Use of mangosteen extract for the preparation of anti-inflammatory and anti-tumor drugs

Abstract

This invention provides the use of mangosteen extract in the preparation of anti-inflammatory and anti-tumor drugs. The mangosteen extract (α-dextrin, β-dextrin, and γ-dextrin) from the Clusiaceae family exhibits strong inhibitory activity against cancer cell proliferation and regulates the expression of inflammation-related factors. The mangosteen extract also demonstrates strong inhibitory activity against malignant tumor cells such as breast cancer, colorectal cancer, and lung cancer, while simultaneously regulating the expression of inflammation-related factors in inflammatory diseases, thereby effectively exerting anti-tumor and anti-inflammatory effects.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the use of a mangosteen extract in the preparation of anti-inflammatory and anti-tumor drugs. Background Technology

[0002] Mangosteen, the fruit of the mangosteen plant (Garcinia oblongifolia), belonging to the genus Garcinia in the family Clusiaceae, is known as the "Queen of Fruits." It is neutral in nature, sweet in taste, and slightly cooling. It enters the lung, spleen, liver, heart, large intestine, and kidney meridians, and has the effects of clearing heat and reducing fire, nourishing yin and moistening dryness, resolving phlegm and relieving cough, and strengthening the spleen and removing dampness. α-Daucusin, β-Daucusin, and γ-Daucusin are important medicinal active ingredients in mangosteen.

[0003] To date, there have been no reports of the use of mangosteen extract in the treatment of malignant tumors such as breast cancer, colorectal cancer, and lung cancer, as well as inflammation-related diseases. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a use of mangosteen extract in the preparation of anti-inflammatory and anti-tumor drugs, which can regulate the expression of inflammatory factors in inflammatory-related diseases, thereby effectively exerting anti-tumor and anti-inflammatory effects.

[0005] To achieve the above and other related objectives, the present invention provides the use of mangosteen extract as a pharmaceutically active ingredient in the preparation of anti-inflammatory drugs.

[0006] Preferably, the mangosteen extract includes one or more of α-trachytin, β-trachytin, and γ-trachytin.

[0007] Preferably, the mangosteen extract includes at least α-dextrin, and the anti-inflammatory drug is selected from one of the anti-KPL-4 inflammatory breast cancer drugs and the anti-SUM149PT inflammatory breast cancer drugs.

[0008] Preferably, the amount of mangosteen extract added to the anti-inflammatory drug is 8-15 μmol / L.

[0009] Preferably, the amount of α-dextrin added to the anti-inflammatory drug is 7-12 μmol / L.

[0010] The present invention also provides the use of mangosteen extract as a pharmaceutically active ingredient in the preparation of anticancer drugs.

[0011] Preferably, the mangosteen extract includes one or more of α-trachytin, β-trachytin, and γ-trachytin.

[0012] Preferably, the anticancer drug is selected from one of the following: anti-breast cancer drugs, anti-colorectal cancer drugs, and anti-lung cancer drugs.

[0013] Preferably, the anti-breast cancer drug is used to inhibit the growth and proliferation of inflammatory breast cancer cells, including KPL-4 inflammatory breast cancer cells and SUM149PT inflammatory breast cancer cells.

[0014] Preferably, the amount of mangosteen extract added to the anti-breast cancer drug is 10-160 μmol / L; the amount of mangosteen extract added to the anti-colorectal cancer drug is 5-40 μmol / L; and the amount of mangosteen extract added to the anti-lung cancer drug is 10-130 μmol / L.

[0015] As described above, the present invention has the following beneficial effects: α-dextrin, β-dextrin, and γ-dextrin, extracts of mangosteen (Garcinia cambogia), a plant of the Garcinia genus in the Clusiaceae family, have strong inhibitory activity against cancer cell proliferation and regulate the expression of inflammation-related factors; mangosteen extract has strong inhibitory activity against malignant tumor cells such as breast cancer, colorectal cancer, and lung cancer, and can also regulate the expression of inflammation-related factors in inflammation-related diseases, thereby effectively exerting anti-tumor and anti-inflammatory effects. Attached Figure Description

[0016] Figure 1 The graph shows the effect of mangosteen extract α-dextrin on the proliferation of normal human breast cells and human breast cancer cells.

[0017] Figure 2 The graph shows the effect of mangosteen extract β-dextrin on the proliferation of normal human breast cells and human breast cancer cells.

[0018] Figure 3 The graph shows the effect of mangosteen extract γ-dextrin on the proliferation of normal human breast cells and human breast cancer cells.

