A marigold extract and its application in preparing anti-prostate cancer drugs

Abstract

The present invention relates to the field of medical technology, and in particular to a marigold extract and an application thereof in the preparation of an anti-prostate cancer drug. The extract is obtained by extracting effective ingredients from marigold roots or stems using ethyl acetate as an extractant. The marigold extract inhibits the survival and proliferation of tumor cells by inducing cell pyroptosis, thereby curbing the development of tumors, and can be used to prepare a drug for preventing and / or treating prostate cancer.

Description

[Technical field]

[0001] The invention belongs to the technical field of medicines, and particularly relates to a marigold extract and application thereof in preparing an anti-prostate cancer drug. [Background technology]

[0002] Marigold is a plant of the genus Tagetes erecta L. in the Asteraceae family. As a traditional Chinese medicine, marigold is often used as a root or flower. The plant marigold is cultivated in various parts of my country. The flower has the effects of clearing away heat and detoxifying, resolving phlegm and relieving cough, and the root detoxifies and reduces swelling. The flower is mainly used to treat upper respiratory tract infections, whooping cough, tracheitis, keratitis, pharyngitis, stomatitis, and toothache; it is used externally to treat mumps, mastitis, and carbuncle. Fresh herb is used externally to treat mastitis, unidentified swelling, and furuncle.

[0003] Prostate cancer (PCa) refers to an epithelial malignant tumor that occurs in the prostate gland. The early clinical manifestations of prostate cancer are atypical. As the disease progresses, symptoms and signs such as bladder irritation, hematuria, and dysuria may gradually appear. It can be classified into the category of "urinary retention" and "stranguria" in traditional Chinese medicine. It has the characteristics of deficiency of vital energy and the presence of phlegm, dampness, heat, blood stasis, and toxins.

[0004] In recent years, traditional Chinese medicine has gradually shown its advantages and prospects in cancer treatment, which can improve patient-related syndromes, delay disease progression, and reduce the risk of complications. Moreover, compared with chemotherapy drugs, traditional Chinese medicine has lower costs and milder side effects, which can not only reduce the burden on patients' families but also improve the quality of life of patients to a certain extent. At present, no relevant experimental studies on the anti-tumor effect of marigold extract have been reported, which to a certain extent limits the clinical expansion of marigold extract against prostate cancer. [Summary of the invention]

[0005] In view of the above, it is necessary to provide a marigold extract and its use in the preparation of anti-prostate cancer drugs. The marigold extract inhibits the survival and proliferation of tumor cells by inducing cell pyroptosis, thereby curbing the development of tumors, and can be used to prepare drugs for the prevention and / or treatment of prostate cancer.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A marigold extract, characterized in that the preparation method of the marigold extract comprises the following steps:

[0008] (1) Pretreatment: Take the root or stem of marigold plant (powder, passed through No. 3 sieve) and put it into a stoppered conical flask, add ethyl acetate, cover with a stopper and seal, and shake to mix well;

[0009] (2) Extraction: Ultrasonic extraction is performed under light-proof conditions to obtain an ethyl acetate extract of marigold;

[0010] (3) Concentration: The ethyl acetate extract of marigold is filtered under reduced pressure and then evaporated to concentrate. The concentrated medicinal solution is then placed in an evaporating dish and the solvent is evaporated in a water bath to form a dry extract, thereby obtaining the marigold extract.

[0011] In the present invention, further, in the step (1), the liquid ratio of the root or stem of the marigold plant to ethyl acetate is 1 g:10 mL.

[0012] In the present invention, it is more preferred to take the root of the marigold plant.

[0013] In the present invention, further, the ultrasonic extraction conditions in step (2) are: ultrasonic power of 500 W, ultrasonic frequency of 40 kHz, extraction time of 30 min, and oscillation once every 10 min during the extraction process.

