TWI842129B - A chrysanthemum morifolium ramat extract and a use of the extract for the manufacture of a pharmaceutical composition for inhibiting inflammation and promoting wound healing - Google Patents

Abstract

A Chrysanthemum morifolium Ramat extract and a use of the extract for the manufacture of a pharmaceutical composition for inhibiting inflammation and promoting wound healing is provided, wherein the Chrysanthemum morifolium Ramat extract was obtained by extracting Chrysanthemum morifolium Ramat sample by subcritical water extraction under a high temperature and a high pressure condition, wherein the said “wound healing” including chronic wound healing and wound healing in diabetes.

Description

A chrysanthemum extract and its use in preparing a pharmaceutical composition for inhibiting inflammatory response and promoting wound healing

The present invention relates to a chrysanthemum ( Chrysanthemum morifolium Ramat) extract and its use in preparing a pharmaceutical composition for inhibiting inflammatory response and promoting wound healing, in particular to a chrysanthemum extract obtained by extracting a chrysanthemum sample at a high temperature and a high pressure using a subcritical water extraction method and its use in preparing a pharmaceutical composition for inhibiting inflammatory response and promoting wound healing.

Inflammation is the healthy host's response to damage or infection caused by toxic chemicals, dead cells, pathogens, irritants or allergens. The important role played by neutrophils in various infectious and inflammatory diseases makes them an attractive target for therapeutic intervention.

When activated, neutrophils produce many inflammatory responses, including respiratory burst to produce superoxide anions (O ₂ ^- ) and reactive oxygen species (ROS), degranulation to release elastase, trending to inflamed areas, releasing cytokines, and forming neutrophil extracellular traps (NETs). Therefore, inhibiting the inflammatory response of neutrophils is an effective strategy for treating neutrophil inflammatory diseases.

Wound healing is the process of epidermal tissue regeneration after skin trauma, which generally includes four stages: hemostasis, inflammation, proliferation, and tissue remodeling or regression (Gosain and DiPietro, 2004). However, there are many factors that affect wound healing, such as wound care and wound sugar. Diabetic wound healing disorders are related to multiple factors such as blood vessels, neuropathies, immunity, and biochemical components. High blood sugar can cause vascular hardening, which in turn leads to slow circulation and microvascular dysfunction, and reduces tissue oxygenation; high blood sugar can also reduce the migration of white blood cells to the wound, making patients more susceptible to infection. Peripheral neuropathy in diabetes mellitus (DM) may lead to numbness in the area and a reduced ability to feel pain, which may result in the wound not being noticed immediately and treated appropriately (Spampinato SF, Caruso GI, De Pasquale R, Sortino MA, Merlo S. The Treatment of Impaired Wound Healing in Diabetes: Looking among Old Drugs. Pharmaceuticals (Basel). 2020; 13(4): 60.). Of particular note, diabetic foot ulcer (DFU) is a major complication of DM, occurring in 15% of diabetic patients. Risk factors associated with DFU are believed to be neuropathy, vascular disease, and infection. Past studies have also found that the risk of lower limb amputation in diabetic patients is 15 to 46 times that of non-diabetic patients.

Natural compounds have the advantages of rich sources and diverse skeletons, and are an important basis for drug development. From 1981 to 2019, nearly half of the newly approved drugs by the U.S. Food and Drug Administration came from natural products or their derivatives, such as anesthetics derived from cocaine, analgesics derived from morphine, vincristine, doxorubicin and paclitaxel used to treat cancer, and penicillin derived from fungi used as an antibiotic, etc. Therefore, the present invention actively studies which natural compounds have the potential to develop into new drugs that inhibit inflammatory responses caused by neutrophils and promote wound healing.

The present invention selects chrysanthemum ( Chrysanthemum morifolium Ramat) as a research subject.

The invention relates to a chrysanthemum ( Chrysanthemum morifolium Ramat) extract. The chrysanthemum extract is prepared by extracting a chrysanthemum sample at a high temperature and a high pressure using a subcritical water extraction method to obtain a subcritical water extract.

In the present invention, the chrysanthemum sample is fresh or dried chrysanthemum.

