CN115463164B - Preparation method of ephedra root ethyl acetate part, and medicine and application thereof - Google Patents

Abstract

The invention discloses a preparation method of ephedra root ethyl acetate (ERE) and a medicine and application thereof, and particularly relates to application of ephedra root ethyl acetate in preparing a medicine for preventing and treating colonitis. According to the invention, ERE is adopted as a medicament for preventing and treating colonitis for the first time, and in-vivo and in-vitro efficacy experiments prove that the prepared medicament can inhibit the generation of Nitric Oxide (NO) of mouse mononuclear macrophage leukemia cells RAW264.7 induced by Lipopolysaccharide (LPS) and prevent acute colonitis of mice induced by DSS. ERE is able to significantly inhibit DSS-induced weight loss, elevation of DAI score, elevation of spleen index and pathological lesions of colon tissue, which are associated with its ability to inhibit expression of pro-inflammatory factors such as TNF- α, IL-6, promote elevation of IL-10 levels and regulate oxidative stress.

Description

Preparation method of ethyl acetate fraction of Ephedra root and its medicines and uses

Technical field

The present invention relates to the technical field of traditional Chinese medicine applications, specifically to a preparation method of the ethyl acetate fraction of Ephedra root and its medicines and uses, and in particular to the application of the ethyl acetate fraction of Ephedra root in the preparation of medicines for preventing or treating colitis.

Background technique

my country is rich in ephedra resources, but due to the restricted use of ephedrine components in ephedra, it is necessary to further develop the use of ephedra roots and promote the full utilization of ephedra plant resources. Ephedra root is the dried root and rhizome of Ephedra sinica Stapf or Ephedra intermedia Schrenk et C.A. Mey. Excavate in late autumn, remove residual stems, fibrous roots and silt, and dry. Traditional Chinese medicine believes that the roots of ephedra are neutral in nature, sweet and astringent in taste, and are directed to the heart and lung meridian. The chemical composition of ephedra root is complex, mainly containing alkaloids, flavonoids, volatile oils, organic acids, polysaccharides and trace elements. Modern pharmacological research shows that ephedra root has antihypertensive, antiperspirant, anti-inflammatory, anticancer, and antibacterial effects.

Ulcerative colitis (UC) is a multi-factorial and multi-layered non-specific inflammatory disease of the digestive tract of unknown etiology. The lesions are mainly located in the colorectum and manifest as diffuse, continuous and superficial lesions in the mucosa and submucosa. Superficial inflammation, accompanied by corresponding histological changes; the main clinical manifestations are abdominal pain, diarrhea, mucus, pus and bloody stools, and tenesmus; the course of the disease is characterized by being chronic, lifelong, and easy to relapse. In recent years, the incidence of ulcerative colitis in my country has been on the rise. Currently, there is no ideal drug for the treatment of ulcerative colitis. Therefore, further research and development of corresponding new therapeutic drugs for ulcerative colitis have important theoretical and practical significance.

So far, there are no reports on the use of ephedra root and its extracts in the prevention and treatment of ulcerative colitis.

Contents of the invention

Purpose of the invention: In order to solve the above technical problems, the purpose of the present invention is to provide a preparation method of the effective part of Ephedra root (ERE) and its new medicinal use. The effective part of the medicine is mainly used in the preparation of medicines for preventing or treating colitis.

Technical solution: The present invention provides a method for preparing the ethyl acetate fraction of Ephedra root, which includes the following steps:

1) Take the ephedra root medicinal material and crush it into granules;

2) Soak the granular ephedra root medicinal materials in 95% ethanol at room temperature for 12-20 hours, then conduct ultrasonic extraction with thermal reflux at 55-65°C, collect the extract, filter, recover the solvent under reduced pressure, and dry to obtain the extract;

3) Add distilled water to the extract and soak it for 12-20 hours, then extract 5 to 10 times with 10 to 15 times the volume (w/v) of petroleum ether and 5 to 10 times with ethyl acetate, each extraction for 0.5 to 1.5 hours. , combine the extracted ethyl acetate fractions, and after drying, the ethyl acetate fraction of ephedra root is obtained.

