Application of nine-steamed nine-sun-dried polygonum multiflorum extract in preparation of medicine for treating kidney deficiency

Abstract

The invention relates to the technical field of medicines, in particular to application of a nine-steamed nine-sun-dried polygonum multiflorum extract in preparation of a medicine for treating kidney deficiency. In the specific embodiment of the invention, a kidney yin deficiency animal model is constructed, the kidney tonifying effect of the nine-steamed and nine-sun-dried polygonum multiflorum extract is evaluated by observing the change condition of the hormone levels of HPT axis, HPA axis, HPG axis and the like after model animal administration, and the result shows that the nine-steamed and nine-sun-dried polygonum multiflorum extract can remarkably regulate the functions of the HPT axis, the HPA axis and the HPG axis and play the kidney tonifying effect, so that the effect of tonifying the kidney is achieved. The method can be used for preparing kidney-tonifying medicines. Therefore, the invention provides a scientific basis for research and development of kidney tonifying medicines. Meanwhile, the specific embodiment of the invention evaluates the hepatotoxicity of the nine-steamed and nine-sun-dried polygonum multiflorum extract by using a special liver injury model, and proves that the medication safety of the nine-steamed and nine-sun-dried polygonum multiflorum extract is superior to that of raw polygonum multiflorum.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, and in particular to application of a nine-steamed and nine-dried Polygonum multiflorum extract in preparing a medicine for treating kidney deficiency. Background Art

[0002] Traditional Chinese medicine believes that the kidney stores innate essence, is the basis of yin and yang of the internal organs, and the source of life, so the kidney is called the "innate basis". The kidney is responsible for bone and marrow production, and has an important influence on the growth and development of the human body and reproductive function. Therefore, kidney deficiency will lead to a series of symptoms such as loss of qi and blood, decreased body function, and premature aging; at the same time, the kidney and bladder are the exterior and interior of each other, and the orifices are in the ears and two yin organs. Kidney deficiency will also affect the functions of related internal organs, leading to insomnia, frequent urination, osteoporosis and other symptoms. At the same time, modern research has also confirmed that Chinese medicine has the characteristics of "multi-component, multi-pathway, multi-target" synergistic effects, and has unique advantages in adjusting the functions of internal organs.

[0003] Polygonum multiflorum is a commonly used tonic Chinese medicine, and its clinical application is mainly based on processed products. In ancient times, the processing method of nine steaming and nine drying was mostly used, while in modern times, the method of stewing / steaming with black bean juice is mainly used to exert its effects of nourishing the liver and kidney, benefiting the essence and blood, and blackening the hair. Most of the prescriptions for nourishing the liver, kidney and benefiting the essence and blood included in the "Chinese Pharmacopoeia" contain processed Polygonum multiflorum. However, Polygonum multiflorum processed by modern methods often causes hepatotoxicity due to non-standard processes, and leads to clinical adverse events. Nine steaming and nine drying is a traditional special processing process. Polygonum multiflorum processed by this method has a long history of application and has better safety than modern process products. However, there is no report on the kidney-tonifying effect of nine steaming and nine drying Polygonum multiflorum. It is urgent to provide a safe and effective preparation method of Polygonum multiflorum slices and its active substances to provide support for the development of modern Chinese medicine for kidney tonification. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a nine-steamed and nine-dried Polygonum multiflorum extract in the preparation of a drug for treating kidney deficiency, so as to solve the problems existing in the above-mentioned prior art. The present invention finds that the nine-steamed and nine-dried Polygonum multiflorum extract has a significant effect in treating kidney deficiency, and its medication safety is better than that of raw Polygonum multiflorum.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides application of a nine-steamed and nine-dried Polygonum multiflorum extract in preparing a medicine for treating kidney deficiency.

[0007] Preferably, the method for preparing the nine-steamed and nine-dried Polygonum multiflorum extract comprises the following steps:

[0008] The nine-steamed and nine-dried Polygonum multiflorum was extracted by percolation with a methanol solution to obtain an extract;

[0009] After the extract is decompressed to recover the methanol solution, it is freeze-dried to obtain the nine-steamed and nine-dried Polygonum multiflorum extract.

[0010] Preferably, the concentration of methanol in the methanol solution is 80%;

[0011] The amount of the methanol solution used is 10-30 times the mass of the nine-steamed and nine-dried Polygonum multiflorum.

[0012] Preferably, the method for preparing nine-steamed and nine-dried Polygonum multiflorum comprises the following steps:

[0013] After laying raw Polygonum multiflorum and black beans at intervals, they are steamed and air-dried, with one steaming and one air-dried as one cycle, which is repeated 9 times to obtain the nine-steamed and nine-dried Polygonum multiflorum.

[0014] Preferably, the mass ratio of the raw Polygonum multiflorum slices to the black beans is 1:(1-4);

[0015] The steaming time is 1-5h.

[0016] Preferably, the black beans are obtained by soaking in water for 12 hours.

