CN111467367A

CN111467367A - Plant monomer composition for inhibiting tumor cell growth and preparation method and application thereof

Info

Publication number: CN111467367A
Application number: CN202010439015.3A
Authority: CN
Inventors: 陈炯; 向廷秀
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-05-22
Filing date: 2020-05-22
Publication date: 2020-07-31
Anticipated expiration: 2040-05-22
Also published as: CN111467367B

Abstract

The invention discloses a plant monomer composition for inhibiting tumor cell growth, and a preparation method and application thereof. The plant monomer composition consists of the following plant monomers in parts by weight: 20(R) -ginsenoside Rg 30.1-10 parts, oridonin 7-30 parts and ganoderan 5-30 parts. Preferably, the plant monomer composition consists of the following plant monomer-phospholipid complexes in parts by weight: 0.1-10 parts of 20(R) -ginsenoside Rg 3-phospholipid complex, 7-30 parts of oridonin-phospholipid complex and 5-30 parts of ganoderan-phospholipid complex. In vivo and in vitro tumor proliferation inhibition experiments prove that the plant monomer composition has the effective rate of 100 percent for inhibiting tumor proliferation and the inhibition rate of 86.13 percent. In conclusion, the plant monomer composition of the invention has obvious effect of inhibiting proliferation on tumors, and is a good drug choice for clinically treating solid tumors.

Description

Plant monomer composition for inhibiting tumor cell growth and preparation method and application thereof

Technical Field

The invention relates to a plant monomer composition, a preparation method and application thereof, in particular to a plant monomer composition for inhibiting the growth of tumor cells, a preparation method and application thereof. The invention belongs to the technical field of medicines.

Background

The american cancer society official journal "the journal of clinicians" published an "2018 report of global cancer statistics" online, and 1810 million new cancer cases and 960 million cancer death cases are predicted to be the second killer of humans after cardiovascular diseases in 2018. The number of newly added cases accounts for 380.4 ten thousands and the number of dead cases accounts for 229.6 thousands in China. The total number of tumor patients in the whole country is about 550 ten thousand at present.

The existing anti-tumor drugs and treatment methods in the world are various, and comprise chemical tumor inhibitors, natural anti-tumor drugs, cytotoxic anti-tumor drugs, anti-tumor small molecule targeted therapy, anti-tumor gene therapy, immune anti-tumor therapy and the like. But is not satisfactory because of the defects of low tumor cell inhibition rate, great toxic and side effects, long treatment period, high drug resistance of tumor cells, high treatment cost and the like.

The natural medicine is from nature, has wide source, simple extraction and relatively small toxic and side effect, and is more and more valued and favored by people. The application of natural medicine effective components in the anti-tumor clinical treatment by utilizing modern scientific technology is a research hotspot of the anti-tumor medicine at present. Although many natural plant active ingredients (plant monomers, single molecules) have been demonstrated to have good antitumor effects, their specific application to clinical treatments has been faced with the following difficulties.

① due to the low solubility of some plant antitumor effective components (plant monomers) (such as paclitaxel and oridonin with good antitumor effect), the bioavailability of the medicine is very poor, including low in vivo absorption rate, short half-life time, insufficient effective treatment concentration, etc., which causes poor antitumor treatment effect and is difficult to be practically applied in clinic.

② most of non-target antineoplastic agents have no specific inhibition effect on tumor cells (such as antineoplastic chemotherapeutic drugs and untreated natural antineoplastic drugs), and have inhibition effect on tumor tissues and damage to normal tissues of the whole body in the course of antineoplastic therapy, with the side effects of strong gastrointestinal reaction, alopecia, leukopenia, etc.

③ the pathological mechanism is very complex because the tumorigenesis is the result of multigenic mutation, and the single antitumor drug component is only used for attacking a certain part of tumor cells, so the tumor cell growth can not be completely and effectively inhibited, the effectiveness and inhibition rate of the tumor cell inhibition are relatively low, and the recurrence and metastasis rates are high.

Therefore, aiming at the problems in the prior art, the invention provides a plant monomer composition for inhibiting the growth of tumor cells and a preparation method thereof.

Disclosure of Invention

The invention aims to provide a plant monomer composition for inhibiting the growth of tumor cells, and a preparation method and application thereof.

