Application of extracts from Herba Lycopodii in the preparation of anti-tumor drugs

Abstract

The invention discloses an application of a soft spot grass extract in the preparation of anti-tumor drugs. The preparation method is to take the dry whole grass of the soft spot grass, wash it with water, dry it and cut it into small pieces, extract it with 5-10 times the amount of solvent at constant temperature reflux for 2-4 times, each time for 1-2.5h, combine the extracts, and concentrate under reduced pressure to obtain a total extract; add 1-3 times the mass of distilled water to the obtained total extract for suspension, use an ultrasonic oscillator to assist the suspension, obtain a suspension, then add 2-5 times the volume of petroleum ether to extract 2-4 times, and combine the petroleum ether extracts; the obtained water layer is extracted 2-4 times with 2-5 times the volume of ethyl acetate, and the ethyl acetate extracts are combined, and the solvent is recovered under reduced pressure to obtain a soft spot grass extract. The invention provides a preparation method of a soft spot grass extract, and the extracted extract is applied to the preparation of anti-tumor drugs, the medicinal value of the soft spot grass is developed, and a theoretical basis is provided for the treatment of tumors and the research and development of new drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor drug preparation, and more specifically to the application of a Herba Lysimachiae extract in the preparation of anti-tumor drugs. Background Art

[0002] The main treatment methods for tumors include surgery, drug chemotherapy, radiotherapy, immunotherapy, and traditional Chinese medicine treatment, and chemotherapy is currently the main method for treating malignant tumors. The main types of chemotherapy drugs include apoptosis inducers, tumor cytotoxic agents, cell senescence inducers, tumor resistance reversal agents, cell differentiation inducers, tumor chemoprevention agents, and tumor metastasis inhibitors. The reason why anti-tumor treatment is difficult is that the efficacy of chemotherapy is uncertain and the adverse reactions are significant. Therefore, it is particularly important to find new therapeutic targets, anti-cancer active drugs, and research methods.

[0003] The main sources for screening new anti-tumor drugs include active ingredients from natural products, synthesis of new chemical substances, biotherapeutic drugs, and new uses of old drugs. The outstanding efficacy of traditional Chinese medicine in tumor prevention and treatment has made it increasingly popular among patients. Traditional Chinese medicine is trying to change the status quo of tumor prevention and treatment with its unique theories. As the main material basis for the efficacy of traditional Chinese medicine, traditional Chinese medicine is currently one of the main sources of natural products for screening new anti-tumor drugs.

[0004] The whole herb of the Boraginaceae plant is used as medicine. It tastes slightly bitter and astringent, is flat in nature, has a little poison, belongs to the lung meridian, and has the effects of relieving cough and stopping bleeding. It has a long history of folk application and has a definite effect. During the visit and data collection, it was found that many old Chinese medicine practitioners used it in clinical treatment, and the effect was remarkable. Some experienced elderly people would often pick it themselves for emergency use. In addition, a survey of wheat field weeds was conducted in many areas such as Yangzhou City and Guanzhong, Jiangsu Province. The results showed that there were weak spot grass, which was not harmful in itself and had certain medicinal value, but was regarded as a weed that harmed farmland, which seriously subverted the medicinal value of weak spot grass. As a medicinal material included in the "Compilation of Common Folk Herbs" and "Chinese Materia Medica", weak spot grass has now fallen into the ranks of weeds and lost its use.

[0005] Therefore, how to bring out the medicinal value of the herb and prepare extracts for use in tumor treatment is a technical problem that those skilled in the art need to solve urgently. Summary of the invention

[0006] In view of this, the present invention provides a method for preparing an extract of the herb, and applies the extracted extract to the preparation of anti-tumor drugs, thereby developing the medicinal value of the herb, and providing a theoretical basis for the treatment of tumors and the development of new drugs.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] Application of extracts of Herba Lycopodii in the preparation of anti-tumor drugs.

[0009] Preferably, the tumor includes leukemia cells HL-60, lung cancer cells A549, liver cancer cells SMMC-7721, breast cancer cells MCF-7 and colon cancer cells SW480.

