A cat's claw extract and its application in cancer treatment

Abstract

The present invention discloses a cat's claw extract and its application in the preparation of cancer therapeutic drugs. The cat's claw extract contains active ingredients of RTE, and the main targets are AKT1, EGFR and HSP90AA1. The preparation method of the cat's claw extract of the present invention comprises soaking the cat's claw coarse powder with ethanol for reflux extraction, and then extracting with petroleum ether, ethyl acetate and n-butanol in sequence, and selecting the ethyl acetate extract. Based on the network pharmacology method, it is confirmed that the anti-cancer active targets in the active ingredients of the RTE of cat's claw are AKT1, EGFR and HSP90AA1 active targets. The RTE of the present invention has an anti-esophageal squamous cell carcinoma effect, and therefore has a wide application value.

Description

Technical Field

[0001] The present invention relates to the field of plant extracts, and in particular to a cat's claw extract. More particularly, the present invention provides a cat's claw extract with AKT1, HSP90AA1 and EGFR as main anti-cancer targets and uses thereof. Background Art

[0002] Cancer is the number one killer threatening human health today. In 2020, more than 10 million people died of cancer worldwide. Cancer has biological characteristics such as abnormal cell differentiation and proliferation, uncontrolled growth, invasiveness and metastasis. Its occurrence is a complex process with multiple factors and steps, which is divided into three processes: carcinogenesis, cancer promotion and evolution. It is closely related to smoking, infection, occupational exposure, environmental pollution, unreasonable diet and genetic factors. Among the current treatment methods, surgical resection is still the preferred treatment method for early or relatively early solid tumors, followed by chemotherapy, radiotherapy and other treatment methods. The above treatment methods have good effects, but they have certain requirements on the physical fitness of patients. They have a certain tolerance and can withstand the greater physiological pain caused by the above treatment methods. As a complementary and alternative medicine, traditional Chinese medicine has the advantage of low side effects. It can reduce the toxic side effects of radiotherapy and chemotherapy, promote patient recovery and enhance tolerance to radiotherapy and chemotherapy. It is widely used together with anti-tumor drugs to treat various forms of cancer.

[0003] Cat's claw (scientific name: Ranunculus ternatus Thunb.) is an annual herbaceous plant of the Ranunculaceae family, Ranunculaceae, mainly distributed in Henan and Anhui regions of China. It is a neutral plant, light-loving, shade-tolerant, and warm and humid climate. It grows in hills, dry slopes, ridges, roadsides, and wet places in wasteland, and has strong adaptability. It is not strict with soil requirements and is suitable for growing in loose, moist, fertile humus loam cultivation. It is relatively tolerant to water and humidity. Cat's claw is warm in nature, sweet and pungent in taste, enters the liver and lung meridians, and has the effects of clearing away heat and detoxifying, reducing swelling and dispersing knots, relieving cough and removing phlegm. It is clinically used to treat pulmonary tuberculosis, lymph node tuberculosis, pharyngitis, malaria and various cancers, but their active ingredients and potential targets are still unclear. Summary of the invention

[0004] In view of the problems that the active ingredients and potential targets of cat's claw extract in cancer treatment are unclear, the present invention explores the potential targets of RTE and the mechanism of inducing cancer cell apoptosis through network pharmacology methods, and uses different solvents to extract different parts of cat's claw alcohol extract. The effects of cat's claw petroleum ether extract (RTP) and ethyl acetate extract (RTE) in inhibiting cancer cell proliferation are confirmed through experiments.

[0005] The present invention provides a cat's claw extract, and the preparation method thereof comprises the following steps:

[0006] Step a: adding ethanol to the crude powder of cat's claw according to a certain proportion, soaking, refluxing extraction and filtering, repeating the extraction for several times, combining the filtrate, and concentrating under reduced pressure until there is no ethanol smell, thereby obtaining a concentrated solution;

[0007] Step b: adding ultrapure water to the concentrated solution to dissolve it, adding petroleum ether, ethyl acetate and n-butanol in sequence and letting it stand overnight, extracting after stratification, spinning dry and pre-freezing after a certain number of extractions, and vacuum freeze-drying to obtain different parts of cat's claw alcohol extract (cat's claw petroleum ether part, cat's claw ethyl acetate part and cat's claw n-butanol part), which are then sealed and stored.