In the present invention, preferably, the high temperature is between 120 and 250°C.

In the present invention, preferably, the high pressure is between 5 and 20 MPa.

In the present invention, preferably, the subcritical water extraction method is performed for 5 to 60 minutes.

Preferably, the present invention further comprises step c: dissolving the subcritical water extract in pure water and placing it in a column, and using pure water and different proportions of alcohol aqueous solutions as mobile phases for separation; and step d: collecting the alcohol aqueous solution with the largest yield and freeze-drying it to obtain the chrysanthemum extract of the present invention.

In the present invention, preferably, the alcohol aqueous solution of different proportions in step c is selected from the group consisting of 20% alcohol aqueous solution, 40% alcohol aqueous solution, 60% alcohol aqueous solution and 95% alcohol aqueous solution.

In the present invention, preferably, the stratification in step c is performed sequentially using pure water, 20% alcohol aqueous solution, 40% alcohol aqueous solution, 60% alcohol aqueous solution and 95% alcohol aqueous solution as the mobile phase.

The present invention also relates to the use of a chrysanthemum extract for preparing a pharmaceutical composition for inhibiting an inflammatory response caused by neutrophils, which comprises an effective dose of a chrysanthemum extract and a medically acceptable carrier thereof.

Preferably, the present invention is also a use of a chrysanthemum extract for preparing a pharmaceutical composition for removing free radicals in the body, which comprises an effective dose of a chrysanthemum extract and a medically acceptable carrier thereof.

Preferably, the present invention is also a use of a chrysanthemum extract for preparing a pharmaceutical composition for promoting wound healing in an individual, which comprises an effective dose of a chrysanthemum extract and a medically acceptable carrier thereof.

Preferably, the wound healing includes chronic wound healing.

More preferably, the wound healing includes diabetic wound healing or diabetic foot.

In each use of the present invention, the effective dose is 0.1 mg/kg body weight to 50 mg/kg body weight.

In the present invention, the individual referred to in the present invention is a human or a mammal.

Figure 1 is a schematic diagram of different extraction methods for chrysanthemum sample BQ01.

Figures 2A to 2B show the free radical scavenging effects of crude chrysanthemum extracts under Soxhlet extraction, water extraction, alcohol extraction, ethyl acetate extraction and subcritical water extraction conditions. Figure 2A shows that crude chrysanthemum extracts under 200°C subcritical water extraction conditions have better DPPH and ABTS scavenging rates; Figure 2B shows that the free radical scavenging rate of crude chrysanthemum extracts under 200°C subcritical water extraction conditions is dose-dependent.

Figure 3 shows the UPLC component analysis of crude extracts extracted by BQ01 using different extraction methods.

Figure 4 shows the scavenging rates of DPPH and ABTS in the extracts of each layer of BQ01-(200)_30.

Figure 5 shows that the extracts from each layer of BQ01-(200)_30 have no cytotoxicity to neutrophils.

Figure 6 shows that BQ01-(200)_30 and BQ01-(200)_30-60%EtOH effectively stimulate the cell migration of the human normal fibroblast cell line Hs68.

Figures 7A to 7E show the effects of BQ01-(200)_30 and BQ01-(200)_30-60%EtOH on diabetic wound healing. Figure 7A shows the wound healing time course and photos of the diabetic wound healing animal model; Figures 7B and 7C show the diabetic wound healing results after low and high doses of BQ01-(200)_30 treatment; Figures 7D and 7E show the diabetic wound healing results after low and high doses of BQ01-(200)_30-60%EtOH treatment. * indicates P < 0.05 and ** indicates P < 0.01 , which are significantly different from the untreated group; and @ indicates P < 0.05 and @@ indicates P < 0.01 , which are significantly different from the control group (Pluronic acid group).

Figure 8 shows that during the diabetic wound healing experiment of the present invention, all diabetic animals were in a diabetic state.

