Nano-suspension formulation of juglone and its preparation method and anti-tumor application

Abstract

The application discloses a nano-suspension preparation of juglone, which is mainly prepared from 5-15 parts of juglone and 1-20 parts of a stabilizer by weight. The application also adopts a reverse solvent method-high pressure homogenization method to establish a corresponding preparation method, which is simple in process and can obtain smaller drug particle size, thereby improving the drug bioavailability and enhancing the anti-tumor effect. Therefore, the application prepares a novel traditional Chinese medicine nano-preparation of juglone through the means of pharmacy, effectively improves the solubility and bioavailability of juglone. The in-vitro anti-tumor activity experimental results show that the nano-suspension preparation has significant inhibitory activity on the growth of various tumor cells, can be used for preparing drugs for preventing and treating related tumors, increases the targeting and bioavailability of the drug on tumors, reduces the toxicity, provides a new method and strategy for clinical treatment, and has good market prospect and high social value and economic value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a juglone nano-suspension preparation, a preparation method thereof and anti-tumor application. BACKGROUND

[0002] Colon cancer ranks first in the incidence of cancer, only next to lung cancer and gastric cancer, and has shown an increasing trend in recent years. The incidence of digestive tract tumors is grim, and the main cause is that colon mucosa adenoma or atypical growth and other lesions gradually evolve and eventually develop into cancer. The commonly used classic chemotherapy drug for colon cancer is 5-fluorouracil (5-FU). 5-FU is metabolized into 5-fluorodeoxyuridine nucleotide in the liver, showing an anticancer effect, the main mechanism of which is to inhibit the synthesis of thymidine nucleotide synthetase, thereby inhibiting DNA synthesis. Due to long-term clinical application, some patients have shown 5-FU insensitivity, so it is necessary to seek new chemotherapy drugs for colon cancer to gradually replace 5-FU. At present, the anti-colon cancer drugs used in clinical practice have considerable drug resistance and certain toxicity, so it is urgent and necessary to find a highly effective and low-toxicity anti-colon cancer drug preparation.

[0003] The molecular formula of juglone is C10H6O3, the relative molecular weight is 174.15, the chemical name is 5-hydroxy-1,4 naphthoquinone or 5-hydroxy-1,4 naphthalene dione, the melting point is 155℃, and it exists in the leaves, bark, green dragon clothes and root bark of Juglans plants such as fresh walnuts and Juglans mandshurica. The content in the fresh walnut bark can reach more than 0.02-0.4mg / g. It is mainly distributed in Northeast China, and there is a small amount of distribution in Hebei, Beijing, Inner Mongolia and Shanxi, and it is a precious medicinal plant in China. It has been reported at home and abroad that juglone has an anti-tumor effect, and can have obvious anti-cancer activity on ascitic hepatocarcinoma and spontaneous gastric cancer.

[0004] Juglone has an inducing effect on the detoxification enzymes-quinone reductase and glutathione transferase in the tissues of the mouse cecum, duodenum, colon, gastric antrum and jejunum, so as to enhance the activity thereof and prevent the generation of intestinal cancer induced by chemicals. Juglone is considered to be an inhibitor of peptidyl-prolyl isomerase (Pin1), Pin1 is overexpressed in many tumor tissues, and plays an important role in the occurrence of tumors, and is called a "catalytic molecule" of tumor occurrence, and juglone can become a promising tumor prevention and treatment drug.

