CN113116886A

CN113116886A - Pharmaceutical composition for treating brain glioma

Info

Publication number: CN113116886A
Application number: CN201910812481.9A
Authority: CN
Inventors: 查晓明; 李昌明
Original assignee: Shenzhen Mingshi Medical Biotechnology Co ltd
Current assignee: Shenzhen Mingshi Medical Biotechnology Co ltd
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2021-07-16

Abstract

The invention provides a pharmaceutical composition for treating brain glioma, which is used for treating brain glioma or melanoma and belongs to the technical field of tumor drugs. The pharmaceutical composition contains the compounds shown in the formulas I, II and III. The compounds shown in the formula I, the formula II and the formula III and the extract (including effective parts) prepared by the preparation method have high killing effect on brain glioma and melanoma. In particular, the IC50 value of the compound shown in the formula I is only 0.4044nM for brain glioma U87-MG, the IC50 value of the compound is only 0.5152nM for melanoma SK-MEL-28, and the IC50 value of the compound for most other tumors is often between 1uM and 100uM and differs by more than three orders of magnitude, so that the compound shows selective and efficient killing effects on brain glioma U87-MG and melanoma SK-MEL-28.

Description

Pharmaceutical composition for treating brain glioma

Technical Field

The invention belongs to the technical field of tumor treatment and biomedicine, and particularly relates to a pharmaceutical composition for treating brain glioma.

Background

Malignant tumors are serious diseases seriously harming human life and health, and become one of the leading causes of death due to diseases nowadays. The morbidity and mortality of tumors in China are continuously high, and the situation is severe. At present, the main treatment means of brain glioma include surgical treatment, radiotherapy, chemotherapy and the like. Chemotherapy remains the current primary means of addressing tumors because of the need for chemotherapy for tumor recurrence and metastasis following surgery. The clinician can optimize to obtain the maximum curative effect and the minimum toxicity by adjusting the dosage of the medicine, thereby helping the patient to prolong the survival time and improve the prognosis life quality. However, chemotherapy drugs generally have the problem of overlarge toxic and side effects, which often cause one thousand of killers and eight hundred of killers to be damaged, so that physical ability and resistance of patients are sharply reduced, and the life cycle is greatly shortened. One of the main reasons for this result is that most chemotherapy drugs have poor drug effect selectivity, and the killing effect on tumor cells is not much different from that of normal cells of the body; another reason is that the distribution of antitumor drugs in vivo is not selective and is widely distributed in both tumor tissues and normal tissues of the body. Therefore, screening chemotherapeutic drugs with selective killing effect on certain tumor cells is one of the ways to improve the tumor treatment effect.

Among the various tumor entities, including brain gliomas, Acute Myelogenous Leukemia (AML), chondrosarcomas, cholangiocellular carcinomas, melanomas, prostate cancer, T-cell lymphomas of angioimmunoblasts, and the like, IDH1 or IDH2 are mutated at independent amino acid positions (Balss J. acta Neuropathithol.2008Dec; 116(6): 597. D602, Mardis ER, N Engl J Med.2009Sep 10; 361(11):1058-66, Amary MF, J Pathol.2011Jul; 224(3):334-43, Borger DR, oncologg.2012; 17(1):72-9, Shibata, Am J Pathol.2011; 178(3): 1395: (402, GhiAF, Oncogene.20116; Aubaira.20133, Cairi.20133: 19, Feid # 20123, 23, J.2011; 19: 19). Such mutations are always heterozygous and mutually exclusive. Most of these point mutations have been found at key positions in the catalytic domain of the enzyme (responsible for 2-oxoglutarate coordination), e.g., IDH1R100, IDH1R132, IDH1G97 and IDH2R140, IDH2R172(Dang L., Nature,2009Dec 10; 462(7274): 739-44). In gliomas, more than 70% of all non-primary malignant gliomas were IDH1 mutated, and arginine was replaced by histidine in 92.7% of IDH1 mutated tumors (IDH1R132H) (Hartmann C, Acta neuropathohol. 2009 Oct; 118(4): 469-74).

Substitution of wild-type amino acids at those catalytic residues results in a new viability of the enzyme, converting alpha-ketoglutarate to R-2-hydroxyglutarate (2-HG). 2-HG is a metabolic waste product, but also a tumor metabolite (oncomelalate), and is thought to contribute to tumorigenesis (Dang L., Nature,2009Dec 10; 462(7274): 739-44). 2-HG is produced only at very low levels in normal cells, but cells harboring IDH mutations produce high levels of 2-HG. High amounts of 2-HG were also found in tumors with IDH mutations. IDH mutations have also been described in patients with other disorders with high 2-HG levels, e.g., in rare neurometabolic diseases characterized by supraphysiological levels of 2-HG (2-HG sour urine) (krannendijk M, science.2010oct 15; 330(6002): 336).

Thus, inhibition of IDH mutations and its neoactivity is a potential treatment for glioma and other IDH mutation-related disorders.

Disclosure of Invention

In view of the problems of the background art, the present invention aims to provide a pharmaceutical composition for treating brain glioma.

The pharmaceutical composition for treating brain glioma provided by the invention comprises a compound shown in any one or more of a formula I, a formula II and a formula III and other pharmaceutical active components;

specifically, the invention provides an application of a compound shown as a formula I in preparing a medicament for treating brain glioma or melanoma;

preferably, the medicament for treating the brain glioma or the melanoma further comprises a compound shown as a formula II and/or a formula III.

Preferably, the dosage form of the medicament comprises: tablet, capsule, granule, microcapsule, injection, lyophilized powder for injection, gel, topical preparation or cavity administration preparation.

The invention provides a preparation method of an annona squamosa seed extract for treating brain glioma or melanoma, which comprises the following steps:

(1) crushing sweetsop seeds to obtain particles; (ii) a

(2) Mixing the particles with a degreasing solvent, and carrying out solid-liquid separation after degreasing to obtain filter residues;

(3) mixing the filter residue with an extraction solvent, and carrying out solid-liquid separation after extraction to obtain a liquid phase;

(4) removing the solvent from the liquid phase to obtain the annona squamosa seed extract.

Preferably, the particle size of the particles in the step (1) is 2-25 meshes.

Preferably, the degreasing solvent in the step (2) comprises one or more of petroleum ether, n-hexane and cyclohexane; the mass ratio of the use amount of the degreasing solvent to the particulate matter is 200-10000 mL: 20-250 g; the degreasing time is 3-8 h.

Preferably, the extraction solvent of step (3) comprises one or more of ethanol, methanol, acetone and ethyl acetate; the mass ratio of the usage amount of the extraction solvent to the particulate matter is 200-2000 mL: 20 to 250 g.

Preferably, the preparation of the effective part is further included after the sweetsop seed extract is obtained in the step (4).

Preferably, the preparation method of the effective part comprises the following steps:

mixing the sweetsop seed extract with water to obtain an aqueous suspension of the extract;

② mixing the water suspension of the extract with ethyl acetate, extracting to obtain ethyl acetate extract;

removing the solvent in the ethyl acetate extract to obtain an extract;

mixing the extract with silica gel, stirring, loading on a column, and sequentially mixing the extract with n-hexane in a volume ratio of 5:95 of acetone-n-hexane, in a volume ratio of 15:85, sufficiently eluting with acetone-n-hexane, and collecting the eluate at a volume ratio of 15:85 of acetone-n-hexane eluent, and recovering the solvent under reduced pressure to obtain the effective part.

