Method for preparing nano-composite containing pan jade extract and application of nano-composite

Abstract

The invention relates to the technical field of mineral extraction and compound preparation thereof, in particular to a preparation method and application of a nano compound containing a pan jade extract. The method comprises the following steps: S1, crushing and sieving pan jade to obtain pan jade powder, then adding a first solvent, carrying out ultrasonic treatment, centrifuging, taking supernatant liquid, filtering, and carrying out spin-drying, redissolving and vacuum drying on filtrate to obtain pan jade extract; s2, preparing a ZIF-8 nano material from zinc nitrate hexahydrate and 2-methylimidazole; s3, loading the pan jade extract onto the ZIF-8 nano material, so as to obtain ZIF-8 loaded with the pan jade extract; and S4, carrying out functional modification on the ZIF-8 loaded with the pan jade extract by utilizing folic acid to obtain the nano-composite containing the pan jade extract. The nano-composite containing the pan jade extract prepared by the invention is used for resisting tumor proliferation.

Description

Technical Field

[0001] The invention belongs to the technical field of mineral extraction and preparation of composites thereof, and specifically relates to a preparation method and application of a nano composite containing a Panlong jade extract. Background Art

[0002] Mineral medicines are referred to as mineral medicines. They are the part of mineral resources that have medicinal effects. They are very rich in resources and have unique therapeutic effects. my country has been using mineral medicines to treat various difficult and complicated diseases for more than 2,000 years. The Classic of Mountains and Seas is the earliest work that records mineral medicines, which records more than 60 kinds of mineral medicines. The Supplement to Compendium of Materia Medica also records 269 kinds of mineral medicines in detail. So far, there are more than 370 kinds of mineral medicines that have been recorded.

[0003] Among mineral medicines, jade, as a rare natural mineral, has important medical and health care value. In particular, the Panlong jade and Panlong black jade produced in Longgang Mountains, Panshi City, Jilin Province, my country in recent years are unique in composition and contain Sn, Ba, B, Zr, P, Sr, Zn, As, Pb, Mo, Cr, Ni, V, Cu, Cd, Se and other mineral elements, which are recognized by the industry as having extremely high medicinal development potential. The patent application with announcement number CN112022872A discloses a Panlong black jade extract and its preparation method and use. The application uses methanol, ethanol or water to extract Panlong black jade to obtain Panlong black jade extract, and the Panlong black jade extract is used to prepare anti-tumor drugs. However, when the Panlong black jade extract prepared by the application treats tumors, the mineral element active ingredients contained in it quickly enter the blood and participate in human metabolism. Not only does it have a short action time in the human body, but it also damages normal human tissues and brings side effects when acting on tumor cells. Therefore, it is urgent to provide a Panlong jade complex that can slowly release the active ingredients in the drug and only act on tumor cells.

[0004] ZIF-8 nanomaterials, namely zeolite imidazolate framework materials, are a kind of nanomaterials composed of zinc ions (Zn 2+ ) and 2-methylimidazole (2-MiM) through coordination reaction to form a porous, crystalline metal organic framework (MOF) material. Zn is a trace element necessary for the survival of organisms, imidazole is a component of the amino acid histidine, and the components of ZIF-8 are endogenous substances with good biocompatibility, low toxicity, high drug loading rate, large specific surface area, high porosity and good stability. Therefore, it has certain potential in drug sustained release. Moreover, there is no report on the use of ZIF-8 nanomaterials to load Panlongyu extract.

[0005] In view of this, the present application loads the Panlong jade extract onto the ZIF-8 nanomaterial and further functionalizes the ZIF-8 nanomaterial loaded with Panlong jade to prepare a new type of complex containing Panlong jade extract, so as to achieve the slow release of the active ingredients in Panlong jade in the human body, improve its tumor inhibition effect and reduce side effects. Summary of the invention

[0006] In order to solve the technical problems in the prior art that the active ingredients in the Panlongyu extract are rapidly released in the human body, have a short action time and large side effects, the present invention provides a preparation method of a nanocomposite containing the Panlongyu extract and its application.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a method for preparing a nanocomposite containing a Panlongyu extract, comprising the following steps:

[0009] Step S1: crushing and sieving the Panlong jade to obtain Panlong jade powder, then adding the first solvent, ultrasonically treating, centrifuging, filtering the supernatant, spinning the filtrate to dryness, re-dissolving, and finally vacuum drying to obtain the Panlong jade extract;

[0010] Step S2: dissolving zinc nitrate hexahydrate and 2-methylimidazole in a second solvent respectively, adding the zinc nitrate solution dropwise to the 2-methylimidazole solution under stirring, continuing stirring to obtain a mixed solution containing ZIF-8, centrifuging, washing with a second solvent, and vacuum drying to obtain a ZIF-8 nanomaterial;

