Application of wild jujube leaf thorn extract in preparation of antioxidant product and / or enzyme inhibitor

Abstract

The invention relates to the field of biological medicines, in particular to application of a wild jujube leaf thorn extract in preparation of an antioxidant product and / or an enzyme inhibitor. A wild jujube leaf thorn water extract, a wild jujube leaf thorn absolute ethyl alcohol extract and a wild jujube leaf thorn ethyl acetate extract are prepared through reflux extraction, the content of total phenols, total flavonoids, total sugar and total triterpenes of the wild jujube leaf thorn water extract, the wild jujube leaf thorn absolute ethyl alcohol extract and the wild jujube leaf thorn ethyl acetate extract is measured, and the in-vitro antioxidant activity is evaluated through four methods including DPPH free radicals, ABTS cationic free radicals, hydroxyl free radicals and FRAP. The invention also researches the inhibition effect of the wild jujube leaf thorn extract on four enzymes, namely alpha-glycosidase, acetylcholin esterase, tyrosinase and xanthine oxidase. The result shows that the wild jujube leaf thorn extract has certain antioxidant activity and enzyme inhibition effect, and can be developed into a natural antioxidant of a plant source and an inhibitor of alpha-glucosidase, acetylcholin esterase, tyrosinase and xanthine oxidase.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the application of wild jujube leaf thorn extract in the preparation of antioxidant products and / or enzyme inhibitors. Background Art

[0002] Wild jujube is a deciduous tree or shrub of the genus Ziziphus in the family Rhamnaceae. It was anciently called Ji or Er, and also known as "wild jujube". It is a unique wild fruit tree in China and is the original species of cultivated jujube, with a history of 12 to 14 million years. Wild jujube has the characteristics of drought tolerance, waterlogging tolerance, and barren tolerance, and is often used as a resource for germplasm innovation and the most important rootstock of cultivated jujube. Wild jujube belongs to the original wild resources and has evolved into rich and diverse variants with different flavors through long-term evolution, which is the source basis for exploring excellent genes and seed selection and breeding.

[0003] Wild jujube has high economic utilization value and development potential. In recent years, more and more practitioners in industries such as agriculture, processing, and medicine have begun to research and develop wild jujube germplasm resources and have made certain progress, laying a solid foundation for the sustainable development of the wild jujube industry. The wild jujube germplasm resources in China are extremely rich and diverse, and the germplasm traits of wild jujube in different regions vary greatly. Xingtai City, Hebei Province, has rich wild jujube resources and is known as "Xingtai wild jujube is the best in the world", and is called the largest wild jujube industry base in China. The wild jujube produced there is of high quality, plump, and has high medicinal efficacy. In addition, wild jujube is also a pioneer tree species for wind prevention and sand fixation and soil and water conservation.

[0004] Wild jujube has rich nutritional and medicinal values. The pulp of wild jujube is sweet and sour, appetizing, spleen-strengthening, and digestion-promoting, and is very nutritious, containing a large amount of vitamin C, fat, protein, and organic acids. In addition, the pulp of wild jujube is also rich in trace elements such as iron, calcium, magnesium, phosphorus, and zinc. Although the pulp of wild jujube is thin, it is rich in a large amount of vitamins and can be eaten raw or processed into unique-flavored foods such as wild jujube juice, wild jujube cakes, wild jujube noodles, wild jujube jams, and wine, which are deeply favored by consumers; the wild jujube seed is sweet, flat, and slightly warm in nature and is a commonly used traditional Chinese medicine for sedation and sleep. It is often used in medicine to treat symptoms such as night sweats and thirst. In addition to the seeds being used as medicine, the flowers of wild jujube tree have the functions of improving eyesight and wound healing; the pollen of wild jujube tree can soften blood vessels and enhance lung vitality, and has a good effect on improving immunity, and is generally processed into high-grade supplements for consumption; the bark, root bark, and leaf thorns of wild jujube tree have the function of astringing and stopping bleeding and are often used to treat symptoms such as hematochezia, metrorrhagia, and hypertension. Wild jujube contains a variety of bioactive chemical components, but the components and contents contained in the seeds, pulp, roots, and leaves are not the same. It can be seen that wild jujube has high research value, but at present, the research and development of wild jujube are still focused on the seeds, and there is little existing technology for researching wild jujube leaf thorns.

[0005] Leaf thorns are formed by the metamorphosis of leaves or stipules. The thorns of the sour jujube are a self-protection mechanism formed by wild sour jujube trees during the long evolution process. The leaf thorns can protect their fruits (sour jujube) from being preyed on by animals, thus maintaining the growth and reproduction of the species itself. The sour jujube tree is covered with sharp thorns all over its body. The leaf thorns have the functions of defense, protection and energy storage, so the leaf thorns of sour jujube are rich in nutritional value and have great research prospects.

[0006] Diabetes is a common chronic disease in clinical practice, and its main feature is the abnormal increase in blood sugar levels. Due to insufficient insulin secretion in the body, metabolic disorders such as glucose, protein, and electrolytes are caused, which eventually induce the occurrence of diseases, mainly type 2 diabetes. The incidence of diabetes in my country is about 12%, and more than 50% of the adult population has potential type 2 diabetes. Diabetes can cause intraretinal vascular leakage, glial dysfunction, and neuronal apoptosis. Diabetes is moderately correlated with deaths from liver cancer, pancreatic cancer, ovarian cancer, colorectal cancer, lung cancer, bladder cancer, and breast cancer. In addition to cancer and vascular diseases, diabetes is also associated with deaths from kidney disease, liver disease, pneumonia, and other infectious diseases. α-Glucosidase can cut the α-1,4 glycosidic bond of the non-reducing end of oligosaccharides digested by a series of enzymes in the gastrointestinal tract, convert it into glucose, and then be absorbed into the blood circulation by the small intestinal epithelial cells to become blood sugar. Therefore, reducing the activity of α-glycosidase can achieve effective sugar control. Gout is a metabolic disease characterized by elevated uric acid. Reducing the activity of xanthine oxidase in the human body can reduce the probability of gout. Vitiligo is an acquired skin disease caused by the loss of skin and mucous membrane pigments due to the loss of epidermal melanocytes, and the activation of tyrosinase can promote the production of melanin. Alzheimer's disease is a chronic progressive degenerative disease of the central nervous system and the most common form of dementia in the elderly. Acetylcholinesterase inhibitors can be used in the treatment of Alzheimer's disease by inhibiting the activity of acetylcholinesterase to increase acetylcholine levels and neuronal excitability. Among enzyme inhibitors, acarbose, allopurinol, febuxostat, donepezil and tacrine are widely used in clinical practice, but long-term use will have side effects and are expensive, so there is a need to develop safer and non-toxic enzyme inhibitors.