It should be understood that the detailed description of the embodiments is to illustrate the preferred embodiments of the present invention, rather than to limit the present invention to certain embodiments. It should be noted that the present invention is intended to cover all alternative embodiments within the same spirit and scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

Preparation of Chrysanthemum Extract

The present invention uses fresh or dried chrysanthemum ( Chrysanthemum morifolium Ramat) as an extraction sample, especially the flower as an extraction sample, and grinds the chrysanthemum sample into powder, which is named BQ01.

In the present invention, the chrysanthemum sample BQ01 is extracted by Soxhlet extraction, water extraction, alcohol extraction, ethyl acetate (EA) extraction and subcritical water extraction, and the schematic diagram of the process is shown in Figure 1.

Among them, the Soxhlet extraction method is to mix 5g of BQ01 with 70mL of water, perform Soxhlet extraction at 100℃ for 60 minutes, and then filter the suspension with filter paper to obtain the crude extract BQ01-S.

Water extraction, alcohol extraction and ethyl acetate extraction were performed on a Dionex ASE 350 system (ThermoFisher Scientific, US). 5 g of BQ01 was placed in a 66 mL stainless steel extraction tank with filter paper at the bottom. The stainless steel extraction tank was placed in an oven and filled with solvents for 30 seconds. The solvents were water, 50% ethanol, 95% ethanol and ethyl acetate, respectively. The tank was then heated under high pressure (about 1500 psi) to the required extraction time (water extraction conditions were 100°C for 15 minutes; ethanol extraction and ethyl acetate extraction conditions were 50°C for 1 minute). 5 minutes), the extraction is completed, the extract is poured into the collection bottle from the extraction tank, and the residue is blown under a pressure of about 10MPa for 1 minute (usually using nitrogen), and then the residual pressure is released, and the final extract is collected in the collection bottle. The organic solvent part is removed by solvent in a rotary vacuum evaporator at 35°C, and then placed in a freeze dryer for drying. The water extract part is frozen and then dried in a freeze dryer to obtain crude extracts BQ01- _H2O , BQ01-50%E, BQ01-95%E and BQ01-EA, respectively.

Subcritical water extraction was performed on a Dionex ASE 350 system (ThermoFisher Scientific, US). 5 g of BQ01 was placed in a 66 mL stainless steel extraction cell with filter paper at the bottom and extracted with pure water. The stainless steel extraction cell was placed in an oven, water was added to the cell for 30 seconds, and then 25 different extraction conditions were performed under high pressure (about 1500 psi), which combined 5 different extraction temperatures (120°C, 140°C, 160°C, 180°C and 200°C) and 5 different extraction times (5, 10, 1 5, 30 and 60 minutes), after which the residue was purged at a pressure of about 10 MPa for 1 minute (usually using nitrogen), and then the residual pressure was released, and the final extract was collected in a collection bottle. The extract was frozen and dried in a freeze dryer for 24 hours, and finally 25 crude extracts were obtained: BQ01-(120)_5, BQ01-(120)_10, BQ01-(120)_15, BQ01-(120)_2 _30, BQ01-(120)_60, BQ01-(140)_5, BQ01-(140)_10, BQ01-(140)_15, BQ01-(140)_30, BQ01-(140)_60, BQ01-(160)_5, BQ01-(160)_10, BQ01-(160)_15, BQ01-(160)_30, BQ01- (160)_60, BQ01-(180)_5, BQ01-(180)_10, BQ01-(180)_15, BQ01-(180)_30, BQ01-(180)_60, BQ01-(200)_5, BQ01-(200)_10, BQ01-(200)_15, BQ01-(200)_30 and BQ01-(200)_60.

Free radical scavenging ability test

1,1-Diphenyl-2-trinitrophenylhydrazyl (DPPH) and 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) are stable free radicals associated with inflammation. In the present invention, the ability of 10 μg/mL of each chrysanthemum crude extract to scavenge free radicals was further tested, wherein DMSO was used as a control group. When each chrysanthemum crude extract had free radical scavenging ability, after mixing with ethanol-prepared DPPH (100 μM) or ABTS (7 mM) reaction solution for 30 minutes, its ΔA ₅₁₇ or ΔA ₇₃₄ would be significantly decreased, which was used as an activity indicator for scavenging free radicals.