[0008] Furthermore, the ethanol used in step a is 95% ethanol, the mass ratio of the cat's claw powder to ethanol is 1:5-1:10, preferably 1:10; the soaking and reflux time is 1-3 hours, preferably, the soaking time is 1 hour, and the reflux time is 3 hours; the number of repeated extractions is 1-3 times, preferably 3 times.

[0009] Furthermore, the volume ratio of ultrapure water used in step b to the concentrated solution is 1:1-1:5, preferably 1:1; the number of extractions is 3-5 times, preferably 3 times; the pre-freezing temperature is -80°C, the pre-freezing time is 6-18h, preferably 12h; the vacuum freeze-drying time is 24-72h, preferably 48h.

[0010] Furthermore, when used, different parts of the cat's claw alcohol extract were added to DMSO, ultrasonicated until completely dissolved and fixed to 1 ml, and stored at 4°C for later use.

[0011] The present invention also provides a method for exploring the mechanism of the cat's claw extract as mentioned above inducing cancer cell apoptosis based on network pharmacology methods, which mainly includes the following steps: step (1) obtaining effective ingredients and matching targets, step (2) collecting cancer-related targets, step (3) constructing a component target network diagram, step (4) constructing a target interaction diagram, and step (5) GO biological process enrichment and KEGG signal pathway analysis. The above steps all require the use of corresponding network databases and / or software.

[0012] Furthermore, the network databases include Pubchem (https: / / pubchem.ncbi.nlm.nih.gov / ), TCMSP (https: / / old.tcmsp-e.com / tcmsp.php), SEA (http: / / sea.bkslab.org / ), TargetNet (http: / / targetnet.scbdd.com / ), PharmMapper (http: / / lilab-ecust.cn / pharmmapper / index.html), SwissTargetPrediction (http: / / swisstargetprediction.ch / ), Uniprot (https: / / www.chemsrc.com / ), GeneCard (https: / / www.genecards.org / ), OMIM (https: / / www.omim.org / ), DAVID (https: / / david.ncifcrf.gov / ) and STRING (https: / / string-db.org / ); the software includes CAS (https: / / www.chemsrc.com / ), Venny 2.1.0 (https: / / bioinfogp.cnb.csic.es / tools / venny / ) and Cytoscape (Version 3.7.2, http: / / www.cytoscape.org) or more thereof.

[0013] Furthermore, the step (1) comprises: collecting the chemical components of RTE through literature retrieval, verifying and converting them through Pubchem database and CAS website, summarizing the action targets and predicted targets of each active ingredient through TCMSP, SEA, TargetNet, Pubchem, PharmMapper, SwissTargetPrediction database, and obtaining all relevant targets of RTE after screening and summarizing according to degree value, and uniformly converting gene abbreviations using Uniprot database.

[0014] Furthermore, the step (2) includes: collecting cancer-related target information in the GeneCard and OMIM databases, deleting duplicate values ​​from the data obtained from the two databases, and obtaining the final disease-related targets; matching and mapping the RTE-related targets and the cancer-related targets through Venny2.1.0 to obtain a common target, which is the main anti-cancer target of RTE.

[0015] Furthermore, the step (3) includes: importing the effective ingredients and main anti-cancer targets of RTE into Cytoscape software, constructing a drug-active ingredient-main target-disease relationship network diagram (ingredient target network diagram), wherein "node" represents the attribute type, such as Chinese medicine, compound, target and disease, and "edge" represents the interaction relationship. The size and color of each node in the network diagram represent the number of edges, that is, the size of the degree value, and the targets and active ingredients are sorted by degree value to show the connection between the drug-active ingredient-main target-disease in the entire network.

[0016] Furthermore, the step (4) includes: importing the main anti-cancer targets of RTE into the STRING database, selecting humans as the research species, deleting free targets without interaction relationships, and obtaining a target interaction network diagram (protein-protein interaction, PPI).