The invention provides an annona squamosa seed extract prepared by the preparation method, wherein the annona squamosa seed extract comprises compounds shown in formula I, formula II and formula III.

The invention also provides application of the sweetsop seed extract prepared by the preparation method in preparing a medicament for treating brain glioma or melanoma.

Advantageous effects: the invention provides application of a compound shown in any one or more of a formula I, a formula II and a formula III in preparation of a medicine for treating brain glioma or melanoma. The compounds shown in the formula I, the formula II and the formula III are monomer components in the total lactone extract of the sweetsop respectively. In the screening process of sensitive cell strains of the total lactones and the monomer components of the annona squamosa, the invention unexpectedly discovers that the half inhibitory concentration IC of the annona squamosa on most tumor cells₅₀Under the condition that the value is up to several mu M to nearly hundred mu M, the compounds shown in the formulas I, II and III and the extract (including the effective part) prepared by the preparation method have high-efficiency killing effect on brain glioma and melanoma. IC of the extract prepared by the preparation method of the invention on U87-MG₅₀The highest value is 19.25ng/mL, and the lowest value is 2.167 ng/mL; IC on melanoma SK-MEL-28₅₀The highest value is 35.96ng/mL, and the lowest value is 3.593 ng/mL. The total lactone effective part prepared by the preparation method of the invention is IC of U87-MG₅₀IC at a value of 1.645nM for the melanomas SK-MEL-5 and SK-MEL-28₅₀Values were 0.5797nM and 2.635nM, respectively. In particular to the IC of the compound shown as the formula I on brain glioma U87-MG₅₀IC of value of 0.4044nM only, for melanoma SK-MEL-28₅₀The value was only 0.5152 nM. And IC for other majority of tumors₅₀Often between 1uM and 100uM, differing by more than three orders of magnitude, showing selective and highly effective killing of brain glioma U87-MG and melanoma SK-MEL-28.

The invention also provides a preparation method of the sweetsop seed extract, and the sweetsop seed extract (including the effective part) prepared by the method also shows specific selectivity and high-efficiency killing effect on glioma U87-MG or melanoma SK-MEL-28. The method provided by the invention does not need to accurately screen monomer components in the total lychee lactones, can achieve the effect of inhibiting glioma U87-MG or melanoma SK-MEL-28 by using the compound similar to that shown in the formula I on the premise of omitting complicated purification steps, is relatively more economic and has wide application prospect.

Drawings

FIG. 1 is an HPLC chromatogram of an ethanol extract of Annona squamosa seeds, wherein the total content of 5 main compounds K20, K437, K19, K16 and K109 is 8%;

FIG. 2 is an HPLC chromatogram of the ethyl acetate fraction of an annona squamosa seed alcohol extract, wherein the total content of 5 main compounds K20, K437, K19, K16 and K109 is 20%;

FIG. 3 is an HPLC chromatogram of the total lactone-effective fraction of Annona squamosa L.in example 6, wherein the total content of 5 main compounds K20, K437, K19, K16 and K109 is 62%, and the total content of K19 and K16 is about 51%;

FIG. 4 shows the results of in vitro growth inhibition tests of Annona squamosa total lactones according to example 15 of the present invention on human malignant brain glioma U87-MG, human melanoma SK-MEL-5 and SK-MEL-28;

FIG. 5 shows the results of the in vitro growth inhibition test of annonaceous acetogenins monomeric compound of example 15 in human malignant glioma U87-MG cells;

FIG. 6 shows the results of in vitro growth inhibition test of Annona squamosa Linn lactone monomer compound of example 15 on human melanoma SK-MEL-28 cells;

FIG. 7 shows the results of in vitro growth inhibition tests of annona squamosa total lactone nanoparticles of example 15 for human malignant glioma U87-MG;

FIG. 8 shows the results of in vitro growth inhibition test of Annona squamosa L.var.squamosa;

FIG. 9 shows the results of the in vitro growth inhibition test of Annona squamosa Linn seed alcoholic extract for human malignant brain glioma U87-MG in example 15 of the present invention.

FIG. 10 shows the results of in vitro growth inhibition test of Annona squamosa Linn seed alcoholic extract on human melanoma SK-MEL-28 according to example 15 of the present invention.

FIG. 11 shows the in vitro growth inhibition test results of the annona squamosa seed alcohol extract nanoparticles of example 15 of the present invention on human malignant glioma U87-MG.

FIG. 12 shows the in vitro growth inhibition test results of sweetsop seed alcohol extract nanoparticles on human melanoma SK-MEL-28 according to example 15 of the present invention.

FIG. 13 is a graph showing the effect of temperature on annonacin in example 20 of the present invention, wherein the temperature condition in FIG. 13- (1) is 60 ℃ and the temperature condition in FIG. 13- (2) is 100 ℃.

Detailed Description

The invention provides application of a compound shown in any one or more of a formula I, a formula II and a formula III in preparation of a medicament for treating brain glioma or melanoma;

the compound shown in formula I is a monomer component Bullatacin (K16) in Annona squamosa total lactone extract, and IC of Bullatacin in Annona squamosa total lactone extract on glioma U87-MG₅₀IC of human melanoma SK-MEL-28 with a value of 0.4044nM₅₀A value of 0.5152 nM; the compound represented by formula II is Squamocin (K19) as monomer component in Annona squamosa total lactone extract, and has effect in treating glioma U87-MG IC₅₀IC of human melanoma SK-MEL-28 with a value of 1.058nM₅₀The value was 1.058 nM; the compound shown in formula III is isoannonaceetin (K109) in Annona squamosa total lactone extract, and IC of Isoannonacein in Annona squamosa total lactone extract on glioma U87-MG₅₀The value was 0.5102nM, and the IC50 value was 1.31nM for human melanoma SK-MEL-28. The three compound monomer components have selective and efficient killing effect on brain glioma U87-MG or human melanoma SK-MEL-28.

In the present invention, the dosage form of the drug preferably includes: tablet, capsule, granule, microcapsule, injection, lyophilized powder for injection, gel, topical preparation or cavity administration preparation. The present invention is not particularly limited to the preparation method of each of the above formulations, and any method can be used as is conventional in the art.

The invention provides a preparation method of an annona squamosa seed extract, which comprises the following steps:

(1) crushing sweetsop seeds to obtain particles;

(4) recovering the solvent from the liquid phase to obtain the annona squamosa seed extract.

The invention firstly crushes the sweetsop seeds to obtain fine particles. In the present invention, the variety of Annona squamosa seeds preferably comprises Annona squamosa, Annona spinosa and/or Annona glabra. The particle size of the particles is preferably 2-25 meshes, and more preferably 10-18 meshes.

After the particles are obtained, the particles are mixed with a degreasing solvent, and solid-liquid separation is carried out after degreasing to obtain filter residues. In the present invention, the degreasing solvent preferably includes one or more of petroleum ether, n-hexane, and cyclohexane; more preferably n-hexane. The mass ratio of the volume of the degreasing solvent to the particles is preferably 200-10000 mL: 20-250 g; more preferably 8000 mL: 200 g. The degreasing time is preferably 3-8 h, and more preferably 5 h. In the present invention, the number of times of degreasing is preferably two, and the solid matter obtained by separating the degreasing solvent after the first degreasing is subjected to the second degreasing and the solid-liquid separation to obtain the residue. The method of the solid-liquid separation in the present invention is not particularly limited, and any conventional method in the art such as filtration with filter paper may be used.