Specifically, as a preference, in step 2), the granular ephedra root medicinal material is soaked in 95% ethanol at room temperature for 12 hours, followed by ultrasonic extraction at 60° C., collecting the extract, filtering, recovering the solvent under reduced pressure, and drying to obtain an extract. ;

Specifically, as a preference, step 3) add distilled water to the extract and soak it for 12 hours, then extract it with 10 times the volume (w/v) of petroleum ether 5 times and ethyl acetate 9 times, each extraction for 0.5h, and combine The extracted ethyl acetate fraction is dried to obtain the ethyl acetate fraction of Ephedra root.

The present invention also includes the ethyl acetate fraction of ephedra root prepared by the preparation method.

The present invention includes the application of the ethyl acetate fraction of Ephedra root in inhibiting lipopolysaccharide (LPS)-induced NO production in macrophages.

Preferably, the ethyl acetate fraction of Ephedra root has anti-inflammatory activity in vitro and can inhibit the production of NO in RAW264.7 cells induced by LPS.

The content of the present invention also includes the application of the ethyl acetate fraction of Ephedra root in the preparation of drugs for treating and/or preventing colitis.

Among them, the ethyl acetate fraction of Ephedra root is used in the preparation of a drug that inhibits the expression of TNF-α and IL-6 in mouse colon tissue and promotes the increase of IL-10 levels.

Among them, the ethyl acetate fraction of Ephedra root is used in the preparation of drugs that inhibit the production of MPO and MDA in mouse colon tissue, promote the increase of SOD, and regulate the expression of oxidative stress response.

Wherein, the medicine is a single component or a compound preparation.

Wherein, the dosage form of the drug is one of tablets, capsules, oral liquids, syrups, dripping pills, injection dosage forms or freeze-dried powder dosage forms.

Beneficial effects: Compared with the existing technology, the present invention has the following advantages: The present invention uses ERE as a drug to prevent and treat ulcerative colitis for the first time. Through cell and animal efficacy experiments, ERE can not only inhibit LPS-induced RAW264.7 cells The production of nitric oxide (NO) in the medium can significantly alleviate the weight loss, DAI score increase, spleen index increase and pathological damage of colon tissue induced by DSS, which is due to its ability to inhibit pro-inflammatory factors such as TNF-α, The expression of IL-6 is related to promoting the increase of IL-10 levels and regulating oxidative stress.

Description of the drawings

Figure 1. The effect of ERE on the survival rate of RAW264.7 cells and the effect on NO production in cells under LPS stimulation (Mean±SD, n=3);

Figure 2. Effect of ERE on body weight, DAI score and feces of DSS-induced UC mice (Mean± SD, n=6);

Figure 3. Effect of ERE on colon length of DSS-induced UC mice (Mean±SD, n=6);

Figure 4. Effect of ERE on spleen index of DSS-induced UC mice (Mean±SD, n=6);

Figure 5. Effect of ERE on colon histopathology in DSS-induced UC mice (Mean±SD, n=6);

Figure 6. Effect of ERE on TNF-α, IL-6 and IL-10 in colon tissue of DSS-induced UC mice (Mean±SD, n=6);

Figure 7. Effect of ERE on the contents of MPO, MDA and SOD in the colon tissue of DSS-induced UC mice (Mean±SD, n=6).

Detailed ways

The experimental cells used in the examples of the present invention: RAW264.7 were purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai. Culture in DMEM medium containing 10% fetal calf serum at 37°C and 5% _CO2 .

Experimental animals used in the examples of the present invention: Kunming mice (SPF grade, male, 6-8 weeks old, 30-35g) were purchased from Changzhou Cavins Experimental Animal Co., Ltd. (SCXK (Su) 2021-0013). Animal quality certificate number: NO.202207710. All mice were fed in a mouse room with a room temperature of 22-25°C and an air humidity of 45%-55%. The mouse room was illuminated alternately for 12 hours between day and night. During the experiment, the mice were free to drink water and eat. The mice were adaptively fed for one week before the start of the experiment, and the body weight of the mice was recorded daily.

