CN111467367B - Plant monomer composition for inhibiting tumor cell growth and preparation method and application thereof - Google Patents
Plant monomer composition for inhibiting tumor cell growth and preparation method and application thereof Download PDFInfo
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- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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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
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.
Part of the plant anti-tumor active ingredients (plant monomers) have low solubility (such as taxol, oridonin and the like with good anti-tumor effect), so that the bioavailability of the medicament is extremely poor, the absorption rate in vivo is low, the half-life period is short, the effective treatment concentration is insufficient, and the like, so that the anti-tumor treatment effect is poor, and the difficulty is caused because the anti-tumor active ingredients cannot be practically applied to clinic.
Secondly, most of non-targeted antitumor agents have no specific inhibition effect on tumor cells (such as antitumor chemotherapeutic drugs, untreated natural antitumor drugs and the like), and in the process of antitumor treatment, the non-targeted antitumor agents have the inhibition effect on tumor tissues and damage to normal tissues of the whole body, and have toxic and side effects of strong gastrointestinal reaction, alopecia, leukopenia and the like.
And thirdly, because the tumorigenesis is the result of polygenic mutation, the pathological mechanism is very complex, the tumor cells can not be comprehensively and effectively inhibited from growing only by attacking a certain part of the tumor cells by means of a single antitumor drug component, the effectiveness and inhibition rate of the tumor cells 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:
the 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. The clinical treatment efficacy of inhibiting or killing tumor cells, inhibiting tumor angiogenesis, improving the active immunity of the organism and the like is achieved, and the treatment effect of 1+1+ 1-4 or 5 is achieved.
Secondly, the total curative effect of the compound preparation is greatly improved, the individual plant monomer compatibility 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:
the bioavailability (slow release property) of the medicine after oral administration is improved, including the absorption rate of the medicine in small intestine, the half-life time of the medicine in blood is prolonged, the effective treatment concentration of the medicine in blood is maintained, and the like.
Secondly, after the plant monomer-phospholipid complex is taken orally, the plant monomer-phospholipid complex is emulsified by bile in the small intestine to form micro emulsion, the diameter of the micro emulsion is between 100 and 300nm, the micro emulsion is absorbed by capillary lymphatic vessels in mucosa of the small intestine, enters lymphatic circulation and then enters blood circulation. Because the gap (6nm) between the endothelial cells of the normal capillary is smaller than the diameter of the monomer compound, the monomer compound does not enter normal tissues, but tumor neocapillary has irregular shape, disordered cell arrangement and wider gap between the cells, and the monomer compound enters tumor tissues through the tumor neocapillary, thereby realizing the targeted inhibition of tumor cells (physical targeting and lymphatic directionality), and achieving the purposes of quickly and effectively inhibiting and killing the tumor cells, reducing the tumor cell metastasis, tumor recurrence and the like.
And thirdly, because the plant monomer-phospholipid complex does not enter most normal tissues of the human body, the medicine is mainly and intensively distributed on tumor tissues, reticuloendothelial organs such as the liver and the spleen, brain and other parts, the damage to the normal tissues is reduced, and the toxic and side effects of the medicine are indirectly reduced. Meanwhile, the curative effect on liver and brain tumors is more obvious.
The affinity (histocyte compatibility) of the plant monomer-phospholipid complex to the tumor cells is improved after the plant monomer is subjected to lipidization treatment, 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:
the results of various solid tumor cell inhibition experiments show that the effective rate is 100 percent and the inhibition rate is 87 percent. The inhibition rate of the tumor cell inhibition experiment of a single oridonin preparation which is large in dose and is not subjected to lipidization treatment is only 41 percent.
② the experimental result of a tumor-bearing mouse of a solid tumor model shows that the effective rate is 100 percent and the inhibition rate is 86.13 percent. The single oridonin preparation has low, medium and high content (67, 100, 150 mg/kg) in the result of tumor-bearing mouse experiment for inhibiting solid tumor model-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.
The results of normal hepatocyte inhibition experiments show that no abnormal change of normal tissue cells occurs.