[0014] In the present invention, further, the evaporation concentration method of step (3) is specifically to put the marigold extract into a 250 ml round-bottom flask, and then use a rotary evaporator to evaporate and recover water at 60° C. under reduced pressure to obtain 10 ml of concentrated solution.

[0015] In the present invention, further, the temperature of the water bath in step (3) is 95°C.

[0016] The present invention also provides the use of marigold extract in preparing a drug for preventing and / or treating prostate cancer.

[0017] The present invention also provides a pharmaceutical composition, which comprises the marigold extract and a pharmaceutically acceptable carrier or excipient.

[0018] In the present invention, further, the amount of the marigold extract is 40-60% of the total mass of the pharmaceutical composition.

[0019] In the present invention, further, the pharmaceutically acceptable carrier or excipient is selected from one or any combination of solvents, diluents, dispersants, suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, adhesives, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavoring agents, sweeteners, ion exchangers, release agents, coating agents, flavoring agents and antioxidants.

[0020] In the present invention, further, the pharmaceutical composition is formulated into any dosage form of powder, tablet, capsule, pill, pellet, injection, emulsion, suspension or tincture.

[0021] The present invention has the following beneficial effects:

[0022] 1. The present invention proposes for the first time to use marigold extract as an active ingredient to prepare a drug for treating prostate cancer. Experiments have shown that the marigold extract of the present invention has a significant inhibitory effect on the growth and migration of prostate cancer cells, providing a variety of new ways for the application of marigold extract, and greatly improving the market application prospects of marigold.

[0023] 2. The marigold extract of the present invention is particularly used in the preparation of drugs for treating and / or preventing prostate cancer. Based on the application, the applicant has developed for the first time an extraction method suitable for the application, and found that the marigold extract obtained by using ethyl acetate as an extractant to extract the roots and stems of marigold is the best component of the pharmaceutical composition, which is of great significance for the development of new pharmaceutical compositions for prostate cancer.

Brief Description of the Drawings

[0024] Figure 1 The figure is the effect of marigold acetic acid extract on the proliferation of 22Rv1 cells;

[0025] In the figure: the upper part A shows the effect of the extract of the acetic acid root of marigold on the clone formation of 22Rv1 cells; the lower part B shows the effect of the extract of the acetic acid stem of marigold on the clone formation of 22Rv1 cells;

[0026] Figure 2 This is a diagram of 22Rv1 cell pyroptosis induced by ethyl acetate extracts from marigold roots and stems;

[0027] In the figure: nucleus (N), mitochondria (Mi), rough endoplasmic reticulum (RER), primary lysosomes (Ly);

[0028] Figure 3 The figure shows the effect of marigold acetic acid extract on the growth of xenograft prostate cancer in mice;

[0029] In the figure: A is the change of mouse body weight; B is the change of mouse tumor volume; C is the mouse tumor volume at the end point of the experiment; D is the mouse tumor weight at the end point of the experiment; E is the photo of the mouse tumor at the end point of the experiment. [Specific implementation method]

[0030] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0031] Example 1: Preparation of marigold root extract

[0032] This embodiment provides a marigold extract, and the preparation method of the marigold extract is as follows:

[0033] (1) Pretreatment: Take 10 g of marigold root, put it into a conical flask with a stopper, add 100 mL of ethyl acetate, cover with a stopper, and shake to mix well;

[0034] (2) Extraction: Perform ultrasonic extraction for 30 min in a dark environment with an ultrasonic power of 500 W and a frequency of 40 kHz, and oscillate once every 10 minutes to obtain an ethyl acetate extract of marigold;

[0035] (3) Concentration: The ethyl acetate extract of marigold is filtered under reduced pressure and then placed in a 250 ml round-bottom flask, and then evaporated under reduced pressure at 60° C. using a rotary evaporator to recover water to obtain 10 ml of concentrated solution. The 10 ml concentrated solution is placed in a 100 ml evaporating dish and the solvent is evaporated in a 95° C. water bath to form a dry extract, thereby obtaining the marigold extract.

