Preparation method and application of nano traditional Chinese medicine compound

Abstract

The invention discloses a preparation method and application of a nano traditional Chinese medicine compound. The nano traditional Chinese medicine compound NCMCCE2 is prepared on the basis of deep analysis of active ingredients of traditional Chinese medicines, the proportion of the active ingredients is simulated, the medicine effect is verified, meanwhile, active ingredients possibly packaged through self-assembly after decoction are considered, and three single components including camptotheca acuminate bark, taxus chinensis bark and radix ranunculi ternati are taken as examples. Anticancer active molecules are packaged in the prepared NCMCCE2, antitumor activity tests in liver cancer (Hepa1-6) and lung cancer (A549) cell models show that the NCMCCE2 has the effect of inhibiting growth of liver cancer and lung cancer cells, it is indicated that the NCMCCE2 has broad-spectrum antitumor activity, and the prepared nano traditional Chinese medicine compound has high medicinal value and good clinical application prospects.

Description

Technical Field

[0001] The invention relates to a preparation method and application of a nano traditional Chinese medicine compound, belonging to the technical field of biomedicine. Background Art

[0002] At present, the research on nanotechnology of traditional Chinese medicine has achieved certain results. Through the application of nanocrystal technology, liposomes, polymers, supramolecular assembly, solid lipid nanoparticles and other technologies, gratifying results have been achieved in the research of multiple monomer components and effective parts of traditional Chinese medicine. However, nanotechnology is rarely involved in traditional Chinese medicine compound. Due to its multi-target synergistic treatment advantages, traditional Chinese medicine compound has attracted more and more attention in recent years. Traditional Chinese medicine compound has a wide range of applications in cancer, infection, metabolism, regeneration and other fields; these compounds can exert the unique therapeutic effects of traditional Chinese medicine through exquisite compatibility and decoction process, providing strong support for the treatment of various diseases.

[0003] However, the preparation of compound nanoformulations in the prior art is simply to crush the entire compound medicinal material particles to the nanoscale, which is challenged by the differences in the physical and chemical properties of the medicinal materials that make up the Chinese medicine compound, and the fact that the Chinese medicine compound also has large toxic side effects (mainly toxins) and lacks characterization of its ingredients. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing a nano-TCM compound in view of the shortcomings of existing TCM compound prescriptions, such as unstable ingredients, high toxicity and difficulty in characterization. The nano-TCM compound prepared by the method is expected to be applied to the treatment of cancer, infection, metabolism, regeneration and other diseases.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a method for preparing a nano-TCM compound, comprising: based on disease characteristics and information in a TCM database, as well as preset assembly results, selecting one or more TCMs according to the disease characteristics and designing a ratio, adding the TCMs into distilled water, boiling them, filtering to remove insoluble matter, and then removing toxins and small molecule impurities through dialysis to obtain the nano-TCM compound.

[0007] Preferably, the dialysis uses a dialysis bag with a molecular weight cutoff of 3.5 to 14 KDa, and the dialysis time is 3 to 5 days.

[0008] Preferably, the dialysis further comprises a freeze-drying step.

[0009] In a second aspect, the present invention provides a nano Chinese medicine compound prepared by the above preparation method.

[0010] The third aspect of the present invention provides the use of the above preparation method in the preparation of broad-spectrum anticancer drugs.

[0011] The fourth aspect of the present invention provides the use of the nano-TCM compound prepared by the above-mentioned preparation method in the preparation of drugs for treating diseases.

[0012] Preferably, the disease comprises any one of cancer, infection, metabolism and regeneration.

[0013] The metabolic disease refers to a disease caused by the accumulation or deficiency of certain metabolic substances such as sugar, fat, protein, purine, calcium and copper when the biochemical process in the body is disturbed.

[0014] The regenerative disease refers to a disease caused by failure of bone marrow hematopoietic function or impaired tissue regeneration ability due to some reason. Among regenerative diseases, the most typical one is aplastic anemia (AA), which is a bone marrow hematopoietic function failure caused by various causes. The main manifestations are decreased proliferation of bone marrow hematopoietic cells and decreased peripheral blood cells, and anemia, bleeding and infection are the main clinical symptoms.

[0015] The fifth aspect of the present invention provides a method for preparing a broad-spectrum anticancer drug, comprising: adding yew bark, camptotheca acuminata bark and cat's scratch grass into water and boiling them, wherein the mass ratio of the three Chinese medicinal herbs, yew bark, camptotheca acuminata and cat's scratch grass, is 1:1:1; after boiling, filtering to remove insoluble matter; and then removing toxins and small molecule impurities by dialysis to obtain the drug.

