European verbena herb extract with anti-radiation effect as well as preparation method and application of European verbena herb extract

Abstract

The invention discloses a verbena extract with an anti-radiation effect and a preparation method and application thereof.The preparation method comprises the specific steps that the overground part of verbena is taken, ethyl alcohol is added for heating reflux operation, and then the obtained mixed solution is filtered to obtain an extracting solution; the extraction process is repeated for 1-3 times, then extracting solutions obtained in multiple times are combined, concentration and drying treatment are carried out, and finally an extractum-like substance, namely the verbena extract, is obtained. The traditional Chinese medicine composition can effectively improve the morphology and pathological change of spleen tissues, significantly increase the counting level of peripheral blood leukocytes (WBC), neutrophils (NE) and lymphocytes (LY), and reduce the release of inflammatory factors such as tumor necrosis factor alpha (TNF-alpha), interleukin 17 (IL-17) and interleukin 6 (IL-6); in this way, the tissue damage degree caused by radiation can be practically reduced, and a potential effective intervention means is provided for treating radiation damage.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a verbena extract with anti-radiation effect, a preparation method and an application thereof. Background Art

[0002] Radiation damage has become an important factor endangering people's health. Hematopoietic and immune function damage after radiation is the main cause of death; the recovery of hematopoietic function and the reconstruction of immune function after radiation exposure become the key to its treatment;

[0003] Clinically, many cancer patients receiving radiotherapy are affected by the hematopoietic and immune function damage caused by radiation, which also affects the treatment of the disease. This is also a thorny problem in radiation therapy. Currently, the commonly used radiation protection and treatment methods in clinical practice include biological therapy, cytokines, gene transfection, etc. Among them, those with good protection effects often have obvious toxic and side effects, severely limiting their use. Therefore, finding safe and effective radiation protection agents has become a hot topic and a difficult point in the current radiation field research.

[0004] With the development of traditional Chinese medicine, traditional Chinese medicine has achieved remarkable curative effects in the treatment of various diseases. Searching for radiation protection drugs from traditional Chinese medicine treatment has become an important direction in radiation research. In view of the existing technical problems such as poor curative effect and toxic and side effects, the present application provides a verbena extract with anti-radiation effect, a preparation method and an application thereof. Summary of the Invention

[0005] In view of the above-mentioned defects or deficiencies in the prior art, it is desirable to provide a verbena extract with anti-radiation effect, a preparation method and an application thereof.

[0006] A preparation method of a verbena extract with anti-radiation effect provided by the present invention includes the following steps:

[0007] 1) Weigh a specified weight portion of verbena and an ethanol solution;

[0008] 2) Perform alcohol extraction on the verbena and the ethanol solution at a material-liquid ratio of 1:5 to 1:40, heat and reflux for extraction 1 to 3 times, each time for 1.5 to 3 hours;

[0009] 3) Combine, concentrate and dry the products obtained from each heat reflux extraction in step 2) to obtain an extract, that is, obtain the verbena extract.

[0010] Further, the ethanol solution is an ethanol solution with a concentration range of 20 to 100%.

[0011] Further, the ethanol solution is an ethanol solution with a concentration of 100%.

[0012] Further, the material-liquid ratio of verbena to ethanol solution is 1:20.

[0013] Further, in step 2), the heating reflux extraction time each time is 2 h, the extract is obtained by filtration, the extraction is repeated 3 times, and the extracts obtained 3 times are combined, concentrated and dried.

[0014] The verbena extract prepared by the preparation method of the verbena extract with anti-radiation effect.

[0015] An application of verbena in anti-radiation drugs, wherein the anti-radiation drug is a preparation composed of a traditional Chinese medicine extract and a pharmaceutically acceptable carrier.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] For the verbena extract with anti-radiation effect, preparation method and application of the present invention, experiments were carried out on the verbena extract. Using 137 Csγ rays were used to irradiate the whole body of rats once to establish a radiation injury model; the verbena extract can effectively improve the radiation injury of rats established by γ-ray irradiation, improve the morphological and pathological changes of spleen tissue, significantly increase the levels of peripheral blood white blood cells (WBC), neutrophils (NE) and lymphocytes (LY), and reduce the expression of tumor necrosis factor-α (TNF-α), interleukin 17 (IL-17) and interleukin 6 (IL-6);

[0018] Therefore, in summary, the verbena extract prepared by the production method of the present application can effectively improve the morphological and pathological changes of spleen tissue, significantly increase the levels of peripheral blood white blood cells (WBC), neutrophils (NE) and lymphocytes (LY), and reduce the release of inflammatory factors such as tumor necrosis factor α (TNF-α), interleukin 17 (IL-17) and interleukin 6 (IL-6); in this way, it can effectively reduce the degree of tissue damage caused by radiation and provide a potentially effective intervention means for dealing with radiation injury.