[0010] As the same inventive concept as the above technical solution, the present invention also claims a method for preparing a Herba Lysimachiae extract, the process comprising:

[0011] (1) Extraction: Take the dried whole herb of the soft spot grass, wash it with water, dry it, and cut it into small pieces. Extract it with 5-10 times the amount of solvent under constant temperature reflux for 2-4 times, each time for 1-2.5 hours. Combine the extracts, concentrate under reduced pressure, and obtain the total extract;

[0012] (2) Extraction: add 1-3 times the mass of distilled water to the total extract obtained in step (1) for suspension, use an ultrasonic oscillator to assist the suspension, and then add 2-5 times the volume of petroleum ether to extract 2-4 times, and combine the petroleum ether extracts; the obtained water layer is extracted 2-4 times with 2-5 times the volume of ethyl acetate, and the ethyl acetate extracts are combined. The solvent is recovered under reduced pressure to obtain a Herba Lysimachiae extract.

[0013] Preferably, in step (1), the length of the small segment is 0-3 cm.

[0014] Preferably, in step 1), the drying is carried out in a constant temperature drying oven at 50°C.

[0015] Preferably, in step (1), the solvent is one of 95% ethanol, 75% ethanol and methanol.

[0016] Preferably, in step (1), 1 kg of the whole herb of the Herba Lycopodii is added with 10 L of extraction solvent, and the extraction is performed under constant temperature reflux for 3 times, with each reflux for 2 hours.

[0017] Preferably, in step 1), the temperature of the constant temperature reflux is 80°C.

[0018] It can be known from the above technical scheme that compared with the prior art, the extract of the present invention is made of a single dried whole herb of the soft spot grass as a raw material (in the fields of Caoji Township, Suyu District, Suqian City, Jiangsu Province, identified as the soft spot grass of the Boraginaceae family by Associate Researcher Huang Yunfeng of Guangxi Traditional Chinese Medicine Research), and is prepared by the steps of reflux, extraction, concentration, etc., and its extraction process is simple and low in cost. The results of the anti-tumor activity screening experiment showed that the extract of the present invention has a significant inhibitory effect on the 5 screened tumor cell strains, among which the inhibitory effect on the lung cancer A459 and liver cancer SMMC-7721 tumor cell strains is stronger, and has great development potential. It can not only solve the "weed problem", which is beneficial to environmental protection, but also drive local economic development, and provide a large number of cheaper and more effective Chinese medicinal materials for clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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 or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0020] Figure 1 The attached figure is a diagram showing the inhibition of tumor cells by extract solutions of different concentrations;

[0021] Figure 2 Accompanying drawing is the total ion current chromatogram of ethyl acetate portion;

[0022] Figure 3 The attached figure shows the cleavage pathway of Calycosin (flavonoids);

[0023] Figure 4 The attached figure shows the cleavage pathway of 5,7-Dihydroxy-4-methylcoumarin (Coumarins);

[0024] Figure 5 The attached figure shows the cleavage pathway of Caffeic Acid (organic acids);

[0025] Figure 6 The attached figure shows the cleavage pathway of Vitexin2"-O-β-D-Glucoside (glycosides). DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Example 1

[0028] A preparation method of a soft spot grass extract, the process is: take 2kg of dry whole grass of soft spot grass, wash it lightly with water to remove sand, dry it in a constant temperature drying oven at 50℃, cut it into small pieces, the length of which is controlled within 3cm, add 10 times the amount of 95% ethanol extraction solvent per kilogram of medicinal material, extract it 3 times with constant temperature reflux at 80℃, each time for 2h, and obtain 95% ethanol solvent extract; combine the extracts, and concentrate under reduced pressure to obtain 242.75g of concentrate. Add 1.5 times the weight of distilled water to suspend the concentrate, use an ultrasonic oscillator to assist in suspension, obtain a suspension, add 2 times the volume of petroleum ether of the suspension to extract 3 times, combine the petroleum ether extracts; extract the obtained water layer with 2 times the volume of ethyl acetate 3 times, and recover the solvent under reduced pressure to obtain 8.73g of ethyl acetate extract, which is the Chinese medicine extract of the present invention.