[0017] Preferably, the drug achieves the effect of treating kidney deficiency by improving the hormone levels of the HPT axis, HPA axis and HPG axis.

[0018] Further preferably, improving HPT axis dysfunction specifically includes lowering the levels of triiodothyronine (T3) and thyroxine (T4), and increasing the level of thyroid stimulating hormone (TSH); improving HPA axis dysfunction specifically includes lowering the levels of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and cortisol (CORT); improving HPG axis hormone levels specifically includes increasing the levels of estradiol (E2) and testosterone (T); improving cell function specifically includes lowering the level of cyclic adenosine monophosphate (cAMP), increasing the level of cyclic guanosine monophosphate (cGMP), and significantly reducing the cAMP / cGMP ratio.

[0019] The invention provides a medicine for treating kidney deficiency. The effective component of the medicine comprises a nine-steamed and nine-dried Polygonum multiflorum extract.

[0020] Preferably, the method for preparing the nine-steamed and nine-dried Polygonum multiflorum extract comprises the following steps:

[0021] The nine-steamed and nine-dried Polygonum multiflorum was extracted by percolation with a methanol solution to obtain an extract;

[0022] After the extract is decompressed to recover the methanol solution, it is freeze-dried to obtain the nine-steamed and nine-dried Polygonum multiflorum extract.

[0023] Further preferably, the concentration of methanol in the methanol solution is 80%;

[0024] The amount of the methanol solution used is 10-30 times the mass of the nine-steamed and nine-dried Polygonum multiflorum.

[0025] Further preferably, the preparation method of the nine-steamed and nine-dried Polygonum multiflorum comprises the following steps:

[0026] After laying raw Polygonum multiflorum and black beans at intervals, steaming and air-drying are performed, with steaming and air-drying as one cycle, which is repeated 9 times to obtain the nine-steamed and nine-dried Polygonum multiflorum.

[0027] Further preferably, the mass ratio of the raw Polygonum multiflorum to the black bean is 1:(1-4);

[0028] The steaming time is 1-5h.

[0029] Further preferably, the black beans are obtained by soaking in water for 12 hours.

[0030] Further preferably, the drug also includes a pharmaceutically acceptable excipient.

[0031] Further preferably, the auxiliary materials include one or more of a binder, a plasticizer, a glidant, a disintegrant, a lubricant and a propellant.

[0032] Preferably, the dosage form of the drug includes capsules, pills, tablets, granules, oral liquids, ointments, aerosols or sprays.

[0033] The present invention discloses the following technical effects:

[0034] The present invention provides the use of a nine-steamed and nine-dried Polygonum multiflorum extract in the preparation of a drug for treating kidney deficiency. In a specific embodiment of the present invention, a kidney yin deficiency animal model was constructed, and the kidney-tonifying effect of the nine-steamed and nine-dried Polygonum multiflorum extract was evaluated by observing the changes in hormone levels such as the thalamus-pituitary-thyroid (HPT) axis, the hypothalamus-pituitary-adrenal cortex (HPA) axis, and the hypothalamus-pituitary-gonad (HPG) axis after administration of the model animal. The results show that the nine-steamed and nine-dried Polygonum multiflorum extract can significantly regulate the functions of the HPT axis, the HPA axis, and the HPG axis, exert a kidney-tonifying effect, and can be used for the preparation of kidney-tonifying drugs. It can be seen that the present invention provides a scientific basis for the research and development of kidney-tonifying drugs. At the same time, the specific embodiment of the present invention evaluated the hepatotoxicity of the nine-steamed and nine-dried Polygonum multiflorum extract in a peculiar liver injury model, confirming that the drug safety of the nine-steamed and nine-dried Polygonum multiflorum extract is better than that of raw Polygonum multiflorum. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 This is a comparison chart of extraction effects of different extraction solvents;

[0037] Figure 2 The results of the HPT axis hormone test in kidney yin deficiency model mice; A is the level of triiodothyronine (T3), B is the level of thyroxine (T4), and C is the level of thyroid stimulating hormone (TSH). Compared with the control group, * P<0.05, ** P<0.01; compared with the model group, △ P<0.05, △△ P<0.01; compared with raw Polygonum multiflorum, # P<0.05, ## P < 0.01;

[0038] Figure 3 The results of cAMP and cGMP determination in mice with kidney yin deficiency model; A is the level of cyclic adenosine monophosphate (cAMP), B is the level of cyclic guanosine monophosphate (cGMP), and C is the ratio of cAMP / cGMP; compared with the control group, * P<0.05, ** P<0.01; compared with the model group, △ P<0.05, △△ P<0.01; compared with raw Polygonum multiflorum, # P<0.05, ## P < 0.01;

