CN111150754B

CN111150754B - Application of chestnut flower extract in preparation of food or anti-inflammatory drugs

Info

Publication number: CN111150754B
Application number: CN202010112764.5A
Authority: CN
Inventors: 彭飞; 杨越冬; 王同坤; 尹洪洋; 牛奎; 李晓菁; 杜彬; 解莹
Original assignee: Hebei Normal University of Science and Technology
Current assignee: Hebei Normal University of Science and Technology
Priority date: 2020-02-24
Filing date: 2020-02-24
Publication date: 2021-06-22
Anticipated expiration: 2040-02-24
Also published as: CN111150754A

Abstract

The invention relates to the technical field of medicines, in particular to application of a chestnut flower extract in preparing anti-inflammatory medicines or foods, wherein the chestnut flower extract is prepared by at least the following preparation steps: extracting the water extract of flos Castaneae with ethyl acetate. The chestnut flower extract can effectively inhibit macrophages induced by LPS from generating excessive inflammatory mediators NO and three inflammatory cytokines: TNF-alpha, IL-1 beta, IL-6. And also effectively inhibit two key enzymes in the inflammatory signaling pathway of LPS-induced cellular overexpression: nitric oxide induces synthase (iNOS) and cyclooxygenase 2 (COX-2). In addition, the chestnut flower composition can also effectively inhibit mouse ear swelling caused by xylene and mouse foot swelling caused by carrageenan, further proves the anti-inflammatory effect of the chestnut flower composition, and can provide scientific basis for the application of the chestnut flower composition in preparing anti-inflammatory products.

Description

Application of chestnut flower extract in preparation of food or anti-inflammatory drugs

Technical Field

The invention relates to the technical field of medicines, in particular to application of a chestnut flower extract in preparation of anti-inflammatory medicines or foods.

Background

Chinese chestnut (Castanea mollissima Blume), also known as chestnut, chestnut and dried Chinese fan, belongs to the plant of Castanea (Castanea Mill) of Fagaceae, is native to China, is one of the earliest fruit trees cultivated according to history records in China, and has cultivation history of more than two thousand years. The chestnut trees are isogenic, cross pollination is carried out, the number ratio of male flowers to female flowers can reach 1:2349, and researches show that only a small amount of male flowers can meet the pollination requirement, a large amount of male flowers are redundant, the tree nutrition is consumed by white flowers, about 90% of male inflorescences are removed, and the yield of chestnuts can be improved. At the end of the last century, with the further development of Chinese chestnut processing industry, the consumption of Chinese chestnuts is increased year by year, and the planting area of the Chinese chestnuts is also rapidly enlarged. Most of the chestnut staminate flowers are discarded as waste, so that huge waste of resources is caused.

The chestnut flower has sweet and mild taste and is nontoxic. As folk medicine, the Chinese medicinal composition has early application in clinic and has certain treatment effect. According to the records of Ben Cao gang mu, the chestnut flower can be used for treating scrofula, red and white dysentery and other symptoms by eating, or externally used, and pounding and taking juice can be used for treating sore pain. The recorded Chinese chestnut flower in Yunnan herbal medicine can treat red and white dysentery and large intestine bleeding for a long time. The flos Castaneae is decocted with water for oral administration, and can be used for treating intestinal tract diseases such as infantile emesis. The pharmacological active parts of the chestnut flowers reported in the prior literature are mostly alcohol extracts, and the activities mainly comprise four aspects of oxidation resistance, melanin resistance, tumor resistance and bacteria resistance. However, the extraction of the chestnut flowers adopts different solvents and different purification methods, and the chemical components and the active functions of the chestnut flowers are obviously different, so that the medicinal efficacy of the chestnut flowers is not fully developed.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide application of a chestnut flower extract in preparing anti-inflammatory drugs so as to provide a new medicinal effect of chestnut flowers.

