CN115463164A - Preparation method of ephedra root ethyl acetate part, medicine and application thereof - Google Patents
Preparation method of ephedra root ethyl acetate part, medicine and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/17—Gnetophyta, e.g. Ephedraceae (Mormon-tea family)
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A61K2236/50—Methods involving additional extraction steps
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Abstract
The invention discloses a preparation method of an ephedra root ethyl acetate part (ERE), a medicine and an application thereof, and particularly relates to an application of the ephedra root ethyl acetate part in preparing medicines for preventing and treating colitis. The invention adopts ERE as the medicine for preventing and treating colitis for the first time, and the medicine prepared by the invention can inhibit the generation of mouse mononuclear macrophage leukemia cell RAW264.7 Nitric Oxide (NO) induced by Lipopolysaccharide (LPS) and prevent the mouse acute colitis induced by DSS through the verification of in vivo and in vitro efficacy experiments. ERE can remarkably inhibit DSS-induced weight loss, DAI score increase, spleen index increase and colon tissue pathological damage, which are related to the effects of inhibiting the expression of proinflammatory factors such as TNF-alpha and IL-6, promoting the increase of IL-10 level and regulating oxidative stress.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine application, in particular to a preparation method of an ethyl acetate part of ephedra roots, a medicine and application thereof, and especially relates to application of the ethyl acetate part of the ephedra roots in preparing a medicine for preventing or treating colitis.
Background
China has rich ephedra resources, but because of the limited use of ephedrine components in ephedra, the use of ephedra roots needs to be further developed, and the full utilization of ephedra plant resources is promoted. The radix Ephedrae is dried root and rhizome of Ephedra sinica Stapf or Ephedra sinica intermedia Schrenk et C.A.Mey. Collected in late autumn, removed the residual stem, fibrous root and silt, and dried. The traditional Chinese medicine considers that the ephedra roots are neutral in nature, sweet and astringent in taste, enter heart and lung channels, have the effects of strengthening superficies and arresting sweating, and are clinically used for treating spontaneous sweating and night sweat. The chemical components of the ephedra root are complex and mainly contain alkaloids, flavonoids, volatile oils, organic acids, polysaccharides, trace elements and the like. Modern pharmacological research shows that the ephedra root has the functions of reducing blood pressure, arresting sweating, resisting inflammation, resisting cancer, resisting bacteria and the like.
Ulcerative Colitis (UC) is a multifactorial, multilayered, nonspecific inflammatory disease of the digestive tract with unknown etiology, with lesions mainly located in the colorectal, manifested by diffuse, continuous and superficial inflammation of the mucosa and submucosa, accompanied by corresponding histological changes; clinically, abdominal pain, diarrhea, mucopurulent bloody stool and tenesmus are the main clinical manifestations; the disease course has the characteristics of chronic, lifelong and easy recurrence. In recent years, the incidence rate of ulcerative colitis in China is on the rise, and at present, no ideal medicine for treating ulcerative colitis exists, so that further research and development of a corresponding new therapeutic medicine for treating ulcerative colitis have important theoretical and practical significance.
So far, no research on the use of the ephedra root and the extract thereof for preventing and treating ulcerative colitis is reported.
Disclosure of Invention
The purpose of the invention is as follows: in order to solve the technical problems, the invention aims to provide a preparation method of an effective part (ERE) of ephedra roots and a new application of the ERE, wherein the effective part of the ERE is mainly used for preparing a medicine for preventing or treating colitis.
The technical scheme is as follows: the invention provides a preparation method of an ethyl acetate part of ephedra root, which comprises the following steps:
1) Pulverizing radix Ephedrae into granules;
2) Soaking granular radix Ephedrae in 95% ethanol at room temperature for 12-20 hr, performing hot reflux ultrasonic extraction at 55-65 deg.C, collecting extractive solution, filtering, recovering solvent under reduced pressure, and drying to obtain extract;
3) Adding distilled water into the extract, soaking for 12-20h, sequentially extracting for 5-10 times by using petroleum ether with volume (w/v) being 10-15 times, extracting for 5-10 times by using ethyl acetate, extracting for 0.5-1.5 h each time, combining extracted ethyl acetate parts, and drying to obtain the ephedra root ethyl acetate part.
