Marigold extract, and application of the same to preparation of Anti-prostatic cancer medicine

Abstract

To provide a marigold extract, and application thereof to preparation of an anti-prostatic cancer medicine.SOLUTION: The present invention relates to a medical technical field, particularly application of marigold extract and preparation thereof into an anti-prostatic cancer medicine, where the extract uses ethyl acetate as an extraction agent, is obtained by extracting active ingredients of roots or stems of marigold, marigold extract suppresses survival and proliferation of a tumor cell by inducing pyrokinesis of a cell, can suppress progress of a tumor, and can be used for preventing prostatic cancer and / or preparing a therapeutic drug.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to marigold extract and its application in preparing anti-prostate cancer drugs. [Background technology]

[0002] Marigold is a plant of the Asteraceae family, Tagetes erecta L. (Tagetes erecta L.). In traditional Chinese medicine, marigolds are often used as roots or flowers. The plant is cultivated throughout China. The flowers have the effects of clearing heat, expelling toxins, dissolving phlegm, and relieving coughs, while the roots detoxify and reduce swelling. The flowers are primarily used to treat upper respiratory tract infections, whooping cough, bronchitis, ocular keratitis, pharyngitis, stomatitis, and toothache, and are applied externally to treat mumps, mastitis, carbuncles, and swellings. The fresh herb is primarily used externally to treat mastitis, nameless swellings, and pustulosis.

[0003] Prostate cancer (PCa) is an epithelial malignant tumor that develops in the prostate gland. Early clinical symptoms of prostate cancer are atypical, and as the disease progresses, symptoms such as bladder irritation, hematuria, and difficulty urinating gradually appear. PCa falls under the category of "urinary retention" and "gonorrhea" in traditional Chinese medicine, and also shares the pathogenesis of qi deficiency, phlegm, dampness, heat, blood stasis, and toxins.

[0004] In recent years, traditional Chinese medicine has gradually highlighted its advantages and potential in cancer treatment, improving patient-related symptoms, delaying disease progression, and reducing the risk of complications. Furthermore, compared with chemotherapy drugs, traditional Chinese medicine is less expensive and has fewer side effects, which can reduce the burden on patients' families and improve the quality of life to some extent. Currently, no experimental studies have been reported on the antitumor properties of marigold extract, which to some extent limits the expanded clinical application of marigold extract in the treatment of prostate cancer. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, it is necessary to provide a marigold extract and its application in the preparation of anti-prostate cancer drugs.Marigold extract can suppress tumor cell survival and proliferation by inducing cellular pyrokinesis, thereby inhibiting tumor progression, and can be used to prepare drugs for the prevention and / or treatment of prostate cancer. [Means for solving the problem]

[0006] In order to achieve the above object, the technical scheme adopted by the present invention is as follows.

[0007] A marigold extract, comprising the steps of:

[0008] (1) Pretreatment: Take the roots or stems of marigold plants and place them in a stoppered Erlenmeyer flask, add ethyl acetate, seal the flask with a stopper, and shake well by vibrating;

[0009] (2) Extraction: ultrasonic extraction under light-shielding conditions to obtain marigold ethyl acetate extract;

[0010] (3) Concentration: The ethyl acetate extract of marigold is subjected to vacuum suction filtration, and then evaporated and concentrated. The concentrated solution is then placed in an evaporating dish, and the solvent is shaken over a water bath to dry the solution to form a dry extract, i.e., the marigold extract is obtained.

[0011] In the present invention, the ratio of the raw material solution of the roots or stems of the marigold plant to ethyl acetate in the step (1) is 1 g:10 mL.

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

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

[0014] In the present invention, the evaporation concentration method in step (3) is specifically carried out by placing the marigold extract in a 250 ml round-bottom flask, and then evaporating it under reduced pressure at 60°C using a rotary evaporator to recover the water, thereby obtaining 10 ml of a concentrated liquid.