The invention is realized by the following steps:

in a first aspect, the embodiments of the present invention provide an application of a chestnut flower extract in the preparation of anti-inflammatory drugs, wherein the chestnut flower extract is prepared by at least the following preparation steps: extracting the water extract of flos Castaneae with ethyl acetate.

The ingredients are extracted by ethyl acetate of the water extract of the specific chestnut flowers, so that the chestnut flowers can effectively and fully have anti-inflammatory properties.

Alternatively, the aqueous extract of chestnut flowers is prepared by the following steps: the chestnut flower is extracted in water at 60-100 ℃, and active ingredients can be fully and effectively extracted at the temperature. Further, in order to achieve better water extraction effect, when the raw material for water extraction is selected, the fresh chestnut flowers are dried and crushed, and then extracted, and optionally, the crushed chestnut flowers are sieved by a 60-mesh sieve.

In order to achieve the purpose of full extraction, intermittent extraction is adopted for three times during water extraction, optionally, each extraction time is 2-4 hours, and the liquid-solid mass ratio of each extraction is 10-20: 1. That is, the time for each extraction may be 2 hours, 2.5 hours, 3 hours, 3.5 hours, or 4 hours; the mass ratio of water to chestnut flower may be 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1 or 20: 1.

Further, when ethyl acetate is used for extraction, the volume ratio of the ethyl acetate to the ethyl acetate is 1-2: 1, extracting the liquid after water extraction by ethyl acetate, concentrating the ethyl acetate extract under reduced pressure after extraction, and concentrating the ethyl acetate extract under reduced pressure until the solvent is evaporated to dryness.

In order to further improve the activity of the chestnut flower aqueous extract, the preparation method further comprises the step of purifying the extract obtained by the extraction of the ethyl acetate by sequentially passing through a polyamide column and a macroporous resin. The polyamide is a high molecular polymer containing a repeating unit amido bond (-CO-NH-) in the structure, O, N atoms on the amido group are combined with protons in an acid cut to carry positive charges, and form anions in an adsorption solution by classical attraction, so that the anions form hydrogen bonds with compounds rich in phenolic hydroxyl groups such as phenols, flavonoids and quinones to be adsorbed, and the compounds which cannot form the hydrogen bonds are separated. And the macroporous resin is further purified and decontaminated on the basis of the adsorption of the polyamide, so that the activity of the extract is improved.

Specifically, the step of enabling the extract obtained by ethyl acetate extraction to pass through a polyamide column comprises the steps of dissolving the extract in 1-3 times of water by volume, eluting the extract on the polyamide column by using water until the extract is colorless, and eluting the extract by using ethanol, wherein the mass fraction of the optional ethanol solution is 30-60%, and the using amount of the ethanol solution is 6-10 column volumes;

optionally, the purification by macroporous resin comprises hydrolyzing the concentrate purified by the polyamide column, loading the hydrolyzed concentrate into macroporous resin, washing the hydrolyzed concentrate to be colorless, eluting the washed concentrate by using an ethanol solution, and then drying the eluted concentrate, wherein the ethanol solution preferably accounts for 30-60% by mass and is used in an amount of 6-10 column volumes.

Optionally, the preparation method of the chestnut flower extract specifically comprises the following steps: drying and crushing fresh chestnut flowers, sieving with a 60-mesh sieve, using water as a solvent, taking 10-20 times of water for extraction at the extraction temperature of 60-100 ℃ for 2-4 hours, intermittently extracting for three times, filtering and collecting filtrate, extracting with ethyl acetate, concentrating the ethyl acetate extract under reduced pressure until the solvent is completely evaporated to obtain an extract A, dissolving the extract A with 1-3 times of water to a polyamide column, eluting with water to be colorless, then eluting with 30-60% of ethanol in the volume of 6-10 columns, combining the filtrates, concentrating under reduced pressure to obtain an extract B, dissolving the extract B with water to be macroporous resin, eluting with 30-60% of ethanol after washing to be colorless, concentrating and removing ethanol, and drying to obtain a chestnut flower extract C.