[0039] Figure 4 The results of the HPA axis hormone test in kidney yin deficiency model mice; A is the level of adrenocorticotropic hormone (CRH), B is the level of adrenocorticotropic hormone (ACTH), and C is the level of cortisol (CORT); compared with the control group, * P<0.05, ** P<0.01; compared with the model group, △ P<0.05, △△ P<0.01; compared with raw Polygonum multiflorum, # P<0.05, ## P < 0.01;

[0040] Figure 5The results of the HPG axis hormone test in kidney yin deficiency model mice; A is the level of estradiol (E2), B is the level of testosterone (T); compared with the control group, * P<0.05, ** P<0.01; compared with the model group, △ P<0.05, △△ P<0.01; compared with raw Polygonum multiflorum, # P<0.05, ## P < 0.01;

[0041] Figure 6 The results of plasma transaminase determination in rats with idiosyncratic hepatotoxicity model; A is the ALT level, B is the AST level; compared with the control group, * P<0.05, ** P < 0.01;

[0042] Figure 7 The results of the determination of inflammatory factors in rats with idiosyncratic hepatotoxicity model; A is the level of TNF-α, B is the level of IL-1β, C is the level of IL-6, and D is the level of IFN-γ; * P<0.05, ** P<0.01; compared with the model group, △ P<0.05, △△ P<0.01; compared with raw Polygonum multiflorum, # P<0.05, ## P<0.01

[0043] Figure 8 Pathological sections of liver tissues of rats with idiosyncratic hepatotoxicity model (200×); among them, A is Control; B is LPS; C is HSW-L; D is HSW-M; E is HSW-H; F is ZSSW-9-L; G is ZSSW-9-M; H is ZSSW-9-H; I is LPS+HSW-L; J is LPS+HSW-M; K is LPS+HSW-H; L is LPS+ZSSW-9-L; M is LPS+ZSSW-9-M; N is LPS+ZSSW-9-H. DETAILED DESCRIPTION

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0046] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0047] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0048] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0049] Example 1: Investigation of extraction solvent

[0050] 1. Instruments and test drugs

[0051] 1.1 Instruments

[0052] High performance liquid chromatograph (LC-20AT, Shimadzu Corporation, Japan), 1 / 100,000 electronic analytical balance (XSR105DU, Mettler-Toledo), 1 / 10,000 electronic balance (FA2204B, Shanghai Precision Scientific Instrument Co., Ltd.), ultrasonic cleaner (KQ-100DE, Kunshan Ultrasonic Instrument Co., Ltd.), small high-speed pulverizer (ZN-08L, Beijing Zhongke Naichi Technology Co., Ltd.).

[0053] 1.2. Drug testing

[0054] 5-Hydroxymethylfurfural, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (hereinafter referred to as stilbene glycoside), physophanol methyl ether-8-O-β-D-glucoside (Chengdu Pufeide Biotechnology Co., Ltd., batch numbers 20090201, 19070305, 20120702, purity ≥98%), gallic acid, physophanol-8-O-β-D-glucoside (Sichuan Weiqiqi Biological Science Co., Ltd., batch numbers 20090201, 19070305, 20120702, purity ≥98%), Technology Co., Ltd., batch numbers wkq21090111 and wkq21060404, purity ≥98%), rhubarb (Beijing Rongcheng Xinde Technology Development Co., Ltd., batch number D-029-190713, purity ≥98%), rhubarb methyl ether (Chengdu Pusi Biotechnology Co., Ltd., batch number PS0291-0020, purity>98.5%), acetonitrile was chromatographic grade, water was purified water, and other reagents were of analytical grade.

[0055] 2. Methods and Results

[0056] 2.1 Chromatographic conditions

[0057] Agilent ZORBAX SB-C 18 Chromatographic column (4.6 mm × 250 mm, 5 μm), mobile phase acetonitrile (A)-0.1% formic acid water (B), gradient elution (0-20 min, 5%-25% A; 20-26 min, 25%-35% A; 26-30 min, 35%-50% A; 30-35 min, 50%-70% A; 35-45 min, 70%-90% A; 45-50 min, 90% A), detection wavelength 280 nm, flow rate 1.00 mL / min, column temperature 30°C, injection volume 10 μL. Under these conditions, the components measured can be well separated.

[0058] 2.2 Preparation of reference solution

[0059] Take appropriate amount of gallic acid, 5-hydroxymethylfurfural, stilbene glycoside, rhubarb-8-O-β-D-glucoside, physcion-8-O-β-D-glucoside, rhubarb, and physcion methyl ether reference substances, accurately weigh them, and add 80% methanol to prepare reference substance solutions containing 57.6 mg / L of gallic acid, 68.4 mg / L of 5-hydroxymethylfurfural, 625.0 mg / L of stilbene glycoside, 50.2 mg / L of rhubarb-8-O-β-D-glucoside, 8.6 mg / L of physcion methyl ether-8-O-β-D-glucoside, 180.0 mg / L of rhubarb, and 0.216 mg / L of physcion methyl ether.