In order to achieve the purpose, the invention adopts the following technical means:

firstly, the invention selects plant monomers (single molecules) with strong anti-tumor effect, low toxic and side effect or no toxic and side effect as main components.

Secondly, aiming at the problems of complex pathological mechanism of multiple gene mutation of malignant tumor and toxic and side effect of the medicine to human body, a plurality of plant monomers (single molecules) with anti-tumor effect are adopted as the effective component combination formula to achieve the following purposes:

①, multiple plant monomers respectively act on the tumor cells from different target positions of the tumor cells in multiple points (biological targeting), inhibit the growth of the tumor cells and induce the apoptosis of the tumor cells, thereby achieving the clinical treatment effects of inhibiting or killing the tumor cells, inhibiting the angiogenesis of the tumor, improving the active immunity of the organism and the like, and achieving the treatment effect of 1+1+ 1-4 or 5.

②, the total curative effect of the compound preparation is greatly improved, the individual plant monomer compatible dosage with larger toxic and side effects on human bodies is reduced, the toxic and side effects of the preparation on the human bodies are reduced, and the high-efficiency and low-toxicity treatment effect of the medicinal preparation on the tumor treatment process is realized.

Thirdly, the problems of insolubility, targeting and toxic and side effects of the plant monomer are solved. The plant monomer is combined with phospholipid substances to carry out plant monomer lipidization treatment to form a plant monomer-phospholipid complex, so that the plant monomer which is difficult to dissolve exists in the plant monomer-phospholipid complex in a molecular state (noncrystalline state), and the following purposes are achieved:

①, improving bioavailability (sustained release) of the medicine after oral administration, including improving the absorption rate of the medicine in small intestine, prolonging the half-life time of the medicine in blood, and maintaining the effective therapeutic concentration of the medicine in blood.

②, because the plant monomer-phospholipid complex is emulsified by bile in small intestine after oral administration to form micro emulsion with diameter of 100-300 nm, absorbed by lymphatic capillary in small intestine mucosa, enter lymphatic circulation, and then enter blood circulation, because the gap (6nm) between endothelial cells of normal capillary is smaller than the diameter of the monomer complex, the monomer complex does not enter normal tissue, but the shape of the tumor new capillary is irregular, the cell arrangement is disordered, the gap between cells is wider, the monomer complex enters tumor tissue through the tumor new capillary, the target inhibition of tumor cells (physical targeting and lymphatic directionality) is realized, and the purposes of quickly and effectively inhibiting and killing tumor cells, reducing tumor cell metastasis and tumor recurrence and the like are achieved.

③, because the plant monomer-phospholipid complex does not enter most normal tissues of human body, the medicine is mainly distributed on tumor tissues, reticuloendothelial organs such as liver and spleen, brain and other parts, thereby reducing the damage to normal tissues and indirectly reducing the toxic and side effects of the medicine.

④, the affinity (histocyte compatibility) of the plant monomer-phospholipid complex to tumor cells is improved after lipidization treatment of the plant monomer, and the plant monomer-phospholipid complex can be tightly attached to the surface of the tumor cell membrane or enter the tumor cells to persistently generate an inhibiting effect.

Finally, on the basis of the above research, the present invention provides a phytomer composition (abbreviated as "sanmate (RGO)") for inhibiting tumor cell growth, the phytomer composition comprising the following phytomers by weight: 20(R) -ginsenoside Rg 30.1-10 parts, oridonin 7-30 parts and ganoderan 5-30 parts. Wherein:

ginsenoside Rg 3: inhibiting tumor angiogenesis by inhibiting proliferation and migration of tumor vascular endothelial cells, inhibiting VEGF activity and signal transduction pathway, and inhibiting degradation of vascular extracellular matrix. Has molecular targeting property, and has no obvious toxic and side effects.

Oridonin: inducing tumor cell apoptosis, resisting mutation, inhibiting tumor growth, enhancing therapeutic effect of other antitumor drugs, and inhibiting telomerase activity. Has cytotoxicity when being used in large dose clinically.

Ganoderma lucidum polysaccharide: has effects in regulating immunity, destroying tumor blood vessel, and resisting cancer. The food and the medicine have the same source, and no obvious toxic or side effect is seen.