Optionally, the anti-inflammatory agent is an anti-inflammatory agent produced by macrophages.

In a second aspect, the present embodiments provide the use of a chestnut flower extract in the manufacture of an inhibitor for inhibiting the production of proinflammatory cytokines by macrophages, said proinflammatory cytokines comprising at least one of TNF- α, IL-1 β and IL-6, preferably said proinflammatory cytokines comprising TNF- α, IL-1 β and IL-6.

In a third aspect, the embodiments of the present invention provide the use of an extract of chestnut flowers for the preparation of an inhibitor for the production of NO, an inflammatory mediator by macrophages.

In a fourth aspect, the embodiments of the present invention provide the use of a chestnut flower extract in the preparation of an inhibitor for inhibiting the expression of nitric oxide-induced synthase.

In a fifth aspect, the embodiments of the present invention provide the use of a chestnut flower extract in the preparation of an inhibitor for inhibiting the expression of cyclooxygenase 2.

In a sixth aspect, the embodiments of the present invention provide the use of a chestnut flower extract in the preparation of a medicament for the treatment of inflammatory ear swelling.

In a seventh aspect, the embodiments of the present invention provide an application of a chestnut flower extract in preparing a medicament for treating inflammatory foot swelling.

The invention has the following beneficial effects:

the chestnut flower extract provided by the invention can effectively inhibit macrophages induced by LPS from generating excessive inflammatory mediators NO and three inflammatory cytokines: TNF-alpha, IL-1 beta, IL-6. And also effectively inhibit two key enzymes in the inflammatory signaling pathway of LPS-induced cellular overexpression: nitric oxide induces synthase (iNOS) and cyclooxygenase 2 (COX-2). In addition, the chestnut flower composition can also effectively inhibit mouse ear swelling caused by xylene and mouse foot swelling caused by carrageenan, further proves the anti-inflammatory effect of the chestnut flower composition, and can be used as a raw material or an auxiliary agent for preparing anti-inflammatory drugs or foods. Furthermore, the chestnut flower composition has the advantages of rich raw material sources, simple and convenient process method operation, low cost, easy industrialization, no toxic or side effect and the like.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the MTT assay to determine the effect of compounds on cell proliferation;

FIG. 2 is a graph showing the effect of chestnut flower extract on the expression level of iNOS gene;

FIG. 3 shows the effect of chestnut flower extract on COX-2 gene expression level.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The following is a specific description of the use of the chestnut flower extract according to the embodiments of the present invention in the preparation of anti-inflammatory drugs.

The preparation method of the chestnut flower extract of the embodiment of the invention comprises the following steps:

drying and crushing fresh chestnut flowers, sieving with a 60-mesh sieve, using water as a solvent, extracting with 15 times of water as much as the raw materials at 80 ℃ for 3 hours, extracting for three times intermittently, filtering and collecting filtrate, extracting with ethyl acetate, concentrating the ethyl acetate extract under reduced pressure until the solvent is completely evaporated to obtain an extract A, dissolving the extract A with 2 times of volume of water to a polyamide column, eluting with water to be colorless, then eluting with 8 column volumes of 50% ethanol, combining the filtrates, concentrating under reduced pressure to obtain an extract B, dissolving the B with water to be macroporous resin, eluting with 50% ethanol after being colorless, concentrating and removing ethanol, and drying to obtain a chestnut flower extract C.

