CN115814003B

CN115814003B - Application of Ampelopsis grossedentata extract in improving NAD level

Info

Publication number: CN115814003B
Application number: CN202211694708.2A
Authority: CN
Inventors: 汪玉芳; 张旭光; 贺瑞坤; 吴新星; 岳中宝
Original assignee: BY Health Co Ltd
Current assignee: BY Health Co Ltd
Priority date: 2022-12-28
Filing date: 2022-12-28
Publication date: 2024-04-09
Anticipated expiration: 2042-12-28
Also published as: CN115814003A

Abstract

The present application relates to the use of ampelopsis grossedentata in the preparation of a pharmaceutical composition for the prevention and/or treatment of diseases and/or symptoms in a subject benefiting from increased NAD levels. The application also relates to the use of ampelopsis grossedentata in the preparation of a composition for increasing NAD levels in at least 1 tissue of a subject.

Description

Application of Ampelopsis grossedentata extract in improving NAD level

Technical Field

Background

Nicotinamide adenine dinucleotide NAD is widely present in various tissues of the body and is the most important electron carrier for cells. NAD exists in two forms, one is NAD+ in an oxidized form and the other is NADH in a reduced form. The main functions of NAD are important coenzymes required for cellular redox reactions, assisting hydrogen transfer in key metabolic pathways, including processes such as glyceraldehyde-3-phosphate dehydrogenation in glycolysis, partial oxidation in tricarboxylic acid cycle, and oxidation of fatty acids and amino acids in mitochondria. During oxidation of fatty acids and amino acids in the mitochondria, NAD+ is converted to reduced nicotinamide adenine dinucleotide (reduced nicotinamide adenine dinucleo-tide, NADH), which is used for oxidative phosphorylation and as an electron donor for ATP synthesis. In addition, NAD+ is also an important substrate for class 3 enzymes (SIRT, ADP ribosyl transferase (ADPRT) or polyribosyl polymerase (poly ADP-ribose polymerase, PARP)) and cyclic ADP ribose synthetase (CD 38 and CD 157), and is involved in regulating apoptosis, tumor progression, cell senescence, DNA repair, mitochondrial biosynthesis and other processes.

Thus, maintaining proper levels of NAD is important for cellular energy metabolism, redox balance, and signal transduction in the body. There are three NAD synthesis pathways in mammals: (1) a de novo synthesis pathway, where tryptophan is the precursor to nad+, where indole 2, 3-dioxygenase is the rate limiting enzyme; (2) the Preiss-Handler pathway, wherein the synthetic material is niacin taken from the food, and wherein nicotinamide riboside transferase catalyzed reaction is the rate limiting step; (3) the salvage synthesis pathway, which uses the NAD products nicotinamide, nicotinamide riboside or nicotinamide mononucleotide taken up or degraded from food as precursors to produce nad+, plays an important key role in nad+ synthesis by mammalian cells.

Studies have shown that with age, NAD levels decrease markedly in a number of tissues and organs such as brain, liver, pancreas, etc. Whereas down-regulation of NAD levels can induce the appearance of aging and related phenotypes, thereby triggering the generation of various aging-related diseases, such as degenerative diseases of the central nervous system, cardiovascular diseases, tumors, and the like; restoring NAD levels in the body can improve the disease condition and even prolong life.

Disclosure of Invention

The inventor of the application confirms that the ampelopsis grossedentata and the extract thereof can obviously improve the NAD level through in vitro experiments and in vivo experiments, and also confirms that the ampelopsis grossedentata and the extract thereof can improve the NAD level in heart, liver, spleen, lung, kidney, muscle and/or brain of a subject. Moreover, the effect of the ampelopsis grossedentata and the extract thereof on improving the NAD level is obviously superior to that of the extracts of other plants of the same Vitaceae.

Thus, in a first aspect, the present application provides the use of ampelopsis grossedentata in the preparation of a pharmaceutical composition for the prevention and/or treatment of diseases and/or symptoms in a subject benefiting from increased NAD levels.

In certain embodiments, wherein the pharmaceutical composition is used for preventing and/or treating reproductive dysfunction (e.g., male reproductive dysfunction, female reproductive dysfunction), neurological dysfunction, and/or debilitating disease in a subject.

In certain embodiments, the female reproductive dysfunction comprises premature ovarian failure.

In certain embodiments, the male reproductive dysfunction includes a decrease in sperm quality (e.g., motility) and/or number.

In certain embodiments, the neurological disorder and/or debilitating disease comprises sleep disorders, memory impairment.

In certain embodiments, the pharmaceutical composition is used for preventing and/or treating premature ovarian failure, male reproductive dysfunction, sleep disorders and/or hypomnesis in a subject.

In certain embodiments, the preventing and/or treating premature ovarian failure comprises increasing the weight of the ovary, the estradiol content, and/or decreasing follicular stimulating hormone in the subject.

In certain embodiments, the preventing and/or treating male reproductive dysfunction comprises improving sperm quality (e.g., motility) and/or number in a subject.

In certain embodiments, the preventing and/or treating sleep disorders comprises improving the quality and/or duration of sleep in the subject.

In certain embodiments, wherein the Ampelopsis grossedentata in the pharmaceutical composition is in the form of Ampelopsis grossedentata extract.

In certain embodiments, the pharmaceutical composition further comprises additional components.

In certain embodiments, the additional component is capable of increasing NAD levels, or helps ampelopsis grossedentata to increase NAD levels.

In certain embodiments, the additional component is selected from the group consisting of pagodatree flower bud extract, ginkgo leaf extract, olive leaf extract, pine bark extract, cowberry fruit extract, turmeric extract, melatonin, gamma-aminobutyric acid, theanine, nicotinamide riboside, nicotinamide mononucleotide, or any combination thereof.

In certain embodiments, the pharmaceutical composition comprises ampelopsis grossedentata extract and pagodatree flower bud extract.

In certain embodiments, the ratio of ampelopsis grossedentata leaf extract to pagodatree flower bud extract is 1:2 to 2:1 (e.g., 1:2;1:1.5;1:1;1.5:1; 2:1).

In certain embodiments, the ampelopsis grossedentata extract is prepared from stems and/or leaves of ampelopsis grossedentata.

In certain embodiments, the ampelopsis grossedentata extract is obtained from stems and/or leaves of ampelopsis grossedentata by solvent extraction.