[0005] The nanosuspension is a non-uniform liquid preparation formed by dispersing a poorly water-soluble solid drug in a microparticle state in a dispersion medium. The drug microparticles in the suspension are generally between 0.5-10 μm, and the smaller ones can be 0.1 μm, and the larger ones can be up to 50 μm or more. The nanocrystal, also known as nanosuspension, is a nanosized drug or drug compound dispersed in a medium, usually water, under the action of a stabilizer, and the particle size of the drug is reduced to less than 1 μm by nanosizing technology such as mechanical grinding, high-pressure homogenization or controlled crystallization, to form a drug nanocolloid dispersion system, which can significantly improve the solubility and bioavailability of poorly water-soluble drugs. Compared with other methods for solving the problem of poor water solubility, this method does not need to add any carrier matrix, only needs to add a stabilizer during preparation to stabilize the prepared nanocrystals, and the production process is simple. The prepared drug nanosuspension has the advantages of good solubility and high bioavailability, and thus can effectively solve the problem of clinical application of poorly water-soluble drugs, facilitate the absorption of drugs in the intestinal tract and prolong the in vivo action time of drugs, thereby improving the bioavailability of poorly water-soluble drugs. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a simple process, good performance, and anti-tumor application of a nanosuspension preparation of juglone, which overcomes the defects of poor water solubility, low bioavailability, and toxicity of juglone.

[0007] To solve the above technical problems, the present application adopts the following technical solutions:

[0008] The nanosuspension preparation of juglone is mainly prepared from 5-15 parts of juglone and 1-20 parts of a stabilizer by weight.

[0009] The nanosuspension preparation of juglone further comprises 0.01-1 parts of a lyophilization protective agent.

[0010] The stabilizer is one or more of sodium dodecyl sulfate, sodium carboxymethyl starch, Tween 80, polyethylene glycol 400, and poloxamer 188; and the lyophilization protective agent is one or more of sucrose, lactose, glucose, maltose, mannitol, and sorbitol.

[0011] The nanosuspension preparation of juglone is mainly prepared from 10 parts of juglone, 10 parts of sodium carboxymethyl starch, and 0.05 parts of mannitol by weight.

[0012] The preparation method of the nanosuspension preparation of juglone comprises the following steps:

[0013] (1) Dissolve the juglone in an organic solvent, and ultrasonicate to obtain an organic solution of completely dissolved juglone as an organic phase;

[0014] (2) the stabilizer is dissolved in distilled water to obtain an aqueous phase; the organic phase obtained in step (1) is slowly added into the aqueous phase under stirring to recrystallize juglone, and the organic solvent is removed by rotary evaporation to obtain a crude juglone suspension;

[0015] (3) the crude suspension obtained in step (2) is transferred into a high-pressure homogenizer for homogenization to obtain a nanocrystal suspension of juglone.

[0016] The organic solvent is one or a combination of anhydrous ethanol and methanol; the ultrasonic treatment is performed at 35-40℃ for 2 min; the stirring speed is 400-1000 r / min for 10-25 min; the homogenization temperature is 20-50℃, the homogenization pressure is 30-150 MPa, and the homogenization times is 10-30.

[0017] The organic solvent is anhydrous ethanol; the ultrasonic treatment is performed at 40℃ for 2 min; the stirring speed is 600 r / min for 20 min; the homogenization temperature is 30℃, the homogenization pressure is 140 MPa, and the homogenization times is 14.

[0018] The preparation method of the above-mentioned nanosuspension of juglone further comprises the following steps:

[0019] (4) the obtained nanosuspension of juglone is added with a freeze-drying protective agent, is divided into portions, is freeze-dried, is protected by nitrogen after the freeze-drying is completed, is plugged, and is capped to obtain a freeze-dried product of the nanosuspension of juglone.

[0020] The above-mentioned nanosuspension of juglone is used in the preparation of an antitumor drug.

[0021] The tumor is colon cancer, liver cancer, bladder cancer, malignant melanoma, breast cancer, cervical cancer, brain tumor, intestinal cancer, rectal cancer, kidney cancer, gastrointestinal stromal tumor, prostate tumor, lung cancer, and gastric cancer.