[0011] Step S3: dissolving the Panlongyu extract prepared in step S1 in a first solvent to obtain a Panlongyu extract solution, adding the ZIF-8 nanomaterial prepared in step S2 to the Panlongyu extract solution, performing ultrasonic dispersion, stirring, centrifugation, solvent washing, and vacuum drying to obtain ZIF-8 loaded with the Panlongyu extract;

[0012] Step S4: ultrasonically disperse folic acid in PBS, add EDC and NHS, stir, and obtain activated folic acid; ultrasonically disperse the ZIF-8 loaded with Panlongyu extract prepared in step S3 in PBS to obtain a ZIF-8 dispersion, dropwise add activated folic acid, stir, centrifuge, wash with deionized water, and vacuum dry to obtain a nanocomposite containing Panlongyu extract.

[0013] Preferably, in step S1, the Panlong jade powder is sieved through a 400-mesh sieve, the first solvent is any one of methanol and ethanol, the ratio of the Panlong jade powder to the first solvent is 1 g:(50-100 mL), the ultrasonic power of the ultrasonic treatment is 150-250 W, the time is 1-2 h, and the filtration is a 0.22 μm filter membrane.

[0014] Preferably, in step S2, the second solvent is any one of methanol aqueous solution and ethanol aqueous solution, and the concentration of methanol aqueous solution or ethanol aqueous solution is 40-60% (v / v), and the ratio of zinc nitrate hexahydrate, 2-methylimidazole and the second solvent is 1g:2g:(150-300mL).

[0015] Preferably, in step S2, the stirring speed is 180-250 r / min, the dropping speed of the 2-methylimidazole solution is 100-200 d / min, and the vacuum drying is performed at 50-80° C. for 8-12 h.

[0016] Preferably, in step S3, the ratio of the Panlongyu extract, the ZIF-8 powder and the first solvent is 1 g: 1 g: (50-200 mL).

[0017] Preferably, the ultrasonic power of the ultrasonic dispersion in step S3 is 80-120 W and the time is 5-15 min, and the vacuum drying is carried out at 50-80° C. for 8-12 h.

[0018] Preferably, in step S4, when preparing activated folic acid, the ratio of folic acid, EDC, NHS and PBS is 2g:1g:1g:(80-120mL); the ratio of ZIF-8 loaded with Panlongyu extract and PBS is 10mg:(1-2mL); the mixed volume ratio of the ZIF-8 dispersion and activated folic acid is 4:1.

[0019] Preferably, the pH value of PBS in step S4 is 7.2-7.4, the dropping speed of the activated folic acid is 2-5 d / s, and the vacuum drying is performed at 50-80° C. for 8-12 h.

[0020] In a second aspect, the present invention provides an application of a nanocomposite containing a Panlongyu extract for inhibiting tumor proliferation.

[0021] Preferably, the tumor includes breast cancer, lung cancer, endometrial cancer, melanoma, ovarian tumor and mesothelioma.

[0022] Based on the above technical solutions, the present invention has the following beneficial effects:

[0023] (1) The Panlongshi extract of the present application includes a variety of trace elements related to human physiological functions, such as Se, Sr, V, Zn, Cu, etc. These trace element active ingredients can directly interfere with the metabolism of carcinogens, and can also inhibit the growth and spread of tumor cells through indirect pathways such as enhancing the body's immune function.

[0024] (2) The carrier ZIF-8 in the present invention is composed of 2-methylimidazole and Zn 2+Both are endogenous substances in the human body, so they have good biocompatibility and low toxicity. The porous structure of ZIF-8 nanoparticles gives them the advantages of large specific surface area and high porosity. The active ingredients in the Panlongshi extract are more easily loaded into the pores and surface of ZIF-8, thereby improving the drug loading rate.

[0025] (3) The glycolysis rate in tumor cells is high, and the external and lysosomal environments are acidic. ZIF-8 is stable under physiological conditions (pH = 7.4), but easily decomposes under acidic conditions (pH = 5.0-6.0). This is conducive to the disintegration of ZIF-8 in the acidic tumor microenvironment (pH 5.5-6.0) and the release of the drugs loaded on it, thereby increasing the drug concentration in the tumor site, thereby improving the drug use effect and reducing the side effects of the drug on normal tissues.

[0026] (4) The metal ion active ingredients in the Panlongyu extract can weakly interact with the imidazole in the ZIF-8 structure (for example, metal active substances such as Sn, Ba, Zr, Sr, Pb, and Mo can accept the lone pair of electrons from the nitrogen atom on the imidazole ring to form a coordination bond; while metal active substances such as B, P, As, and Se can form a covalent bond with the N on the imidazole), further increasing the loading rate of the effective active ingredients in the Panlongyu extract on ZIF-8.

