CN109364130B

CN109364130B - Application of spina gleditsiae counterfeit product in preparation of antioxidant

Info

Publication number: CN109364130B
Application number: CN201811518828.0A
Authority: CN
Inventors: 殷果; 王珏; 王铁杰; 江坤; 李菁; 金一宝; 王丽君; 王思明; 刘凯双
Original assignee: Shenzhen Institute For Drug Control (shenzhen Testing Center Of Medical Devices)
Current assignee: Shenzhen Institute For Drug Control (shenzhen Testing Center Of Medical Devices)
Priority date: 2018-12-12
Filing date: 2018-12-12
Publication date: 2021-11-09
Anticipated expiration: 2038-12-12
Also published as: CN109364130A

Abstract

The invention provides an application of a spina gleditsiae counterfeit product in preparing an antioxidant, and the spina gleditsiae counterfeit product shows equivalent activity or even better activity compared with rutin, hawthorn extract and other substances through detection of the antioxidant activity of the spina gleditsiae counterfeit product, the DPPH free radical clearance rate can reach 60-75%, the ORAC value can reach 1.20-1.30, a new strategy is provided for researching and developing related preparations, medicines and the like, and the spina gleditsiae counterfeit product has a good application prospect and a high application value.

Description

Application of spina gleditsiae counterfeit product in preparation of antioxidant

Technical Field

The invention belongs to the field of medicinal chemistry, and relates to application of a spina gleditsiae counterfeit product in preparation of an antioxidant.

Background

In the pharmacopoeia of China (2015 edition), spina Gleditsiae is recorded to have the effects of relieving swelling, expelling toxin, expelling pus and killing parasites; can be used for treating early carbuncle and cellulitis or suppurative abscess; it is indicated for external treatment of scabies and leprosy. In recent years, the market demand for spina gleditsiae has increased dramatically with the development and use of drugs. At present, spinas of gleditsiae produced by gleditsia sinensis trees growing in different areas are uneven in quality, and spina of other plants are also mixed in the spinas of gleditsia sinensis trees for sale. Common counterfeits include mountain spina gleditsiae (Gleitsia japonica Miq.), wild spina gleditsiae (Gleitsia microphylla Gordon ex Y.T.Lee), spina date seed (Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H.F.Chow), and the like, with mountain spina gleditsiae and wild spina gleditsiae being the most common.

The occurrence of the above-mentioned diseases associated with the production of spina Gleditsiae pseudopeptides is largely related to the metabolic disorder of active oxygen in vivo. Therefore, two in vitro antioxidant activity evaluation methods, namely a DPPH free radical scavenging method (DPPH method) and an oxygen radical scavenging ability method (ORAC method), are adopted.

Although most of the free radicals have extremely active chemical properties and extremely short lives, few of the free radicals have stable chemical properties, and DPPH (1, 1-diphenyl-2-piperidinylhydrazyl) is one of the free radicals. Its stability mainly comes from resonance stabilizing action and steric hindrance of three benzene rings, so that the unpaired electron sandwiched on middle nitrogen atom can not exert its proper electron pairing action. DPPH.in ethanol is purple with strong absorption at 517 nm. When a radical scavenger is present, the single electrons of DPPH are paired to lighten the solution color, and the absorbance at the maximum absorption wavelength becomes small, and the degree of the decrease in absorbance is linear with the degree of radical scavenging. Therefore, the absorbance at this wavelength can be used to detect the scavenging of free radicals, thereby evaluating the antioxidant capacity of the test sample. The antioxidant has a mode of action AH + DPPH → DPPH + A, and the scavenging activity is related to the number of available phenolic hydroxyl groups in the antioxidant molecule and the stability of the newly formed antioxidant free radicals (A). In 1958, Blois applied the antioxidant screening study, and since then, many scholars at home and abroad studied the property of the substance to remove free radicals by using the method.