[0022] In view of the problems of poor solubility and low bioavailability of juglone, the inventors use nanocrystal technology to develop a juglone nanosuspension preparation, which is mainly prepared from 5-15 parts of juglone and 1-20 parts of stabilizer by weight. The inventors also use the anti-solvent method-high pressure homogenization method to establish the corresponding preparation method, which is simple in process and can obtain smaller drug particle size. Compared with the raw material, the reduction of particle size makes it easier to rapidly and uniformly disperse in the gastrointestinal fluid. Under the action of the stabilizer, the drug overcomes the obstacle of passing through the epithelial cell membrane of the gastrointestinal tract, easily passes through the gastrointestinal wall hydration layer to the absorption site, is beneficial to improve the drug dissolution and permeability, and the drug can continuously dissolve from the matrix and penetrate into the absorption site to enhance the anti-tumor effect. The juglone nanosuspension lyophilizate developed accordingly has good reconstitution, can be rapidly reconstituted into a colloidal dispersion after dilution with distilled water, has high drug content, uniform particle size and good stability. Therefore, the inventors prepare a new type of traditional Chinese medicine nanosuspension of juglone by means of pharmacy, which effectively improves the solubility and bioavailability of juglone. The in vitro and in vivo anti-tumor activity experimental results show that the nanosuspension of the application has significant inhibitory activity on the growth of human hepatoma cell H22, bladder cancer cell UM-UC-3, colon cancer cell CT-26, colon cancer cell HT116, cervical cancer cell Hela and other tumor cells, and can be used to prepare drugs for preventing and treating related tumors, increase the targeting and bioavailability of the drug for tumors, reduce the toxicity, provide a new method and strategy for clinical treatment, and have good market prospect and high social value and economic value. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a juglone nanosuspension reconstitution stability result graph.

[0024] Figure 2 is a mouse weight change graph.

[0025] Figure 3 is a tumor volume change graph in tumor-bearing mice in vivo.

[0026] Figure 4 is a tumor weight change graph in tumor-bearing mice in vivo. DETAILED DESCRIPTION

[0027] The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available unless otherwise specified.

[0028] Main test method

[0029] 1 Prescription screening of juglone nanosuspension

[0030] Juglone Nanosuspension (60ml:10mg), juglone was used as the main drug in this study. According to the requirements of the preparation of suspension, the selection of stabilizer and freeze-drying protective agent in nanosuspension was carried out by single factor test to select the optimal formula.

[0031] 2 Preparation process

[0032] According to the formula proportion, juglone was first dissolved in organic solvent, heated to 35-40℃, and kept at the heating temperature to completely dissolve in the organic solvent to obtain the organic phase. The surfactant was dissolved in distilled water to obtain the aqueous phase. The organic phase was slowly added to the aqueous phase and stirred to make juglone recrystallize and precipitate. The organic solvent was removed by rotary evaporation to obtain a crude suspension. The obtained crude suspension was transferred into a high-pressure homogenizer and homogenized at a certain temperature and pressure for a certain number of times to obtain a nanocrystal suspension. The nanocrystal suspension obtained by homogenization was added with an appropriate amount of freeze-drying protective agent, stirred and mixed uniformly, divided, freeze-dried, and then nitrogen protection, plug sealing and capping were carried out to obtain the juglone nanosuspension lyophilizate.

[0033] 3 Quality evaluation of nanosuspension (appearance, color and particle size)

[0034] The color and microstructure of the nanosuspension were observed, and the particle size distribution of the nanosuspension was measured by Malvern laser particle size analyzer. Before measurement, the nanosuspension was shaken well and diluted to an appropriate concentration with deionized water.

[0035] Example 1 Preparation of juglone nanosuspension by stirring at different speeds

[0036] Formulation

[0037] Juglone 0.017% (w / v)

[0038] Sodium carboxymethyl starch 0.017% (w / v)

[0039] Distilled water to 60ml

[0040] Process

[0041] Juglone was dissolved in anhydrous ethanol, and ultrasonic treatment was carried out at 40℃ for 2min to obtain completely dissolved juglone organic solvent as the organic phase. The stabilizer was dissolved in distilled water to obtain the aqueous phase. The organic phase was slowly added to the aqueous phase at a certain speed (such as Table 1) and stirred to make juglone recrystallize and precipitate. The organic solvent was removed by rotary evaporation to obtain a crude suspension. The obtained crude suspension was transferred into a high-pressure homogenizer and homogenized at a certain temperature and pressure for a certain number of times to obtain a nanocrystal suspension. The homogenization process parameters were homogenization temperature 30℃, homogenization pressure 130Mpa, and each cycle 13 times. The nanosuspension was obtained.