Medicines and reagents used in the examples of the present invention: DMEM culture medium: Hyclone Company (USA); Fetal calf serum (04-001-1ACS): Israel BI Company; MTT powder (ST1537) and BCA protein concentration determination kit (P0012) : Product of Shanghai Biyuntian Biological Company. LPS: Sigma (Merck Life Sciences Co., Ltd.), product number: L8274; Dimethyl sulfoxide (DMSO): Beijing Solebao Technology Co., Ltd., product number D8371; DSS: Abe Medical Equipment Trading Co., Ltd., batch number: S5148; Ephedra Root: Xuzhou Guandong Changjiu Traditional Chinese Medicinal Planting Co., Ltd.; Mouse TNF-α ELISE kit: Yikesai Biotechnology Co., Ltd., item number: EM008; Mouse IL-10 ELISE kit: Yikesai Biotechnology Co., Ltd., item number: EM005; Myeloperoxidase (MPO) determination kit: Nanjing Jiancheng Bioengineering Institute, Catalog No.: A044-1-1; Malondialdehyde (MDA) test kit: Nanjing Jiancheng Bioengineering Institute, Catalog No.: A003-1 ; Superoxide dismutase (SOD) assay kit: Nanjing Jiancheng Bioengineering Institute, Cat. No.: A001-3.

The data in the embodiments of the present invention are all processed statistically, and the data are expressed as mean ± standard deviation (x ± S). The differences between groups are t-tested. P<0.05 indicates that the difference is statistically significant.

Example 1 Extraction and drug preparation of ERE

1) Take 220g of ephedra root medicinal material and crush it into granules (particle size less than 2mm). The granular ephedra root medicinal materials were soaked in 95% ethanol (solid-liquid mass ratio 1:10) at room temperature for 12 hours, and then subjected to thermal reflux ultrasonic extraction at 60°C twice for 2 hours each time, for a total of 2 times. Collect the extract, filter it, recover the solvent under reduced pressure, and obtain 15g of extract after drying at 60°C.

2) Add 150mL distilled water to 15g extract, soak for 12 hours, extract 5 times with 150mL petroleum ether and 9 times with ethyl acetate, each extraction time is 0.5h, combine the extracted ethyl acetate parts, and dry them. 5g of ethyl acetate fraction (ERE) of Ephedra root was obtained.

3) Dissolve the ethyl acetate fraction (ERE) of Ephedra root in DMSO and dilute it with culture medium to the corresponding concentration. For example, during cell experiments, ERE is dissolved in DMSO, and the concentration of the ERE stock solution is 96 mg/mL for later use, and then diluted with 10% fetal calf serum DMEM medium to obtain concentrations of 5, 10, 20, 40, 80, 160, respectively. 320μg/mL ERE solution.

4) Dissolve the ethyl acetate fraction (ERE) of Ephedra root in 0.3% CMC-Na solution to prepare a suspension to obtain the drug. For example, in animal experiments, ERE is dissolved in 0.3% CMC-Na solution, and high-concentration ERE 40mg/ml and low-concentration ERE 20mg/mL are prepared for use.

Example 2 Pharmaceutical uses of ERE

1. ERE anti-inflammatory activity in vitro

Inflammatory response is one of the key factors in the continued development of UC, and is mainly mediated by macrophages. Inducible nitric oxide synthase (iNOS) is one of the important markers after macrophage activation, and it is not expressed in resting cells. Expression, when cells are attacked by inflammatory reactions and cytokines, such as LPS, they are induced to be synthesized, which then catalyzes the synthesis of large amounts of NO, resulting in a series of pathological reactions.

The MTT method was used to determine the effects of different doses of ERE (the ERE solutions prepared in step 3 of Example 1 at concentrations of 5, 10, 20, 40, 80, 160, and 320 μg/mL) on mouse mononuclear macrophage leukemia cells RAW264.7. Effect on cell viability, the results (Figure 1A) show that ERE has no toxicity to cells when the concentration is in the range of 5-160 μg/mL. The Griess method was used to determine the effect of different doses of ERE (5, 10, 20, 40, 80, 160 μg/mL) on the NO content of RAW264.7 stimulated by LPS (0.5 μg/mL). The results are shown in Figure 1B. The results show that ERE ( 5-160μg/mL) can dose-dependently inhibit the production of NO in RAW264.7 cells.

2. Preparation of DSS-induced colitis model and group administration

The experiment was conducted using the 40 mg/ml ERE solution and the 20 mg/ml ERE solution prepared in step 4) in Example 1.