Specifically, preferably, in the step 2), the granular ephedra root medicinal material is soaked in 95% ethanol at room temperature for 12 hours, then is subjected to hot reflux ultrasonic extraction at 60 ℃, and an extracting solution is collected, filtered, subjected to reduced pressure recovery of a solvent and dried to obtain an extract;
specifically, preferably, in the step 3), distilled water is added into the extract for soaking for 12 hours, petroleum ether with 10 times of volume (w/v) is sequentially used for extraction for 5 times, ethyl acetate is used for extraction for 9 times, each time for 0.5 hour, the extracted ethyl acetate parts are combined, and the ephedra root ethyl acetate part is obtained after drying.
The invention also comprises the ethyl acetate part of the ephedra root prepared by the preparation method.
The invention comprises the application of the ephedra root ethyl acetate part in inhibiting NO generation in macrophages induced by Lipopolysaccharide (LPS).
Preferably, the ethyl acetate part of the ephedra roots has an in vitro anti-inflammatory activity function and can inhibit the generation of NO in RAW264.7 cells induced by LPS.
The invention also comprises the application of the ephedra root ethyl acetate part in preparing a medicament for treating and/or preventing colitis.
Wherein the ephedra root ethyl acetate part is applied to preparing a medicament for inhibiting the expression of TNF-alpha and IL-6 in colon tissues of mice and promoting the increase of IL-10 level.
Wherein, the application of the ephedra root ethyl acetate part in preparing the medicine for inhibiting the generation of MPO and MDA in the colon tissue of mice, promoting the increase of SOD and regulating the expression of oxidative stress reaction.
Wherein the medicament is a single-component or compound preparation.
Wherein, the dosage form of the medicine is one of tablets, capsules, oral liquid, syrup, dropping pills, injection or freeze-dried powder injection.
Has the beneficial effects that: compared with the prior art, the invention has the following advantages: the invention adopts ERE as the medicine for preventing and treating ulcerative colitis for the first time, and the cell and animal pharmacodynamic experiments prove that the ERE can not only inhibit the generation of Nitric Oxide (NO) in RAW264.7 cells induced by LPS, but also can obviously relieve the weight loss, the increased DAI score, the increased spleen index and the pathological damage of colon tissues caused by DSS induction, which is related to the inhibition of the expression of proinflammatory factors such as TNF-alpha and IL-6, the promotion of the increase of IL-10 level and the regulation of oxidative stress.
Drawings
Figure 1, effect of ERE on RAW264.7 cell survival and on NO production in cells under LPS stimulation (Mean ± SD, n = 3);
figure 2, effect of ERE on DSS-induced UC mouse body weight, DAI score and feces (Mean ± SD, n = 6);
figure 3, effect of ERE on DSS-induced UC mouse colon length (Mean ± SD, n = 6);
figure 4, effect of ERE on DSS-induced UC mouse spleen index (Mean ± SD, n = 6);
figure 5, effect of ERE on DSS-induced UC mouse colon histopathology (Mean ± SD, n = 6);
FIG. 6, effects of ERE on TNF- α, IL-6 and IL-10 in DSS-induced UC mouse colon tissue (Mean + -SD, n = 6);
FIG. 7, effect of ERE on MPO, MDA and SOD content in colon tissue of DSS-induced UC mice (Mean + -SD, n = 6).
Detailed Description
Experimental cells used in the examples of the present invention: RAW264.7 was purchased from shanghai academy of sciences cell bank. DMEM medium containing 10% fetal bovine serum at 37 deg.C and 5% CO 2 Culturing under the condition.
Experimental animals used in the examples of the present invention: kunming mice (SPF grade, male, 6-8 weeks old, 30-35 g) were purchased from Kyoho Kavens laboratory animals Co., ltd (SCXK (threo) 2021-0013). Animal quality certification number: NO.202207710. All mice are fed in a mouse room with the room temperature of 22-25 ℃ and the air humidity of 45% -55%, the mouse room is irradiated with light for 12 hours alternately day and night, and the mice take water and eat freely during the experiment. Mice were acclimatized for one week prior to initiation of the experiment and their body weights were recorded daily.