After the filter residue is obtained, the filter residue is mixed with the extraction solvent, and the solid-liquid separation is carried out after the extraction to obtain the liquid phase. In the present invention, the extraction solvent preferably includes one or more of ethanol, methanol, acetone, and ethyl acetate, more preferably ethanol; the concentration of the ethanol is preferably more than or equal to 95 percent. In the present invention, the amount of the extraction solvent used is based on the fine particles obtained by the above pulverization, and the ratio of the volume of the extraction solvent to the mass of the fine particles is preferably 200 to 2000 mL: 20-250 g; more preferably 1500 mL: 200 g. The extraction method is not particularly limited in the present invention, and conventional extraction methods in the art such as ultrasonic extraction, tissue disruption stirring extraction, and reflux extraction may be used. When ultrasonic extraction is used, the power of the ultrasonic is preferably 200-300W, and more preferably 250W; the extraction temperature is preferably 10-60 ℃, more preferably 20-30 ℃, and more preferably 25 ℃; the extraction time is preferably 0.5-12 h, more preferably 1-3 h, and more preferably 2 h. When tissue crushing, stirring and extraction are used, flash extraction equipment produced by Beijing gold advanced science and technology development Inc. or Henan gold advanced science and technology development Inc. is preferably used in the invention; the extraction temperature is preferably 10-60 ℃, more preferably 20-30 ℃, and more preferably 25 ℃; the extraction time is preferably 1-120 min, more preferably 10-30 min, and still more preferably 20 min. When reflux extraction is used, the extraction temperature is preferably 60 to 80 ℃, and more preferably 70 ℃. The method of the solid-liquid separation in the present invention is not particularly limited, and any conventional method in the art such as filtration with filter paper may be used.

After obtaining the liquid phase, the invention recovers the solvent in the liquid phase, and the residual solid is the sweetsop seed extract. The present invention is not particularly limited in the method for recovering the solvent, and conventional recovery methods in the art such as reduced pressure rotary evaporation, flash concentration, spray drying, freeze drying and the like may be used. In the present invention, the temperature of the solvent during the recovery process is preferably 70 ℃ or less, more preferably 60 ℃ or less, and still more preferably 50 ℃ or less. If flash concentration or spray drying is adopted, the heating temperature of the solvent is preferably 60-150 ℃, and the heating time is preferably less than or equal to 2min, more preferably less than or equal to 1min, and more preferably less than or equal to 30 s.

IC of the extract prepared by the preparation method of the invention on U87-MG₅₀The highest value is 19.25ng/mL, and the lowest value is 2.167 ng/mL; IC on melanoma SK-MEL-28₅₀The highest value is 35.96ng/mL, the lowest value is 3.593ng/mL, and the specific selectivity and the efficient killing effect on the glioma U87-MG are shown.

After the sweetsop seed extract is obtained, the invention preferably carries out fine extraction and purification on the extract to prepare the effective part. In the present invention, the preparation method of the effective part preferably includes the steps of:

③ recovering the solvent in the ethyl acetate extract to obtain an extract;

The sweetsop seed extract is firstly mixed with water to obtain the aqueous suspension of the extract. The aqueous suspension of the extract was then mixed with ethyl acetate and extracted to give an ethyl acetate extract. In the invention, the volume (liter) of the water is preferably 2-6 times of the weight (kilogram) of the sweetsop seed extract, and more preferably 3-4 times; the volume of the ethyl acetate is preferably 1 to 3 times, and more preferably 2 times of the volume of the aqueous extract suspension. The invention preferably extracts for 3 times, and the ethyl acetate extract liquid is combined after extraction.

After the ethyl acetate extract is obtained, the solvent in the ethyl acetate extract is recovered to obtain the extract. The present invention is not particularly limited in the method for recovering the solvent, and conventional recovery methods in the art such as reduced pressure rotary evaporation, flash concentration, spray drying, freeze drying and the like may be used. In the present invention, the temperature of the solvent during the recovery process is preferably 70 ℃ or less, more preferably 60 ℃ or less, and still more preferably 50 ℃ or less. If flash evaporation concentration or spray drying is adopted, the heating temperature of the solvent is preferably 60-150 ℃, the heating time is preferably less than or equal to 2min, more preferably less than or equal to 1min, and more preferably less than or equal to 30 s.

After the extract is obtained, the extract and silica gel are mixed and stirred, the mixture is loaded on a column, and n-hexane and silica gel in a volume ratio of 5:95 of acetone-n-hexane, in a volume ratio of 15:85, sufficiently eluting with acetone-n-hexane, and collecting the eluate at a volume ratio of 15:85 of acetone-n-hexane eluent, and recovering the solvent under reduced pressure to obtain the effective part.

The effective part prepared by the preparation method is annona squamosa total lactone (effective part) which is IC of human malignant glioma U87-MG₅₀An IC value of 1.645nM for human melanomas SK-MEL-5 and SK-MEL-28₅₀The values are 0.5797nM and 2.635nM, respectively, and the specific selectivity and high-efficiency killing effect on brain glioma U87-MG and human melanoma SK-MEL are shown.

The invention provides an sweetsop seed extract (or effective part) prepared by the preparation method. In the invention, the sweetsop seed extract (or the effective part) comprises the compounds shown in the formulas I, II and III, and the inhibitory effect of the compounds shown in the formulas I, II and III on brain glioma U87-MG and human melanoma SK-MEL-28 can be achieved approximately under the condition that the compounds shown in the formulas I, II and III are not purified. The method provided by the invention omits complicated purification steps, and the prepared product is relatively more economical and has wide application prospect.

The invention also provides one or more compounds shown in any one of the formula I, the formula II and the formula III, and application of the sweetsop seed extract (or effective part) prepared by the preparation method in preparation of a medicament for treating brain glioma or human melanoma. In the invention, the medicine can be added with pharmaceutically acceptable auxiliary materials to prepare various forms convenient for clinical administration, such as tablets, capsules, granules, microcapsules, injection, freeze-dried powder, gel, external preparations or cavity administration preparations and the like; the drug may be present in the formulation in the form of molecular states, nanoparticles, liposomes, clathrates, microparticles, and the like.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Round sweetsop, ethanol ultrasonic extraction (round-ethanol ultrasonic extract)

Taking 20g of dried Annona squamosa Linn seeds, pulverizing into 15-mesh particles, adding n-hexane, soaking and degreasing for 2 times (200 mL of n-hexane is used each time, and shaking intermittently for 5 hours), filtering with filter paper, adding 200mL of absolute ethyl alcohol into filter residues, ultrasonically extracting the filter residues at 25 ℃ and 250W for two times for 30min each time, filtering with filter paper, combining the filtrates, heating at 55 ℃ under reduced pressure and carrying out rotary evaporation to remove the organic solvent, and drying in vacuum to obtain the ethanol ultrasonic extract.

Example 2

Annona squamosa, ethanol ultrasonic extraction (soil-ethanol ultrasonic extract)

Taking 20g of dried Annona squamosa L.var.chaeta seeds, crushing into 18-mesh particles, adding n-hexane, soaking and degreasing for 2 times (200 mL of n-hexane is used each time, shaking intermittently for 5 hours), filtering by using filter paper, adding 200mL of absolute ethyl alcohol into filter residues, carrying out ultrasonic extraction twice at 25 ℃ and 250W for 30min each time, filtering by using filter paper, combining the filtrates of the two times, carrying out reduced pressure heating and rotary evaporation at 60 ℃ to remove an organic solvent, and carrying out vacuum drying to obtain an ethanol ultrasonic extract.