The fluorescent substance fluorescein disodium can emit 527nm fluorescence under the excitation of 485nm light. AAPH [2,2'-azobis (2-amidinopropane) dichloride, 2,2' -azo (diamidinopropane) dichlorine ] can release peroxy Radicals (ROO) in aqueous solution and oxidize fluorescein disodium, so that the fluorescence characteristic is lost. In the presence of an antioxidant, it competes with fluorescein disodium for the oxidizing agent, slowing its fluorescence decay. Based on this property, the oxygen radical scavenging activity (ORAC) of the sample can be measured. The ORAC method is a method for determining the total antioxidant capacity of a substance, and uses Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water-soluble analog of alpha-tocopherol, as a standard control. The method can provide stable and controllable free radical sources, is suitable for hydrophilic and lipophilic compounds, has accurate fluorescent quantitative result and can realize trace and high-throughput automatic detection. After the anti-oxidation activity studies by Glaz and Ghiselli et al, the method has been widely used to evaluate the anti-oxidation ability of biological products and foods through a series of improvements.

The spina gleditsiae counterfeit product also has some pharmacological effects similar to those of spina gleditsiae.

CN104087487A discloses a brewing process of spina gleditsiae wine, which takes spina gleditsiae as a raw material and sweet potato as a substrate, and is processed by the steps of raw material treatment, soaking, superfine grinding, sweet potato treatment, raw material mixing, medicine adding, after-fermentation, squeezing and filtering, wine liquid mixing and the like; the invention adopts the brewing process combining the raw material soaking and the fermentation, not only can improve the utilization rate of the raw materials, but also can lead the nutrient substances of the finished product spina gleditsiae wine to be more balanced, the taste to be mellow and the aftertaste to be long, and simultaneously has various health care efficacies of detumescence, pus discharge, wind dispelling, collateral activating, heat clearing, detoxifying and the like. The method discloses that spina Gleditsiae is combined with other products for use, and has certain health promotion effect.

CN102940672B discloses an application of spina gleditsiae total flavonoids in preparing medicaments for preventing and treating tumors, in particular to an application of spina gleditsiae total flavonoids as a cell junction communication enhancer in preparing medicaments for preventing and treating tumors, especially in the aspects of tumor prevention, tumor chemotherapy synergy and tissue hyperplasia, such as prevention of chemical carcinogenesis, improvement of tumor chemotherapy index, reversal of tumor chemotherapy resistance, prevention of mammary gland hyperplasia and prostatic hyperplasia and the like. The efficacy of spina gleditsiae against tumors is disclosed in this application.

CN107383151A discloses an anti-breast cancer active pentacyclic triterpenoid saponin compound of spina gleditsiae and an extraction method thereof, and the method provides the potential value of the spina gleditsiae in the aspect of anti-breast cancer.

At present, no related research and related reports exist about the application of the spina gleditsiae counterfeit product in the aspect of oxidation resistance.

Disclosure of Invention

The invention aims to provide the application of the spina gleditsiae counterfeit product in preparing the antioxidant, so as to promote the expansion of the application field of the spina gleditsiae counterfeit product and improve the application value of the spina gleditsiae counterfeit product.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides an application of a spina gleditsiae counterfeit product in preparing an antioxidant.

According to the invention, the spina gleditsiae counterfeit product has a strong antioxidation effect and can be used as an antioxidant, and through the detection of the antioxidation activity of the spina gleditsiae counterfeit product, the spina gleditsiae counterfeit product shows equivalent activity or even better activity compared with rutin, hawthorn extract and other substances, so that a new strategy is provided for researching and developing related preparations, medicines and the like.

In the current research, no report is made about the effect of the spina gleditsiae counterfeit product on the oxidation resistance, and the spina gleditsiae counterfeit product has more excellent oxidation resistance activity compared with a single flavonoid substance.

Preferably, the spina gleditsiae counterfeit product comprises any one of spina gleditsiae, spina gleditsiae or spina date seed.