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

[0016] The present invention also provides the application of marigold extract in the preparation of a medicament for the prevention and / or treatment of anti-prostate cancer.

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

[0018] Furthermore, in the present invention, the amount of the marigold extract used is 40 to 60% of the total mass of the pharmaceutical composition.

[0019] In the present invention, the pharmaceutically acceptable carrier or excipient is further selected from one or any combination of solvents, diluents, dispersants, suspension aids, surfactants, isotonicity agents, thickeners, emulsifiers, preservatives, binders, lubricants, stabilizers, water mixtures, emulsification enhancers, buffers, absorbents, colorants, fragrances, sweeteners, ion exchange agents, release agents, coating agents, flavoring agents, and antioxidants.

[0020] In the present invention, the pharmaceutical composition is further prepared in any of the following dosage forms: powder, tablet, capsule, pill, drop pill, injection, emulsion, suspension, or tincture. [Effects of the Invention]

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

[0022] 1. This invention is the first to propose using marigold extract as a viability ingredient to prepare a prostate cancer treatment drug. Experiments have proven that the marigold extract of this invention has significant inhibitory effects on the growth and metastasis of prostate cancer cells, providing various new routes 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 to prepare drugs for the treatment and / or prevention of prostate cancer. Based on this application, the applicant has for the first time researched an extraction method suitable for this application and discovered an extraction method using ethyl acetate as an extractant to extract the roots and stems of marigold. The resulting marigold extract is an optimal ingredient for pharmaceutical compositions, which is of great significance for the development of new pharmaceutical compositions for prostate cancer. [Brief explanation of the drawings]

[0024] [Figure 1] This figure shows the effect of ethyl acetate extract of marigold on the proliferation of 22Rv1 cells. In the figure, the upper part (A) shows the effect of ethyl acetate extract of marigold root on the clonal formation of 22Rv1 cells, and the lower part (B) shows the effect of ethyl acetate extract of marigold stem on the clonal formation of 22Rv1 cells. [Figure 2] Induction of cellular pyrokinesis by ethyl acetate extract of marigold roots and stems. Figure shows nuclei (N), mitochondria (Mi), rough endoplasmic reticulum (RER), and primary lysosomes (Ly). [Figure 3] This figure shows the effect of marigold ethyl acetate extract on the growth of mouse xenograft prostate cancer. In the figure, A is the change in mouse weight, B is the change in mouse tumor volume, C is the tumor volume at the end of the experiment, D is the tumor weight at the end of the experiment, and E is a photograph of the tumor at the end of the experiment. DETAILED DESCRIPTION OF THE INVENTION

[0025] In order to make the above-mentioned objects, features, and advantages of the present invention more clearly comprehensible, specific embodiments of the present invention will be described in detail below with reference to the drawings. In the following description, many specific details are set forth to facilitate a thorough understanding of the present invention. However, the present invention can be implemented in many ways other than those described herein, and those skilled in the art can make similar modifications without violating the spirit and scope of the present invention, and therefore the present invention is not limited to the specific implementations disclosed below.

[0026] Example 1: Preparation of Marigold Root Extract

[0027] This example provides a marigold extract, and the preparation method of the marigold extract is as follows:

[0028] (1) Pretreatment: Take 10 g of marigold roots and place them in a stoppered Erlenmeyer flask. Add 100 mL of ethyl acetate, seal the flask with a stopper, and shake well.

[0029] (2) Extraction: Ultrasonic extraction was performed for 30 minutes under light-shielded conditions, with an ultrasonic power of 500 W and a frequency of 40 kHz, oscillating every 10 minutes to obtain an ethyl acetate extract of marigold.

[0030] (3) Concentration: The ethyl acetate extract of marigold was filtered under reduced pressure, then placed in a 250 ml round-bottom flask, and further evaporated under reduced pressure at 60 °C using a rotary evaporator to recover water, obtaining 10 ml of concentrated solution. 10 ml of concentrated solution was placed in a 100 ml evaporating dish, and the solvent was shaken and dried on a water bath at 95 °C to form a dry extract, that is, the marigold extract was obtained.