[0007] In order to better study the sour jujube resources, the present invention uses anhydrous ethanol, ethyl acetate and water three solvents to reflux extract the sour jujube leaf thorn extract, and determines the contents of total phenol, total flavonoids, total sugar and total triterpenes, evaluates its inhibitory ability on α-glycosidase, xanthine oxidase, tyrosinase and acetylcholinesterase, and uses DPPH free radical, ABTS cation free radical, hydroxyl free radical and FRAP methods to evaluate the in vitro antioxidant capacity of the sour jujube leaf thorn extract, in the hope of providing new value in the development of sour jujube resources. Summary of the invention

[0008] The purpose of the present invention is to provide an application of a thorny leaf extract in the preparation of an antioxidant product and / or an enzyme inhibitor to solve the problems of the above-mentioned prior art. The thorny leaf extract provided by the present invention has a good antioxidant effect and can also effectively inhibit the enzyme activities of α-glucosidase, xanthine oxidase, tyrosinase and acetylcholinesterase.

[0009] To achieve the above object, the present invention provides the following solutions:

[0010] The invention provides application of a thorny leaf extract of Ziziphus jujuba in preparing antioxidant products and / or enzyme inhibitors.

[0011] Preferably, the method for preparing the extract of Ziziphus jujuba leaf thorn comprises the following steps:

[0012] After mixing the thorns of the Chinese jujube leaf and the extractant, the thorns of the Chinese jujube leaf are subjected to reflux extraction, vacuum filtration, concentration and drying in sequence to obtain the thorns of the Chinese jujube leaf extract;

[0013] The mass volume ratio of the sour jujube leaf thorn to the extractant is 1g:10mL.

[0014] Preferably, the extractant comprises one of water, anhydrous ethanol and ethyl acetate.

[0015] Preferably, the reflux extraction time is 4 hours and the temperature is 105°C;

[0016] The vacuum degree of the vacuum filtration is 0.095 MPa, and the time is 30 min;

[0017] The concentration temperature is 60°C and the rotation speed is 100r / min;

[0018] The drying temperature is 60° C. and the drying time is 12 hours.

[0019] Preferably, the enzyme inhibitor is a preparation that inhibits α-glucosidase, acetylcholinesterase, tyrosinase and xanthine oxidase.

[0020] The invention provides an antioxidant product, wherein the effective component of the antioxidant product is a thorny leaf extract of Ziziphus jujuba.

[0021] Preferably, the method for preparing the extract of Ziziphus jujuba leaf thorn comprises the following steps:

[0022] After mixing the thorns of the Chinese jujube leaf and the extractant, the thorns of the Chinese jujube leaf are subjected to reflux extraction, vacuum filtration, concentration and drying in sequence to obtain the thorns of the Chinese jujube leaf extract;

[0023] The mass volume ratio of the sour jujube leaf thorn to the extractant is 1g:10mL; the extractant includes one of water, anhydrous ethanol and ethyl acetate.

[0024] The invention provides an enzyme inhibitor. The active ingredient of the enzyme preparation is a thorny leaf extract of Ziziphus jujuba.

[0025] Preferably, the method for preparing the extract of Ziziphus jujuba leaf thorn comprises the following steps:

[0026] After mixing the thorns of the Chinese jujube leaf and the extractant, the thorns of the Chinese jujube leaf are subjected to reflux extraction, vacuum filtration, concentration and drying in sequence to obtain the thorns of the Chinese jujube leaf extract;

[0027] The mass volume ratio of the sour jujube leaf thorn to the extractant is 1g:10mL; the extractant includes one of water, anhydrous ethanol and ethyl acetate.

[0028] Preferably, the enzyme inhibitor is a preparation that inhibits α-glucosidase, acetylcholinesterase, tyrosinase and xanthine oxidase.

[0029] The present invention discloses the following technical effects:

[0030] The invention prepares a water extract of the thorny leaf of jujube, an anhydrous ethanol extract of the thorny leaf of jujube and an ethyl acetate extract of the thorny leaf of jujube by reflux extraction, and determines the contents of total phenol, total flavonoids, total sugar and total triterpenes thereof, evaluates the in vitro antioxidant activity by four methods, namely, DPPH free radical, ABTS cation free radical, hydroxyl free radical and FRAP, and studies the inhibitory effect of the thorny leaf of jujube on four enzymes, namely, α-glucosidase, acetylcholinesterase, tyrosinase and xanthine oxidase. The results show that the thorny leaf of jujube has certain antioxidant activity and enzyme inhibitory effect, and can be developed as a natural antioxidant from plant sources, as well as an inhibitor of α-glucosidase, acetylcholinesterase, tyrosinase and xanthine oxidase. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] 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.

[0032] Figure 1 is the standard curve of total phenols;

[0033] Figure 2 is the standard curve of flavonoids;

[0034] Figure 3 is the standard curve of total sugar;

[0035] Figure 4 is the standard curve of total triterpenes;

[0036] Figure 5is the DPPH free radical scavenging rate;

[0037] Figure 6 is the ABTS cation radical scavenging rate;

[0038] Figure 7 is the FRAP clearance rate;

[0039] Figure 8 α-glucosidase activity inhibition rate

[0040] Fig. 9 is the acetylcholinesterase activity inhibition rate;

[0041] Fig.10 is the tyrosinase activity inhibition rate;

[0042] Fig.11 is the xanthine oxidase activity inhibition rate. DETAILED DESCRIPTION

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0048] Example 1

[0049] 1. Experimental materials, reagents and instruments

[0050] Materials: Ziziphus jujuba leaf thorn (Neiqiu, Xingtai, Hebei).