②, the results of the acute toxicity test of normal mice show that half of lethal dose (LD50) is not detected, only the maximum tolerated dose is detected, and the dosage is only 1/140 times of the maximum tolerated dose. Meanwhile, the morphological abnormal change is not found in the section examination of the important organ histological cells of the mouse.
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 C57BL/6 mouse;
FIG. 3 is a statistical graph of transplanted tumor weights of C57BL/6 mice;
FIG. 4 is a representative graph of HE staining of transplanted tumor tissue of C57BL/6 mouse;
FIG. 5 shows the body weight change of LD50 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;
2. freeze drying the above plant monomers, pulverizing into powder, mixing, and making into capsule, soft capsule, tablet or oil preparation.
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;
2. freeze drying the above plant monomers, pulverizing into powder, mixing, and making into capsule, soft capsule, tablet or oil preparation.
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:
processing oridonin lipidization: taking 50ml of tetrahydrofuran, adding 20mg of oridonin and 50mg of soybean lecithin, placing in an environment temperature of 60 ℃, stirring at a constant speed, and stirring at a rotation speed of 400 r/min until the oridonin is completely dissolved and reacts with the soybean lecithin. Thereafter, the tetrahydrofuran was removed by rotary evaporation and the remaining solid fraction formed the oridonin-phospholipid complex.
② the lipidization treatment of 20(R) -ginsenoside Rg 3: adding 20(R) -ginsenoside Rg320mg and soybean lecithin 50mg into tetrahydrofuran 50ml, stirring at 60 deg.C at constant speed at 400R/min until 20(R) -ginsenoside Rg3 is completely dissolved and reacts with soybean lecithin. Thereafter, the tetrahydrofuran was removed by rotary evaporation, and the remaining solid portion formed 20(R) -ginsenoside Rg 3-phospholipid complex.
③ lipidization treatment of ganoderma lucidum polysaccharide: adding 50ml of tetrahydrofuran, 20mg of ganoderan and 50mg of soybean lecithin into the tetrahydrofuran, placing the mixture in an environment with the temperature of 60 ℃, stirring at a constant speed, and stirring at the rotating speed of 400 r/min until the ganoderan is completely dissolved and reacts with the soybean lecithin. Thereafter, the tetrahydrofuran was removed by rotary evaporation, and the remaining solid portion formed ganoderan-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. Lipidation of the plant monomer to form a plant monomer-phospholipid complex:
processing oridonin lipidization: taking 60ml of tetrahydrofuran, adding 30mg of oridonin and 50mg of soybean lecithin, placing in an environment temperature of 55 ℃, stirring at a constant speed, and stirring at a rotating speed of 300 r/min until the oridonin is completely dissolved and reacts with the soybean lecithin. Thereafter, the tetrahydrofuran was removed by rotary evaporation and the remaining solid fraction formed the oridonin-phospholipid complex.
Fourthly, carrying out lipidization treatment on 20(R) -ginsenoside Rg 3: adding 20(R) -ginsenoside Rg 330 mg and soybean lecithin 60mg into tetrahydrofuran 60ml, stirring at 55 deg.C at uniform speed at 300R/min until 20(R) -ginsenoside Rg3 is completely dissolved and reacts with soybean lecithin. Thereafter, the tetrahydrofuran was removed by rotary evaporation, and the remaining solid portion formed 20(R) -ginsenoside Rg 3-phospholipid complex.
Ganoderma lucidum polysaccharide lipidization treatment: taking 60ml of tetrahydrofuran, adding 30mg of ganoderan and 60mg of soybean lecithin, placing in an environment temperature of 55 ℃, stirring at a constant speed, and stirring at a rotating speed of 300 r/min until the ganoderan is completely dissolved and reacts with the soybean lecithin. Thereafter, the tetrahydrofuran was removed by rotary evaporation, and the remaining solid portion formed ganoderan-phospholipid complex.
