Preparation method and application of alkaloid compound sephaline B
阅读说明：本技术 一种生物碱化合物sophaline B的制备方法及用途 (Preparation method and application of alkaloid compound sephaline B ) 是由 代小勇 张玉波 于 2021-07-20 设计创作，主要内容包括：一种生物碱化合物sophaline B的制备方法及用途,属于天然药物化学技术领域。本发明一方面提供了生物碱化合物sophaline B在制备非小细胞肺癌疾病药物中的用途,一种用于预防或治疗非小细胞肺癌细胞的药物制剂,另一方面提供了生物碱化合物sophaline B的制备方法。本发明从中药材苦豆子中分离的生物碱类化合物sophaline B对肿瘤细胞有很好抑制作用,能够显著抑制非小细胞肺癌细胞A549,PC9的增殖。本发明提供的化合物可为新型抗肿瘤药物的开发提供物质基础,有利于中药材苦豆子药用价值的进一步开发。为非小细胞肺癌等提供有效的治疗小分子药物,能够在医学与生物学领域得到广泛的应用。(A preparation method and application of alkaloid compound sephaline B, belonging to the technical field of natural medicinal chemistry. The invention provides an application of an alkaloid compound, namely, sephaline B in preparation of a non-small cell lung cancer disease medicament, a medicinal preparation for preventing or treating non-small cell lung cancer cells, and provides a preparation method of the alkaloid compound, namely, sephaline B. The alkaloid compound, namely the sephaline B, separated from the traditional Chinese medicine sophora alopecuroides has a good inhibition effect on tumor cells, and can obviously inhibit the proliferation of non-small cell lung cancer cells A549 and PC 9. The compound provided by the invention can provide a material basis for the development of novel antitumor drugs, and is beneficial to the further development of the medicinal value of the Chinese medicinal herb sophora alopecuroides. Provides effective small molecule treatment medicine for non-small cell lung cancer and the like, and can be widely applied in the fields of medicine and biology.)
1. Use of alkaloid compound sephaline B in preparation of medicines for resisting non-small cell lung cancer diseases.
2. The use according to claim 1, wherein the sepharose B inhibits proliferation, migration, invasion and cloning of non-small cell lung cancer cell A549 and promotes apoptosis of non-small cell lung cancer cell A549.
3. The use according to claim 1, characterized in that said sephaline B inhibits the proliferation of non-small cell lung cancer cell PC 9.
4. Use according to claim 1, characterized in that said sephaline B inhibits HUVEC angiogenesis of non-small cell lung cancer cells.
5. A pharmaceutical preparation for preventing or treating non-small cell lung cancer cells, characterized in that the pharmaceutical preparation contains an alkaloid compound, sephane B.
6. The pharmaceutical preparation for preventing or treating non-small cell lung cancer cells according to claim 5, wherein the pharmaceutical preparation is prepared by adding conventional adjuvants according to a conventional process;
the conventional auxiliary materials comprise one or more of starch, lactose, microcrystalline cellulose, dextrin, calcium phosphate, polyethylene glycol-4000, polyethylene glycol-6000, sodium carboxymethylcellulose, hydroxypropyl cellulose, crospovidone, micropowder silica gel, hydrogenated vegetable oil, talcum powder, polyethylene glycols, magnesium lauryl sulfate, magnesium stearate, croscarmellose sodium, sodium bicarbonate, citric acid and sodium carboxymethyl starch;
the preparation comprises tablets, capsules, dripping pills, granules, powder, solutions, emulsions and suspensions.
7. A preparation method of an alkaloid compound, sephaline B, is characterized by comprising the following steps:
(1) weighing dried herba Sophorae Alopecuroidis seed, pulverizing, percolating with 95% ethanol for 3 times, mixing extractive solutions, and concentrating under reduced pressure to obtain extract;
(2) dispersing the obtained extract in water, adjusting pH to 3 with 1% hydrochloric acid, adding chloroform, extracting, removing organic layer, adjusting pH of the remaining water layer to 8 with 2% ammonia water, and extracting with chloroform to obtain total alkaloids of herba Sophorae Alopecuroidis;
(3) suspending the total aloperine obtained in the step (2) in water, applying D101 macroporous resin, and performing gradient elution with water, 40%, 65% and 95% ethanol in sequence to obtain a water part, a 40% ethanol part, a 65% ethanol part and a 95% ethanol part;
(4) the 95% ethanol fraction was further analyzed by ODS column using methanol: gradient elution is carried out by a water system to obtain 5 fractions Fr.1-Fr.5, and the Fr.5 fraction is subjected to preparative high performance liquid phase treatment by adopting acetonitrile: water: and purifying by using a diethylamine system to obtain the compound sephaline B.