In certain embodiments, the method of preparing the ampelopsis grossedentata extract comprises: providing dried ampelopsis grossedentata stems and/or leaves (e.g., dried ampelopsis grossedentata stems and/or leaves), pulverizing the stems and/or leaves, and extracting with a solvent; optionally, concentrating the extracted solution to obtain Ampelopsis grossedentata extract.

In certain embodiments, the solvent is water. In certain embodiments, the ratio of the mass of the dry product to the volume of water is 1:10 to 1:60 (e.g., 1:10,1:20,1:30,1:40,1:50, 1:60).

In certain embodiments, the pagodatree flower bud extract is prepared from the flower buds of pagodatree.

In certain embodiments, the pagodatree flower bud extract is obtained from the flower buds of pagodatree by solvent extraction.

In certain embodiments, the method of preparing the pagodatree flower bud extract comprises: providing dried flower buds of Sophora japonica (for example, dried flower buds of Sophora japonica), pulverizing the flower buds, and extracting with solvent; optionally, concentrating the extracted solution to obtain the flos Sophorae Immaturus extract.

In certain embodiments, the solvent is water. In certain embodiments, the dried flower bud of sophora japonica has a mass to water volume ratio of 1:10 to 1:60 (e.g., 1:10,1:20,1:30,1:40,1:50, 1:60).

In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.

In certain embodiments, the pharmaceutical composition is in a form selected from the group consisting of a pill, a powder, a capsule, a tablet (e.g., an effervescent tablet), a caplet, a granule, a liquid, or a suppository.

In certain embodiments, the pharmaceutical composition is administered to the subject in a manner selected from the group consisting of orally, intravenously, by application, respiratory inhalation, and urogenital introduction.

In certain embodiments, the subject is a mammal, e.g., a human.

In a second aspect, the application provides the use of ampelopsis grossedentata in the preparation of a composition for increasing NAD levels in at least 1 (e.g., 2, 3, 4, 5, 6, 7) tissues of a subject.

In certain embodiments, the tissue is selected from heart, liver, spleen, lung, kidney, muscle and/or brain.

In certain embodiments, wherein the Ampelopsis grossedentata in the composition is in the form of Ampelopsis grossedentata extract.

In certain embodiments, the composition further comprises additional components.

In certain embodiments, the composition comprises ampelopsis grossedentata extract and pagodatree flower bud extract.

In certain embodiments, the composition further comprises a food additive, or prebiotic.

In certain embodiments, the prebiotic is selected from the group consisting of fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soy oligosaccharides, inulin, spirulina, arthrospira, coriolus versicolor polysaccharides, nitrogen-containing polysaccharides from the group consisting of the plant, casein hydrolysates, alpha-lactalbumin, lactoferrin, or any combination thereof.

In certain embodiments, the composition is in a form selected from the group consisting of a pill, a powder, a capsule, a tablet (e.g., an effervescent tablet), a caplet, a granule, a liquid, or a suppository.

In certain embodiments, the composition is administered to the subject in a manner selected from oral, intravenous, topical, or respiratory inhalation.

In certain embodiments, the subject is a mammal, e.g., a human.

In a third aspect, the present application provides a method of increasing NAD levels in a cell, the method comprising administering to a subject an effective amount of a ampelopsis grossedentata extract or a composition comprising the ampelopsis grossedentata extract.

In certain embodiments, the cells are obtained from tissue of the heart, liver, spleen, lung, kidney, muscle and/or brain.

In such embodiments, the methods described above can be used to maintain cell viability for the time of cell survival in a cell suspension (e.g., plasma, serum, cell culture medium), or to maintain cell viability. In certain embodiments, the cells may be cells that are preserved for a long period of time.

In certain embodiments, the composition further comprises: pagodatree flower bud extract, ginkgo leaf extract, olive leaf extract, pine bark extract, cowberry fruit extract, turmeric extract, melatonin, gamma-aminobutyric acid, theanine, nicotinamide riboside, nicotinamide mononucleotide, or any combination thereof.

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Further, the procedures of molecular genetics, nucleic acid chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA, etc., as used herein, are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, the term "ampelopsis grossedentata" refers to a woody vine whose branches are small into cylinders with significant longitudinal ribs; the fruits are spherical or nearly spherical; the seeds are in inverted oval shape. The main production areas of the plant comprise Jiangxi, fujian, hubei, hunan, guangdong, guangxi, guizhou and Yunnan.

With the development of genomics, some researchers recently reclassify some plants after a systematic overall comparison of their genomics. Accordingly, "ampelopsis grossedentata" is named "large tooth Niu Guoteng (Nekemias grossedentata (hand-mazz.) j.wen & z.l.nie)", and belongs to the genus niujia of the family Vitaceae. However, what is still described in some published documents is the common name "Ampelopsis grossedentata" of "Datooth Niu Guoteng". Thus, in the text, "big tooth Niu Guoteng" and "ampelopsis grossedentata" both represent the same plant and are used interchangeably.

As used herein, the term "pagodatree flower bud" refers to a flower bud of the legume plant pagodatree (sophra japonica l.). Usually, the flower buds are collected in summer and dried to be called pagodatree flower buds. The pagodatree flower bud is oval or elliptic, has the length of 2-6 mm and the diameter of about 2mm. A plurality of longitudinal patterns are arranged at the lower part of the calyx; yellow and white petals which are not opened are arranged above the calyx; the pedicel is tiny; light weight, and can be broken by twisting with hands. No smell, slightly bitter and astringent taste. As used herein, the term "pagodatree flower bud extract" refers to an extract of the flower buds of pagodatree.

As used herein, the term "stem" refers to an organ between the root and leaf of a plant. In plants, the stems generally have a function of supporting leaves, a function of transporting and storing nutrients, a reproductive function, and photosynthesis.

As used herein, the term "leaf" refers to an organ supported by the stem of a plant. In general, in plants, leaves have photosynthesis, transpiration, absorption and secretion, and reproduction.

As used herein, the term "premature ovarian failure" (premature ovarian failure, POF) refers to a disease that stops menstrual shutdown associated with secondary amenorrhea, sex steroid hormone deficiency, and elevated gonadotrophin serum levels before the age of 40. Physiological indexes are Follicle Stimulating Hormone (FSH) elevation, luteinizing Hormone (LH) elevation and estradiol (E2) reduction. The clinical symptoms are amenorrhea, infertility, etc., and can be accompanied with low estrogen symptoms such as cheekbone flush, night sweat, low libido, vaginal dryness, etc.