[0031] Example 2: Preparation of Marigold Stem Extract

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

[0033] (1) Pretreatment: Take 10 g of marigold stems and place them in a stoppered Erlenmeyer flask. Add 100 mL of ethyl acetate, seal with a stopper, and shake well.

[0034] (2) Extraction: Ultrasonic extraction was performed for 30 minutes under light-shielded conditions, with an ultrasonic power of 500 W and a frequency of 40 kHz, oscillating every 10 minutes to obtain an ethyl acetate extract of marigold.

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

[0036] Test example:

[0037] Test Example 1:

[0038] In this test example, different parts of the marigold plant were extracted with different solvents to obtain different marigold extracts.

[0039] The marigold extract was prepared as follows.

[0040] (1) Pretreatment: Take 10 g of marigold plants (roots, stems, and flower powder, passed through a No. 3 sieve) and place it in a stoppered Erlenmeyer flask. Add 100 mL of extractant (water, methanol, ethyl acetate), seal the flask with the stopper, and shake well.

[0041] (2) Extraction: Ultrasonic extraction was performed for 30 minutes under light-shielded conditions, with an ultrasonic power of 500 W and a frequency of 40 kHz, oscillating every 10 minutes to obtain a marigold extract (water, methanol, ethyl acetate).

[0042] (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 recover the water, obtaining 10 ml of a concentrated solution. 10 ml of the concentrated solution was placed in a 100 ml evaporating dish, and the solvent was shaken and dried on a water bath at 95°C to form a dry extract, i.e., the marigold extract was obtained.

[0043] The extraction combinations are shown in Table 1.

[0044] [Table 1]

[0045] Test Example 2:

[0046] In this test example, the inhibitory effects of marigold extracts in different solvents in Test Example 1 on the survival rate of prostate cancer cells were compared.

[0047] 22Rv1 prostate cancer cells in logarithmic growth phase were harvested and seeded in a 96-well cell culture plate with 5,000 cells per well in complete medium containing 10% FBS. After all cells had adhered to the wells, marigold extracts extracted with different solvents were added to the wells at concentrations of 120 μg / ml, 60 μg / ml, 30 μg / ml, 15 μg / ml, and 7.5 μg / ml. DMSO was added to the control group at a concentration of 0.1%. After treating the cells with compounds for 72 hours, 10 μL / well of MTT solution (5 mg / mL) was added to the culture medium. The cells and MTT were incubated in an incubator for 4 hours. After the cell culture plate was removed, the OD was measured at 570 nm and the IC was calculated using GraphPad Prism 8.0. 50 The values were fitted.

[0048] The recorded data is shown in Table 2.

[0049] [Table 2]

[0050] Table 2 shows that marigold extracts in different solvents inhibit the viability of prostate cancer cells to different degrees. IC of marigold root, stem, and flower aqueous extracts 50 The IC values of the methanol extracts of marigold roots, stems, and flowers were 116.2 μg / ml, 120 μg / ml, and 120 μg / ml, respectively. 50 were 119.8ug / ml, 67.3ug / ml, and 102.1ug / ml, respectively, and the IC of ethyl acetate extracts of marigold roots, stems, and flowers 50 The concentrations were 11.7 μg / ml, 18.5 μg / ml, and over 120 μg / ml, respectively. These data suggest that the ethyl acetate extract of marigold root and stem is superior to the aqueous and methanol extracts in inhibiting the viability of prostate cancer cells.

[0051] Test Example 3:

[0052] In this test example, the inhibitory effects on the viability of prostate cancer cells of marigold extracts obtained by extracting different parts with ethyl acetate in Test Example 1 were compared.