The medicines and reagents used in the embodiment of the invention are as follows: DMEM medium: hyclone corporation (USA); fetal bovine serum (04-001-1 ACS): israel BI Inc.; MTT powder (ST 1537) and BCA protein concentration determination kit (P0012): shanghai Biyuntian bio corporation. LPS: sigma (merck life science, ltd), cat #: l8274; dimethyl sulfoxide (DMSO): beijing Sorbao science and technology, inc., part number D8371; DSS: amdoubly medical instruments trade limited, lot number: s5148; ephedra root: xuzhou kandongtoujiu traditional Chinese medicine planting limited; mouse TNF- α ELISE kit: science and technology ltd, cat #: EM008; mouse IL-10ELISE kit: science and technology ltd, cat #: EM005; myeloperoxidase (MPO) assay kit: nanjing was established as a bioengineering institute, with a cargo number: a044-1-1; malondialdehyde (MDA) test kit: nanjing was established as a bioengineering institute, with a cargo number: a003-1; superoxide dismutase (SOD) determination kit: nanjing was built into the institute of bioengineering, cargo number: a001-3.
The data in the embodiment of the invention are all statistically processed, the data are expressed by mean value + -standard deviation (x + -S), the difference between groups is t-tested, and P < 0.05 represents that the difference has statistical significance.
Example 1 extraction and pharmaceutical preparation of ERE
1) Taking 220g of ephedra root medicinal material, and crushing into granules (the grain diameter is less than 2 mm). Soaking granular radix Ephedrae in 95% ethanol (solid-liquid mass ratio of 1: 10) at room temperature for 12 hr, and performing ultrasonic extraction at 60 deg.C for 2 times (2 hr each time for 2 times). Collecting extractive solution, filtering, recovering solvent under reduced pressure, and drying at 60 deg.C to obtain 15g extract.
2) Adding 150mL of distilled water into 15g of the extract, soaking for 12 hours, sequentially extracting for 5 times by using 150mL of petroleum ether and 9 times by using ethyl acetate, wherein the extraction time is 0.5 hour each time, combining the extracted ethyl acetate parts, and drying to obtain 5g of ephedra root ethyl acetate part (ERE).
3) The ethyl acetate fraction (ERE) of the ephedra roots was dissolved in DMSO and diluted to the corresponding concentration with medium. For example, in cell experiments, ERE is dissolved in DMSO to prepare ERE stock solution with a concentration of 96mg/mL for later use, and then diluted with 10% fetal bovine serum DMEM medium to obtain ERE solutions with concentrations of 5, 10, 20, 40, 80, 160 and 320. Mu.g/mL.
4) Dissolving the ethyl acetate part (ERE) of radix Ephedrae in 0.3% CMC-Na solution to obtain suspension. For example, in animal experiments, ERE was dissolved in 0.3% CMC-Na solution and prepared to have a high ERE concentration of 40mg/mL and a low ERE concentration of 20mg/mL for further use.
EXAMPLE 2 pharmaceutical use of ERE
1. ERE in vitro anti-inflammatory Activity
Inflammatory response is one of the key factors for the continuous development of UC, and is mainly mediated by macrophages, and Inducible Nitric Oxide Synthase (iNOS) is one of the important markers after macrophage activation, and is not expressed in resting cells, and is induced and synthesized when cells are attacked by inflammatory response and cytokines, such as LPS, so as to catalyze and synthesize a large amount of NO, thereby generating a series of pathological responses.
MTT assay measures the effect of varying doses of ERE (example 1, step 3) formulated in ERE solutions at concentrations of 5, 10, 20, 40, 80, 160, 320. Mu.g/mL) on the survival of mouse monocyte macrophage leukemia cells RAW264.7 cells, and the results (FIG. 1A) indicate that ERE concentrations in the range of 5-160. Mu.g/mL are not toxic to cells. The Griess method measures the effect of different dosages of ERE (5, 10, 20, 40, 80, 160 μ g/mL) on the NO content of RAW264.7 stimulated by LPS (0.5 μ g/mL), and the results are shown in fig. 1B, which shows that ERE (5-160 μ g/mL) can inhibit the generation of NO in RAW264.7 cells in a dose-dependent manner.
2. DSS-induced colitis model preparation and grouped drug delivery
The experiment was carried out using the 40mg/ml ERE solution and the 20mg/ml ERE solution prepared in step 4) of example 1.