2. Weighing each plant monomer-phospholipid complex according to the following parts by weight:
7 parts of 20(R) -ginsenoside Rg 3-phospholipid complex, 10 parts of oridonin-phospholipid complex and 20 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 6 preparation of a phytomer composition for inhibiting tumor cell growth and formulation thereof
1. Lipidation of the plant monomer to form a plant monomer-phospholipid complex:
processing oridonin lipidization: taking 60ml of tetrahydrofuran, adding 30mg of oridonin and 50mg of soybean lecithin, placing in an environment temperature of 55 ℃, stirring at a constant speed, and stirring at a rotating speed of 300 r/min until the oridonin is completely dissolved and reacts with the soybean lecithin. Thereafter, the tetrahydrofuran was removed by rotary evaporation and the remaining solid fraction formed the oridonin-phospholipid complex.
Sixthly, lipidization treatment of 20(R) -ginsenoside Rg 3: adding 20(R) -ginsenoside Rg 330 mg and soybean lecithin 60mg into tetrahydrofuran 60ml, stirring at 55 deg.C at uniform speed at 300R/min until 20(R) -ginsenoside Rg3 is completely dissolved and reacts with soybean lecithin. Thereafter, the tetrahydrofuran was removed by rotary evaporation, and the remaining solid portion formed 20(R) -ginsenoside Rg 3-phospholipid complex.
Seventhly, lipidization treatment of ganoderma lucidum polysaccharide: taking 60ml of tetrahydrofuran, adding 30mg of ganoderan and 60mg of soybean lecithin, placing in an environment temperature of 55 ℃, stirring at a constant speed, and stirring at a rotating speed of 300 r/min until the ganoderan is completely dissolved and reacts with the soybean lecithin. Thereafter, the tetrahydrofuran was removed by rotary evaporation, and the remaining solid portion formed ganoderan-phospholipid complex.
2. Weighing each plant monomer-phospholipid complex according to the following parts by weight:
20(R) -ginsenoside Rg 3-phospholipid complex 10 weight parts, rubescensin A-phospholipid complex 10 weight parts, and ganoderan-phospholipid complex 10 weight parts;
3. freeze drying the above plant monomer-phospholipid complex, pulverizing into powder, mixing, and making into capsule, soft capsule, tablet or oil.
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 LO2, 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 C57BL/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
Human hepatoma cells Hepa3B and HepG2, human colon cancer cells HCT116 and human normal liver cells LO2 were cultured to logarithmic phase of growth, respectively. Counting cells in logarithmic growth phase, and adjusting cell concentration to 1 × 103Adding each cell per 100 mu L, adding a 96-well plate, adding plant monomer composition (sanmate) diluent with different concentrations according to the requirement after the cells adhere to the wall for 4 hours, adding CCk 810 mu L into each well when acting 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
C57BL/6 mice, 4-6 weeks old, were purchased in female and male halves. Culturing in animal room for 1 week, adapting to environment, culturing Hepa1-6 tumor cells, and diluting cell suspension to 1-2 × 10 per ml according to counting result70.2ml of each cell was inoculated subcutaneously. Observe the animal to have a tumor of 0.5mm3When the size is small, the mice are randomly divided into a control group and a Shendongling group, wherein each group comprises 10 mice, and the mice are half female and half male. The control group was intragastrically administered with 200 μ L of edible soybean oil; the SHENDONGLING group adopts 0.25ml/kg SHENDONGLING (RGO) stock solution (diluted soybean oil of plant monomer composition) for intragastric administration once per day, and continuously perfusingStomach for 14 days. Mice were observed daily for appetite, mental state, mobility, and defecation, and tumor volume was measured with a vernier caliper every 3 days. The tumor size calculation formula is tumor length × tumor width 2 × 0.52. When the animal experiment is finished, the mouse is killed by a cervical dislocation method, the subcutaneous tumor tissue of the mouse is weighed, the size of the tumor is measured, and the tumor inhibition rate is calculated; fixing with 4% paraformaldehyde, embedding in paraffin, HE staining, observing with light microscope, and taking photograph.