The mice were randomly divided into 4 groups according to the body weight recorded on the last day of the adaptation period, with 6 mice in each group: control group, model group, ERE low-dose group (400 μg/g) (ERE-LD) and ERE high-dose group ( 800μg/g) (ERE-HD). The mice in the control group were given sterile distilled water throughout the entire experiment and could drink it freely; the mice in the model group, low-dose group, and high-dose group were given sterile distilled water for the first 14 days, and the drinking water given on days 15-21 was DSS powder. and 4% DSS solution prepared with distilled water; on days 15-21, mice in the ERE dose group were given corresponding doses of Ephedra root ethyl acetate extract suspension by gavage according to body weight, and mice in the control group and The mice in the model group were given an equal dose of 0.3% CMC-Na solution by gavage.

3. Disease activity index (DAI) scoring criteria

During the administration of DSS, the mice's weight, stool characteristics, and blood in the stool were observed and recorded at 8:00 am every day. The DAI score is the average score of weight change, stool characteristics and blood in the stool. For specific scoring rules, see Table 1.

Table 1 DAI scoring criteria

During the experiment, the status of the mice was observed every day, and the body weight and DAI score of the mice were recorded. During the DSS intervention period, the weight of the mice on day 0 was used as the initial weight, and the body weight change line chart and DAI score were drawn. As shown in Figure 2A, during the first three days of DSS intervention, the weight changes of the mice in the model group and the drug group fluctuated erratically. From the fourth day, the weight began to decrease significantly. On the 7th day of DSS intervention, as shown in Figure 2B, except for the control group, the body weight of mice in all other groups decreased to varying degrees, and the differences between each dose group of ERE and the model group were significant (P<0.01). The disease activity index (DAI) score reflects changes in the mouse's weight, stool characteristics and blood in the stool. The higher the DAI score, the more serious the colon disease is. During the DSS intervention period, the DAI scores of different groups are shown in Figure 2C. The mice in the control group had no weight loss, loose stools, or bloody mucus stools, and the DAI scores were around 0. After administration of DSS, the mice in the model group gradually developed symptoms such as listlessness, unresponsiveness, disheveled hair, fatigue and lethargy, bunching up, back arching, weight loss, and loose stools and bleeding. The related symptoms of mice in the drug group were significantly improved compared with the model group, and the DAI score was lower than that of the model group. Figure 2D is a picture of the feces of mice in each group before and after DSS intervention.

3. Colon length measurement

Figure 3A is a photo of the colon of mice in each group. The colon length of mice in each group is shown in Figure 3B. Compared with the control group, the colon of mice in the model group was the shortest, and there was a very significant difference (P<0.001). The colon length of mice in each dose group of ERE was longer than that of the model group, which was statistically significant (P<0.05).

4. Calculation of spleen index

After washing the dissected mouse spleen with PBS solution, use filter paper to absorb the surface moisture, weigh it, and calculate the mouse spleen index according to formula (1-1).

ERE can reduce spleen index in mice with DSS-induced ulcerative colitis. As shown in Figure 4B, after intervention with DSS, the spleen index of mice in the model group was significantly higher than that of the control group (P<0.001). The spleen index of mice in the drug group was lower than that of the model group, which was statistically significant (P<0.05). Figure 4A is a photo of the spleen of mice in each group. It can be seen that the spleen of mice in the model group is larger than that of the other groups.

5. Histopathological analysis of colon

The fixed colon specimens were dehydrated with ethanol, permeabilized with xylene, stained with hematoxylin and eosin in 5 μm-thick serial sections, and sealed with neutral gum. The pathological conditions of the colon tissue were observed under a microscope. The pathology score was evaluated by two professionals without knowing the experimental protocol and grouping, referring to the pathology score sheet (Table 2).

