granulosa cells is reduced, and the reproductive function is protected. In terms of morphological structure, the tissue morphology gradually improves, the abnormal atretic follicles are reduced, the normal development and discharge of follicles at all levels are promoted, and the organ index tends to be normal. In terms of hormones, by down-regulating the levels of serum sex hormones FSH and LH, and up-regulating the levels of E2 and P4, the development and maturation of reproductive cells are promoted, thereby improving the reproductive ability of mice.

[0019] The research of the present application proves that angelica oligosaccharide can be used for the preparation of a treatment for diseases caused by PRV, and can also be used as a PRV rehabilitation drug, thereby providing a new treatment drug for organ damage and rehabilitation after PRV. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The present application is a method for regulating PRV-induced mouse body weight in different experimental groups of example 2; Figure 2 The present application is a method for regulating PRV-induced mouse body weight in different experimental groups of example 2; Figure 3 The present application is a method for regulating PRV-induced mouse body weight in different experimental groups of example 2; Figure 4 The present application is a method for regulating PRV-induced mouse body weight in different experimental groups of example 2; Figure 5 The present application is a method for regulating PRV-induced mouse body weight in different experimental groups of example 2; Figure 6 The present application is a method for regulating PRV-induced mouse body weight in different experimental groups of example 2; Figure 7is the ovary index situation of different experimental groups in the embodiment 2 of the present application; Figure 8 is the ovary H&E staining situation comparison of different experimental groups in the embodiment 2 of the present application; Figure 9 is the uterus index situation of different experimental groups in the embodiment 2 of the present application; Figure 10 is the uterus H&E staining situation comparison of different experimental groups in the embodiment 2 of the present application; Figure 11 is the influence of angelica oligosaccharide on the liver function and kidney function of PRV infected mice in the embodiment 2 of the present application; wherein, A figure is AST, B figure is ALT, C figure is SCR, and D figure is BUN; Figure 12 is the determination of the inflammation level of angelica oligosaccharide on each group in the embodiment 2 of the present application; wherein, A figure is IL-6, B figure is TNF-α, C figure is IL-4, and D figure is IL-10; Figure 13 is the influence of angelica oligosaccharide on the reproductive hormone level of mice in the embodiment 3 of the present application; wherein, A figure is E2 level, B figure is P4 level, C figure is FSH level, and D figure is LH level.

[0021] Figure 14 is the determination of the tissue virus load of mice; wherein, A figure is uterus, B figure is ovary, C figure is liver, and D figure is kidney; Remark: Figure 4 , 6 , 8 and 10, C is a normal group; P is a PRV model group; T is a tamoxifen+PRV group; L is a low angelica oligosaccharide+PRV group; M is a medium angelica oligosaccharide+PRV group; and H is a high angelica oligosaccharide+PRV group. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical scheme and beneficial effects of the present application more clear, the technical scheme of the present application will be described in detail below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0023] According to the present application, the concept of “prevention and / or treatment” means any measure suitable for preventing and / or treating PRV liver and kidney damage and reproductive function disorder related diseases, or treating such diseases or symptoms, or avoiding the recurrence of such diseases, such as recurrence after the end of the treatment period or treating the symptoms of the disease that has already occurred, or preventing or inhibiting or reducing the occurrence of such diseases or symptoms in advance.

[0024] The angelica oligosaccharide in the application is an ingredient extracted from angelica. In the application, the angelica oligosaccharide is prepared by water extraction.

[0025] In order to more clearly illustrate the application, the following embodiments are described in detail. Embodiment 1

[0026] Preparation of angelica oligosaccharide, including the following steps: S1, dry angelica (ASP) is extracted by ethanol reflux to remove fat-soluble substances, and the residue is obtained; S2, the residue of step S1 is boiled with water for extraction treatment, and the water extract is obtained. The insoluble components are removed by centrifugation, and the supernatant is collected; S3, the supernatant obtained in step S2 is subjected to alcohol precipitation treatment, and the supernatant is collected after standing overnight; S4, the supernatant obtained in step S3 is removed after ethanol to obtain crude oligosaccharide; S5, the crude oligosaccharide obtained in step S4 is subjected to freeze-drying treatment.

