Anti-tumor alkaline land dandelion root extract as well as preparation method and application thereof

Abstract

The invention provides an anti-tumor alkaline land dandelion root extract as well as a preparation method and application thereof, and belongs to the technical field of biology. The alkaline land dandelion root extract with an obvious inhibiting effect on tumor generation and metastasis is extracted from the root of alkaline land dandelion, can obviously reduce the invasion rate of tumor cells, inhibits in-situ growth of the tumor cells, has an obvious inhibiting effect on tumor generation and metastasis, and can be used for preparing an anti-tumor drug. Moreover, a theoretical basis is provided for development and utilization of the medicinal value of the alkali land dandelion resource, and a new thought is provided for clinical treatment of related diseases such as cancers and finding of potential drug targets.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an anti-tumor alkaline dandelion root extract, a preparation method and application thereof. Background Art

[0002] Due to factors such as environmental pollution and bad living habits, the incidence of cancer is increasing. As of 2023, there will be more than 20 million new cancer patients each year worldwide, which has become a major global public health issue. Studies have shown that benign in situ tumor proliferation can affect the normal physiological functions of certain organs and systems, but the mortality rate is not high. More than 90% of cancer deaths are caused by late metastasis, recurrence, or the formation of discontinuous secondary foci. Therefore, how to effectively control or even reverse the growth and metastasis of tumor cells is of great significance to the clinical treatment of patients.

[0003] Dandelion (Taraxacum mongolicum Hand.-Mazz.), alkali dandelion (Taraxacum sinicumKitag., also known as Chinese dandelion-Taraxacum borealisinense Kitam.) or several species of the same genus, belong to the Asteraceae family, Taraxacum genus, perennial herb, strong adaptability, wide distribution range, with extremely high edible and medicinal value, as a traditional Chinese medicine. Dandelion was first recorded in the Tang Dynasty's "New Compendium of Materia Medica". It is a dried whole plant with roots. It tastes bitter and sweet, is cold in nature, and enters the liver and stomach meridians. It has the effects of clearing away heat and detoxification, reducing swelling and dispersing knots, and diuresis and relieving stranguria. It is widely used in clinical practice and can be used to treat breast abscess, lung abscess, intestinal abscess, scrofula, red eyes and many other diseases.

[0004] Modern pharmacological studies have shown that dandelion (extract) and its active ingredients have certain therapeutic effects on the occurrence, invasion and metastasis of tumors. Dandelion thiazolinone can inhibit the migration and invasion levels of TNBC MDA-MB-231 cells through the ERK / Slug signaling axis. At the same time, dandelion total flavonoids have an inhibitory effect on lung cancer, which may be due to the improvement of the host's protective immune response through a milder tumor growth inhibition effect than cyclophosphamide. In addition, network pharmacology analysis and cell culture experiments have confirmed that dandelion thiazolinone can treat gastric cancer through a multi-target-multi-pathway approach. The above studies have confirmed the anti-tumor efficacy of dandelion, but which active ingredients of dandelion are at work and the potential molecular mechanism need to be further explored.

[0005] At present, due to factors such as the harvesting origin, harvesting method, and processing method, the quality of dandelion medicinal materials on the market varies, and no further distinction and labeling is made between ordinary dandelions and alkaline dandelions. In view of the impact of saline-alkali environment on plant secondary metabolites and nutrients, previous studies have found that dandelion strains grown under salinized soil or salt water stress can accumulate more effective ingredients and have better quality. Therefore, it is crucial to further develop the medicinal effects of alkaline dandelions. Summary of the invention

[0006] The purpose of the present invention is to provide an anti-tumor alkaline dandelion root extract, a preparation method and an application. The present invention extracts an alkaline dandelion root extract that has a significant inhibitory effect on tumor occurrence and metastasis, provides a theoretical basis for the development and utilization of the medicinal value of alkaline dandelion resources, and provides a new idea for the clinical treatment of cancer and other related diseases and the search for potential drug targets.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing an anti-tumor alkaline dandelion root extract, comprising the following steps:

[0009] (1) drying and grinding the roots of dandelions grown in alkaline soil to obtain root dry powder;

[0010] (2) Soaking the root dry powder in ethanol, filtering to separate the dry powder residue and the filtrate, keeping the dry powder residue for later use, centrifuging the filtrate, and taking the supernatant;

[0011] (3) adding the dry powder residue from step (2) into deionized water, boiling over high heat, filtering, and centrifuging to obtain the supernatant;

[0012] (4) The supernatants obtained in steps (2) and (3) are combined, centrifuged, and filtered to obtain an alkaline dandelion root extract.