As used herein, the term "subject" refers to mammals, including, but not limited to, humans, rodents (mice, rats, guinea pigs), dogs, horses, cows, cats, pigs, monkeys, chimpanzees, etc. In certain embodiments, the subject is a human.

As used herein, the term "disease and/or symptom of a subject that benefits from increased NAD levels" refers to a disease that can be prevented and/or treated, or a symptom that can be ameliorated and/or alleviated by the elevation of NAD levels in the subject.

As used herein, the term "pharmaceutically acceptable" means that the pharmaceutical art recognizes that it is useful for animals, particularly humans. As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and active ingredient, which is well known in the art (see, e.g., remington's Pharmaceutical sciences. Mediated by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995), and includes, but is not limited to: pH modifiers (including but not limited to phosphate buffers), surfactants (including but not limited to cationic, anionic or nonionic surfactants such as Tween-80), adjuvants, ionic strength enhancers (including but not limited to sodium chloride), diluents, excipients, vehicles for containing or administering therapeutic agents, and any combination thereof.

As used herein, pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. When the pharmaceutical composition is administered intravenously, water is the preferred carrier. Saline solutions as well as aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.

As used herein, pharmaceutically acceptable excipients may include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried milk, glycerol, propylene, glycol, water, ethanol and the like. The pharmaceutical composition may also contain a wetting agent, or an emulsifying agent, such as sodium hyaluronate, or a pH buffering agent, if desired. The pharmaceutical composition may take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release formulation, or the like.

Advantageous effects of the invention

In-vitro and in-vivo experiments prove that the ampelopsis grossedentata and the extract thereof can remarkably improve the NAD level, in-vivo experiments also prove that the ampelopsis grossedentata and the extract thereof, and the combination of the ampelopsis grossedentata extract and the pagodatree flower bud extract can improve the NAD level in the heart, liver, spleen, lung, kidney, muscle and/or brain of a subject. And, the effect of the combination of the ampelopsis grossedentata extract and the pagodatree flower bud extract on improving the NAD level is obviously superior to that of the extracts of other plants of the grape family.

Further, the applicant has experimentally confirmed that ampelopsis grossedentata and its extract, and a combination of ampelopsis grossedentata extract and pagodatree flower bud extract can prevent and/or treat male dysfunction, sleep disorder, hypomnesis, and female reproductive dysfunction (e.g., premature ovarian failure). Thus, ampelopsis grossedentata and extracts thereof of the present application have the potential to prevent and/or treat diseases and/or symptoms in a subject that benefit from increased NAD levels.

Drawings

FIG. 1 shows a standard curve of dihydromyricetin obtained by measurement with an aluminum ion chromogenic method.

Figure 2 shows a standard curve of gallic acid obtained by the Fu Lin Fenfa assay.

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

The experiments and methods described in the examples were performed substantially in accordance with conventional methods well known in the art and described in various references unless specifically indicated. For example, for the conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention, reference may be made to Sambrook (Sambrook), friech (Fritsch) and manitis (Maniatis), molecular cloning: laboratory Manual (MOLECULAR CLONING: A LABORATORY MANUAL), edit 2 (1989); the handbook of contemporary molecular biology (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY) (edited by f.m. ausubel (f.m. ausubel) et al, (1987)); series (academic publishing company) of methods in enzymology (METHODS IN ENZYMOLOGY): PCR2: practical methods (PCR 2: A PRACTICAL APPROACH) (M.J. MaxPherson (M.J. MacPherson), B.D. Thoms (B.D. Hames) and G.R. Taylor (G.R. Taylor) editions (1995)), and animal cell CULTURE (ANIMAL CELL CULTURE) (R.I. French Lei Xieni (R.I. Freshney) editions (1987)).

In addition, the specific conditions are not specified in the examples, and the process is carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

Example 1 preparation of the extract

The Ampelopsis grossedentata extract is obtained by taking stems and/or leaves of Ampelopsis grossedentata of Vitaceae as raw materials, extracting, filtering and drying. The preparation method comprises the following steps: pulverizing stem and/or She Ganpin of Ampelopsis grossedentata (purchased from the market of Qingping Chinese medicinal materials in Liwan region of Guangzhou, city), sieving (mesh number is greater than or equal to 10 mesh, preferably 30 mesh), placing into an extraction tank, extracting with water and/or ethanol (preferably pure water as extraction solvent, wherein the ratio of dry product mass of Ampelopsis grossedentata to water mass is 1:10-60, preferably 1:40) at 90-95deg.C for 15min-3h (preferably 2 h), and extracting for 1-3 times (preferably 2 times). Collecting the extractive solution, centrifuging (at a centrifugal speed of 3000-10000r/min, preferably 5000 r/min), filtering to obtain supernatant, concentrating to 1/3-1/10 volume of concentrated solution, and spray drying at 60-80deg.C to obtain Ampelopsis grossedentata extract.

The method comprises extracting stems and/or leaves of Ampelopsis japonica (Ampelopsis japonica (thunder.) Makino), filtering, and spray drying to obtain Ampelopsis japonica extract. The stem and/or leaf of Ampelopsis japonica are purchased to the market of Qingping Chinese medicinal materials in Liwan area in Guangzhou city, and the specific preparation method is the same.

The stem and/or leaf of Niu Guoteng (Nekemias cantoniensis (Hook. & Arn. & Z.L.Nie) (commonly known as Guangdong Ampelopsis grossedentata) is used as raw material, and the extract of Butyrospermum parkii is obtained by extraction, filtration and spray drying. Niu Guoteng stem and/or leaf dry product is purchased to the Qingping traditional Chinese medicine market in the Liwan area of Guangzhou city, and the specific preparation method is the same.

Flower buds of Sophora japonica L.are used as raw materials, and the extract of the Sophora flower bud is obtained through extraction, filtration and drying. The preparation method comprises the following steps: pulverizing flos Sophorae Immaturus, sieving with sieve (mesh number not less than 10 mesh, preferably 30 mesh), placing into extraction tank, and extracting with water and/or ethanol (preferably ethanol as extraction solvent, wherein the ratio of dry flos Sophorae Immaturus mass to water mass is 1:10-60, preferably 1:40) at 90-95deg.C for 15min-3 hr (preferably 2 hr) for 1-3 times (preferably 2 times). Collecting extractive solution, centrifuging (at rotation speed of 3000-10000r/min, preferably 5000 r/min), filtering to obtain supernatant, concentrating to 1/3-1/10 volume of concentrated solution, and spray drying at 60-80deg.C to obtain flos Sophorae Immaturus extract.