[0029] Example 2

[0030] A preparation method of a soft spot grass extract, the process is: take 2kg of dry whole grass of soft spot grass, wash it lightly with water to remove sand, dry it in a constant temperature drying oven at 50℃, cut it into small pieces, the length of which is controlled within 3cm, add 10 times the amount of 75% ethanol extraction solvent per kilogram of medicinal material, extract it 3 times at 80℃ constant temperature reflux, each time for 2h, and obtain 75% ethanol solvent extract; combine the extracts, and concentrate under reduced pressure to obtain 280.90g of concentrate. Add 1.5 times distilled water for suspension, use an ultrasonic oscillator to assist suspension, obtain a suspension, add 2 times the volume of petroleum ether of the suspension for extraction 3 times, combine the petroleum ether extracts; extract the obtained water layer with 2 times the volume of ethyl acetate 3 times, and recover the solvent under reduced pressure to obtain 7.02g of ethyl acetate extract, which is the Chinese medicine extract of the present invention.

[0031] Example 3

[0032] A preparation method of a soft spot grass extract, the process is: take 2kg of dry whole grass of soft spot grass, wash it lightly with water to remove sand, dry it in a constant temperature drying oven at 50℃, cut it into small pieces, the length of which is controlled within 3cm, add 10 times the amount of methanol extraction solvent per kilogram of medicinal material, extract it 3 times at 80℃ constant temperature reflux, each time for 2h, and obtain a methanol solvent extract; combine the extracts, and concentrate under reduced pressure to obtain 234.95g of a concentrate. Add 1.5 times the weight of the concentrate to suspend it, use an ultrasonic oscillator to assist in suspension, obtain a suspension, add 2 times the volume of petroleum ether of the suspension to extract 3 times, combine the petroleum ether extracts; extract the obtained water layer with 2 times the volume of ethyl acetate 3 times, and recover the solvent under reduced pressure to obtain 5.26g of ethyl acetate extract, which is the Chinese medicine extract of the present invention.

[0033] Example 4 Verification of the effect of the soft spot grass prepared in Example 1 on the proliferation of five tumor cell strains

[0034] Tumor cell strains

[0035] Leukemia cells HL-60, lung cancer cells A549, liver cancer cells SMMC-7721, breast cancer cells MCF-7, and colon cancer cells SW480 were all purchased from China General Microbiological Culture Collection Center.

[0036] Test drug

[0037] Cisplatin (Mei Lun Biotech, N1001A) and paclitaxel (Mei Lun Biotech, D1106A) were dissolved in 0.9% saline. Cisplatin was prepared into 0.064, 0.32, 1.60, 8.00, and 40 μg / mL solutions, and paclitaxel was prepared into 0.008, 0.04, 0.20, 1.00, and 5.00 μg / mL solutions. The extract of the herb was prepared with DMSO and PBS as solvents and sterilized water to make up to volume, and the concentrations were 100.00, 20.00, 4.00, 0.80, and 0.16 μg / mL solutions, respectively.

[0038] Test methods

[0039] Inoculation of cells: Take cells in logarithmic growth phase, observe them under an inverted microscope and see that they are in good condition, prepare a single cell suspension with culture medium containing 10% fetal bovine serum, and inoculate 3000 to 15000 cells per well into a 96-well plate with a volume of 100 μL per well. The cells are inoculated and cultured 12 to 24 hours in advance. The plates are divided into 4 groups: a blank group (a blank group refers to a group in which nothing is added, no cells are present, and only background subtraction is performed, which is not recorded in the table), an extract group of the present invention, a cisplatin control group, and a paclitaxel control group.

[0040] Add the test solution: the extract of the weak spot grass was screened at concentrations of 100, 20, 4, 0.8, and 0.16ug / ml, with a final volume of 200μL in each well, and 3 replicate wells were set up. The positive control was tested according to the configured concentration, and 3 replicate wells were also set up.

[0041] Color development: After culturing at 37°C for 48 hours, discard the culture medium in the wells for adherent cells, add 20 μL of MTS solution and 100 μL of culture medium to each well; discard 100 μL of culture supernatant for suspended cells, add 20 μL of MTS solution to each well; set up 3 blank replicate wells (a mixture of 20 μL MTS solution and 100 μL culture medium), continue incubation for 2 to 4 hours, and measure the light absorbance value after the reaction is sufficient.