[0060] 2.3. Preparation of test solution

[0061] 2.3.1. Preparation of Nine-steamed and Nine-dried Polygonum multiflorum

[0062] Take raw Polygonum multiflorum slices and lay them out with black beans. Before laying out, the black beans should be soaked in water for 12 hours. The thickness of the black beans should be 1 cm. Steam them at 100℃ for 4 hours, take them out and air-dry them. Repeat this process nine times to obtain nine-steamed and nine-dried Polygonum multiflorum. The ratio of Polygonum multiflorum to black beans is 1:3.

[0063] 2.3.2 Preparation of test solution

[0064] Take about 1 g of the above-prepared nine-steamed and nine-dried Polygonum multiflorum powder (passed through a 40-mesh sieve), accurately weigh it, a total of 10 portions, place it in a stoppered conical flask, and accurately add 50% (volume percentage) methanol, 80% (volume percentage) methanol, anhydrous methanol, 50% (volume percentage) ethanol, and ethanol, respectively, in an amount of 25 mL, seal it, weigh it, ultrasonically extract it for 30 minutes, cool it, seal it, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, and filter the filtrate with a microporous filter membrane (0.45 μm) to obtain it.

[0065] 2.4. Sample determination and results

[0066] Accurately pipette 10 μL of the sample and reference solution respectively and inject them into the liquid chromatograph for determination. Figure 1 As shown, the results show that the extraction content is the highest with 80% (volume percentage) methanol, so 80% (volume percentage) methanol is selected as the extraction solvent.

[0067] Example 2 Preparation of Nine-steamed and Nine-dried Polygonum multiflorum Extract

[0068] (1) Take raw Polygonum multiflorum slices and lay them with black beans. Before laying, the black beans should be soaked in water for 12 hours. The black beans are laid with a thickness of 1 cm. Steam them at 100°C for 4 hours, take them out and air-dry them. Repeat this process nine times to obtain nine-steamed and nine-dried Polygonum multiflorum slices. The ratio of Polygonum multiflorum to black beans is 1:1.

[0069] (2) taking the nine-steamed and nine-dried Polygonum multiflorum slices, adding 80% methanol solution to perform percolation extraction to obtain an extract, wherein the extraction temperature is room temperature; the amount of 80% (volume percentage) methanol solution is 10 times the mass of the nine-steamed and nine-dried Polygonum multiflorum slices;

[0070] (3) After the extract is decompressed to recover the methanol solution, the resultant is freeze-dried to obtain the nine-steamed and nine-dried Polygonum multiflorum slice extract, wherein the freeze-drying time is 12 hours and the temperature is -65°C.

[0071] Example 3 Preparation of Nine-steamed and Nine-dried Polygonum multiflorum Extract

[0072] (1) Take raw Polygonum multiflorum slices and lay them with black beans. Before laying, the black beans should be soaked in water for 12 hours. The black beans are laid with a thickness of 2 cm. Steam them at 100°C for 3 hours, take them out and air-dry them. Repeat this process nine times to obtain nine-steamed and nine-dried Polygonum multiflorum slices. The ratio of Polygonum multiflorum to black beans is 1:2.

[0073] (2) taking the nine-steamed and nine-dried Polygonum multiflorum slices, adding 80% (volume percentage) methanol solution to perform percolation extraction to obtain an extract, wherein the extraction temperature is room temperature; the amount of 80% (volume percentage) methanol solution is 30 times the mass of the nine-steamed and nine-dried Polygonum multiflorum slices;

[0074] (3) After the extract is decompressed to recover the methanol solution, the resultant is freeze-dried to obtain the nine-steamed and nine-dried Polygonum multiflorum slice extract, wherein the freeze-drying time is 30 hours and the temperature is -65°C.

[0075] Example 4 Preparation of Nine-steamed and Nine-dried Polygonum multiflorum Extract

[0076] (1) Take raw Polygonum multiflorum slices and lay them with black beans. Before laying, the black beans should be soaked in water for 12 hours. The black beans are laid with a thickness of 3 cm. Steam them at 100°C for 4 hours, take them out and air-dry them. Repeat this process nine times to obtain nine-steamed and nine-dried Polygonum multiflorum slices. The ratio of Polygonum multiflorum to black beans is 1:3.

[0077] (2) taking the nine-steamed and nine-dried Polygonum multiflorum slices, adding 80% (volume percentage) methanol solution to perform percolation extraction to obtain an extract, wherein the extraction temperature is room temperature; the amount of 80% (volume percentage) methanol solution is 21 times the mass of the nine-steamed and nine-dried Polygonum multiflorum slices;

[0078] (3) After the extract is decompressed to recover the methanol solution, the obtained product is freeze-dried to obtain the nine-steamed and nine-dried Polygonum multiflorum slice extract, wherein the freeze-drying time is 24 hours and the temperature is -65°C.