EXAMPLE 2 analysis of Ampelopsis grossedentata extract composition

1. Experimental instrument

Agilent 1290 ultra-high performance liquid chromatograph including quaternary gradient pump, on-line degasification system, autosampling system, column oven, diode Array Detection (DAD), agilent G6470QQQ MS (Agilent Technologies, santa Clara, calif., USA), agilent G6546QTOF MS (Agilent Technologies, santa Clara, calif., USA), KQ-250DB type ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.).

2. Experimental reagent and standard substance

The experimental reagent mainly comprises: methanol and formic acid were both chromatographically pure, purchased from Sigma-Aldrich (St.Louis, MO, USA)), ultrapure water, 0.22um filtration membrane (Pall Corporation).

Standard substance: protocatechuic acid (CAS: 99-50-3), gallocatechin (CAS: 3371-27-5), (+) -catechin (CAS: 154-23-4), chlorogenic acid (CAS: 327-97-9), epicatechin (CAS: 490-46-0), dihydroquercetin (CAS: 111003-33-9), rutin (CAS: 153-18-4), phloretin (CAS: 60-82-2), myricetin (CAS: 529-44-2), quercetin (CAS: 117-39-5), naringenin (CAS: 480-41-1), kaempferol (CAS: 520-18-3), alpinetin (CAS: 36052-37-6), caffeic acid (CAS: 331-39-5), gallic acid (CAS: 149-91-7), p-coumaric acid (CAS: 501-98-4), procyanidin B1 (CAS: 20315-25-7), procyanidin B2 (CAS: 29106-49-8), myricetin (CAS: 17912-87-7), myricetin (CAS: 27200-12-0), betuline (CAS: 15-1), apigenin (CAS: 520-35-37-6), caffein (CAS: 35-35), caffein (CAS: 331-39-5), emodin (CAS: 20315-35) and 35-35) and daidzein (CAS: 35-35) Phlorizin (CAS: 60-81-1), and bergamot (CAS: 480-20-6) were added at about 5mg (0.01 mg accurate) to a methanol solution to prepare a 1mg/mL stock solution.

3. Experimental method

3.1 preparation of sample solutions

Accurately weighing about 500mg (accurate to 0.01 mg) of the Ampelopsis grossedentata extract obtained in example 1 by a ten-thousandth balance, adding 5mL of 70% methanol solution, mixing the samples, refrigerating at 4 ℃ for 2h, performing ultrasonic-assisted extraction for 30min at low temperature, centrifuging for 10min with 10000g, collecting the supernatant, and filtering with a 0.22 μm microporous filter membrane to obtain an Ampelopsis grossedentata extract test solution.

3.2 method for measuring total flavone content

(1) Diluting the Ampelopsis grossedentata extract test solution 800 times, taking 0.6mL of the Ampelopsis grossedentata extract test solution and 0.3mL of 5% sodium nitrite solution, mixing uniformly, and standing for 6min.

(2) 0.3mL of 10% aluminum nitrate solution is added, mixed evenly and stood for 6min.

(3) 4mL of 4% sodium hydroxide solution was added.

(4) The absolute methanol is fixed to volume to 10mL and is mixed uniformly.

(5) The mixture was supplemented with methanol to a volume of 25mL, and the mixture was thoroughly mixed and allowed to stand at room temperature for 15min.

(6) The absorbance value is measured by a spectrophotometer, and the maximum absorption wavelength of the ampelopsis grossedentata is 415nm.

(7) The method comprises the steps of preparing samples with the concentration of 5, 10, 20, 40, 60 and 100ug/mL by taking dihydromyricetin as a standard substance, measuring absorbance values at 415nm, drawing a standard curve, and calculating the total flavone content of the ampelopsis grossedentata extract by taking dihydromyricetin as an equivalent.

3.3 determination of Total Polyphenol content

The total polyphenol content is determined by using a Fu Lin Fen method, and the specific steps are as follows:

(1) The prepared test solutions were diluted 10, 100 and 1000 times respectively, and then the following reactions were performed with the test solution stock solution, diluted 10, 100 and 1000 times of the test solutions (as a preliminary experiment to determine the dilution of the sample, the preliminary experiment was not repeated, and the final experiment required three biological replicates).

(2) 0.5mL of the test solution and 0.5mL of Fu Lin Fen reagent (concentration of 0.25 mol/L) were mixed uniformly and allowed to stand for 30min. (preparation of 0.25mol/L Fu Lin Fen reagent: 2mL of original Fu Lin Fen reagent (2 mol/L) was taken, and 14mL of distilled water was added thereto and thoroughly mixed).

(3) 1mL of 15% sodium carbonate solution is added, mixed evenly and stood for 30min.

(4) After the color development was stabilized, 2mL of the product mixture was centrifuged at 3500r/min for 3min, and the supernatant (which should be uniformly blue) was taken.

(5) The absorption wavelength was measured at 760nm using a spectrophotometer (curve plotting gallic acid).

(6) Gallic acid is used as a standard substance, and the concentration is prepared: 100. 80, 60, 40, 20, 10 and 5ug/ml, sequentially measuring absorbance values, drawing a standard curve, and calculating the total polyphenol content of the ampelopsis grossedentata extract by taking gallic acid as an equivalent.

3.4 specific component detection

Chromatographic column: zorbax Eclipse Plus C18,1.8 μm, 2.1X100 mm (cat# 959758-902)

Mobile phase: phase A: pure water (0.1% formic acid); phase B, methanol.

Elution gradient: 0min 5% B,3min 95%,6min 20%,15min 40%,20min 60%,24min 80%,28min 95%,28.1min 5%,30min 5%.

Detection wavelength 200-600nm, flow rate: the sample injection amount is 3 mu L at 0.3 mL/min.

Agilent 1290II-G6546 QTOF (Agilent Technologies, santa Clara, calif., USA) 100-1,000 mass/charge (m/z) range, mass spectrometry detection was performed using positive and negative ion modes, respectively, condition dimensions of the ion source: the dry gas flow rate (N2) was 12.0L/min ^-1 The method comprises the steps of carrying out a first treatment on the surface of the Sprayer pressure 40psi, gas drying temperature 370 ℃; capillary voltage 4000V. Broad-spectrum qualitative analysis was performed in Auto MS/MS scanning mode. Data acquisition and processing uses agilent Qualititative Analysis software analysis software.