Example 1

(1) Preparation and culture of mouse abdominal cavity macrophage RAW264.7

C57BL/6 mice, male, weighing 20 g, were intraperitoneally injected with 4% sodium thioglycolate solution, and after 4 days, were sacrificed by cutting the ends, and the skin was peeled off by scissors along the mouse axilla. The needle was inserted into the abdominal cavity from the second intercostal space with a 10mL syringe (carefully avoiding poking the liver and spleen), the cells were washed twice with 8 mL of ice-cold PBS buffer, the cell eluate was collected, centrifuged at 1000r/min for 5 minutes, washed once with ice-cold RPMI1640 medium, the cells were counted, and the cells were adjusted to 1.2X 10 with a medium 1640 containing 10% FCS⁶cells·mL^-1。

(2) Griess method for detecting influence of compound on generation of mouse abdominal cavity macrophage NO

The cells grown logarithmically were plated on 48-well ELISA plates at 37 ℃ in a volume of 500. mu.L per well with CO₂The cells were allowed to stand in the incubator for 2.5 hours to adhere to the walls. After draining the supernatant, washing the plate for 2 times by using a 1640 culture medium, adding 490 mu L of the 1640 culture medium again, adding 5 mu L of bacterial lipopolysaccharide LPS, respectively adding 5 mu L of the extract A, the extract B and the chestnut flower extract C with different concentrations (1 mu g/mL, 5 mu g/mL, 25 mu g/mL, 125 mu g/mL and 625 mu g/mL), and simultaneously arranging a blank control group (without LPS and a sample to be detected) and a model group (without LPS and without LPS)Plus the sample to be tested), CO₂Incubation was continued for 24 hours in the incubator. The supernatant was taken out and mixed with an equal amount of Griess reagent (1% sulfanilamide solution containing 2.5% phosphoric acid and 0.1% naphthylethylenediamine solution mixed at 1:1 temporarily) on a micro-shaker, and then left to stand at room temperature for 10 minutes. Reading by 550nm filter microplate reader according to NO^2-The standard curve calculates the NO content. Results were expressed as half inhibitory concentration IC₅₀The effect on NO production is shown and the results are shown in Table 1.

TABLE 1 Effect of different extracts on NO production

NO inhibition	Extract A	Extract B	Chestnut flower extract C
				IC₅₀(μg/mL)	464.3	197.1	65.4

The results in table 1 show that the chestnut flower extract C of the present invention has a significantly significant inhibitory effect on the generation of NO in the LPS-induced mouse macrophage RAW264.7 inflammation model, compared to the crude extracts a and B.

(3) MTT method for detecting toxicity of compound to cells

MTT method for detecting the influence of compound on the survival of mouse abdominal cavity macrophage RAW 264.7: each well was filled with 100. mu.L and 5. mu.L of cell suspension at different concentrations (1. mu.g/mL, 5. mu.g/mL, 25. mu.g/mL, 125. mu.g/mL,625 μ g/mL) of chestnut flower extract C, using cell-free wells as full blank control, setting four multiple wells at 37 deg.C and 5% CO₂After culturing in the cell culture chamber of (3) for 24 hours, 5 hours of MTT (mg.mL) was added thereto^-1) 20 mu L of the suspension was cultured for 4 hours, 10% SDS solution was added to each well, the mixture was shaken and mixed, the mixture was left in a cell incubator for 12 hours, and the O.D. value at 570nm was measured. The effect of MTT method for detecting compound on cell proliferation is shown in figure 1, from which it can be seen that the concentration of chestnut flower extract C is in the range of 1-625 μ g/mL, and has no toxic effect on the growth of mouse macrophage RAW264.7 (figure 1).

Example 2

ELLISA method for detecting contents of proinflammatory cytokines TNF-alpha, IL-1 beta and IL-6

The cells grown logarithmically were plated on 48-well microplate, 500. mu.l of each well, 37 ℃ C., CO₂The incubator is kept still for 24 hours. After draining the supernatant, the plate was washed 2 times with 1640 medium, and then 490. mu.l of 1640 medium was added again for culture. The experiment was divided into blank, LPS and experimental groups. The experimental group was prepared by adding 5 μ l of chestnut flower extract C (1, 5, 25, 125, 625 μ g/mL) and LPS (1 μ g/mL) at different concentrations to each well, adding culture medium to LPS group (without adding the sample to be tested), adding blank control group to each well, setting 3 replicate wells for each sample, and adding CO₂Incubation was continued for 16 hours in the incubator. The supernatant was removed and the procedure was performed exactly as described for the ELISA kit.