Preferably, the antioxidant has a DPPH radical scavenging rate of 60% to 75%, and may be, for example, 60%, 62%, 64%, 65%, 66%, 70%, 71%, 72%, 73%, 74%, or 75%.

Preferably, the antioxidant has an ORAC value of 1.20 to 1.30, and may be, for example, 1.20, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, or the like.

The ORAC value of the present invention refers to the magnitude of the protected integrated area on the fluorescence decay curve (i.e., the integrated area under the fluorescence decay curve minus the area under the blank curve without antioxidant) in the presence of an antioxidant, as shown in FIG. 1. The protective areas of different antioxidants were uniformly converted to the protective area of 1 μ M Trolox for comparison. Thus, 1 ORAC unit is defined as: the corresponding protection integration area of Trolox at a final concentration of 1 μ M on the fluorescence decay curve.

Preferably, the antioxidant acts as an antioxidant drug.

In the invention, the antioxidant can be further developed into a related antioxidant drug.

Preferably, the antioxidant drug further comprises pharmaceutically acceptable auxiliary materials.

The antioxidant medicine provided by the invention is prepared from an antioxidant and optional auxiliary materials.

Preferably, the pharmaceutically acceptable auxiliary materials include any one of or a combination of at least two of a filler, a binder, a disintegrant, a lubricant, a glidant, a pressurizing agent, an effervescent agent, a surfactant, a film forming agent, a toner, a flavoring agent, a preservative, a dispersing agent or a flavoring agent.

The filler can be auxiliary materials with filling effect such as starch, saccharide, cellulose or inorganic salt. The adhesive can be microcrystalline cellulose, gelatin, saccharide, polyethylene glycol, polyvinylpyrrolidone, pregelatinized starch, hydroxypropyl cellulose or hydroxypropyl methyl cellulose, etc. with viscosity improving effect and easy preparation forming effect. The disintegrant may be starch sodium glycolate, sodium carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, crospolyvinylpyrrolidone, polyvinylpyrrolidone, methylcellulose, microcrystalline cellulose, low carbon number alkyl substituted hydroxypropyl cellulose, starch, pregelatinized starch or sodium alginate, and other adjuvants with disintegrating effect. The lubricant hard can be one or more of magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, magnesium stearate, and sodium lauryl sulfate. The glidant can be silicon dioxide or talcum powder and other auxiliary materials with the function of improving fluidity. The surfactant can be lecithin or fatty glyceride, etc. with solubility increasing effect. One skilled in the art can select suitable pharmaceutically acceptable adjuvants for processing according to the dosage form to be prepared.

In addition, the study of the invention also finds that the spina gleditsiae counterfeit product also has a certain effect of inhibiting nitric oxide, the inhibition rate of inhibiting nitric oxide can reach more than 60%, and the spina gleditsiae counterfeit product has activity equivalent to that of resveratrol.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, the spina gleditsiae counterfeit product has a strong antioxidation effect and can be used as an antioxidant, through the detection of the antioxidation activity of the spina gleditsiae counterfeit product, the spina gleditsiae counterfeit product shows equivalent activity or even better activity compared with rutin, hawthorn extracts and other substances, the DPPH free radical clearance rate can reach 60-75%, the ORAC value can reach 1.20-1.30, and a new strategy is provided for researching and developing related preparations, medicines and the like.

Drawings

FIG. 1 is a schematic representation of the protected integrated area on the fluorescence decay curve of the present invention.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

In the following examples of the present invention, the samples used are listed in table 1 below, the numbers of which correspond to those in the examples.

TABLE 1

The samples are prepared by extraction in the following way before testing to obtain the test sample.

Precisely weighing about 1.0g of sample powder (passing through a No. 3 sieve), placing the sample powder into a conical flask with a plug, adding 10mL of 70% ethanol solution, carrying out ultrasonic treatment (power 250W and frequency 40kHz) for 2 times, each time for 30min, filtering, combining filtrates, placing the mixture into an evaporating dish, volatilizing in a water bath at 50 ℃ until the volume of the solution is 1-2 mL, adding 70% ethanol solution into residues, dissolving the residues into a 5mL measuring flask, diluting the residues to a scale with 70% ethanol solution, shaking up, and filtering through a 0.45 mu m microporous filter membrane to obtain a sample solution.