Example 3

Annona muricata, ethanol ultrasonic extraction (thorn-ethanol ultrasonic extract)

Taking 20g of dried Annona spinosa seeds, crushing the seeds into 10-mesh particles, adding n-hexane, soaking and degreasing for 2 times (200 mL of n-hexane is used each time, shaking intermittently for 5 hours), filtering by using filter paper, adding 200mL of absolute ethyl alcohol into filter residues, carrying out ultrasonic extraction twice at 25 ℃ and 250W for 30min each time, filtering by using filter paper, combining filtrates obtained in two times, carrying out reduced pressure heating and rotary evaporation at 50 ℃ to remove an organic solvent, and carrying out vacuum drying to obtain an ethanol ultrasonic extract.

Example 4

Round Annona squamosa, ethanol reflux extraction (round-ethanol reflux extract)

Taking 185 g of dried Annona squamosa seeds, crushing the Annona squamosa seeds into 15-mesh particles, adding n-hexane for degreasing for 2 times (7 liters of n-hexane is used each time, and shaking is carried out intermittently for 5 hours), filtering by filter paper, adding 1400ml of 95% ethanol into filter residues, carrying out reflux extraction twice at 70 ℃ for 30min each time, filtering by filter paper, combining filtrates of the two times, carrying out reduced pressure heating and rotary evaporation at 60 ℃ for removing organic solvent to obtain 95% ethanol reflux extract, pre-freezing in a refrigerator at 20 ℃ for 12 hours, carrying out freeze-drying at 40 ℃ and 0.12Pa to obtain two samples, and obtaining a tan extract at the bottom of a bottle, which is named as; the upper oily liquid is named as 'round-alcohol reflux extract-liquid'.

Example 5

Smooth annona squamosa, dynamic extraction by tissue disruption (flash)

Taking 200g of dried smooth annona squamosa seeds, crushing the seeds into 15-mesh particles, placing the particles in a flash extractor with the volume of 2.5 liters, adding 1.5 liters of n-hexane for degreasing for 3 times (room temperature, the rotating speed of 5000rpm, the time of 15 minutes, the interval of 3 times), centrifuging for 10 minutes at 5000rpm, adding 1 liter of n-hexane into the precipitate for degreasing by the same method, and degreasing for three times; discarding supernatant, adding 1.0L 95% ethanol into precipitate, performing flash extraction for 3 times (room temperature, rotation speed of 5000rpm, time of 15 min each time, interval of 2min each time of extraction 5min, centrifuging at 5000rpm for 10min, and subjecting the precipitate to next extraction), mixing the three extractive solutions, and performing rotary evaporation at 60 deg.C under reduced pressure to remove organic solvent to obtain 95% ethanol flash extract.

Example 6

Annona squamosa L total lactones ACGs, effective part

The extract of example 5 was uniformly dispersed in 3 times by weight of water to obtain an aqueous suspension, and 2 times by volume of ethyl acetate was added to the aqueous suspension and mixed by shaking, followed by extraction by standing 3 times. Combining ethyl acetate extract, recovering solvent under reduced pressure to obtain ethyl acetate extract, mixing the extract with 1.3 times of silica gel, loading on a column filled with 1 time of clean silica gel (containing sample silica gel), eluting with 10 times of bed volume of n-hexane (discarding eluent), eluting with 10 times of bed volume of acetone-n-hexane (5:95, v/v) (discarding eluent), eluting with 40 times of bed volume of acetone-n-hexane (15:85, v/v), collecting the eluent, and recovering solvent under reduced pressure to obtain total lactone effective parts (ACGs).

Example 7

Chromatographic analysis and content determination of sweetsop seed extract and effective part

HPLC chromatographic conditions: waters hplc (Waters model 2695 pump, Waters 2996 detector, Empower workstation); the chromatographic column is Diamonsil C18(2), 5um, 4.6 x 250 mm; the mobile phase is as follows: a is acetonitrile, B is 0.3% phosphoric acid water solution, and gradient elution is carried out: 29-29% of B in 0-10 min, 27-27% of B in 10-35 min, 25-25% of B in 35-45 min, 15-15% of B in 45-55 min, and 29-29% of B in 55-70 min; flow rate: 1.0 mL/min; column temperature: 30 ℃; detection wavelength: 210 nm; sample introduction amount: 20 μ L.

Precisely weighing 1mg of each of K20 (squarostatin-B), K437(cherinolin), K19 (squarocin), K16(bullatacin) and K109 (isoannoretic) in a 5mL volumetric flask, adding acetonitrile to a constant volume, sequentially taking out 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 0.5mL of the solution, placing the solution in a 1mL volumetric flask, and diluting the solution to a scale with a mobile phase. Obtaining a solution with the mass concentration of 1.6, 4, 8, 20, 40, 60 and 100 mu g/mL, feeding a sample to determine a peak area A, and drawing a standard curve by taking the concentration of each index component of the annona squamosa as a horizontal coordinate and the peak area as a vertical coordinate to obtain a regression equation.

The sweetsop seed extract in example 5, the ethyl acetate extract in example 6 and the obtained total lactone effective parts are prepared into methanol solution with proper concentration, sample injection is carried out according to HPLC chromatographic conditions to obtain a chromatogram, and the total content of 5 index components in the sweetsop seed extract in example 5, the ethyl acetate extract in example 6 and the obtained total lactone effective parts is determined according to chromatographic peaks and regression modes of the obtained 5 reference substances.

The results are shown in FIGS. 1 to 3, and FIGS. 1 to 3 show that: the impurities are lower and lower from the alcohol extract, the ethyl acetate extract to the total lactone effective part, and the lactone components are enriched. In the effective part, the total content of 5 index components exceeds 60%, and the total content of two main components K19 and K16 is about 50%.

Example 8

Total lactone nanoparticles, P188 as carrier (P188-ACGs nanoparticles)

Weighing 5mg of annona squamosa total lactones, dissolving the annona squamosa total lactones in 0.5mL of absolute ethyl alcohol, dissolving 5mg of poloxamer P188 in 5mL of deionized water, and slowly dripping the ethanol solution into the deionized water in which the P188 is dissolved under the ultrasonic condition of 250HZ at normal temperature. And after finishing dripping, removing the ethanol by rotary evaporation to obtain the ACGs nanoparticles. The average particle diameter was 169.8nm, the polydispersity index (PDI) was 0.108 and the potential value was-17.8 mV, as determined by dynamic light scattering.

Example 9

Total lactone nanoparticles, TPGS as carrier (TPGS-ACGs nanoparticles)

Weighing 5mg of annona squamosa total lactones and 5mg of vitamin E polyethylene glycol succinate TPGS, dissolving in 0.5mL of absolute ethyl alcohol, and slowly dripping the ethanol solution into 5mL of deionized water at normal temperature under the ultrasonic condition of 250 HZ. And after finishing dripping, removing the ethanol by rotary evaporation to obtain the ACGs nanoparticles. The mean particle diameter was found to be 136.0nm by dynamic light scattering, the polydispersity index (PDI) was 0.195 and the potential value was-19.6 mV.

Example 10

Total lactone nanoparticles, PCL2000-PEG2000 as carrier (PCL-PEG-ACGs nanoparticles)

Weighing 5mg of annona squamosa total lactones, dissolving in 0.5mL of absolute ethyl alcohol, dissolving 5mg of polyethylene glycol 2000-polycaprolactone 2000PEG2k-PCL2k in 0.2mL of acetone, mixing the two solutions uniformly, and slowly dripping the mixed solution into 5mL of deionized water at normal temperature under the ultrasonic condition of 250 HZ. And after finishing instillation, removing the organic solvent by rotary evaporation to obtain the ACGs nanoparticles. The mean particle diameter was 133.9nm, the polydispersity index (PDI) was 0.175 and the potential value was-16.2 mV, determined by dynamic light scattering.