[0051] Reagents: 2,2-biphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS), foline phenol, ascorbic acid, 2,4,6-tripyridine triazine, xanthine, allopurinol, and levodopa (Shanghai MacLean Biochemical Technology Co., Ltd.); vanillin and acarbose (Shanghai Bid Pharmaceutical Technology Co., Ltd.); acetylcholinesterase, 5,5'-dithiobis(2-nitrobenzoic acid), iodinated thioacetylcholine, huperzine A, rutin, gallic acid, β-D-galacturonic acid, oleanolic acid, xanthine oxidase, and tyrosinase (Shanghai Yuanye Biotechnology Co., Ltd.); other chemical reagents were of analytical grade: Sinopharm Chemical Reagent Co., Ltd.

[0052] Instruments: PLS-10LAN ultrafine pulverizer (Jinan Pulaishen Machinery Equipment Co., Ltd.); ZX800A multifunctional pulverizer (Yongkang Zhanfan Industry and Trade Co., Ltd.); RE-52AA rotary evaporator (Shanghai Yarong Biochemical Instrument Factory); electronic balance (Shanghai Huachao Electric Co., Ltd.); ZNHW digital display electric heating jacket (Tianjin Gongxing Laboratory Instrument Co., Ltd.); DZTW temperature-adjustable electric heating jacket (Shanghai Lichen Bangxi Instrument Technology Co., Ltd.); T-6 UV-visible spectrophotometer (Beijing Puxi General Instrument Co., Ltd.) Co., Ltd.); electric blast drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd.); microplate reader (Beijing Pulang New Technology Co., Ltd.); WKB-200 microplate incubator (Tuohe Electromechanical Technology (Shanghai) Co., Ltd.); ZW-A micro-oscillator (Changzhou Runhua Electric Co., Ltd.); 85-2 constant temperature magnetic stirrer (Guohua Electric Co., Ltd.); SHZ-III circulating water vacuum pump (Shanghai Yarong Biochemical Instrument Factory); HH-2 electric constant temperature water bath (Beijing Kewei Yongxing Instrument Co., Ltd.).

[0053] 2. Experimental methods

[0054] 2.1. Preparation of extract from the thorny leaves of Ziziphus jujuba

[0055] Use a multifunctional crusher to crush the jujube leaf thorns for 5 minutes, grind into powder and pass through a 40-mesh sieve. Accurately weigh 10g of jujube leaf thorn powder in a 250mL round-bottom flask, add 100mL of pure water at a solid-liquid ratio of 1g:10mL, connect a condenser and place it in a boiling water bath, and extract at 105℃ for 4h. After cooling to room temperature in an ice-water bath, vacuum filtration (vacuum degree of 0.095MPa, temperature of room temperature, time of 30min) is performed to obtain the filtrate, and then transferred to a rotary evaporator, 60℃, 100r / min speed, reduced pressure evaporation and concentration, concentrated to viscosity, dried in an electric heating blast drying oven at 60℃ for 12h to remove a small amount of water in the jujube leaf thorns, and finally dried to obtain the water extract of jujube leaf thorns (WT), and placed in a sealed state at room temperature away from light for later use.

[0056] Accurately weigh two portions of 10 g of Jujube leaf thorns powder, add 100 mL of anhydrous ethanol and 100 mL of ethyl acetate respectively according to the above method to prepare anhydrous ethanol extract of Jujube leaf thorns (AET) and ethyl acetate extract of Jujube leaf thorns (EAT).

[0057] The extraction rate (%) of the thorny leaf extract refers to the mass percentage of the mass of the extract after drying to the total mass of the thorny leaf powder.

[0058] 2.2. Determination of active ingredient content

[0059] 2.2.1. Determination of total phenol content

[0060] The method of Folin phenol colorimetry was slightly modified. The specific steps are as follows: weigh 2.51 mg of gallic acid reference substance, place it in a 25 mL volumetric flask, dissolve it in methanol and prepare 1 mL of gallic acid reference substance stock solution with a content of 0.1004 mg. Accurately measure 1, 2, 3, 4 and 5 mL of gallic acid reference substance stock solution, place them in 100 mL volumetric flasks, dilute to the mark with methanol, shake well, and obtain a series of gallic acid reference substance solutions. Accurately measure 1 mL of each series of gallic acid reference substance solution, place them in 10 mL volumetric flasks, add 0.5 mL of Folin phenol, react for 5 min, add 2 mL of 7.5% Na2CO3, dilute to the mark with purified water, react for 30 min in a dark environment, measure the absorbance at 765 nm, use blank reagent as reference, draw a standard curve with absorbance as the ordinate and mass concentration of gallic acid as the abscissa, and obtain a linear regression equation.

[0061] Weigh 2.51 mg of WT, AET and EAT respectively, dissolve in methanol in a 25 mL volumetric flask and prepare 1 mL of a 0.1004 mg stock solution of the thorny leaf extract of the jujube. Accurately measure 5 mL of the stock solution of the thorny leaf extract of the jujube, place it in a 100 mL volumetric flask, and dilute to the mark with methanol to obtain a solution of the thorny leaf extract of the jujube. Prepare the solution according to the above steps, and measure it in the same way as above. Each test is repeated 3 times, and the results are expressed as the gallic acid equivalent value (mg) per gram of water extract (mg / g).