[0019] It should be understood that the content described in the invention content part is not intended to limit the key or important features of the embodiments of the present invention, nor is it used to limit the scope of the present invention. Other features of the present invention will become easy to understand through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] By reading the detailed description of the non-limiting embodiments with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:

[0021] Figure 1 The chromatographic mobile phase gradient elution program of the verbena extract of this application;

[0022] Figure 2 is the base peak chromatogram of the verbena extract of this application;

[0023] Figure 3 is the chemical composition identification result of the verbena extract of this application;

[0024] Figure 4 is the diagram of the appearance of the spleen of rats with radiation injury affected by the verbena extract of this application;

[0025] Figure 5 is the diagram of the effect of the verbena extract of this application on the peripheral blood cells of rats with radiation injury;

[0026] Figure 6 is the diagram of the effect of the verbena extract of this application on inflammatory factors of rats with radiation injury;

[0027] Figure 7 is the diagram of the effect of the verbena extract of this application on the spleen sections of rats with radiation injury. Detailed implementation manners

[0028] The present invention will be further described in detail below with reference to the drawings and embodiments. It can be understood that the specific embodiments described herein are only used to explain the related invention, rather than limiting the invention. Additionally, it should be noted that, for the sake of description, only the parts related to the invention are shown in the drawings.

[0029] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the drawings and embodiments.

[0030] Please refer to Figures 1-7 , the embodiments of the present invention provide a preparation method of a verbena extract with anti-radiation effect, including the following steps:

[0031] 1) Weigh a specified weight portion of verbena and an ethanol solution; wherein, the ethanol solution is an ethanol solution with a concentration range of 20-100%;

[0032] 2) Perform alcohol extraction on verbena and the ethanol solution in a ratio of solid-to-liquid of 1:5-1:40, heat under reflux for extraction 1-3 times, each time for 1.5-3 h;

[0033] 3) Combine, concentrate and dry the products obtained from each heat reflux extraction in step 2) to obtain an extract, that is, obtain the verbena extract.

[0034] Preferably, the ethanol solution is an ethanol solution with a concentration of 100%; the solid-to-liquid ratio of verbena and the ethanol solution is 1:20.

[0035] In step 2), the heating reflux extraction time is 2 hours each time, and the extract is obtained by filtration. The extraction is repeated 3 times, and the extracts obtained 3 times are combined, concentrated and dried.

[0036] The invention relates to an application of verbena in an anti-radiation drug. The anti-radiation drug is a preparation composed of a traditional Chinese medicine extract and a pharmaceutically acceptable carrier.

[0037] Example 1

[0038] 1 Materials and Methods

[0039] 1.1 Experimental Animals

[0040] Male SD rats, weighing (200±20) g, were purchased from Beijing Weitonglihua Experimental Animal Company and housed in the Animal Center of the Institute of Radiation Medicine, Chinese Academy of Sciences, at an ambient temperature of 20-25°C and a humidity of 40%-60%, with free access to water and food every day.

[0041] 1.2 Drugs and reagents

[0042] Ethanol (analytical grade domestic reagent); all Chinese medicinal materials were purchased from Kangmei Pharmaceutical Co., Ltd., paraformaldehyde fixative (specification: 4%, Feijing Biotechnology Co., Ltd., batch number: 20240419).

[0043] 2 Extract preparation

[0044] Weigh 500g of verbena, extract it with 20%-100% ethanol solution at a solid-liquid ratio of 1:5-1:40, heat and reflux extraction 1-3 times, each time for 1.5-3h, combine and shake, concentrate and dry to obtain an extract, also known as verbena extract.

[0045] Among them, after combining and shaking, take 1 ml of the extract, filter through a 0.22 μm filter membrane, and take the filtrate as the test sample solution.