[0079] Example 5 Preparation of Nine-steamed and Nine-dried Polygonum multiflorum Extract

[0080] (1) Take raw Polygonum multiflorum slices and lay them with black beans. Before laying, the black beans should be soaked in water for 12 hours. The black beans are laid with a thickness of 2 cm. Steam them at 100°C for 5 hours, take them out and air-dry them. Repeat this process nine times to obtain nine-steamed and nine-dried Polygonum multiflorum slices. The ratio of Polygonum multiflorum to black beans is 1:3.

[0081] (2) taking the nine-steamed and nine-dried Polygonum multiflorum slices, adding 80% (volume percentage) methanol solution to perform percolation extraction to obtain an extract, wherein the extraction temperature is room temperature; the amount of 80% (volume percentage) methanol solution is 15 times the mass of the nine-steamed and nine-dried Polygonum multiflorum slices;

[0082] (3) After the extract is decompressed to recover the methanol solution, the resultant is freeze-dried to obtain the nine-steamed and nine-dried Polygonum multiflorum slice extract, wherein the freeze-drying time is 18 hours and the temperature is -65°C.

[0083] Example 6

[0084] 1. Animals and Grouping

[0085] KM mice, male, 90, weighing 19-21 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., license number SYXK (Beijing) 2021-0011. They were kept in the Animal Experiment Center of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences [license number SYXK (Beijing) 2019-0003]. The experiment was approved by the Animal Ethics Committee of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences (ethics approval number: 2023B138) and complied with the 3R principle.

[0086] The mice were randomly divided into 9 groups according to their body weight, namely, a control group (Control), a model group (Model), a positive drug group (Liuwei Dihuang Pills, LWDHW), low, medium and high dose groups of raw Polygonum multiflorum (HSW-L, HSW-M and HSW-H), and low, medium and high dose groups of nine-steamed and nine-dried Polygonum multiflorum extract (prepared in Example 4) (ZSSW-9-L, ZSSW-9-M and ZSSW-9-H), with 10 mice in each group.

[0087] 2. Reagents and instruments

[0088] 2.1 Reagents

[0089] Thyroid tablets (Shanghai Zhonghua Pharmaceutical Co., Ltd., batch number 220502), reserpine (China Food and Drug Inspection Institute, batch number 100041-202015, purity 99.9%), Liuwei Dihuang Pills (Zhongjing Wanxi Pharmaceutical Co., Ltd., batch number 220937), mouse T3, T4, TSH, cAMP, cGMP, CRH, ACTH, CORT, E2, T enzyme-linked immunosorbent assay kit (Jiangsu Jingmei Biotechnology Co., Ltd., catalog numbers JM-02857M1, JM-02858M1, and JM-02827M2, respectively). 1. JM-02304M1, JM-02849M1, JM-02852M1, JM-02837M1, JM-02848M1, JM-11776M1, JM-02792M1, all with batch number 202308), 4% paraformaldehyde (Biosharp, product number BL539A, batch number 23206277), anhydrous ethanol, xylene (Sinopharm Chemical Reagent Co., Ltd.), HE staining kit (Genepool, product number GPB1826), neutral gum (Solabo, product number G8590).

[0090] 2.2 Instruments

[0091] 352 microplate reader (Labsystems Multiskan MS, Finland), AC8 plate washer (ThermoLabsystems, Finland), TG16W microcentrifuge (Hunan Xiangyi Group), GNP-9080 water-tight constant temperature incubator (Shanghai Jinghong Experimental Equipment Co., Ltd.), JJ-12J dehydrator, JB-P5 embedding machine, JB-L5 freezing table (Wuhan Junjie Electronics Co., Ltd.), RM2016 pathology slicer (Shanghai Leica Instrument Co., Ltd.), KD-P tissue slide machine (Zhejiang Jinhua Kedi Instrument Equipment Co., Ltd.), DHG-9140A oven (Shanghai Huitai Instrument Manufacturing Co., Ltd.), Pannoramic MIDI panoramic scanner (3DHISTECH, Hungary), 10127105P-G cover glass and slide (Jiangsu Shitai Experimental Equipment Co., Ltd.), 1730R micro high-speed refrigerated centrifuge (Hong Kong Gene Co., Ltd.), XSR105DU one-hundred-thousandth electronic analytical balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.), FA2204B one-ten-thousandth electronic balance (Shanghai Precision Scientific Instrument Co., Ltd.).