8. The method according to claim 7, wherein the ratio of the sophora alopecuroides to the 95% ethanol in the step (1) is 1:10 by mass.
9. The method according to claim 7, wherein the ratio of methanol in the gradient elution system in the step (4): the water volume ratio is 30:70-100: 0.
10. The method according to claim 7, wherein in the step (4), the ratio of acetonitrile: water: the diethylamine volume ratio was 35:65: 0.01.
Technical Field
The invention belongs to the technical field of natural pharmaceutical chemistry, and particularly relates to a preparation method and application of an alkaloid compound, namely, sephaline B.
Background
Lung cancer (Lung cancer) is one of the most common malignant tumors in the world, and the incidence of Lung cancer is increasing continuously with the increase of the global population and the aging of the population, so that the Lung cancer becomes the tumor with the highest fatality rate. The 2015 statistical data released by the world health organization showed that 2012 about 180 million new cases of lung cancer account for 13% of all tumor types. About 55 million deaths of the Chinese lung cancer in 2013 account for 1/3 worldwide deaths of the cancer. In China, the incidence and the fatality rate of lung cancer of men account for the 1 st position of all malignant tumors, the incidence of lung cancer of women account for the 2 nd position, and the fatality rate of lung cancer accounts for the 1 st position. The statistics of the national cancer center shows that about 73 thousands of cases occur in 2015 Chinese lung cancer, wherein about 51 thousands of cases are male and about 22 thousands of cases are female; in the same year, about 61 ten thousand cases of lung cancer die in China, of which about 43 ten thousand cases are male and about 18 ten thousand cases are female. The incidence and mortality of lung cancer increases with age, generally increasing significantly after age 40, reaching a peak around age 75, and then decreasing.
Non-Small Cell Lung Cancer (NSCLC) is a common Lung malignancy, with the vast majority of Non-Small Cell Lung cancers originating in the bronchial mucosal epithelium. Refers to a disease in which cell growth in lung tissue is out of control. This cell growth may cause metastasis, i.e., invasion of adjacent tissues and penetration outside the lungs. Accounting for about 80-85% of the total lung cancer. It is a heterogeneous tumor that can be further classified as squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. The NSCLC is already advanced when most patients are diagnosed. The NSCLC patients with locally advanced and good physical condition can reach 8 months disease-free survival (DFS) stage after a series of chemotherapy, radiotherapy and/or surgical treatment, and the 5-year survival rate is less than 15%. Therefore, it is necessary to find new effective therapeutic drugs!
Sophora alopecuroides L is a seed of Sophora alopecuroides L belonging to Leguminosae. The plants or base parts of Sophora are lignified into shrub-like plants, and the height can reach 1 m. Distributed in North China, northwest China, Henan and Tibet. Has the effects of clearing heat, drying dampness, relieving pain and killing parasites. It is commonly used for dysentery, stomachache, leukorrhagia, eczema, sore, furuncle, and intractable tinea. Has effects in inhibiting central nervous system, relieving inflammation, resisting allergy, inhibiting immunity, resisting arrhythmia and positive muscle strength, and protecting liver. The sophaline B compound is an alkaloid compound separated from sophora alopecuroides, and the prior art does not report that the compound has the effect of treating the non-small cell lung cancer.
Disclosure of Invention
In view of the problems in the prior art, the present invention aims to provide a preparation method and use of alkaloid compound sephaline B. The invention discovers for the first time that the traditional Chinese medicine sophora alopecuroide or the extract thereof has obvious anti-non-small cell lung cancer tumor activity and the patent medicine potential of preparing the medicinal preparation for preventing or treating the non-small cell lung cancer and the related diseases thereof, and the preparation method of the medicinal preparation is simple, the cost is low, and the patent medicine economic benefit is good.