Example 11

Round sweetsop seed ethanol ultrasonic extract-P188 nanoparticles (P188-round-ethanol ultrasonic extract)

Weighing 10mg of Annona glabra Linn seed ethanol ultrasonic extract, dissolving in 1mL of absolute ethanol, dissolving 10mg of poloxamer P188 in 10mL of deionized water, and slowly dripping the ethanol solution into the deionized water in which the P188 is dissolved under the ultrasonic condition of 250HZ at normal temperature. And after finishing dripping, removing the ethanol by rotary evaporation to obtain the ACGs nanoparticles. The mean particle diameter was 177.3nm, the polydispersity index (PDI) was 0.112 and the potential value was-33.8 mV, determined by dynamic light scattering.

Example 12

Annona squamosa L.seed ethanol ultrasonic extract-P188 nanoparticles (P188-ethanol ultrasonic extract)

Weighing 5mg of annona squamosa seed ethanol ultrasonic extract, dissolving the annona squamosa seed ethanol ultrasonic extract in 0.5mL of absolute ethanol, dissolving 5mg of poloxamer P188 in 5mL of deionized water, and slowly dripping the ethanol solution into the deionized water in which the P188 is dissolved under the ultrasonic condition of 250HZ at normal temperature. And after finishing dripping, removing the ethanol by rotary evaporation to obtain the ACGs nanoparticles. The average particle diameter was 177.9nm, the polydispersity index (PDI) was 0.128 and the potential value was-12.8 mV, determined by dynamic light scattering.

Example 13

'round-alcohol reflux extract-solid' -P188 nano-particle (P188-round-alcohol reflux extract-solid)

Weighing 10mg of round-alcohol, refluxing, freeze-drying and dissolving in 1mL of absolute ethanol, dissolving 10mg of poloxamer P188 in 10mL of deionized water, and slowly dripping the ethanol solution into the deionized water in which the P188 is dissolved under the ultrasonic condition of 250HZ at normal temperature. And after finishing dripping, removing the ethanol by rotary evaporation to obtain the ACGs nanoparticles. The average particle diameter was 197.3nm, the polydispersity index (PDI) was 0.286, and the potential value was-24.6 mV, as determined by dynamic light scattering.

Example 14

"round-alcohol reflux extract-liquid" -P188 nano-particle (P188-round-alcohol reflux extract-liquid)

Weighing 10mg of round-alcohol reflux freeze-drying solution, dissolving the round-alcohol reflux freeze-drying solution in 1mL of absolute ethyl alcohol, dissolving 10mg of poloxamer P188 in 10mL of deionized water, and slowly dripping the ethanol solution into the deionized water in which the P188 is dissolved under the ultrasonic condition of 250HZ at normal temperature. And after finishing dripping, removing the ethanol by rotary evaporation to obtain the ACGs nanoparticles. The average particle diameter was 279.6nm, the polydispersity index (PDI) was 0.370 and the potential value was-19.6 mV, determined by dynamic light scattering.

Example 15

Direct killing assay for tumor cells

In vitro growth inhibition test of Annona squamosa Total lactones on human malignant brain glioma U87-MG, human melanoma SK-MEL-28 and SK-MEL-5

The experimental method comprises the following steps:

1) adjusting the growth states of human malignant brain glioma U87-MG, human melanoma SK-MEL-28 and SK-MEL-5 cells, harvesting the cells when the cells are in logarithmic phase, digesting and washing, staining with trypan blue, and counting;

2) adherent cell density was adjusted to 5.56X 10 using complete medium⁴Per mL;

3) inoculating 96-well cell culture plate at 90 μ l/well to ensure that the number of adherent cells per well is 5 × 10³A plurality of;

4) placing the inoculated cell plate in an incubator at 37 ℃ for overnight incubation;

5) dissolving Annona squamosa total lactones (effective fraction prepared in example 6) in DMSO, preparing 100mM stock solution (reduced according to molecular weight 622), and diluting the sample with culture medium to obtain 50. mu.M, 15.8. mu.M, 5. mu.M, 1.58. mu.M, 0.5. mu.M, 0.158. mu.M, 0.05. mu.M, 0.0158. mu.M, and 0.005. mu.M;

6) taking out the cell culture plate, adding the medicinal working solution with the concentration into the cell culture plate, wherein each hole is 10 mu l, a blank culture medium is used as a control, and each concentration is provided with three multiple holes;

7) placing the cell culture plate in an incubator for further incubation for 72 hours;

8) after 72 hours, CellTiter was added

The AQueous One Solution reagent was thawed at room temperature and equilibrated to room temperature, and 20. mu.l/well of CellTiter was added to the cell culture plate

An AQueous One Solution reagent;

9) placing the cell culture plate in an incubator at 37 ℃ and continuously incubating for 3 hours;

10) reading the absorbance (OD value) of each well at 490nm wavelength by using a microplate reader;

11) processing and analyzing the data to obtain IC of the object to be measured₅₀The value is obtained.

The test substance was then further diluted with a medium to give a series of working solutions at concentrations of 50nM, 15.8nM, 5nM, 1.58nM, 0.5nM, 0.158nM, 0.05nM, 0.0158nM, 0.005nM and re-run. The half inhibitory concentration (IC50 value) of the total lactone effective part and three monomeric compounds of Annona squamosa Linn in the cell growth of human malignant glioma U87-MG, human melanoma SK-MEL-28 and SK-MEL-5 is shown in Table 1.

TABLE 1 Total Annona squamosa lactone effective fraction and half inhibitory concentration (IC50 value) of three monomeric compounds against cell growth of human malignant glioma U87-MG, human melanoma SK-MEL-28 and SK-MEL-5

The results are shown in FIG. 4 and Table 1, for annona squamosaHalf inhibitory concentration IC of total lactone effective component for inhibiting growth of human malignant glioma cells U87-MG, human melanoma cells SK-MEL-28, and SK-MEL-5₅₀1.645nM, 2.635nM, and 0.5797nM, respectively, show high killing effect on human glioblastoma cells and melanoma.

(II) in vitro growth inhibition test of Annona squamosa Linn lactone monomer compound on human malignant brain glioma U87-MG cell and human melanoma cell SK-MEL-28

The experimental method comprises the following steps:

1) adjusting the growth states of human malignant brain glioma U87-MG cells and human melanoma SK-MEL-28 cells, harvesting the cells when the cells are in logarithmic growth phase, digesting and washing, staining with trypan blue, and counting;

5) dissolving total lactone monomer compounds of Annona squamosa Linn (K16, formula I), Squalocin (K19, formula II) and Isoannoretic (K109, formula III) in DMSO respectively to prepare 100mM stock solution, and diluting the stock solution with culture medium to obtain a series of working solutions of 50nM, 15.8nM, 5nM, 1.58nM, 0.5nM, 0.158nM, 0.05nM, 0.0158nM and 0.005 nM;

8) after 72 hours, CellTiter was added

An AQueous One Solution reagent;

The results are shown in fig. 5, fig. 6 and table 1. Fig. 5 and table 1 show that: annona squamosa lactone monomer compounds K16, K19 and K109 half inhibition concentration IC for growth inhibition of human malignant brain glioma U87-MG₅₀0.4044nM, 1.058nM and 0.5102nM respectively, which all show high-efficiency killing effect on human malignant brain glioma U87-MG; fig. 6 and table 1 show that: half inhibitory concentration IC of annonaceous acetogenin monomer compounds K16, K19 and K109 for inhibiting growth of human melanoma SK-MEL-28₅₀0.5152nM, 1.571 nM and 1.31nM respectively, all show high killing effect on human melanoma SK-MEL-28.