[0042] Table 1 Particle size and PDI of nanosuspension of juglone prepared with different rotation speed in prescription

[0043] Rotational speed (r / min) 400 600 800 1000 Average particle size 142 117.2 119.4 114.6 Average PDI 0.265 0.167 0.213 0.233

[0044] From Table 1, different rotation speed can make different changes in particle size, can increase the contact area of organic phase and water phase, increase the chance of drug crystallization, but this effect is not proportional trend within 400-1000r / min rotation speed, because the rotation speed is too fast to easily lead to the production of bubbles and make the drug crystallization efficiency decline, thus reducing the PDI, the importance of rotation speed for nanosuspension can be seen, the preferred rotation speed is 600r / min.

[0045] Example 2 Preparation of nanosuspension of juglone - use of different stabilizers

[0046] Prescription

[0047] Juglone 0.017% (w / v)

[0048] Stabilizer 0.017% (w / v)

[0049] Distilled water to 60ml

[0050] Process

[0051] Take juglone raw material, further screening with the preferred value determined by the process method in Example 1, select different stabilizers to prepare nanosuspension of juglone, select different stabilizers to prepare nanosuspension in prescription and compare particle size and polydispersity index.

[0052] Table 2

[0053] Stabilizer Tw80 P188 SDS CMS-Na PEG400 Average particle size 197.3 172 142.1 118.12 165.5 Average PDI 0.433 0.221 0.243 0.165 0.236

[0054] From Table 2, the use of different stabilizers can help the drug more stable dispersion in the solvent, increase the drug effect, different stabilizers for nanosuspension have different effects.

[0055] Example 3 Preparation of nanosuspension of juglone - use of different ratios of stabilizer and drug

[0056] Prescription

[0057] Juglone 0.017% (w / v)

[0058] Sodium carboxymethyl starch 0.017% (w / v)

[0059] Distilled water to 60ml

[0060] Process

[0061] Take juglone raw material, using the preferred values determined in the process of Example 2 further screening, select different stabilizer and drug ratio preparation of juglone nanosuspension, the selection of different stabilizer and drug ratio in the preparation of nanosuspension and comparison.

[0062] Table 3

[0063] Drug to stabilizer ratio 1:1 1:2 1:3 1:4 Average particle size 113.3 185.3 109.6 1189.3 Average PDI 0.164 0.331 0.268 0.316

[0064] From table 3, in the use of different proportion of stabilizer, in a certain range is not proportional, should be selected with the preparation of the ratio of more in line with the requirements, preferably the ratio of drug and stabilizer is 1:1 for preparation.

[0065] Example 4 preparation of juglone nanosuspension - the influence of different homogenization pressure on particle size

[0066] Prescription

[0067] Juglone 0.017% (w / v)

[0068] Sodium carboxymethyl starch 0.017% (w / v)

[0069] Distilled water to 60ml

[0070] Process

[0071] Take juglone raw material, using the preferred values determined in the process of Example 3 further screening, select different homogenization pressure preparation of juglone nanosuspension, the selection of different homogenization pressure in the preparation of nanosuspension and comparison.

[0072] Table 4

[0073] Homogenization pressure (Mpa) 60 80 100 120 140 160 Average particle size 187.8 168.11 133.8 109.77 102.8 105.7 Average PDI 0.346 0.331 0.268 0.223 0.161 0.162

[0074] From table 4, using different homogenization pressure preparation of nanosuspension, in the same process in a certain range, with the increase of pressure can obtain smaller particle size and smaller dispersion coefficient. But when greater than 140Mpa, the average particle size and average polydispersity index change little, in the premise of reducing energy consumption, the preferred homogenization pressure is 140Mpa

[0075] Example 5 preparation of juglone nanosuspension - the influence of different homogenization times on particle size

[0076] Prescription

[0077] Juglone 0.017% (w / v)

[0078] Sodium carboxymethyl starch 0.017% (w / v)

[0079] Distilled water to 60ml

[0080] Process

[0081] Take juglone raw material, further screening with the preferred values determined in the process method of Example 4, select different homogenization times to prepare juglone nanosuspension, select different homogenization times to prepare nanosuspension in the prescription and compare.