[0053] Logarithmic growth phase PC-3, 22Rv1, and DU145 prostate cancer cells were harvested and seeded in complete medium containing 10% FBS onto 96-well cell culture plates at 5,000 cells per well. After all cells had adhered to the wells, various concentrations of ethyl acetate marigold extract were added to the wells at concentrations of 120 μg / ml, 60 μg / ml, 30 μg / ml, 15 μg / ml, and 7.5 μg / ml. DMSO was added to the control group at a concentration of 0.1%. After 72 hours of compound treatment, 10 μL / well of MTT solution (5 mg / mL) was added to the culture medium. The cells and MTT were incubated in an incubator for 4 hours. After 4 hours, the cell culture plates were removed and the OD readings at 570 nm were measured. IC values were calculated using GraphPad Prism 8.0. 50 The values were fitted.

[0054] [Table 3]

[0055] As shown in Table 3, the ethyl acetate extract of marigold can inhibit the viability of prostate cancer cells to different degrees. The IC of ethyl acetate extract of marigold root, stem, and flower inhibits the viability of 22Rv1 prostate cancer cells. 50 The IC values were 11.71 μg / ml, 18.5 μg / ml, and 120 μg / ml, respectively, indicating that the ethyl acetate extract of marigold roots, stems, and flowers inhibited the viability of PC3 prostate cancer cells. 50 The IC values were 17.63 μg / ml, 54.29 μg / ml, and 120 μg / ml, respectively, indicating that the ethyl acetate extract solution of marigold roots, stems, and flowers inhibited the viability of DU145 prostate cancer cells. 50 The concentrations were 36.40 μg / ml, 78.59 μg / ml, and over 120 μg / ml, respectively. These data suggest that ethyl acetate extracts of marigold roots and stems are superior to the flowers in inhibiting the viability of prostate cancer cells. Furthermore, ethyl acetate extracts of marigold roots were the most effective in inhibiting the viability of prostate cancer cells.

[0056] Test Example 4:

[0057] From the above results, it was found that the ethyl acetate extract of marigold root was the most effective in inhibiting the survival rate of prostate cancer cells, and this test example further verified this effect.

[0058] 1. Effect of ethyl acetate extract of marigold on proliferation of 22Rv1 cells

[0059] Prostate cancer 22Rv1 cells in logarithmic growth phase were harvested and seeded into 6-well plates at 1,000 cells per well in a 37°C, 5% CO2 incubator. After 24 h of incubation, the cells were treated with different treatments: a blank control, 40 μM enzalutamide, ethyl acetate extract of marigold root (10 μg / ml, 20 μg / ml, 40 μg / ml), and ethyl acetate extract of marigold stem (10 μg / ml, 20 μg / ml, 40 μg / ml). The cells were cultured for two weeks, after which the formation of cell clones was observed under a microscope. The cells were then fixed, stained, photographed, and counted using a cell clone counter. The results are shown in Figure 1.

[0060] As shown in Figure 1, upper A, 20 μg / ml and 40 μg / ml of ethyl acetate extract of marigold roots significantly inhibited the clonal formation of 22Rv1 cells, and as shown in Figure 1, lower B, 20 μg / ml and 40 μg / ml of ethyl acetate extract of marigold stems significantly inhibited the clonal formation of 22Rv1 cells. These data suggest that both ethyl acetate extract of marigold roots and stems have the effect of significantly inhibiting the proliferation of prostate cancer cells.

[0061] 2. Induction of pyrokinesis in 22Rv1 cells by ethyl acetate extract of marigold

[0062] Prostate cancer 22Rv1 cells in good logarithmic growth phase were harvested and seeded onto 10 cm cell culture dishes at 1 × 10 cells per well. 6 Cells were cultured in a 37°C, 5% CO2 incubator. After 24 hours of culture, three 10cm cell culture dishes were placed in each of three different treatment groups: a blank control, a 40µg / ml ethyl acetate extract of marigold root, and a 40µg / ml ethyl acetate extract of marigold stem. After 72 hours of culture, the cells were harvested and fixed with 0.5% glutaraldehyde. Cell samples were prepared according to the sample preparation method for transmission electron microscopy, and the internal morphology of the cells was observed using a transmission electron microscope.