2 results
2.1CCK8 method for detecting the proliferation inhibiting effect of the plant monomer composition (sanmate (RGO)) on tumor cells
Human hepatoma cells Hepa3B and HepG2, human colon cancer cells HCT116 and human normal liver cells LO2 were treated with different concentrations (0, 0.15. mu.M, 0.30. mu.M, 0.60. mu.M, 1.2. mu.M) of ginseng radix Rubrum (RGO) dilutions, respectively, and the results showed that Hepa3B and HCT116 cells did not exhibit inhibitory effect at a concentration less than 0.3. mu.M, exhibited a significantly enhanced inhibitory effect with increasing concentration, and the inhibitory effect was stronger than that of HepG 2. The half-lethal concentration (IC50) of sanmate (RGO) preparation 48h to Hepa3B was 0.6223. mu.M, the IC50 of HCT116 was 0.6059, and the IC50 of HepG2 was 1.311. mu.M. Whereas human normal hepatocytes LO2 showed no proliferation-inhibiting effect at concentrations less than 1.2. mu.M (FIG. 1). Therefore, the sanmate (RGO) stock solution (diluted soybean oil as a plant monomer composition) shows the effect of inhibiting tumor proliferation and causing less damage to normal cells at a concentration of 0.3-1.2. mu.M, which is the optimum therapeutic concentration.
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
Murine hepatoma cells Hepa1-6 were subcutaneously transplanted into C57BL/6 mice, tumor growth was observed, and tumor size was measured. Tumor tissues were harvested 14 days after administration, tumor weights were weighed, and immunohistochemical analysis was performed on the tumor tissues. As a result of the study, the volume and weight of the tumor implanted subcutaneously in the sanmate (RGO) preparation group were significantly reduced as compared with the control group (FIG. 2, FIG. 3). The average tumor volume of the Shendongling (RGO) treated group is determined to be 80.87mm3Mean 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 are as follows: SPF grade C57BL/6 mice.
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 LD50 preliminary experiment
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 LD50 preliminary experiment
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 ]
The LD50 could not be detected by experiment, so the experiment of maximum dose of mouse was carried out. After the maximum dosage drenching group is dredged after gastric lavage, the feed consumption and the body weight are reduced, the recovery is realized within two days, and other observation indexes among the groups have no obvious difference, which shows that the plant monomer composition (ginseng radix ophiopogonis (RGO)) has lower acute toxicity and the clinically planned dosage is only 1/140 times of the maximum dosage. The plant monomer composition (ginseng radix panacis quinquefolii (RGO)) provided by the invention is suggested to have certain safety in clinical application.
Claims (7)
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 2, wherein the phytomer-phospholipid complex 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 pharmaceutical preparation according to claim 5, wherein the preparation is a powder, capsule, granule, tablet or oil.
7. The pharmaceutical formulation of claim 6, wherein the capsule is a soft capsule.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101495108A (en) * | 2006-05-11 | 2009-07-29 | 活性植物科技有限公司 | Composition for the treatment of resistant cancers comprising oridonin |
| CN102579883A (en) * | 2012-03-14 | 2012-07-18 | 周永刚 | Medicament-Shenxianling granules for preventing and treating cancers and preparation method of same |
| CN105796638A (en) * | 2016-05-20 | 2016-07-27 | 中山大学 | Applications of mixed oridonin and cryptotanshinone in preparing medicine for treating leukemia |
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| CN101695513B (en) * | 2009-10-28 | 2011-12-14 | 上海永神生物科技有限公司 | Composition with anti-tumor effect and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101495108A (en) * | 2006-05-11 | 2009-07-29 | 活性植物科技有限公司 | Composition for the treatment of resistant cancers comprising oridonin |
| CN102579883A (en) * | 2012-03-14 | 2012-07-18 | 周永刚 | Medicament-Shenxianling granules for preventing and treating cancers and preparation method of same |
| CN105796638A (en) * | 2016-05-20 | 2016-07-27 | 中山大学 | Applications of mixed oridonin and cryptotanshinone in preparing medicine for treating leukemia |
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