(III) in vitro growth inhibition test of Annona squamosa total lactone nanoparticles on human malignant brain glioma U87-MG and human melanoma SK-MEL-28

The experimental method comprises the following steps:

1) adjusting the growth states of human malignant brain glioma U87-MG and human melanoma SK-MEL-28 cells, harvesting the cells when the cells are in logarithmic growth phase, digesting and washing, staining with trypan blue, and counting;

5) preparing 100mM stock solutions (the molecular weight of the total lactones is reduced by 622) respectively for the three total lactone nanoparticles in the embodiments 8, 9 and 10, and diluting the substance to be detected with a culture medium to obtain a series of working solutions with the concentrations of 50nM, 15.8nM, 5nM, 1.58nM, 0.5nM, 0.158nM, 0.05nM, 0.0158nM and 0.005 nM;

8) after 72 hours, CellTiter was added

An AQueous One Solution reagent;

The half inhibitory concentration (IC50 value) of the annona squamosa total lactone nanoparticles on the cell growth of human malignant glioma U87-MG and human melanoma SK-MEL-28 is shown in Table 2.

TABLE 2 half inhibitory concentration (IC50 value) of annona squamosa total lactone nanoparticles against human malignant glioma U87-MG and human melanoma SK-MEL-28 cell growth

The results are shown in fig. 7, fig. 8 and table 2. FIG. 7 and Table 2 show that the half inhibition concentration IC of annona squamosa total lactone nanoparticles taking poloxamer P188, TPGS and PCL2000-mPEG2000 as a carrier for inhibiting the growth of human malignant glioma U87-MG cells₅₀1.37, 1.524, 3.448nM, respectively; compared with the IC50 value (1.645nM) of U87-MG of total lactone in Table 1, the nanoparticle can solve the problem that the total lactone is difficult to dissolve and is difficult to be used as a medicineWhen the preparation is used, the high-efficiency killing effect of the total lactones on human malignant glioma cells is completely maintained. FIG. 8 and Table 2 show that the half inhibitory concentration IC of annona squamosa total lactone nanoparticles using poloxamer P188, TPGS, PCL2000-mPEG2000 as carrier on the growth inhibition of human melanoma SK-MEL-28 cells₅₀0.9717, 1.289 and 2.059nM respectively; compared with the IC50 value (2.635nM) of the total lactone to SK-MEL-28 in Table 1, the nanoparticle solves the problem that the total lactone is difficult to dissolve and difficult to use, and meanwhile slightly improves the efficient killing effect of the total lactone on human melanoma cells.

(IV) in vitro growth inhibition test of Annona squamosa seed alcohol extract on human malignant brain glioma U87-MG and human melanoma SK-MEL-28

The experimental method comprises the following steps:

1) adjusting the growth states of human malignant brain glioma U87-MG cells and human melanoma SK-MEL-28, harvesting the cells when the cells are in logarithmic growth phase, digesting and washing, staining with trypan blue, and counting;

3) inoculating 96-well cell culture plate at 90 μ l/well to ensure that the number of adherent cells per well is 5 × 10³A plurality of; 4) placing the inoculated cell plate in an incubator at 37 ℃ for overnight incubation;

5) the three ethanol extracts of examples 1, 2 and 3 were prepared as 100mM stock solutions (total lactone molecular weight is estimated at 622), and the samples were diluted with medium to obtain a series of working solutions with concentrations of 50nM, 15.8nM, 5nM, 1.58nM, 0.5nM, 0.158nM, 0.05nM, 0.0158nM and 0.005 nM;

8) after 72 hours, CellTiter was added

AQueous One SThe solution reagent was thawed at room temperature and equilibrated to room temperature, and 20. mu.l/well CellTiter was added to the cell culture plate

An AQueous One Solution reagent;

The test substance was then further diluted with a medium to give a series of working solutions at concentrations of 50nM, 15.8nM, 5nM, 1.58nM, 0.5nM, 0.158nM, 0.05nM, 0.0158nM, 0.005nM and re-run.

The half inhibitory concentration (IC50 value) of the ethanol extract of sweetsop seeds on the growth of human malignant glioma U87-MG and human melanoma SK-MEL-28 cells is shown in Table 3.

TABLE 3 half inhibitory concentration (IC50 value) of Annona squamosa seed ethanol extract on the growth of human malignant glioma U87-MG and human melanoma SK-MEL-28 cells

The results are shown in fig. 9, fig. 10 and table 3. Fig. 9 and table 3 show that: half inhibition concentration IC of ethanol ultrasonic extracts of Annona glabra Linn and Annona squamosa Linn seeds for inhibiting growth of human malignant brain glioma U87-MG₅₀2.459ng/mL and 2.167ng/mL respectively; while two extracts of Annona glabra Linn with ethanol thermal reflux have half inhibition concentration IC for inhibiting growth of human malignant brain glioma U87-MG₅₀3.325ng/mL and 19.25ng/mL, respectively. Fig. 10 and table 3 show that: half inhibitory concentration IC of Annona glabra Linn and Annona squamosa Linn seed ethanol ultrasonic extract for inhibiting human melanoma SK-MEL-28 growth₅₀3.593ng/mL and 4.006ng/mL respectively; while two extracts of Annona glabra Linn with ethanol thermal reflux have half inhibitory concentration IC on growth inhibition of human malignant brain glioma SK-MEL-28₅₀7.814ng/mL and 35.96ng/mL, respectively.

These data show that the ethanol extract of defatted seeds also has high killing effect on human malignant brain glioma U87-MG and SK-MEL-28. The extract has simple preparation process, easy industrial preparation, no complicated step of enriching total lactone, low cost and high commercial value.

(V) in vitro growth inhibition test of sweetsop seed alcohol extract nanoparticles on human malignant brain glioma U87-MG and human melanoma SK-MEL-28

The experimental method comprises the following steps:

5) preparing 100mM stock solutions (calculated according to the concentration of the extracts) of the nanoparticles of the three ethanol extracts in the embodiments 11, 13 and 14, respectively, and diluting the to-be-detected substance with a culture medium to obtain a series of working solutions with the concentrations of 50nM, 15.8nM, 5nM, 1.58nM, 0.5nM, 0.158nM, 0.05nM, 0.0158nM and 0.005 nM;

8) after 72 hours, CellTiter was added

The AQueous One Solution reagent was thawed at room temperature and equilibrated to room temperature, and 20. mu.l was added to the cell culture plateCellTiter/pore

An AQueous One Solution reagent;

The half inhibitory concentration (IC50 value) of the annona squamosa seed ethanol extract nanoparticles on the growth of human malignant glioma U87-MG and human melanoma SK-MEL-28 cells is shown in Table 4.

TABLE 4 half inhibitory concentration IC50 values of sweetsop seed ethanol extract nanoparticles on human malignant glioma U87-MG and human melanoma SK-MEL-28 cell growth

The results are shown in fig. 11, fig. 12 and table 4. Fig. 11 and table 4 show that: half inhibition concentration IC of three annona squamosa extracts nanoparticles for human malignant brain glioma U87-MG cell growth₅₀1.576ng/mL, 3.175ng/mL and 11.20ng/mL, respectively; half inhibitory concentration IC on human melanoma SK-MEL-28 cell growth₅₀5.261ng/mL, 14.84ng/mL and 36.79ng/mL, respectively.