[0046] 3UPLC-Q-TOF / MS detection conditions

[0047] 3.1 Chromatographic conditions: Waters Acquity UPLC BEH C18 column (2.1×100 mm, 1.7 μm), mobile phase system consisting of A (acetonitrile) and B (0.1% formic acid in water), flow rate 0.3 mL / min, column temperature 35°C, injection volume 5 μL; gradient elution was used, the gradient program was as follows: Figure 1 As shown;

[0048] 3.2 Mass spectrometry conditions: Electrospray ionization source (ESI), capillary voltage 3.0 kV in positive ion mode and 2 kV in negative ion mode; ion source temperature 120 °C, sample cone voltage 40 V, cone gas flow rate 50 L / h, desolvation temperature 450 °C, desolvation gas flow rate 800 L / h, scanning range m / z 50 - 1500, and leucine enkephalin as internal reference calibration solution for real-time molecular weight calibration.

[0049] 4 Data collection and data processing

[0050] Retrieve through databases (such as CNKI, PubMed, TCMSP, etc.), compare and analyze MS and MS2 data information under positive and negative modes, combine retention time, peak intensity, etc. to conduct structural identification of each chromatographic peak, clarify the chemical components contained in the verbena extract, and set the error value between the theoretical molecular weight and the actual molecular weight to 10 ppm during the analysis process.

[0051] 5. Animal experiments

[0052] 5.1 Establishment of radiation - damaged rat model and dosing groups

[0053] Five days before the experiment, 24 male SD rats were given free access to food and water for adaptive feeding; they were randomly divided into a normal group, a model group, a low - dose verbena extract group, and a high - dose verbena extract group, with 6 rats in each group.

[0054] According to the dosing methods and doses shown in Table 1, rats were administered drugs by gavage daily for 14 consecutive days.

[0055] Except for the normal group, the remaining groups were irradiated with 5 Gy 137 Csγ - rays once all over the body of the rats on the 7th day to establish a radiation - damaged model; the rats were fasted for 12 h (allowed free access to water) before sampling.

[0056] Table 1 Construction of radiation - damaged rat model and dosing scheme

[0057] Group Administration method and dosage Quantity Normal group (Group C) 2 mL distilled water 6 Model group (Group M) 2 mL distilled water 6 Low-dose verbena extract group (Group L) 2 mL verbena extract solution (21.3 mg / ml) 6 High-dose verbena extract group (Group H) 2 mL verbena extract solution (106.6 mg / ml) 6

[0058] 5.2 Evaluation of anti - radiation effect

[0059] Collect rat blood and spleen samples for subsequent research, observe the spleen tissue morphology, and fix it with paraformaldehyde solution; section and observe the fixed spleen tissue, and measure the complete blood count.

[0060] 5.3 Peripheral blood cell count

[0061] Take part of the collected rat blood into a centrifuge tube, and detect peripheral blood cells such as white blood cells (WBC), neutrophils (NE), lymphocytes (LY) with an automatic blood cell analyzer.

[0062] 5.4 ELISA detection of inflammatory factor levels

[0063] Take a part of the collected rat blood into a centrifuge tube, centrifuge at 4 °C to separate the serum; prepare each working solution according to the ELISA detection kit instructions, operate according to the instructions, add the stop solution after incubation in the dark; use an enzyme-linked immunosorbent assay reader to detect the absorbance value at 450 nm, substitute the absorbance value into the standard curve, and calculate the concentrations of TNF-α, IL-6 and IL-17 in the sample.

[0064] Experimental results

[0065] 1 Mass spectrometry analysis results

[0066] A total of 34 components were found in the verbena extract, including 6 iridoid compounds, 11 phenylethanoid glycoside compounds, 12 flavonoid compounds, 4 triterpenoid compounds, and 1 diterpenol compound; the base peak chromatogram of the verbena extract is shown in Figure 2 , (A) is the negative ion mode, (B) is the positive ion mode; the UPLC-Q-TOF / MS data are summarized in Figure 3 .

[0067] 2 Animal experiments

[0068] 2.1 Changes in animal status

[0069] After the model was established, the rats in the model group, the low-dose verbena extract group, and the high-dose verbena extract group all showed a listless state.

[0070] 2.2 Effects on spleen tissue

[0071] The spleen of the rats in the model group showed significant atrophy and a significant decrease in volume;

[0072] Compared with this model group, the volume of the spleen of the rats intervened with low-dose verbena extract and high-dose verbena extract was significantly increased;

[0073] As Figure 4 shown, C is the normal group, M is the model group, L is the low-dose verbena extract group, and H is the high-dose verbena extract group.