[0062] 2.2.2 Determination of flavonoid content

[0063] The method of sodium nitrite-aluminum nitrate colorimetry is slightly modified. The specific steps are as follows: weigh 5.20 mg of rutin reference substance, place it in a 25 mL volumetric flask, dissolve it in methanol and prepare 1 mL of rutin reference substance stock solution with a content of 0.208 mg. Weigh 2, 3, 4, 5 and 6 mL of rutin reference substance stock solution, place them in 25 mL volumetric flasks respectively, add water to 6 mL, add 1 mL of 5% sodium nitrite solution, place it for 6 min, add 1 mL of 10% aluminum nitrate solution, place it for 6 min, add 10 mL of 4% sodium hydroxide solution, add water to the scale, place it for 10 min, and measure its absorbance at 510 nm, with blank reagent as reference. With absorbance as the ordinate and rutin mass concentration as the abscissa, draw a standard curve to obtain a linear regression equation.

[0064] Weigh 5.20 mg of WT, AET and EAT respectively, dissolve in methanol in a 25 mL volumetric flask and prepare 1 mL of reference stock solution of 0.208 mg of jujube leaf thorn extract. Accurately measure 6 mL of reference stock solution of jujube leaf thorn extract and place in a 25 mL volumetric flask. Prepare the solution according to the above steps, and measure the same method as above. Each test is repeated 3 times, and the results are expressed as the equivalent value of rutin (mg) per gram of water extract (mg / g).

[0065] 2.2.3. Determination of total sugar content

[0066] Refer to the phenol-sulfuric acid method and make slight changes. The specific steps are as follows: accurately weigh 10 mg of glucose into a 250 mL volumetric flask, add water to the mark to obtain a glucose standard solution. Pipette 0.00, 0.20, 0.40, 0.60, 0.80 and 1 mL of glucose standard solution respectively, make up to 1 mL with distilled water, then add 1 mL of 5% phenol and 5 mL of concentrated sulfuric acid, shake well and cool. After leaving at room temperature for 20 minutes, measure the optical density at 490 nm. Use the blank reagent as a reference. Plot a standard curve with the optical density value as the ordinate and the glucose mass concentration as the abscissa to obtain a linear regression equation.

[0067] Weigh 10 mg each of WT, AET and EAT into a 250 mL volumetric flask, add water to the mark, and prepare the solution according to the above steps. The determination is the same as above. Each test is repeated 3 times. The results are expressed as the glucose equivalent value (mg) per gram of extract (mg / g).

[0068] 2.2.4. Determination of total triterpenoid content

[0069] Accurately weigh 10 mg of oleanolic acid in a 100 mL volumetric flask, dilute with anhydrous ethanol to prepare a 0.1 mg / mL oleanolic acid reference standard solution. Pipette 0.2, 0.4, 0.6, 0.8 and 1.0 mL of the oleanolic acid reference standard solution into a 10 mL stoppered test tube, evaporate the solvent in a water bath, add 0.2 mL of 5% vanillin-glacial acetic acid solution, add 0.8 mL of perchloric acid, heat in a 60 ° C water bath for 15 minutes, then quickly cool to room temperature, add 5 mL of glacial acetic acid, let stand at room temperature for 10 minutes, and measure its absorbance at an absorbance wavelength of 547 nm. Use the blank reagent as a reference. Draw a standard curve with absorbance as the ordinate and oleanolic acid concentration as the abscissa to obtain a linear regression equation.

[0070] Weigh 10 mg of WT, AET and EAT respectively, dissolve them in anhydrous ethanol to prepare a 1 mg / mL sample solution, take 0.5 mL in a stoppered test tube, and measure the absorbance values ​​according to the above standard curve determination method. Each test was repeated 3 times, and the results were expressed as galacturonic acid equivalent (mg) per gram of jujube leaf thorn extract (mg / g).

[0071] 2.2.5. In vitro antioxidant activity assay

[0072] 2.2.5.1. Determination of DPPH free radical activity

[0073] Weigh 0.01288 g of DPPH, dissolve it in anhydrous ethanol, and transfer it quantitatively into a 50 mL volumetric flask. Add anhydrous ethanol to the mark and shake to obtain a concentration of 6.5 × 10 -4 mol / L DPPH stock solution was placed in a refrigerator for later use. When used, 10 mL of DPPH stock solution was diluted to 50 mL with anhydrous ethanol to obtain a concentration of 1.3×10 -4 4 mg of WT, AET, EAT and ascorbic acid were weighed respectively and prepared into 1.6 mg / mL solution with anhydrous ethanol, and then diluted into solutions with concentrations of 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8 and 1.6 mg / mL.

[0074] Experimental operation: In a 1.5mL EP tube, the reaction solution of each experimental group was added according to the order and volume of addition in Table 1. After addition, it was mixed. 200μL of the final solution was aspirated into each reaction tube and added to a 96-well plate. The absorbance of each sample at λ=517nm was determined by an enzyme marker. Three groups of different concentrations were tested in parallel to calculate the DPPH radical scavenging rate. The calculation formula of DPPH radical scavenging rate was: DPPH radical scavenging rate=[1-(As-Ar) / (Am-Ao)]×100% (Ao: anhydrous ethanol, λ=517nm, absorbance was measured, i.e.: blank group; Am: DPPH+anhydrous ethanol, λ=517nm, absorbance was measured, i.e.: DPPH group; Ar: anhydrous ethanol+drug, λ=517nm, absorbance was measured, i.e.: ginseng group; As: DPPH+drug, λ=517nm, absorbance was measured, i.e.: drug group).

[0075] Table 1 DPPH method sample addition order

[0076] Reagents Blank group DPPH group Ginseng group Drug group Anhydrous ethanol 200 40 160 - DPPH - 160 40 160 Drugs (WT, AET, EAT, or ascorbic acid) - - - 40

[0077] 2.2.5.2. Determination of ABTS cation radical activity

[0078] 5 mg of ascorbic acid was weighed and prepared into a 0.05 mg / mL stock solution, which was then diluted to obtain solutions with final concentrations of 0, 0.0078125, 0.0015625, 0.003125, 0.00625, 0.0125, 0.025, and 0.05 mg / mL.