Compared with the IC50 values of the corresponding extracts in the table 3 on the total U87-MG and SK-MEL-28, the nanoparticles can solve the problem that the total lactones are difficult to dissolve and difficult to use, and effectively keep the efficient killing effect of the extracts on human malignant glioma and melanoma cells. The extract has simple preparation process, easy industrial preparation, no complicated step of enriching total lactone and low cost, so the extract has wide clinical application prospect and high commercial value.

Example 16

Oral acute toxicity experimental study of annona squamosa total lactone nanoparticles (P188 is a carrier).

Preparing nanoparticles: referring to the method of example 8, the carrier P188 was used to prepare a carrier ratio of 1: 5. 1: 1. 5:1 total lactone nanoparticles (ACGs-P188-NPs)

The administration scheme is as follows: kunming mice with a weight of 20g are selected and randomly divided into 7 groups, 10 mice in each group are divided into two halves, after adaptive feeding is carried out for 2 days, water is not forbidden for 12 hours in one day before administration, and 60, 80, 110, 140, 170 and 200mg/kg of ACGs-NSps (which are prepared by an equal ratio dilution method) with a drug loading ratio of 1:5 and P188 blank auxiliary materials (200mg/kg) are respectively administered by single gastric gavage.

Kunming mice with a weight of 20g are selected and randomly divided into 7 groups, 10 mice in each group are divided into two halves, after adaptive feeding is carried out for 2 days, water is not forbidden for 12 hours in one day before administration, and 60, 80, 110, 140, 170 and 200mg/kg of ACGs-NSps (which are prepared by an equal ratio dilution method) with a drug loading ratio of 1:1 and P188 blank auxiliary materials (200mg/kg) prepared according to example 8 are respectively administered by single gastric gavage.

Kunming mice with the weight of 20g are simultaneously selected and randomly divided into 7 groups, 10 mice in each group are divided into two halves, after adaptive feeding is carried out for 2 days, the mice are fasted for 12 hours before administration, 30, 40, 60, 80, 110, 140 and 170mg/kg of ACGs-NSps (which are prepared by an equal ratio dilution method) and P188 blank auxiliary materials (200mg/kg) in the drug loading ratio of 5:1 are respectively administered by single gastric gavage.

And (4) investigation indexes are as follows: the physiological state and toxic reaction condition of the mice after administration are observed, the death time and number are recorded, the body weight of the mice is detected every 2 days, and the observation is continued for 2 weeks.

The results are shown in tables 5-7:

TABLE 5 ACCS-P188-NPs mice acute toxicity experiment death rate table (mean + -SD, n 10) with drug loading ratio 1:5

As can be seen from table 5, the drug loading ratio 1:5 ACGs-P188-NPs high dose group shows toxic symptom within 1h, mice gradually slow moving, stop eating and death prophaseSlight convulsion appears, and the limbs are stiff gradually in the later period. However, the mice of the adjuvant group have no adverse reaction and abnormal expression under the dosage of 200 mg/kg. LD calculation from mouse mortality in Table 1₅₀With a value of 36.86mg/kg, and is mixed with annonaceous acetogenin as bulk drug LD₅₀The value is increased by 1.9 times compared with 19.21mg/kg, which is 37 times of the highest effective dose (1 mg/kg). The annonaceous acetogenins prepared into the nano-particles can reduce the drug toxicity to a certain extent.

TABLE 6 ACCS-P188-nsps mice acute toxicity experiment death rate table (mean + -SD, n 10) at drug loading ratio 1:1

As can be seen from Table 6, the P188-ACGs nanoparticle high-dose group with the drug loading ratio of 1:1 also has the poisoning symptom within 1h, the mouse gradually acts slowly, stops eating and breathing rapidly, slight convulsion appears in the early death period, and the phenomenon of limb stiffness gradually appears in the later period. In the adjuvant group, the mice also have no adverse reaction and abnormal expression under the dosage of 200 mg/kg. LD calculation from mouse mortality in Table 6₅₀With a value of 135.51mg/kg, and is mixed with annonaceous acetogenin as bulk drug LD₅₀The value is increased by 6.3 times compared with 19.21 mg/kg. The nanoparticles prepared by P188 with the drug loading ratio of 1:1 can obviously reduce the oral toxicity of ACGs. LD for comparing mouse acute toxicity experiment₅₀The data show that under the condition of the same auxiliary materials and similar particle sizes, the toxicity of the nanoparticles with different drug loading ratios is greatly different due to the reason that the nanoparticle structure, the surface property, the action mode with intestinal epithelial cells and the like are different. The average body weight of the surviving mice continuously increased, suggesting that the toxic and side effects of higher doses on the mice are shorter in duration.

TABLE 7 ACCS-P188-nsps mice acute toxicity experiment death rate table (mean + -SD, n 10) at drug loading ratio 5:1

As can be seen from Table 7: the mortality rate is positively correlated with the dosage, wherein the mortality rate of the highest dosage group reaches 90 percent, and the mortality rate of the lowest dosage group and the adjuvant group does not die. Calculation of LD of ACGs-P188-NPs on mice by Bliss software₅₀The value is 125.51mg/kg, while the tumor inhibition rate of ACGs-P188-NPs orally taken is more than 60% in the study, and LD₅₀The difference of the values is more than 44 times, which indicates that the annonaceous acetogenins nano-particles prepared by P188 with the drug loading ratio of 5:1 also obviously expand the treatment window.

In this example, the nanoparticles all use poloxamer P188 as an adjuvant, and have similar particle sizes, but the drug loading ratios are 1:5, 1:1, and 5:1, respectively, which results in oral administration of LD in mice₅₀36.86mg/kg, 135.51mg/kg and 121.51mg/kg, respectively. The LD that the difference of the drug loading ratio can influence the oral acute toxicity of the nanoparticles₅₀This fact.

Example 17

Annona squamosa total lactone nanoparticles (PCL2000-mPEG2000 as a carrier) are used for oral acute toxicity experimental study.

Referring to the method in example 10, the annona squamosa total lactones ACGs and the same weight of PEG2000-PCL2000 are prepared in a drug loading ratio of 1:1 of PCL-PEG-ACGs nanoparticles.

Kunming mice with the weight of 20g are selected and randomly divided into 6 groups, each group comprises 10 mice, each group comprises a male mouse and a female mouse, after adaptive feeding is carried out for 2 days, fasting is carried out for 12 hours before administration, and PCL-PEG-ACGs nanoparticles (drug loading ratio is 1:1) which take PEG2000-PCL2000 as an auxiliary material and are administered with 15, 26.15, 37.5, 48.75 and 60mg/kg of PCL-PEG-ACGs with PEG2000-PCL2000 as an auxiliary material and P188 blank auxiliary materials (60mg/kg) are respectively administered by single gastric lavage on the basis of a pre-experiment.

And (4) investigation indexes are as follows: the physiological state and toxic reaction condition of the mice after administration are observed, the death time and number are recorded, the body weight of the mice is detected every 2 days, and the observation is continued for 2 weeks. The mortality status of the mice is shown in table 8.

TABLE 8 death rate table of Annona squamosa total lactone nanoparticle mice acute toxicity experiment prepared by using PCL2000-mPEG2000 as adjuvant

The LD of PCL-PEG-ACGs nanoparticles is calculated according to the table 8₅₀The value was 58.25 mg/kg.