In order to further improve the bioavailability of the plant monomer, the invention also provides a plant monomer composition for inhibiting the growth of tumor cells, wherein the plant monomer composition consists of the following plant monomer-phospholipid complexes in parts by weight: 0.1-10 parts of 20(R) -ginsenoside Rg 3-phospholipid complex, 7-30 parts of oridonin-phospholipid complex and 5-30 parts of ganoderan-phospholipid complex.

Wherein, preferably, the phospholipid complex of the plant monomer is prepared by the following method: taking 10-70 ml of tetrahydrofuran, adding 10-30 mg of the plant monomer and 10-100 mg of the soybean lecithin, placing the mixture in an environment with the temperature of 55-65 ℃, stirring at a constant speed of 100-500 r/min until the plant monomer is completely dissolved and reacts with the soybean lecithin. And then, carrying out rotary evaporation to remove tetrahydrofuran, forming a plant monomer-phospholipid complex on the residual solid part, and respectively preparing a 20(R) -ginsenoside Rg 3-phospholipid complex, an oridonin-phospholipid complex and a ganoderan-phospholipid complex according to the method.

The administration dosage of the plant monomer composition of the invention is as follows: 20(R) -ginsenoside Rg3 (0.1-10 mg/kg), oridonin (7-30 mg/kg), ganoderan (5-30 mg/kg), and soybean lecithin (15-100 mg/kg).

Furthermore, the invention also provides application of the plant monomer composition in preparing a medicament for inhibiting the growth of tumor cells.

A medicinal preparation for inhibiting tumor cell growth is prepared by adding auxiliary materials required by preparation forming into the plant monomer composition, and preparing various clinically suitable preparations according to a conventional method of medicinal preparations.

Wherein, the preparation is preferably powder, capsule, soft capsule, granule, tablet or oil solution.

The present invention is designed and improved by the above-mentioned "drawbacks and disadvantages of the prior art" in combination with various key technical points in the technical solution, and the advantages of the present invention for tumor treatment are shown below.

1. The inhibition effect on the tumor is obvious and mainly reflected in that:

①, the results of multiple solid tumor cell inhibition experiments show that the effective rate is 100% and the inhibition rate is 87%, while the inhibition rate of the tumor cell inhibition experiment is only 41% for a single oridonin preparation with large dose and without lipidization treatment.

②, the experimental results of mice bearing tumor of solid tumor model show that the effective rate is 100%, and the inhibition rate is 86.13%^-1) The tumor inhibition rate of 3 dosage groups is only 43.94%, 54.65% and 59.57%;

2. has low toxic and side effects.

①, the result of the inhibition experiment of normal liver cells shows that no abnormal change of normal tissue cells occurs.

②, the results of acute toxicity tests on normal mice show that half of lethal dose (L D50) is not detected, only the maximum tolerated dose is detected, and the dosage is only 1/140 times of the maximum tolerated dose.

3. The targeting of the medicine is obvious, and the medicine is mainly and intensively distributed in tumor tissues, reticuloendothelial organs such as liver and spleen, brain and other parts according to the experimental speculation and literature data. The technical scheme of the invention has obvious inhibition effect on tumor cells, and has extremely low toxic and side effects on human tissue cells.

4. The invention is widely used for treating various solid tumors, is a multi-target tumor inhibitor, does not need tumor gene detection, and can save treatment cost and time for patients.

5. The invention has simple and clear prescription and conforms to the research, production and sale standards of new medicines of the FDA and European Union in the United states.

6. The invention has simple production process, and can be produced by common pharmaceutical production equipment.

7. The oral administration is simple and convenient in treatment mode.

Drawings

FIG. 1 shows the cell proliferation inhibition effect of the phytomer composition of the present invention (sanmate (RGO)) at various concentrations;

FIG. 2 is a photograph of a transplanted tumor body of a C57B L/6 mouse;

FIG. 3 is a statistical graph of transplanted tumor weights of C57B L/6 mice;

FIG. 4 is a representative graph of HE staining of transplanted tumor tissue of C57B L/6 mouse;

FIG. 5 shows the body weight changes of L D50 pre-experimental mice;

FIG. 6 is a representation of pathological HE staining of various organ tissues;