[0079] 2 mg of WT, AET and EAT were weighed respectively, prepared into 0.2 mg / mL solutions, and then diluted into solutions with concentrations of 0.003125, 0.00625, 0.0125, 0.025, 0.05, 0.1 and 0.2 mg / mL, respectively.

[0080] 7mmol / L ABTS cation radical solution and 2.45mmol / L potassium persulfate solution were mixed in a volume ratio of 1:1, and kept at 4°C in the dark for 12h to obtain a blue-green ABTS cation radical solution. Using water as a reference, the solution was diluted with distilled water to an absorbance of 0.70±0.020 at a wavelength of 734nm to obtain a 7mmol / LABTS cation radical application solution.

[0081] Experimental operation: In a 1.5mL EP tube, the reaction solution of each experimental group was added according to the order and volume of addition in Table 2. After addition, the mixture was mixed. 250μL of the final solution was drawn from each reaction tube and added to a 96-well plate. The absorbance of each sample at λ=734nm was determined by an enzyme marker. Three groups of different concentrations were tested in parallel. Ascorbic acid was used as a positive control to calculate the ABTS cation radical scavenging rate. The calculation formula of the ABTS cation radical scavenging rate was: ABTS cation radical scavenging rate = [1-(Ai-Aj) / A] × 100% (A: pure water + ABTS, λ = 734nm, absorbance was measured, i.e.: blank group; Ai: ABTS + drug, λ = 734nm, absorbance was measured, i.e.: drug group; Aj: pure water + drug, λ = 734nm, absorbance was measured, i.e.: ginseng group)

[0082] Table 2 ABTS method sample addition order

[0083] Reagents Blank group μL Ginseng group μL Drug group μL Pure water 50 200 - ABTS 200 - 200 Drugs (WT, AET, EAT, or ascorbic acid) - 50 50

[0084] 2.2.5.3 FRAP activity assay

[0085] Weigh 5.1g of sodium acetate and place it in a 250mL volumetric flask, add 20mL of glacial acetic acid, add water to make up to 250mL, then add an appropriate amount of 1mmol / L sodium hydroxide solution and adjust the pH value to 3.6 to prepare acetic acid-sodium acetate buffer; weigh 0.0312g of TPTZ and place it in a 10mL volumetric flask, add 40mmol / L hydrochloric acid solution to make up to a 10mmol / L solution; weigh 0.1625g of FeCL3 and place it in a 50mL volumetric flask, add water to make up to a 20mmol / L solution; mix the above three solutions evenly in a volume ratio of 10:1:1 to obtain FRAP working solution.

[0086] Drawing of standard curve: Weigh 3.2mg FeSO4, prepare 0.32mg / mL FeSO4 solution, dilute with pure water to concentrations of 0.01, 0.02, 0.04, 0.08, 0.16 and 0.32mg / mL. Add the reaction solution of each group to the 1.5mL EP tube according to the order and volume of Table 3, and mix immediately after adding each reagent. Incubate at 37℃ for 10min. After the reaction is completed, take 240μL of the final solution from each reaction tube and add it to a 96-well plate. Use an enzyme marker to measure the absorbance of each sample at 593nm. Repeat the whole operation 3 times. Use blank reagent as reference. Use absorbance as the ordinate and FeSO4 content (mmol / L) as the abscissa to draw a standard curve and obtain the linear regression equation.

[0087] Weigh 16 mg of WT, AET, EAT and ascorbic acid respectively and prepare a 3.2 mg / mL solution. The sample solution is determined in the same way as the FeSO4 solution. The reducing capacity of the sample is expressed as the FRAP value (1 FRAP unit = 1 mmol / LFeSO4, i.e. the antioxidant capacity of the sample is equivalent to the mmol / L number of FeSO4).

[0088] Table 3 FRAP method sample addition order

[0089] Reagents Blank Group Ginseng group Drug group Pure water 50 150 FRAP 150 150 Drugs (WT, AET, EAT, or ascorbic acid) 50 50

[0090] 2.2.5.4 Determination of Hydroxyl Radical Activity

[0091] Prepare the solution: 0.1 mmol / L acetic acid solution; 7.5 mmol / L phenanthroline ethanol solution; 7.5 mmol / L ferrous sulfate solution (prepare before use); 1% (volume fraction) hydrogen peroxide.

[0092] Hydroxyl radical activity assay: The hydroxyl radical activity of the target compound was detected with ascorbic acid as a positive control. 5 mg WT, AET, EAT and ascorbic acid were weighed respectively, prepared into a 0.5 mg / mL solution, and then diluted into solutions with concentrations of 0.015625, 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg / mL.

[0093] Experimental method: Each drug was divided into 6 dose groups. Afterwards, the reaction solutions of each group were added to the 1.5mL EP tubes according to the order and volume of addition in Table 4. After the prepared reaction solution was incubated at 37°C for 30 minutes, 240μL of the final solution was drawn from each reaction tube and added to a 96-well plate. The absorbance of each sample at 536nm was measured by an ELISA instrument. The whole operation was repeated 3 times, and the hydroxyl radical scavenging rate was calculated. The calculation formula of the hydroxyl radical scavenging rate was: hydroxyl radical scavenging rate = [(A(drug group)-A(ginseng group))-(A(damage group)-A(blank group)] / (A(undamaged)-A(damage group))×100%.

[0094] Table 4 Hydroxyl radical addition order

[0095]

[0096] 2.2.6. α-Glucosidase Inhibitory Activity Assay

[0097] The test was carried out on a 96-well plate, and 8 mg of WT, AET, EAT and positive control acarbose were weighed to prepare a 0.4 mg / mL stock solution, and diluted to concentrations of 0, 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2 and 0.4 mg / mL for use. Potassium phosphate buffer (PBS buffer, pH = 6.8) was then prepared, and 0.0094 g of PNPG was weighed and diluted to a 50 mL volumetric flask with the prepared PBS buffer to prepare a 0.3125 mmol / L PNPG solution; and 1.0 U / mL α-glucosidase solution was then prepared with PBS buffer.