[0042] Colorimetry: Select 492nm wavelength, use a multifunctional microplate reader to read the light absorption value of each well, record the results, and after data processing, draw a cell growth curve with concentration as the horizontal axis and cell proliferation inhibition rate as the vertical axis. Apply the two-point method (Reed and Muench method) to calculate the IC of the compound 50 value.

[0043] Experimental Results

[0044] The results of in vitro anti-tumor activity screening showed that it had a significant inhibitory effect on the proliferation of 5 tumor cell strains, with the strongest inhibitory effect on lung cancer and liver cancer cells, and all inhibitory effects were at the microgram level. As shown in Table 1, the IC 50 The content of 56.72μg / mL, A549 is 41.13μg / mL, SMMC-7721 is 36.69μg / mL, MDA-MB-231 is 48.63μg / mL, and SW480 is 44.48μg / mL. Figure 2 As shown, when the extract concentration reached 100 μg / mL, the inhibitory effect on lung cancer cell lines was the strongest.

[0045] Table 1 In vitro antitumor activity screening

[0046]

[0047] Example 5 Liquid Mass Spectrometry Analysis of Ethyl Acetate Extract

[0048] Sample preparation

[0049] Weigh 30 mg of ethyl acetate extract, add 500 μl 75% methanol water, vortex for 60 seconds, and ultrasonicate for 30 minutes to fully dissolve it. Centrifuge at 17000g for 15 minutes, take the supernatant and vacuum dry it at room temperature. Add 100 μl 50% methanol aqueous solution, vortex for 60 seconds, filter with 0.22um organic filter membrane, and wait for testing.

[0050] Instrument parameter settings

[0051] Mobile phase conditions:

[0052] Column temperature 40°C, sample volume 3 μl

[0053] Positive ion mode: A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile; Negative ion mode: A: water (2 mM ammonium acetate); B: acetonitrile

[0054] The mobile phase elution gradient is shown in Table 2;

[0055] Table 2

[0056]

[0057] Mass spectrometry conditions:

[0058] The AB 5600Triple TOF mass spectrometer can perform primary and secondary mass spectrometry data acquisition based on the IDA function under the control of the control software (Analyst TF 1.7, AB Sciex). In each data acquisition cycle, the molecular ions with the strongest intensity greater than 100 are screened out to collect the corresponding secondary mass spectrometry data. The primary acquisition range is 50-1200, the bombardment energy is 30eV, and 10 secondary spectra are taken every 50ms. The ESI ion source parameters are set as follows: nebulizer pressure (GS1): 60Psi, auxiliary gas pressure: 60Psi, air curtain pressure: 35Psi, temperature: 650℃, spray voltage: 5000V (positive ion mode) or -4000V (negative ion mode).

[0059] Compound structure analysis

[0060] 1) Format conversion: The data is first converted to .abf format using AnalysisBaseFileConverter

[0061] 2) The converted abf files were processed by peak finding, peak alignment and other data processing using MSDIAL ver4.24 software, and the databases of Metlin, MassBank, MoNA and HMDB were independently integrated based on primary and secondary map searches, version V6.0, to obtain the identification results.

[0062] 3) MSDIAL software setting parameters:

[0063] Identification:Accurate mass tolerance(MS1):

[0064] 0.01Da(MS2):0.05Da Identification score cut off: 60％；Retention time tolerance: 0.2min;

[0065] 4) Search for relevant literature reports on the chemical composition of fine-stemmed spot grass and its plants of the same family at home and abroad, and use the PubChem database to collect and organize various chemical component data in the weak spot grass. After collecting data using a liquid chromatography-mass spectrometer, extract the mass spectrum of each chromatographic peak, judge and obtain the precise relative molecular mass of the primary mass spectrum based on information such as quasi-molecular ions, compare it with the literature database, and make a preliminary guess for each chromatographic peak. Screen the secondary mass spectrum with good signal-to-noise ratio to obtain the secondary mass spectrum information of the chromatographic peak, and obtain the corresponding fragment ions of the compound. According to the fragmentation of the ions and combined with the literature data, further compare and guess the chemical composition.