[0027] (5) The present application also uses folic acid to functionalize the ZIF-8 nanomaterial loaded with Panlongyu. The obtained nanocomplex has an inhibitory effect on common tumors and has a better inhibitory effect on tumor cells expressing folate receptors. Therefore, it is more suitable for the treatment of tumors that can express folate receptors, such as breast cancer, lung cancer, ovarian tumors, mesothelioma, etc.

[0028] In summary, the nanocomplex containing Panlongyu extract prepared by the method of the present application can target tumor tissue, disintegrate in the tumor tissue and release the active ingredients of the drug, thereby achieving the slow release of the active ingredients in the Panlongyu extract, improving the tumor treatment effect and reducing the side effects on normal tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The process flow chart of the preparation method of the nanocomposite containing the Panlongyu extract of the present invention;

[0030] Figure 2 It is a line graph of the drug release amount of the nanocomposite containing the Panlongyu extract prepared by the present invention during the dialysis process;

[0031] Figure 3 It is a line graph of the drug release amount of the Panlongyu extract prepared by the present invention during the dialysis process. DETAILED DESCRIPTION

[0032] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the following is combined with the attached Figure 1-3 The specific preparation method, ingredients and effects of the present invention are described in detail below with preferred embodiments and test examples.

[0033] Example 1

[0034] This embodiment provides a method for preparing a nanocomposite containing a Panlongyu extract, such as Figure 1 As shown, the specific steps include:

[0035] Step S1: Mechanically crush the Panlong jade collected from Longgang Mountains in Panshi City, Jilin Province, and pass it through a 400-mesh sieve to obtain Panlong jade powder. Then, take 10 g of Panlong jade powder and add it to 750 mL of methanol. Ultrasonic treatment is performed for 1.5 h at an ultrasonic power of 200 W, and centrifuged at 4000 rpm for 30 min. The supernatant is filtered with a 0.22 μm filter membrane, and the filtrate is spin-dried at 55° C., and then re-dissolved with 1 mL of methanol, and finally vacuum-dried at 80° C. to obtain a Panlong jade extract.

[0036] Step S2: dissolving 1 g of zinc nitrate hexahydrate in 150 mL of a 50% (v / v) methanol aqueous solution, dissolving 2 g of 2-methylimidazole in 70 mL of a 50% (v / v) methanol aqueous solution, adding the zinc nitrate solution dropwise to the 2-methylimidazole solution at a speed of 140 d / min under stirring at 220 r / min, continuing stirring for 2 h to obtain ZIF-8 nanoparticles, centrifuging at 4000 rpm for 10 min, washing the solid matter with 30 mL of a methanol aqueous solution, and drying in a vacuum drying oven at 65° C. for 10 h to obtain ZIF-8 nanomaterials;

[0037] Step S3: 500 mg of the Panlongyu extract prepared in step S1 was dissolved in 75 mL of methanol to obtain a Panlongyu extract solution, 500 mg of the ZIF-8 nanomaterial prepared in step S2 was added, ultrasonic dispersion was performed at an ultrasonic power of 100 W for 10 min, stirring at 200 r / min for 24 h, centrifuging at 4000 r / min for 10 min, washing with 20 mL of methanol, and drying in a vacuum drying oven at 65° C. for 10 h to obtain ZIF-8 loaded with Panlongyu extract;

[0038] Step S4: ultrasonically disperse 2 g of folic acid in 100 mL of PBS (0.1 mol / L, pH = 7.3), add 1 g of EDC and 1 g of NHS, and stir at 600 r / min for 12 h to obtain activated folic acid; weigh 500 mg of ZIF-8 loaded with Panlongyu extract prepared in step S3 and ultrasonically disperse it in 80 mL of PBS (0.1 mol / L, pH = 7.3) to obtain a ZIF-8 dispersion, and drop 20 mL of the prepared activated folic acid into the ZIF-8 dispersion at a speed of 3 d / s, stir at 200 r / min for 6 h, centrifuge at 4000 r / min for 10 min, wash with 10 mL of deionized water, and dry in a vacuum drying oven at 65°C for 10 h to obtain a nanocomposite containing Panlongyu extract.

[0039] Example 2

[0040] This embodiment provides a method for preparing a nanocomposite containing a Panlongyu extract, which specifically comprises the following steps:

[0041] Step S1: Mechanically crush the Panlong jade collected from Longgang Mountains in Panshi City, Jilin Province, and pass it through a 400-mesh sieve to obtain Panlong jade powder. Then, take 10 g of Panlong jade powder and add it to 500 mL of ethanol. Ultrasonic treatment is performed for 2 h at an ultrasonic power of 150 W, and centrifuged at 4000 rpm for 30 min. The supernatant is filtered with a 0.22 μm filter membrane, and the filtrate is spin-dried at 55° C., and then re-dissolved with 1 mL of ethanol, and finally vacuum-dried at 80° C. to obtain a Panlong jade extract.