[0098] There are 4 groups in the experiment, namely E blank group, D control group, C ginseng group, B drug group, each group has three wells. PBS buffer, test sample solution, and α-glucosidase solution were added to the 96-well plate in sequence as shown in Table 5, mixed for 1 min, and incubated at 37°C for 10 min. Then, PNPG solution was added to each group, mixed for 1 min, and incubated at 37°C for 5 min. The absorbance of the samples was measured at 405 nm, and the α-glucosidase activity inhibition rate was calculated. The calculation formula of the α-glucosidase activity inhibition rate is: α-glucosidase activity inhibition rate = [1-(absorbance of B-absorbance of C) / (absorbance of D-absorbance of E)] × 100%.

[0099] Table 5 α-glucosidase inhibitory activity operation sequence

[0100]

[0101]

[0102] 2.2.7. Assay for acetylcholinesterase inhibitory activity

[0103] The test was carried out on a 96-well plate, and 0.3U / mL acetylcholinesterase solution, 5mmol / L 5,5-dithiobis-(2-nitrobenzoic acid) and 10mmol / L iodinated thioacetylcholine solution were prepared using PBS buffer (50mmol / L, pH=8.0), and the 10mmol / L iodinated thioacetylcholine solution was diluted to a 5mmol / L solution. 10mg of WT, AET, EAT and positive control huperzine A were weighed respectively, and 8mg / mL sample solutions were prepared, and diluted to 4, 2, 1, 0.5, 0.25, 0.125, 0.0625 and 0mg / mL sample solutions for use.

[0104] There were 4 groups in the experiment, namely blank group, control group, experimental blank group and experimental group, each group had three wells. PBS buffer, sample solution, acetylcholinesterase solution and DTNB solution were added to the 96-well plate in sequence as shown in Table 6. After incubation at 37°C for 5 min, ATCl solution was added to each group. After further incubation at 37°C for 20 min, the absorbance of the samples was measured at 405 nm, and the acetylcholinesterase activity inhibition rate was calculated. The calculation formula for the acetylcholinesterase activity inhibition rate was: acetylcholinesterase activity inhibition rate = [1-(absorbance of F-absorbance of G) / (absorbance of H-absorbance of J)] × 100%.

[0105] Table 6 Acetylcholinesterase inhibitory activity operation sequence

[0106]

[0107] 2.2.8. Tyrosine activation activity assay

[0108] PBS buffer (50mmol / L, pH=6.8) was used to prepare 50U / mL tyrosinase solution and 8mmol / L L-DOPA solution, and the L-DOPA solution was diluted to 1mmol / L solution. WT, AET and EAT 2.5mg were weighed respectively to prepare 0.5mg / mL sample solution; and diluted to 0.5, 0.25, 0.125, 0.0625, 0.03125 and 0mg / mL sample solutions for use.

[0109] There were 4 groups in the experiment, namely blank group, control group, experimental blank group and experimental group, each group had three wells. PBS buffer and sample solution were added to the 96-well plate in sequence as shown in Table 7. L-DOPA solution was added to each group. After incubation at 37°C for 10 min, tyrosinase solution was added. After further incubation at 37°C for 20 min, the sample absorbance was measured at 475 nm, and the tyrosinase activation rate was calculated. The calculation formula of tyrosinase activation rate was: tyrosinase activation rate = [(absorbance of K-absorbance of L) / (absorbance of M-absorbance of N)-1] × 100%.

[0110] Table 7 Tyrosinase activation properties Operation sequence

[0111]

[0112]

[0113] 2.2.9. Xanthine oxidase inhibitory activity assay

[0114] The detection method in the literature "Study on the in vitro xanthine oxidase inhibition activity and in vivo uric acid lowering of extracts from Prunella vulgaris and Perilla leaves" ([1]. Wen Wen, Hu Haifeng, Wu Chunzhen. Study on the in vitro xanthine oxidase inhibition activity and in vivo uric acid lowering of extracts from Prunella vulgaris and Perilla leaves [J]. Shaanxi Agricultural Sciences, 2023, 69(04): 39-42.) was slightly modified. The specific steps are as follows: PBS buffer (50mmol / L, pH = 7.2) was used to prepare 0.1U / mL xanthine oxidase solution and 1.6mmol / L xanthine solution respectively. WT, AET and EAT and positive control allopurinol 16mg were weighed respectively to prepare 1.6mg / mL sample solution; and diluted to 0, 0.0625, 0.125, 0.25, 1 and 2mg / mL sample solutions for later use. There were 4 groups in the experiment, namely blank group, control group, experimental blank group and experimental group, each group had three wells. PBS buffer, sample solution and xanthine oxidase solution were added to the 96-well plate in sequence as shown in Table 8. After incubation at 37°C for 10 min, xanthine solution was added to each group. After further incubation at 37°C for 25 min, the absorbance of the samples was measured at 290 nm, and the xanthine oxidase activity inhibition rate was calculated. The calculation formula for the xanthine oxidase activity inhibition rate was: xanthine oxidase activity inhibition rate = [1-(absorbance of S-absorbance of P) / (absorbance of Q-absorbance of R)] × 100%.

[0115] Table 8 Xanthine oxidase inhibition activity operation sequence

[0116]

[0117] 2.3 Data Processing

[0118] All tests were performed in triplicate and the results were expressed as average values. Excel 2019 software was used for basic data statistics, and Origin 2019 software was used for chart drawing.

[0119] 3. Results

[0120] 3.1 Analysis of active components in the extract of Ziziphus jujuba leaf thorn

[0121] The extract of the thorn of the jujube leaf was prepared by boiling water extraction and its active ingredient content was determined. The results are shown in Table 9. The extraction rate of WT was 6.68%, and the contents of total phenols, total flavonoids, total sugars and total triterpenes were 88.73 mg / g, 93.68 mg / g, 205.54 mg / g and 54.95 mg / g, respectively; the extraction rate of AET was 8.179%, and the contents of total phenols, total flavonoids, total sugars and total triterpenes were 108.3 mg / g, 120.06 mg / g, 259.88 mg / g and 70.97 mg / g, respectively; the extraction rate of EAT was 1.333%, and the contents of total phenols, total flavonoids, total sugars and total triterpenes were 83.51 mg / g, 94.94 mg / g, 102.35 mg / g and 251.98 mg / g, respectively. The results showed that the extract of the thorn of the jujube leaf was rich in active components such as polyphenolic compounds and polysaccharides.