FIG. 7 shows the feed consumption of mice with maximal tolerance in acute toxicity test;

FIG. 8 shows the body weight changes of mice with maximal tolerance in acute toxicity test;

fig. 9A and 9B are representative pictures of HE staining of each organ histopathology.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. The examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

EXAMPLE 1 preparation of a phytomonomer composition for inhibiting tumor cell growth and its formulation

1. Weighing each plant monomer according to the following parts by weight:

20(R) -ginsenoside Rg 35, 20 rubescensin A and 15 ganoderan;

2. freeze drying the above plant monomers, pulverizing into powder, mixing, and making into capsule, soft capsule, tablet or oil preparation.

EXAMPLE 2 preparation of a phytomonomer composition for inhibiting tumor cell growth and a formulation thereof

1. Weighing each plant monomer according to the following parts by weight:

20(R) -ginsenoside Rg 37, rubescensin A10 and ganoderan 20;

EXAMPLE 3 preparation of a phytomonomer composition for inhibiting tumor cell growth and preparation thereof

1. Weighing each plant monomer according to the following parts by weight:

20(R) -ginsenoside Rg 310, rubescensin A10 and ganoderan 10;

EXAMPLE 4 preparation of a phytomonomer composition for inhibiting tumor cell growth and its formulation

1. Lipidation of the plant monomer to form a plant monomer-phospholipid complex:

① A lipidization treatment of oridonin comprises adding oridonin 20mg and soybean lecithin 50mg into tetrahydrofuran 50ml, stirring at 60 deg.C at constant speed at 400 rpm until oridonin is completely dissolved and reacts with soybean lecithin, and rotary evaporating to remove tetrahydrofuran to obtain oridonin-phospholipid complex.

② 20 lipidization of (R) -ginsenoside Rg3, adding 50ml tetrahydrofuran, 20(R) -ginsenoside Rg320mg and 50mg soybean lecithin, placing at 60 deg.C, stirring at constant speed, stirring at 400R/min to 20(R) -ginsenoside Rg3 completely dissolving and reacting with soybean lecithin, and removing tetrahydrofuran by rotary evaporation to obtain 20(R) -ginsenoside Rg 3-phospholipid complex.

③ A process for lipidization of Ganoderma polysaccharide comprises collecting 50ml tetrahydrofuran, adding 20mg Ganoderma polysaccharide and 50mg soybean lecithin, standing at 60 deg.C, stirring at constant speed at 400 rpm until Ganoderma polysaccharide is completely dissolved and reacts with soybean lecithin, and rotary evaporating to remove tetrahydrofuran to obtain Ganoderma polysaccharide-phospholipid complex.

2. Weighing each plant monomer-phospholipid complex according to the following parts by weight:

5 parts of 20(R) -ginsenoside Rg 3-phospholipid complex, 20 parts of oridonin-phospholipid complex and 15 parts of ganoderan-phospholipid complex;

3. freeze drying the above plant monomer-phospholipid complex, pulverizing into powder, mixing, and making into capsule, soft capsule, tablet or oil.

EXAMPLE 5 preparation of a phytomer composition for inhibiting tumor cell growth and formulation thereof

① A lipidization treatment of oridonin comprises adding 30mg of oridonin and 50mg of soybean lecithin into 60ml of tetrahydrofuran, stirring at a constant speed of 300 r/min at 55 deg.C until oridonin is completely dissolved and reacts with soybean lecithin, and removing tetrahydrofuran by rotary evaporation to obtain oridonin-phospholipid complex.

④ 20 lipidization of (R) -ginsenoside Rg3, adding 20(R) -ginsenoside Rg330mg and soybean lecithin 60mg into tetrahydrofuran 60ml, placing at 55 deg.C, stirring at uniform speed, stirring at 300R/min to completely dissolve 20(R) -ginsenoside Rg3 and reacting with soybean lecithin, and removing tetrahydrofuran by rotary evaporation to obtain 20(R) -ginsenoside Rg 3-phospholipid complex.

⑤ A process for lipidization of Ganoderma polysaccharide comprises collecting tetrahydrofuran 60ml, adding Ganoderma polysaccharide 30mg and soybean lecithin 60mg, standing at 55 deg.C, stirring at uniform speed at 300 r/min until Ganoderma polysaccharide is completely dissolved and reacts with soybean lecithin, and rotary evaporating to remove tetrahydrofuran to obtain Ganoderma polysaccharide-phospholipid complex.