[0027] In step S1, the drying can be drying treatment, such as drying treatment at 40-60℃ (preferably 50℃); the specific method of ethanol reflux extraction includes: using high concentration ethanol (ethanol with a concentration greater than 75%) for reflux extraction 1-5 times, preferably 3 times, each time for 1-5 hours, preferably 2 hours.

[0028] It should be noted that: the drying temperature mainly affects the drying time, and the reflux extraction times mainly affect the extraction rate of angelica oligosaccharide, and has no substantial effect on the extracted components, and cannot be understood as a necessary limitation of the technical scheme of the application.

[0029] In step S2, the specific conditions of boiling with water include: the solid-liquid ratio is 1:1-10 (kg / L), preferably 1:5, the boiling treatment time is 1-3h, preferably 2h, and the boiling with water times is 1-5 times, preferably 3 times.

[0030] It should be noted that: the solid-liquid ratio and the boiling times mainly affect the extraction rate of angelica oligosaccharide, and have no substantial effect on the extracted components, and the solid-liquid ratio and the extraction times during extraction cannot be regarded as a necessary limitation of the technical scheme of the application.

[0031] In step S3, the alcohol precipitation treatment method includes: according to the ratio obtained by pre-experiment, 95% ethanol is added to the supernatant, stirred uniformly, and the supernatant is collected after standing; the standing treatment time is 10-40 hours, preferably 24 hours.

[0032] Note: The pre-experiment is to pre-take about 10ml of concentrated liquid, and the pre-experiment is carried out with 95% ethanol: concentrated liquid 3:1, to observe whether the flocculent polysaccharide can be seen, and the flocculent polysaccharide is too little to reduce the proportion, In step S4, the supernatant is recovered by a rotary evaporator to obtain a crude oligosaccharide.

[0033] In step S5, the storage condition is -80°C refrigerator storage, and the freeze-drying machine is pre-cooled for 2h, and the freeze-dried powder is collected after freeze-drying for 72h for standby.

[0034] Note: The ordinary low-temperature-resistant plastic (such as PP) has a similar effect to the special freeze-drying bottle, and does not need to be strictly screened. The pre-cooling time of the freeze-drying machine is 2h and 4h, and the cold trap temperature can finally meet the standard, and does not affect the sample quality.

[0035] The preparation method further comprises freeze-drying the angelica oligosaccharide obtained in step S5 to obtain a freeze-dried product.

[0036] The glucose standard product is prepared by the phenol-sulfuric acid method, a standard curve (R²≥0.999) is prepared, quantitative analysis is carried out, and the sugar content of the prepared angelica oligosaccharide freeze-dried product is 64.67±1.32%.

[0037] The following provides an application example of the angelica oligosaccharide prepared in this embodiment. Example 2

[0038] The liver protection effect of angelica oligosaccharide in the treatment of mouse liver and kidney injury induced by PRV was tested. 144 6-week-old ICR female mice (24.56±0.75 g) were randomly divided into 6 groups, and a control group, a PRV model group, a tamoxifen+PRV group, a low angelica oligosaccharide+PRV group, a medium angelica oligosaccharide+PRV group and a high angelica oligosaccharide+PRV group were set. The low angelica oligosaccharide (50mg / kg)+PRV group, the medium angelica oligosaccharide (100mg / kg)+PRV group, the high angelica oligosaccharide (200mg / kg)+PRV group and the tamoxifen (3mg / kg)+PRV group were administered to the mice by gavage according to the corresponding dose, and the control group was administered with normal saline by gavage, and the mice were allowed to freely eat during the experiment. After 7 days, the mice in the PRV model group, the tamoxifen+PRV group, the low angelica oligosaccharide+PRV group, the medium angelica oligosaccharide+PRV group and the high angelica oligosaccharide+PRV group were respectively infected with 20μL of PRV suspension of 10 50 mL 5 The control group was treated with the same volume of PBS (phosphate buffered saline) by nose drop, and the clinical symptoms of the mice were observed at any time.