[0082] Table 5

[0083] Homogenization times 6 8 10 12 14 16 18 Average particle size 231.1 222.6 189.77 122.7 109.2 103 101.5 Average PDI 0.311 0.281 0.226 0.201 0.151 0.146 0.144

[0084] From Table 5, it can be seen that different homogenization times are used to prepare nanosuspension, within a certain range under the same process, when the homogenization times reach 14 or more, the particle size and dispersion coefficient obtained will not change much. From the energy consumption point of view, the preferred homogenization times is 14 times.

[0085] Example 6 Preparation of juglone nanosuspension - effect of different lyophilization protectants on particle size

[0086] Prescription

[0087] Juglone 0.017% (w / v)

[0088] Sodium carboxymethyl starch 0.017% (w / v)

[0089] Distilled water to 60 ml

[0090] Process

[0091] Take juglone raw material, further screening with the preferred values determined in the process method of Example 5 to prepare juglone nanosuspension.

[0092] The obtained nanosuspension is added with lyophilization protectant, divided and packaged, lyophilized, after the lyophilization is completed, nitrogen protection, plug compression and capping are carried out, to obtain juglone nanosuspension lyophilized preparation. The lyophilization process is as follows:

[0093] Pre-freezing stage: the semi-finished product filled and semi-capped is placed on the shelf in the lyophilization machine box for pre-freezing, and the semi-finished product reaches the pre-freezing temperature in the shortest possible time, the pre-freezing temperature range is between -20 and -30°C, and the time is 24h;

[0094] Sublimation drying stage: when the vacuum degree in the drying box reaches 150-200 mbar, the main drying temperature is reduced to -40 to -50°C, which takes 6-10h.

[0095] Table 6 Average particle size of juglone nanosuspension lyophilized product after adding different types and amounts of single lyophilization protectant (the average particle size before lyophilization is 106.2 nm)

[0096] Average particle size Sucrose Trehalose Mannitol Maltose Lactose 1％ 274.1 239.3 199.7 176.4 147.8 5％ 192.8 223.8 120.4 167.8 134.7 10％ 187.3 200.7 137.6 197.3 138.8

[0097] Appearance and morphology of the nanosuspension of juglone

[0098] The nanosuspension of juglone prepared according to Example 6 was freeze-dried, and when the freeze-drying protective agent was sucrose, the 1%, 5%, and 10% proportions all showed caking, and were difficult to reconstitute. When the freeze-drying protective agent was trehalose, the 1%, 5%, and 10% proportions all showed varying degrees of foaming and sticking, and were difficult to reconstitute. When the freeze-drying protective agent was maltose, the 1%, 5%, and 10% proportions all showed sticking, and were difficult to reconstitute. When the freeze-drying protective agent was lactose, the 1%, 5%, and 10% proportions all obtained relatively flat and loose freeze-dried powders, and were good at reconstitution. When the freeze-drying protective agent was mannitol, the 1%, 5%, and 10% proportions of the juglone freeze-dried product were all white cakes, flat and loose, without stratification, foaming, and other phenomena. Reconstitution with distilled water resulted in a uniform yellowish nanosuspension. The 5% mannitol freeze-dried product was flatter and looser, reconstituted better, and had a smaller change in particle size and a lower polydispersity index after reconstitution, and could maintain a good state, so the preferred freeze-drying protective agent was 5% mannitol.