7 parts of 20(R) -ginsenoside Rg 3-phospholipid complex, 10 parts of oridonin-phospholipid complex and 20 parts of ganoderan-phospholipid complex;

EXAMPLE 6 preparation of a phytomer composition for inhibiting tumor cell growth and formulation thereof

⑥ 20 lipidization of (R) -ginsenoside Rg3, adding 20(R) -ginsenoside Rg330mg and soybean lecithin 60mg into tetrahydrofuran 60ml, placing at 55 deg.C, stirring at uniform speed, stirring at 300R/min to completely dissolve 20(R) -ginsenoside Rg3 and reacting with soybean lecithin, and removing tetrahydrofuran by rotary evaporation to obtain 20(R) -ginsenoside Rg 3-phospholipid complex.

⑦ A process for lipidization of Ganoderma polysaccharide comprises collecting tetrahydrofuran 60ml, adding Ganoderma polysaccharide 30mg and soybean lecithin 60mg, standing at 55 deg.C, stirring at uniform speed at 300 r/min until Ganoderma polysaccharide is completely dissolved and reacts with soybean lecithin, and rotary evaporating to remove tetrahydrofuran to obtain Ganoderma polysaccharide-phospholipid complex.

20(R) -ginsenoside Rg 3-phospholipid complex 10 weight parts, rubescensin A-phospholipid complex 10 weight parts, and ganoderan-phospholipid complex 10 weight parts;

Example 7 tumor proliferation inhibition assay of the plant monomer composition of the present invention (sanmate (RGO))

1 materials and methods

1.1 materials

1.1.1 Experimental drugs

Plant monomer composition: prepared by the method of example 3

1.1.2 cells and animals

Human hepatoma cells Hepa3B and HepG2, human colon carcinoma cells HCT116, human normal hepatocytes L O2 and murine hepatoma cells Hepa1-6 were all purchased from ATCC (American Type Culture Collection) in the United states.

SPF grade 4-6 week old female/male C57B L/6 mice were provided by Chongqing university of medicine laboratory animal center.

1.2 methods

1.2.1CCK8 method for detecting the proliferation inhibition effect of the plant monomer composition (sanmate (RGO)) on different cells

Culturing human liver cancer separatelyThe cells Hepa3B and HepG2, human colon cancer cell HCT116 and human normal liver cell L O2 are counted in logarithmic phase, and the cell concentration is adjusted to 1 × 10³Adding the plant monomer composition (sanmate) diluent with different concentrations according to the needs after cells adhere to the wall for 4 hours per 100 mu L, adding CCk 810 mu L into each well when the plant monomer composition (sanmate) diluent acts for 0 hour, 24 hours, 48 hours or 72 hours respectively, culturing for 1 hour, and detecting the absorbance at 450 nm.

1.2.2 subcutaneous tumor formation experiment for liver cancer

Purchasing 4-6 weeks old C57B L/6 mice, breeding in animal room for 1 week, adapting to environment, culturing Hepa1-6 tumor cells, and diluting cell suspension to × 10 per ml (1-2) according to counting result⁷0.2ml of each cell was inoculated subcutaneously. Observe the animal to have a tumor of 0.5mm³When the size is large, mice are randomly divided into a control group and a Shendongling group, each group comprises 10 mice, the female part and the male part are half respectively, the control group adopts edible soybean oil of 200 mu L for intragastric administration, the Shendongling group adopts 0.25ml/kg Shendongling (RGO) stock solution (plant monomer composition soybean oil diluent) for intragastric administration, the intragastric administration is carried out once every day, the intragastric administration is continuously carried out for 14 days, the appetite condition, the mental state, the activity and the defecation condition of the mice are observed every day, the tumor volume is measured every 3 days by a vernier caliper, the tumor size calculation formula is equal to the tumor length of ×, the tumor width is 2 × 0.52.52, when the animal experiment is finished, the mice are killed by a cervical dislocation method, subcutaneous tumor tissues of the mice are weighed, the tumor size is measured, the tumor inhibition rate is calculated, the mice are fixed by 4% paraformaldehyde, and are sliced by conventional paraffin embedding, HE.