Table 2 Pathology score sheet

The results of colon sections after H&E staining are shown in Figure 5. According to Figure 5A, the colon tissue in the control group was highly structured, showing dense columnar epithelium and complete intestinal crypts, and no abnormalities were found in histology. In contrast, the DSS model group showed epithelial destruction, mucosal damage, crypt disappearance, and inflammatory cell infiltration in the submucosa and muscularis. However, after treatment with ERE, a significant reduction in damage in colon tissue sections from the ERE-LD and ERE-HD groups was observed. In addition, the severity of colon injury was scored. As shown in Figure 5B, the DSS model group (11.08 ± 0.47) had the highest score among each group, and the score was significantly higher compared with the control group (P < 0.001). However, the histological scores of the ERE-LD (5.92±0.98) and ERE-HD (6.33±1.22) groups were significantly lower than those of the DSS model group (P<0.01). Our results indicate that ERE treatment has a clear protective effect against DSS-induced colitis.

6. Determination of TNF-α, IL-6 and IL-10 inflammatory factors

Take a piece of colon tissue of about 100 mg, rinse it in pre-cooled saline to remove residual blood, weigh it and cut the tissue into pieces on ice. Add the chopped colon tissue and physiological saline into a centrifuge tube at a weight-to-volume ratio of 1:9, homogenize thoroughly with an electric homogenizer, take the supernatant from the homogenate, and measure according to the requirements of the kit.

The levels of TNF-α, IL-6 and IL-10 in the colon tissues of mice in each group were measured by ELISA. The results are shown in Figure 6. Compared with the control group, the mice in the model group had higher levels of TNF-α and IL-6 and lower levels of IL-10, which was statistically significant (P<0.001). The levels of TNF-α and IL-6 in the colon tissue of mice in each dose group of ERE were significantly reduced compared with the model group (P＜0.001), and the level of IL-10 was significantly increased compared with the model group (P＜0.05). This shows that ERE can significantly inhibit the expression of TNF-α and IL-6 in the colon tissue of DSS-induced UC mice and promote the increase of IL-10 levels.

7. Determination of SOD, MDA and MPO

Take the colon tissue homogenate and perform the measurement according to the requirements of the kit. The expression levels of MPO, MDA and SOD in mouse colon tissue were measured, and the results are shown in Figure 7. Compared with the control group, the levels of MPO and MDA in the colon tissue of mice in the model group were significantly increased (P<0.001), and the levels of SOD were significantly decreased (P<0.001). The MPO and MDA levels in the colon tissue of mice in each dose group of ERE were significantly lower than those in the model group (P<0.05), and the SOD levels were significantly higher than those in the model group (P<0.01). The above results indicate that ERE can inhibit the production of MPO and MDA, promote the increase of SOD, and thereby regulate oxidative stress.

Claims (6)

1. The application of the ephedra root ethyl acetate part in preparing the medicine for treating and/or preventing colonitis is characterized in that the preparation method of the ephedra root ethyl acetate part comprises the following steps:

1) Taking 220g ephedra root medicinal material, crushing the medicinal material into particles with the particle size smaller than 2mm, and using the solid-liquid mass ratio of the granular ephedra root medicinal material to be 1:10, soaking the mixture in 95% ethanol for 12 hours at room temperature, performing heat reflux ultrasonic extraction for 2 times at 60 ℃ and 2 times each time, collecting an extracting solution, filtering, recovering a solvent under reduced pressure, and drying at 60 ℃ to obtain 15g extractum;

2) Adding 150mL distilled water into 15g extract, soaking for 12 hr, sequentially extracting with 150mL petroleum ether for 5 times and ethyl acetate for 9 times, each time for 0.5h, mixing the extracted ethyl acetate parts, and drying to obtain 5g herba Ephedrae root ethyl acetate parts.

2. The use according to claim 1, wherein the concentration of the ethyl acetate fraction of ephedra root is 5-160 μg/mL.

3. The use according to claim 1, wherein the ephedra root ethyl acetate site is used for preparing a medicament for inhibiting TNF- α and IL-6 expression in colon tissue of a mouse and promoting an increase in IL-10 level.

4. The use according to claim 1, wherein the ephedra root ethyl acetate site is used for preparing a medicament for inhibiting the production of MPO and MDA in colon tissue of a mouse, promoting the increase of SOD and regulating the expression of oxidative stress reaction.

5. The use according to claim 1, wherein the medicament is a single component or a compound formulation.

6. The use according to claim 1, wherein the pharmaceutical dosage form is one of a tablet, a capsule, an oral liquid, a syrup, a drop pill, an injection or a lyophilized powder injection.