The inhibition ratio was calculated by the following formula, and the results are shown in table 2.

Inhibition rate ═ 100% of (LPS group-experimental group)/(LPS group-blank group)%

TABLE 2 influence of chestnut flower extract C on the production of inflammatory factors

The results in the table 2 show that the chestnut flower extract C has obvious improvement effect on an LPS-induced mouse macrophage RAW264.7 inflammation model, effectively inhibits the release of inflammatory factors TNF-alpha, IL-1 beta and IL-6, and shows obvious in-vitro anti-inflammatory effect.

Example 3

Detection of COX-2 and iNOS mRNA expression level by chestnut flower extract

(1) Extraction of Total RNA from RAW264.7 macrophages

Taking log-phase RAW264.7 cells, and performing cell selection at 2 × 10⁵Individual cells/well were seeded in 6-well plates, incubated for 24h, and the supernatant decanted. Three groups are simultaneously arranged: blank group, LPS group, experimental group (LPS added with different concentrations of flos Castaneae extract C5, 25, 125 μ M), and placing at 37 deg.C and CO₂Incubate for 12h, aspirate supernatant and wash 2 times with PBS. Cells were lysed using Trizol reagent (see Trizol extraction instructions) and RNA concentration was measured using a microplate method. And RNA reverse transcription was performed using a reverse transcription kit (RT-PCR) (performed according to Prime Script TMRT Master Mix kit). The fluorescent quantitative PCR reaction was carried out using the Step One plus Real-Time PCR System kit, and the reaction systems are shown in the following tables 3 and 4:

TABLE 3 RT-PCR reaction solution preparation

Composition (I)	Volume (μ L)
		SYBRGreen	10
Fprimer	0.8
		Rprimer	0.8
Rox Reference Dye	0.4
		dH₂O	6
cDNA	2

TABLE 4 fluorescent quantitative PCR primer sequence Listing

NO is an important inflammation medium, is mainly induced and produced by nitric oxide-induced synthase (iNOS), and the generation and the development of inflammation can be effectively controlled by inhibiting the expression of the NO. In order to clarify the anti-inflammatory effect of the chestnut flower extract, the expression of iNOS was examined. The results are shown in FIG. 2: the iNOS expression level of the negative control is set as 1, the iNOS expression level of the cells can be obviously increased by LPS co-culture, the addition of the chestnut flower extract C with different concentrations can obviously inhibit the iNOS up-regulation induced by LPS, and the inhibition rate and the concentration are in direct proportion.

Prostaglandin PGE2 is an inflammatory mediator that is induced by LPS and affects the inflammatory response, while cyclooxygenase 2(COX-2) catalyzes the synthesis of PGE 2. Therefore, inhibition of COX-2 expression is effective in inhibiting the development of inflammation. As can be seen from FIG. 3, while the expression level of COX-2 in normal cells not induced by LPS was 1, the expression level of COX-2 in LPS-induced cells was increased to about 27 times, and the different concentrations of the chestnut flower extract C could inhibit the expression of COX-2 induced by LPS, and the higher the concentration, the better the inhibition effect.

The RT-PCR detection shows that the relative expression quantity of the m RNA of inflammatory genes iNOS and COX-2 can be obviously increased after LPS treatment, and compared with a blank group, the difference has statistical significance (P is less than 0.05); the chestnut flower extract C can reduce the relative expression of i NOS and COX-2m RNA after being treated, and has statistical significance (P <0.05) compared with a model group. The chestnut flower composition shown in fig. 2 and 3 is the chestnut flower extract C.