2 results

2.1CCK8 method for detecting the proliferation inhibiting effect of the plant monomer composition (sanmate (RGO)) on tumor cells

When the human hepatoma cells Hepa3B and HepG2, the human colon cancer cells HCT116 and the human normal liver cells L O2 are respectively treated by the panaxatilin (RGO) diluents with different concentrations (0, 0.15 mu M, 0.30 mu M, 0.60 mu M and 1.2 mu M), the results show that the panaxatilin (RGO) diluents do not show inhibition effect when the concentration is less than 0.3 mu M and show obvious inhibition enhancement effect along with the increase of the concentration on the Hepa3B and HCT116 cells, the inhibition effect is stronger than that of the panaxatilin (RGO) preparation 48h has half lethal concentration (IC50) of 0.6223 mu M on Hepa3B, the IC50 of HCT116 is 0.6059, the IC50 of HepG2 is 1.311 mu M, while the panaxatilin (RGO) diluents do not show inhibition effect on proliferation when the concentration is less than 1.2 mu M on the Hepa3, the normal liver cells L O2 (figure 1. mu M), and show optimal inhibition effect on the plant proliferation when the concentration is less than 0.2. mu.1. M.

2.2 the growth inhibition of the phytomer composition (ginseng radix asparagi (RGO)) of the present invention on subcutaneous tumor formation of liver cancer mice

The liver cancer cell Hepa1-6 of the mouse is transplanted to the C57B L/6 mouse subcutaneously, the growth of the tumor is observed, the size of the tumor is measured, the tumor tissue is picked up after 14 days of drug application, the weight of the tumor is weighed, and the immunohistochemical analysis is carried out on the tumor tissue, the research result shows that compared with the control group, the volume and the weight of the tumor transplanted to the subcutaneous tissue of the sanmate (RGO) preparation group are obviously reduced (figure 2, figure 3), and the average volume of the tumor of the sanmate (RGO) treatment group is 80.87mm after being measured³Mean tumor volume of 583.19mm in control group³. The data are put into a tumor inhibition rate formula, and the tumor inhibition rate of the sandong (RGO) reaches 86.13 percent.

The results of hematoxylin-eosin staining (HE staining) of tumor tissues of tumor bodies of a ginseng and Chinese dongling (RGO) treatment group and a control group show that the HE staining of the control group shows that tumor cells have typical characteristics of nuclear enlargement, irregular nuclear morphology, deep nuclear staining, nuclear cytoplasmic ratio disorder, disordered arrangement, mutual extrusion, stacking or mosaic and the like, and rarely have a vascular-like state. HE staining of tumor bodies of the Shendongling group shows that cell nuclei are solidified and condensed, chromatin is concentrated, gathers at the same time, and clings to the periphery of a nuclear membrane in a crescent shape; morphological changes in apoptosis, such as nuclear fragmentation and nuclear membrane invagination, were organized mostly with more lymphocyte infiltration (FIG. 4).

3 small knot

In vitro experiments show that the plant monomer composition (ginseng radix ophiopogonis (RGO)) has stronger effect of inhibiting tumor proliferation and smaller damage to normal cells within 0.3-1.2 mu M dosage, and reaches a peak value after 48 hours of action; in vivo experiments show that the plant monomer composition (ginseng radix ophiopogonis (RGO)) can obviously inhibit the growth of subcutaneous transplanted tumors of liver cancer mice. The experiment of in vivo and in vitro tumor proliferation inhibition effect proves that the effective rate of inhibiting tumor proliferation is 100 percent, and the inhibition rate is 86.13 percent. In conclusion, the plant monomer composition (ginseng radix panacis quinquefolii (RGO)) shows a remarkable proliferation inhibiting effect on tumors and is a good drug choice for clinically treating solid tumors.

Example 8 acute toxicity test in mice of the phytomonomer composition of the present invention (sanmate (RGO))

[ Experimental purpose ] the safety of the plant monomer composition (sanmate (RGO)) of the present invention was preliminarily evaluated by observing its acute toxicity in mice.