As shown in Figure 2A, the results show that the crude extracts of chrysanthemum under subcritical water extraction conditions at 200°C have better scavenging rates of DPPH and ABTS.

In addition, as shown in Figure 2B, the free radical scavenging rate of the chrysanthemum crude extract under subcritical water extraction conditions at 200°C is also dose-dependent and can achieve a free radical scavenging ability equivalent to that of vitamin E (Vit E).

Superoxide anion and elastin inhibition test

The present invention evaluates the anti-inflammatory ability of the above-mentioned various chrysanthemum crude extracts by superoxide anion and elastic enzyme inhibition test.

First, neutrophils are prepared.

Take healthy blood donors (20-35 years old; normal work and rest and no medication for more than one week) who are willing to assist or are interested in this project. Use a vacuum sterile blood collection tube to collect about 50mL of blood from the elbow vein, add it to an equal volume of 3% dextran solution (Dextran) and mix it evenly. Let it stand to precipitate red blood cells. Take the supernatant containing neutrophils, cover it in a centrifuge tube containing Ficoll-Paque solution and centrifuge it. Use density gradient to separate different blood cells, and obtain neutrophils and a few red blood cells that precipitate at the bottom. Use NaCl solutions of different concentrations to expand and rupture the remaining red blood cells, and use the difference in osmotic pressure tolerance between the two to retain the required neutrophils.

The evaluation of the anti-inflammatory response of the various chrysanthemum crude extracts mentioned above in the present invention is carried out by respiratory burst test and degranulation function evaluation.

Respiratory burst mainly produces superoxide anions (O ₂ ^．－ ) and reactive oxygen species ( ROS ). The detection of superoxide anions (O ₂ ^．－ ) was performed by adding the above-mentioned chrysanthemum crude extracts to a neutrophil suspension (6×10 ⁵ cells/mL) containing 0.6 mg/mL ferricytochrome c at 37°C for 2 minutes, and then treating the cells with 1 μg/mL cytochalasin B (CB) for about 3 minutes. Then, N -formyl-methionyl-leucyl-phenylalanine (fMLF) was used as a stimulant to activate the cells for about 10-30 minutes . Then use an ultraviolet spectrophotometer to measure its absorbance at a wavelength of 550nm.

In the elastase release experiment, a neutrophil suspension (6×10 5 cells/mL) containing 100 μM elastase substrate MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide was preheated to 37°C for 5 minutes, and then the above-mentioned chrysanthemum crude extracts were added for 2 minutes. The cells were treated with 0.5 μg/mL cytochalasin ^B (CB) for about 3 minutes, and then 0.1 μM fMLF was used to stimulate neutrophil activation for about 10-30 minutes. Finally, the absorbance was measured at a wavelength of 405 nm using a UV spectrophotometer. The experimental results are expressed as the percentage of elastin release.

The results are shown in Table 1.

Table 1

IC₅₀：半數最大抑制濃度；統計結果以平均值±標準誤差顯示；統計結果是否具有顯著差異係以各菊花粗萃取物的實驗結果與對照組(DMSO)的實驗結果進行比較，*P<0.05,**P<0.01,***P<0.005。

IC ₅₀ : half maximal inhibitory concentration; statistical results are presented as mean ± standard error; statistical results were compared with the experimental results of each chrysanthemum crude extract and the control group (DMSO) to determine whether they were significantly different, * P < 0.05 , ** P < 0.01 , *** P < 0.005 .

From the results of Figure 2A to Figure 2B and Table 1, it can be found that BQ01-(200)_30 has the best performance.

BQ01(200)30 concentrated distillate preparation

The present invention further uses UPLC to analyze the components of crude extracts of BQ01-(200)_30 and chrysanthemum obtained by other extraction methods. The results are shown in Figure 3. It can be seen that BQ01-(200)_30 seems to contain more components worth exploring compared with other crude extracts. Therefore, the present invention prepares a concentrated distillate of BQ01-(200)_30.