[0013] Preferably, in step (2), the mass volume ratio of the root dry powder to ethanol is 0.5-1.5 g: 5-15 mL; and the ethanol has a volume fraction of 60-80%.

[0014] Preferably, in step (2), the soaking time is 36 to 48 hours, and the soaking temperature is 3 to 5°C; the centrifugation time is 1.5 to 2.5 hours, and the centrifugal speed is 4000 to 4500 r / min.

[0015] Preferably, in step (3), the mass volume ratio of the dry powder residue to deionized water is 0.5-1.5 g: 5-15 mL; the high-fire decoction time is 1-2 h; the centrifugation time is 1.5-2.5 h, and the centrifugal speed is 4000-4500 r / min.

[0016] The invention also provides a method for preparing an anti-tumor alkaline dandelion root extract and the alkaline dandelion root extract is prepared.

[0017] The present invention also provides an application of the alkaline soil dandelion root extract in the preparation of a drug for treating tumors.

[0018] The beneficial effects of the present invention compared with the prior art are:

[0019] The invention extracts an alkaline dandelion root extract having a significant inhibitory effect on tumor occurrence and metastasis from the alkaline dandelion root. The alkaline dandelion root extract can significantly reduce the invasion rate of tumor cells, inhibit the in situ growth of tumor cells, and has a significant inhibitory effect on tumor occurrence and metastasis. It also provides a theoretical basis for the development and utilization of the medicinal value of alkaline dandelion resources, and provides a new idea for the clinical treatment of cancer and other related diseases and the search for potential drug targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0021] Figure 1 This is a schematic diagram of the correlation of Binpu-3 root extract samples in positive ion mode in Example 1 of the present invention;

[0022] Figure 2 Schematic diagram of the correlation of Binpu-3 root extract samples in negative ion mode in Example 1 of the present invention

[0023] Figure 3 The base peak chromatogram under positive ion mode in Example 1 of the present invention Figure 1 Mark peak;

[0024] Figure 4 Base peak chromatogram under negative ion mode in Example 1 of the present invention Figure 1 Mark peak;

[0025] Figure 5 Ras in Experimental Example 1 of the present invention V12 / lgl - / - Schematic diagram of tumor hybridization;

[0026] Figure 6 The Binpu-3 root extract in Experimental Example 1 of the present invention can inhibit Ras V12 / lgl - / -Figure 2 shows the induced tumor growth and invasion. a is a schematic diagram of the head tissue complex of the third instar larvae of the normal control group, in which the eye imaginal disc and brain hemisphere cells are marked by green fluorescent protein (GFP); bf is Ras V12 / lgl - / - Schematic diagram of the head tissue complex of the third instar larvae in the tumor model group and each drug intervention group; g-1 is a schematic diagram of the corresponding dissected abdominal ganglion (VNC) in af, which is affected by Ras V12 / lgl - / - Tumor cells invade to varying degrees. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this article are open-ended terms, meaning including but not limited to.

[0032] Example 1

[0033] Example 1 of the present invention provides a method for preparing an anti-tumor alkaline dandelion root extract, and detects its components. The specific steps are as follows:

[0034] (1) Alkaline soil dandelion (Binpu-3) mildly saline-alkali soil field planting:

[0035] The seeds of the alkaline dandelion resource Binpu-3 (variety identification number: Jirenyao (2023) 015) are sown in the greenhouse and transplanted to the base field when 3-4 leaves grow. The field planting time is from April to October each year. The furrow depth is 2-3cm and the furrow distance is 30cm. During the cultivation period, a negative pressure drip irrigation automatic irrigation system is adopted, in which the soil matrix potential controlled by negative pressure is -15kPa. When the leaves grow to 10cm-15cm, 10kg-15kg of urea is applied per mu. The field soil is coastal slightly saline-alkali soil (salt content 0.2%-0.3%), and the soil bulk density is 1.4-1.6g / cm 3 , groundwater mineralization is >10g / L, soil pH is 7.8-8.5, and organic matter content is 1.426%.