[0122] Table 9 Extraction rate of WT, AET and EAT and the contents of total phenols, total flavonoids, total sugars and total triterpenes

[0123] Extraction rate (%) Total phenols (mg / g) Total flavonoids (mg / g) Total sugar (mg / g) Total triterpenes (mg / g) WT 6.68 88.73 93.98 205.54 205.75 AET 8.179 108.3 120.06 259.88 261.52 E-A-T 1.333 83.51 94.94 102.35 251.98

[0124] The total phenol content in WT, AET and EAT was determined by Folin phenol colorimetry. Figure 1 As shown. With blank reagent as reference, absorbance as ordinate, and mass concentration of gallic acid as abscissa, a standard curve was drawn, and the linear regression equation was y=12.251x+0.03749, R 2 =0.9979.

[0125] The sodium nitrite-aluminum nitrate colorimetric method was used to determine the content of flavonoids in WT, AET and EAT. Figure 2 As shown. With blank reagent as reference, absorbance as ordinate, and rutin mass concentration as abscissa, a standard curve was drawn, and the linear regression equation was y=5.6887x+0.04759, R 2 =0.9917.

[0126] The phenol-sulfuric acid method was used to determine the total sugar content in WT, AET, and EAT. Figure 3 As shown. With blank reagent as reference, absorbance as ordinate, glucose mass concentration as abscissa, draw a standard curve, linear regression equation y = 49.52x + 0.1054, R 2 =0.9938.

[0127] The content of total triterpenes in WT, AET and EAT was determined. Figure 4 As shown. With blank reagent as reference, absorbance as ordinate, and oleic acid mass concentration as abscissa, a standard curve was drawn, and the linear regression equation was y=0.002x-0.0053, R 2 =0.9912.

[0128] 3.2 Analysis of the antioxidant activity of extracts from the leaves of Ziziphus jujuba

[0129] The in vitro antioxidant capacity of the water extract, anhydrous ethanol extract and ethyl acetate extract of Ziziphus jujuba leaf thorn was evaluated by four methods: DPPH free radical, ABTS cation free radical, hydroxyl free radical and FRAP.

[0130] The extract of jujube leaf thorn has a certain scavenging ability on DPPH free radicals, which increases with the increase of concentration. Figure 5 As shown in the figure, WT, EAT and AET have strong ability to scavenge DPPH free radicals, but their scavenging ability is weaker than that of ascorbic acid. It may be that the low purity and impurities of the extract of Ziziphus jujuba leaf thorn affect its activity. IC values ​​of WT, EAT, AET and positive control ascorbic acid 50 The values ​​were 0.0235 mg / mL, 0.0789 mg / mL, 0.0961 mg / mL and 0.02287 mg / mL respectively.

[0131] The extract of jujube leaf thorn has a certain scavenging ability for ABTS cationic free radicals, which increases with the increase of concentration. Figure 6 As shown. Within the concentration range, the scavenging rates of the ABTS cationic free radicals by the extract of the thorny leaves of Ziziphus jujuba and ascorbic acid were positively correlated with their mass concentrations, showing strong antioxidant capacity. The IC values ​​of WT, EAT, AET and the positive control ascorbic acid were 50 The values ​​were 0.01466 mg / mL, 0.01039 mg / mL, 0.008604 mg / mL and 0.01267 mg / mL respectively.

[0132] The extract of Ziziphus jujuba leaf thorn has a certain scavenging ability for hydroxyl free radicals, which increases with the increase of concentration. Within the measured concentration range, the scavenging rates of EAT, AET and ascorbic acid for hydroxyl free radicals are positively correlated with their mass concentrations, showing strong antioxidant capacity, while WT has no scavenging ability for hydroxyl free radicals. The IC values ​​of EAT, AET and positive control ascorbic acid are 50 The values ​​were 0.002117 mg / mL, 0.003112 mg / mL, and 0.008897 mg / mL respectively.

[0133] Under acidic conditions, Fe 3+ It can form an orange complex with TPTZ (Fe 3+ -TPTZ), when reducing substances are added to it, Fe 3+ -TPTZ is reduced to Fe 2+ -TPTZ is blue. A standard curve was drawn with FeSO4 as the standard substance ( Figure 7) and the regression equation was obtained. The antioxidant capacity of the samples was expressed as FRAP value (1 FRAP unit = 1 mmol / LFeSO4, that is, the antioxidant capacity of the sample is equivalent to the mmol / L number of FeSO4). The statistical results are shown in Table 10.

[0134] Table 10 Antioxidant capacity of extracts from jujube leaves

[0135] ascorbic acid WT E-A-T AET Antioxidant Capacity 109.04FRAP / mg 149.64FRAP / mg 153.66FRAP / mg 159.21FRAP / mg

[0136] The IC values ​​of the extracts from the leaves of Ziziphus jujuba were determined by DPPH free radical, ABTS cation free radical, hydroxyl free radical and FRAP. 50 The FRAP method used FeSO4 as the standard sample to make a standard curve, and the inhibition rate of the extract of the thorn of the jujube leaf and ascorbic acid was calculated by the formula. Among the four antioxidant methods, the IC values ​​of DPPH free radical scavenging ability of WT, EAT and AET were 50 The antioxidant capacity of WT, EAT and AET was better than that of ascorbic acid (0.02287 mg / mL). The IC values ​​of ABTS free radical scavenging ability of WT, EAT and AET were 50 The antioxidant capacity of WT was lower than that of ascorbic acid (0.01267 mg / mL), while that of EAT and AET was better than that of ascorbic acid (0.01466 mg / mL, 0.01039 mg / mL and 0.008604 mg / mL). The FRAP antioxidant capacity of WT, EAT and AET (149.64 FRAP / mg, 153.66 FRAP / mg, 159.21 FRAP / mg) was better than that of ascorbic acid (109.04 FRAP / mg), while the IC 50 The value is relatively large. The results show that the extract of the thorn leaf of the jujube has a certain antioxidant capacity, and the extract of the thorn leaf of the jujube has the strongest scavenging activity against water-soluble ABTS cation radicals and fat-soluble DPPH radicals, while the hydroxyl radical scavenging ability (HRSA) is the weakest. This may be due to the hydrogen atom transfer mechanism of the thorn leaf extract of the jujube, which is rich in polyphenols and flavonoids, forming highly branched polyhydroxyl active sites in its water system. This configuration promotes the formation of metal complexes and antioxidant reactions based on electron transfer.