[0017] Furthermore, the step (5) includes: performing GO biological process enrichment and KEGG signaling pathway analysis on the core action targets of cat's claw (the intersection of targets whose component target network diagram has a value greater than the average and targets whose PPI has a value greater than the average) through the DAVID database to obtain the main signaling pathways and biological processes involved in which RTE exerts its effects.

[0018] The invention provides an application of a cat's claw extract in preparing a cancer treatment drug.

[0019] Furthermore, the cancer is esophageal squamous cell carcinoma.

[0020] Furthermore, the cancer treatment inhibits the proliferation of esophageal squamous cell carcinoma cancer cells by reducing the expression of related core targets.

[0021] Furthermore, the relevant core targets are AKT1, EGFR and HSP90AA1.

[0022] Furthermore, the reduction of the expression of the core target is achieved by regulating the MEK / ERK and PI3K / Akt pathways and / or reducing the expression of downstream related oncogenic factors HSP90 and c-Myc.

[0023] Furthermore, the esophageal squamous cell carcinoma cancer cells are TE-1, TE-13 and / or EC-109.

[0024] Furthermore, the drug is in oral dosage form.

[0025] The present invention obtains the active ingredients of RTE through literature retrieval, and explores the potential targets of RTE and the mechanism of inducing cancer cell apoptosis through network pharmacology methods, and finds three main anti-cancer targets: AKT1, EGFR and HSP90AA1. At the same time, the present invention obtains different extracts of cat's claw by using different solvents for extraction, and confirms the effects of RTP and RTE in inhibiting the proliferation of cancer cells (ESCC) by conducting cell experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the RTE component target network diagram;

[0027] Figure 2 This is the PPI network diagram of RTE-related targets;

[0028] Figure 3 This is the result of GO enrichment analysis of RTE-related targets;

[0029] Figure 4 This is the result diagram of KEGG enrichment analysis of RTE-related targets;

[0030] Figure 5 This is a graph showing the experimental results of the effect of RTE on the expression of related core targets. DETAILED DESCRIPTION

[0031] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The present invention is specifically introduced below in conjunction with the specific embodiments.

[0032] Example 1 Preparation of extracts of cat's claw from different organic solvents:

[0033] Weigh 100 g of crude powder of cat's claw root (purchased from a medicinal material company), add 10 times the amount of 95% ethanol, soak for 1 hour, reflux at 90°C for 3 hours, filter the liquid while hot, repeat the extraction 3 times, combine the filtrate, and concentrate at a constant temperature of 60°C under reduced pressure until there is no ethanol smell (which can be determined by smelling) to obtain a concentrated solution. Add ultrapure water with the same volume as the concentrated solution to dissolve it, and extract it with petroleum ether, ethyl acetate and n-buOH in sequence. The three solvents are used for extraction 3 times each. The different extracts of cat's claw obtained above are spin-dried and pre-frozen in a -80°C refrigerator for 12 hours. After being placed in a vacuum freeze dryer for low-temperature sublimation drying for 48 hours, they are sealed and stored at -20°C to obtain the petroleum ether part (RTP) of cat's claw ethanol extract, the ethyl acetate part (RTE) of cat's claw ethanol extract and the n-butanol part (RTB) of cat's claw ethanol extract.

[0034] Example 2 Experiment on RTE-induced apoptosis of cancer cells based on network pharmacology:

[0035] (1) Acquisition of effective ingredients and matching targets of RTE:

[0036] First, all active ingredients of RTE were analyzed, and the results are shown in Table 1:

[0037] Table 1. Active ingredients of RTE

[0038]

[0039]

[0040]

[0041] The 32 active ingredients of RTE in Table 1 were input into TCMSP, Pubchem, SEA, TargetNet, PharmMapper, and SwissTargetPrediction databases for retrieval, and the related targets of each active ingredient were summarized and duplicate values ​​were deleted. After deleting duplicate values, a total of 472 RTE-related targets were obtained.