A total of 11.4498 g of BQ01-(200)_30 was used as a column filled with a porous styrene adsorption resin (Diaion HP-20, Mitsubishi Chemical Co., Ltd., Japan) as the stationary phase. The column was 42 cm high and 3.5 cm in diameter. BQ01-(200)_30 was dissolved in pure water and placed in a column. Pure water, 20% alcohol, 40% alcohol, 60% alcohol, and 95% alcohol were used as mobile phases for washing and layering. The volume of each mobile phase was 2000 mL. After each layer was recovered, it was concentrated at 38°C using a reduced pressure concentrator (Buchi Rotary Evaporator: Interface I-300, Vacuum pump V-300, Rotavapor R-210, Heating Bath B-491) and then dried using a freeze dryer.

After drying, 5.3721 g (46.9%) of H ₂ O layer extract, 1.6554 g (14.5%) of 20% alcohol layer extract, 1.0693 g (9.3%) of 40% alcohol layer extract, 2.0332 g (17.8%) of 60% alcohol layer extract and 0.6617 g (5.8%) of 95% alcohol layer extract were obtained, with a total recovery rate of 94.3%. They were named BQ01-(200)_30-H ₂ O, BQ01-(200)_30-20%EtOH, BQ01-(200)_30-40%EtOH, BQ01-(200)_30-60%EtOH and BQ01-(200)_30-95%EtOH.

Anti-inflammatory and cytotoxicity tests of extracts from each layer

The superoxide anion and elastin inhibition tests of the extracts from each layer were the same as the above steps, and the results are shown in Table 2.

IC₅₀：半數最大抑制濃度；統計結果以平均值±標準誤差顯示；統計結果是否具有顯著差異係以各菊花粗萃取物的實驗結果與對照組(DMSO)的實驗結果進行比較，*P<0.05,**P<0.01,***P<0.005。 Table 2

IC ₅₀ : half maximal inhibitory concentration; statistical results are presented as mean ± standard error; statistical results were compared with the experimental results of each chrysanthemum crude extract and the control group (DMSO) to determine whether they were significantly different, * P < 0.05 , ** P < 0.01 , *** P < 0.005 .

From the data shown in Table 2, it can be found that the BQ01-(200)_30-60%EtOH and BQ01-(200)_30-95%EtOH groups have the best ability to inhibit superoxide anions and elastic enzymes.

In the free radical scavenging ability test, the test steps are the same as above, and the results are shown in Figure 4. The free radical scavenging rates of the three groups BQ01-(200)_30-40%EtOH, BQ01-(200)_30-60%EtOH and BQ01-(200)_30-95%EtOH are better, among which the free radical scavenging rate of BQ01-(200)_30-60%EtOH has the best performance.

The toxicity of each fraction to neutrophils was further tested.

Since lactate dehydrogenase (LDH) exists in various cells, when the cell membrane is broken, LDH will be released into the environment. Therefore, measuring the amount of LDH released by cells is used as a basis for evaluating whether drugs are toxic to cells.

After the separated neutrophil suspension was treated with each fraction extract of BQ01-(200)_30, the supernatant was obtained by centrifugation. The LDH content in the supernatant was detected by CytoTox 96 non-radioactive cytotoxicity assay (Promega). The results are shown in Figure 5. Each fraction extract was non-toxic to cells.

Based on cost-effectiveness considerations, since the yield of BQ01-(200)_30-60%EtOH is higher than that of BQ01-(200)_30-95%EtOH, subsequent experiments will be conducted using BQ01-(200)_30-60%EtOH.

In vitro wound healing test

The present invention further tests the wound healing ability of BQ01-(200)_30 and BQ01-(200)_30-60%EtOH.

Normal human fibroblast cell line Hs68 (purchased from Bioresource Collection and Research Center) was cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and 1% penicillin/streptomycin in an incubator at 37°C and 5% CO ₂ .

The cells were cultured in a 24-well plate and divided into four groups, namely the control group (Basal), the BQ01-(200)_30 group (given 0.1μg/mL of BQ01-(200)_30), the BQ01-(200)_30-60%EtOH group (given 0.1μg/mL of BQ01-(200)_30-60%EtOH) and the positive control group (given 20ng/mL of PDGF-AA), and then an in vitro wound healing test was performed using IBIDI Culture-Inserts (purchased from GmbH, Germany) according to the manufacturer's instructions.