[0042] Step S2: dissolving 1 g of zinc nitrate hexahydrate in 150 mL of 40% (v / v) ethanol aqueous solution, dissolving 2 g of 2-methylimidazole in 150 mL of 40% (v / v) ethanol aqueous solution, adding the zinc nitrate solution dropwise to the 2-methylimidazole solution at a speed of 200 d / min under stirring at 220 r / min, continuing stirring for 2 h to obtain ZIF-8 nanoparticles, centrifuging at 4000 rpm for 10 min, washing the solid matter with 30 mL of ethanol aqueous solution, and drying in a vacuum drying oven at 50° C. for 12 h to obtain ZIF-8 nanomaterials;

[0043] Step S3: 500 mg of the Panlongyu extract prepared in step S1 was dissolved in 25 mL of ethanol to obtain a Panlongyu extract solution, 500 mg of the ZIF-8 nanomaterial prepared in step S2 was added, ultrasonic dispersion was performed for 15 min under an ultrasonic power of 80 W, stirring was performed at 200 r / min for 24 h, centrifuged at 4000 r / min for 10 min, washed with 10 mL of ethanol, and dried in a vacuum drying oven at 50° C. for 12 h to obtain ZIF-8 loaded with Panlongyu extract;

[0044] Step S4: ultrasonically disperse 2 g of folic acid in 80 mL of PBS (0.1 mol / L, pH = 7.2), add 1 g of EDC and 1 g of NHS, and stir at 600 r / min for 12 h to obtain activated folic acid; weigh 500 mg of ZIF-8 loaded with Panlongyu extract prepared in step S3 and ultrasonically disperse it in 50 mL of PBS (0.1 mol / L, pH = 7.2) to obtain a ZIF-8 dispersion, and drop 12.5 mL of the prepared activated folic acid into the ZIF-8 dispersion at a speed of 2 d / s, stir at 200 r / min for 6 h, centrifuge at 4000 r / min for 10 min, wash with 10 mL of deionized water, and dry in a vacuum drying oven at 50°C for 12 h to obtain a nanocomposite containing Panlongyu extract.

[0045] Example 3

[0046] This embodiment provides a method for preparing a nanocomposite containing a Panlongyu extract, which specifically comprises the following steps:

[0047] Step S1: Mechanically crush the Panlong jade collected from Longgang Mountains in Panshi City, Jilin Province, and pass it through a 400-mesh sieve to obtain Panlong jade powder. Then, take 10 g of Panlong jade powder and add it to 1000 mL of methanol. Ultrasonic treatment is performed at an ultrasonic power of 250 W for 1 hour, and centrifuged at 4000 rpm for 30 minutes. The supernatant is filtered with a 0.22 μm filter membrane, and the filtrate is spin-dried at 55° C., and then re-dissolved with 1 mL of methanol, and finally vacuum-dried at 80° C. to obtain a Panlong jade extract.

[0048] Step S2: dissolving 1 g of zinc nitrate hexahydrate in 80 mL of a 60% (v / v) methanol aqueous solution, dissolving 2 g of 2-methylimidazole in 70 mL of a 60% (v / v) methanol aqueous solution, dropping the zinc nitrate solution into the 2-methylimidazole solution at a speed of 100 d / min under stirring at 250 r / min, continuing stirring for 2 h to obtain ZIF-8 nanoparticles, centrifuging at 4000 rpm for 10 min, washing the solid matter with 30 mL of a methanol aqueous solution, and drying in a vacuum drying oven at 80° C. for 8 h to obtain ZIF-8 nanomaterials;

[0049] Step S3: 500 mg of the Panlongyu extract prepared in step S1 was dissolved in 100 mL of methanol to obtain a Panlongyu extract solution, 500 mg of the ZIF-8 nanomaterial prepared in step S2 was added, ultrasonic dispersion was performed for 5 min under an ultrasonic power of 120 W, stirring was performed at 200 r / min for 24 h, centrifuged at 4000 r / min for 10 min, washed with 20 mL of methanol, and dried in a vacuum drying oven at 80° C. for 8 h to obtain ZIF-8 loaded with Panlongyu extract;

[0050] Step S4: ultrasonically disperse 2 g of folic acid in 120 mL of PBS (0.1 mol / L, pH = 7.4), add 1 g of EDC and 1 g of NHS, and stir at 600 r / min for 12 h to obtain activated folic acid; weigh 500 mg of ZIF-8 loaded with Panlongyu extract prepared in step S3 and ultrasonically disperse it in 100 mL of PBS (0.1 mol / L, pH = 7.4) to obtain a ZIF-8 dispersion, and drop 25 mL of the prepared activated folic acid into the ZIF-8 dispersion at a speed of 5 d / s, stir at 200 r / min for 6 h, centrifuge at 4000 r / min for 10 min, wash with 10 mL of deionized water, and dry in a vacuum drying oven at 80°C for 8 h to obtain a nanocomposite containing Panlongyu extract.