[0063] As shown in Figure 2, after cells were treated with 40 μg / ml of ethyl acetate extracts of marigold roots and stems, the nuclei (indicated by N in the figure) were irregularly shaped, with uniform chromatin distribution, predominantly euchromatin, and deep heterochromatin staining, mainly distributed along the nuclear membrane. Mitochondria (indicated by Mi in the figure) were massively swollen, i.e., their volume increased, their ridges decreased, ruptured, or disappeared, their matrix became less electron-dense, and their contents were partially dissolved, appearing flocculent or vacuolated. The rough endoplasmic reticulum (indicated by RER in the figure) was dilated (blue arrows) and saccular, with ribosomes attached to its surface. The cell membrane ruptured (orange arrows), releasing its contents. A small number of primary lysosomes (indicated by Ly in the figure), autophagy (indicated by purple arrows), and numerous vesicles (indicated by yellow arrows) were also observed in the cytoplasm. The above intracellular morphological changes were consistent with the characteristics of cellular pyrokinesis, and transmission electron microscopy revealed that both ethyl acetate extracts of marigold roots and stems had the ability to induce pyrokinesis in 22Rv1 cells.

[0064] 3. Effect of ethyl acetate extract of marigold on the growth of xenografted prostate cancer in mice

[0065] Fifty male Balb / c nude mice (5-6 weeks old, Hunan Slake Jingda Laboratory Animal Co., Ltd.) were purchased. After one week of adaptation, 1 × 10 22Rv1 cells were injected subcutaneously into the armpit of each mouse's right upper limb. 6 The tumor volume of the mice was 50-100 mm 3When tumor size reached 100 mg / kg, 36 mice were selected from the 50 model mice and administered medication. Thirty-six Balb / c nude mice were randomly divided into six groups (n=6) based on tumor volume: the model group, 1 mg / kg docetaxel group, 150 mg / kg marigold root ethyl acetate extract group, 75 mg / kg marigold root ethyl acetate extract group, 150 mg / kg marigold stem ethyl acetate extract group, and 75 mg / kg marigold stem ethyl acetate extract group. The docetaxel group received intraperitoneal injection twice weekly for four weeks. The model group and marigold extract group received intragastric administration five days a week for four weeks. During the treatment period, tumor volume was measured once a week, and mice were weighed once a week. After four weeks of treatment, tumors were harvested, weighed, fixed in 4% paraformaldehyde, and / or flash-frozen and stored in liquid nitrogen for storage. This animal experiment was approved by the Experimental Animal Welfare and Ethics Committee of Guangxi University of Traditional Chinese Medicine (approval number: DW20231016-218).

[0066] The results are shown in Figure 3. Figure 3A shows that during the administration period, there were no significant differences in mouse weights in the 1 mg / kg docetaxel group, 150 mg / kg marigold root ethyl acetate extract group, 75 mg / kg marigold root ethyl acetate extract group, 150 mg / kg marigold stem ethyl acetate extract group, and 75 mg / kg marigold stem ethyl acetate extract group compared to the model group. Figure 3B shows that during the administration period, the tumor volumes in the 1 mg / kg docetaxel group, 150 mg / kg marigold root ethyl acetate extract group, 75 mg / kg marigold root ethyl acetate extract group, 150 mg / kg marigold stem ethyl acetate extract group, and 75 mg / kg marigold stem ethyl acetate extract group were significantly smaller than those in the model group. As shown in Figure 3C, at the end of the experiment, the tumor volumes in mice in the 1 mg / kg docetaxel group, the 150 mg / kg ethyl acetate extract of marigold root group, the 75 mg / kg ethyl acetate extract of marigold root group, the 150 mg / kg ethyl acetate extract of marigold stem group, and the 75 mg / kg ethyl acetate extract of marigold stem group were significantly smaller than those in the model group. As shown in Figure 3D, at the end of the experiment, the tumor weights in mice in the 1 mg / kg docetaxel group, the 150 mg / kg ethyl acetate extract of marigold root group, the 75 mg / kg ethyl acetate extract of marigold root group, the 150 mg / kg ethyl acetate extract of marigold stem group, and the 75 mg / kg ethyl acetate extract of marigold stem group were significantly smaller than those in the model group. As shown in Figure 3E, the mouse tumors at the end of the experiment are shown, among which: row 1: mouse tumors in the model group; row 2: mouse tumors in the 1 mg / kg docetaxel group; row 3: mouse tumors in the 150 mg / kg marigold root ethyl acetate extract group; row 4: mouse tumors in the 75 mg / kg marigold root ethyl acetate extract group; row 5: mouse tumors in the 150 mg / kg marigold stem ethyl acetate extract group; row 6: mouse tumors in the 75 mg / kg marigold stem ethyl acetate extract group.As can be seen from the figure, at the end of the experiment, the sizes of the mouse tumors in the 1 mg / kg docetaxel group, 75 mg / kg marigold root ethyl acetate extract group, 150 mg / kg marigold root ethyl acetate extract group, 75 mg / kg marigold stem ethyl acetate extract group, and 150 mg / kg marigold stem ethyl acetate extract group were significantly smaller than the sizes of the mouse tumors in the model group.