4. Experimental results

4.1 measurement of Total Flavonoids content

The reaction is carried out by utilizing an aluminum ion chromogenic method, dihydromyricetin is used as a standard substance of the ampelopsis grossedentata, the concentration is 5, 10, 20, 40, 60 and 100ug/mL, the absorbance value is measured at 415nm, and a standard curve is drawn. The total content of ampelopsis grossedentata extract was calculated on the basis of dihydromyricetin equivalent, the average value of the content was 205.93mg/g, and the content RSD (standard deviation sd/average value 100%) was 8.72% (Table 1).

TABLE 1 Total flavone content (mg/g)

4.2 measurement of total phenolic acid content

After the ampelopsis grossedentata extracts are diluted by 1000 times, OD value at 750nm is measured after the reaction by using the Fulin phenol method. And simultaneously, gallic acid is selected as a standard substance, the concentrations of the gallic acid are 100, 80, 40, 20, 10 and 5 mug/mL, the OD value of the gallic acid is measured after the reaction by using a Fulin phenol method, a standard curve is drawn (figure 2 and table 2), the total phenolic acid content of the extract is calculated by taking gallic acid as an equivalent, the total phenolic acid content of the extract is shown, the average value of the content is 604.35mg/g, and the RSD value of the content is 2.14%.

TABLE 2 Total phenolic acid content (mg/g)

4.3 detection results of specific Components

After the data are collected, the Agilent Qualititative Analysis software software is used for carrying out peak extraction and redundancy elimination, then the self-built database is used for comparison, and the substances are identified according to the fragment information, the retention time and the accurate molecular mass of the substances in the self-built database. And finally, manually correcting the identification result. On the basis, 20 compounds were identified in Ampelopsis grossedentata extract, including 16 flavonoids, 2 phenolic acids, one terpene and one anthraquinone (Table 3, table 4).

TABLE 3 identification of substances in Ampelopsis grossedentata extract under Positive ion mode

TABLE 4 identification of substances in Ampelopsis grossedentata extract under anion mode

Each standard was configured as a working solution with a concentration of 2, 1, 0.5, 0.25, 0.125, 0.0635, 0.03125ug/ml, data were collected using the developed methods described above, peak areas were extracted, and then standard curves for each standard were drawn. And then under the same condition, carrying out data acquisition on the ampelopsis grossedentata, extracting peak area, repeating for three times, and carrying out quantitative analysis on active substances by utilizing a corresponding standard curve.

The quantitative analysis of 20 compounds in the ampelopsis grossedentata extract shows that the total content of 20 compounds measured by ampelopsis grossedentata leaves is 218179.8 mug/g, the total content of 20 compounds measured by ampelopsis grossedentata stems is 204612.46 mug/g, and the RSD of the content of each compound is less than 10%, which indicates that the repeatability of the measured result is good and the result is reliable.

Example 3 in vitro modulation of NAD level assay

Increasing intracellular NAD levels is a promising approach to delay aging and prevent age-related degenerative diseases. This example compares the effect of ampelopsis grossedentata extract and extracts of other two Vitaceae plants on NAD+.

In eukaryotic cells, NAD+ synthetases and NAD+ consuming enzymes are highly regionalized. For many years, weak endogenous fluorescence of NADH has been used for the study of mitochondrial metabolic status; however, nad+ lacks intrinsic fluorescence, which prevents its direct imaging in living cells and in vivo. NAD was detected according to the fluorescent probe mCherry-FiNad disclosed in published literature (which is published in the paper "Illuminating NAD+ Metabolism in Live Cells and In Vivo Using a Genetically Encoded Fluorescent Sensor" by Yejun Zou et al, DEVELOPMETAL CELL). mCherry-FiNad is a fluorescent probe capable of specifically responding to NAD (including its oxidized form nad+ and reduced form NADH), without reacting to other adenine nucleotides (e.g., nadp+, NADPH, ATP, or ADP). The fluorescent probe has the structure that: mcherry-T-Rex (1-77) -T-Rex (78-189) -cpYFp-T-Rex (190-211). The probe comprises two fluorescent proteins, mcherry and cpYFp, and T-Rex (derived from a protein that the bacteria are able to specifically bind to NAD). When NAD binds to the T-Rex protein, the configuration of the cpYFp fluorescent protein changes, the green fluorescence intensity changes, while the configuration of the mcherry fluorescent protein is not affected, and the red fluorescence does not change. Thus, the green/red fluorescence ratio of the mCherry-FiNad fluorescent probe can be used to report NAD dynamics that are not affected by probe concentration.

1. Preparation of sample solutions for sample preparation

Test group 1: dissolving Ampelopsis grossedentata leaf extract prepared in example 1 in cell culture medium to obtain 0.01, 0.02 and 0.05mg/ml solution;

test group 2: dissolving the Ampelopsis japonica leaf extract prepared in the example 1 in cell culture solution to prepare 0.01, 0.02 and 0.05mg/ml solution;

test group 3: dissolving Niu Guoteng leaf extract prepared in example 1 in cell culture solution to obtain 0.01, 0.02 and 0.05mg/ml solution;

test group 4: the ampelopsis grossedentata leaf extract prepared in the example 1 and the pagodatree flower bud extract prepared in the example 1 are mixed according to the proportion of 1.5:1, and then are dissolved by cell culture solution to prepare 0.01, 0.02 and 0.05mg/ml solution;

2. test method

293 cells (human kidney epithelial cells) were cultured in vitro, transfected with plasmid DNA containing mCherry-FiNad fluorescent probes and expressed for 36 hours. The sample was then added for stimulation for 24 hours, after careful removal of the medium, the cells were washed twice with PBS. 100 μLPBS was added at 37deg.C and fluorescence was detected by Synergy 2Multi-Mode Microplate Reader (Bio Tek), excitation filters were 4815 BP20nm and 780 BP20nm, emission filters 528BP20nm (green fluorescence) and 640 BP 40nm (red fluorescence). Calculating the Ratio green fluorescence/red fluorescence values of the blank control group and each experimental group, and carrying out standardized processing on the Ratio values of each experimental group by taking the Ratio value of the blank control group as a reference to obtain Ratio' values.