[0092] 3. Modeling and drug administration

[0093] Except for the control group, mice in the other groups were gavaged with thyroxine (112.5 mg kg -1 ) and reserpine (0.75 mg kg -1 ), and the control group was gavaged with an equal amount of distilled water. Except for the control group and the model group, the mice in the other groups were gavaged with the set dose every afternoon (the dosage of Polygonum multiflorum, nine-steamed and nine-dried Polygonum multiflorum extract and positive drugs were calculated according to the clinical dosage of humans, and three dose groups were set up: low, medium and high dose groups, and the raw Polygonum multiflorum was 0.78 g·kg -1 、1.56g herbal medicine kg -1 、3.12g herbal medicine kg -1 , nine steamed and nine dried Polygonum multiflorum are 1.56g crude drug kg -1 、3.12g herbal medicine kg -1 、6.24g herbal medicine kg -1 , Liuwei Dihuang Wan is 4.68g·kg -1 ), the control group and the model group were gavaged with an equal amount of distilled water for 10 consecutive days. After the last administration, the animals were fasted but not watered for 12 hours. On the 11th day, the eyeballs were removed and blood was collected. The blood was allowed to stand for 2 hours and centrifuged at 4°C and 3500r / min for 15 minutes. The upper serum was aspirated and the ELISA method was used to detect the contents of T3, T4, TSH, cAMP, cGMP, CRH, ACTH, CORT, E2 and T in the mouse serum.

[0094] 4. Results

[0095] 4.1 Effects of nine-steamed and nine-dried Polygonum multiflorum extract on HPT axis function in mice with kidney yin deficiency model

[0096] The results of the investigation on the effect of nine-steamed and nine-dried Polygonum multiflorum extract on the HPT axis function in mice with kidney yin deficiency model are shown in Figure 2 .Depend on Figure 2 It can be seen that compared with the model group, raw Polygonum multiflorum and nine-steamed and nine-dried Polygonum multiflorum extracts can significantly reduce T3 and T4 levels, and significantly increase TSH levels (P<0.01). Among them, the T3 level of the high-dose group of nine-steamed and nine-dried Polygonum multiflorum extract was most significantly reduced, indicating that the nine-steamed and nine-dried Polygonum multiflorum extract can improve the HPT axis function of mice with kidney yin deficiency model.

[0097] 4.2 Effects of nine-steamed and nine-dried Polygonum multiflorum extract on cellular function in mice with kidney-yin deficiency model

[0098] The results of the investigation on the effect of nine-steamed and nine-dried Polygonum multiflorum extract on the cell function of mice with kidney yin deficiency model are shown in Figure 3 .Depend on Figure 3 It can be seen that compared with the model group, the medium and high dose groups of the nine-steamed and nine-dried Polygonum multiflorum extract and all dose groups of raw Polygonum multiflorum can significantly reduce the cAMP level, increase the cGMP level, and reduce the cAMP / cGMP ratio, and the effect of the nine-steamed and nine-dried Polygonum multiflorum extract is better, indicating that the nine-steamed and nine-dried Polygonum multiflorum can improve the cellular function of mice with kidney yin deficiency.

[0099] 4.3 Effects of nine-steamed and nine-dried Polygonum multiflorum extract on HPA axis function in mice with kidney yin deficiency

[0100] The results of the investigation on the effect of nine-steamed and nine-dried Polygonum multiflorum extract on the HPA axis function in mice with kidney yin deficiency model are shown in Figure 4 .Depend on Figure 4 It can be seen that compared with the model group, the nine-steamed and nine-dried Polygonum multiflorum extract and raw Polygonum multiflorum can significantly reduce the levels of CRH, ACTH, and CORT (P<0.01), and the nine-steamed and nine-dried Polygonum multiflorum extract has the most significant reduction, indicating that the nine-steamed and nine-dried Polygonum multiflorum extract can significantly improve the HPA axis function of mice with kidney yin deficiency model.

[0101] 4.4 Effects of nine-steamed and nine-dried Polygonum multiflorum extract on HPG axis function in mice with kidney-yin deficiency model

[0102] The results of the investigation on the effect of nine-steamed and nine-dried Polygonum multiflorum extract on the HPG axis function in mice with kidney yin deficiency model are shown in Figure 5 .Depend on Figure 5It can be seen that compared with the model group, the nine-steamed and nine-dried Polygonum multiflorum extract and raw Polygonum multiflorum can extremely significantly increase the E2 level (P<0.01); the nine-steamed and nine-dried Polygonum multiflorum extract can extremely significantly increase the T level (P<0.01), and low-dose administration of raw Polygonum multiflorum can significantly increase the T level (P<0.05), indicating that the nine-steamed and nine-dried Polygonum multiflorum extract has a better effect on improving the HPG axis function of mice with kidney yin deficiency model.

[0103] Example 7

[0104] 1. Animals and Grouping

[0105] 140 male SD rats, weighing 170-220 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., license number SYXK (Beijing) 2021-0006. They were kept in the Animal Experiment Center of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences [license number SYXK (Beijing) 2019-0003]. The experiment was approved by the Animal Ethics Committee of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences (ethics approval number: 2023B197) and complied with the 3R principle.