[0137] 3.3. α-Glucosidase inhibitory activity

[0138] The inhibitory effect of the extract of Ziziphus jujuba leaf thorn on α-glucosidase is shown in Figure 8As shown in the figure, the inhibitory effect of the extract of Ziziphus jujuba leaf thorn on α-glucosidase showed an overall upward trend in the mass concentration range of 0.0-0.4 mg / mL. The IC 50 The values ​​were 0.03950 mg / mL, 0.02033 mg / mL and 0.05520 mg / mL respectively. EAT showed higher activity, which may be related to the higher active substances or content in EAT.

[0139] 3.4 Acetylcholinesterase inhibitory activity

[0140] The inhibitory effect of the extract from the leaves of Ziziphus jujuba on the activity of acetylcholinesterase Fig. 9 As shown in the figure, the acetylcholinesterase inhibitory activity of the extract of the thorny jujube leaf was significantly positively correlated with its mass concentration within the mass concentration range of 0.0625-2 mg / mL. IC 50 The values ​​were 0.2269 mg / mL, 0.7252 mg / mL and 0.3707 mg / mL respectively. When the mass concentration was 0.0625 mg / mL, the acetylcholinesterase inhibition rate of the extract of the thorn of the jujube leaf was small, but with the increase of the mass concentration, the inhibition rate increased significantly; when the mass concentration exceeded 2 mg / mL, the inhibition rate increased slowly and the difference was not significant.

[0141] 3.5 Tyrosinase inhibitory activity

[0142] The inhibitory effect of the extract from the leaves of Ziziphus jujuba on tyrosinase activity is shown in Fig.10 As shown in the figure, the tyrosinase inhibitory activity of the extract of the jujube leaf thorn was significantly positively correlated with its mass concentration in the mass concentration range of 0.03125-0.5 mg / mL. IC 50 The values ​​were 0.4384 mg / mL, 0.2197 mg / mL and 0.2217 mg / mL respectively.

[0143] 3.6. Xanthine oxidase inhibitory activity

[0144] The inhibitory effect of the extract from the leaves of Ziziphus jujuba on the activity of xanthine oxidase Fig.11 As shown in the figure, the acetylcholinesterase inhibitory activity of the extract of the thorny jujube leaf was significantly positively correlated with its mass concentration within the mass concentration range of 0.0625-2 mg / mL. IC 50 The values ​​were 0.4321 mg / mL, 0.9443 mg / mL and 1.324 mg / mL respectively.

[0145] 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 the extract of Ziziphus jujuba leaf thorn in the preparation of antioxidant products and / or enzyme inhibitors.

2. The use according to claim 1, characterized in that: The preparation method of the thorny jujube leaf extract comprises the following steps: After mixing the thorns of the Chinese jujube leaf and the extractant, the thorns of the Chinese jujube leaf are subjected to reflux extraction, vacuum filtration, concentration and drying in sequence to obtain the thorns of the Chinese jujube leaf extract; The mass volume ratio of the sour jujube leaf thorn to the extractant is 1g:10mL.

3. The use according to claim 2, characterized in that: The extractant includes one of water, anhydrous ethanol and ethyl acetate.

4. The use according to claim 2, characterized in that: The reflux extraction time is 4 hours and the temperature is 105°C; The vacuum degree of the vacuum filtration is 0.095 MPa, and the time is 30 min; The concentration temperature is 60°C and the rotation speed is 100r / min; The drying temperature is 60° C. and the drying time is 12 hours.

5. The use according to claim 1, characterized in that: The enzyme inhibitor is a preparation that inhibits α-glucosidase, acetylcholinesterase, tyrosinase and xanthine oxidase.

6. An antioxidant product, characterized in that: The active ingredient of the antioxidant product is the extract of thorny leaves of Ziziphus jujuba.

7. The antioxidant product according to claim 6, characterized in that The preparation method of the thorny jujube leaf extract comprises the following steps: After mixing the thorns of the Chinese jujube leaf and the extractant, the thorns of the Chinese jujube leaf are subjected to reflux extraction, vacuum filtration, concentration and drying in sequence to obtain the thorns of the Chinese jujube leaf extract; The mass volume ratio of the sour jujube leaf thorn to the extractant is 1g:10mL; the extractant includes one of water, anhydrous ethanol and ethyl acetate.

8. An enzyme inhibitor, characterized in that The active ingredient of the enzyme preparation is the extract of thorns of wild jujube leaves.

9. The enzyme inhibitor according to claim 8, characterized in that The preparation method of the thorny jujube leaf extract comprises the following steps: After mixing the thorns of the Chinese jujube leaf and the extractant, the thorns of the Chinese jujube leaf are subjected to reflux extraction, vacuum filtration, concentration and drying in sequence to obtain the thorns of the Chinese jujube leaf extract; The mass volume ratio of the sour jujube leaf thorn to the extractant is 1g:10mL; the extractant includes one of water, anhydrous ethanol and ethyl acetate.

10. The enzyme inhibitor according to claim 8, characterized in that The enzyme inhibitor is a preparation that inhibits α-glucosidase, acetylcholinesterase, tyrosinase and xanthine oxidase.

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