3. Test results

TABLE 5 Effect of Ampelopsis grossedentata extract on NAD levels

Wherein, p < 0.05, p < 0.01 compared to the blank.

As shown in Table 5, in vitro test results show that compared with the blank control group, the extract of Ampelopsis grossedentata leaf and the composition of Ampelopsis grossedentata leaf extract and flos Sophorae Immaturus extract can significantly improve NAD level (the improvement of NAD level has statistical difference (p < 0.01)). And, as the concentration increases, so does the increase in NAD level. The effect of improving NAD by ampelopsis japonica leaf extract and Niu Guoteng leaf extract of ampelopsis japonica of Ampelopsis is not obvious.

Example 4 modulation of NAD animal tests

To further investigate the effect of the Ampelopsis grossedentata extract on NAD promotion in vivo, the applicant further investigated the effect of Ampelopsis grossedentata on NAD in various organs and tissues of mice. Test set 1 (Ampelopsis grossedentata leaf extract), test set 2 (Ampelopsis grossedentata leaf extract), test set 3 (Ampelopsis grossedentata leaf extract and flos Sophorae Immaturus extract in a 1.5:1 compound), positive control (NMN) and blank control.

Wherein, the test group and the positive control group (NMN) were administered with 75mg/kg BW (body weight) and 83.3mg/kg BW gastric lavage mice, respectively, and the extract and NMN, and the blank group was administered with solvent water. The administration was performed by gavage for 28 days, 1 time for 1 day. After the end of the administration, the NAD+ level of several tissues and organs of heart, liver, spleen, lung, kidney, brain and muscle of the mice is detected.

The specific detection method comprises the following steps: the tissue samples of the mice were collected, added with 480uL of lysate, allowed to stand on ice for 5 minutes, and centrifuged at 10,000 rpm after neutralization with the neutralization solution, at 4℃for 5 minutes. The supernatant was applied to a 10KD centrifuge column and centrifuged at 10,000 rpm for 10 minutes at 4 ℃. The column was discarded, the centrifuged liquid was retained and placed on ice. To the sample were added alcohol dehydrogenase (available from Kang Ruina Biotech Co., ltd., cat No. 9031-72-5) and colorless tetrazolium salt molecule WST (available from Biyun Tian), which reduced NAD+ in the sample to NADH, and at the same time converted colorless WST to water-insoluble blue-violet crystalline formazan, which was read by a spectrophotometer or an enzyme-labeled instrument. A standard curve is set during detection, and each sample is provided with a complex hole. The results are shown in Table 6.

TABLE 6 Effect of Ampelopsis grossedentata extract on NAD levels of individual organs

Compared with blank group, p < 0.05, p < 0.01

The test results showed (table 6): the Ampelopsis grossedentata extract can remarkably improve the NAD content of livers, kidneys, muscles and brains. Wherein the improvement of NAD content of muscle and brain is even significantly better than that of positive control NMN. Whereas ampelopsis japonica leaf extract has significantly less or substantially equal effect in enhancing NAD content than ampelopsis japonica leaf extract, it has no effect on NAD content of kidney, muscle and brain, although it can enhance NAD content of liver. The composition of the ampelopsis grossedentata leaf extract and the pagodatree flower bud extract can remarkably improve the content of NAD of heart, liver, spleen, lung, kidney, muscle and brain, wherein the content of NAD of the lung, muscle and brain is improved even remarkably better than that of NMN of a positive control group.

EXAMPLE 5 evaluation of efficacy of Ampelopsis grossedentata extract in improving Male function

Modern researches show that NAD+ can be used as neurotransmitter to relieve smooth muscle cells, and NAD+ can be used for helping male mice to produce testosterone through mediating SIRT1 reaction, so that sexual function is improved, and the research on improving male function is carried out by using a zebra fish model. Test setup: test group 1 (Ampelopsis grossedentata leaf extract), test group 2 (Ampelopsis grossedentata leaf extract and flos Sophorae Immaturus extract are mixed according to 1.5:1), blank control group, model control group, and positive control group (WUZIYAZONG pill).

Zebra fish strain: adult male zebra fish of wild AB strain. The evaluation index is the number of sperm. Samples were formulated with 100% DMSO at 200mg/mL and stored at-20 ℃. Positive control: wuzi Yanzong pills (brown pills, lot number 21011185, beijing Tongren Tang Co., ltd., tongren Tang pharmaceutical factory) are stored in a cool, dry state. Preparing mother liquor of 6.00mg/mL with ultrapure water, and sub-packaging and storing at-20deg.C.

Instrument, consumable and reagent: electrokinetically focused continuously variable magnification fluorescence microscope (AZ 100, nikon, japan); CCD camera (VertA 1, shanghai Tusen Vision technologies Co., ltd.); precision electronic balances (CP 214, OHAUS, USA); a high-speed refrigerated centrifuge (PICO 17/21, thermo, USA). Cyclophosphamide (lot H2121240, china, shanghai aladine biochemical technologies inc.); dimethyl sulfoxide (DMSO, lot BCCD8942, sigma, switzerland); sperm motility fluorescent staining kit (lot number 1-426511-10, shanghai Jiemei Gene medicine Co., ltd., china); PBS (cat. BL601A, biosharp, china).

The molding method and the principle are as follows: cyclophosphamide induced oligospermia model. Cyclophosphamide is a commonly used clinical immunosuppressant and antitumor drug, and is one of chemotherapeutic drugs which can definitely impair the spermatogenic function. It can kill the spermatogenic cells in differentiation, resulting in long-term oligospermia or oligospermia, and is a method for experimental adoption of more models of induced oligospermia, oligospermia or reproductive system injury. 10mpf wild type AB strain male zebra fish were randomly selected in culture jars, and 8 zebra fish were treated per jar (experimental group). The sample was given a concentration of 50.0. Mu.g/mL in water, the positive control Wuzi Yanzong pill of 6.00. Mu.g/mL, and a blank control group and a model control group were simultaneously set, with a capacity of 1.2L per cylinder. After 8 days of sample treatment, the rest of the experimental groups except the blank control group are all water-soluble to administer cyclophosphamide to establish a zebra fish oligospermia model. And (3) continuously treating the same cyclophosphamide for 16 days in each experimental group, using a sperm motility fluorescent staining kit to stain the sperm, photographing under a fluorescent microscope after the staining is finished, analyzing and collecting data by using Image J advanced Image processing software, analyzing the number of the sperm of the zebra fish, and evaluating the male sexual function improvement effect of the sample according to the statistical analysis result of the index. Statistical treatment results are expressed in mean+ -SE. Statistical analysis was performed with SPSS26.0 software, p <0.05 indicated that the differences were statistically significant. And (3) continuously treating the test groups with cyclophosphamide for 16 days, placing the test groups under a video camera for observation, video recording and storage, evaluating the rear-end collision condition of the zebra fish of each test group on normal female zebra fish, and evaluating the male sexual function improvement effect of the sample according to the statistical analysis result of the index.