[0067] The above examples illustrate some embodiments of the present invention, and the explanations are more specific and detailed, but the scope of the present invention should not be understood as being limited thereby.

Claims

1. A marigold extract, comprising the steps of: (1) Pretreatment: Take the roots or stems of marigold plants and put them into a stoppered Erlenmeyer flask, add ethyl acetate, seal the flask with a stopper, and shake well by vibrating; (2) Extraction: ultrasonic extraction under light-shielding conditions to obtain an ethyl acetate extract of marigold; (3) Concentration: The ethyl acetate extract of marigold is filtered under reduced pressure, evaporated and concentrated, and then the concentrated solution is placed in an evaporating dish and dried by shaking the solvent over a water bath to form a dry extract, i.e., the marigold extract is obtained.

2. 2. The marigold extract according to claim 1, wherein the ratio of the roots or stems of the marigold plant to the raw material liquid of ethyl acetate in step (1) is 1 g:10 mL.

3. 2. The marigold extract according to claim 1, wherein the ultrasonic extraction conditions in step (2) are an ultrasonic power of 500 W, an ultrasonic frequency of 40 kHz, an extraction time of 30 minutes, and oscillation every 10 minutes during the extraction process.

4. The marigold extract according to claim 1, characterized in that the evaporation and concentration method in step (3) is specifically performed by placing the marigold extract in a 250 ml round-bottom flask and evaporating it under reduced pressure at 60°C in a rotary evaporator to recover the water and obtain 10 ml of a concentrated liquid.

5. 2. The marigold extract according to claim 1, wherein the temperature of the water bath in step (3) is 95°C.

6. Use of the marigold extract according to any one of claims 1 to 5 in the preparation of a medicament for the prevention and / or treatment of anti-prostate cancer.

7. A pharmaceutical composition for the prevention and / or treatment of prostate cancer, comprising the marigold extract according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier or excipient.

8. 8. The pharmaceutical composition according to claim 7, wherein the amount of the marigold extract used is 40 to 60% of the total mass of the pharmaceutical composition.

9. 8. The pharmaceutical composition according to claim 7, wherein the pharmaceutically acceptable carrier or excipient is selected from one or any combination of solvents, diluents, dispersants, suspension aids, surfactants, isotonicity agents, thickeners, emulsifiers, preservatives, binders, lubricants, stabilizers, water mixtures, emulsification enhancers, buffers, absorbents, colorants, fragrances, sweeteners, ion exchange agents, release agents, coating agents, flavoring agents, and antioxidants.

10. The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition is prepared in any one of the following dosage forms: powder, tablet, capsule, pill, drop pill, injection, emulsion, suspension, or tincture.

Images