[ Experimental test articles ]

1. Plant monomer composition: prepared by the method of example 3

2. The clinical application amount is 0.25ml/kg stock solution (soybean oil diluent) of plant monomer composition (sanmate (RGO)).

3. Dose, route of administration set basis: the mouse is used for intragastric administration, the maximum administration volume is 0.35ml/10g, and the administration is carried out by intragastric administration once. Reference is made to clinical planned routes, guidelines for new drug research on plant monomers (pharmacy, pharmacology, toxicology).

4. Preparation: PBS was administered to the blank control group, and PBS diluted stock solution was administered to the administration group.

[ Experimental animals ]

1. The source is provided by animal experiment center of Chongqing university of medicine.

2. The species is SPF grade C57B L/6 mouse.

3. Strain: an inbred line.

4. Weight: 12-16 g.

5. Sex: the male and female are half.

6. Number of animals: 80 animals were kept in 5 cages each.

7. The experimental environment is as follows: in an IVC animal feeding room of animal experiment center of Chongqing medical university, sterile water is drunk, sterile feed is fed, and sterile padding is adopted, wherein the temperature of the laboratory is 20-24 ℃, the relative humidity is 40-70%, and the light and the shade alternate day and night.

[ Experimental methods ]

1. Mouse L D50 preliminary experiments

The method comprises the steps of numbering 50 mice with half of each of the male and female, separating the male and female, feeding the mice in cages with 5 mice in cages, randomly dividing the mice into 10 groups, wherein each group of 5 mice is respectively a PBS female group, a PBS male group, a 50ul female group, a 50ul male group, a 100ul female group, a 100ul male group, a 200ul female group, a 200ul male group, a 500ul female group and a 500ul male group. After fasting for 12 hours without water prohibition, 200ul PBS is fed into a PBS female group and a PBS male group for intragastric administration, 50ul stock solution is fed into 50ul female group and 50ul male group for 50ul and is diluted to 200ul for intragastric administration, 100ul stock solution is fed into 100ul female group and 100ul male group for 100ul and is diluted to 200ul for intragastric administration, 200ul stock solution is directly fed into 200ul female group and 200ul male group for 200ul, and 500ul stock solution is directly fed into 500ul female group and 500ul male group for 500 ul. Continuously feeding and observing for 14 days, observing the toxic reaction condition of the mice every day, and weighing the body weight of the mice one day before, 7 days after and 14 days after the gavage. Mice were sacrificed on day 14 after gavage.

2. Maximum dose test in mice

30 mice, each half of male and female, were taken and randomly divided into 4 groups, PBS female group and PBS male group, 5 mice each, 500ul female group and 500ul male group, 10 mice each. After fasting for 12h without water prohibition, 500ul stock solution of a female group and 500ul stock solution of a male group are subjected to one-time intragastric administration, and PBS is applied to the female group and the male group in an equal amount. Continuously feeding and observing for 14 days, observing the toxic reaction condition of the mice every day, and weighing the weight and the feed consumption of the mice one day before the gavage, 1 day after the gavage, 3 days after the gavage, 5 days after the gavage, 7 days after the gavage, 9 days after the gavage, 11 days after the gavage and 13 days after the gavage. Mice were sacrificed on day 14 after gavage.

3. Systematic autopsy and histopathological examination

3.1 systematic dissection

All experimental mice were dissected gross at the end of the experiment. Animals sacrificed due to dying during the experiment and dead animals are subjected to systematic dissection and pathological tissue examination in time.

3.2 histopathological examination

The brain, heart, liver, spleen, lung, kidney, stomach and intestine of all experimental animals were examined histopathologically. All tissues were fixed with 4% paraformaldehyde, sectioned by conventional paraffin embedding, HE stained, light-microscopic and slide-shot. No obvious abnormality was observed under an optical microscope.

[ statistical treatment ]

The measurement data are all expressed by x + s, and the differences among the comparison groups are analyzed by SPSS 25.0 software package multi-factor variance.

[ Experimental results ]

1. Mouse L D50 preliminary experiments

1.1 toxicity of the animal

After administration, the mice in 500ul of the administration group had relatively reduced activity, poor spirit, recovery within 2-3 hours, slight contamination of the fur, and wet sweat. There was no significant difference between the other indices. No obvious difference exists among groups in the aspects of continuously observing the behavior, activity, mental state and the like of the mice within 14 days.