[0066] Result analysis:

[0067] like Figure 2 As shown in Table 1, a total of 34 chemical components were identified, including 12 flavonoids, 9 coumarins, 7 organic acids, 1 naphthoquinone, 2 glycosides and 3 other compounds. Among them, the components that have been reported to have anti-lung cancer activity mainly include 4-Hydroxybenzoic acid, Pinocembrin, Luteolin, 3'-O-Methylluteolin, Hesperetin, Shikonin, 4-Hydroxycoumarin, etc.

[0068] Table 3 Liquid mass analysis results

[0069]

[0070]

[0071]

[0072]

[0073]

[0074] Structural analysis

[0075] The results showed that the ethyl acetate fraction contained 12 flavonoid compounds, the largest number. The quasi-molecular ion peak of compound 3 was m / z 285.04 [MH]-. After removing one molecule of C7H2O4 or one molecule of C3O2, fragment ion peaks of m / z 133.03 and m / z 217.05 were formed. After removing one molecule of O or one molecule of C2H2O, fragment ion peaks of m / z 107.01 and m / z 175.04 were formed. After removing one molecule of C2, a fragment ion peak of m / z 151.00 was formed, which was identified as luteolin. The excimer ion peak of compound 2 was m / z 283.06 [MH]-, the fragment ion peak of m / z 268.04 was formed after removing one molecule of CH3, and the fragment ion peak of m / z 148.01 was formed after removing one molecule of O, which was identified as Calycosin. The fragmentation pathways are as follows: Figure 3 shown.

[0076] There are 9 coumarin compounds, the second largest in number. The quasi-molecular ion peak of compound 20 is m / z 191.03 [MH]-. After removing one molecule of CO or one molecule of CO2, fragment ion peaks of m / z 163.04 and m / z 147.05 are formed, which are identified as 5,7-Dihydroxy-4-methylcoumarin. Figure 4 shown.

[0077] There are 7 organic acid compounds, ranking third. The quasi-molecular ion peak of compound 29 is m / z 179.04 [MH]-. After removing one molecule of C8H7O2 or one molecule of C3H3O3 or one molecule of C3H2O2, fragment ion peaks of m / z 43.99, m / z 93.03 and m / z 109.03 are formed. After removing one molecule of CO or one molecule of H2O, fragment ion peaks of m / z 65.04 and m / z 91.02 are formed, which are identified as Caffeic Acid. The fragmentation pathway is as follows Figure 5 shown.

[0078] Two compounds were identified as glycosides and ranked fourth. The ion peak of compound 17 was m / z 593.15 [MH]-. After removing one molecule of glucoside, a fragment ion peak of m / z 283.06 was formed. After removing one molecule of O or one molecule of -C9H8O2 or one molecule of C8H7O2, fragment ion peaks of m / z 267.07, m / z 135.01 and m / z 148.02 were formed. After removing the CH2 molecule, a fragment ion peak of m / z 253.05 was formed. After removing one C9H5O3 molecule, a fragment ion peak of m / z 93.03 was formed, which was identified as Vitexin 2"-O-β-D-Glucoside. The fragmentation pathways are as follows. Figure 6 shown.

[0079] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0080] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of extracts of Herba Lysimachiae in the preparation of anti-tumor drugs; wherein: The preparation method of the extract of Herba Lysimachiae includes: (1) Extraction: Take the dried whole herb of the soft spot grass, wash it with water, dry it, and then cut it into small pieces of 0-3 cm, extract it with 5-10 times the amount of solvent under constant temperature reflux for 2-4 times, each time for 1-2.5 hours, combine the extracts, and concentrate under reduced pressure to obtain the total extract; the drying is carried out in a constant temperature drying oven at 50°C; the solvent is one of 95% ethanol, 75% ethanol, and methanol; the constant temperature reflux temperature is 80°C; (2) Extraction: add 1-3 times the mass of distilled water to the total extract obtained in step (1) to suspend, use an ultrasonic oscillator to assist the suspension, and then add 2-5 times the volume of petroleum ether to extract 2-4 times, and combine the petroleum ether extracts; the obtained water layer is extracted 2-4 times with 2-5 times the volume of ethyl acetate, and the ethyl acetate extracts are combined, and the solvent is recovered under reduced pressure to obtain a herba spatholobifolia extract; The tumors are leukemia cells HL-60, lung cancer cells A549, liver cancer cells SMMC-7721, breast cancer cells MCF-7 and colon cancer cells SW480.

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