[0036] (2) Preparation of Alkaline Dandelion Root Extract

[0037] When alkaline dandelions grow and mature in a field with slightly saline-alkali soil, collect the whole dandelions and wash them, separate the underground part (referred to as roots) from the aboveground part (containing a small amount of flowers, referred to as leaves), place them in an oven and dry them at 60°C until the moisture content is less than 13%, then grind them through a No. 4 sieve to obtain root dry powder.

[0038] Weigh 100g of Binpu-3 root powder, put it into a gauze bag, soak it in 1000mL 70% ethanol at 4℃ for 48 hours, filter it after soaking, centrifuge the alcohol filtrate at 4200 rpm for 2 hours, and collect the alcohol precipitation supernatant; then, soak the dry powder residue in 1000mL deionized water, boil it for 1h over high heat, filter it after decoction, centrifuge the decoction at 4200r / min for 2 hours, and collect the water extraction supernatant by filtration; finally, combine the two extracts, centrifuge them, filter them through a 0.45μm filter membrane, and adjust the volume to 100mL to prepare an alkaline dandelion root extract with a final concentration of 1g / mL. The dandelion root liquid was divided into centrifuge tubes, 45mL / tube, and frozen at -80℃ for subsequent experimental studies.

[0039] (3) Analysis of components of dandelion root extract (UPLC-HRMS)

[0040] 1) Root extract sample preparation

[0041] Take 600 μL of the sample into a 1.5 mL centrifuge tube, add 400 μL of pure methanol, and vortex mix for 10 seconds; take 200 μL of the above solution, add 200 μL of 40% methanol aqueous solution, vortex mix for 10 seconds, and centrifuge for 15 minutes (16000g, 4°C) to obtain the supernatant.

[0042] 2) Chromatography-mass spectrometry conditions

[0043] A. Chromatographic conditions

[0044] The samples were separated using a Vanquish UHPLC (Thermo Fisher Scientific, Bremen, Germany) ultra-high performance liquid chromatography system combined with an ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) column; the column temperature was 35° C.; the flow rate was 0.3 mL / min; the mobile phase composition was A 0.1% formic acid aqueous solution, B 0.1% formic acid acetonitrile solution; and the gradient elution was performed according to Table 1.

[0045] Table 1 Chromatographic elution conditions

[0046] Time (minutes) Mobile phase A (%) Mobile phase B (%) Initial 95 5 3 75 25 8.5 55 45 14 5 95 17 2 98 17.2 95 5 20.0 95 5

[0047] B. Mass spectrometry conditions

[0048] The Q-Exactive HFX mass spectrometer was used to collect the primary and secondary spectra of the samples.

[0049] The Q-Exactive HFX mass spectrometer sample was coupled to the UHPLC system, and mass spectra were collected in the electrospray ionization (ESI) positive and negative ion modes. The spray voltage was 3800V (ESI+) / 3500V (ESI-), the sheath gas pressure was 45arb, the auxiliary gas pressure was 20arb, the ion transfer tube temperature was 320℃, and the nebulization temperature was 350℃; the detection mode was full scan / data-dependent secondary scan (Full-MS / dd-MS2) mode, the primary and secondary resolutions were 60000 and 15000, respectively, the top 10MS1 ions were used to obtain MS / MS spectra, and the collision energies (CEs) used step-normalized energy levels of 20, 40, and 60; the primary mass-to-charge ratio scanning range was 90-1300.

[0050] C. Sample testing and analysis

[0051] Accurately pipette 6 μL of the prepared sample solution and inject it into LC-MS for analysis. Repeat the injection of the sample 5 times.