[0106] Rats were randomly divided into 14 groups according to body weight, namely a control group (Control), an LPS group, low, medium and high dose groups of raw Polygonum multiflorum (HSW-L, HSW-M and HSW-H), low, medium and high dose groups of steamed and dried Polygonum multiflorum extract (ZSSW-9-L, ZSSW-9-M and ZSSW-9-H), low, medium and high dose groups of LPS combined with raw Polygonum multiflorum (LPS+HSW-L, LPS+HSW-M and LPS+HSW-H), low, medium and high dose groups of LPS combined with steamed and dried Polygonum multiflorum extract (LPS+ZSSW-9-L, LPS+ZSSW-9-M and LPS+ZSSW-9-H), the steamed and dried Polygonum multiflorum extract was prepared according to Example 4, and there were 10 rats in each group.

[0107] 2. Reagents and instruments

[0108] 2.1 Reagents

[0109] Lipopolysaccharide (LPS, Sigma, USA, catalog number L2880), rat TNF-α, IL-1β, IL-6, IFN-γ enzyme-linked immunosorbent assay kits (Jiangsu Jingmei Biotechnology Co., Ltd., catalog numbers JM-01587R1, JM-01454R1, JM-01597R1, JM-01605R1, batch number 202309), 4% paraformaldehyde (Biosharp, catalog number BL539A, batch number 232345 96), anhydrous ethanol, xylene (Sinopharm Chemical Reagent Co., Ltd.), HE stain (Zhuhai Beso Biotechnology Co., Ltd., catalog number BA4022), differentiation solution, blueing solution (Servicebio, catalog numbers G1005-3 and G1005-4 respectively), neutral gum (Sinopharm Chemical Reagent Co., Ltd., catalog number 10004160), slides, coverslips (Jiangsu Shitai Experimental Equipment Co., Ltd., catalog numbers 188105W and 10212432C respectively).

[0110] 2.2 Instruments

[0111] TBA-40FR fully automatic biochemical analyzer (Toshiba Corporation, Japan), 352 type microplate reader (Labsystems Multiskan MS, Finland), AC8 type plate washer (Thermo Labsystems, Finland), TG16W type micro high-speed centrifuge (Hunan Xiangyi Group), GNP-9080 type water-insulated constant temperature incubator (Shanghai Jinghong Experimental Equipment Co., Ltd.), JT-12S type dehydrator, JB-L7 type embedding machine, JB-L5 type freezing table, JK-6 type tissue spreader (Wuhan Junjie Electronics Co., Ltd.), RM2016 / RM2235 type pathology slicer (Shanghai Leica Instrument Co., Ltd.), DHG-9070A type oven (Shanghai Yiheng Scientific Instrument Co., Ltd.), E100 / CX23 type microscope (Nikon Corporation, Japan), Pannoramic Scan panoramic scanner (3DHISTECH, Hungary), 1730R micro high-speed refrigerated centrifuge (Hong Kong Gene Co., Ltd.), XSR105DU 1 / 100,000 electronic analytical balance (Mettler Toledo Instrument (Shanghai) Co., Ltd.), FA2204B 1 / 10,000 electronic balance (Shanghai Precision Scientific Instrument Co., Ltd.).

[0112] 3. Modeling and drug administration

[0113] After 12 h of fasting but not water deprivation, SD rats were weighed and intragastrically administered with the set doses in each drug-treated group and LPS combined drug-treated group. The control and LPS groups were intragastrically administered with the same amount of distilled water. LPS (2.8 mg kg -1), the other groups were injected with an equal amount of saline by tail vein. After 7 hours, 5 mL of blood was collected from the abdominal aorta of the animals under anesthesia, and the blood was allowed to stand for 2 hours. The blood was centrifuged at 4°C and 3500 r / min for 15 minutes, and the upper plasma was drawn. The ALT and AST levels of rat plasma were detected by biochemical analyzer, and the TNF-α, IL-1β, IL-6, and γ-IFN levels in rat plasma were detected by ELISA. The rat liver was fixed in 4% paraformaldehyde for 24 hours, dehydrated with gradient ethanol, routinely paraffin-embedded, sectioned, and HE stained. The morphology of liver tissue was observed under an optical microscope.

[0114] 4. Results

[0115] 4.1 Effects of nine-steamed and nine-dried Polygonum multiflorum extract on plasma transaminases in rats with idiosyncratic hepatotoxicity model

[0116] The results of the investigation on the effect of nine-steamed and nine-dried Polygonum multiflorum extract on plasma transaminases in rats with idiosyncratic hepatotoxicity model are shown in Figure 6 .Depend on Figure 6 It can be seen that compared with the control group, the tail vein injection of non-toxic doses of LPS alone had no significant effect on the plasma ALT and AST levels of rats (P>0.05); raw Polygonum multiflorum alone had no significant effect on the plasma ALT and AST levels of rats, but after the administration of medium doses of raw Polygonum multiflorum combined with LPS, the plasma ALT and AST levels of rats were significantly increased (P<0.05), and after the administration of high doses of raw Polygonum multiflorum combined with LPS, the plasma ALT and AST levels of rats were extremely significantly increased (P<0.01); the nine-steamed and nine-dried Polygonum multiflorum extract alone or in combination with LPS administration had no significant effect on the plasma ALT and AST levels of rats (P>0.05). This shows that the nine-steamed and nine-dried Polygonum multiflorum has a better safety than raw Polygonum multiflorum.