In order to achieve the purpose, the invention adopts the following technical scheme:
use of alkaloid compound sephaline B in preparation of medicines for resisting non-small cell lung cancer diseases.
The application is characterized in that the sephaline B inhibits the proliferation, migration, invasion and cloning of the non-small cell lung cancer cell A549 and promotes the apoptosis of the non-small cell lung cancer cell A549.
The use, characterized in that the sephaline B inhibits the proliferation of non-small cell lung cancer cell PC 9.
The use, characterized in that the sephaline B inhibits the angiogenesis of non-small cell lung cancer cells HUVEC.
A pharmaceutical preparation for preventing or treating non-small cell lung cancer cells, characterized in that the pharmaceutical preparation contains an alkaloid compound, sephane B.
The medicinal preparation for preventing or treating the non-small cell lung cancer cells is characterized in that the medicinal preparation is prepared by adding conventional auxiliary materials according to a conventional process;
the conventional auxiliary materials comprise one or more of starch, lactose, microcrystalline cellulose, dextrin, calcium phosphate, polyethylene glycol-4000, polyethylene glycol-6000, sodium carboxymethylcellulose, hydroxypropyl cellulose, crospovidone, micropowder silica gel, hydrogenated vegetable oil, talcum powder, polyethylene glycols, magnesium lauryl sulfate, magnesium stearate, croscarmellose sodium, sodium bicarbonate, citric acid and sodium carboxymethyl starch;
the preparation comprises tablets, capsules, dripping pills, granules, powder, solutions, emulsions and suspensions.
A preparation method of an alkaloid compound, sephaline B, is characterized by comprising the following steps:
(1) weighing dried herba Sophorae Alopecuroidis seed, pulverizing, percolating with 95% ethanol for 3 times, mixing extractive solutions, and concentrating under reduced pressure to obtain extract;
(2) dispersing the obtained extract in water, adjusting pH to 3 with 1% hydrochloric acid, adding chloroform, extracting, removing organic layer, adjusting pH of the remaining water layer to 8 with 2% ammonia water, and extracting with chloroform to obtain total alkaloids of herba Sophorae Alopecuroidis;
(3) suspending the total aloperine obtained in the step (2) in water, applying D101 macroporous resin, and performing gradient elution with water, 40%, 65% and 95% ethanol in sequence to obtain a water part, a 40% ethanol part, a 65% ethanol part and a 95% ethanol part;
(4) the 95% ethanol fraction was further analyzed by ODS column using methanol: gradient elution is carried out by a water system to obtain 5 fractions Fr.1-Fr.5, and the Fr.5 fraction is subjected to preparative high performance liquid phase treatment by adopting acetonitrile: water: and purifying by using a diethylamine system to obtain the compound sephaline B.
The preparation method is characterized in that the mass ratio of the sophora alopecuroide to the 95% ethanol in the step (1) is 1: 10.
The preparation method is characterized in that in the step (4), methanol in a gradient elution system: the water volume ratio is 30:70-100: 0.
The preparation method is characterized in that in the step (4), acetonitrile: water: the diethylamine volume ratio was 35:65: 0.01.
The invention has the beneficial effects that:
1. the invention discovers for the first time that the alopecuroide extract sophaline B has obvious anti-non-small cell lung cancer tumor activity, has the patent medicine potential of preparing the medicinal preparation for preventing or treating the non-small cell lung cancer and the related diseases thereof, provides a new material basis for developing anti-tumor innovative medicines, and has potential and huge social and economic benefits.
2. The sophora alopecuroide extract has low cost and little pollution, is beneficial to large-scale production under the conditions of energy conservation and emission reduction, and has good industrialization prospect.
3. The invention strongly proves that the sophora alopecuroides extract has obvious inhibition and apoptosis effects on tumor cells by researching the proliferation inhibition effect and apoptosis induction effect of the sophora alopecuroides extract sophylline B on non-small cell lung cancer cells A549 and PC9 and the inhibition effect on cell migration capacity and invasion capacity.
4. The sophora alopecuroide or the sophora alopecuroide extract also has the effects of dispelling wind, removing dampness, relaxing muscles and tendons, promoting blood circulation and the like, can be used for treating symptoms such as rheumatic arthritis, traumatic injury, stasis, swelling and pain, postpartum paralysis and the like, and can play a role in treating other complications while preventing or treating tumors.