Mice were randomly divided into 4 groups of 6 mice per group, based on body weight recorded on the last day of the acclimation period: control, model, ERE low dose (400. Mu.g/g) (ERE-LD) and ERE high dose (800. Mu.g/g) (ERE-HD). Sterile distilled water is given to the control group of mice in the whole experiment process, and the mice can be freely drunk; sterile distilled water was administered 14 days before the mice in the model group, the low dose group, and the high dose group, and drinking water was administered at 15-21 days as a 4% DSS solution formulated with DSS powder and distilled water; on days 15-21, mice in the ERE dose group were gavage with the corresponding dose of the suspension of the ethyl acetate extract of the ephedrine root, and mice in the control group and the model group were gavage with the equivalent dose of 0.3% CMC-Na solution.
3. Disease Activity Index (DAI) scoring criteria
During DSS administration, mice were observed and recorded daily at 8 am for body weight, fecal traits, and hematochezia status. DAI scores are the average scores for body weight change, stool traits and hematochezia. The detailed score is shown in Table 1.
TABLE 1DAI scoring criteria
The status of the mice was observed daily during the experiment and the body weight and DAI score of the mice were recorded. During DSS intervention, body weight change line plots and DAI scores were plotted, with mouse day 0 body weight as the initial body weight. As shown in fig. 2A, the body weight change of the model group and drug group mice was unstable three days before DSS intervention, and body weight began to drop significantly from day four. On day 7 of DSS intervention, mice in each of the other groups, except the control group, showed a different decrease in body weight average, with the ERE dose groups differing significantly from the model groups (P < 0.01), as shown in figure 2B. Disease Activity Index (DAI) scores reflect mouse weight change, stool characteristics, and hematochezia. A higher DAI score indicates a more severe colon lesion. During DSS intervention, the DAI scores for the different groups were shown in figure 2C, with no weight loss, no loose stools and mucous bloody stools in the control group mice, and around 0. After the mice in the model group are given with DSS, the symptoms of lassitude, slow response, messy and lusterless hair, lassitude, sleepiness, hunched and hunched back, weight loss, bleeding in loose stool and the like gradually appear. The drug group mice showed significantly improved symptoms compared to the model group, and had a lower DAI score than the model group. Fig. 2D is a picture of stool from each group of mice before and after DSS intervention.
3. Colon length measurement
FIG. 3A is a photograph of the colon of each group of mice. The length of the colon of each group of mice is shown in FIG. 3B, and compared with the control group, the colon of the model group of mice is the shortest and has a very significant difference (P < 0.001). The colon length of the mice in each dosage group of ERE is larger than that of the model group, and the mice have statistical significance (P is less than 0.05).
4. Spleen index calculation
After the spleen of the mouse obtained by dissection was washed with a PBS solution, surface water was blotted with filter paper, and the weight was weighed, and the mouse spleen index was calculated according to the formula (1-1).
ERE is able to reduce the spleen index in DSS-induced ulcerative colitis mice. As shown in FIG. 4B, the spleen index of the model group mice was significantly higher than that of the control group (P < 0.001) after the dry-state with DSS. The spleen index of the mice in the drug group is lower than that of the mice in the model group, and the statistical significance is achieved (P is less than 0.05). FIG. 4A is a photograph of spleens of mice in each group, showing that spleens of mice in the model group are larger than those in other groups.
5. Colon histopathological analysis
And (3) dehydrating the fixed colon specimen with ethanol, carrying out dimethylbenzene permeabilization, carrying out hematoxylin and eosin staining on a continuous section with the thickness of 5 mu m, sealing the section with neutral gum, and observing the pathological condition of the colon tissue under a microscope. Pathology scores were assessed by two professionals without knowledge of the protocol and group, with reference to the pathology score table (table 2).
TABLE 2 Pathology grading Table
The results of colon sections after H & E staining are shown in fig. 5. According to fig. 5A, the colon tissue of the control group is highly structured, presents a dense columnar epithelium and an intact intestinal crypt, and has no histology abnormality. In contrast, the DSS model group showed epithelial destruction, mucosal damage, crypt disappearance, and inflammatory cell infiltration in the submucosa and muscularis. However, a significant reduction in colon tissue section damage from the ERE-LD and ERE-HD groups was observed following treatment with ERE. In addition, when the severity of colon injury was scored, as shown in FIG. 5B, the DSS model group (11.08. + -. 0.47) scored the highest among the groups, and the score was significantly higher (P < 0.001) compared to the control group. 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 significant protective effect against DSS-induced colitis.