[0019] Figure 4 The graph shows the effects of mangosteen extracts α-dextrin, β-dextrin, and γ-dextrin on the proliferation of colorectal cancer cells.

[0020] Figure 5 The graph shows the effects of mangosteen extracts α-dextrin, β-dextrin, and γ-dextrin on lung cancer cell proliferation.

[0021] Figure 6 To demonstrate the effect of mangosteen extract α-dextrin on the expression of inflammatory-related factors in KPL-4 inflammatory breast cancer cells using real-time quantitative polymerase chain reaction (q-PCR).

[0022] Figure 7 The results, presented as quantitative real-time polymerase chain reaction (q-PCR), show the effect of mangosteen extract α-thujone on the expression of inflammatory-related factors in SUM149PT inflammatory breast cancer cells. Detailed Implementation

[0023] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0024] It should be noted that the process equipment or apparatus not specifically mentioned in the following embodiments are all conventional equipment or apparatus in the art.

[0025] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0026] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0027] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature; see Sambrook et al., *MOLECULAR CLONING: A LABORATORY MANUAL*, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., *CURRENT PROTOCOLS IN MOLECULAR BIOLOGY*, John Wiley & Sons, New York, 1987 and periodic updates; these series *METHODS IN ENZYMOLOGY*, Academic Press, San Diego; Wolffe, *CHROMATINSTRUCTURE AND FUNCTION*, Third edition, Academic Press, San Diego, 1998; *METHODS IN ENZYMOLOGY*, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and *METHODS IN MOLECULAR*. BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0028] The following one or more pharmaceutically acceptable excipients refer to drug-acceptable carriers or excipients, such as those added to conventional diluents (water, etc.), fillers (lactose, etc.), binders (starch, dextrin, etc.), transdermal absorption enhancers, flavoring agents, thickeners, solvents (ethanol, etc.) and other excipients.

[0029] The following topical preparations include ointments, creams, gels, films, patches, tinctures, and aerosols; the following oral preparations include tablets, granules, capsules, pills, powders, oral liquids, and syrups.

[0030] This application provides an example of using mangosteen extract as a pharmaceutically active ingredient in the preparation of an anticancer drug. The mangosteen extract includes α-dextrin, β-dextrin, and γ-dextrin. The anti-breast cancer drug is used to inhibit the growth and proliferation of inflammatory breast cancer cells, including KPL-4 and SUM149PT inflammatory breast cancer cells. The anticancer drug is an anti-breast cancer drug, an anti-colorectal cancer drug, and an anti-lung cancer drug. The amount of mangosteen extract added to the anti-breast cancer drug is 10–160 μmol / L; the amount added to the anti-colorectal cancer drug is 5–40 μmol / L; and the amount added to the anti-lung cancer drug is 10–130 μmol / L.

[0031] 1. Experimental Materials

[0032] 1.1 Reagents

[0033] α-Dystolin, β-Dystolin, and γ-Dystolin (Yuan Ye Company); 10% fetal bovine serum (Vistec, SA); DMEM culture medium (Gino Biopharmaceutical Technology Co., Ltd.); 0.25% trypsin (Shanghai Yuanpei Biotechnology Co., Ltd.); CCK8 kit (Dalian Meilun Biotechnology Co., Ltd.); Trizol (Invitrogen); Chloroform (Sinopharm Group); Isopropanol (Sinopharm Group); Anhydrous ethanol (Sinopharm Group); Enzyme-free ultrapure water (Meilun Biotechnology Co., Ltd.); Reverse transcription kit (Vinozyme Biotechnology Co., Ltd.)

[0034] 1.2 Cell lines

[0035] The normal human breast cell line MCF-10A, breast cancer cell lines MCF-7, MDA453, BT474, MDA-MB-231, Bcap-37, and inflammatory breast cancer cell lines KPL-4, SUM149PT, colorectal cancer cells HCT-8, SW480, and lung cancer cells H420 and H1299 were purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0036] 2 Experimental Methods

[0037] 2.1 Cell Counting Kit 8 (CCK8) for Detecting Cell Proliferation

[0038] (1) Select normal human breast cells MCF-10A, breast cancer cells, inflammatory breast cancer cells, colon cancer cells and lung cancer cells that are in good growth state and in the logarithmic growth phase. Digest them with trypsin, add complete culture medium containing 10% FBS to stop digestion, pipette into cell suspension, transfer to EP tube, centrifuge at 800 rpm for 5 min, discard the supernatant, add complete culture medium to make cell suspension.