[0051] Sustained release performance test of nanocomposite

[0052] Test method: Weigh 10 mg of the nanocomposite containing Panlongyu extract, put it into a pre-treated dialysis bag, add 3 mL of PBS buffer (pH=5.6 / 7.4, containing 1% Tween-80 by volume), put the dialysis bag into a beaker with a capacity of 100 mL, and at the same time measure 30 mL of PBS buffer solution of the corresponding pH (containing 1% Tween-80 by volume) and put it outside the dialysis bag in the beaker. Stir the solution in the beaker at 37°C at 100 rpm, take out 1 mL of the mixed solution outside the dialysis bag every 2 hours (after taking out 1 mL, add 1 mL of the above PBS buffer), and use Agilent7700X inductively coupled plasma mass spectrometer to detect the concentration of Sn in the mixed solution (Sn represents the effective active ingredient in the Panlongyu extract) until the concentration of Sn in the mixed solution no longer changes. The Sn release amount was calculated according to the following formula (1), and the drug release curve was plotted with time as the horizontal axis and the Sn release amount at the corresponding time as the vertical axis. Figure 2 .

[0053] Sn release amount = volume × Sn concentration (1)

[0054] In formula (1), the Sn release amount is the mass of Sn in the PBS outside the dialysis bag, in ug;

[0055] The volume is the volume of PBS outside the dialysis bag. It can be measured by lifting the dialysis bag and using the scale value of the beaker. The unit is mL.

[0056] The concentration of Sn is the concentration of Sn detected at time t, and the unit is ug / mL.

[0057] Depend on Figure 2It can be seen that the release of Sn, the active ingredient of the drug, from the nanocomplex containing Panlongyu extract at different pH values ​​increases with time, and the release gradually reaches equilibrium after 20 hours, and the release amount is larger (50ug) at pH = 5.6, indicating that Panlongyu extract is released more under acidic conditions. Since the lysosomes in tumor cells and their microenvironment are under this acidic condition, the nanocomplex containing Panlongyu extract can be better used for drug release in tumor sites.

[0058] Take 5 mg of the Panlongyu extract prepared in step S1, repeat the above test, and draw a drug release curve. Figure 3 .

[0059] Depend on Figure 3 It can be seen that the release amount of Sn, the active ingredient in the Panlongyu extract, at different pH values ​​increases with time, and the release gradually reaches equilibrium after 6 hours. Compared with the release curve of Sn in the nanocomposite containing Panlongyu extract, the Panlongyu extract reaches equilibrium earlier, indicating that the nanocomposite containing Panlongyu extract prepared by the present invention can achieve the slow release of active ingredients of the drug (20h release reaches equilibrium), which can prolong the drug action time. In addition, there is no obvious difference in the release curve of the active ingredient Sn of the Panlongyu extract without ZIF-8 loading in PBS at different pH values, indicating that the effect of the drug is not affected by the pH value, and can act on tumor cells in a slightly acidic environment, and can also damage normal tissue cells at pH 7.4, thereby causing side effects.

[0060] In vitro tumor inhibition assay

[0061] The test materials involved in this test case mainly include:

[0062] Test sample: the nanocomposite containing the Panlongyu extract prepared in Example 1;

[0063] Positive control 1: Cisplatin injection (6ml:30mg, Hausen Pharmaceuticals)

[0064] Positive control substance 2: the Panlongyu extract prepared in step S1 of Example 1;

[0065] Preparation of test products and positive control products

[0066] Preparation of test sample: Weigh 0.6 g of test sample powder, open the lid and irradiate with ultraviolet light for 30 min in a clean bench, add 2 mL of complete culture medium, prepare a suspension with a concentration of 300 mg / mL, and dilute it in multiple ratios to suspensions with concentrations of 60 mg / mL and 12 mg / mL to obtain high, medium and low dose groups, respectively.

[0067] Preparation of positive control 1: Take 36 μL of cisplatin stock solution in a 5 mL sterile EP tube, add 1964 μL of complete culture medium, mix well, and prepare a solution with a concentration of 300 μM, and dilute it in multiple ratios to solutions with concentrations of 60 μM and 12 μM to obtain high, medium, and low dose groups, respectively.