6. TNF-alpha, IL-6 and IL-10 inflammatory factor assay
A section of colon tissue of about 100mg is taken, the colon tissue is rinsed in precooled physiological saline, residual blood is removed, and the tissue is cut into pieces on ice after weighing. The minced colon tissue is mixed with normal saline according to the ratio of l: 9. adding the mixture into a centrifugal tube according to the weight-volume ratio, fully homogenizing by using an electric homogenizer, taking supernatant from the homogenate liquid, and measuring according to the requirements of a kit.
The levels of TNF- α, IL-6 and IL-10 in colon tissue were determined by ELISA in each group of mice. As shown in FIG. 6, the levels of TNF-. Alpha.and IL-6 were higher and IL-10 was lower in the model group mice than in the control group, and they were statistically significant (P < 0.001). The significant reduction of TNF-alpha and IL-6 levels in colon tissues of mice in each dose group of ERE compared to the model group (P < 0.001) and the significant increase of IL-10 levels compared to the model group (P < 0.05) indicate that ERE can significantly inhibit the expression of TNF-alpha and IL-6 in colon tissues of DSS-induced UC mice and promote the increase of IL-10 levels.
7. SOD, MDA and MPO assays
Taking colon tissue homogenate to perform measurement according to the requirements of the kit. The expression levels of MPO, MDA and SOD in colon tissues of mice were measured, and the results are shown in fig. 7. Compared with the control group, the levels of MPO and MDA in colon tissues of the mice in the model group are obviously increased (P is less than 0.001), and the level of SOD is obviously reduced (P is less than 0.001). The levels of MPO and MDA in colon tissues of mice in each dose group of ERE are obviously lower than those in a model group (P < 0.05), and the level of SOD is obviously higher than that in the model group (P < 0.01). The results show that ERE can inhibit the production of MPO and MDA and promote the increase of SOD, thereby regulating oxidative stress.
Claims (9)
1. The preparation method of the ethyl acetate part of the ephedra root is characterized by comprising the following steps:
1) Pulverizing radix Ephedrae into granules;
2) Soaking granular radix Ephedrae in 95% ethanol at room temperature for 12-20 hr, performing hot reflux ultrasonic extraction at 55-65 deg.C, collecting extractive solution, filtering, recovering solvent under reduced pressure, and drying to obtain extract;
3) Adding distilled water into the extract, soaking for 12-20h, sequentially extracting for 5-10 times by using petroleum ether with volume (w/v) being 10-15 times, extracting for 5-10 times by using ethyl acetate, extracting for 0.5-1.5 h each time, combining extracted ethyl acetate parts, and drying to obtain the ephedra root ethyl acetate part.
2. The ethyl acetate fraction of ephedra produced by the process of claim 1.
3. Use of the ethyl acetate moiety of ephedra roots of claim 2 to inhibit LPS-induced NO production in macrophages.
4. The use of claim 3, wherein the concentration of the ethyl acetate fraction of the ephedra roots is 5-160 μ g/mL.
5. Use of the ethyl acetate fraction of ephedra roots according to claim 2 for the preparation of a medicament for the treatment and/or prevention of colitis.
6. The use according to claim 5, wherein the ethyl acetate fraction of ephedra roots is used for preparing a medicament for inhibiting the expression of TNF- α and IL-6 in colon tissues of mice and promoting the increase of IL-10 level.
7. The use of claim 5, wherein the ethyl acetate fraction of the ephedra roots is used for preparing a medicament for inhibiting the production of MPO and MDA in colon tissues of mice, promoting the increase of SOD and regulating the expression of oxidative stress.
8. The use according to any one of claims 5 to 7, wherein the medicament is a mono-component or a combination formulation.
9. The use of any one of claims 5 to 7, wherein the dosage form of the medicament is one of tablet, capsule, oral liquid, syrup, dripping pill, injection or lyophilized powder injection.
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