[0099] Example 8 Verification of the optimal process of the nanosuspension of juglone and its dilution stability

[0100] Prescription:

[0101] Juglone 0.017% (w / v)

[0102] Sodium carboxymethyl starch 0.017% (w / v)

[0103] Distilled water to 60 ml

[0104] Add after preparation of the preparation:

[0105] 5% mannitol

[0106] Process:

[0107] First, 10 mg of juglone was dissolved in anhydrous ethanol, heated to 35-40°C, and maintained at the heating temperature to completely dissolve in anhydrous ethanol to obtain an organic phase. 10 mg of sodium carboxymethyl starch was dissolved in distilled water to obtain an aqueous phase. The organic phase was slowly added to the aqueous phase and stirred (at a speed of 600 r / min) to recrystallize and precipitate the juglone. Anhydrous ethanol was removed by rotary evaporation to obtain a crude suspension. The obtained crude suspension was transferred to a high-pressure homogenizer and homogenized 14 times at a set temperature and a pressure of 140 Mpa to obtain a nanosuspension. The nanocrystal suspension obtained by homogenization was added with 5% mannitol as a freeze-drying protective agent, pre-frozen for 24 h after dispensing, and freeze-dried to obtain a nanosuspension freeze-dried product, which was reconstituted with distilled water, left still, and the average particle size and polydispersity index were measured at 0, 2, 4, 6, 8, 10, and 12 h.

[0108] As Figure 1 shown, the results show that the average particle size of the juglone nanosuspension obtained by the optimal process before freeze-drying is 101.78, and the average PDI is 0.164, and the particle size and polydispersity index obtained are good, and the stability of the reconstituted juglone nanosuspension freeze-dried product is good.

[0109] Example 9 Cell cytotoxicity activity experiment

[0110] 1 Experimental method

[0111] In this example, the cell lines used were cultured in complete culture medium (DMEM medium containing 10% fetal bovine serum, 1% L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin) and incubated in a 5% CO2, 37°C constant temperature incubator. The anti-proliferative activity screening was carried out according to the following operation method:

[0112] (1) Collect the cancer cell lines (HepG2, SH-SY5Y, UM-UC-3, A375, HT116, HCT-116 / L, CT26 or Hela) in the logarithmic phase, adjust the concentration of the cell suspension, and inoculate 3-5×10 3 cells / well in a 96-well plate, and add 100 μL of cell suspension to each well.

[0113] (2) Incubate in a cell culture incubator, and after adhering, add drugs (juglone nanosuspension prepared in the above examples) in a concentration gradient, so that the final concentration of juglone nanosuspension is 5, 10, 15, 20, 30, 40, 60, 80, 100, 120 μM, and 3 replicate wells are set for each concentration, with the addition of an equal volume of DMSO as a blank control, and doxorubicin (DOX) as a positive control drug.

[0114] (3) Incubate at 37°C in 5% CO2, and after 48 h, add 20 μL of 0.5% MTT solution and continue to incubate for 4 h; discard the culture medium and terminate the culture.

[0115] (4) Add 150 μL / well of DMSO, shake for 5-10 min, and after the crystals are fully dissolved, use a microplate reader to detect the OD value at 570 nm, calculate the cell proliferation inhibition rate according to the following formula, and use SPSS software to analyze and calculate to obtain the half inhibitory concentration (IC50) of the measured compound on the corresponding cancer cells.

[0116] Cell proliferation inhibition rate = [OD 570 (blank control group) - OD 570 (drug group)] / OD 570 (blank control group) x 100%.

[0117] 2 Experimental results

[0118] 2.1 Juglone and the nano-suspension of juglone (Example 8) showed different degrees of inhibitory activity on two colon cancer cell lines HCT116 and HCT116 / L, with a half inhibitory concentration IC50 of 20-35 μM. The results are shown in Tables 7 and 8.

[0119] Table 7 Growth inhibitory activity of juglone raw material and nano-suspension of juglone on HCT116

[0120]

[0121] Table 8 Growth inhibitory activity of juglone raw material and nano-suspension of juglone on HCT116 / L

[0122]

[0123] The nano-suspension of juglone showed different degrees of growth inhibitory activity on nine tumor cell lines MDA-MB-231, MCF-7, HepG2, SH-SY5Y, UM-UC-3, A375, BGC-823, HCT-116 and Hela, and the effect on breast cancer cells MDA-MB-231 and MCF-7 was obviously stronger than that on other tumor cell lines, and the inhibitory activity on colon cancer cell line CT26 was the strongest, with an IC50 value of 16.1 ± 1.7 μM. The results are shown in Table 9 below.