[0042] (2) Collecting cancer-related targets

[0043] ESCC (esophageal squamous cell carcinoma) related target information was collected from Genecard and OMIM databases. The data obtained from the two databases were deleted with duplicate values ​​to obtain a total of 4838 disease-related targets. The 4838 ESCC-related targets and 472 RTE-related targets were imported into Venny 2.1.0. By mapping and matching the Venn diagram, a total of 281 ESCC and RTE overlapping targets were obtained, which are the main anti-cancer targets of RTE.

[0044] (3) Constructing a component-target network diagram

[0045] The 32 active ingredients and 281 major anticancer targets of RTE were imported into Cytoscape software to construct a drug-active ingredient-main target-disease relationship network diagram (ingredient target network diagram). Figure 1 As shown, all target nodes are represented by diamonds. All targets and active ingredients in the network diagram are ranked by degree. The average degree of compounds and targets is 9.47. There are 32 compounds and 55 targets with degrees greater than the average (see Table 2).

[0046] Table 2. Key nodes and topological characteristics of the RTE component-target network

[0047]

[0048]

[0049]

[0050] (4) Construction of target interaction map

[0051] Upload 281 major anti-cancer targets to the STRING database, set the Combined Score > 0.7, select humans as the research species, hide unrelated free targets, and obtain the target site interaction network diagram (PPI), as shown in the figure: Figure 2 As shown, there are 254 targets and 1357 edges. The PPI network diagram obtained by STRING was imported into Cytoscape3.7.2, and the PPI network diagram was analyzed. The average degree value was calculated to be 10.68, and there were 81 nodes with a degree value greater than the average value, as shown in Table 3. The top three targets were AKT1, EGFR, and HSP90AA1. These three targets were taken as target targets in subsequent experiments.

[0052] Table 3. PPI key node ranking table

[0053]

[0054]

[0055] (5) GO biological process enrichment and KEGG signaling pathway analysis

[0056] The 81 targets with a greater than average degree in the PPI network diagram and the 55 targets with a greater than average degree in the component target network diagram were intersected to obtain a total of 14 intersection targets of the two groups (Table 4). The 14 core targets were uploaded to the DAVID database (updated in 2021) for GO biological process enrichment analysis. After setting the conditions count ≥ 2 and P < 0.05, 52 GO biological processes, 9 molecular functions, and 30 cellular components were screened out. The top 24 BPs, top 5 CCs, and top 5 MFs were selected to generate visualization results of GO biological process enrichment analysis, as shown in Figure 4. Figure 3 As shown in the figure, it can be seen that biological processes involving signal transduction, cytokine-mediated signaling pathways and drug response are important biological processes for RTE target intervention. KEGG analysis was also performed in the DAVID database. After setting the conditions of count ≥ 2 and P < 0.05, 36 KEGG pathways were screened out and a bubble chart was generated as shown below. Figure 4 ,From the figure, we can see that the cancer pathway, TNF signaling pathway, MAPK signaling pathway, and PI3K / Akt pathway are potential signaling pathways that may be important targets for RTE intervention. Therefore, these cell pathways were selected for study in subsequent experiments.

[0057] Table 4. Ranking of core targets

[0058] Serial number Target Serial number Target 1 HSP90AA1 8 PTGS2 2 MAPK1 9 CDK2 3 RelA 10 F2 4 ESR1 11 MAPK10 5 CASP3 12 ESR2 6 PPARG 13 PGR 7 AR 14 MMP3

[0059] Example 3 Cell experiment:

[0060] (1) Experiments on the inhibition of cell proliferation by RTE, RTP and RTB:

[0061] Different concentrations of RTE (0, 50, 100, 200, 400, 600 and 800 μg / mL), RTP (0, 50, 100, 200, 400, 600 and 800 μg / mL) and RTB (0, 200, 400, 600, 800, 1000 and 2000 μg / mL) were respectively applied to ESCC cell lines TE-1, TE-13 and EC-109 for 48 h, and then the MTT assay was used to detect the results. The results are shown in Table 5. As can be seen from Table 5, RTE and RTP can significantly inhibit the proliferation of the three different cell lines (P<0.001 and P<0.05), and the IC of the EC-109 cell line is higher than that of the RTP. 50 The value (half inhibitory concentration) was the smallest, but there was no significant difference in the proliferation inhibition of the three cell lines by the extracts of these two different reagents (P>0.05); and when the concentration of RTB reached 2000 mg / mL, the proliferation inhibition of the three cell lines did not exceed 50%, indicating that RTB had no anti-tumor activity (results not shown). Therefore, excluding RTB extracts, RTE and RTP can be used.