[0039] (2) Use a cell counting chamber to count cells. Seed 5000 cells per well in 6 replicates per group of cells into a 96-well plate with 100 μL per well. Add an equal amount of PBS solution to the wells around the perimeter to prevent edge effects.

[0040] (3) Incubate the conventional culture medium overnight in a 37℃, 5% CO2 incubator; remove the liquid culture medium from each well, and prepare 10μM α-dextrin, β-dextrin and γ-dextrin culture solutions under light-protected conditions, and add them to each well for continued incubation.

[0041] (4) After culturing for 48 hours, remove the culture medium from the well plate under dark conditions, prepare the CCK8 solution required for the experiment, add 100 μL of the solution to each well (prevent air bubbles from being generated during the addition process to avoid inaccurate measurement by the microplate reader), and incubate in the incubator for 1-2 hours; (5) Adjust the wavelength of the fully automated microplate reader to 450 nm and measure the OD value of each well.

[0042] (6) Based on the OD values, the growth curves of each group of cells were plotted, and the cell proliferation was calculated and analyzed using Graphpad 7.0.

[0043] 2.2 Real-time quantitative polymerase chain reaction (q-PCR) detection of expression of inflammation-related factors in cells

[0044] 2.2.1 Extraction of total RNA from cells

[0045] (1) Take inflammatory breast cancer cells with a growth density of about 80%, digest them with trypsin, and then store the cells at 4 × 10⁻⁶ cells per dish. 5 One cell was seeded in a small culture dish and incubated at 37°C in an incubator containing 5% CO2.

[0046] (2) After the cells adhered to the cell wall, the culture medium was aspirated, 1 mL of PBS was added along the side wall to wash the cells, and culture medium containing 10 μM of drug was added. The cells were placed in an incubator containing 5% CO2 and cultured at 37°C. Microscopic observation was performed at intervals of 12 h, 24 h, 36 h, and 48 h.

[0047] (3) When obvious changes in cell morphology are observed under a microscope, collect the cells with a cell scraper and collect them together with the cell culture medium into a 15mL centrifuge tube. Centrifuge at 4℃ and 2000rpm for 5min and discard the supernatant.

[0048] (4) Add 1 mL of PBS to resuspend the cells, transfer the cells to a 1.5 mL centrifuge tube, centrifuge at 4°C and 2000 rpm for 5 min, and discard the supernatant;

[0049] (5) Add 1 mL of Trizol solution to each tube, resuspend the cells, add 300 μL of chloroform, shake vigorously, and place on ice for 5 min; (6) Centrifuge at 4℃ (pre-cooled centrifuge required) and 12000g for 15 min;

[0050] (7) After centrifugation, the liquid in the centrifuge tube is divided into three layers. Use a pipette to carefully transfer the upper layer of liquid to a new 1.5mL centrifuge tube, add an equal amount of isopropanol, and gently invert the centrifuge tube 3-5 times. Then place it in a -20℃ refrigerator for 30 minutes.

[0051] (8) Centrifuge at 12000g for 10min using a centrifuge with 4℃ degraded cooling, prepare 75% ethanol, pre-cool at 4℃ for later use, discard the supernatant after centrifugation, and try to remove the residual liquid from the tube wall.

[0052] Add 1 mL of 75% ethanol to each tube, vortex for 15 seconds to fully wash the precipitate with ethanol, and centrifuge at 4°C and 7500g for 5 minutes.

[0053] (10) Discard the supernatant, centrifuge at 4℃ and 12000g for 5 minutes, discard the supernatant again, aspirate the remaining liquid from the tube wall, open the cap and place in a fume hood for about 5 minutes, until the RNA precipitate on the tube wall changes from white to translucent;

[0054] (11) Dissolve the obtained RNA precipitate in 20-200 μL of enzyme-free water (preheated at 65℃) until the precipitate dissolves; take 1 μL of sample and measure the RNA concentration using a NanoDrop micro spectrophotometer, and store it in a -80℃ refrigerator.

[0055] 2.2.2 Reverse Transcription PCR

[0056] Add each component to a 200 μL centrifuge tube according to Table 1, gently mix with a 20 μL pipette, centrifuge briefly using a microcentrifuge, place the centrifuge tube in a PCR instrument, and set the reaction conditions to 42℃ for 2 min.

[0057] Table 1. GDNA Removal Reagent Dosing Table

[0058] reagents volume 4×gDNA wiper Mix 4μL template RNA Total RNA 1μg <![CDATA[RNase-free ddH2O]]> To 16μL

[0059] After completing the above steps, remove the centrifuge tube, add each component in order according to Table 2, gently mix with a 20μL pipette, centrifuge briefly using a microcentrifuge, and place the centrifuge tube in the PCR instrument again, setting the reaction conditions as follows: 37℃ for 15min, 85℃ for 5s.