5. The sophora alopecuroide can be compatible with other medicinal materials to form a compound medicine for preventing or treating tumors, and the sophora alopecuroide extract can be mixed with auxiliary materials to be prepared into tablets, capsules, dripping pills or granules.
Drawings
FIG. 1 is the chemical structural formula of alkaloid sephaline B compound;
FIG. 2 shows the effect of alkaloid sephaline B compounds on the proliferation of non-small cell lung cancer cells A549 and PC9, wherein A is the effect of the sephaline B compounds on the proliferation of non-small cell lung cancer cells A549 under different action time, and B is the effect of the sephaline B compounds on the proliferation of non-small cell lung cancer cells PC9 under different action time;
FIG. 3 shows the effect of alkaloid sephaline B compounds on the apoptosis of non-small cell lung cancer cells A549, wherein A is the effect of the sephaline B compounds on the apoptosis of non-small cell lung cancer cells A549, and B is the apoptosis statistics;
fig. 4 shows the effect of alkaloid sephaline B compounds on migration and invasion of non-small cell lung cancer cells a549, wherein a is the influence of the sephaline B compounds on migration and invasion of non-small cell lung cancer cells a549, B is migration statistics, and C is invasion statistics;
FIG. 5 shows the effect of alkaloid sephaline B compounds on the formation of non-small cell lung cancer cell A549 clones, wherein A is the influence of the sephaline B compounds on the formation of non-small cell lung cancer cell A549 clones, and B is the clone statistics;
FIG. 6 shows the effect of the alkaloid sephaline B compound on angiogenesis of non-small cell lung cancer cell A549, wherein A is the influence of the sephaline B compound on angiogenesis of HUVEC cells, B is the statistics of blood vessel length, C is the statistics of blood vessel percentage, and D is the statistics of blood vessel junction length.
Detailed Description
In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.
Example 1: extraction of Sophora alopecuroides L
Pulverizing dried herba Sophorae Alopecuroidis seed (30kg), percolating with 10 times of 95% ethanol for 3 times, mixing extractive solutions, and concentrating under reduced pressure to obtain extract (1.9 kg). Dispersing the obtained extract in water, adjusting pH to 3 with 1% hydrochloric acid, adding chloroform, extracting thoroughly, and discarding organic layer. The remaining aqueous layer was adjusted to pH 8 with 2% aqueous ammonia and extracted with chloroform to give total alkaloids of Sophora alopecuroides (897 g). Suspending herba Sophorae Alopecuroidis total alkaloids in water, loading onto D101 macroporous resin, sequentially gradient-eluting with water, 40%, 65% and 95% ethanol to obtain water part (302g), 40% ethanol part (317g), 65% ethanol part (177g) and 95% ethanol part (35 g). And analyzing the 95% ethanol part by using an ODS column, wherein the mass ratio of methanol: gradient eluting with water (volume ratio of 30:70-100:0) to obtain 5 fractions (Fr.1-Fr.5). And (5) purifying the Fr.5 part by using a preparative high-performance liquid phase (the volume ratio of acetonitrile to water to diethylamine is 35:65:0.01) to obtain a compound sephaline B, wherein the structure of the compound is shown in figure 1.
Example 2: the sephaline B compound can obviously inhibit the proliferation of non-small cell lung cancer cells A549 and PC9
1. Non-small cell lung cancer cells A549 and PC9 at 5 × 103Inoculating each well into a 96-well cell culture plate, culturing for 24h with the culture medium volume of 200 mu L per well, and then starving overnight;
2. adding compounds with different concentration gradients (100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M) and culturing for 24 hours, 48 hours and 72 hours respectively;
3. adding 20 mu L of MTT working solution into each hole, and continuously putting the mixed solution into a carbon dioxide incubator to be cultured for 4 hours;
4. discarding the supernatant in the culture plate, adding 150 mu LDMSO (dimethyl sulfoxide), shaking for 10 minutes, selecting 490nm wavelength on a microplate reader for detection, and drawing the growth curve of the cells.