[0052] (4) Data analysis: The raw data file in .raw format was imported into proteoWizard and converted into .mzXML format. XCMS software was used for peak alignment, retention time correction and peak extraction. The chemical substances in the sample were structurally identified by searching the local high-resolution mass spectrometry database of traditional Chinese medicine. Among them, the primary mass error was less than 25ppm, and the secondary fragmentation spectrum matching score was greater than 0.7. The higher the score, the higher the spectrum similarity. It is generally believed that the identification result is reliable if it is above 0.7.

[0053] (5) Experimental data quality evaluation

[0054] This experiment evaluates the stability of the instrument and the reliability of the data. Pearson correlation analysis is performed on the test results of traditional Chinese medicine samples. Figure 1 and 2 As shown. Generally, a correlation coefficient greater than 0.9 indicates good correlation. The results of this experiment show that the correlation coefficients between samples are all above 0.9, indicating that the experimental repeatability is good and the test data is stable and reliable.

[0055] (6) Compound identification

[0056] 1) Compound Quantity Identification

[0057] The compound identification was performed by searching the local high-resolution mass spectrometry database of traditional Chinese medicine. The primary mass error was less than 25 ppm, and the secondary fragmentation spectrum matching score was greater than 0.7. The chemical components in the dandelion root extract samples were analyzed and identified, and the statistical results are shown in Table 2.

[0058] Table 2 Statistics of the number of compounds identified in positive and negative ion modes

[0059]

[0060]

[0061] Note: Total of positive and negative ion modes: If the same compound is identified in positive and negative ion modes, it will not be counted repeatedly

[0062] 2) Sample base peak chromatogram (BPC) analysis

[0063] In the sample positive and negative ion base peak chromatograms (BPC), the peaks with higher abundance are confirmed by peak shape and secondary spectrum inspection, and then the positive and negative ion graphs are marked with chromatographic peak numbers in numerical order, such as Figure 3 , Figure 4 shown.

[0064] Figure 3 and Figure 4The 33 high-abundance chromatographic peaks marked in the CCP-labeled alkaline soil dandelion root extract samples, the corresponding identification compounds and their content information are shown in Table 3.

[0065] Table 3 Chemical composition identification results of BPC icon peaks

[0066]

[0067] Test Example 1

[0068] Test Example 1 of the present invention detected the anti-tumor effect of the dandelion root extract prepared in Example 1, and the specific steps were as follows:

[0069] (1) Effect of dandelion root extract on Drosophila Ras V12 / lgl - / - Tumor suppression

[0070] 1) Fruit fly breeding

[0071] The fruit fly strain was raised at 25°C, 60% relative humidity, in normal culture medium. Conventional fruit fly culture medium (1L) contains: 50g sucrose, 10g agar powder, 60g corn flour, 8g yeast, 6mL propionic acid, and 1000mL ultrapure water. On this basis, the corresponding volume of Binpu-3RE mother solution was added according to the final concentration to obtain the drug-doped culture medium. After anesthesia on a plate with CO2, experimental operations such as selection of male and female fruit flies, collection of virgin flies, hybridization, and phenotypic observation were performed.

[0072] 2) Ras V12 / lgl - / - Model building, use and intervention

[0073] Select w 1118 Female flies and w 1118- ,ey-Flp act>y + >GAL4 UAS-GFP / Cyo male flies were crossed and raised in normal culture medium, and the third-instar larvae of the offspring with the genotype of ey-Flp act>y+>GAL4 UAS-GFP / + were collected. The head tissue complex was dissected and recorded under a stereofluorescence microscope. The tissue labeled with green fluorescent protein (GFP) was normal cell and served as the control group.

[0074] Select yw ey-Flp; tub-GAL80 FRT40A; act>y+>GAL4 UAS-GFP female flies and w; lgl 4 FRT40A UAS-Ras V12 / Cyo male flies were hybridized and reared in normal medium or drug-added medium. The genotype of the flies was yw ey-Flp; tub-GAL80 FRT40A / lgl 4 FRT40A UAS-Ras V12 ; act>y+>GAL4 UAS-GFP / + third instar larvae (see Figure 5 ), which can produce Ras under the induction of ey-Flp / FRT V12 / lgl - / - Tumor tissues of somatic chimeric clones (GFP-labeled) were used as the model group or the drug-added group.