Example 4

Influence of chestnut flower extract on mouse ear swelling caused by xylene

Mice with the weight of 20-22g are randomly divided into 5 groups, a model group control group, a positive control group and a chestnut flower extract high, medium and low measurement group. Respectively carrying out subcutaneous injection to give medicine (chestnut flower extract C) (100, 200 and 400mg/mL) with set dose, giving equal volume of medicine solvent to control group, smearing dexamethasone ointment on positive control group, after 1h administration, coating xylene 0.1mL on both sides of right ear of mouse to cause inflammation, and taking left ear as normal control. After 4h of inflammation, the mice were sacrificed, ears were cut off along the base line of auricle, the ears at the same positions of the left and right ears were removed with a punch having a diameter of 6mm, respectively, weighed, the difference in mass between the left and right ears indicated the degree of swelling of inflammatory tumor, and the percent inhibition of swelling (%) was calculated as (degree of swelling of control group-degree of swelling of administration group)/degree of swelling of control group ═ 100.

TABLE 5 evaluation results of the ear swelling of mice caused by p-xylene extracted from chestnut flowers

The results in table 5 show that the chestnut flower extract administration group has obvious inhibition effect on mouse ear swelling caused by dimethylbenzene, the inhibition rates of swelling of three different dose groups are in obvious dose-dependent relation, and the inhibition effect is increased along with the increase of the dose. Compared with a blank control group, the positive control group can obviously inhibit the ear swelling of the mice, and has obvious statistical difference (P is less than 0.01), and the low-dose group and the control group have no statistical difference (P is more than 0.05) on the swelling.

Example 5

Effect of chestnut flower extract on carrageenan-induced swelling of mouse feet

Mice with the weight of 20-22g are randomly divided into 5 groups, a model group control group, a positive control group and a chestnut flower extract C high, medium and low measurement group. The normal foot volume of each mouse was determined by a toe swelling tester using a pen-drawn line as a measurement marker at the prominent point of the right ankle joint of each mouse. The medicine (chestnut flower extract C) (100, 200 and 400mg/mL) is respectively injected and given to groups in a subcutaneous way, the control group is given with the same volume of normal saline, the positive control group is dexamethasone injection, 1 percent of carrageenan is injected under the tendon membrane of the right hind foot of the mouse after 45min of administration to cause inflammation, the volume of the right hind foot is measured after 6h, and data are recorded and the swelling inhibition rate is calculated.

The swelling inhibition ratio (%) was (degree of swelling in control group-degree of swelling in administered group)/degree of swelling in control group × 100%.

TABLE 6 evaluation of Castanea mollissima flower extract on carrageenan-induced swelling of mouse feet

As can be seen from Table 6, the low, medium and high doses of the chestnut flower extract can reduce the volume of the feet of the mice, and compared with the blank control group, the positive control group and the high doses of the chestnut flower extract can obviously inhibit the swelling of the feet of the mice, and have obvious statistical difference (P is less than 0.01), and the low dose group and the control group have no statistical difference (P is more than 0.05) in swelling.

Therefore, the chestnut flower extract can reduce ear swelling induced by xylene and foot swelling inflammatory reaction of mice caused by carrageenan, further proves the in-vivo anti-inflammatory effect of the chestnut flower extract, and can provide a new target for irritant or contact dermatitis.

In conclusion, the chestnut flower extract provided by the invention has no influence on the proliferation of RAW264.7, and is safe and nontoxic; can effectively inhibit mouse macrophage RAW264.7 induced by LPS from generating excessive inflammatory mediator NO and three inflammatory cytokines: TNF-alpha, IL-1 beta, IL-6, and their effects present a dose dependence. In addition, two key enzymes in the target inflammatory signaling pathway of LPS-induced RAW264.7 cell excess can be effectively inhibited: nitric oxide induces synthase (iNOS) and cyclooxygenase 2 (COX-2). The chestnut flower extract can also effectively inhibit the ear swelling of mice caused by xylene and the foot swelling of mice caused by carrageenan. Further proves that the anti-inflammatory effect of the composition can provide a new target for irritant or contact dermatitis.