[0036] Example 2: Preparation of marigold stem extract

[0037] This embodiment provides a marigold extract, and the preparation method of the marigold extract is as follows:

[0038] (1) Pretreatment: Take 10 g of marigold stems, put them into a stoppered conical flask, add 100 mL of ethyl acetate, cover with a stopper, and shake to mix well;

[0039] (2) Extraction: Perform ultrasonic extraction for 30 min in a dark environment with an ultrasonic power of 500 W and a frequency of 40 kHz, and oscillate once every 10 minutes to obtain an ethyl acetate extract of marigold;

[0040] (3) Concentration: The ethyl acetate extract of marigold is filtered under reduced pressure and then placed in a 250 ml round-bottom flask, and then evaporated under reduced pressure at 60° C. using a rotary evaporator to recover water to obtain 10 ml of concentrated solution. The 10 ml concentrated solution is placed in a 100 ml evaporating dish and the solvent is evaporated in a 95° C. water bath to form a dry extract, thereby obtaining the marigold extract.

[0041] Test example:

[0042] Test Example 1:

[0043] In this test example, different solvents were used to extract different parts of the marigold plant to obtain different marigold extracts;

[0044] The preparation method of the marigold extract is as follows:

[0045] (1) Pretreatment: Take 10 g of marigold plant (root, stem, and flower powder, passed through a No. 3 sieve), put it into a stoppered conical flask, add 100 mL of extractant (water, methanol, ethyl acetate), cover with a stopper, and shake to mix well;

[0046] (2) Extraction: Ultrasonic extraction was performed for 30 min in a dark environment with an ultrasonic power of 500 W and a frequency of 40 kHz, with oscillation once every 10 minutes to obtain a marigold extract (water, methanol, ethyl acetate);

[0047] (3) Concentration: The marigold extract (water, methanol, ethyl acetate) was filtered under reduced pressure and then placed in a 250 ml round-bottom flask. The water was then evaporated under reduced pressure at 60° C. using a rotary evaporator to obtain 10 ml of concentrated solution. The 10 ml concentrated solution was placed in a 100 ml evaporating dish and the solvent was evaporated in a 95° C. water bath to form a dry extract, thereby obtaining the marigold extract.

[0048] The extraction combination is shown in Table 1:

[0049]

[0050] Test Example 2:

[0051] This test example compares the inhibitory effects of marigold extracts of different solvents on the activity of prostate cancer cells in Test Example 1

[0052] Take 22Rv1 prostate cancer cells in the logarithmic growth phase, inoculate the cells into 96-well cell culture plates with complete culture medium containing 10% FBS, and inoculate 5000 cells per well. After all the cells are attached. Add marigold extracts extracted with different solvents to the cell culture wells to make the concentration of marigold extract solution reach 120ug / ml, 60ug / ml, 30ug / ml, 15ug / ml, and 7.5ug / ml; add DMSO to the control group to make the concentration reach 0.1%. After the cells are treated with the compound for 72 hours, add 10μL / well MTT solution (5mg / mL) to the culture medium, let the cells react with MTT in the incubator for 4h, then take out the cell culture plate, detect the OD value at a wavelength of 570nm, and fit the IC with GraphPad Prism 8.0. 50 value.

[0053] The recorded data are shown in Table 2:

[0054] Table 2 Effect of different solvent marigold extracts on the inhibition of prostate cancer cell viability

[0055]

[0056] According to Table 2, different solvents of marigold extract inhibited the activity of prostate cancer cells to different degrees. IC50 The IC values ​​of methanol extracts of marigold root, stem and flower were 116.2ug / ml, greater than 120ug / ml and greater than 120ug / ml respectively. 50 The IC values ​​of ethyl acetate extracts from marigold roots, stems and flowers were 119.8ug / ml, 67.3ug / ml and 102.1ug / ml respectively. 50 The values ​​were 11.7ug / ml, 18.5ug / ml and greater than 120ug / ml respectively; the above data indicated that the ethyl acetate extract of marigold root and stem inhibited the activity of prostate cancer cells better than water extract and methanol extract.