Place the IBIDI Culture-Inserts into the wells of a 24-well cell culture plate and press gently on the top to ensure tight attachment. IBIDI Culture-Inserts consist of two wells separated by a 500 μm thick wall. Dispense equal amounts of Hs68 cells (4.5×10 ⁵ cells/mL) into the two wells of the IBIDI Culture-Inserts. After 24 hours of culture, switch to FBS-free medium, gently remove the IBIDI Culture-Inserts to create a 500 μm gap, and wash with PBS.

The cell nuclei were stained with 2μg/mL Hoechst 33342 for 10 minutes and washed with PBS. Then, the cells were treated with 0.1μg/mL BQ01-(200)_30, 0.1μg/mL BQ01-(200)_30-60%EtOH and 20ng/mL PDGF-AA according to the groups. The cell migration was observed and photographed using the Citation 5 imaging system (Biotek, England) at 0, 6, 9, 12 and 24 hours.

As shown in Figure 6, BQ01-(200)_30 and BQ01-(200)_30-60%EtOH produced significant cell migration effects, proving that BQ01-(200)_30 and BQ01-(200)_30-60%EtOH can effectively promote wound healing.

Diabetic Wound Healing Trial

All animal experiments in this invention were approved by the Institutional Animal Care and Use Committee (IACUC) of Chang Gung University.

First, a diabetic animal model was established. The present invention selected 8-week-old C57BL/6 male mice weighing 20-25g. After fasting for 4 hours, the mice were intraperitoneally injected with 55mg/kg-body weight of streptozotocin (STZ) dissolved in pH 4.5 citrate buffer for five consecutive days. After the injection of STZ for the first 4 days, normal food and 3% glucose water were given to induce diabetes. On the last day, glucose water was no longer given, and ordinary drinking water was given instead.

During the above schedule, 72 hours after STZ injection, the mice were fasted for 4 hours, and the fasting blood glucose levels were measured by piercing the tails of the mice and bleeding using a Contour Plus portable blood glucose meter (Ascensia, UK). Animals with fasting blood glucose levels of 150-250 mg/dL were considered prediabetic, while those >250 mg/dL were considered diabetic and used in subsequent experiments. All mice had fasting blood glucose levels >250 mg/dL.

After the diabetic animal model was established, the diabetic wound healing animal model was established.

A total of 48 C57BL/6 male diabetic mice were used in the experiment and randomly divided into 5 groups: 13 mice in the control group (given 25% Pluronic acid), 8 in the BQ01-(200)_30 low-dose group (given 10 mg/kg of BQ01-(200)_30), 10 in the BQ01-(200)_30-60%EtOH low-dose group (given 10 mg/kg of BQ01-(200)_30-60%EtOH), 7 in the BQ01-(200)_30 high-dose group (given 25 mg/kg of BQ01-(200)_30), and 10 in the BQ01-(200)_30-60%EtOH high-dose group (given 25 mg/kg of BQ01-(200)_30-60%EtOH).

The hair of each diabetic mouse was removed by shaving and using depilatory cream under anesthesia, the back skin was fixed with medical tape to prevent skin stretching, and two full-thickness wounds with a diameter of 6 mm were made on the left and right sides using a sterile biopsy punch.

During the experiment, all wounds were fixed with medical tape with a circular notch to keep the wounds at the notch. The medical tape fixed the skin around the wound to prevent the skin from shrinking during wound healing and affecting the calculation of the wound area. The left wounds were not treated, while the right wounds were given 25% Pluronic Acid, 10mg/kg BQ01-(200)_30, 10mg/kg BQ01-(200)_30-60%EtOH, 25mg/kg BQ01-(200)_30 and 25mg/kg BQ01-(200)_30-60%EtOH were used for daily topical treatment for 2 weeks, and the wounds were photographed daily.

Wound area was calculated using Image J analysis software based on photos taken every day and expressed as the percentage of wound healing compared with day 0.