[0124] Table 9 Inhibitory activity of nano-suspension of juglone and positive control drug doxorubicin (DOX) on various cells

[0125]

[0126] 3 Experimental conclusion

[0127] The nano-suspension of juglone with hydroxyl group has good tumor growth inhibitory activity, and the nano-suspension of juglone shows good growth inhibitory activity on human colon cancer cells HCT116, human colon cancer cells HCT116 / L, human colon cancer cells HT29, human colon cancer cells CT26, liver cancer cells HepG2, bladder cancer cells UM-UC-3, malignant melanoma cells A375 and colon cancer cells HCT116. Therefore, the nano-suspension of juglone described in the present application can be used for preparing a drug for treating or preventing cancer, especially a drug for treating colon cancer.

[0128] Example 10 In vivo activity experiment of human colon cancer cell line subcutaneously transplanted tumor in nude mice

[0129] 1 Establishment of human colon cancer cell line subcutaneously transplanted tumor in nude mice and administration scheme

[0130] (1) Nude mice breeding: The animals used in this test example are SPF female BALB / c-nu mice. The breeding temperature of the nude mice is 25-28℃, with good ventilation environment, and the light and dark are alternated for 12h light / 12h dark every day. The sterile bedding and drinking water are changed every 2-3 days. Within 2 weeks of breeding, the growth conditions of the nude mice are closely observed: the brightness of the skin color, whether the body weight is reduced, whether the drinking and eating are normal, and whether there are pathological manifestations.

[0131] (2) Preparation of cell suspension: The human colon cancer CT26 cells in the logarithmic phase and in excellent growth state are selected, centrifuged at 1000 rpm for 5 min, resuspended with sterile PBS, counted under a microscope, and adjusted to a cell concentration of about 1×10 7 / mL, and placed on ice to maintain cell viability.

[0132] (3) Inoculation: The nude mice are fixed, and the local skin of the nude mice needs to be disinfected before inoculation. The CT26 cells are inoculated into the right back of the nude mice near the axillary by subcutaneous injection using a disposable sterile syringe, the needle is slowly pulled out, and at the same time a medical cotton swab is pressed for about 30 s. The inoculation volume of each nude mouse is about 200 μL, and the cell number is about 2×10 6 .

[0133] (4) Tumorigenic state and grouping: After the inoculation of cancer cells is completed, the growth state of the nude mice is observed at all times. After 2-3 days, different degrees of tumor protrusions can be seen at the inoculation site of each nude mouse. After 4 days of observation, the tumor volume is about 60 mm3 or the tumor length is about 5-6 mm. It is indicated that the transplanted tumor model is successfully established. The nude mice are randomly divided into three groups: a control group (5% DMSO and 95% physiological saline), a juglone raw material drug group (mg / kg), and a juglone nanosuspension group (mg / kg), with 5 in each group.

[0134] (5) Drug administration and index determination: According to the dose scheme in (4), the intravenous injection administration method is used for administration, once every 4 days, for a total of 3 weeks. Before each administration, the body weight of the nude mice is weighed, and the tumor size is measured with a vernier caliper. Make a good record, and calculate the tumor volume according to the following formula.

[0135] V (tumor volume, mm3) = L (long diameter, mm) × W 2 (short diameter, mm) × 0.5

[0136] (6) Tumor peeling and sampling: 24 h after the last administration, the experiment is terminated, and the mouse body weight is weighed. Subsequently, the nude mice are sacrificed by cervical dislocation, and the tumor is peeled off and weighed in turn.