[0057] Test Example 3:

[0058] This test example compares the effect of marigold extracts obtained from different parts of the marigold extracted with ethyl acetate in test example 1 on inhibiting the activity of prostate cancer cells

[0059] PC-3, 22Rv1, and DU145 prostate cancer cells in the logarithmic growth phase were taken and inoculated into 96-well cell culture plates with complete culture medium containing 10% FBS, with 5,000 cells in each well. After all the cells were attached to the wall, different concentrations of marigold ethyl acetate extract solutions were added to make the concentrations of marigold ethyl acetate extract solutions reach 120ug / ml, 60ug / ml, 30ug / ml, 15ug / ml, and 7.5ug / ml; DMSO was added to the control group to make the concentration reach 0.1%. After the cells were treated with the compound for 72 hours, 10μL / well MTT solution (5mg / mL) was added to the culture medium, and the cells and MTT were allowed to react in the incubator for 4h, and then the cell culture plate was taken out, the OD value at a wavelength of 570nm was detected, and the IC was fitted using GraphPad Prism 8.0 50 value.

[0060] Table 3 Effects of ethyl acetate extracts from different parts of marigold on the viability of different prostate cancer cells

[0061]

[0062]

[0063] As shown in Table 3, the ethyl acetate extract of marigold can inhibit the viability of prostate cancer cells to varying degrees. The ethyl acetate extract solution of marigold roots, stems, and flowers inhibits the viability of 22Rv1 prostate cancer cells IC 50 The IC values ​​of marigold root, stem and flower ethyl acetate extracts inhibited the activity of PC3 prostate cancer cells. 50The IC values ​​of marigold root, stem and flower ethyl acetate extracts inhibited the activity of DU145 prostate cancer cells. 50 The above data show that the ethyl acetate extracts of marigold roots and stems are better than flowers in inhibiting the activity of prostate cancer cells. Furthermore, the ethyl acetate extract of marigold roots is the best in inhibiting the activity of prostate cancer cells.

[0064] Test example 4:

[0065] Based on the above results, it is known that the ethyl acetate extract of marigold root is the best for inhibiting the activity of prostate cancer cells. This test example further verifies its effect;

[0066] 1. Effect of marigold acetic acid extract on 22Rv1 cell proliferation

[0067] Prostate cancer 22Rv1 cells with good growth in the logarithmic growth phase were taken and inoculated into a 6-well plate with 1000 cells per well. The plates were placed in a constant temperature incubator at 37°C and 5% CO2 for culture. After culturing the cells for 24 hours, different treatments were adopted, including a blank control group, a 40uM Enzalutamide group, a marigold root acetic acid extract group (10ug / ml, 20ug / ml, 40ug / ml), and a marigold stem acetic acid extract group (10ug / ml, 20ug / ml, 40ug / ml). The cells were cultured for 2 weeks, and the cell clone formation was observed under a microscope after two weeks. The cells were then fixed and stained, and then photographed and counted using a cell clone counter. The results are shown in the figure. Figure 1 shown.

[0068] like Figure 1 As shown in the upper part A, 20ug / ml and 40ug / ml marigold root acetic acid extract significantly inhibited the colony formation of 22Rv1 cells; Figure 1 As shown in the lower part B, 20ug / ml and 40ug / ml ethyl acetate extracts from marigold stems significantly inhibited the colony formation of 22Rv1 cells; the above data show that both ethyl acetate extracts from marigold roots and stems have a significant inhibitory effect on the proliferation of prostate cancer cells.