Example 18

The annona squamosa total lactone nanoparticles have an in vivo tumor inhibiting effect on human malignant glioma U87-MG tumor-bearing mice when orally taken.

Culturing human malignant glioma cell U87-MG in vitro to logarithmic growth phase, digesting adherent cell with pancreatin, adjusting cell suspension concentration to 6 × 10 with sterile PBS⁷one/mL. Inoculating 0.2mL of the above cell suspension subcutaneously into the right axilla of Balb/c nude mice (6 weeks old, female) until the tumor volume reaches 100mm³On the left and right, 40 mice with relatively consistent tumor sizes were selected for the experiment.

Randomly dividing the screened tumor-bearing mice into 4 groups, 10 mice in each group, except for normal diet, grouping according to the following groups, wherein a positive drug group is intravenously injected with 10mg/kg of commercial paclitaxel injection every 2 days, a negative control group is intragastrically injected with 0.2mL of physiological saline every day, and an experimental group is administered and intragastrically injected every day to give a drug loading ratio of 1:1 (60 mu g/kg) and the PCL-PEG nanoparticles (drug loading ratio is 1:1) (200 mu g/kg) of the ethanol reflux extract of the sweetsop seeds, and the experiment duration is 12 days. Observing whether the mice have abnormality or not and death conditions every day; the body weight and tumor volume (V ═ ab) of each group of mice were measured every two days ²2; a is a long side and b is a short side). On day 12, the mice were sacrificed by cervical dislocation, dissected to obtain each group of tumors and the tumor inhibition rate was calculated according to the following formula:

the tumor inhibition rate is (1-tumor weight of administration group/tumor weight of physiological saline) x 100%

The experimental results show that: the total lactone nanoparticles have the tumor inhibition rate of 65 percent on human malignant glioma U87-MG tumor-bearing mice by oral administration under the low dose of 60 mu g/kg; the tumor inhibition rate of the extract nanoparticles in a 200 mu g/kg oral administration on human malignant glioma U87-MG tumor-bearing mice reaches 71 percent, and the high-efficiency anti-tumor effect on human malignant glioma is shown. Meanwhile, the body weight of each group in hours is not different from that of the normal saline group, and none of mice in the experimental group die in the whole test process and is in a good state, so that the administration dosage of the mice in the experimental group is better in safety.

Example 19

The annona squamosa total lactone nanoparticles have an in vivo tumor inhibiting effect on human melanoma SK-MEL-28 tumor-bearing mice.

Culturing human melanoma cell SK-MEL-28 in vitro to logarithmic growth phase, digesting adherent cell with pancreatin, and adjusting cell suspension concentration to 6 × 10 with sterile PBS⁶one/mL. Inoculating 0.2mL of the above cell suspension subcutaneously into the right axilla of Balb/c nude mice (5 weeks old, female) until the tumor volume reaches 100mm³On the left and right, 40 mice with relatively consistent tumor sizes were selected for the experiment.

Randomly dividing the screened tumor-bearing mice into 4 groups, 10 mice in each group, except for normal diet, grouping according to the following groups, wherein a positive drug group is intravenously injected with 10mg/kg of commercial paclitaxel injection every 2 days, a negative control group is intragastrically injected with 0.2mL of physiological saline every day, and an experimental group is administered and intragastrically injected every day to give a drug loading ratio of 1:1 (0.1mg/kg) of PCL-PEG total lactone nanoparticles and 0.3mg/kg of PCL-PEG nanoparticles (drug loading ratio is 1:1) of annona squamosa seed ethanol reflux extract, and the experiment duration is 12 days. Observing whether the mice have abnormality or not and death conditions every day; the body weight and tumor volume of each group of mice were measured every two days (V ═ ab 2/2; a for the long side and b for the short side). On day 12, the mice were sacrificed by cervical dislocation, dissected to obtain each group of tumors and the tumor inhibition rate was calculated according to the following formula:

The experimental results show that: the total lactone nanoparticles have the tumor inhibition rate of 63 percent on human malignant black upper road tumor U87-MG tumor-bearing mice by oral administration at a low dose of 0.1 MG/kg; the tumor inhibition rate of the extract nanoparticles to human malignant glioma U87-MG tumor-bearing mice after 0.3g/kg oral administration reaches 71%, and the high-efficiency anti-tumor effect to human malignant glioma is shown. Meanwhile, the body weight of each group in hours is not different from that of the normal saline group, and none of mice in the experimental group die in the whole test process and is in a good state, so that the administration dosage of the mice in the experimental group is better in safety.

Example 20

Investigation of thermal stability

Placing the Annona squamosa total lactone effective part obtained in example 6 in an oven at 60 ℃, standing for 10 days, sampling on the 5 th and 10 th days respectively, precisely weighing an appropriate amount, detecting the content of annonaceous acetogenins after dissolving acetonitrile, and inspecting the influence of humidity on the stability of the annonaceous acetogenins.

The results are shown in fig. 13, and fig. 13 shows: the content of each index component in the annona squamosa total lactone is not obviously changed at the temperature of 60 ℃, and after the annona squamosa total lactone is placed at the high temperature of 100 ℃ for 12 hours, the content of the four components is reduced to be below 30 percent. It is suggested that the treatment temperature should not be too high during the preparation of the extract and effective part, and is preferably not higher than 70 deg.C, more preferably below 60 deg.C.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A pharmaceutical composition for the treatment of brain glioma; which comprises the following compounds

2. The use of claim 1, wherein the medicament is in a dosage form comprising: tablet, capsule, granule, microcapsule, injection, lyophilized powder for injection, gel, topical preparation or cavity administration preparation.

3. A preparation method of an annona squamosa seed extract for treating brain glioma or melanoma is characterized by comprising the following steps:

(1) crushing sweetsop seeds to obtain particles;

(2) mixing the particles with a degreasing solvent, degreasing, and carrying out solid-liquid separation to obtain filter residues;

(3) mixing the filter residue with an extraction solvent for extraction, and carrying out solid-liquid separation to obtain a liquid phase;

4. The method according to claim 3, wherein the particle size of the particulate matter in the step (1) is 2 to 25 mesh.

5. The method according to claim 4, wherein the degreasing solvent of step (2) comprises one or more of petroleum ether, n-hexane and cyclohexane; the mass ratio of the volume of the degreasing solvent to the particles is 200-10000 mL: 20-250 g; the degreasing time is 3-8 h.

6. The method according to claim 5, wherein the extraction solvent of step (3) comprises one or more of ethanol, methanol, acetone and ethyl acetate; the mass ratio of the volume of the extraction solvent to the particles is 200-2000 mL: 20 to 250 g.

7. The method according to any one of claims 3 to 6, wherein the preparation of the effective fraction is further included after the annona squamosa seed extract is obtained in the step (4).

8. The method for preparing the effective part according to claim 7, wherein the method for preparing the effective part comprises the steps of:

removing the solvent in the ethyl acetate extract to obtain an extract;

mixing the extract with silica gel, stirring, loading on a column, and sequentially mixing the extract with n-hexane in a volume ratio of 5:95 of acetone-n-hexane, in a volume ratio of 15:85, and collecting the mixture in a volume ratio of 15:85 of acetone-n-hexane eluent, and recovering the solvent under reduced pressure to obtain the effective part.

9. The Annona squamosa Linn seed extract prepared by the method of any one of claims 3 to 8, wherein the Annona squamosa Linn seed extract comprises compounds represented by formula I, formula II and formula III.

10. Use of an annona squamosa seed extract prepared by the method of any one of claims 3 to 8 in the preparation of a medicament for the treatment of glioma or melanoma.