[0068] Preparation of positive control 2: Calculate the required mass of Panlongyu extract according to the loading amount of Panlongyu extract on the nanocomplex in Example 1 (33.2%) (which should be consistent with the mass of Panlongyu extract contained in the test sample), that is, 0.6g×33.2%=0.1992g; weigh 0.1992g of Panlongyu extract and prepare it according to the preparation method of the test sample to obtain suspensions with concentrations of 300mg / mL, 60mg / mL and 12mg / mL, respectively, to obtain high, medium and low dose groups, respectively.

[0069] Safety protection for preparation personnel: to protect the safety of test operators, such as wearing protective clothing, goggles, gloves, masks, etc.

[0070] Other major reagents include:

[0071] Fetal bovine serum (Cat. No. 11011-8611; Sijiqing);

[0072] 20XPBS buffer (lot number: 20191029, Solebio);

[0073] Pancreatin (lot number: 20191226, Solebo);

[0074] Penicillin-streptomycin mixture (batch number: 20191018, Solebo);

[0075] CCK-8 kit (cell proliferation and toxicity detection kit, catalog number: CA1210-100T, Solebo)

[0076] The names, models and numbers of the main equipment involved in this test example are shown in Table 1.

[0077] Table 1 Equipment information

[0078] Device Name Manufacturer model Instrument No. Centrifuge Hunan Kecheng H4-20KR YQ050002 CO2 Incubator Hong Kong Likang HF151 YQ010012 Pipette Big Dragon 0.1-1ml YQ100009 Multifunctional ELISA reader BMGLABTE CH FLUOstar Omega YQ010060 Clean bench Kazakhstan East Union DJ-CJ-2NDI YQ070006

[0079] The cell lines, cell sources and cell culture conditions used in this experiment are shown in Table 2 below:

[0080] Table 2 Cell information

[0081]

[0082]

[0083] Test methods

[0084] 1. Cell plating and drug incubation

[0085] (1) Human breast cancer MCF-7 cells in the logarithmic growth phase were digested with 0.25% trypsin, centrifuged at 1000 rpm / min for 5 min, and the cells were counted;

[0086] (2) Use complete culture medium to adjust the cell concentration to 2.5×10 4 ~1.25×10 5 cell / mL;

[0087] (3) First, add 400 μL of 1×10 4 ~5×10 4 Cell suspension, cultured overnight;

[0088] (4) The next day, remove the 24-well plate with the chambers from 37°C and gently add 200 μL of samples of different concentrations to each upper chamber, avoiding the formation of bubbles.

[0089] (5) Place in a 37°C and 5% CO2 incubator for 72 h. The specific layout of the 24-well plate is as follows, see Table 3:

[0090] Table 3 24-well plate layout

[0091]

[0092]

[0093] 4 hours before the end of drug incubation, the upper chamber of the chamber was removed, the culture medium of the lower chamber was discarded, 270 μL of culture medium was added to each well of the lower chamber, and then 30 μL of CCK-8 detection reagent was added. After the plate was placed in the incubator and cultured for another 4 hours, the 24-well plate was removed, 100 μL of the test solution was transferred from each well to a 96-well ELISA plate, and the absorbance was read at a wavelength of 450 nm using an ELISA reader. The cell viability of each group of human breast cancer cell MCF-7 is shown in Table 4-1:

[0094] Cell viability = (OD value of test well / OD value of blank control well) × 100% (2)

[0095] Table 4-1 Statistics of viability of human breast cancer cell MCF-7 (%)

[0096]

[0097] As shown in Table 4-1, under the experimental conditions, the cell viabilities of the low-dose, medium-dose and high-dose cisplatin groups were approximately 27%, 52% and 74% respectively (the average value of the parallel tests was taken, similarly below); the cell viabilities of the low-dose, medium-dose and high-dose groups of the nanocomposite containing Panlongyu extract were approximately 32%, 58% and 96% respectively; the cell viabilities of the low-dose, medium-dose and high-dose groups of Panlongyu extract were approximately 84%, 92% and 93% respectively. It is shown that both cisplatin and the nanocomposite containing Panlongyu extract have a significant inhibitory effect on the in vitro growth of human breast cancer cells MCF-7, but the inhibitory effect of the nanocomposite of the present invention is slightly lower than that of the anticancer drug cisplatin. In addition, the in vitro growth inhibitory effect of Panlongyu extract on human breast cancer cells MCF-7 is not obvious, and only high-concentration Panlongyu extract shows a weaker cancer cell inhibition effect. This proves that the nanocomposite obtained by Panlongyu extract after being loaded with ZIF-8 and folic acid modified by the present invention has a significantly enhanced anti-breast cancer effect and can be used to fight breast cancer.