[0069] 2. Marigold acetic acid extract induces pyroptosis in 22Rv1 cells

[0070] Prostate cancer 22Rv1 cells with good growth status in the logarithmic growth phase were selected and inoculated into a 10 cm cell culture dish, with 1×10 6Cells were placed in a constant temperature incubator at 37°C and 5% CO2 for culture. After 24 hours of cell culture, three 10 cm cell culture dishes were treated differently, and were set as a blank control group, a 40ug / ml marigold root acetic acid extract group, and a 40ug / ml marigold stem acetic acid extract group. After 72 hours of continuous culture, the cells were collected, fixed with 0.5% glutaraldehyde, and then cell samples were prepared according to the transmission electron microscopy sample preparation method, and the internal morphology of the cells was observed with a transmission electron microscope.

[0071] like Figure 2 As shown in the figure, after the cells were treated with 40ug / ml ethyl acetate extracts from the roots and stems of marigold, the nuclei (marked as N in the figure) were irregular in shape, the chromatin was evenly distributed, mainly euchromatin, and the heterochromatin was stained darker and mainly distributed at the nuclear membrane; a large number of mitochondria (marked as Mi in the figure) swelled, that is, the volume became larger, the cristae decreased, broke or disappeared, the matrix electron density decreased, and the contents were partially dissolved, showing flocculent or even vacuolar shapes; the rough endoplasmic reticulum (marked as RER in the figure) expanded (marked by blue arrows) and was cystic, with ribosomes attached to the surface; the cell membrane ruptured (marked by orange arrows), and the contents flowed out; a small amount of primary lysosomes (marked as Ly in the figure), autophagy (marked by purple arrows) and a large number of vesicles (marked by yellow arrows) were also seen in the cytoplasm; the above intracellular morphological changes were consistent with the characteristics of cell pyroptosis, and the results of transmission electron microscopy showed that the ethyl acetate extracts from the roots and stems of marigold had the effect of inducing pyroptosis in 22Rv1 cells.

[0072] Effect of marigold acetic acid extract on the growth of xenograft prostate cancer in mice

[0073] Fifty male Balb / c nude mice (5-6 weeks old, Hunan Slake Jingda Experimental Animal Co., Ltd.) were purchased. After one week of adaptive feeding, 1×10 6 22Rv1 cells. When the tumor volume of mice reaches 50-100mm 3At the same time, 36 mice that met the requirements were selected from 50 modeling mice for drug intervention. Thirty-six Balb / c nude mice were randomly divided into 6 groups (n=6) according to tumor volume, namely model group, 1 mg / kg docetaxel group, 150 mg / kg marigold root acetic acid extract group, 75 mg / kg marigold root acetic acid extract group, 150 mg / kg marigold stem acetic acid extract group, and 75 mg / kg marigold stem acetic acid extract group. The docetaxel group was administered twice a week by intraperitoneal injection for four weeks. The model group and marigold extract group were administered 5 days a week by gavage for 4 weeks. During the administration period, the tumor volume was measured once a week and the mouse weight was measured once a week. After 4 weeks of administration, the tumors in the mice were collected, weighed, fixed with 4% paraformaldehyde and / or quickly frozen and stored in liquid nitrogen for later use. This animal experiment was approved by the Experimental Animal Welfare Ethics Committee of Guangxi University of Chinese Medicine (approval number: DW20231016-218).