The test results showed (tables 7 and 8) that the number of sperm in the model control group (oligospermia model) was significantly reduced as compared with the blank control group. After the composition of the wuzi yanzong pill, the ampelopsis grossedentata leaf extract and the pagodatree flower bud extract is used, the number of sperms and the number of rear-end collisions are obviously improved, and the number of the sperms and the number of rear-end collisions are statistically different from the number of models. The composition of the ampelopsis grossedentata leaf extract and the pagodatree flower bud extract has the effect of improving male functions. And compared with a positive control group, the combination of the ampelopsis grossedentata leaf extract and the pagodatree flower bud extract further improves the number of sperms and the rear-end collision frequency, and the difference is obvious.

Table 7. Results of evaluation of male sexual function improving efficacy test (n=8)

P <0.05, < p <0.01 compared to model control.

TABLE 8 evaluation of results of test for improving the efficacy of male sexual function (sperm motility)

EXAMPLE 6 evaluation of sleep improvement efficacy of Ampelopsis grossedentata extract

Modern research has shown that nad+ regulates biological clocks through SIRT1, helping to sleep disorders caused by circadian rhythm reversal or age increase. The present example used a zebra fish model for sleep improvement functional studies. Test setup: test group 1 (Ampelopsis grossedentata extract) and test group 2 (Ampelopsis grossedentata leaf extract and flos Sophorae Immaturus extract in 1.5:1), blank control group, model control group and positive control group (diazepam).

The zebra fish strain used in the test is wild type AB strain zebra fish, and the evaluation index is behavioral analysis (wakefulness and wakefulness total time).

The molding method and the principle are as follows: PTZ is an inhibitor of gamma-aminobutyric acid (GABA for short), a naturally occurring non-protein amino acid, which is an important inhibitory neurotransmission substance in the central nervous system of mammals. Small doses of PTZ can induce both mania and depression resulting in insomnia. The 5dpf wild type AB strain zebra fish were randomly selected in 6 well plates, and 30 zebra fish were treated per well (experimental group). The sample was given 100. Mu.g/mL in water, and the positive control drug diazepam was given a concentration of 10.0. Mu.g/mL, while the blank control and model control were set at a capacity of 3mL per well. After the treatment at 28 ℃ for 1 day, the PTZ is given to each of the rest experimental groups except the blank control group in a water-soluble manner to establish a zebra fish insomnia model, 10 zebra fish are randomly selected from each experimental group and placed in a behavior analyzer to collect data, the activity and total time of wakefulness of the zebra fish are analyzed, and the sleep improvement effect of the sample is evaluated according to the statistical analysis results of the indexes. Statistical treatment results are expressed in mean+ -SE. Statistical analysis was performed with SPSS 26.0 software, p <0.05 indicated that the differences were statistically significant.

The experimental results are shown in table 9. Under the experimental condition, compared with a blank control group, the model group (zebra fish insomnia model) has obviously increased wakefulness total time and wakefulness activity, which indicates successful modeling. The positive medicine (diazepam) has reduced wakefulness time and wakefulness activity, and the combination of Ampelopsis grossedentata leaf extract, ampelopsis grossedentata leaf and flos Sophorae Immaturus extract has reduced wakefulness time and wakefulness activity, and has statistical difference compared with model group. The composition of the extract of the ampelopsis grossedentata leaves and the extract of the sophora japonica has the effect of improving the sleep of the zebra fish.

TABLE 9 behavioral analysis of sleep improvement efficacy

Wherein p <0.05, < p <0.01 compared to model group.

EXAMPLE 7 study of memory improving efficacy of Ampelopsis grossedentata extract

Studies have shown that nad+ plays a vital role in cell life regulation, metabolism and cell health, however, nad+ content decreases with age, increasing nad+ content, increasing mitochondrial activity, allowing mitochondria to produce more energy, increasing blood flow, and restoring signal pathways between neurons. The present example used a zebra fish model for the study of improving memory function. Test setup: test group 1 (Ampelopsis grossedentata extract) and test group 2 (Ampelopsis grossedentata leaf extract and flos Sophorae Immaturus extract are combined according to 1.5:1), blank control group, model control group and positive control group (edaravone).

The molding method and the principle are as follows: zebra fish of 5dpf wild type AB strain were randomly selected in beakers, and 30 zebra fish were treated per beaker (experimental group). The sample was given 100. Mu.g/mL in water, the positive control edaravone was 5.00. Mu.g/mL, and a blank control group and a model control group were simultaneously set, each beaker having a capacity of 20mL. The other experimental groups except the blank control group were all water-soluble to administer bisphenol AF to establish a zebra fish memory impairment model. After the treatment at 28 ℃ for 1 day, each experimental group randomly selects 5 zebra fish to be put into a cross-shaped module, the modules are divided into four areas of yellow, blue, red and green, 6 modules are respectively put into each group, data are collected by a behavior analyzer, the percentage (%) of the total movement distance of the zebra fish in the blue area to the total movement distance of the whole cross-shaped module area within 20 minutes is analyzed, and the effect of improving the memory of the sample is evaluated according to the statistical analysis result of the index. Statistical treatment results are expressed in mean+ -SE. Statistical analysis was performed with SPSS26.0 software, p <0.05 indicated that the differences were statistically significant.

Table 10 sample memory improvement efficacy evaluation test results (n=6)

P <0.05, < p <0.01 compared to model control.

The experimental results show (table 10) that the blue duty ratio of the model control group (zebra fish memory impairment model) is obviously reduced compared with that of the blank control group under the experimental conditions, which indicates that the modeling is successful. The blue ratio of the positive medicine edaravone group is obviously increased, the blue ratio of the composition of the ampelopsis grossedentata leaf extract group, the ampelopsis grossedentata leaf and the pagodatree flower bud extract is obviously increased more than that of the model group, and the composition has statistical difference, so that the composition of the ampelopsis grossedentata leaf extract, the ampelopsis grossedentata leaf extract and the pagodatree flower bud extract has the effect of improving memory.