Example 1

This example measures DPPH radical scavenging Activity by the following method

(1) Preparation of DPPH solution and test solution

DPPH is 2X 10 prepared by absolute ethyl alcohol^-4The solution of M is stored at 4 ℃ in the dark. All the monomer compounds and the positive drugs are prepared into 1mg/mL stock solution by absolute ethyl alcohol, and the stock solution is diluted into the required concentration by absolute ethyl alcohol before testing.

(2) Determination of Activity

Referring to the literature, the experimental method is improved, and the test solution with different concentrations is 100 mu L and 2X 10^-4Adding 100 mu L of M DPPH solution into each well of a 96-well plate, taking each concentration of a test solution without DPPH (replacing DPPH with 100 mu L of absolute ethyl alcohol) as a control to eliminate the interference of the color of the test sample on the test result, setting DPPH negative control (replacing the test sample with 100 mu L of absolute ethyl alcohol), and setting 4 multiple wells in parallel in each group. Placing the 96-well plate into an enzyme labeling instrument, shaking for 1min, storing under the condition (room temperature and dark), testing the absorbance OD value of the 96-well plate at 517nm after 30min, and calculating the free radical clearance rate of the test sample according to the following formula.

Radical scavenging rate ═ OD_{DPPH·control}-(OD_sample-OD_{sample control})]/OD_{DPPH·control}×100％

Wherein: OD_{DPPH·control}The average value of the DPPH negative control group OD values is obtained;

D_samplethe OD values of the sample groups are averaged;

OD_{sample control}is the average value of OD values of the sample ethanol control group。

(3) Preliminary test results for radical scavenging ability

The ability of each extract to scavenge DPPH free radicals at a concentration of 50. mu.g/mL was tested using the above experimental method, while the positive controls, rutin, a flavonoid (at a concentration of 10. mu.g/mL) and hawthorn extract (at a concentration of 40. mu.g/mL), were used, and activity was considered to be present with a free radical scavenging rate higher than 50%. The results of the experiment are shown in Table 2 (where the sample numbers correspond to the samples represented by the sample numbers in Table 1):

TABLE 2

Sample numbering	Clearance rate
		1	60.1％
2	62.1％
		3	65.6％
4	61.7％
		5	68.7％
6	65.3％
		7	64.4％
Hawthorn extract	52.7％
		Rutin	63.2％

Example 2

This example measures the oxygen radical scavenging ability by the following method

(1) Preparation of reaction reagent and test solution

The fluorescein disodium salt is dissolved in 75mM potassium phosphate buffer (75mM KH)₂PO₄、75mM K₂HPO₄) Prepare 63 μ M stock solution, store at 4 ℃ in the dark, and dilute 100 times with this buffer solution before testing. AAPH in the experiment before 75mM potassium phosphate buffer solution to make 18.3mM solution. Trolox was made up to 10. mu.M stock with 75mM potassium phosphate buffer and diluted to the desired concentration with this buffer before testing. Each monomeric compound and the positive drug VC were prepared as 100mM stock solutions in 75mM potassium phosphate buffer, and diluted to the desired concentrations with this buffer before testing.

(2) Determination of Activity

The ORAC assay used in this experiment was referenced and modified to the method of Ronald l. In the experiment, 20 mu L of sample solution to be detected with different concentrations is added into each well of a 96-well plate, 20 mu L of 75mM potassium phosphate buffer solution and 140 mu L of AAPH (final concentration is 12.8mM) are added, 20 mu L of fluorescein disodium (final concentration is 63nM) is added finally to start reaction, the 96-well plate is quickly placed in a fluorescence microplate reader (preset temperature is 37 ℃) to start measurement, and 3 multiple wells are arranged in parallel in each group. One point was measured every 2min in a kinetic manner until the fluorescence intensity decay was zero.