[0098] According to the above test method, human non-small cell lung cancer cells A549, human malignant melanoma cells A375, human mesothelioma cells NCI-H28, human ovarian cancer cells SKOV-3, and human endometrial cancer cells Ishikawa in the logarithmic growth phase were tested, and the viability statistics of different cells were obtained, as shown in Table 4-2, Table 4-3, Table 4-4, Table 4-5 and Table 4-6, respectively.

[0099] Table 4-2 Statistics of viability of human non-small cell lung cancer cell A549 (%)

[0100]

[0101] As shown in Table 4-2, under the experimental conditions, the cell viabilities of the low-dose, medium-dose and high-dose cisplatin groups were about 8%, 22% and 50%, respectively; the cell viabilities of the low-dose, medium-dose and high-dose groups of the nanocomposite containing Panlongyu extract were about 37%, 66% and 98%, respectively; the cell viabilities of the low-dose, medium-dose and high-dose groups of Panlongyu extract were all above 90%. This indicates that both cisplatin and the nanocomposite containing Panlongyu extract have a significant inhibitory effect on the in vitro growth of human non-small cell lung cancer cells A549, and since cisplatin is a drug specifically used to treat non-small cell lung cancer, its cancer cell inhibition function is significantly better than that of the nanocomposite in the present invention. In addition, the in vitro growth inhibitory effect of Panlongyu extract on non-small cell lung cancer cells A549 is not obvious. This proves that the nanocomposite obtained by Panlongyu extract after being loaded with ZIF-8 and modified with folic acid in the present invention has a significantly enhanced anti-breast cancer effect and can be used to fight non-small cell lung cancer.

[0102] Table 4-3 Statistics of viability of human malignant melanoma cell A375 (%)

[0103]

[0104] As shown in Table 4-3, under the experimental conditions, the cell viabilities of the low-dose, medium-dose and high-dose cisplatin groups were approximately 14%, 42% and 74%, respectively; the cell viabilities of the low-dose, medium-dose and high-dose groups of the nanocomposite containing Panlongyu extract were approximately 42%, 63% and 90%, respectively; the cell viabilities of the low-dose, medium-dose and high-dose groups of Panlongyu extract were all above 90%. It is shown that both cisplatin and the nanocomposite containing Panlongyu extract have a significant inhibitory effect on the in vitro growth of human malignant melanoma cells A375, but the inhibitory effect of the nanocomposite of the present invention is lower than that of cisplatin, a special anticancer drug for melanoma. In addition, the in vitro growth inhibitory effect of Panlongyu extract on human malignant melanoma cells A375 is not obvious. This proves that the nanocomposite obtained by Panlongyu extract after being loaded with ZIF-8 and modified with folic acid of the present invention has a significantly enhanced anti-melanoma effect and can be used for anti-melanoma.

[0105] Table 4-4 Statistics of human mesothelioma cell NCI-H28 viability (%)

[0106]

[0107] Table 4-5 Statistics of the survival rate of human ovarian cancer cell SKOV-3 (%)

[0108]

[0109] Table 4-6 Statistics of Ishikawa viability of human endometrial cancer cells (%)

[0110]

[0111]

[0112] As shown in Tables 4-5, 4-6 and 4-7, the nanocomplexes prepared in the present application all exhibited certain in vitro inhibitory effects on different cancer cells, which were superior to the unloaded and unmodified Panlongyu extracts, indicating that the nanocomplexes prepared in the present invention all have the function of inhibiting the above tumor cells and can be used to prepare anti-tumor drugs. It is worth noting that among several cancer cells, the nanocomplexes had the most obvious inhibitory effect on human mesothelioma cells NCI-H28, which was also higher than the inhibitory effect of the positive control cisplatin on cells NCI-H28.

[0113] In addition, we also noticed that although the nanocomplex had a certain inhibitory effect on all tumor cells involved in the experiment, the nanocomplex had the worst inhibitory effect on melanoma cells. This is because the remaining five tumor cells (human breast cancer cells MCF-7, human non-small cell lung cancer cells A549, human mesothelioma cells NCI-H28, human ovarian cancer cells SKOV-3, and human endometrial cancer cells Ishikawa) can express folate receptors on their surfaces. After the nanocomplex was modified with folic acid, the folic acid loaded on it could target the folate receptors on the surfaces of the five tumor cells, making the nanocomplex easier to approach tumor cells and disintegrate in the weakly acidic environment of the tumor, releasing the active ingredients in the Panlongyu extract, so it has a better inhibitory effect on these five tumors.