[0075] Larvae samples from each group were collected, dissected, observed and counted. V12 / lgl - / - Tumor growth and metastasis.

[0076] Drosophila Ras V12 / lgl - / - The tumor invasion model was used to more comprehensively evaluate the anti-tumor effect of Binpu-3RE. First, the activated form of Ras, a member of the EGFR signaling pathway, was specifically expressed in the eye-antenna imaginal cells of Drosophila using ey-FLP / FRT-mediated somatic cell chimeric cloning technology and the GAL4 / UAS expression system (also known as the ey-Flp / MARCM system). V12 , and homozygous mutation of cell polarity gene lethal giant larvae (lgl, tumor suppressor gene), establishing Ras V12 / lgl - / - Tumor invasion model. Compared with the control group (the eye-antennae imaginal disc cells of the wild type were marked by GFP, Figure 6 a, g), Ras V12 / lgl - / - Chimeric cells (GFP-labeled) can overgrow in situ, produce tumor-like proliferation, and break through the basement membrane to invade the adjacent ventral nerve cord (VNC), with an invasion rate of 71.3% ( Figure 6 b, h, m, n).

[0077] In addition, these invasive tumor cells also affected the normal function of the brain hemisphere (BH) of Drosophila larvae, resulting in the obstruction of the normal development process of the larvae to the pupal stage, and finally stagnation in the third instar larvae and death. Compared with the above tumor invasion model group, Binpu-3RE intervention can significantly inhibit Ras V12 / lgl - / -Induced tumorigenesis and invasion, but the inhibitory effect was not dose-dependent with the drug concentration ( Figure 6 b, c, d, e, f, h, i, g, k, l, m, n), among which 12.50 mg / mL, 25.00 mg / mL and 50.00 mg / mL concentration groups could significantly inhibit the in situ growth of tumor cells (GFP labeled) ( Figure 6 d, e, f), 12.50mg / mL and 25.00mg / mL can significantly inhibit the proportion of tumor cells invading VNC ( Figure 6 j, k). Based on the statistical results of tumor size and invasion rate, Binpu-3RE had an inhibitory effect on Ras V12 / lgl - / - The induced tumorigenesis and invasion inhibition effects were the best.

[0078] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing an anti-tumor alkaline dandelion root extract, characterized in that: The steps include: (1) drying and grinding the roots of dandelions grown in alkaline soil to obtain dry root powder; (2) Soak the root dry powder in ethanol, filter and separate the dry powder residue and the filtrate, keep the dry powder residue for later use, centrifuge the filtrate and take the supernatant; (3) adding the dry powder residue from step (2) into deionized water, boiling over high heat, filtering, and centrifuging to obtain the supernatant; (4) The supernatants obtained in steps (2) and (3) are combined, centrifuged, and filtered to obtain an alkaline dandelion root extract.

2. The method for preparing the anti-tumor alkaline dandelion root extract according to claim 1, characterized in that: In step (2), the mass volume ratio of the root dry powder to ethanol is 0.5-1.5 g:5-15 mL; and the ethanol has a volume fraction of 60-80%.

3. The method for preparing the anti-tumor alkaline dandelion root extract according to claim 1, characterized in that: In step (2), the soaking time is 36-48 hours, and the soaking temperature is 3-5°C; the centrifugation time is 1.5-2.5 hours, and the centrifugation speed is 4000-4500 r / min.

4. The method for preparing the anti-tumor alkaline dandelion root extract according to claim 1, characterized in that: In step (3), the mass volume ratio of the dry powder residue to deionized water is 0.5-1.5 g:5-15 mL; the high-fire decoction time is 1-2 h; the centrifugation time is 1.5-2.5 h, and the centrifugal speed is 4000-4500 r / min.

5. An alkaline dandelion root extract prepared according to the method for preparing the anti-tumor alkaline dandelion root extract according to any one of claims 1 to 4.

6. Use of the alkaline dandelion root extract according to claim 5 in the preparation of drugs for treating tumors.

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