(3) The results are expressed as an antioxidant capacity index, which is the size of the protected integrated area on the fluorescence decay curve (i.e., the integrated area under the fluorescence decay curve minus the area under the blank curve without antioxidant) in the presence of antioxidant, as shown in fig. 1, which is a schematic representation only. The protective areas of different antioxidants were uniformly converted to the protective area of 1 μ M Trolox for comparison. Thus, 1 ORAC unit is defined as: the corresponding protection integration area of Trolox at a final concentration of 1 μ M on the fluorescence decay curve.

The above experimental method is adopted to test the oxygen free radical scavenging ability of each extract under the concentration of 50 mug/mL, and simultaneously, the flavonoid compound rutin (with the concentration of 30 mug/mL) is used as a positive control, and the antioxidant ability index is equivalent to or superior to that of rutin and is considered to have activity. Specific results are shown in table 3:

TABLE 3

Example 3

In this example, the contents of the respective components of the counterfeit Chinese honeylocust spine whose sample numbers are 1 to 7 in Table 1 were measured by liquid chromatography

An Agilent ZORBAX SB-Aq C18(250mm multiplied by 4.6mm,5 mu m) chromatographic column is adopted, the mobile phase is methanol-0.1 percent acetic acid solution for gradient elution, the detection wavelength is 254nm, the column temperature is 35 ℃, the flow rate is 1.0mL/min, and the chromatogram obtained when the sample amount is 10 mu L has better separation degree and peak type, more chromatographic peaks and uniform distribution. Recording chromatogram till 2 times retention time (180min), no chromatographic peak appears after 90min, so analysis time is 90min, and recording peak area. The results are shown in Table 4 (where the sample numbers correspond to the samples represented by the sample numbers in Table 1):

TABLE 4

As can be seen from table 4, the pseudolarix and fustin in the spina gleditsiae product have high content, and the existence of these substances greatly promotes the antioxidant activity of spina gleditsiae.

From the test results of examples 1 to 3, it is known that the spina gleditsiae counterfeit product contains abundant active substances, and mutual promotion among the active substances enables the spina gleditsiae counterfeit product to have higher antioxidant activity, and to have equivalent activity or even higher activity compared with rutin, hawthorn extract and the like.

Example 4

This example measures the inhibition of NO release activity from rat macrophages by the following method

Since NO is extremely unstable, it is oxidized very quickly

Has stable properties, and can be measured

Indirectly reflect the amount of NO produced. The method adopts the Griess method to determine the content of the solution

The content of (a) is determined by the principle that under acidic conditions,

diazotizing with Sulfanilamide, and coupling reacting with N-1-naphthyl-ethylenediamine hydrochloride (N-1-naphthylethylene-diamine dichloride) to obtain mauve azo dye with maximum absorption spectrum at 570nm, and measuring the absorbance with enzyme linked immunosorbent detector. Determination by Griess method

Has the characteristics of high sensitivity, high accuracy and reproducibility, and the method is simple and effective and is the most common determination at present

Content, method for measuring NO level.

(1) Cell culture and preparation of test sample and reaction reagent

RAW264.7 rat macrophages (Abelson Leukemia virus transformed) were derived from ATCC (American Tissue Culture Collection) USA. At 37 ℃ 5% CO₂The wet heat incubator of (1), was cultured in HAM's F12 medium containing 10% FBS.

HAM's F12 medium (Sigma, N4888): glutamic acid (Sigma) and 50mL fetal bovine serum were added to 500 mL.

IFN-gamma: Genzyme/Techne Inc.

Sulfanilamide, N-1-naphthylethylene-diamine dihydrate, MTT: wako corporation.