[0062] Table 5. IC of RTE and RTP in three different ESCC cell lines 50 value

[0063]

[0064]

[0065] Since the chemical components in RTP have poor polarity, which limits its practical application (for preparing drugs), only RTE is selected for experiment in the next embodiment.

[0066] (2) Experiments on the effect of RTE on the expression of relevant core targets:

[0067] After RTE was applied to EC109 and TE-13 cell lines for 48 h (corresponding to the experimental group and the control group), the protein expression or status levels of various cytokines were detected by western blotting. The results are as follows: Figure 5As shown. Compared with the control group, the expression of p-ERK1 / 2 (phosphorylation state of extracellular signal-regulated kinase 1 / 2, whose expression is closely related to lymph node metastasis and tumor differentiation), p-Akt (also known as phosphorylated protein kinase B or PKB, whose expression is closely related to cancer differentiation, stage, and lymph node metastasis), HSP90 (heat shock protein 90, one of the core members of heat shock protein, many client proteins regulated by it are closely related to the occurrence and development of diseases such as tumors) and c-Myc (c-myc gene is both a translocation gene and a regulated gene regulated by multiple substances. It is also a gene that can make cells proliferate indefinitely, acquire immortalization function, and promote cell division) was detected to be decreased. For EC109 cell line, the expression of p-ERK1 / 2 decreased most significantly, followed by p-Akt, and then c-Myc. The expression of HSP90 also decreased, while the expression of β-Actin did not decrease. For TE -13 cell line, the expression of p-ERK1 / 2 decreased most significantly, followed by p-Akt, c-Myc and HSP90, while the expression of β-Actin did not decrease; while the expression of total ERK1 / 2 and Akt did not change significantly (the results of EC109 and TE-13 cell lines were the same, with no significant changes). This confirms that RTE inhibits the proliferation of ESCC by regulating the MEK / ERK and PI3K / Akt pathways and reducing the expression of downstream related carcinogenic factors HSP90 and c-Myc.

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A use of a cat's claw extract in the preparation of a drug for treating esophageal squamous cell carcinoma, characterized in that: The preparation method of the cat's claw extract comprises the following steps: Step a: soaking the crude powder of cat's claw root in ethanol, refluxing and extracting it, filtering it, repeating the extraction, combining the filtrates, and concentrating under reduced pressure to obtain a concentrate; Wherein, the mass ratio of the cat's claw root coarse powder to the ethanol is 1:5-1:10, and the soaking and refluxing time is 1-3 hours; Step b: adding water to the concentrate to dissolve it, extracting it with ethyl acetate, prefreezing it, and then vacuum freeze-drying it to obtain the cat's claw extract; The volume ratio of the water to the concentrated solution is 1:1-1:5, the pre-freezing temperature is -80°C, the pre-freezing time is 6-18 hours, and the vacuum freeze-drying time is 24-72 hours; The esophageal squamous cell carcinoma treatment inhibits the proliferation of esophageal squamous cell carcinoma cancer cells by reducing the expression of related core targets; The relevant core targets are AKT1, EGFR and HSP90AA1; The reduction in the expression of the relevant core targets is achieved by regulating the MEK / ERK and PI3K / Akt pathways and reducing the expression of downstream related carcinogenic factors HSP90 and c-Myc.

2. The use according to claim 1, characterized in that The esophageal squamous cell carcinoma cancer cells are TE-1, TE-13 and / or EC-109.

3. The use according to claim 1 or 2, characterized in that: The drug is in oral dosage form.

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