As shown in fig. 2, the sophaline B compound at different concentrations can significantly inhibit the proliferation of non-small cell lung cancer cells a549 and PC9, and the effect of the compound on inhibiting the proliferation of non-small cell lung cancer cells is more significant as the action time increases.
Example 3: alkaloid Sophaline B compound can promote apoptosis of non-small cell lung cancer cell A549
1. Non-small cell lung cancer cell A549 at 5 × 105Inoculating each cell/well in 6-well cell culture plates, culturing for 24h with the volume of culture medium per well being 1mL, and then starving overnight;
2. adding compounds with different concentration gradients (25. mu.M, 50. mu.M) for culturing for 48 hours;
3. the supernatant was collected in a centrifuge tube, and the cell culture solution was collected by careful digestion with pancreatin without EDTA. Centrifuging about 500g for 5 minutes to precipitate cells;
4. washing the cells twice by using precooled PBS, and centrifuging about 500g for 5 minutes to collect the cells;
5. adding 100. mu.L of precooled 1 × annexin V Binding Buffer, and resuspending the cells;
6. adding 5 mu LannexinV-FITC and 5 mu LPI, mixing evenly, and reacting for 15 minutes at room temperature in a dark place;
7. add 400. mu.L of precooled 1 × annexin V Binding Buffer, mix gently, place the sample on ice in the dark, and detect it with flow cytometer within 1 h. And analyzing the detection result.
As shown in figure 3, flow cytometry analysis shows that the Sophaline B compound can obviously promote the apoptosis of the non-small cell lung cancer cell A549 with the increase of the action concentration.**P<0.01 compared to the control group.
Example 4: the alkaloid Sophaline B compound can obviously inhibit the migration and invasion of non-small cell lung cancer cell A549
Transwell upper chamber seed 2X 105Adding compounds with different concentration gradients (25 μ M, 50 μ M) to each cell, and culturing for 24 hours;
2. the lower chamber was filled with 800 μ L of medium containing 10% FBS;
3. washing with PBS for 3 times, and fixing with 4% paraformaldehyde for 30 min;
4. washing with PBS for 3 times, and staining with 0.5% crystal violet for 1 h;
5. carefully scraping the upper layer of cells by using a cotton swab, and taking a picture under a microscope;
as shown in figure 4, experiments show that the Sophaline B compound can obviously inhibit the migration and invasion of the non-small cell lung cancer cell A549 along with the increase of the action concentration.**P<0.01 compared to the control group.
Example 5: the alkaloid Sophaline B compound can obviously inhibit the in-vitro clone forming capability of non-small cell lung cancer cell A549
1. Inoculating the non-small cell lung cancer cell A549 into a 6-well cell culture plate at 1000/well, wherein the volume of a culture medium in each well is 1mL, culturing for 10 days, and then starving overnight;
2. adding compounds with different concentration gradients (25 μ M, 50 μ M) for 24 h;
washing with PBS for 3 times, and fixing with 4% paraformaldehyde for 30 min;
4. washing with PBS for 3 times, adding 0.5% crystal violet, and dyeing for 1 h;
5. washed 3 times with PBS and photographed microscopically.
As shown in figure 5, a cloning experiment shows that the Sophaline B compound can obviously inhibit the cloning of the non-small cell lung cancer cell A549 with the increase of the action concentration.**P<0.01 compared to the control group.
Example 6: alkaloid Sophaline B compound can obviously inhibit HUVEC (human vascular endothelial cell factor) angiogenesis capacity
1. Firstly, paving a 24-hole plate by using Matrigel overnight;
2. HUVEC cells were cultured at 5X 104Inoculating each well into a 24-well cell culture plate, adding compounds with different concentration gradients (25 mu M and 50 mu M) and culturing for 6 h;
3. washed 3 times with PBS and photographed microscopically.
As shown in figure 6, the Sophaline B compound can obviously inhibit HUVEC angiogenesis along with the increase of action concentration.**P<0.01 compared to the control group.
It will be appreciated by those skilled in the art that the use of the present invention is not limited to the specific applications described above. The invention is also not limited to the preferred embodiments thereof with respect to the specific elements and/or features described or depicted herein. It should be understood that the invention is not limited to the disclosed embodiment or embodiments, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the invention as set forth and defined by the following claims.