[0016] The sixth aspect of the present invention provides the use of the broad spectrum anticancer drug prepared by the above preparation method in the preparation of drugs or preparations for treating cancer.

[0017] Preferably, the cancer includes liver cancer and lung cancer.

[0018] The present invention is based on an in-depth analysis of the active ingredients of traditional Chinese medicine, simulates its proportion and verifies the efficacy, and considers the active ingredients that may be encapsulated by self-assembly after decoction. Taking three single prescriptions such as camptothecin bark, yew bark, and cat's claw as examples, a nano-TCM CCE2 is prepared. Among them, camptothecin bark contains a variety of active ingredients, such as camptothecin, which has significant anti-cancer activity; the most famous medicinal ingredient in yew bark is paclitaxel, which has significant anti-cancer activity. In addition, it also contains taxane compounds, flavonoid compounds, polysaccharide compounds, etc., which also have anti-cancer and immunomodulatory effects; cat's claw is a toxin-removing, swelling-removing, and sore-healing medicine. Its dried tubers are used as medicine, mainly containing saponins, flavonoid compounds, etc., which have the effects of expectoration, dispersing nodules, detoxification, and swelling. It contains a variety of anti-cancer active ingredients, such as baicalin and quercetin. Based on the nano-TCM compound preparation technology of the present invention, a new anticancer drug NCMCCE2 was prepared according to the characteristics of common cancer incidence. The nano-TCM compound is expected to be used in the treatment of various cancers.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention utilizes modern separation technology to prepare nano-scale Chinese medicine compound, which avoids the toxicity and instability of traditional Chinese medicine compound. In addition, the present invention combines traditional Chinese medicine theory and clinical practice experience to design the proportion of Chinese medicine compound, ensuring that small molecule active ingredients can be successfully assembled by macromolecules to form nano drugs (i.e., ensuring that nano drug particles can be successfully prepared), and the interaction and efficacy between drugs are maximized. The proportioned Chinese medicine compound is more targeted. At the same time, the nano Chinese medicine compound integrates the advantages of nanotechnology, such as targeting (nanoparticles have targeting due to the EPR effect (Enhanced Permeability and Retention)) and long circulation.

[0021] (2) The NCMCCE2 prepared by the present invention encapsulates anticancer active molecules. Anti-tumor activity test experiments in liver cancer (Hepa1-6) and lung cancer (A549) cell models show that it has the effect of inhibiting the growth of liver cancer and lung cancer cells, indicating that NCMCCE2 has a broad-spectrum anticancer activity. The nano-TCM compound prepared by the present invention has high medicinal value and good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 General preparation method and application of nano-TCM compound NCMCCE2;

[0023] Figure 2 The infrared spectrum of the nano-TCM compound NCMCCE2 of the present invention;

[0024] Figure 3 This is the nuclear magnetic spectrum (hydrogen spectrum) of a nano-Chinese medicine compound NCMCCE2 of the present invention;

[0025] Figure 4 The mass spectrum fitting diagram of the nano-TCM compound NCMCCE2 of the present invention is used to identify quercetin (a), camptothecin (b), baicalin (c), and paclitaxel (d);

[0026] Figure 5 The transmission electron microscopy analysis of the nano-TCM compound NCMCCE2 of the present invention is shown;

[0027] Figure 6 Dynamic light scattering analysis of the nano-TCM compound NCMCCE2 of the present invention;

[0028] Figure 7 Potential analysis of the nano-TCM compound NCMCCE2 of the present invention;

[0029] Figure 8The in vitro anticancer activity of the nano Chinese medicine compound NCMCCE2 of the present invention. DETAILED DESCRIPTION

[0030] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0031] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0032] Example

[0033] Example 1 Preparation of Nano Traditional Chinese Medicine Compound NCMCCE2

[0034] The preparation route of nano-TCM compound is as follows Figure 1 As shown, the specific preparation method includes: after the single Chinese medicine ratio is optimized, it is boiled directly for 3-5 hours without crushing, insoluble matter is removed by filtration, dialysis bag (3.5-14KDa) is dialyzed for 3-5 days to remove small molecules, and freeze-dried by freeze dryer to prepare nano Chinese medicine compound. According to this general method, the feed ratio of three Chinese medicines of yew bark, camptotheca acuminata bark and cat's scratch grass in this embodiment is 30g:30g:30g, 600mL of double distilled water is added, boiled for 3 hours, filtered with medium-speed filter paper, and dialysis bag (3.5-14KDa) is dialyzed for 3 days and then freeze-dried to obtain a nano Chinese medicine compound, which is called NCMCCE2.