EXAMPLE 8 study of improvement of ovarian efficacy by Ampelopsis grossedentata extract

Studies have shown that NAD decline is associated with oocyte dysfunction during reproductive aging and nad+ supplementation can rescue female fertility during reproductive aging. This example used a zebra fish model to conduct an study of improving ovarian function. Test setup: test group 1 (Ampelopsis grossedentata extract) and test group 2 (Ampelopsis grossedentata leaf extract and flos Sophorae Immaturus extract are combined according to 1.5:1), blank control group, model control group and positive control group (KUNTAI Capsule).

The molding method and the principle are as follows: female zebra fish of 6mpf wild type AB strain were randomly selected in culture jars, and 16 zebra fish were treated per beaker (experimental group). The samples were given 50. Mu.g/mL in water, the positive control Kuntai capsule was 250. Mu.g/mL, and the blank and model control groups were set at the same time, with a capacity of 4.8L per beaker. After 8 days of sample treatment, the cyclophosphamide is water-soluble to establish a zebra fish premature ovarian failure model in all experimental groups except for a blank control group. Treatment with cyclophosphamide was continued for 16 days for each experimental group: 1) Measuring and counting the weight; 2) Measuring and counting the weight of the ovary; 3) Collecting the zebra fish ovary tissue samples of each experimental group according to the specification of the estradiol kit, collecting data by utilizing the multifunctional enzyme-labeled instrument software, analyzing the content of the zebra fish ovary estradiol of each experimental group, and evaluating the influence of the samples and NMN on the content of the zebra fish estradiol (estrogen (E2)) of the premature ovarian failure by using the statistical analysis result of the index; 4) Collecting the zebra fish ovary tissue samples of each experimental group according to the specification of the follicle stimulating hormone kit, collecting data by using the multifunctional enzyme-labeled instrument software, analyzing the activity of the zebra fish ovary FSH of each experimental group, and evaluating the influence of the samples and NMN on the activity of the zebra fish FSH of the premature ovarian failure by using the statistical analysis result of the index. Statistical treatment results are expressed in mean+ -SE. Statistical analysis was performed with SPSS26.0 software, p <0.05 indicated that the differences were statistically significant.

Table 11 sample ovarian protection efficacy (weight of ovaries) evaluation test results (n=8)

P <0.001 compared to model control.

Table 12 sample ovarian protection efficacy (estradiol content) evaluation test results (n=8)

P <0.01 and p <0.001 compared to model control.

Table 13. Sample ovarian protection efficacy (FSH viability) evaluation test results (n=8)

P <0.05, p <0.01, p <0.001 compared to model control.

The experimental results show (tables 11, 12 and 13) that under the experimental conditions, compared with a blank control group, the ovarian weight and the estradiol content of a model control group (zebra fish premature ovarian failure model) are obviously reduced, and the FSH activity is increased, so that the modeling is successful. The weight of the ovary and the content of the estradiol of the positive control group are obviously increased, and the activity of FSH is reduced; the composition of the ampelopsis grossedentata leaf and the pagodatree flower bud extract has the advantages that the weight of ovaries and the content of estradiol of two groups of ampelopsis grossedentata leaf and pagodatree flower bud extract are obviously increased, the activity of FSH is reduced, and compared with a model group, the composition has statistical differences. The composition of the ampelopsis grossedentata leaf extract, the ampelopsis grossedentata leaf and the pagodatree flower bud extract has the function of ovary protection.

While specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the invention is intended to be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims

1. Use of ampelopsis grossedentata in the preparation of a pharmaceutical composition, wherein the pharmaceutical composition comprises the components in a ratio of 1:2 to 2:1 Ampelopsis grossedentata leaf extract and flos Sophorae Immaturus extract; wherein the Ampelopsis grossedentata leaf extract is obtained by extracting Ampelopsis grossedentata leaves with water; the flos Sophorae Immaturus extract is obtained by extracting flower buds of flos Sophorae Immaturus with water or ethanol;

wherein the pharmaceutical composition is for preventing and/or treating a reproductive dysfunction in a subject, wherein the reproductive dysfunction is selected from the group consisting of male reproductive dysfunction and female reproductive dysfunction, wherein the female reproductive dysfunction comprises premature ovarian failure and the male reproductive dysfunction comprises reduced sperm quality and/or number.

2. The use of claim 1, wherein the preventing and/or treating premature ovarian failure comprises increasing the weight of the ovary, estradiol content and/or lowering follicular stimulating hormone in the subject.

3. The use of claim 1, wherein the preventing and/or treating male reproductive dysfunction comprises improving sperm quality and/or number in a subject.

4. The use of claim 1, wherein the ratio of ampelopsis grossedentata leaf extract to pagodatree flower bud extract is 1:2;1:1.5;1:1, a step of; 1.5:1, a step of; or 2:1.

5. The use of claim 1, wherein the preparation method of ampelopsis grossedentata leaf extract comprises: providing dried ampelopsis grossedentata leaves, crushing the leaves and extracting with water; concentrating the extracted solution to obtain Ampelopsis grossedentata leaf extract.

6. The use according to claim 5, wherein the dried Ampelopsis grossedentata leaf is a dried Ampelopsis grossedentata leaf.

7. The use of claim 6, wherein the ratio of the mass of the dried ampelopsis grossedentata leaves to the volume of water is 1:10 to 1:60.

8. the use of claim 7, wherein the preparation method of the pagodatree flower bud extract comprises the following steps: providing dried flower buds of Sophora japonica, pulverizing the flower buds, and extracting with water or ethanol; concentrating the extracted solution to obtain flos Sophorae Immaturus extract.

9. The use of any one of claims 1-8, having one or more features selected from the group consisting of:

(1) The pharmaceutical composition further comprises a pharmaceutically acceptable excipient;

(2) The pharmaceutical composition is in the form of pill, powder, capsule, tablet, granule, liquid or suppository;

(3) The mode of administration of the pharmaceutical composition to a subject is selected from oral administration, intravenous injection, application, respiratory inhalation or genitourinary introduction;

(4) The subject is a mammal.