[0039] After challenge, the mice were taken blood from the eye vein at 24, 48, 72 h, 1.5 mL of ordinary blood, 3000 r / min centrifugal 15 min to separate serum for storage. After the last blood collection, the mice were sacrificed by CO2 asphyxia, the head was dissected, and the uterus, ovary, liver, kidney and other organs were collected, one part was stored in a -80°C refrigerator, and the other part was fixed with 4% paraformaldehyde solution. After 24 h of 4% paraformaldehyde fixation, the waste liquid was discarded, and the fixed material was again stored in new 4% paraformaldehyde solution for subsequent experiments.

[0040] The body weight of each experimental group of mice was weighed, and the average daily body weight of each group of mice was calculated (such as Figure 1 ), and the results showed that angelica oligosaccharide slowed down the trend of weight loss and liver index of mice induced by PRV.

[0041] The organ index of body weight, liver, kidney, ovary, uterus and other organs was analyzed, and the organ tissue was collected and stored in 4% paraformaldehyde fixing solution for more than 48 h. The fixed tissue was gradient dehydrated, embedded in paraffin, sliced into 5 μm thick sections, stained with H&E, and observed under a microscope for morphological changes in organ tissue (results are shown in Figures 3 to 10 ). The results showed that the liver lobule structure of the normal group of mice was clear, the liver cell cord was arranged radially around the central vein, the cytoplasm of the liver cells was uniform, the portal area structure was clear, there was no fibrous connective tissue hyperplasia, and no inflammatory cell infiltration was observed. The kidney tissue structure remained normal, and no inflammatory cell infiltration and necrosis was observed; the liver cell cord of the PRV model group was arranged in disorder, the size of the liver cells was uneven, the liver cells were swollen, the cytoplasm was loose, and there were scattered punctate hemorrhage and lymphocyte infiltration. The kidney structure showed abnormalities, a large number of mesangial cells in the glomerulus were reduced, the structure of the renal tubular lumen was not clear, and a large number of inflammatory cells were observed in the interstitium; the liver cell cord of the tamoxifen group was arranged in order, and the inflammatory infiltration cells in the cells were significantly reduced; the cell damage degree and inflammatory cell infiltration of the angelica drug group were reduced, and showed a dose-dependent effect. Compared with the PRV model group, the damage of the glomerulus and renal tubule of each drug group was reduced.

[0042] Liver function index determination: After blood collection, centrifugation was performed at 4°C for 2 h (3500 rpm, 15 min), and the supernatant was collected. Then the detection was performed according to the instructions of the ALT and AST kits. ELISA results (Figures Figure 11 A-B) showed that angelica oligosaccharide could reduce the levels of ALT and AST.

[0043] Kidney function index determination: The results showed (Figures Figure 11 C-D) that angelica oligosaccharide could improve PRV-induced kidney damage, inhibit weight loss, and reduce the levels of SCR and BUN.

[0044] Example 3: Anti-inflammatory effect of angelica oligosaccharide in vivo Experimental methods, animal models: same as example 2.

[0045] Index determination: After standing at 4℃ for 2h, centrifugation (3500 rpm, 15 min) was performed to collect the supernatant. Then detection was performed according to the IL-6, IL-10, IL-4, TNF-a kit instructions. ELISA results showed that (Fig. 1A-D), angelica oligosaccharide could increase the expression levels of IL-4 and IL-10, and reduce the expression levels of TNF-a and IL-6. Figure 12

[0046] Example 4: Angelica oligosaccharide improves PRV-induced reproductive dysfunction in mice Experimental methods, animal models: same as example 2.

[0047] Index determination: The hormone levels and viral load in the mouse organs were determined.