[0035] Through infrared ( Figure 2 )、NMR( Figure 3 ) characterizes the active ingredients contained in NCMCCE2, indicating that it contains macromolecular glycosides. Figure 2 ), 1000cm -1 It is the characteristic peak of glycoside molecules. Figure 3 ), 4.3-3.5ppm is the characteristic peak of sugar molecules; by dynamic light scattering ( Figure 6 ) to measure the size and distribution of nanoparticles, and electron microscopy ( Figure 5 ) characterizes its microscopic morphology, from Figure 5 and Figure 6 It can be seen that the prepared NCMCCE2 is nanoscale. Figure 7 The potential analysis of the nano-TCM compound NCMCCE2 showed that the nanoparticles were negatively charged. Since the surface charge of human blood and body fluids is weakly negative, the negatively charged nanoparticles can form a stable dispersion in a physiological environment, and the repulsive force between the negatively charged nanoparticles is small, which helps to reduce the aggregation and precipitation of the nanoparticles in body fluids, thereby maintaining their stability in the blood and prolonging the circulation time.

[0036] Example 2 Prediction of biological activity and testing of antitumor activity of NCMCCE2

[0037] Broad-spectrum anti-tumor activity is the main goal of NCMCCE2 research. Through molecular weight fitting of mass spectrometry, it was found that NCMCCE2 encapsulates quercetin (a), camptothecin (b), baicalin (c), paclitaxel (d), etc. (e.g. Figure 4 shown), combined Figure 2 and Figure 3 The results of the analysis show that the NCMCCE2 prepared by the present invention is a nanoparticle formed by encapsulating small molecules with anticancer activity by amphiphilic macromolecular glycosides during the decoction process. Because camptothecin and paclitaxel are widely used in clinical anticancer drugs, recent literature has shown that quercetin and baicalin have good anti-tumor activity. The present embodiment takes NCMCCE2 as an example to introduce the preparation of anticancer drugs according to the method of the present invention. Based on this example, nano-TCM compounds for treating cancer, infection, metabolism, regeneration and other diseases can be prepared according to this method. The preparation of nano-TCM compounds by this method has a relatively broad application prospect.

[0038] Because NCMCCE2 encapsulates more active ingredients of anticancer drugs, this example uses liver cancer cell Hepa1-6 and lung cancer cell A549 as models to test the anticancer activity of NCMCCE2 at different concentrations. The results are as follows: Figure 8 As shown, Figure 8 The horizontal axis represents the concentration of NCMCCE2 (unit: mg / mL), and the vertical axis represents the cell survival rate, where the cell survival rate is the survival rate data obtained by dividing the number of live cells measured by the MTT method after treatment with different concentrations of NCMCCE2 by the number of untreated live cells (i.e., control cells); Figure 8 The results showed that NCMCCE2 could inhibit the growth of liver cancer and lung cancer cells.

[0039] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a nano-traditional Chinese medicine compound, characterized in that: include: Based on the characteristics of the disease, the information in the traditional Chinese medicine database, and the preset assembly results, one or several traditional Chinese medicines are selected according to the characteristics of the disease and the ratio is designed. The traditional Chinese medicine is added to distilled water, boiled, and then filtered to remove insoluble matter. Toxins and small molecule impurities are then removed through dialysis to obtain a nano-traditional Chinese medicine compound.

2. The preparation method according to claim 1, characterized in that: The dialysis uses a dialysis bag with a molecular weight cutoff of 3.5 to 14 KDa, and the dialysis time is 3 to 5 days.

3. The preparation method according to claim 1, characterized in that: The dialysis further includes a freeze-drying step.

4. The nano-TCM compound prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the preparation method according to any one of claims 1 to 3 in the preparation of broad-spectrum anticancer drugs.

6. Use of the nano-TCM compound prepared by the preparation method according to any one of claims 1 to 3 in preparing drugs for treating diseases.

7. The use according to claim 5, characterized in that: The disease includes any of cancer, infection, metabolism and regeneration.

8. A method for preparing a broad-spectrum anticancer drug, characterized in that: include: Add yew bark, camptotheca acuminata bark and cat's scratch grass into water and boil them in a mass ratio of 1:1:

1. After boiling, filter to remove insoluble matter, and then remove toxins and small molecular impurities through dialysis to obtain the product.

9. Use of the broad-spectrum anticancer drug prepared by the preparation method of claim 8 in the preparation of drugs or preparations for treating cancer.

10. The use according to claim 9, characterized in that: The cancers include liver cancer and lung cancer.

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