The results of wound healing are shown in Figures 7A to 7E. Figure 7A shows wound healing photos of the untreated group, control group, low-dose group and high-dose group (including BQ01-(200)_30 and BQ01-(200)_30-60%EtOH). Figures 7B to 7E quantify the wound area and display it in a line graph. The results show that compared with the untreated group, the wounds of mice treated with BQ01-(200)_30 and BQ01-(200)_30-60%EtOH low-dose and high-dose groups showed significant wound healing.

The above dosage is converted into human dosage of 0.8mg/kg body weight and 2mg/kg body weight.

In order to further confirm that BQ01-(200)_30 and BQ01-(200)_30-60%EtOH only promote wound healing in diabetic patients, but not treat diabetes, the fasting blood glucose level of each mouse was measured in the first and second weeks. The results are shown in Figure 7. The fasting blood glucose level of each mouse continued to increase, proving that BQ01-(200)_30 and BQ01-(200)_30-60%EtOH only promote wound healing in diabetic patients, but not treat diabetes.

Although the present invention has been described and illustrated in sufficient detail so that a person skilled in the art can make and implement the present invention, various substitutions, modifications and improvements should be obvious without departing from the spirit and scope of the present invention.

It is readily understood by those skilled in the art that the present invention is well adapted to achieve the objects and advantages mentioned and those inherent therein. The cells, animals, and processes and methods for producing them represent only the best modes of implementation, are exemplary, and are not intended to limit the scope of the invention. Those skilled in the art will be able to conceive of modifications and other uses thereof, which are included within the spirit of the invention and are limited by the scope of the scope of the patent application.

Claims (13)

A method for preparing a chrysanthemum extract comprises the following steps: a. taking a chrysanthemum sample; and b. extracting the sample by subcritical water extraction at a high temperature and a high pressure to obtain a subcritical water extract; wherein the high temperature is between 120 and 250°C, and wherein the high pressure is between 5 and 20 MPa. The method as described in claim 1, wherein the subcritical water extraction method is performed for 5 to 60 minutes. The method as described in claim 1 further comprises the following steps: c. dissolving the subcritical water extract in pure water and placing it into a column, and performing separation using pure water and different proportions of alcohol aqueous solutions as mobile phases; and d. collecting the alcohol aqueous solution layer with the largest yield and freeze-drying it to obtain a chrysanthemum extract. The method as described in claim 3, wherein the different proportions of the alcohol aqueous solution in step c are selected from the group consisting of 20% alcohol aqueous solution, 40% alcohol aqueous solution, 60% alcohol aqueous solution and 95% alcohol aqueous solution. The method as described in claim 3, wherein the stratification in step c is performed sequentially using pure water, 20% alcohol aqueous solution, 40% alcohol aqueous solution, 60% alcohol aqueous solution and 95% alcohol aqueous solution as the mobile phase. A chrysanthemum extract, which is produced by the production method described in any one of claims 1 to 5. A use of a chrysanthemum extract for preparing a pharmaceutical composition for inhibiting an inflammatory response caused by neutrophils in a body, comprising an effective dose of a chrysanthemum extract and a medically acceptable carrier thereof, wherein the chrysanthemum extract is prepared by the manufacturing method as described in any one of claims 1 to 5. A use of a chrysanthemum extract for preparing a pharmaceutical composition for removing free radicals in the body, comprising an effective dose of a chrysanthemum extract and a medically acceptable carrier thereof, wherein the chrysanthemum extract is prepared by the manufacturing method as described in any one of claims 1 to 5. A use of a chrysanthemum extract for preparing a pharmaceutical composition for promoting wound healing in a body, comprising an effective dose of a chrysanthemum extract and a medically acceptable carrier thereof, wherein the chrysanthemum extract is prepared by the manufacturing method as described in any one of claims 1 to 5. The use as described in claim 9, wherein the wound healing includes chronic wound healing. The use as described in claim 9, wherein the wound healing includes diabetic wound healing or diabetic foot. The use as described in any one of claims 7 to 11, wherein the effective dose is 0.1 mg/kg body weight to 50 mg/kg body weight. The use as described in any one of claim 7 to 11, wherein the individual is a human or a mammal.

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