[0074] The results are as follows Figure 3 As shown, Figure 3 As shown in A, during the administration period, compared with the model group, there was no significant difference in the body weight of mice in the 1 mg / kg docetaxel group, the 150 mg / kg marigold root acetic acid extract group, the 75 mg / kg marigold root acetic acid extract group, the 150 mg / kg marigold stem acetic acid extract group, and the 75 mg / kg marigold stem acetic acid extract group. Figure 3 As shown in B, during the administration period, the tumor volumes of mice in the 1 mg / kg docetaxel group, the 150 mg / kg marigold root extract group, the 75 mg / kg marigold root extract group, the 150 mg / kg marigold stem extract group, and the 75 mg / kg marigold stem extract group were significantly smaller than those of mice in the model group. Figure 3 As shown in C, at the end point of the experiment, the tumor volumes of mice in the 1 mg / kg docetaxel group, the 150 mg / kg marigold root acetic acid extract group, the 75 mg / kg marigold root acetic acid extract group, the 150 mg / kg marigold stem acetic acid extract group, and the 75 mg / kg marigold stem acetic acid extract group were significantly smaller than those of mice in the model group. Figure 3 As shown in D, at the end of the experiment, the tumor weights of mice in the 1 mg / kg docetaxel group, the 150 mg / kg marigold root acetic acid extract group, the 75 mg / kg marigold root acetic acid extract group, the 150 mg / kg marigold stem acetic acid extract group, and the 75 mg / kg marigold stem acetic acid extract group were significantly smaller than those of mice in the model group. Figure 3E shows the mouse tumors at the experimental endpoint, among which, the first row: mouse tumors in the model group; the second row: mouse tumors in the 1 mg / kg docetaxel group; the third row: mouse tumors in the 150 mg / kg marigold root acetate extract group; the fourth row: mouse tumors in the 75 mg / kg marigold root acetate extract group; the fifth row: mouse tumors in the 150 mg / kg marigold stem acetate extract group; the sixth row: mouse tumors in the 75 mg / kg marigold stem acetate extract group; it can be seen from the figure that at the experimental endpoint, the tumor sizes of mice in the 1 mg / kg docetaxel group, 75 mg / kg marigold root acetate extract group, 150 mg / kg marigold root acetate extract group, 75 mg / kg marigold stem acetate extract group, and 150 mg / kg marigold stem acetate extract group were significantly smaller than those of mice in the model group.

[0075] The above-mentioned embodiments merely express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it should not be understood as limiting the scope of the present invention.

Claims

1. Use of a marigold extract in the preparation of a drug for preventing and / or treating prostate cancer, characterized in that: The preparation method of the marigold extract comprises the following steps: (1) Pretreatment: Take the root or stem of marigold plant and put it into a conical flask with a stopper, add ethyl acetate, cover with a stopper and seal, and shake to mix well; (2) Extraction: Ultrasonic extraction is performed under light-proof conditions to obtain ethyl acetate extract of marigold; (3) Concentration: The ethyl acetate extract of marigold is filtered under reduced pressure and then evaporated to concentrate. The concentrated medicinal solution is then placed in an evaporating dish and the solvent is evaporated in a water bath to form a dry extract, thereby obtaining the marigold extract.

2. The use according to claim 1, characterized in that: In the step (1), the liquid ratio of the root or stem of the marigold plant to ethyl acetate is 1 g:10 mL.

3. The use according to claim 1, characterized in that: The ultrasonic extraction conditions in step (2) are as follows: ultrasonic power of 500 W, ultrasonic frequency of 40 kHz, extraction time of 30 min, and oscillation once every 10 min during the extraction process.

4. The use according to claim 1, characterized in that: The evaporation concentration method in step (3) is specifically to put the marigold extract into a 250 ml round-bottom flask, and then use a rotary evaporator to evaporate and recover water at 60° C. under reduced pressure to obtain 10 ml of concentrated solution.

5. The use according to claim 1, characterized in that: The temperature of the water bath in step (3) is 95°C.

6. A pharmaceutical composition for preventing and / or treating prostate cancer, characterized in that: The pharmaceutical composition comprises the marigold extract according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier or excipient.

7. The pharmaceutical composition according to claim 6, characterized in that The amount of the marigold extract is 40-60% of the total weight of the pharmaceutical composition.

8. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutically acceptable carrier or excipient is selected from one or any combination of solvents, diluents, dispersants, suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, adhesives, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, ion exchangers, release agents, coating agents, flavoring agents and antioxidants.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition is prepared in any dosage form of powder, tablet, capsule, pill, injection, emulsion, suspension or tincture.

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