[0074] 2.3 Effects on peripheral blood cell count

[0075] Compared with the normal group, the contents of white blood cells (WBC), neutrophils (NE), and lymphocytes (LY) in the peripheral blood of the rats in the model group were significantly decreased;

[0076] Compared with the model group, after intervention with low-dose verbena extract and high-dose verbena extract, the contents of WBC, NE, and LY in the peripheral blood of the rats showed an increasing trend. The results are shown inFigure 5 , C is the normal group, M is the model group, L is the low-dose verbena extract group, and H is the high-dose verbena extract group; ## indicates P < 0.01 compared with the normal group, * indicates P < 0.05 compared with the model group, where P is the p value of the t-test.

[0077] 2.4 Effects on inflammatory factor levels

[0078] Compared with the normal group, the serum levels of TNF-α, IL-6, and IL-17 in the model group rats were significantly increased;

[0079] Compared with the model group, the low-dose and high-dose verbena extract groups could reduce the levels of TNF-α, IL-6, and IL-17 in serum in a dose-dependent manner. Verbena has a significant anti-inflammatory effect.

[0080] See the results Figure 6 , C is the normal group, M is the model group, L is the low-dose verbena extract group, and H is the high-dose verbena extract group; # indicates P < 0.05 compared with the normal group, ## indicates P < 0.01 compared with the normal group, * indicates P < 0.05 compared with the model group, and ** indicates P < 0.01 compared with the model group, where P is the p value of the t-test.

[0081] 2.5 Splenic histopathological effects

[0082] In the normal group, the red and white pulp of the spleen tissue were clearly demarcated, and no obvious pathological changes were observed; in the model group, the red and white pulp were not clearly demarcated, the red pulp was congested, and the white pulp lymphocyte structure was disordered; in the low-dose and high-dose verbena extract groups, the red and white pulp of the spleen were well demarcated, and the degree of damage was significantly reduced compared with the model group, indicating that verbena extract can alleviate the pathological changes of the spleen caused by radiation damage; Figure 7 As shown, C is the normal group, M is the model group, L is the low-dose verbena extract group, and H is the high-dose verbena extract group.

[0083] In this specification, the terms "connect," "install," and "fix" should be understood broadly. For example, "connect" can refer to a fixed connection, a detachable connection, or an integral connection; it can refer to a direct connection or an indirect connection through an intermediary. Those skilled in the art will understand the specific meanings of these terms in this application based on the specific circumstances.

[0084] In the description of this specification, the descriptions of the terms "one embodiment", "some embodiments", etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or instance. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0085] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A preparation method of a verbena extract with anti-radiation effect, characterized in that, It includes the following steps: 1) Weigh specified weight portions of verbena and ethanol solution; 2) Perform alcohol extraction on the verbena and ethanol solution at a material-liquid ratio of 1:5 to 1:40, heat under reflux for extraction 1 to 3 times, each time for 1.5 to 3 hours; 3) Combine, concentrate, and dry the products obtained from each heat reflux extraction in step 2) to obtain an extract, that is, obtain the verbena extract.

2. The preparation method of the verbena extract with anti-radiation effect according to claim 1, characterized in that, The ethanol solution is an ethanol solution with a concentration range of 20 to 100%.

3. The preparation method of the verbena extract with anti-radiation effect according to claim 1, characterized in that, The ethanol solution is an ethanol solution with a concentration of 100%.

4. The preparation method of the verbena extract with anti-radiation effect according to claim 1, characterized in that, The material-liquid ratio of the verbena to the ethanol solution is 1:

20.

5. The preparation method of the verbena extract with anti-radiation effect according to claim 1, characterized in that, In step 2), the heat reflux extraction time each time is 2 hours, filter to obtain the extract, repeat the extraction 3 times, and combine, concentrate, and dry the extracts obtained 3 times.

6. A verbena extract prepared by the preparation method of the verbena extract with anti-radiation effect according to any one of claims 1-5.

7. Use of verbena according to claim 6 in a medicament for anti-radiation effect, characterized in that, The anti-radiation effect drug is a preparation composed of a traditional Chinese medicine extract and a pharmaceutically acceptable carrier.

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