[0117] 4.2 Effects of nine-steamed and nine-dried Polygonum multiflorum on plasma inflammatory factors in rats with idiosyncratic hepatotoxicity model

[0118] The results of the investigation on the effects of nine-steaming and nine-drying Polygonum multiflorum on plasma inflammatory factors in rats with idiosyncratic hepatotoxicity model are shown in Figure 7 .Depend on Figure 7It can be seen that compared with the same dose of raw Polygonum multiflorum, the nine-steamed and nine-dried Polygonum multiflorum extract has different degrees of reducing effects on the levels of various inflammatory factors. Low-dose administration of nine-steamed and nine-dried Polygonum multiflorum extract alone can significantly reduce the level of TNF-α (P<0.05); medium and high-dose administration of nine-steamed and nine-dried Polygonum multiflorum extract alone can extremely significantly reduce the levels of TNF-α, IL-6, IFN-γ, and IL-1β (P<0.01); LPS combined with nine-steamed and nine-dried Polygonum multiflorum extract can extremely significantly reduce the levels of TNF-α, IL-1β, IL-6, and IFN-γ (P<0.01). The regulatory effect of nine-steamed and nine-dried Polygonum multiflorum extract alone or in combination with LPS on the levels of inflammatory factors is generally better than that of raw Polygonum multiflorum at the same dose.

[0119] 4.3 Effects of nine-steamed and nine-dried Polygonum multiflorum on liver tissue lesions in rats with idiosyncratic hepatotoxicity model

[0120] The results of the investigation on the effect of nine-steaming and nine-drying Polygonum multiflorum on liver tissue lesions in rats with idiosyncratic hepatotoxicity model are as follows: Figure 8 Described by Figure 8 It can be seen that after the administration of LPS combined with raw Polygonum multiflorum, pathological changes such as fatty degeneration in local hepatocytes, narrowing of the hepatic sinusoidal spaces, more dilation and congestion of the interlobular veins, inflammatory cell infiltration in the portal area, hyperplasia of the interlobular bile duct, congestion of the interlobular arteries, hyperplasia of the fibrous tissue of the duct wall, and infiltration of inflammatory cells occurred. No obvious pathological changes were observed after the combined administration of LPS and the nine-steamed and nine-dried Polygonum multiflorum extract, indicating that the nine-steamed and nine-dried Polygonum multiflorum extract has better safety than raw Polygonum multiflorum.

[0121] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of nine-steamed and nine-dried Polygonum multiflorum extract in the preparation of medicines for treating kidney deficiency.

2. The use according to claim 1, characterized in that: The preparation method of the nine-steaming and nine-drying Polygonum multiflorum extract comprises the following steps: The nine-steamed and nine-dried Polygonum multiflorum was extracted by percolation with a methanol solution to obtain an extract; After the extract is decompressed to recover the methanol solution, it is freeze-dried to obtain the nine-steamed and nine-dried Polygonum multiflorum extract.

3. The use according to claim 2, characterized in that: The concentration of methanol in the methanol solution is 80%; The amount of the methanol solution used is 10-30 times the mass of the nine-steamed and nine-dried Polygonum multiflorum.

4. The use according to claim 2, characterized in that: The preparation method of nine-steamed and nine-dried Polygonum multiflorum comprises the following steps: After laying raw Polygonum multiflorum and black beans at intervals, they are steamed and air-dried, with one steaming and one air-dried as one cycle, which is repeated 9 times to obtain the nine-steamed and nine-dried Polygonum multiflorum.

5. The use according to claim 4, characterized in that: The mass ratio of the raw Polygonum multiflorum to the black beans is 1:(1-4); The steaming time is 1-5h.

6. The use according to claim 4, characterized in that: The black beans are obtained by soaking in water for 12 hours.

7. The use according to claim 1, characterized in that: The drug achieves the effect of treating kidney deficiency by improving the hormone levels of the HPT axis, HPA axis and HPG axis.

8. A medicine for treating kidney deficiency, characterized in that: The active ingredients of the medicine include nine-steamed and nine-dried Polygonum multiflorum extract.

9. The drug according to claim 8, characterized in that The preparation method of the nine-steaming and nine-drying Polygonum multiflorum extract comprises the following steps: The nine-steamed and nine-dried Polygonum multiflorum was extracted by percolation with a methanol solution to obtain an extract; After the extract is decompressed to recover the methanol solution, it is freeze-dried to obtain the nine-steamed and nine-dried Polygonum multiflorum extract.

10. The drug according to claim 8, characterized in that The dosage form of the drug includes capsules, pills, tablets, granules, oral liquids, ointments, aerosols or sprays.

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