[0114] Based on the above technical solutions, the nanocomplex containing Panlongyu extract prepared by the present invention can target tumor tissue. After reaching the weakly acidic environment of the tumor, ZIF-8 disintegrates in the weakly acidic environment and releases the active ingredients of the drug, thereby realizing the slow release of the active ingredients in the Panlongyu extract, increasing the drug concentration near the tumor, and then improving the tumor treatment effect and reducing the side effects on normal tissues.

[0115] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a nanocomposite containing a Panlongyu extract, characterized in that: The steps include: Step S1: crushing and sieving the Panlong jade to obtain Panlong jade powder, then adding the first solvent, ultrasonically treating, centrifuging, filtering the supernatant, spinning the filtrate to dryness, re-dissolving, and finally vacuum drying to obtain the Panlong jade extract; Step S2: dissolving zinc nitrate hexahydrate and 2-methylimidazole in a second solvent respectively, adding the zinc nitrate solution dropwise to the 2-methylimidazole solution under stirring, continuing stirring to obtain a mixed solution containing ZIF-8, centrifuging, washing with a second solvent, and vacuum drying to obtain a ZIF-8 nanomaterial; Step S3: dissolving the Panlongyu extract prepared in step S1 in a first solvent to obtain a Panlongyu extract solution, adding the ZIF-8 nanomaterial prepared in step S2 to the Panlongyu extract solution, performing ultrasonic dispersion, stirring, centrifugation, solvent washing, and vacuum drying to obtain ZIF-8 loaded with the Panlongyu extract; Step S4: ultrasonically disperse folic acid in PBS, add EDC and NHS, stir, and obtain activated folic acid; ultrasonically disperse the ZIF-8 loaded with Panlongyu extract prepared in step S3 in PBS to obtain a ZIF-8 dispersion, dropwise add activated folic acid, stir, centrifuge, wash with deionized water, and vacuum dry to obtain a nanocomposite containing Panlongyu extract.

2. The method for preparing a nanocomposite containing a Panlongyu extract according to claim 1, characterized in that: In the step S1, the Panlong jade powder is passed through a 400-mesh sieve, the first solvent is any one of methanol and ethanol, the ratio of the Panlong jade powder to the first solvent is 1g:(50-100mL), the ultrasonic power of the ultrasonic treatment is 150-250W, the time is 1-2h, and the filtration is a 0.22μm filter membrane.

3. The method for preparing a nanocomposite containing a Panlongyu extract according to claim 1, characterized in that: In step S2, the second solvent is any one of methanol aqueous solution and ethanol aqueous solution, and the concentration of methanol aqueous solution or ethanol aqueous solution is 40-60% (v / v), and the ratio of zinc nitrate hexahydrate, 2-methylimidazole and the second solvent is 1g:2g:(150-300mL).

4. The method for preparing a nanocomposite containing a Panlongyu extract according to claim 1, characterized in that: In the step S2, the stirring speed is 180-250 r / min, the dropping speed of the 2-methylimidazole solution is 100-200 d / min, and the vacuum drying is carried out at 50-80° C. for 8-12 h.

5. The method for preparing a nanocomposite containing a Panlongyu extract according to claim 1, characterized in that: In the step S3, the ratio of the Panlongyu extract, the ZIF-8 powder and the first solvent is 1g:1g:(50-200mL).

6. The method for preparing a nanocomposite containing a Panlongyu extract according to claim 1, characterized in that: The ultrasonic power of the ultrasonic dispersion in step S3 is 80-120 W and the time is 5-15 min. The vacuum drying is carried out at 50-80° C. for 8-12 h.

7. The method for preparing a nanocomposite containing a Panlongyu extract according to claim 1, characterized in that: In the step S4, when preparing activated folic acid, the ratio of folic acid, EDC, NHS and PBS is 2g:1g:1g:(80-120mL); the ratio of ZIF-8 loaded with Panlongyu extract and PBS is 10mg:(1-2mL); the mixed volume ratio of the ZIF-8 dispersion and activated folic acid is 4:

1.

8. The method for preparing a nanocomposite containing a Panlongyu extract according to claim 1, characterized in that: In the step S4, the pH value of PBS is 7.2-7.4, the dropping speed of the activated folic acid is 2-5 d / s, and the vacuum drying is performed at 50-80° C. for 8-12 h.

9. An application of a nanocomposite containing a Panlongyu extract, based on a method for preparing a nanocomposite containing a Panlongyu extract according to any one of claims 1 to 8, characterized in that: Used in anti-tumor proliferation.

10. The use of a nanocomposite containing a Panlongyu extract according to claim 9, characterized in that: Such tumors include breast cancer, lung cancer, endometrial cancer, melanoma, ovarian tumors, and mesothelioma.

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