[0048] In terms of hormones, the concentrations of estradiol (E2), progesterone (P4), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in mouse serum were determined by enzyme-linked immunosorbent assay (ELISA) to ensure high specificity and sensitivity. The results showed that (Fig. 2A-D), PRV could cause the levels of E2 and P4 in mouse serum to decrease, and the levels of FSH and LH to increase. Angelica oligosaccharide reversed this change at three time points, down-regulated the levels of serum sex hormones FSH and LH, and up-regulated the levels of E2 and P4, promoting the development and maturation of reproductive cells and thus improving the reproductive ability of mice, especially the high angelica oligosaccharide group, which had a more obvious effect. Figure 13 In terms of viral load (Fig. 3A-D), it was found that in the uterus, ovaries, liver, and kidneys of mice, the viral load of the PRV model group was significantly higher than that of the normal group at three time points (P<0.001) and showed a trend of first increasing and then decreasing. The high angelica oligosaccharide group significantly or extremely significantly reduced the viral load at multiple time points (P<0.01 or P<0.001), and had statistical differences compared with the tamoxifen group and the low angelica oligosaccharide group (P<0.05). The tamoxifen group also significantly reduced the viral load at certain time points (P<0.05).

[0049] Figure 14 A to D), it was found that in the uterus, ovaries, liver, and kidneys of mice, the viral load of the PRV model group was significantly higher than that of the normal group at three time points (P<0.001) and showed a trend of first increasing and then decreasing. The high angelica oligosaccharide group significantly or extremely significantly reduced the viral load at multiple time points (P<0.01 or P<0.001), and had statistical differences compared with the tamoxifen group and the low angelica oligosaccharide group (P<0.05). The tamoxifen group also significantly reduced the viral load at certain time points (P<0.05). Figure 13 A-D). It was found that after PRV infection in mice, the viral load in tissues from high to low was ovary > uterus > liver > kidney, and the change trend of viral load from 24h to 48h, and then to 72h was first increased and then decreased. High angelica oligosaccharide showed significant antiviral effect on the uterus, ovaries, liver, and kidneys of PRV-infected mice, and the effect was better than that of tamoxifen and low angelica oligosaccharide. Tamoxifen also showed certain antiviral effect on the uterus, ovaries, and kidneys of PRV-infected mice, but the effect was weaker.​​

[0050] Finally, it should be pointed out that the above preferred embodiments are only used to illustrate the technical solutions of the present application but not to limit the present application. Although the present application has been described in detail by the above preferred embodiments, those skilled in the art should understand that various modifications can be made in form and details thereof without departing from the scope of the present application defined by the claims.

Claims

1. Application of angelica oligosaccharide as a drug for preventing or treating liver or kidney damage and reproductive dysfunction.

2. The use according to claim 1, characterized in that The liver or kidney damage refers to liver or kidney damage caused by or associated with pseudorabies virus.

3. Application of Angelica oligosaccharide in the preparation of therapeutic drugs for treating or preventing pseudorabies virus-induced or related diseases.

4. The use according to claim 3, characterized in that Application of angelica oligosaccharide in preparing drugs for reducing SCR or BUN levels in serum or increasing IL-4 and IL-10 expression levels.

5. The use according to claim 3, characterized in that Application of angelica oligosaccharide in the preparation of antiviral drugs.

6. The use according to claim 3, characterized in that The disease is one or more of liver damage, kidney damage, and reproductive dysfunction.

7. Application of Angelica oligosaccharide in the preparation of pseudorabies recovery drugs.

8. A drug for preventing or treating liver or kidney damage, characterized in that: The medicine contains angelica oligosaccharide.

9. A drug for preventing or treating a disease induced or caused by pseudorabies, characterized in that: The medicine contains angelica oligosaccharide.

10. A method for preparing angelica oligosaccharide, characterized in that: The following steps are involved: S1. Refluxing the dried angelica with ethanol to remove fat-soluble substances to obtain a residue; S2. The residue obtained in step S1 is boiled with water and then subjected to extraction treatment to obtain a water extract, which is centrifuged to remove insoluble components and the supernatant is collected; S3, subjecting the supernatant obtained in step S2 to alcohol precipitation, and collecting the supernatant after standing overnight; S4, removing ethanol from the supernatant obtained in step S3 to obtain crude oligosaccharides; S5. Freeze-drying the crude oligosaccharide obtained in step S4.

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