[0137] 2 Experimental results

[0138] For example, Figure 2As shown in Table 10, the body weight of the three groups of mice did not change significantly, which indicated that the juglone nano-suspension had no serious toxicity to the mice. Compared with the non-administration tumor-bearing mice, the juglone nano-suspension (Example 8) treatment group could significantly reduce the tumor growth rate in the tumor-bearing mice, the tumor volume grew slowly, and the tumor volume growth was in a dose-dependent manner. Figure 3 After 3 weeks of administration, the inhibition rates of the juglone nano-suspension treatment group on the tumor growth in the tumor-bearing mice were 10.75%, 41.84%, and 60.92%, respectively, which indicated that the juglone nano-suspension had in vivo anti-colon cancer proliferation activity. Figure 4

[0139] Table 10

[0140] Group Dose (mg / kg) Body weight (g) Model group - 20.3±0.115 Juglone raw material drug group 0.6 19.54±0.11*** Juglone nanosuspension low-dose group 0.3 19.12±0.115** Juglone nanosuspension medium-dose group 0.6 19.67±0.14** Juglone nanosuspension high-dose group 1.2 19.66±0.180*

[0141] Table 11

[0142]

[0143] 3. Experimental conclusion

[0144] The above in vivo pharmacological experiment of the tumor-bearing mice indicated that the juglone nano-suspension had good anti-cancer activity. Therefore, the juglone nano-suspension of the present application can be used for preparing a medicine for treating cancer.​

Claims

1. A juglone nanosuspension formulation, characterized in that... The preparation is mainly composed of 10 parts by weight of juglone, 10 parts by weight of sodium carboxymethyl starch, and 0.05 parts by weight of mannitol; the preparation is prepared by the following method: (1) Dissolve juglone in an organic solvent and sonicate to obtain an organic solution in which juglone is completely dissolved as the organic phase; (2) Dissolve the stabilizer sodium carboxymethyl starch in distilled water to obtain an aqueous phase; slowly add the organic phase obtained in step (1) to the aqueous phase and mix and stir to allow juglone to recrystallize and precipitate; remove the organic solvent by rotary evaporation to obtain a crude suspension of juglone. (3) The crude suspension obtained in step (2) is transferred to a high-pressure homogenizer for homogenization to obtain juglone nanocrystal suspension; (4) The obtained juglone nanocrystal suspension was added with mannitol as a freeze-drying protectant, dispensed, freeze-dried, and after the freeze-drying was completed, nitrogen gas was used for protection, and after plugging, the juglone nanocrystal suspension was obtained. The organic solvent is anhydrous ethanol; the ultrasound is performed at 40°C for 2 minutes; the mixing and stirring speed is 600 r / min for 20 minutes; the homogenization temperature is 30°C, the homogenization pressure is 140 MPa, and the homogenization is performed 14 times.

2. The method for preparing the juglone nanosuspension according to claim 1, characterized in that... Includes the following steps: (1) Dissolve juglone in an organic solvent and sonicate to obtain an organic solution in which juglone is completely dissolved as the organic phase; (2) Dissolve the stabilizer sodium carboxymethyl starch in distilled water to obtain an aqueous phase; slowly add the organic phase obtained in step (1) to the aqueous phase and mix and stir to allow juglone to recrystallize and precipitate; remove the organic solvent by rotary evaporation to obtain a crude suspension of juglone. (3) The crude suspension obtained in step (2) is transferred to a high-pressure homogenizer for homogenization to obtain juglone nanocrystal suspension; (4) The obtained juglone nanocrystal suspension was added with mannitol as a freeze-drying protectant, dispensed, freeze-dried, and after the freeze-drying was completed, nitrogen gas was used for protection, and after plugging, the juglone nanocrystal suspension was obtained. The organic solvent is anhydrous ethanol; the ultrasound is performed at 40°C for 2 minutes; the mixing and stirring speed is 600 r / min for 20 minutes; the homogenization temperature is 30°C, the homogenization pressure is 140 MPa, and the homogenization is performed 14 times.

3. The application of the juglone nanosuspension formulation according to claim 1 or the preparation method according to claim 2 in the preparation of antitumor drugs; wherein the tumor is colon cancer, bladder cancer, or malignant melanoma.

Images