Furthermore, the chestnut flower extract provided by the invention has the advantages of rich raw material sources, simple and convenient process method operation, low cost, easy industrialization, NO toxic or side effect and the like, and simultaneously has good in-vivo and in-vitro anti-inflammatory activity, and can be used as an anti-inflammatory drug or raw materials or auxiliary materials thereof for preventing or treating inflammation or irritation or contact dermatitis caused by excessive NO, TNF-alpha, IL-1 beta and IL-6.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The application of the chestnut flower extract in preparing food or anti-inflammatory drugs is characterized in that the chestnut flower extract is prepared by at least the following preparation steps: extracting the water extract of the chestnut flowers by using ethyl acetate;

the preparation step of the chestnut flower extract also comprises the steps of sequentially purifying the extract obtained by ethyl acetate extraction through a polyamide column and macroporous resin;

wherein the step of enabling the extract obtained by ethyl acetate extraction to pass through a polyamide column comprises the steps of dissolving the extract in 1-3 times of water by volume, eluting the extract on the polyamide column by using water until the extract is colorless, and then eluting the extract by using an ethanol solution, wherein the mass fraction of the ethanol solution used in the elution process of the polyamide column is 30-60%, and the using amount of the ethanol solution is 6-10 column volumes;

the macroporous resin purification comprises the steps of hydrolyzing a concentrate purified by a polyamide column, loading the hydrolyzed concentrate into macroporous resin, washing the macroporous resin until the concentrate is colorless, eluting the concentrate with an ethanol solution, and drying the eluted concentrate, wherein the mass fraction of the ethanol solution used in the macroporous resin elution process is 30-60%, and the dosage of the ethanol solution is 6-10 column volumes.

2. Use according to claim 1, characterized in that said aqueous extract of chestnut flowers is prepared by the following steps: extracting the chestnut flowers in water at the temperature of 60-100 ℃.

3. Use according to claim 2, wherein the fresh chestnut flowers are dried and comminuted before extraction.

4. The use of claim 3, wherein the extract is extracted after grinding and sieving with a 60 mesh sieve.

5. Use according to claim 2, wherein the chestnut flowers are extracted in water by: intermittently extracting for three times, wherein each extraction time is 2-4 hours, and the liquid-solid mass ratio of each extraction is 10-20: 1.

6. The use according to claim 2, wherein the ratio by volume is 1-2: 1, extracting the liquid after water extraction by ethyl acetate, and concentrating the ethyl acetate extract under reduced pressure after extraction.

7. The use according to claim 1, wherein the anti-inflammatory agent is an anti-macrophage-derived inflammatory agent.

8. The use according to claim 7, wherein the anti-inflammatory agent is an oral preparation or an external preparation containing a chestnut flower extract as a main active ingredient.

9. Use according to claim 7, characterized in that in the anti-inflammatory agent the chestnut flower extract is used as a raw material or as an adjuvant.

10. Use of a chestnut flower extract for the manufacture of an inhibitor for inhibiting the production of pro-inflammatory cytokines by macrophages according to any one of claims 1 to 9, wherein the pro-inflammatory cytokines comprise at least one of TNF- α, IL-1 β and IL-6.

11. The use according to claim 10, wherein the proinflammatory cytokines comprise TNF-a, IL-1 β and IL-6.

12. Use of an extract of chestnut flowers according to any one of claims 1 to 9 for the preparation of an inhibitor for the production of NO, an inflammatory mediator, by macrophages.

13. Use of a chestnut flower extract for the use according to any one of claims 1 to 9 in the preparation of an inhibitor for inhibiting the expression of nitric oxide-induced synthase.

14. Use of a chestnut flower extract for the use according to any one of claims 1 to 9 in the preparation of an inhibitor for inhibiting the expression of cyclooxygenase 2.

15. Use of a chestnut flower extract according to any one of claims 1 to 9 in the manufacture of a medicament for the treatment of inflammatory ear swelling.

16. Use of a chestnut flower extract according to any one of claims 1 to 9 in the manufacture of a medicament for the treatment of inflammatory foot swelling.