[0060] Table 2 Reverse Transcription Reagent Dosing Table

[0061] reagents volume 5HiscriptⅢqRTsuper Mix 4μL Step 1 reaction solution 16μL

[0062] After the reaction was completed, 1 μL of the sample was taken and the cDNA concentration was measured using a NanoDrop micro spectrophotometer. The sample was then stored at -80°C for later use.

[0063] 2.2.3 Primer Design

[0064] Suitable primers were designed using NCBI and Primerbank databases and synthesized by Qingke Biotechnology Primer Company. The sequences are shown in Table 3.

[0065] Table 3 Primer sequences

[0066]

[0067]

[0068] 2.2.4 Real-time quantitative PCR

[0069] Add the components to the 96-well PCR plate in the order shown in Table 4, with 3 replicates for each sample. Mix all components thoroughly, centrifuge at 2000 rpm for 3 min at 4 °C, and place in a real-time PCR instrument. Set the reaction program conditions as follows: 95 °C for 5 min; 95 °C for 5 s; 60 °C for 30 s; 72 °C for 30 s. Return to step 2 and cycle the reaction 40 times. Finally, extend the reaction at 72 °C for 5 min.

[0070] Table 4. Real-time PCR reaction system

[0071] Components volume 2×ChamQ Universal SYBR q-PCR Master Mix 5μL Forward primer 0.2μL Reverse primer 0.2μL cDNA 0.5μL <![CDATA[ddH2O]]> 4.1μL

[0072] After the reaction, based on the obtained Ct values ​​and using the blank Ct value as a control, the average relative content 2-ΔΔCt value was calculated (ΔCt experimental group = Ct experimental group target gene - Ct experimental group internal reference gene, ΔCt control group = Ct control group target gene - Ct control group internal reference gene, ΔΔCt value = ΔCt experimental group - ΔCt control group).

[0073] 3 Experimental Results

[0074] This invention evaluated the antitumor effects of mangosteen extracts α-trachytin, β-trachytin, and γ-trachytin through the above experiments. The experimental results are shown below. Figures 1 to 5 As shown.

[0075] like Figures 1-3As shown, the effects of different concentrations of α-trachytin, β-trachytin, and γ-trachytin on the growth and proliferation of normal breast cancer cells MCF-10A, breast cancer cells MCF-7, BT474, MDA-MB-231, and inflammatory breast cancer cells KPL-4 and SUM149PT were detected using the CCK8 assay. All experimental concentrations of α-trachytin, β-trachytin, and γ-trachytin inhibited the growth and proliferation of both breast cancer cells and inflammatory breast cancer cells, with the inhibition rate gradually increasing with increasing dose, exhibiting a clear dose-dependent effect. At the same concentration, α-trachytin, β-trachytin, and γ-trachytin did not inhibit the growth of normal human breast cells.

[0076] like Figure 4 , Figure 5 As shown, the effects of different concentrations of α-trachytin, β-trachytin, and γ-trachytin on the growth and proliferation of colorectal cancer cells and lung cancer cells were also detected using the CCK8 assay. The experimental data showed that different concentrations of α-trachytin, β-trachytin, and γ-trachytin all had a certain inhibitory effect on the proliferation of colorectal cancer cells and lung cancer cells. Furthermore, with increasing concentration, the survival rate of colorectal cancer and lung cancer cells continuously decreased, with the half-maximal inhibitory concentration (IC50) at the μM level, indicating good drug efficacy.

[0077] like Figure 6 and Figure 7 As shown, q-PCR was used to detect the effect of mangosteen extract α-mangosteenin on the expression of inflammation-related factors in two types of inflammatory breast cancer cells, KPL-4 and SUM149PT. The results showed that the expression levels of seven genes in both cell types increased or decreased to varying degrees, with the expression level of CXCL13 changing particularly significantly.

[0078] In summary, this invention has found that mangosteen extract exhibits strong inhibitory activity against the proliferation of malignant tumor cells such as breast cancer, colorectal cancer, and lung cancer. Simultaneously, it can regulate the expression of inflammatory factors in inflammation-related diseases, and can be used to prepare anti-inflammatory and anti-tumor drugs, thereby effectively exerting anti-tumor and anti-inflammatory effects. Therefore, this invention effectively overcomes the various shortcomings of existing technologies and has high industrial application value.

[0079] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

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