1.2 weight Change before and after gastric lavage

The body weight changes before and after gavage are shown in table 1 and fig. 5.

TABLE 1 weight changes before and after gastric lavage

1.3 animal mortality

After 50ul, 100ul, 200ul and 500ul of sandongling stock solution (RGO) (plant monomer composition soybean oil diluent) is given to the mice at one time, the mice are observed for 14 days continuously, and the death of the mice is not found, which indicates that half of the death causing amount cannot be caused when the sandongling is given to the mice at one time.

1.4 histopathological examination

The light microscope shows that no obvious pathological changes of organs of each group are found, and no obvious changes caused by drug toxicity of the organ tissues of the experimental animal are found (figure 6).

2. Maximum dose test in mice

2.1 animal toxicity reaction conditions

After the gavage, the activity of the mice is relatively reduced, the fur is slightly polluted, sweat is wetted, and the mice recover within 12 hours.

2.2 feed consumption before and after intragastric administration

The first day after gavage, the relative reduction in the gavage group feed consumption, the loss of appetite in the mice, and recovery within two days, suggest that the phytomonomer composition of the invention (sanmate (RGO)) may have toxicity that could potentially cause a slight loss of appetite (see figure 7).

2.3 weight Change before and after gastric lavage

The body weight changes before and after gavage are shown in table 2 and fig. 8.

TABLE 2 weight changes before and after gastric lavage

After gavage, weight loss occurred in the drench group, which was associated with sweat evaporation and reduced feed consumption, suggesting that the phytomonomer composition of the present invention (sanmate (RGO)) had potential side effects of slight weight loss.

2.4 mortality: no death was observed for 14 consecutive days after administration.

2.5 histopathological examination

The light microscope shows that no obvious pathological changes of organs of each group are found, and no obvious changes caused by drug toxicity are found in the organ tissues of the experimental animal (fig. 9A and 9B).

[ conclusion ]

L D50 cannot be detected through experiments, and therefore, the maximum administration amount experiment of mice is carried out, the maximum administration amount enema is reduced in feed consumption and weight after gastric lavage, the mice recover within two days, and other observation indexes among the groups are not obviously different, so that the plant monomer composition (ginseng winter jasmine (RGO)) is low in acute toxicity, and the clinically planned dose is only 1/140 times of the maximum administration amount.

Claims

1. The plant monomer composition for inhibiting the growth of tumor cells is characterized by consisting of the following plant monomers in parts by weight: 20(R) -ginsenoside Rg 30.1-10 parts, oridonin 7-30 parts and ganoderan 5-30 parts.

2. A plant monomer composition for inhibiting the growth of tumor cells is characterized by consisting of the following plant monomer-phospholipid complexes in parts by weight: 0.1-10 parts of 20(R) -ginsenoside Rg 3-phospholipid complex, 7-30 parts of oridonin-phospholipid complex and 5-30 parts of ganoderan-phospholipid complex.

3. The phytomer composition of claim 1, wherein the phospholipid complex of a phytomer is prepared by: taking 10-70 ml of tetrahydrofuran, adding 10-30 mg of the plant monomer and 10-100 mg of the soybean lecithin, placing in an environment temperature of 55-65 ℃, stirring at a constant speed, and stirring at a rotating speed of 100-500 r/min until the plant monomer is completely dissolved and reacts with the soybean lecithin; and then, carrying out rotary evaporation to remove tetrahydrofuran, forming a plant monomer-phospholipid complex on the residual solid part, and respectively preparing a 20(R) -ginsenoside Rg 3-phospholipid complex, an oridonin-phospholipid complex and a ganoderan-phospholipid complex according to the method.

4. Use of a phytomer composition according to any one of claims 1-3 in the manufacture of a medicament for inhibiting tumor cell growth.

5. A pharmaceutical preparation for inhibiting tumor cell growth, which is characterized in that auxiliary materials required by preparation forming are added into the plant monomer composition of any one of claims 1 to 3, and the plant monomer composition is prepared into various clinically suitable preparations according to a conventional method of pharmaceutical preparations.

6. The method according to claim 5, wherein the preparation is a powder, a capsule, a soft capsule, a granule, a tablet or an oil.