Griess reagent: 5mg of N-1-naphthyl-ethylenediamine hydrochloride (N-1-naphthylethylene-diamine dichloride) was dissolved in 5mL of water for injection; sulfanilamide (sulfaminidamide) 50mg was added to 250. mu.L of phosphoric acid, and then diluted to 5mL with water for injection.

An enzyme-linked immunoassay detector: model 3550Microplate Reader, BIO-RAD.

Each monomeric compound was prepared in DMSO at concentrations of 100mM, 30mM, 10mM and 3mM, and stored at low temperature in the dark.

(2) Determination of Activity

RAW264.7 cells (from ATCC in USA) and adjusted the cell concentration to 1.2X 10⁶one/mL, 200. mu.L per well in 96-well plates, 5% CO₂Incubated at 37 ℃ for 2 hours. Lipopolysaccharide (LPS, 10. mu.g/mL) 2. mu.L, interferon-. gamma. (IFN-. gamma., 33ng/mL) 2. mu.L, and a sample DMSO solution 0.4. mu.L (so that the final concentration of LPS is 100ng/mL, the final concentration of IFN-. gamma., 0.33ng/mL, the DMSO content of the dissolved sample relative to the medium is 0.2%) were added and cultured for 16 hours. Then, 100. mu.L of the supernatant was added to 50. mu.L each of the previously prepared Griess reagents, and the absorbance was measured at 550nm using an enzyme-linked immunosorbent assay. And the cell sap without the sample, the inducer lipopolysaccharide and the interferon-gamma are used as blank control, and the cell sap without the sample is used as negative control. Simultaneous MTT assayEach monomeric compound was tested for cytotoxic effects. Macrophage NO release inhibition rate and cell survival rate were calculated as follows:

cell viability ═ OD mean of sample group ÷ OD mean of negative control group × 100%

(3) Results of the experiment

The experimental method is adopted to test the nitric oxide inhibition capability of each extract under the concentration of 100 mu g/mL, and the resveratrol is selected as a positive control drug, so that the resveratrol has better NO release inhibition activity for inhibiting iNOS induction, the NO release inhibition rate is 62.3% at 50 mu g/mL, and the activity is considered to be achieved when the NO release inhibition rate is higher than 50%. The results obtained are shown in Table 5 (in which the sample numbers correspond to the samples represented by the sample numbers in Table 1):

TABLE 5

Sample numbering	Inhibition ratio (%)
		1	68.1
2	65.2
		3	63.3
4	60.3
		5	62.4
6	63.7
		7	61.1
Resveratrol	62.3

The results in table 5 show that the spina gleditsiae counterfeit product also has high nitric oxide inhibitory activity, and provides a thought for developing a novel nitric oxide inhibitor.

The applicant states that the invention is illustrated by the above examples of the use of the saponaria product of the invention in the preparation of an antioxidant, but the invention is not limited to the above detailed process, i.e. it is not meant that the invention must rely on the above detailed process to be practiced. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims

1. The application of the spina gleditsiae counterfeit product in preparing the antioxidant is characterized in that the spina gleditsiae counterfeit product is treated as follows: 1.0g of sample powder is precisely weighed, placed in a conical flask with a plug, 10mL of 70% ethanol solution is added, the ultrasonic treatment is carried out on the plug for 2 times, each time is 30min, the filtration is carried out, the filtrate is combined, placed in an evaporating dish and volatilized in a water bath at 50 ℃ until the volume of the solution is 1-2 mL, the residue is dissolved in a 5mL measuring flask by adding 70% ethanol solution, the solution is diluted to a scale by 70% ethanol solution, the shaking is carried out uniformly, and the solution is filtered by a 0.45-micrometer microporous membrane to obtain a sample solution;

sieving the sample powder through a No. 3 sieve;

the power of the ultrasonic treatment of the sealing plug is 250W, and the frequency is 40 kHz;

the spina gleditsiae counterfeit product is spina gleditsiae;

the DPPH free radical clearance rate of the antioxidant is 60-75%;

the antioxidant has an ORAC value of 1.20 to 1.30.